Monograph On Fungal Diseases of Wild Animals A guide for postgraduate students in developing countries A lion stalks among the hieroglyphics at the temple of Karnak in Luxor, Egypt. (Niels vanGijn/JAI/Corbis) By Mohamed K. Refai, Abd-Elhamid Z. Fahmy and Heba N. Deif Cairo, 2017 1 Refai et al. (2017) Monograph OnFungal Diseases of Wild Animals A guide for postgraduate students in developing countries. https://www.academia.edu/manuals http://scholar.cu.edu.eg/?q=hanem/book/ https://www.researchgate.net/publication Prof. Dr. Mohamed K. Refai Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza Prof. Dr. Abd-Elhamid Z. Fahmy Department of Animal Hygiene, Faculty of Veterinary Medicine, Cairo University, Giza Dr. Heba N. Deif, Lecturer Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza 2 Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Introduction 6 Ringworm in wild animals 33 Chrysosporium-Related Fungal infections in wild animals 77 Snake fungal disease (SFD) 105 White-nose syndrome (WNS) 124 Chytridiomycosis in wild animals 153 Aspergillosis in wild animals 182 Penicilliosis in wild animals 219 Zygomycosis in wild animals 228 Beauveria bassiana infection in wild animals 239 Fusarium infections in wild animals 245 Paecilomyces lilacinus infection in wild animals 249 Dematiaceous fungal infections in wild animals 253 Scedosporium apiospermum infection in wild animals 266 Sporobolomyces infection in wild animals 267 Pneumocystosis infection in wild animals 269 Adiaspiromycosis infection in wild animals 283 Lobomycosis in wild animals 297 Cryptococcosis in wild animals 313 Candidosis in wild animals 339 Histoplasmosis in wild animals 353 Blastomycosis in wild animals 367 Coccidioidomycosis in wild animals 386 Para coccidioidomycosis in wild animals 400 Sporotrichosis in wild animals 415 3 Preface This monograph is dedicated to all colleagues who helped me to publish studies on wild animals Prof. Wolfgand Bisping Prof. Rudolf Rohde Dr. Mohamed Amer Prof. Hosam Kotb      Prof. Hans Rieth Prof. Omar Tamam My interest in wildlife started in Hannover, Germany, in 1962, when I visited the Zoo together with Prof. Bisping to collect samples from monkeys with ringworm. The paper was published in 1964 (Refai, M. and Bisping, W. : Ueber das Vorkommen von Hautpilzen bei Affen. Kleintier Prax. 9, 147-150, 1964). In Hamburg,in 1964, I accompanied Prof. Rieth in visiting the Circus to inspect 2 tigers with ringworm and the paper was published in 1965(Refai, M. and Rieth, H. : Mikrosporie bei 2 Tigern. Mykosen 8, 62-65 (1965). In Hamburg, during my work in the National Salmonella Center (1964-1965) I had a chance to work on snakes , where I could isolate Salmonella and new Arizona species from the faeces of poisonous snakes and the paper was published in 1965 (Rohde, R. and Refai, M. : Ueber die multiple Infektion einer exotischen Giftschlange mit zwei Arizona- und einer Salmonella species unter besonderer Beruecksichtigung der neuen Arizona 1,4:26:21. Z. Hyg. 151, 178-182 (1965) In 1968, I visited my friend Dr. Mohamed Amer in the Giza Zoo, who kindly collected faecal balls from the snakes, which I took with me in one of my frequent visits to Hamburg, where I could isolate several Arizona and Salmonella serovars, among which one isolate had a new antigenic combinations, which I studied in detail during my Alexander von Humboldt scholarship (1971-1072) and the isolate was approved as a new serovor and was named Salmonell giza and the paper was published in 1971 (Rohde, R. and Refai, M. : Uebr die Isolierung von Salmonella- und Arizona-Bakterien aus Faecesproben von Kaltbluetern in zoologischen Garten von Giza unter besonderer Beruecksichtigung einer neuen Salmonella-species, Salmonella giza 8:y:1,2. Zbl. Vet.-Med. 18, 400-404,1971) The first thesis on wildlife done under my supervision was that of Prof. Hosam Kotb (H.R. Kotb: Studies on fungi of rats. MS thesis, Cairo University, 1986), from which a paper was published in 1988 (Kotb, H., Refai, M. and El-Far, F. : Occurrence and significance of moulds and yeasts isolated from wild rats in Egypt. J. Egypt. Vet. Med. Ass. 48, 243-253 (1988) Lastly I had the chance to cooperate with Prof . Dr. Omar Tamam, Dean of the Institute of Environmental Studies and Research in 2 papers published in 2013 and 2014 (Omar A.S. Tamam* and Mohamed Refai, Dual mycotic pulmonary granulomas caused by alternaria alternata and Aspergillus candidus in the wild egyptian mole rat (Spalax leucodon egyptiacus). Assiut Vet. Med. J. Vol. 59 No. 139 October 2013 , Omar A.S. Tamam and Mohamed Refai, A retrospective analysis of mortalities in Greater Red 4    Musk Shrews (Crocidura flavescens). Egypt. J. Comp. Path &Clinic Path..27,.1, 2014 ; 73- 87). I came in close contact with wild animals in the Animal Park, Hannover and in a private small animal clinic in Hamburg. I came in the vicinity of many wild animals, while I drove my car, when I visited the National parks in Kenya, Zambia and Tanzania during my work as Food Microbiology FAO consultant in East Africa (1976-1978) and in Nigeria, 1981 (Kaduna) and I made a tour in the Zambezi River and enjoyed seeing the hippos., 1977. I spent nights in the within national parks in Mount of Kenya Lodge (1977), In Kilimanjaro lodge Arusha, Tanzania, 1979 and in Berg-en-Dale: cottage, , Kruger National Park, South Africa, 1998 Animal Park Hannover, Germany Private clinic Hamburg, Germany In the street of New Delhi, India Tsavo National Park Hippos in the zambezi river Kruger National Park Mount Kenya Lodge tanzania national park Kamuku National Park, Kaduna kilimanjaro lodge Arusha Berg-en-Dal: cottage, , Kruger National Park Prof. Dr. Mohamed Kamal Refai, Cairo, April 2017 5 1. Introduction Egypt is bordered by the Mediterranean Sea to the north, Libya to the west and Sudan to the south. To the east lies the Red Sea, and the Sinai Peninsula, the Asian part of the country, which is bordered by the Gaza Strip and Israel. Egypt is a transcontinental country, providing a land bridge between Africa and Asia. This results in the flora and fauna having influences from both Africa and Asia, and the marine life from both the Atlantic, Mediterranean Sea, the Red Sea and Indian Ocean. To the west of the Nile lies the Western Desert, occupying about two thirds of the area of the country. It consists largely of high stony and sandy plains with rocky plateaux in places. In the extreme southwest of the country on the border with Libya and Sudan, is Jebel Uweinat, a mountainous region and in the northwest lies the Qattara Depression. Another depression, the Faiyum Oasis lies south west of Cairo and is connected to the Nile by a channel. To the east of the Nile lies the much smaller Eastern Desert, a high mountain ridge running parallel with the Red Sea, seamed with wadis on either flank. At the border with Sudan this rises to the rocky massif of Gebel Elba. Therefore, Egypt have hosted a luxurious variety of native, migratory, hunted and imported species of animal life — the greatest variety in any ecosystem outside the tropical rain forests There are over 200 of wild animals native to, migrating through, or imported into Egypt during the time of the pharaohs. Included are such invertebrates as the dreaded scorpions and the sacred scarab; such amphibians and reptiles as the fearsome Egyptian cobra and the infamous Nile crocodile; and such mammals as the sacred baboon, the grave-robbing jackals, the majestic lion, the formidable hippopotamus, the graceful gazelle, and the host of helpful livestock. Egyption hunter with his hunting dog Exquisite ancient egyptian hunting scene with Rameses III in a chariot hunting wild bulls with spear and arrows in a marsh Luxor in Egypt 6 Ancient Egyptian Trade Boundless Many of these animals have been curved or painted in tombs of Ancient Egypt. Ancient tomb paintings show giraffes, hippopotamuses and crocodiles and the petroglyphs at Silwa Bahari on the upper Nile, between Luxor and Aswan, show elephants, white rhinoceroses, gerenuk and more ostriches, a fauna akin to that of present-day East Africa. Pinterest Giraffe - tomb of Rekhmire ... painted in ancient Egyptian tombs at Saqqara. The crocodile and the hippopotamus were the evil animals which had to be killed to allow the ship of Stock Photo - Bas-relief of a lion on the wall of Karnak Temple in Egypt 7 Bas-relief from Ptahhotep's mastaba, Saqqara, Egypt FeaturePics.com Ancient Egyptian elephant and bear images about Ancient Egypt cultures on Pinterest |... Pinterest Egypt where it all begins 8 The 'walking whales' of Egypt: Fossils in the desert are remains of sea mammals that ruled the oceans 37 million years ago. Dozens of rare fossilised whale skeletons have emerged from the sands of the Egyptian Saharan desert A new $2.17 billion (£1.5 billion) museum has now opened on the site to help preserve the fossils At its centre is a 37 million-year-old legged form of early whale that measures more than 65 feet (20m) long They provide a rare record of how modern whales evolved to swim in the oceans from land mammals Fossilized whale bones are on display outside the Wati El Hitan Fossils and Climate Change Museum The largest intact Basilosaurus isis whale fossil - an early formed of 'legged whale' 9 Egypt's Endangered Mammals Arabian Leopards, Mediterranean Monk Seals, Addaxes, and Nubian Wild Asses are Critically Endangered in Egypt. Foundation for the Protection of the Arabian Leopard in Yemen Mediterranean Monk Seal - Marine Mammal Commission Marine Mammal Commission 10 The Addax (Science) on emaze Emaze Nubian wild ass | Natural History Retrieverman The Western Desert Mammals have been depleted over the years and the addax and scimitar oryx are no longer found there, and the Atlas lion has probably gone as well. 11 Addax, World Atlas Scimitar oryx - Wikipedia Katten | Egypt, Other and Animals Pinterest The Barbary Lion has a much larger mane. Regal. We have made them extinct in the wild. The Western Desert Remaining Mammals include the rhim gazelle, dorcas gazelle, Barbary sheep, Rüppell's fox, lesser Egyptian jerboa and Giza gerbil. . 12 Slender-horned Gazelle, Rhim , Until the 1950s the Dorcas Gazelle was commonly found in large herds. Now, because of poaching and the degradation of its habitat, they are usually just seen in pairs. The Dorcas Gazelle is highly adapted to the desert. the Barbary sheep was widely believed to be extinct in Egypt, and although scientists have proved that the sheep still exists here outside of the Giza Zoo, its small population remains threatened. The Eastern Desert has a quite different range of fauna and has much in common with the Sinai Peninsula, showing the importance of the broad Nile in separating the two desert regions. Here are found the striped hyena, Nubian ibex, bushy-tailed jird, golden spiny mouse, Blanford's fox and Rüppell's fox. The sand partridge, streaked scrub warbler, mourning wheatear and white-crowned wheatear are typical of this region. The high rocky mountains of Gebel Elba in the south have a distinctive range of animals including the aardwolf, striped polecat, and common genet, and there may still be African wild ass in this area. 13 The high rocky mountains of Gabel Elba in the south have a distinctive range of animals including the aardwolf, striped polecat, and common genet, and there may still be African wild ass in this area. About thirty species of snake occur in Egypt, about half of them venomous. These include the Egyptian cobra, false smooth snake and horned viper. There are also numerous species of lizards. Pictures of wild animals in Egypt Amphibians in Egypt Pelophylax ridibundus - Wikipedia Amietophrynus - Wikipedia Nile Delta Toad - Amietophrynus kassasii .. . Alamy , Amietophrynus regularis Nile Soft-Shelled Turtle, Nile Valley Toad, Bufo kassasii Bufo Viridis / Green Toad Dreamstime.com The Little Egyptian Tortoise, Spur-Thighed Mediterranean Tortoise egyptian sand gecko Egyptian Gecko, Tarentola annularis Lizards species of Egypt Egyptian Fan-toed Gecko 14 Qattara Gecko, Tarentola mindiae Desert Agama Spiny Agama, Agama spinosa Egyptian Spiny-Tailed Lizard Savignys Agama, Trapelus savignii Egyptian sandy Lizard Audouin’s Sand Skink, Sphenops sepsoides Desert Monitor, Varanus griseus Nidua Lizard, Acanthodactylus scutellatus Hayden's Animal Facts Nile monitor African chameleon - Wander Lord Desert Monitor, Varanus griseus The Egyptian Snakes The Egyptian Sand Boa The Avicenna Viper, Cobras: The Egyptian Cobra (An “Asp“) The Carpet Viper 15 Horned Desert Viper The Nile Crocodile. Pinterest The Nile Crocodile Hedgehogs The Long-Eared Desert Hedgehog The Desert Hedgehog Shrews The Lesser White-Toothed Shrew The North African Elephant-Shrew Bats The Egyptian Fruit Bat Short-Tailed Tomb Bats 16 Egyptian Slit-Faced Bat | ChiropteraPinterest Primates (Non-Human) The (Hamadryas) Baboon The Anubis Baboon The (Long-Tailed) The North African Banded Weasel Wiki Grass Monkey (A Guenon) Alamy Eurasian River Otter by nitsch Egyptian Mongoose (Ichneumon) Canines The Common (Small-Spotted) Genet Striped Hyena Egyptian Gray Wolf Golden Jackal The Fennec Fox Sand Fox The Aardwolf Black-Backed Jackal Red Fox (Nile Fox 17 Sal3wa. Salawaa Scoop Empire Felines African Wild Cat, Caracal (The Desert ―Lynx―), The Leopard, Jungle Cat (The Swamp ―Lynx―) The Cheetah, The Lion Rhinoceros The African Elephant Dugong Hippopotamus 18 The Coney (The Abyssinian Rock Hyrax) The Wild Boar Wild animals fulfilled spiritual needs of the ancient Egyptians The baboon The baboon was associated with Thoth, Khonsu and Hapy, gods that possessed the qualities of eloquence, strength, fairness and responsibility. Thoth was the god who was responsible for the lunar-based calendar and was often depicted with the head of a baboon in ancient Egyptian pictographs. A baboon, identified with the ancient Egyptian god Thoth, holding an Eye of Horus, Hapy (baboon-headed god) canopic jars contained the lungs of a mummified Egyptian Statue of a scribe writing at the feet of Thoth as a lunar baboon Baboons worshipping the sun god with Ramesses III at Medinet Habu 19 Lions: The power and danger seen in the lion became synonymous with the pharaoh. The lion was a symbol of the pharaoh's power and rulership. As the lions lived in the eastern and western deserts around the Nile, the lion also came to symbolise the rising and the setting sun and its journey through the heavens and the underworld. The lion, though, was hunted by the pharaoh in a show of courage. The lion was a symbol of power and kingship in Egypt from the earliest of times. Pharaohs sometimes liked keeping lions as pets, or hunting them Artist's Sketch of Pharaoh Spearing a Lion, New Kingdom, Ramesside, Dynasty 20, ca. 1186–1070 B.C, Upper Egypt, Thebes, Valley of the Kings, Tomb of Tutankhamun (KV 62), debris near the entrance, Carnarvon/Carter excavations, 1920, Pharaoh Hunting Lions | Q Files Crocodiles Crocodiles were associated with Amnut, Sobek and Taweret, the gods of justice, power and respect. Amnut was a demon that had the head of a crocodile and ate sinners‘ hearts for punishment of their sins. Sobek was depicted as a human that had the head of crocodile, and temples of Sobek were set throughout ancient Egypt and features sacred lakes were crocodiles were fed and cared for. 20 The Crocodile Gods, Sobek Amnut, Taweret The cobra being the dangerous snake of Lower Egypt, came to symbolise Lower Egypt itself. Though a female symbol, the cobra came to mean protection over the ruler. Cobras: the ultimate protection for an Ancient Egyptian Pharaoh The scorpion The goddess Serket was the principal divine personification of the scorpion and was usually depicted with a scorpion perched on her head. She was a protector goddess, perhaps best known to the public at large as one of the four goddesses who's golden statues surrounded the sarcophagus of Tutankhaman in his tomb. Her full name, Serket hetyt itself means "she who causes the throat to breath", referring to the effects of a scorpion sting. However, there were other gods and goddesses also associated with the scorpion. One of the most famous is Isis, who is said to have been protected from her enemies by seven scorpions. 21 Scepter topper with Isis in the form of a scorpion The goddess Serket with a scorpion perched on her head The crocodile was sacred to the god SOBEK, worshiped in temples in the FAIYUM and at KOM OMBO in Upper Egypt. God Sobek was the crocodile god in ancient Egypt. His main cult was at El-Fayoum and Aswan governorates Cobra Goddesses were numerous in Egypt. The most prominent is Wadjet, the Green One. She is the tutelary Deity of Lower Egypt and one of the Two Ladies Who represent the Two Lands of Egypt. The Harvest Goddess, Renenutet, is a Cobra Goddess, as is Meretseger, She Who Loves Silence, the Goddess Who presided over the Theban necropolis. Wadjet (Wadjyt, Wadjit, Uto, Uatchet, Edjo, Buto) was one of the oldest Egyptian goddesses. Her worship was already established by the Predynastic Period, but did change somewhat as time progressed. She began as the local goddess of Per-Wadjet (Buto) but soon became a patron goddess of Lower Egypt. 22 Wadjet The image of Wadjet with the sun disk is called the uraeus, and it was the emblem on the crown of the rulers of Lower Egypt. She was also the protector of kings and of women in childbirth Pinterest Snakes were symbols of new life and resurrection because they shed their skins. One enormous snake, METHEN, guarded the sacred boat of Ré each night, as the god journeyed without end through the HEll. APOPHIS, another charming snake, attacked Ré each night. guarded the sacred boat of Ré each night, as the god journeyed without end through the HEll. APOPHIS, another charming snake, attacked Ré each night. Worship: Apophis, or Apep, was the ancient Egyptian god who embodied chaos The snake goddess Meretseger personified the pyramid shaped peak that rises above the Valley of the Kings. She may have been an object of a domestic cult in the nearby village of the royal tomb builders and their families at Deir el-Medina, because snake figurines were found during excavations, many of which were covered with cooking soot, suggesting she provided protection for the kitchen. 23 Goddess Meretseger at Deir el-Medina Wab, the deceased, is kneeling in adoration before 12 serpents, one above the other, all representing the snake goddess Meretseger. Height: 27 cm Meretseger Although this snake goddess is not named in an inscription, her human face and the two finger-shaped feathers on her crown identify her as ... The Snake Goddess Meretseger "She who Loves Silence" Sandstone, New Kingdom, Dyn. 18, ca 1479-1400 B.C.E. or later Egyptian cobra was represented by the cobra-headed goddess Meretseger. Pharaohs usually had a stylized Egyptian cobra on their headdresses, which showed that they were powerful and divine. Also, the cobra protected the pharaoh from evil by spitting fire at his enemies. Perfil de la máscara de Psusenes I. Foto en J. Malek, Egipto. 4000 años de arte, Barcelona, 2003 Isis from Abydos wearing a uraeus crown (upholding the horns and disk) and a holy cobra upon Her brow 24 From the Cairo Museum, a stela with Isis (left) and Osiris (right) in snake-form, with the griffin of the goddess Nemesis between them. Thoth, the god of wisdom with the head of an ibis, stands recording the result of the weighing of souls. If the dead Egyptian‘s soul was heavier than a feather, then they would be devoured by Ammut, who was one-third crocodile, one-third lion, and one third-hippopotamus. Devourer, detail from the Book of the Dead of Ani 25 The papyrus in the Brooklyn Museum on snakes The papyrus in the Brooklyn Museum which served as a manual for a doctor treating snakebite reveals that the Egyptians had an intimate knowledge of snakes.  Although the beginning of the papyrus is lost, it would have listed the names of some thirty-seven.  At least thirty-six species (some sources say 34, 37, or 40, of which an estimated seven are poisonous) have been identified in modern Egypt, but the ancient typology most likely did not correspond exactly to the modern ones.  The papyrus gives a physical description of each snake and its habitat, along with precise descriptions of the symptoms produced by each snake's venom, whether or not the bite is mortal, and the name of the god or goddess of which the snake is considered to be a manifestation.  Following the list of snakes is a list of remedies to cure bite victims.  Some of the remedies are specific for certain types of snakes, while other were for specific symptoms.  These remedies included    o o o o o o emetics, compresses, unctions, massages, incision of wounds and fumigations. There were also magical incantations that were spoken over the remedies. The ingredients in the remedies include liquids and substances of mineral, animal and vegetable origin. The most common ingredient is onion, still used frequently in Egyptian folk medicine today to treat snakebite. 26 Wild animals in Ancient Egyptian art Head of a Funerary Couch in the Form of a Cheetah or Lion ~ Thebes ~ Egypt Solid gold signet ring of Horemheb Chelsey York saved to Ancient history Gorgeous, solid gold signet ring of Horemheb, last pharaoh of the 18th Dynasty, Ancient Egypt, c. 1069 BCE. In the collection of the Louvre, Paris, France. This solid gold signet ring is exceptional for its size and the quality of its workmanship. Spirals are added toward the rounded ends of the very thick ring, and the four faces of the rectangular, rotating bezel are deeply engraved with a crocodile, a scorpion, a lion, and the coronation name of Horemheb, the last king of the 18th Dynasty. Seated Lion-Egyptian (Artist) PERIOD 525-332 BC (Late Period)| Statue of, Museums and Amenhotep iii Pinterest • The world's catalog of ideas The goddess Sekhmet, daughter of Re, wife of Ptah and mother of Nefertem is Tutankhamen Treasures 27 V Mesopotamian Lizard Pendant, Late 4th ML BCThis creature was carved from a small black stone and probably had inlaid eyes (now lost). The surface is perfectly polished and presents a matte finish. The underside is flat and without anatomical 12th Dynasty copper cult statue of a crocodile from the Faiyum. At the State Museum of Egyptian Art in Munich. Falcon-headed crocodile This statue combines attributes of two great hunters—the keen-eyed falcon and the mighty Nile crocodile. Late Period, 664–332 BCE From MansooraIndurated limestone The Egyptian Museum, Cairo 28 Dynasty 4, life sized Lion Ancient Egypt and Archaeology Web SiteMetropolitan NY Nov-2005 0040 1 Ivory frog This frog may have been more than just a toy, since frogs were associated with fertility, creation, and regeneration.New Kingdom, 1550–1069 BCE Ivory The Egyptian Museum, Cairo. Vessel (aryballos) in the form of a hedgehog Museum of Boston 29 An ancient Egyptian hippo Colorful ceramic Hippopotamus Egypt Middle Kingdom 11th Dynasty 2130-1991 BCE | von mharrsch The Great Cat defeating Apep on the Papyrus of Hunefer Visually Similar Results Button Game of Hounds and Jackals, ca. 1814–1805 B.C. Middle Kingdom, Dynasty 12, reign of Amenemhat IV. Egypt, Upper Egypt; Thebes, el-Asasif, Tomb of Reniseneb. The Metropolitan 30 Museum of Art, New York. Purchase, Edward S. Harkness Gift, 1926 (26.7.1287). Egyptians likened the intricate voyage through the underworld to a game. This made gaming boards and gaming pieces appropriate objects to deposit in tombs. Painting 1986 Egyptian, Cats Egyptian, Ancient Egyptian, Wilkinson Cat, Wilkinson 1897, 1 Cats, Cats In Art, Cat Art, Cat Footing Tefnut and Shu - Twin lion gods Ancient Egyptian Wild Animal Mummies Jackal Anubis, the god of mummification gazelle mummyThe Egyptian may have succeeded in domesticating cranes, ibex, gazelles, oryx and baboons. Bas reliefs show men trying to tame hyenas by tying them up and force feeding them meat. 31 P. Chapuis / MAFB. Copyright Hypogees This mummified lion was found in the tomb on King Tut's wet nurse. The colored scale has been placed next to it for measurement purposes. This coffin, shaped like a baboon, once contained the remains of a baboon as an offering to the god Thoth. Walters Art Museum, Baltimore , X-ray of a baboon mummy Mummified crocodile, for Sobek.OIM 701. Photo by M. LaBarbera 32 2. Ringworm in wild animals      Ringworm is a fungal skin infection that can affect humans and many animal species. Ringworm as a disease that has a worldwide distribution and can be associated with a number of different fungal species. Dermatophytes can be classified into the following three groups: zoophilic, geophilic, and anthropophilic. o Zoophilic dermatophytes are basically lower animal pathogens, but most have the ability to infect humans. o Geophilic dermatophytes are soil fungi that have the additional capacity to cause ringworm in some species of lower animals and also in man. o Anthropophilic dermatophytes are primarily adapted for parasitism of man, but some species occasionally cause ringworm in animals. Transmission Cycle o Dermatophytes produce resistant spores that are adapted to withstand environmental stresses of moisture and temperature. o The dormant spores survive for long periods in soil and can only enter an animal if there are pre-existing cuts or abrasions in the skin. o Once the spores get below the skin surface, they germinate and spread out as branched hyphae, typical of many fungi. o These hyphae can enter hair follicles and weaken each individual hair until it breaks or falls out of the follicle. o This activity can be associated with thin dry flakes or scabs on the skin as well as mild to severe loss of hair, often on the face and lower legs. o The hyphae also produce spores that remain on the skin surface or drop to the ground and have the potential to infect another mammal with an open wound. Lastly, the spores can pass directly during contact with an infected individual, often a carrier animal that does not have any signs of ringworm. o Wildlife ringworm generally is limited to mild infections and small areas of hair loss on the face, muzzle, and lower legs of mule deer. Prevention/Control o Prevention or control of ringworm in wild animals is impractical and not warranted. Diagnosis: Dermatophytosis is diagnosed by  Examination with a Wood’s lamp. The Wood‘s lamp is useful as a screening tool for M canis infections. Infected hairs fluoresce yellow-green; however, only ≤50% of M canis infections fluoresce, and other fungal species in animals do not. Therefore, negative Wood‘s lamp examinations are not meaningful. False-positive examinations may occur and are especially likely in oily, seborrheic skin conditions. Fluorescing hairs should always be cultured to confirm the diagnosis.  Sampling o Selected lesions should have the hair clipped to a length of ~0.3 cm. 33  o The area should be gently patted with an alcohol-moistened sponge and then patted dry to reduce contamination with saprophytic fungi. Direct microscopic examination of hairs or skin scrapings may enhance clinical suspicion by demonstrating characteristic hyphae or arthrospores in the specimen. o The technique is more useful in diagnosing dermatophytosis in large animals than in small animals. o Hairs (preferably white ones) and scrapings from the periphery of lesions are examined for fungal elements in a wet preparation of 20% potassium hydroxide that has been gently warmed or incubated in a humidity chamber overnight. o Fungal culture is the most accurate means of diagnosis using Dermatophyte test medium (DTM) or Sabouraud agar. o Incubation at room temperature is sufficient except when culturing for T verrucosum from food and fiber animals, in which case incubation at 37°C is necessary. T equinum requires nicotinic acid if subcultured from primary growth, and some T verrucosum isolates require thiamine or thiamine and inositol. o Dermatophyte growth is usually apparent within 3–7 days but may require up to 3 wk on any type of DTM. o Dermatophytes growing on DTM cause the medium to change to red at the time of first visible colony formation. o Dermatophyte fungi have white to buff-colored, fluffy to granular mycelia. Saprophytic contaminant colonies are white or pigmented and almost never produce an initial color change on DTM. o Definitive diagnosis and species identification require removal of hyphae and macroconidia from the surface of the colony with acetate tape and microscopic examination with lactophenol cotton blue stain  An ELISA for the serodiagnosis of dermatophytosis has been researched but is not commercially available. o The sensitivity and specificity is high and similar to that of fungal culture with DTM, but positive results can be seen after elimination of the dermatophyte infection. o Cross-reactivity between the various dermatophytes would not allow for species identification, which is important for identification of the source of infection Wild animals reported to be infected withMicrosporum species Microsporum gypseum 1. Rheseus monkey Koch et al. (1964) 2. Ocelot (Felis pardalis) Costa et al. (1995) 3. Lion (Panthera leo) Costa et al. (1995) 4. Tiger (Panthera tigris) Costa et al. (1995) 5. Florida panthers (Felis concolor coryi) Rotstein et al. (1999) 6. hooded gibbon (Hylobates lar) Refai and Bisping (1964) 34 7. Red pandas (Ailurus fulgens fulgens) Kearns et al. (1999) 8. Chinchilla Male and Fritsch (1966) 9. Gray wolf Fisher et al. (1987) 10. Peromyscus polionotus Menges et al. (1957) 11. Rattus norvegiens Menges et al. (1957) 12. Reithrodontomys humulis Menges et al. (1957) 13. Peromyscus gossypinus Menges et al. (1957) 14. Peromyscus nuttalli Menges et al. (1957) 15. Mus musculus Menges et al. (1957) 16. Sigmodon hispidus Menges et al. (1957), Mckeever et al. (1958) 17. Neotomafloridana Menges et al. (1957) 18. Opossums Mckeever et al. (1958) 19. Lionesses Levy et al. (2006) Microsporum. canis 1. 2. 3. 4. 5. 6. Lions (Panther leo) Avram et al. (1958) Chimpanzee (Pan trolodytes Blumenbach) Klokke and De Vries (1963) White- handed gibbon ( (Hylobates lar), Kaben (1964) Hooded gibbon (Hylobates lar) Refai and Bisping (1964) Wild felines Petzoldt and Böhm (1965) Tiger (Panthera tigris) Gierloff and Katic (1961), Refai and Rieth (1964), Sykes and Ramsay (2007) 7. Green iguana Chung et al. (2014) 8. Asian elephants Qiao et al. (2016) Microsporum audouini hooded gibbon (Hylobates lar) BISPING and SEELIGER (1962) Rhesus monkey - Wikipedi hooded gibbon Common chimpanzee - Wikipedia 35 Zazzle Ocelot, Felis pardalis Florida Panther, Tiger (Panthera tigris) Alamy Lion (Panthera leo) CurrenceWiki - Wikispaces Gray Wolf Stock 9 by HOTNStock Asian Elephant HD Wallpapers Green Iguana - wallpaper Red Panda Network Opossum - Wikipedia Chinchilla Hispid Cotton Rat Sigmodon . Alamy = Peromyscus polionotus – Wikipedia Rattus norvegicus Extermínio www.nsrl.ttu.edu (Reithrodontomys humulis) Amer Soc Mammalogists Peromyscus gossypinus 36 (Peromyscus nuttalli) UniProt House mouse - Wikipedia Trichophyton species reported in wild animals Trichophyton mentagrophytes 1. hooded gibbon (Hylobates lar) Refai and Bisping (1964) 2. Capuchin monkeys (Cebus nigrivitatus) Bagnall and Grünberg (1972) 3. White rhinoceroses (Ceratotherium simum, Burchell) Weiss (1974) 4. Florida panthers (Felis concolor coryi) Rotstein et al. (1999) 5. Boselaphus tragocamelus Otcenásek et al. (1978) 6. Wild red fox (Vulpes fulva).Knudtson et al. (1980) 7. Fennec fox Pressanti et al. (2012) 8. Steller sea lion (Eumetopias jubatus) Tanaka et al. (1994) 9. Coquerel's sifaka (Propithecus coquereli) Phair et al. (2011) 10. Red kangaroos (Macropus rufus) Boulton et al. (2013) 11. L'Hoest's monkeys (Cercopithecus lhoesti) Keeble et al. (2010) 12. Guinea-pig Pombier and Kim (1975) 13. Didelphis virginiana Menges et al. (1957) 14. P. polionotus Menges et al. (1957), Mckeever et al. (1958) 15. P. gossypinus Menges et al. (1957) 16. M. musculus Menges et al. (1957) 17. S. hispidus Menges et al. (1957) 18. R. norvegiens Menges et al. (1957) 19. R. rattus. Menges et al. (1957) 20. Opossums Mckeever et al. (1958) 21. Muskrats (Ondatra zibethicus zibethicus) Errington (1942), Charles (1946) 22. Mice in wheat stacks Paul (1917), Lawrence (1918), and Connor (1932) 23. Mink Zimmermann and Haufe (1971) 24. hedgehog Pesterev and Bolshakov (1972) 25. Gray squirrels DeLamater (1939) Trichophyton simii 1. captive chimpanzee Okoshi et al. (1966) Trichophyton persicolor 1. voles (Microtus agrestis) English and Southern (1967) 2. wood mice (Apodemus sylvaticus) English and Southern (1967) 3. shrews (Sorex araneus) English and Southern (1967) Trichophyton rubrum. 1. 2. 3. 4. 5. 6. chimpanzees (Pan troglodytes) Otčenášek et al. (1967) racoon-dog Schonborn (1971) leopard Schonborn (1971) lion Schonborn (1971) jaguar Schonborn (1971) Siberian tiger Schonborn (1971) Trichophyton erinacei 1. Hedgehog Morris and English (1973) Philpot and Bowen (1992), Schauder et al. (2007) Trichophyton quinckeanum 1. Guinea pig Bilek et al. (2005) 37 Trichophyton equinum 1. mink Overy et al. (2015) Trichophyton gallinae 1. Monkey Gordon and Little (1968) Trichophyton tonsurans 1. Malayan tapir (Tapirus indicus) Schonborn (1971) 2. Sibirian tiger (Panthera tigris altaica) Schonborn (1971) Trichophyton benhamiae 1. Canadian porcupines (Erethizon dorsatum) Takahashi et al. (2008) Trichophyton verrucosum 1. deer (Odocoileus hemionus) Koroleva (1976), Wobeser et al. (1983) 2. barking deers Pal and Thapa (1993) 3. red kangaroos (Macropus rufus) Boulton et al. (2013) hooded gibbon white rhinoceros - Wikipedia Capuchin (Cebus nigritus) · iNaturalist.org chimpanzee - Wikipedia Florida panther (concolor coryi)....Tom & Pat Leeson 38 Leopard Siberian snow tiger Jaguar Boselaphus Tragocamelus World News Odocoileus hemionus Steller Sea Lion Archives - Cornforth Red Fox Vulpes Vulpes ...Alamy Muskrat - Wikipedia raccoon dogs Stichting AAP Fennec Fox Zazzle 39 Red Kangaroo - Macropus rufus Opossum Wikipedia Mink .WANT Coquerel's Sifaka Gray squirrel Tradebit N A Porcupine Erethizon dorsatum Hedgehog Peromyscus polionotus – Wikipedia Rattus norvegicus Extermínio Amer Soc Mammalogists Peromyscus gossypinus S. hispidus (Peromyscus nuttalli) UniProt House mouse - Wikipedia Description of main dermatophytes reported in wild animals: Microsporum gypseum (E. Bodin) Guiart & Grigoraki, Lyon Médical 141: 377 (1928) Synonyms:≡Trichophyton gypseum E. Bodin, Les champignons parasites de l'homme: 115 (1902) ≡Achorion gypseum (E. Bodin) E. Bodin, Annales de Dermatologie et Syphilis 8: 585 (1907≡Sabouraudites gypseus (E. Bodin) M. Ota & Langeron, Annales de Parasitologie Humaine Comparée 1: 328 (1923) ≡Closterosporia gypsea (E. Bodin) Grigoraki, Annales des Sciences Naturelles Botanique 7: 411 (1925) ≡Trichophyton mentagrophytes var. gypseum (E. Bodin) Kamyszek, Med. Weteryn.: 146 (1945) =Microsporum flavescens Horta, Memórias do Instituto Oswaldo Cruz 3 (2): 301-308 (1912) =Microsporum scorteum Priestley, Ann. Trop. Med. Parasit.: 113 (1914) =Microsporum xanthodes Fischer, Dermatol. Wochenschr.: 214-247 (1918) =Favomicrosporon pinettii Benedek, Mycopathologia et Mycologia Applicata 31 (2): 111 (1967) On Sabouraud's dextrose agar, colonies are usually flat, spreading, suede-like to granular, with a deep cream to tawny-buff to pale cinnamon coloured red surface. Many cultures 40 develop a central white downy umbo (dome) or a fluffy white tuft of mycelium and some also have a narrow white peripheral boarder. A yellow-brown pigment, often with a central darker brown spot, is usually produced on the reverse, however a reddish-brown reverse pigment may be present in some strains. Cultures produce abundant, symmetrical, ellipsoidal, thin-walled, verrucose, 4-6 celled macroconidia. The terminal or distal ends of most macroconidia are slightly rounded, while the proximal ends (point of attachment to hyphae) are truncate. Numerous clavate shaped microconidia are also present, but these are not diagnostic. Microsporum canis Bodin, Les champignons parasites de l'homme, 1902. Synonyms = Microsporum audouinii Gruby var. canis Bodin in Besnier et al., 1900. = Sabouraudites canis (Bodin) Langer., 1945. = Microsporum felineum Mewborn, 1902. = Microsporum lanosum Sabour., 1907. = Sabouraudites felineus (Mewborn) Ota & Langer., 1923 as '(Fox & Blaxall, 1896)'. = Sabouraudites lanosus (Sabour.) Ota & Langer., 1923. = Closterosporia felinea (Mewborn) Grigoraki, 1925. = Closterosporia lanosa (Sabour.) Grigoraki, 1925. = Microsporum aurantiacum Conant, M. obesum Conant, M. pseudolanosum Conant, and =M. simiae Conant, 1941. Conant also considered M. equinum (Delacr. & Bodin) Gueguen Colonies on Sabouraud's glucose agar are flat, spreading, 55-70 mm diam. after 2 weeks at 25°C, at first mostly submerged, surface very thin and strongly radiating, with a buff, granular to fluffy area in the centre where macroconidia are formed; rapidly mutating to 41 produce patches of dense, fluffy, whitish to pale buff mycelium which eventually grows over the whole colony. Colonies usually have a bright golden yellow to brownish yellow reverse pigment, but non-pigmented strains may also occur. Dysgonic strains are slowgrowing, glabrous, brownish, usually confined to a very small area around the hair stumps from which they are growing; they are not stable and on sub-culture may give rise to colonies typical of M. canis. Macroconidia most abundant in the centre of the colony, fusiform, variable in size, 35-110 x 12-25 μm, with up to 14 septa and thick (up to 4 μm thick at the centre of the cell), verrucose walls; ends remaining narrow and relatively thinwalled. Macroconidia borne terminally on short hyphae, usually at an acute angle along simple hyphae, occasionally on branched hyphae with up to 3 branches, themselves branched, arising at the apex of one cell. Microconidia rare on Sabouraud's glucose agar, more abundant on some other media, 3,5-8·5 x 1,5-3,5 μm, smooth-walled, non-septate or rarely 1-septate, sessile or on short pedicel, borne along the sides of simple hyphae. 42 Trichophyton mentagrophytes (C.P. Robin) R. Blanch., Traité de Pathologie Générale 2: 912 (1896) Synonyms≡Microsporum mentagrophytes C.P. Robin, Histoire naturelle des végétaux parasites qui croissent sur l'homme et sur les animaux vivants: 129 (1853) ≡Sporotrichum mentagrophytes (Robin) Sacc., Sylloge Fungorum 4: 100 (1886) ≡Ectotrichophyton mentagrophytes (Robin) Castell. & Chalm., Manual Trop Med (1919) ≡Ctenomyces mentagrophytes (Robin) Langeron & Miloch., Annls Parasit. hum..: (1930) ≡Spiralia mentagrophytes (Robin) Grigoraki, Compt. Rend. Soc. Biol., Paris: 186 (1932) ≡Sabouraudites mentagrophytes (Robin) M. Ota & Kawats. (1933) [MB#450836] ≡Microides mentagrophytes (Robin) De Vroey, Annales Soc Belge de Méd Trop (1970) =Trichophyton mentagrophytes var. mentagrophytes. =Oidium quinckeanum Zopf, Die Pilze in morphol, physiol , boil system Bez : 481 (1890) =Trichophyton granulosum Sabour., Rev. Gén. Méd. Vét.: 561 (1909) =Trichophyton asteroides Sabour., Maladies du Cuir Chevelu 3: 347 (1910) =Trichophyton denticulatum Sabour., Maladies du Cuir Chevelu 3: 374 (1910) =Trichophyton lacticolor Sabour., Maladies du Cuir Chevelu 3: 362 (1910) =Trichophyton radians Sabour., Maladies du Cuir Chevelu 3: 374 (1910) =Trichophyton depressum MacCarthy, Ann. Dermatol. Syph.: 190 (1925) =Grubyella langeronii E.A. Baudet, Annls Parasitol. Humaine Comp.: 417 (1930) =Trichophyton papilliosum Lebasque (1933) [MB#253799] =Trichophyton papillosum Lebasque, Les Champignons des Teignes 72 (1933) =Trichophyton sarkisovii L.G. Ivanova & I.D. Poljakov, Miko logiya i Fitopatologiya:1983 On Sabouraud's dextrose agar, colonies are generally flat, white to cream in colour, with a powdery to granular surface. Some cultures show central folding or develop raised central tufts or pleomorphic suede-like to downy areas. Reverse pigmentation is usually a yellow-brown to reddish-brown colour. Numerous single-celled microconidia are formed, often in dense clusters. Microconidia are hyaline, smooth-walled, and are predominantly spherical to subspherical in shape, however occasional clavate to pyriform forms may occur. Varying numbers of spherical chlamydoconidia, spiral hyphae and smooth, thinwalled, clavate shaped, multicelled macroconidia may also be present. 43 Trichophyton erinacei (J.M.B. Sm. & Marples) Quaife, Journal of Clinical Pathology 19: 178 (1966) ≡Trichophyton mentagrophytes var. erinacei J.M.B. Sm. & Marples, Sabouraudia 3 (1): 9 (1963) =Trichophyton proliferans M.P. English & Stockdale, Sabouraudia 6: 267 (1968) Colonies (SDA) are white, flat, powdery, sometimes downy to fluffy with a brilliant lemon yellow reverse. Numerous large clavate microconidia are borne on the sides of hyphae. Macroconidia are smooth-walled, two- to six-celled, clavate, variable in size, and may have terminal appendages. Macroconidia are much shorter than those seen in T. mentagrophytes. 44 Trichophyton quinckeanum Colonies are generally flat, white to cream in colour, with a powdery to granular surface. Some cultures show central folding or develop raised central tufts or pleomorphic suede-like to downy areas. Reverse pigmentation is usually a yellowbrown to reddish-brown colour. Numerous microconidia are borne laterally along the sides of hyphae, and are predominantly slender clavate when young. With age the microconidia become broader and pyriform to spherical in shape. Occasional to moderate numbers of smooth, thin-walled, multiseptate, clavate to cigar-shaped macroconidia may be present. Varying numbers of spherical chlamydospores and spiral hyphae may also be present. 45 Trichophyton rubrum (Castell.) Sabour., British Journal of Dermatology: 389 (1911) ≡Epidermophyton rubrum Castell., Philippine Journal of Science Section B Medical Science 5 (2): 203 (1910) ≡Sabouraudites ruber (Castell.) M. Ota & Langeron, Annal Parasitol Humaine Comparée 1: 328 (1923) ≡Sabouraudiella rubra (Castell.) Boedijn, Mycopathologia et Mycologia Applicata 6 (2): 125 (1953) =Trichophyton rosacea Sabour. (1894) =Trichophyton rosaceum Sabour., Trichoph. Hum. F.: 92 (1894) =Trichophyton megninii R. Blanch., Traité de Pathologie Générale 2: 915 (1895) =Trichophyton roseum E. Bodin, Les champignons parasites de l'homme: 120 (1902) =Epidermophyton pernettii Castell., Br. J. Derm. Syph.: 148 (1910) =Trichophyton circonvolutum Sabour., Maladies du Cuir Chevelu 3: 320 (1910) =Trichophyton purpureum H. Bang, Ann. Dermatol. Syph.: 238 (1910) =Trichophyton vinosum Sabour., Maladies du Cuir Chevelu 3: 386 (1910) =Trichophyton rubidum Priestley, Med. J. Aust.: 474 (1917) =Trichophyton marginatum Muijs, Ned. Tijdschr. Geneesk.: 2205 (1921) =Trichophyton pedis M. Ota, Bull. Soc. Pathol. Exot.: 594 (1922) =Epidermophyton lanoroseum MacCarthy, Ann. Dermatol. Syph.: 53 (1925) =Epidermophyton plurizoniforme MacCarthy, Ann. Dermatol. Syph.: 37 (1925) =Trichophyton coccineum Y. Katô, Trans. 6th Congr. Far East Assoc. Trop. Med., Tokyo: 861 (1925) Colonies appear in various shades of white, yellow, brown, and red. It may also be found in various textures, being waxy, cottony, or smooth. Two types may be distinguished: T. rubrum downy type and T. rubrum granular type. On Sabouraud glucose agar, growth is slow to moderately rapid, texture downy, sometimes powdery. Colour white to pale pink on the surface; reverse typically wine red, sometimes brown, violet, yellow or even uncoloured. Intermediate strain between the types occur. Microscopically, microconidia are numerous to rare, club – shaped to pyriform, may be found solitary along the hyphae or sometimes in clusters, and are unicellular; and microconidia are frequently absent; pencil – to cigar – shaped, and are multi - septate. 46 Trichophyton verrucosum E. Bodin, Les champignons parasites de l'homme: 121 (1902) Synonyms:≡Ectotrichophyton verrucosum (E. Bodin) Castell.&Chalm., Manual of Trop Med: 1003 (1919) ≡Favotrichophyton verrucosum (E. Bodin) Neveu-Lem., Précis Parasitol Humaine: 55 (1921)]=Trichophyton verrucosum var. verrucosum =Trichophyton album Sabour., Ann. Dermatol. Syph.: 617 =Trichophyton ochraceum Sabour., Ann. Dermatol. Syph.: 628-834 (1908) =Trichophyton discoides Sabour., Maladies du Cuir Chevelu 3: 408 (1910) Trichophyton verrucosum is very slow-growing compared to other dermatophytes. In culture, it is characterized by being flat, white/cream colour, having an occasional dome, with a glabrous texture, known as the variant album, Trichophyton verrucosum var. ochraceum has a flat, yellow, glabrous colony; Trichophyton verrucosum var. discoides has a gray-white, flat, and tomentose colony; and T. verrucosum var. autotrophicum is rarely seen and is associated with sheep. Under a microscope, macronidia are rare, and have a rat-tail or string bean shape, while micronidia are tear-shaped and have been only observed in laboratories when grown 47 under enriched conditions. At 37 C (the only dermatophyte with an optimum growth temperature this high), chlamydospores become thick-walled and found in long chains. Macronidia are more commonly produced on BCP-milk solids-yeast extract agar, and only on colonies over 7 days old. 48 Trichophyton equinum Gedoelst, Les Champignons parasites de l'homme et des animaux domestiques: 88 (1902) On Sabouraud's dextrose agar, colonies are usually flat, but some may develop gentle folds or radial grooves, white to buff in colour, suede-like to downy in texture, and are similar to T. mentagrophytes. Cultures usually have a deep-yellow submerged fringe and reverse which later becomes dark red in the centre. Microscopically, abundant microconidia which may be clavate to pyriform and sessile or spherical and stalked are formed laterally along the hyphae. Macroconidia are only rarely produced, but when present are clavate, smooth, thin-walled and of variable size. Occasional nodular organs may be present and the microconidia often undergo a transformation to produce abundant chlamydoconidia in old cultures. Reports: Menges et al. (1957) isolated dermatophytes , in a survey of 1, 142 wild animals from hair specimens of 88 (7.7%). Only 16 had skin lesions and dermatophytes were not obtained from those animals. None of the hair specimens showed fluorescence or was positive by microscopy. The organisms encountered were: Microsporum gypseum from Peromyscus polionotus and Rattus norvegiens; M. gypseum ('red variety') from Reithrodontomys humulis, P. polionotus, P. gossypinus, P. nuttalli, Mus musculus, Sigmodon hispidus and Neotomafloridana; Trichophyton mentagrophytes from Didel-phis virginiana, P. polionotus, P. gossypinus, M. musculus, S. hispidus, R. norvegiens and R. rattus. Infection was more common in young rats (12.5%) than in old rats (4.2%). The seasonal peak for all of the species occurred during the spring months. M. gypseum was cultured from 16 (26%) of 62 soil specimens from areas where the animals were trapped. Because of their ubiquity and abundance, rodents probably constitute a more likely source of human infection with T. mentagrophytes than dogs or farm animals. Avram et al. (1958) reported a small epidemic focus of ringworm caused by M. canis in lions (Panther leo). Mckeever et al. (1958) studied the occurrence, abundance, and distribution of ringworm fungi in wild animals in S.W. Georgia by the Newton Field Station, Technology Branch, and the Microbiology Section, Laboratory Branch, of the Communicable Disease centre, Atlanta, Georgia. Of hair specimens cultured from 996 rodents of 8 spp., collected during 1954-56, 114 (11-4%) yielded fungi. There were 17 isolates of Trichophyton mentagrophytes 14 of Microsporum gypseum and 83 49 of Microsporum (red var.), which has not been shown to be pathogenic to either man or lower animals). Lack of any evidence of infection other than positive cultures may indicate that the animals tested were merely carriers of fungus elements picked up from the environment. The range of frequency of isolation from the various species of rodents was 1.2-34.5%. Prevalence of the fungi was found to vary according to the ecological distribution of the specimens, ranging from 64% for animals collected in tall weeds to 47% for those from pine woods. There was some evidence that T. mentagrophytes is most abundant in areas frequented by domestic animals and is most often found on the burrowing rodent, Peromyscus polionotus. The data also indicated that both T. mentagrophytes and M. gypseum were abundant in some localities and scarce or absent in others. The occurrence of the Microsporum (red var.) on Sigmodon hispidus ranged from 28-8% in March to 1.9% in July. In the 2nd part of this investigation hair specimens from 1, 758 large wild mammals from S.W. Georgia and N.W. Florida were similarly tested at the Emory University Field Station. Isolates were obtained from 21 (1.1%); 6 were T. mentagrophytes all from opossums in areas frequented by domestic animals, and 15 were Microsporum (red var.). There was no evidence of infection in animals from which dermatophytes were isolated. M. gypseum was recovered from 13 of 189 soil samples from areas trapped. Gierloff and Katic (1961) isolated dermatophytes from the haircoat of a tiger (Panthera tigris) (13). Bisping and Seeliger (1962) reported Microsporum audouini-infection in a hooded gibbon (Hylobates lar)] in the zoological garden in Hannover. 50 Klokke and De Vries (1963) described tinea capitis in a Liberian chimpanzee (Pan trolodytes Blumenbach) was caused by Microsporum canis Bodin 1902, with characteristics resembling M. obesum. Another chimpanzee became infected with the same fungus. Microscopic and macroscopic descriptions of the isolates were given. Kaben (1964) reported the isolation of atypical Microsporum canis strains from white- handed gibbon ( (Hylobates lar), 51 Koch et al. (1964) isolated Microsorum gypseum from lesions fo ringworm inRheseus monkey imported fron North Vietnam. The animal was treated successfully with Griseofulvin and local application of Leuna liquidum und Brillant green. Refai and Bisping (1964) examined hair and skin scraping samples from 62 monkeys in zoological garden of Hannover with dermatomycosis. Only 13 samples were positive microscopically and in cultures for dermatophytes. Microsporum canis was isolated from the fore legs in 7 monkeys anf from the head and hind legs in 4 animals. Microsporum gypseum was recovered from 2 monkeys and Trichophyton mentagrophytes fron one animal. 52 Refai and Rieth (1964) reported the isolation of Microsporum canis from 2 tigers in a Circus in Hamburg. 53 Petzoldt and Böhm (1965) reported the infestation of wild felines with Microsporum canis. Male and Fritsch (1966) described infection by Microsporum gypseum in a family breeding chinchilla. They discussed the epidemiological, pathogenesis and therapeutic aspects of the infection. 54 Tinea barbae, ectothrix hair invasion of a beard hair 55 Okoshi et al. (1966) studied the clinical and mycological findings on ringworm obtained from a captive chimpanzee. The chimpanzee (Pan satyrus), female, four years old, born in Kenya, Africa was introduced in 1962, from the native country into Japan and kept at the Ueno Zoological Gardens, Tokyo until 1964. Mycological examination revealed that this ringworm had been caused by Trichophyton simii (Pinoy) Stockdale, Mackenzie & Austwick, 1965. The morphological characteristics of the isolate were described. Experimental infection of guinea pigs with the isolate was successful. This case of simian ringworm was treated effectively by the oral administration of griseofulvin. English and Southern (1967) examined animals on Wytham Estate, Berkshire, for T. persicolor by the hairbrush sampling technique: 53% of the 127 bank voles (Clethriomys glareolus), 25% of the 113 field voles (Microtus agrestis), 19% of the 26 wood mice (Apodemus sylvaticus), and 1 of the 6 common shrews (Sorex araneus) examined were infected. More than 1 in 2 infected bank voles were colonized by the fungus compared with 1 in 5 of infected field voles. The infection rate in voles inhabiting woodland and old-established grassland was considerably higher than in those inhabiting new plantations. Otčenášek et al. (1967) reported Trichophyton infection in three two-year-old chimpanzees (Pan troglodytesLINNÉ 1766). The description of cutaneous lesions and detailed mycological characteristic of the causative agent is given. The authors failed in identifying the dermatophyte species definitely, they are, however, of the opinion that the isolate is closely related to Trichophyton rubrum. Gordon and Little (1968) reported spontaneous infection with Trichophyton gallinae in a monkey in the Philippine Islands. The new isolate produced ringworm experimentally in monkeys, guinea pigs and roosters, with typical white-comb lesions and scutula in the birds. Hair involvement was ectothrix in the guinea pig but largely endothrix in the monkey. The significance of verrucose macroconidia in T. gallinae and the proper generic classification of this species are discussed.. Schonborn (1971) reported the incidence of a dermatophyte resembling Trichophyton rubrum in carnivores. From 10 different carnivores (racoon-dog, leopard, lion, jaguar and Siberian tiger) 11 dermatophyte strains -with homogeneous properties were cultured. In 3 young animals there were clinically visible skin changes. The fungus isolated was found to be an intermediate of T. rubrum and T. mentagrophytes and showed a marked similarity to a dermatophyte resembling T. rubrum, isolated from a chimpanzee and described by OTČENÁŠEK et al. As regards the formation of spiral hyphae, virulence and physiology the strains resembled T. mentagrophytes; as regards pigmentation of the colonies, the shape and number of macroconidia and microconidia they resembled T. rubrum. Special characteristics were: brown pigmentation of the primary culture, tendency to submerged mycelial growth and good development at 37o C 56 57 Schonborn (1971) reported ringworm in 2 wild animals in Zoological garden of Leipzig, a Malayan tapir (Tapirus indicus) and a Sibirian tiger (Panthera tigris altaica). The infection was most probably transmitted from a lion imported from East Africa. The isolated dermatophytes from both animals were identified as Trichophyton tonsurans. 58 59 Zimmermann and Haufe (1971) reported generalized dermatomycosis in mink. The lesions were found on the head, back, abdomen,legs and tails. The isolated dermatophyte was identified as Trichophyton mentagrophytes var. granulosum 60 Bagnall and Grünberg (1972) reported a chronic and extensive Trichophyton mentagraphytes infection in 3 Capuchin monkeys (Cebus nigrivitatus). The animals were severely debilitated from internal parasites, pneumonia and enteritis, which led to their deaths within 2 months of purchase by a zoo. Clinically there was widespread hair loss, with skin scaling and crusting. The histological picture was one of marked hyperkeratosis and peri-folliculitis. The disease was not transmitted to 5 other healthy monkeys housed in the same enclosure. Pesterev and Bolshakov (1972) reported the hedgehog as a natural source of trichophytosis in man caused by Trichophton mentagrophtes var gypseum Morris and English (1973) stated that observations on cross infection by Trichophyton erinacei in captive and wild hedgehogs indicate that the fungus is not highly pathogenic. A healthy animal may be exposed to an infected one for some months before itself becoming infected. Infected animals may remain free of lesions for long periods, but the disease eventually increases in extent and severity. No case of regression of infection or of recovery was noted. Possible means of transmission of 61 the fungus are considered, and it is suggested that bodily contact, especially during fights, is the most likely means of cross infection. Low body temperature and reduction of movement during hibernation are both likely to slow down the progress of the disease and reduce the chances of cross infection. Young (1973) reported a high incidence of infection, characterised by thinning and loss of hair, dry skin and hyperkeratosis over the ventral surface of the body, limbs and ears, but no typical 'ringworm' in a colony of Djungarian hamsters at the Centre and in other colonies in England. Chalmers and Barrett (1974) described severe dermatomycosis (ringworm) caused by an unidentified dermatophyte occurred in a mature, debilitated, female mule deer (Odocoileus hemionus) from southwestern Alberta. Lesions involved much of the body surface and were characterized by severe alopecia of the face, lower thoracic wall and abdomen, perineum and limbs. The skin was markedly encrusted and scaly in all areas. The histologic lesions included marked hyperkeratosis and a chronic dermatitis with the presence of numerous spherical ecto- and endothrix arthrospores and segmented mycelial elements. The causative organism could not be grown on artificial media, but the distribution and morphology of arthrospores, the presence of segmented mycelia and the nature of the inflammatory reaction, suggested infection by a Trichophyton species. This is the first report of dermatomycosis in a free-ranging big game animal in North America. Weiss (1974) described an infection by Trichophyton mentagrophytes in a herd of 19 white rhinoceroses (Ceratotherium simum, Burchell). The dermatophyte could be isolated from 3 of 4 examined skin samples and from 1 sample from the surroundings of the animals. Clinical aspects of the infection are also discussed. This is the first report about a dermato-mycosis in rhinoceroses. Pombier and Kim (1975) reported an epizootic outbreak of ringworm in a guinea-pig colony caused by Trichophyton mentagrophytes. The disease was characterized by loss of hair, initially occurring at the tip of the nose and spreading throughout the body. Lesions appeared as circular, scaley alopecia with occasional scarring. Although spread of the infection in the colony was random, the most severe infection occurred in an inbred line with light coat colour. The unusually high temperature and humidity, and the open type of outdoor management, appear to have contributed to the high incidence and severity of infection. Electron microscopic observation indicated that the organism multiplied in the keratin layer of the skin. The hyphae as well as chlamydospores were readily demonstrable by electron microscopy. Koroleva (1976) isolated Union. Trichophyton verrucosum from a deer in the Soviet Otcenásek et al. (1978) described a dermal lesion in seven antelopes of the species Boselaphus tragocamelus, kept in a zoo-park. Mycological examination revealed the dermatophyte Trichophyton mentagrophytes (Robin) Blanchard 1896 as the causative agent of the lesion. The clinical picture of the dermatophytosis was manifested by a non-inflammatory desquamation of the epidermis with focal hair shedding. The lesions, mostly localized on the heads of the animals affected, were successfully treated with local antimycotics and by oral administration of griseofulvin. The authors present a list of the species of Artiodactyla in which Trichophyton mentagrophytes 62 has been found until the present time. They draw attention to the fact that in wild animals the dermatophyte mostly causes symptomless disease. Klingmüller et al. (1979) stated that occasionally the hedgehog (Erinaceus europaeus) is infected by Trichophyton mentagrophytes (Robin) Blanchard var. erinacei. This zoophilic dermatophyte may cause a difficult human phlegmatic trichophytia infection. The thread-fungus grows on the usual culture-medium with a clear-white surface without radius-folding. The lower surface of the culture shows a typical brillant-yellow colour. Microscopically the fungus presents abundant microconidia formation and a few distinct macroconidia. Cross-breeding with the tester strain Arthroderma simii "+" was negative, with "-" showed an increased growth and a formation of cleistothecium-primordia in the combination zone. Knudtson et al. (1980) diagnosed dermatophytosis caused by a zoophilic varient of Trichophyton mentagrophytes in a litter of eight captured wild red fox (Vulpes fulva). The animals had widespread partial alopecia and scattered crusty foci 2 to 3 cm in diameter on the skin. Treatment with 7 mg/kg/body weight/day of griseofulvin in the feed effectively controlled the infection. Wobeser et al. (1983) described 6 mule deer (Odocoileus hemionus) with dermatomycosis. Trichophyton verrucosum was isolated from four. All infections were mild and were not debilitating. The lesions involved the legs in five animals and the face in two. This is the second report of ringworm in a wild ungulate in North America. Fisher et al. (1987) reported infection in a gray wolf caused by Microsporum gypseum 63 Philpot and Bowen (1992) reported 2 related cases of ringworm caused by contact with an infected hedgehog are reported. The causal fungus, Trichophyton erinacei, was isolated from human and animal cases. The epidemiology of hedgehog ringworm is discussed. Pal and Thapa (1993) reported an outbreak of dermatophytosis in 4 barking deers. The animals were 4 months to 8 years old. Lesions were observed on the face including ears and eyes of the 4 months old deer, while the one year old 2 deers showed lesions on the face, head, neck, abdomed and legs. The 8 years old deer suffered from generalized lesions including the tail. Trichophyton verrucosum was the only dermatophyte isolated. 64 65 Tanaka et al. (1994) diagnosed serious dermatophytosis caused by Trichophyton mentagrophytes in a Steller sea lion (Eumetopias jubatus) at Yomiuri Land Marine Aquarium in Tokyo. The external clinical signs were extensive depilation and hyperkeratosis, as well as redness and depigmentation of the skin. Histopathological findings of the skin revealed PAS positive fungal hyphae with septa in the corneum layer of the epidermis. Further microscopic examination suggested that this lesion of the skin was typical chronic dermatophytosis. Based on morphological and growth characteristics, the isolate was identified as Trichophyton mentagrophytes. It was thought that the infection was due to some factors including species and individual specific and environmental factors and so on. Costa et al. (1995) isolated M. gypseum in Brazil from one specimen of each of the following wild felids: ocelot (Felis pardalis), lion (Panthera leo) and tiger (Panthera tigris), in the ocelot. Kearns et al. (1999) identified 14 cases of dermatophytosis from medical records of red pandas (Ailurus fulgens fulgens) housed at the Knoxville Zoo between 1980 and 1996. The median age of affected animals on initial presentation was 8.5 wk (3 wk-11 mo). Clinical signs included crusting, purulent exudate, alopecia, thickening of affected skin, ulceration, and necrosis. Seven animals had mild lesions with signs restricted to crusting and/or alopecia, and six animals had more severe infections, with ulceration, skin necrosis, and purulent exudate. Five of the severely affected pandas had tail involvement. The severity of disease affecting one individual was not recorded. Dermatophytosis was confirmed by culture, cytology, histopathology, or culture followed by histopathology. Microsporum gypseum was the only fungal organism cultured. Six animals were treated for mild disease, and all clinical signs resolved. Partial tail amputation was required as part of the treatment regimen for two of the six severely affected animals, and two others had ulcerated tail lesions that left circumferential scarring after resolution of infection. Itraconazole (5 mg/kg p.o. q 1224 hr) was the most frequently used systemic antifungal agent in animals with severe lesions. All fungal infections resolved, although one panda died from unrelated causes early in the treatment period. Rotstein et al. (1999) diagnosed 3 free-ranging Florida panthers (Felis concolor coryi) were diagnosed with clinical dermatophytosis; two were infected with Trichophyton mentagrophytes, and one was infected with Microsporum gypseum. Two of these panthers were juvenile males that were diagnosed with focal to focally coalescing dermatophytosis; one caused by M. gypseum and the other by T. mentagrophytes. These animals were not treated, and clinical signs resolved spontaneously over 6 mo. The third panther, an adult male from southern Florida, presented with a diffuse dermatophytosis due to T. mentagrophytes infection. Initially, the panther had alopecia, excoriations, ulcerations, and multifocal pyoderma of the head, ears, neck, rear limbs, and abdominal region that progressed to lichenification of the skin and loss of nails from two digits. When topical therapy applied in the field at 45-day intervals was ineffective in clearing the infection, the animal was placed in captivity for intensive oral therapy to prevent further development of dermal mycosis, loss of additional nails, and spread of infection to other panthers. The panther was treated orally with itraconazole (9.5 mg/ kg) in the food s.i.d. for 6 wk. After treatment, nail regrowth occurred but the multifocal areas 66 of alopecia remained. The panther was released back into the wild after two skin biopsy cultures were negative for fungal growth. Bilek et al. (2005) examined in March, 2002, a 52-year-old male patient with a 1- week anamnesis of a solitary, oval, annular focus, 3 cm in diameter, on the right side of his face, located subauricularly. His 12-year-old son had a ,,similar skin disease" with similar annular oval lesion, size about 2 x 3 cm, located in the right chest region. Since January 2002 the family has kept a guinea pig. They have obtained it through a mediator from the Kosice ZOO. The material for mycological examination was taken from peripheral parts of the foci or desquamating lesions from the father, son, and the guinea pig. Scales were examined microscopically in 20 % KOH solution with Parker's blue-black ink. The findings proved the presence of septal hyphae and formation of arthrospores. Thus, dermatomycosis was confirmed in the father and son, caused by T. mentagrophytes var. quinckeanum, the source of which was a pet guinea pig. Levy et al. (2006) determined the presence of dermatophytes on the haircoat of healthy wild felids, kept in captivity at "Fundação Parque Zoológico de São Paulo". Samples were taken from 130 adult animals of both sexes: 25 lions (Panthera leo), 12 tigers (Panthera tigris), 6 jaguars (Panthera onca), 4 leopards (Panthera pardus), 2 snow leopards (Panthera uncia), 2 pumas (Puma concolor), 2 cheetahs (Acinonyx jubatus), 1 ocelot (Leopardus pardalis), 28 tiger cats (Leopardus tigrinus), 10 margays (Leopardus wiedii), 8 geoffroy's cats (Leopardus geoffroyi), 22 jaguarundis (Herpailurus yagouaroundi) and 8 pampas cats (Oncifelis colocolo). The samples were obtained by rubbing the haircoat of the animals with squares of sterile carpet, and then seeded onto Petri dishes containing Mycobiotic agar (Difco™). The plates were incubated at 25°C for 4 weeks. The isolates were subcultured in Sabouraud dextrose agar supplemented with chloramphenicol (100mg/L) and cultured on slides for posterior identification by their macroand microscopic characteristics. Microsporum gypseum was isolated from two apparently healthy lionesses (1.6%), both kept in terrariums. Schauder et al. (2007) reported 8 hedgehog caretakers from Göttingen and the surrounding area with dermatophytosis caused by Trichophyton erinacei. Four patients who handled the animals without gloves developed lesions on the hands that were more in keeping with hand eczema, leading to a delay in diagnosis. The other caretakers who wore gloves presented with typical ringworm on the arms, the big toe, the back, the abdomen, and the thighs. Their typical clinical features led to an early diagnosis and treatment. 67 68 Sykes and Ramsay (2007) reported an outbreak of dermatophytosis caused by Microsporum canis occurred in tigers (Panthera tigris) at an exotic felid sanctuary in 2003. In an attempt to find an effective, practical, safe, and affordable method for controlling this epizootic, a clinical treatment trial was conducted. Nonalopecic tigers were studied to address the inapparent carrier state observed at the facility. The efficacy of three topical and environmental treatment combinations of a 2% lime sulfur solution and a peroxide-based cleaner were evaluated in nonalopecic, culturepositive tigers (n = 18) housed in four separate enclosures. Lime sulfur solution was applied topically to all of these animals. As a control, nonalopecic but culture-positive tigers (n = 6) housed in two other enclosures were not treated. Environmental treatments included lime sulfur solution (n = 1), a peroxide-based cleaner (n = 1), and no treatment (n = 2). All solutions were applied at 2-wk intervals for seven treatments. The 2% lime sulfur solution treatments were unsuccessful in resolving infections in most tigers. Lime sulfur was effective in suppressing environmental fungal growth immediately posttreatment, whereas the peroxide-based cleaner was not effective. A follow-up survey of all study tigers and their enclosures was conducted 2 yr later, at which time 22 of 24 tigers (92%) had attained resolution, defined as two sequential negative hair cultures. Review of the culture results during 69 the clinical trial and follow-up study suggests that nonalopecic dermatophytosis in tigers that are housed outdoors may not warrant aggressive individual or environmental treatment, as the infection may clear with time. Takahashi et al. (2008) studied an intra-familial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) housed in a Japanese zoo. The family consisted of an adult couple and two offspring (a male and a female). The porcupettes, born in Japan, showed severe hair loss while the parent animals, imported from the USA. (male) and Canada (female), showed mild symptoms or were asymptomatic. Morphologically identical Tricophyton spp. isolates were recovered within seven days from quills of all animals on chloramphenicol-supplemented potato dextrose agar plates incubated at 37°C. Two representative colonies from each animal were identified as Arthroderma benhamiae Americano-European race based on mating type (+) and the internal transcribed spacer (ITS) 1-5.5S-ITS 2 region of the rRNA gene sequences (AB236404–AB236408). The present cases constituted the second isolation of dermatophytes from porcupines. There were two different ITS types, i.e., the predominant one isolated from all animals and a secondary one recovered from only the mother porcupine. The sequences have never been recorded in Japan or in the GenBank database to the best of our knowledge. In addition, they were located at a cluster involving the type strain and mating strains of A. benhamiae Americano-European race and its F1 progeny. In contrast, 28 rodents (eight species) and three insectivora (1 species) exhibited in the petting zoo were negative for any dermatophytes as determined by culture. Hair loss accompanied by severe dandruff in a young male Canadian porcupine (Erethizon dorsatum). 70 Colonies of a porcupine isolate IFM 54326. (a) SDA at 25°C for 21 days and (b) PDA at 25°C for 21 days. (a) Pear-shaped or more elongated microconidia attached with a right-angle arrangement to the sides of the mycelium cultured in a microculture system on PDA at 25°C for 21 days (IFM 52326), and (b) a few spiral bodies in a microculture system on PDA at 25°C for 21 days (IFM 52330). The bar indicates 20 µm lactophenol cotton blue staining, ×400. (a) Gymnothecia between the Americano-European race (-) strain of Arthroderma benhamiae (right) and the animal isolate IFM 54330 (left) cultured on salt-added 1/10-diluted Sabouraud agar medium at 25°C for 8 weeks; the bar indicates 1 cm. (b) An enlarged image of gymnothecia; the bar indicates 300 µm (×40). Keeble et al. (2010) reported an outbreak of Trichophyton dermatophytosis was diagnosed in a group of four L'Hoest's monkeys (Cercopithecus lhoesti) housed in the primate section at a zoological collection. The affected animals presented with areas of non-pruritic alopecia, scaling and crusting. The diagnosis was based on culture and direct microscopy of hair plucks. Treatment was commenced with oral terbinafine at a dose of 8.25 mg/kg bodyweight, topical enilconazole washes and disinfectant fogging of the enclosure. Control measures were designed to limit the 71 spread of infection and reduce the zoonotic risk. Treatment was successful, with no further clinical cases being diagnosed and with resolution of the clinical signs after four weeks and mycological cure after eight weeks. Phair et al. (2011) examined 19-yr-old intact male Coquerel's sifaka (Propithecus coquereli) for a crusting facial dermatopathy. Fungal culture and histopathology of skin biopsies were consistent with dermatophytosis caused by Trichophyton mentagrophytes, and treatment with the antifungal medication terbinafine was initiated. After 1 mo of treatment, all clinical signs had resolved and a fungal culture of the skin was negative. The sifaka was treated with terbinafine for a total of 81 days. Two additional fungal cultures were taken and found to be negative for the presence of dermatophytes, the last culture being taken 1 mo after discontinuation of terbinafine. Pressanti et al. (2012) presented a 2-year-old male fennec fox with a 4 month history of nonpruritic, crusty skin lesions on the forehead, the pinnae and the tail tip. Initial investigations, including routine haematology, biochemistry profile, multiple skin scrapings, trichoscopic examination, Wood's lamp examination and fungal culture, failed to reveal any abnormalities. Histopathological examination of a first set of skin biopsies showed an interface dermatitis pattern, with lymphocyte infiltration in the basal layer, a significant lymphocytic exocytosis and occasional apoptotic basal epidermal keratinocytes; periodic acid Schiff stain did not reveal any fungal elements. On further biopsies, there was a pustular neutrophilic dermatitis, with numerous crusts containing high numbers of arthrospores and fungal hyphae. Trichophyton mentagrophytes infection was confirmed on fungal culture and PCR. The fennec fox received oral itraconazole (5 mg/kg once daily for 6 weeks) combined with a miconazole and chlorhexidine shampoo applied on affected areas once weekly, followed with an enilconazole dip. The fox improved dramatically, and a fungal culture performed at 6 weeks was negative. Unfortunately, a few days later the fennec fox developed anorexia, icterus and died. Boulton et al. (2013) analyzed data from Australian and international zoos to evaluate estimated disease prevalence in zoos housing macropods, affected macropod species, causative organisms, predisposing factors, clinical presentations, diagnostics, treatments, and disease risk management. Two questionnaires (initial detailed and subsequent brief) were distributed via email to zoo veterinarians, with an estimated response rate of 23%. The overall estimated disease prevalence from responding zoos was 28%, with 73% of responding Australian zoos and 14% of responding nonAustralian zoos reporting disease. The first cases of confirmed and suspected dermatophytosis in several macropod species and in association with Trichophyton verrucosum and Trichophyton mentagrophytes var. nodulare were reported, with young red kangaroos (Macropus rufus) appearing predisposed. Diagnosis was most commonly based on fungal culture or presumptively on typical clinical signs of minimally/nonpruritic alopecia, crusting, and scaling distributed most frequently on the tail, pinnae, and hind limbs. Both disease resolution without treatment and resolution after an average of 1 to 2 mo of treatment were reported. Chung et al. (2014) a 1-yr-old female green iguana with a nodular, darkly discolored skin lesion surrounded by necrosis in the right ventral abdominal region. A cytologic examination of the fine needle aspiration of the lesion revealed an exuberant proliferation of fibroblasts, macrophages, and multinucleated cells along with 72 frequent filamentous structures consistent with hyphal elements. The necropsy revealed diffuse infiltration of the liver, lung, and cardiac apex with white nodules. A histopathologic examination of the lesions also confirmed a fungal infection associated with granulomatous inflammation. Rapid polymerase chain reaction (PCR) analysis of the chitin synthase 1 gene was conducted for rapid direct detection, and inter-simple sequence repeat fingerprinting was conducted to classify the infectious origin. The PCR analysis definitively demonstrated representative of disseminated Microsporum canis infection with multiorgan involvement in a green iguana. Overy et al. (2015) reported 2 outbreaks of dermatophytosis in 2 different mink ranches. On the first farm, only kits were affected, while on the second farm, small numbers of adults were infected. Affected mink were otherwise clinically healthy and in good body condition. Three animals were euthanized and submitted for autopsy. Grossly, mink exhibited locally extensive to coalescing areas of crusting alopecia but no other significant gross lesions in internal organs. Microscopically, skin lesions were characterized by chronic hyperplastic dermatitis with folliculitis, furunculosis, occasional intracorneal pustules, and large numbers of intrafollicular fungal arthrospores and hyphae. The dermatophyte was cultured and identified as Trichophyton equinum based on molecular barcoding of the internal transcribed spacer region of the ribosomal DNA gene. Qiao et al. (2016) determined the pathogen of skin diseases that occurred in Asian elephants in Chongqing Zoo, China. The isolated fungus was identified through its cultural characteristics, morphology, and polymerase chain reaction (PCR) amplification. The PCR amplification using common fungal primers (ITS1 and ITS4) determined that the pathogen was 99.7% homologous to Microsporum canis. This is the first report on elephants infected with Microsporum canis in China. References: 1. Ainsworth, G.C, and P.K.C. Austwick (1959): Fungal Diseases of Animals. 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[On a Microsporum canis endemy in zoo animals, with occupational infection in man]. Mykosen. 1967 Jan 1;10(1):7-18. 30. Male O, Fritsch P. [Trichophyton mentagrophytes-caused epidemic and enzootic disease in a chinchilla farm]. Mykosen. 1966 Jun 15;4(2):74-84 74 31. Mckeever S, KAPLAN W, AJELLO L. Ringworm fungi of large wild mammals in southwestern Georgia and northwestern Florida. Am J Vet Res. 1958 Oct;19(73):9735. 32. Menges Rw, Love Gj, Smith Ww, Georg Lk. Ringworm in wild animals in southwestern Georgia. Am J Vet Res. 1957 Jul;18(68):672-7. 33. Pal,M., BR Thapa - An outbreak of dermatophytosis in barking deer (Muntiacus muntjak) Veterinary Record 1993;133:347 34. Pesterev PN, Bolshakov VA. [The hedgehog--a source of trichophytosis caused by Trichophyton gypseum]. Vestn Dermatol Venerol. 1972 Sep;46(9):74-6. 35. Petzoldt K, Böhm KH. [Studies on the infestation of wild felines with Microsporum canis]. Dtsch Tierarztl Wochenschr. 1965 Oct 1;72(19):461-4. 36. Phair K, Larsen RS, Wack R. Dermatophytosis (Trichophyton mentagrophytes) in a Coquerel's sifaka (Propithecus coquereli). J Zoo Wildl Med. 2011 Dec;42(4):759-62. 37. Philpot CM, Bowen RG. Hazards from hedgehogs: two case reports with a survey of the epidemiology of hedgehog ringworm. Clin Exp Dermatol. 1992 May;17(3):156-8. 38. Pombier EC, Kim JC. An epizootic outbreak of ringworm in a guinea-pig colony caused by Trichophyton mentagrophytes. Lab Anim. 1975 Jul;9(3):215-21. 39. Pressanti C, Delverdier M, Iriart X, Morcel F, Cadiergues MC. A case of Trichophyton mentagrophytes infection in a fennec fox (Vulpes zerda). Vet Dermatol. 2012 Oct;23(5):456-e87. 40. Qiao X, Hu J, Wu D, Wei L, Yang Y, Chen J, Mi B, Yang S. Isolation and Identification Of Microsporum Canis From Asian Elephants (Elephas Maximus) In The Chongqing Zoo, China. J Zoo Wildl Med. 2016 Sep;47(3):844-845. 41. Okoshi, S., Mitsuo Takashio and Atsuhiko Hasegawa. Ringworm caused by Tnichophyton simii in a captive chimpanzee. Japan. J. Med. Mycol., Vol. 7, 204-208, 1966 42. Otcenásek M, Adámková A, Janecková V, Dvorák J, Lávicka M, Mícek B. [Dermatomycosis of the nilgai antelope (Boselaphus tragocamelus) caused by the dermatophyte trichophyton mentagrophytes]. Vet Med (Praha). 1978 Jun;23(6):37783. 43. Otčenášek, M., Dvořák, J. & Ladzianska, K. Trichophyton rubrum-like dermatophyte as a causative agent of dermatophytosis in chimpanzees. Mycopathologia et Mycologia Applicata (1967) 31: 33. doi:10.1007/BF02050282 44. Overy DP, Marron-Lopez F, Muckle A, Bourque A, Lund L, MacHattie D, Lopez A. Dermatophytosis in farmed mink (Mustela vison) caused by Trichophyton equinum. J Vet Diagn Invest. 2015 Sep;27(5):621-6. 45. Refai, M. and Bisping, W. : Ueber das Vorkommen von Hautpilzen bei Affen. Kleintier Prax. 9, 147-150 (1964) 46. Rotstein, D.S.; Thomas, R.; Heimick, K.; Citino, S.B.; Taylor, S.K.; Dumbar, M.R. Dermatophyte infection in free-ranging Florida panthers (Felis concolor coryi). J. Zoo Wildl. Med., 30, 281-284, 1999. 47. Schauder S, Kirsch-Nietzki M, Wegener S, Switzer E, Qadripur SA. [From hedgehogs to men. Zoophilic dermatophytosis caused by Trichophyton erinacei in eight patients]. Hautarzt. 2007 Jan;58(1):62-7. 48. Schonborn C. A dermatophyte (Trichophyton tonsurans) isolated from zoo animals, Mykosen , 1971, vol. 14 (pg. 19-30) Schonborn C. The occurrence of a Trichophyton rubrum-like dermatophyte in beasts of prey (Carnivora), Mykosen , 1970, vol. 13 (pg. 381-396) 49. Seeliger, H.P.R., W. Bisping and H.P. Brandt (1963): Uber eine MicrosporumEnzootie bei Kappen-Gibbons (Hylobates lar) verursacht durch eine Variante von Microsporum canis. Mykosen, 6, 61- 68. 50. Stockdale, P.M., D.W.R. Mackenzie and P.K.C. Austwick (1965): Arthroderma simii sp, nov., the perfect state of Trichophyton simii (Pinoy) comb, nov. Sabouraudia, 4, 112-123. 75 51. Sykes JM , Ramsay EC. Attempted treatment of tigers (Panthera tigris) infected with Microsporum canis. J Zoo Wildl Med. 2007 Jun;38(2):252-7. 52. Tanaka E, Kimura T, Wada S, Hatai K, Sonoda S. Dermatophytosis in a Steller sea lion (Eumetopias jubatus). J Vet Med Sci. 1994 Jun;56(3):551-3. 53. Weiss R. [Trichophytosis in broad-mouthed rhinos (Ceratotherium simum, Burchell)]. Mykosen. 1974 Jun 1;17(6):137-9 54. Wobeser G, Mitcham SA, Saunders JR, Hunt HM. Dermatomycosis (Ringworm) in Mule Deer (Odocoileus hemionus). The Canadian Veterinary Journal. 1983;24(10):316-317. 55. YOUNG, C. M., 1973: Trichophyton mentagrophytes in the Djungarian hamster (Phodopus sungorus). Zeitschrift fur Versuchstierkunde 15(5/6): 378 76 3. Chrysosporium-Related Fungal infections in wild animals Chrysosporium-Related Fungi: Taxonomy (Cabañes et al., 2014)         The genus Chrysosporium is polyphyletic, having affiliation with at least two orders of the Ascomycota; however, rDNA sequencing studies which included a representative number of reference Chrysosporium and related species indicate that it should be restricted to anamorphs (asexual states) in the order Onygenales [ Vidal et al., 2000]. Relevant fungal pathogens that produce important mycoses such as blastomycosis, coccidioidomycosis, dermatophytosis, histoplasmosis, and paracoccidioidomycosis are also grouped in this order. Species of Chrysosporium are similar to anamorphic genera such as Blastomyces, Emmonsia, Geomyces, Malbranchea, and Myceliophthora, and also to some species in the dermatophyte genus Trichophyton that produce only microconidia. About 65 Chrysosporium species are currently accepted and their sexual morphs (teleomorphs) are found in a variety of genera such as Aphanoascus, Arthroderma, or Nannizziopsis, among others [Seifert et al., 2011]. Most fungal isolates from reptiles have been considered to belong to the Chrysoporium anamorph of N. vriesii because of morphological similarities of the anamorph with those of this ascomycete. This corroborates what was suggested several years ago from preliminary molecular phylogenetic analysis that the Chrysoporium anamorph of N. vriesii actually represented a species complex, rather than a single species, containing members that could be allied to specific hosts [ Pare´et al., 2006]. The latest taxonomic revisions regarding Chrysosporium-related fungi, also noting relationships between specific fungal species and different reptile hosts proposed one new family (Nannizziopsiaceae), two new genera (Ophidiomyces and Paranannizziopsis), and 15 new species [Stchigel et al. 2013 and Sigler et al,. 2013]. Two recent molecular phylogenetic studies of members of the order Onygenales infecting reptiles determined that 96 clinical isolates identified by morphology as Chrysosporium species, Chrysosporium guarroi, C. ophiodiicola or as CANV complex were placed in three lineages of the Onygenales corresponding to the genera Nannizziopsis, Ophidiomyces and Paranannizziopsis (Sigler et al., 2013; Paré and Sigler, 2016). :NCBI Taxonomy Cellular organisms + o Eukaryota +  Opisthokonta +  Fungi +  Dikarya +  Ascomycota +  Saccharomyceta +  Pezizomycotina +  Leotiomyceta +  Eurotiomycetes + 77 o Eurotiomycetidae +  Onygenales +  Nannizziopsiaceae  Nannizziopsis +  Paranannizziopsis +  Ajellomycetaceae +    Ajellomyces +  Histoplasma +  Loboa +  Paracoccidioides + Arthrodermataceae + Gymnoascaceae + Description: Paranannizziopsis australasiensis Sigler, Hambl. & Paré, Journal of Clinical Microbiology 51 (10): 3353 (2013) Colonies on PDA attained 4.5 to 5 cm diam and were powdery or sometimes cottony, flat, faintly zonate. There was no growth at 35ºC. Aleurioconidia were sessile or were subtended by slightly swollen cells from which one or two conidia were produced. The conidia were pyriform to clavate and measured 3.5 to 8 µm long and 1.5 to 2.7 µm wide. Occasional intercalary arthroconidia and undulate hyphae were produced. Ascomatal initials occurred in cottony sectors and appeared as inflated cells with secondary proliferations. Some mycelium surrounding the ascomatal initials demonstrated swollen intercalary cells. Colonial and microscopic morphology of Chrysosporium ophiodiicola R-3923. (A) PYE, front and reverse; (B) potato dextrose agar, front and reverse; (C) PCA, front and reverse; (D) fertile hyphae and conidia; (E) conidia showing remnants of wall following rhexolytic dehiscence; (F) fertile hyphae with arthroconidia and terminal and lateral conidia. Article · Apr 2009 · Journal of clinical microbiolog Species of the genera Nannizziopsis, Paranannizziopsis, and Ophidiomyces reported in wild animals 1. Day geckos (Phelsuma sp.) (Schildger et al., 1991), 78 2. 3. 4. 5. 6. 7. 8. Captive brown tree snakes (Boiga irregularis) Nichols et al. (1999) Adult Parson's chameleon (Chamaeleo parsonii) Paré et al. (1997) Adult jewel chameleon (Chamaeleo lateralis) Paré et al. (1997) Jackson's chameleon (Chamaeleo jacksoni) Paré et al. (1997) Salt-water crocodile (Crocodylus porosus) Thomas et al. (2002) Pigmy rattlesnakes Cheatwood et al. (2003) Tentacled snakes (Erpeton tentaculatum)Bertelsen et al. (2005), Mads et al. (2005) 9. Ameivas (Ameiva sp., Ameiva chaitzami) ( Martell et al., 2006 10. Green anaconda (Eunectes murinus murinus) (Bicknese ,2009) 11. Veiled chameleons (Chamaeleo calyptratus) Paré et al. (2006) 12. Inland bearded dragons (Pogona vitticeps) Bowman et al. (2007) , Abarca et al. (2009), Hedley et al. (2010) 13. Green iguanas (Iguana iguana) Abarca et al. (2008), Abarca et al. (2010) 14. Black rat snake (Elaphe obsoleta obsoleta) Rajeev et al. (2009) 15. Albino Boa constrictor (Constrictor constrictor) Eatwell (2010) 16. Giant girdled lizards (Cordylus giganteus). Hellebuyck et al. (2010) 17. Broad-headed snake (Hoplocephalus bungaroides) McLelland (2010) 18. Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) Allender et al. (2011), Allender et al. (2016) 19. brown anoles (Anolis sagrei) ( Burcham et al., 2011), 20. Leopard geckos (Eublepharis macularius) Toplon et al. (2013) 21. Garter snake (Thamnophis) Vissiennon et al. (1990), Dolinski et al. (2014) 22. Cottonmouths (Agkistrodon piscivorous) Allender et al. (2015b) 23. Free-ranging timber rattlesnakes (Crotalus horridus) McBride et al. (2015) 24. juvenile Nerodia fasciata confluens (Blanchard) (Broad-banded Watersnake) Glorioso et al. (2016) 25. Tuatara Humphrey et al. (2016), Masters et al. (2016) 26. free-ranging mud snake (Farancia abacura) Last et al. (2016) 27. Coastal bearded dragon (Pogona barbata).Masters et al. (2016) 28. free-ranging Lampropeltis triangulum (Eastern Milksnake) Ravesi et al. (2016) Day Gecko Wikipedia Ameiva - WikiVisually 79 Green anaconda - Wikipedia Alamy Eastern milk snake, Lampropeltis triangulum triangulum, timber rattlesnakes (Crotalus horridus) Wikiwand Nerodia fasciata - Banded Water Snake -- Discover Life Mud snake - Wikipedia Florida Cottonmouth www.jaxshells.org Flickr . Broad headed snake (Hoplocephalus bungaroides) Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus) 80 Tentacled Snake - Erpeton tentaculatum Carnivora ForumPinterest Albino Boa Constrictor Alamy Adult male red-sided garter snakes (Thamnophis sirtalis) The Reptile Database Boiga irregularis Pigmy Rattlesnake (Sistrurus miliarius) | SREL. Saltwater Crocodiles, Crocodylus porosus MarineBio.org Alamy Parson's Chameleon, chamaeleo parsonii AlamyJewel chameleon (Chamaeleo lateralis) 81 Alamy Jackson's chameleon, Chamaeleo jacksonii Chamaeleo calyptratus - Verbatim View GBIF Alamy Central Bearded Dragon, Inland Bearded Dragon (Pogona vitticeps) Alamy black rat snake, Elaphe obsoleta obsoleta, Pets4Homes Green Iguanas Shutterstock Giant Girdled Lizard (Cordylus giganteus) Leopard gecko (Eublepharis macularius) - YouTube AZ Animals Tuatara (Sphenodon Punctatus) 82 Brown anole - Wikipedia AROD.com.au Eastern bearded dragon (Pogona barbata) Reports: Vissiennon et al. (1990) reported a male garter snake (Thamnophis) from a private terrarium which was spontaneously and simultaneously infected with Chrysosporium queenslandicum and Geotrichum candidum. The autopsy revealed disseminated mycotic alterations in skin, lungs and liver. Chrysosporium queenslandicum grew well at 28 degrees C, the optimal temperature of the animal. Paré et al. (1997) isolated a dermatophyte-like fungus from skin biopsies of three different species of captive chameleon in which fungal elements had been observed by histologic examination. An adult Parson's chameleon (Chamaeleo parsonii) presented with vesicles that became crusty brown lesions on the limbs and body. Skin biopsies revealed fungal hyphae in the affected epidermis and underlying dermis. The lesions regressed fully after oral administration of itraconazole. An adult jewel chameleon (Chamaeleo lateralis) from the same private collection presented with localized black skin lesions and died while being treated with itraconazole. A pulmonary granuloma was also present in this chameleon at autopsy. Cultures obtained from skin and lung lesions yielded the same fungus. A third isolate was obtained from a skin biopsy of a Jackson's chameleon (Chamaeleo jacksoni) with deep ulcerative cutaneous lesions located at the base of the tail. The fungus, in all three cases, has been identified as the Chrysosporium anamorph of Nannizziopsis vriesii, a poorly known ascomycetous species recorded previously from the skin of a lizard and from soil, on the basis of its keratinolytic activity, resistance to cycloheximide, strongly restricted growth at 37 degrees C, formation of clavate or pyriform single-celled or two-celled aleurioconidia, and alternate and fission arthroconidia. Nichols et al. (1999) described cutaneous fungal infections in four captive brown tree snakes (Boiga irregularis). The ventral scales were most commonly affected, and lesions began as areas of erythema and edema with vesicle formation, followed by development of caseous brown plaques. Lesions usually started where ventral scales overlapped and spread rapidly. All snakes died within 14 days after clinical signs were first noted. The deaths of three of the snakes were directly attributable to the cutaneous disease; the other snake died from renal failure and visceral gout, most likely induced by gentamicin therapy. Histologically, lesions consisted of epidermal hyperplasia and hyperkeratosis, with foci of epidermal necrosis, intraepidermal vesicle formation, and subacute inflammation of the underlying dermis. These lesions were associated with bacteria and numerous septate, branched fungal hyphae within the epidermis and overlying serocelluar crusts. Hyphae that penetrated through the 83 superficial surface of the epidermis often formed terminal arthroconidia. The same species of fungus was isolated in pure culture from the skin of three snakes, but fungal cultures were not performed on samples from the fourth snake. The fungus has been identified as the Chrysosporium anamorph of Nannizziopsis vriesii based on its formation of solitary dermatophytelike aleurioconidia and alternate and fission arthroconidia. The source of the fungus in this outbreak was not determined; however, the warm, moist conditions under which the snakes were housed likely predisposed them to opportunistic cutaneous fungal infections. Thomas et al. (2002) mentioned that the Chrysosporium anamorph of Nannizziopsis vriesii, recently identified as the cause of cutaneous infections in chameleons and brown tree snakes, was associated with skin infections and deaths in salt-water crocodile (Crocodylus porosus) hatchlings on two separate occasions 3 years apart. In all, 48 animals died from the infection. All hatchlings came from the same farm in northern Queensland, Australia. Cheatwood et al. (2003) described a severe skin, eye, and mouth disease in 3 pigmy rattlesnakes. All snakes had severe necrotizing and predominantly granulomatous dermatitis, stomatitis, and ophthalmitis, with involvement of the subadjacent musculature and other soft tissues. Numerous fungal hyphae were seen throughout tissue sections stained with periodic acid Schiff and Gomori's methenamine silver. Samples of lesions were cultured for bacteria and fungi. Based on hyphae and spore characteristics, four species of fungi were identified from culture: Sporothrix schenckii, Pestalotia pezizoides, Geotrichum candidum (Galactomyces geotrichum), and Paecilomyces sp. While no additional severely affected pigmy rattlesnakes were seen at the study site, a garter snake (Thamnophis sirtalis) and a ribbon snake (Thamnophis sauritis) with similar lesions were found. In 1998 and 1999, 42 pigmy rattlesnakes with multifocal minimal to moderate subcutaneous masses were seen at the study site. Masses from six of these snakes were biopsied in the field. Hyphae morphologically similar to those seen in the severe cases were observed with fungal stains. Analysis of a database representing 10,727 captures in previous years was performed after the 1998 outbreak was recognized. From this analysis we determined that 59 snakes with clinical signs similar to those seen during the 1998 outbreak were documented between 1992 and 1997. This study represents the first documented report of a mycotic disease of free-ranging snakes. 84 Bertelsen et al. (2005) identified Chrysosporium anamorph of Nannizziopsis vriesii as the cause of fatal, multifocal, heterophilic dermatitis in four freshwater aquatic captive-bred tentacled snakes (Erpeton tentaculatum). Pale, 1- to 4-mm focal lesions involving individual scales, occurred primarily on the head and dorsum. Histology showed multifocal coagulation necrosis of the epidermis, with marked heterophilic infiltration without involvement of the underlying dermis. Septate, irregularly branched hyphae, and clusters of 4- to 8- by 2- to 3-microm rod-shaped cells (arthroconidia) were present within the lesions and in a superficial crust. Failure to maintain an acidic environment was likely a predisposing factor in the development of these lesions. Mads et al. (2005) identified the fungus Chrysosporium anamorph of Nannizziopsis vriesii as the cause of fatal, multifocal, heterophilic dermatitis in four freshwater aquatic captive-bred tentacled snakes (Erpeton tentaculatum). Pale, 1- to 4-mm focal lesions involving individual scales, occurred primarily on the head and dorsum. Histology showed multifocal coagulation necrosis of the epidermis, with marked heterophilic infiltration without involvement of the underlying dermis. Septate, irregularly branched hyphae, and clusters of 4- to 8- by 2- to 3-μm rod-shaped cells (arthroconidia) were present within the lesions and in a superficial crust. Failure to maintain an acidic environment was likely a predisposing factor in the development of these lesions. Paré et al. (2006) experimentally challenged Veiled chameleons (Chamaeleo calyptratus) with the fungus Chrysosporium anamorph of Nannizziopsis vriesii (CANV). Chameleons were exposed to conidia in their captive environment, or were inoculated by direct application of a conidial suspension inoculum on intact and on abraded skin. The CANV induced lesions in all experimental groups and was recovered from infected animals, fulfilling Koch's postulates and confirming that it may act as a primary fungal pathogen in this species of reptile. A breach in cutaneous integrity, as simulated by mild scarification, increased the risk of infection but was not required for the CANV to express pathogenicity. Initial hyphae proliferation occurred in the outer epidermal stratum corneum, with subsequent invasion of the deeper epidermal strata and dermis. A spectrum of lesions was observed ranging from liquefactive necrosis of the epidermis to granulomatous inflammation in the dermis. CANV dermatomycosis appears to be contagious and can readily spread within a reptile collection, either directly through contact with infective arthroconidia or indirectly via fomites. Dense tufts of arthroconidiating hyphae were demonstrated histologically on the skin surface of many animals that developed dermatomycosis, and these arthroconidia may act as infective propagules involved in the transfer of disease between reptiles. Bowman et al. (2007) isolated he Chrysosporium anamorph of Nannizziopsis vriesii (CANV), a keratinophilic fungus that naturally and experimentally causes severe and often fatal dermatitis in multiple reptile species in pure culture from skin samples of three inland bearded dragons (Pogona vitticeps) with deep granulomatous dermatomycosis. The first animal presented with a focal maxillary swelling involving the skin and gingiva. This lizard died while undergoing itraconazole and topical miconazole therapy. The second presented with focally extensive discoloration and thickening of the skin of the ventrum and was euthanized after 10 weeks of itraconazole therapy. A third lizard presented with hyperkeratotic exudative dermatitis 85 on a markedly swollen forelimb. Amputation and itraconazole therapy resulted in a clinical cure. Histopathology of tissue biopsies in all cases demonstrated granulomatous dermatitis with intralesional hyphae morphologically consistent with those produced by the CANV. The second lizard also had granulomatous hepatitis with intralesional hyphae. Abarca et al. (2008) described the first isolation of a Chrysosporium species as the etiological agent of dermatomycosis in two green iguanas (Iguana iguana). The ITS5.8S rRNA gene of the two strains was sequenced and a search on the GenBank database revealed that the closest match was Nannizziopsis vriesii. Treatment with oral ketoconazole, in combination with topical 2% chlorhexidine solution and terbinafine resulted in clinical cure. Abarca et al. (2009) isolated a Chrysosporium sp. related to Nannizziopsis vriesii in pure culture from squames and biopsies of facial lesions in a pet inland bearded dragon (Pogona vitticeps) in Spain. The presence in histological sections of morphologically consistent fungal elements strongly incriminates this fungus as the aetiological agent of infection. Lesions regressed following treatment with oral ketoconazole and topical chlorhexidine and terbinafine until the lizard was lost to follow up 1 month later. The ITS-5.8S rRNA gene of the isolate was sequenced and a search on the GenBank database revealed a high match with the sequences of two Chrysosporium sp. strains recently isolated from green iguanas (Iguana iguana) with dermatomycosis, also in Spain. Phylogenetic analysis of the sequences revealed that all these strains are related to N. vriesii. This is the first report of dermatomycoses caused by a Chrysosporium species related to N. vriesii in a bearded dragon outside North America. Rajeev et al. (2009) reported a black, male rat snake (Elaphe obsoleta obsoleta) of undetermined age with a history of prolonged anorexia and slow-growing facial masses. The snake was found as an adult at an old home site in an old barn near Sparta, GA, by the current owner, a wildlife educator. The snake had been in his possession for 4 years and was frequently used in public educational performances in the southeast. Upon presentation, the snake had a 1-cm by 1.5-cm subcutaneous, longitudinally ovoid swelling overlying his right ventral mandible area (Fig. (Fig.1A).1A). He also had a 1-cm swelling overlying his right eye and extending down into the orbit, displacing the eyeball laterally and displacing the palate and dorsal limit of the choana ventrally. The masses were lobular, whitish in appearance, and enclosed in a thin capsule. The submandibular mass was removed in its entirety, as its capsule was very discrete. The other mass was very friable and locally extensive. Both masses were surgically removed and submitted for histopathological examination and culture. Not all portions of the second mass could be completely removed, due to the location of this mass, but the area enclosing it was debrided. At the time of surgery, the snake was treated with meloxicam (Metacam; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) at a dose of 0.2 mg/kg of body weight once a day and enrofloxacin (Baytril; Bayer HealthCare, LLC, Animal Health Division, Shawnee Mission, KS) at a dose of 5 mg/kg twice a day. This was continued until the histopathology report indicating a fungal infection was received. Enrofloxacin was discontinued of postoperative swelling at the incision over the orbit. This was treated with warm wet compresses daily, and the swelling decreased. The snake passed away 2 months after surgery., and ketoconazole was initiated. A single 86 oral administration of ketoconazole (Apotex, Inc., Toronto, Ontario, Canada) at 50 mg/kg was administered daily. (A) Cutaneous masses; (B) hematoxylin and eosin-stained section of the lesion; (C) Grocott-Gomori methenamine silverstained section of the lesion. Colonial and microscopic morphology of Chrysosporium ophiodiicola R-3923. (A) PYE, front and reverse; (B) potato dextrose agar, front and reverse; (C) PCA, front and reverse; (D) fertile hyphae and conidia; (E) conidia showing remnants of wall following rhexolytic dehiscence; (F) fertile hyphae with arthroconidia and terminal and lateral conidia. Abarca et al. (2010) isolated Chrysosporium guarroi sp. nov. represented by five strains from cases of dermatomycosis in pet green iguanas (Iguana iguana) in Spain,. This taxon is characterized by its ability to grow at temperatures from 15 to 37 degrees C and by the presence of arthroconidia and aleurioconidia. The latter are unicellular, smooth, pyriform or clavate, sessile or borne at the ends of narrow stalks. The analysis of the sequences of the D1/D2 and ITS regions confirm the separation of this new species from others of the genus Chrysosporium. Eatwell (2010) described a 1-year-old albino Boa constrictor (Constrictor constrictor) with large necrotic areas of skin of two weeks duration. Skin lesions were evident on the mandible, maxilla and dorsally over the spine. Other lesions were developing with mild yellow brown skin discolouration evident on the lateral and ventral aspects of the snake. Skin biopsies were taken under anaesthesia and histopathological changes noted were severe skin necrosis and inflammation likely due to infection with CANV. The snake was treated with itraconazole (Itrafungol ®, Janssen) at 5 mg/kg by mouth once daily and topically 1% silver sulfadiazine cream (Flamazine®, Smith & Nephew) was applied twice daily. The snake died after three weeks of treatment. Hedley et al. (2010) described necotising fungal dermatitis in a group of bearded dragons (Pogona vitticeps).caused by Chrysosporium anamorph of Nannizziopsis vriesii (CANV 87 Hellebuyck et al. (2010) reported the Chrysosporium anamorph of Nannizziopsis vriesii in association with dermatomycosis and high mortality in a group of captive giant girdled lizards (Cordylus giganteus). Treatment of one of the infected girdled lizards with voriconazole, which was selected on the basis of in vitro sensitivity testing of the isolate, resulted in resolution of lesions and negative fungal cultures from the skin. Three hours after oral administration of 10 mg/kg, the plasma level of voriconazole exceeded the 0.25-μg/mL minimal inhibitory concentration tenfold. In conclusion, administration of voriconazole at 10 mg/kg of body weight once daily for 10 weeks resulted in clinical cure and was well tolerated. A longer follow-up time and larger studies will be necessary to determine the long-term efficacy and safety of this treatment in giant girdled lizards. 88 Giant girdled lizard (Cordylus giganteus) with severe cutaneous hyalohyphomycosis caused by the Chrysosporium anamorph of Nannizziopsis vriesii at the time of first clinical examination (a), after 3 weeks (b) and 5 weeks (c) of voriconazole administration at 10 mg/kg once daily and 3 weeks after ceasing voriconazole administration (d). McLelland (2010) described. fatal cutaneous mycosis in a broad-headed snake (Hoplocephalus bungaroides) caused by the Chrysosporium anamorph of Nannizziopsis vriesii, Allender et al. (2011) reported 3 eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) with severe facial swelling and disfiguration which died within 3 weeks after discovery near Carlyle, Illinois, USA. In spring 2010, a similar syndrome was diagnosed in a fourth massasauga; this snake continues to be treated with thermal and nutritional support and antifungal therapy. A keratinophilic fungal infection caused by Chrysosporium sp. was diagnosed after physical examination, histopathologic analysis, and PCR in all 4 snakes. Clinical and gross necropsy abnormalities were limited to the heads of affected animals. In each case, a unilateral subcutaneous swelling completely obstructed the nasolabial pits. In the most severely affected snake, swelling extended to the cranial aspect of the orbit and maxillary fang. Notable histologic lesions were restricted to skin, gingiva, and deeper tissues of the head and cervical region and consisted of cutaneous ulcers with granulomas in deeper tissues. Ulcers had thick adherent serocellular crusts and were delineated by small dermal accumulations of heterophils and fewer macrophages. Crusts contained numerous 4– 6-µm diameter right-angle branching fungal hyphae with terminal structures consistent with spores. In 1 snake, infection was associated with retained devitalized layers of epidermis consistent with dysecdysis. In the same snake, the eye and ventral periocular tissues were effaced by inflammation, but the spectacle and a small fragment of cornea remained; the corneal remnant contained few fungal hyphae. 89 Chrysosporium sp. fungal infection in eastern massasagauga rattlesnake (Sistrurus catenatus catenatus). A) Facial dermatitis and cellulitis caused by Chrysosporium sp. infection in rattlesnake from Carlyle, Illinois, USA; B) close-up showing maxillary fang destruction. C) Maxillary dermal and subcutaneous fungal granuloma (circled area). Hematoxylin and eosin stain, original magnification ×2, scale bar = 5 μ . D Gra ulo a e ter ith large u ers of fu gal h phae. Gro ott ethe a i e sil er stai , origi al ag ifi atio × , s ale ar = μ . Allender et al. (2011) conducted Polymerase chain reaction assays from swabs collected from the faces of 34 snakes. hematologic data were obtained for 31 individuals, plasma biochemical data for 24, and toxicological data for 18. There was no evidence of Chrysosporium in any of the samples. Hematologic and plasma biochemistry parameters were consistent with previous health studies in the Carlyle population. Elemental toxicologic investigation of the plasma indicated variable levels of lead, copper, selenium, strontium, tin, iron, and zinc. Burcham et al.( 2011) reported multiple skin lesions in 2 browon anoles 90 Sigler et al. (2013) compared 49 Chrysosporium anamorph of Nannizziopsis vriesii (CANV from reptiles and six isolates from human sources with N. vriesii based on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the family Onygenaceae of the order Onygenales. One lineage represented the genus Nannizziopsis and comprises N. vriesii, N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genus Ophidiomyces, with the species Ophidiomyces ophiodiicola occurring only in snakes, and Paranannizziopsis gen. nov., with three new species infecting squamates and tuataras. The newly described species are Nannizziopsis dermatitidis, Nannizziopsis crocodili, Nannizziopsis barbata, Nannizziopsis infrequens, Nannizziopsis hominis, Nannizziopsis obscura, Paranannizziopsis australasiensis, Paranannizziopsis californiensis, and Paranannizziopsis crustacea. Chrysosporium longisporum has been reclassified as Paranannizziopsis longispora. N. guarroi causes yellow fungus disease, a common infection in bearded dragons and green iguanas, and O. ophiodiicola is an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis. 91 Colonies of Nannizziopsis, Paranannizziopsis, and Ophidiomyces isolates after 21 days of incubation, except as indicated. Colonies of N. vriesii shown on PDA at 30°C (A) and 35°C (B). N. dermatitidis shown on PDA (C) and on PYE (top) and Mycosel (MYC) (bottom) (D) at 30°C. (E) N. dermatitidis streaked on PDA showing yeast and mold colonies after 16 days at 30°C. N. crocodili shown on PDA (F) and on PYE (top) and MYC (bottom) (G) at 30°C. (H). N. barbata shown on PDA at 30°C. N. guarroi shown on PDA at 30°C (I) and at 35°C (J). (K) N. guarroi streaked on PDA showing yeast and mold colonies after 11 days at 30°C. N. infrequens shown on PDA at 30°C (L) and 35°C (M) and on PYE (top) and MYC (bottom) at 30°C (N). N. hominis shown on PDA at 30°C (O) and 35°C (P) and on PYE (top) and MYC (bottom) at 30°C (Q). N. obscura shown on PDA at 30°C (R) and at 35°C (S) and on PYE (top) and MYC (bottom) (T) at 30°C. Paranannizziopsis australasiensis (U), P. californiensis (V), and P. crustacea (W) shown on PDA at 30°C. (X) Ophidiomyces ophiodiicola shown on PDA at 30°C. Sigler et al. (2013) 92 Microscopic morphology of Nannizziopsis vriesii. (A) Scanning electron micrograph showing wall ornamentation of globose ascospores. (B and C) Slide culture preparations showing aleurioconidia, occasional arthroconidia, and undulate hyphae. (D) Arthroconidia and budding cells produced on PDA. Bars = 10 μm. Microscopic morphology of Nannizziopsis dermatitidis showing aleurioconidia (A), fission arthroconidia (B), and undulate hyphae (C). (D) Arthroconidia and budding cells produced on PDA. (E to I) Microscopic morphology of Nannizziopsis crocodili. (E and F) Scanning electron micrographs showing subglobose aleurioconidia among asperulate hyphae (indicated by arrow) of pseudogymnothecia. (G and H) Slide culture preparation showing aleurioconidia, fission arthroconidia, and an undulate hyphal branch (H inset). (I) Budding cells produced on BCP-MS-G agar. Bars = 10 μm Microscopic morphology of Nannizziopsis barbata showing aleurioconidia (A), fission arthroconidia and undulate hyphae (B), budding cells produced on PDA (C), and asperulate hyphae of a pseudogymnothecium on OAT (D). Microscopic morphology of Nannizziopsis guarroi showing aleurioconidia (E), undulate hyphae (F), and cylindrical arthroconidia, some of which are germinating (G). Microscopic morphology of Nannizziopsis infrequens showing aleurioconidia (H), undulate hyphae and rare intercalary arthroconidia (I), and ascomatal initials (arrow) (J). Bars = 10 μm. Microscopic morphology of Paranannizziopsis australasiensis showing aleurioconidia borne sessile or subtended by a swollen cell (arrows) (A), occasional intercalary arthroconidia (B), undulate hyphae (C), ascomatal initials (D and E), and mycelium with swollen cells produced in the vicinity of the initials (F). Microscopic morphology of Paranannizziopsis californiensis showing aleurioconidia sometimes subtended by a swollen cell (arrow) (G) and large irregularly shaped cells (H) associated with ascomatal initials (I). Bars = 10 μm. 93 Microscopic morphology of Paranannizziopsis crustacea showing aleurioconidia and occasional intercalary arthroconidia (A), fission arthroconidia (B), and an undulate hyphal branch (B inset). (C and D) Microscopic morphology of Ophidiomyces ophiodiicola showing aleurioconidia, fission arthroconidia, and numerous undulate hyphae. Bars = 10 μm. Histopathological sections of skin lesions showing typical arthroconidia of Paranannizziopsis crustacea (A) and aleurioconidia produced at the lesion surface by P. californiensis (B). Image B was used with the permission of A. P. Pessier, San Diego Zoo Institute for Conservation Research, San Diego, CA Toplon et al. (2013) observed an epizootic of ulcerative to nodular ventral dermatitis in a large breeding colony of 8-month to 5-year-old leopard geckos (Eublepharis macularius) of both sexes. Two representative mature male geckos were euthanized for diagnostic necropsy. The Chrysosporium anamorph of Nannizziopsis vriesii (CANV) was isolated from the skin lesions, and identification was confirmed by sequencing of the internal transcribed spacer region of the rRNA gene. Histopathology revealed multifocal to coalescing dermal and subcutaneous heterophilic granulomas that contained septate fungal hyphae. There was also multifocal epidermal hyperplasia with hyperkeratosis, and similar hyphae were present within the stratum corneum, occasionally with terminal chains of arthroconidia consistent with the CANV. In one case, there was focal extension of granulomatous inflammation into the underlying masseter muscle. This is the first report of dermatitis and cellulitis due to the CANV in leopard geckos. Dolinski et al. (2014) described a free-ranging plains garter snake (Thamnophis radix) with a brown, encrusted, cutaneous facial lesion which was presented depressed, emaciated, and dehydrated. It developed respiratory distress and was later euthanized after it failed to respond to antibiotics and supportive treatment. Disseminated granulomatous disease with prominent lung involvement was diagnosed at necropsy. Intralesional hyaline fungal elements, consisting of septate hyphae, were detected histologically among the necrotic core of granulomas and aggregates of epithelioid macrophages. Ophidiomyces ophiodiicola was cultured from tissue specimens and identification was confirmed using 18S rRNA ITS DNA sequencing. Isolation of O. ophiodiicola from lesions that contain morphologically consistent fungal elements strongly incriminates this fungus as the cause of disease in this garter snake. Allender et al. (2015) provided a detailed literature review, introduce new ecological and biological information and consider aspects of O. ophiodiicola that need further investigation. The current biological evidence suggests that this fungus can persist as an environmental saprobe in soil, as well as colonizing living hosts. Not unlike other emerging fungal pathogens, many fundamental questions such as the origin of O. ophiodiicola, mode of transmission, environmental influences, and effective treatment options still need to be investigated. 94 Ophidiomyces ophiodiicola in vipers. (A) Active infection in the ocular region in a cottonmouth (Agkistrodon piscivorous). (B) Crusts within the scales in a massasauga (Sistrurus catenatus). (C, D) More advanced infection of the face and tail in a massasauga. Arrows indicate location of infection. Allender et al. (2015b) designed a cottonmouth snake model to understand the role of O. ophiodiicola in SFD. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted. Facial swelling observed in cottonmouths (Agkistrodon piscivorous) experimentally challenged with Ophidiomyces ophiodiicola. Severity ranged from severe (A, B), moderate (C), and mild (D) 95 Skin lesions in cottonmouths with Snake Fungal Disease.Non-facial skin lesions observed in a cottonmouth (Agkistrodon piscivorous) experimentally challenged with Ophidiomyces ophiodiicola. http://dx.doi.org/10.1371/journal.pone.0140193.g004 Microscopic lesions in cottonmouth snakes inoculated with Ophidiomyces ophiodiicola. A. Normal nasolabial pit. HE stain. B. Nasolabial pit with epithelial thickening and crusts (arrowheads), erosion, ulceration, and dermatitis. Deeper in the head, there is osteomyelitis (asterisk). HE stain. C. Nasolabial pit with crusts and heterophilic dermatitis (arrowheads). HE stain. D. Granuloma deep to the nasolabial pit that contains intralesional fungal hyphae (black linear branching). GMS stain.http://dx.doi.org/10.1371/journal.pone.0140193.g005 Allender et al. (2015c) described the development of a real-time PCR (qPCR) assay for detecting a segment of the internal transcribed spacer 1 region between the 18S and 5.8S ribosomal RNA gene. The assay was able to detect as few as 1.05 × 10(1) gene copies per reaction. An additional 4 positive cases were detected when comparing a conventional PCR (n = 3) and the qPCR (n = 7) when used on swab samples from 47 eastern massasauga rattlesnakes. The newly developed assay is a sensitive and specific tool for surveillance and monitoring in the conservation of freeranging snakes. Guthrie et al.(2015) clinically examined 30 free-ranging snakes on public lands from April to October 2014. Skin biopsy samples were collected from nine snakes that had gross lesions suggestive of SFD; seven of these biopsies were suitable for histologic interpretation, and eight were suitable for culture and PCR detection of O. ophiodiicola. Seven snakes had histologic features consistent with SFD and were positive for O. ophiodiicola by PCR or fungal culture. 96 Gross images and histopathology from free-ranging snakes, Virginia, USA, 2014, infected with the fungus Ophidiomyces ophiodiicola. (A) Skin, rainbow snake (Farancia erytrogramma). Multiple dry, thickened, and slightly raised scales and subcutaneous pustules. (B) Skin, eastern racer (Coluber constrictor). Multiple large areas of dry, thickened, and crusty scales. (C) Skin, eastern racer (Coluber constrictor). The superficial epidermis is thickened by necrotic debris and mixed inflammatory cells with Periodic acid-Schiff (PAS)–positive fungal hyphae (double arrow). Low numbers of granulocytes and macrophages are present in the dermis. PAS. Bar550 mm. (D) Skin, northern water snake (Nerodia sipedon). The epidermis is replaced by a thick layer of necrotic debris admixed with numerous 2–5 mm in diameter PAS-positive fungal hyphae with parallel to undulating walls and occasional septations (arrow) and branching. Arthroconidia (*) are present on the epidermal surface. PAS. Bar520 mm. McBride et al. (2015) reported 8 free-ranging timber rattlesnakes (Crotalus horridus) from two geographically isolated Massachusetts populations with skin lesions located primarily on the head but occasionally also on the lateral and ventral surfaces of the body. The snakes underwent health assessments that included physical examination, clinical pathology, full body radiographs, and full thickness biopsies of skin lesions. Each snake had fungal elements present histologically in tissue sections from skin lesions. Ophidiomyces ophiodiicola was identified from skin lesions using polymerase chain reaction in all eight snakes. Example of dermatologic lesions affecting the lateral aspect of the face. Example of a dermatologic lesion affecting the lateral aspect of the face, caudal to the eye White arrows indicate areas of dermatitis. 97 Timber rattlesnake. Section through affected skin. Marked heterophilic and granulomatous inflammation with reactive fibrosis expands the dermis and is associated with formation of mature dermal granulomas (center). There is intermittent ulceration of the overlying epidermis (left). hematoxylin and eosin (3100). High magnification of dermal granuloma stained with Gomori–Grocott methenamine silver (GMS) stain revealing intralesional argyrophilic fungal hyphal elements. These organisms are 4–6 lm in diameter, septate, and have predominantly parallel cell walls with only rare branching (not present in this micrograph). GMS (3400). Allender et al. (2016) assayed 112 swabs from 102 individual eastern massasaugas (Sistrurus catenatus) at three locations in Michigan in 2014 for Ophidiomyces using quantitative PCR (qPCR). We observed a 12.7% qPCR prevalence of skin lesions. Individuals at each site had lesions, and occurrence of skin lesions was not significantly different between sites. We detected Ophidiomyces DNA at each of the three sites in five individuals (4.9%). We found no difference in detection probabilities between sites; however, snakes with dermatitis had higher Ophidiomyces DNA detection probabilities (P¼0.1560.08 SE) than snakes without dermatitis (P¼0.0260.01 SE, P¼0.026). The emergence of SFD mortalities has potentially serious consequences for the viability of the eastern massasauga in Michigan. Future work should track temporal patterns in vital rates and health parameters, link health data to body condition indices for individual snakes, and conduct a ‗‗hotspot‘‘ analysis to examine health on a landscape scale Eastern massasaugas (Sistrurus catenatus) with Ophidiomyces-positive skin lesions identified by PCR from Camp Grayling (A) and Pierce Cedar Creek Institute (B, C), Michigan, USA, 2014. Glorioso et al. (2016) documented the first documented occurrence of SFD in a freeranging wild snake in Louisiana, and one of the few documented cases in the United States of SFD in juvenile snakes. Clinical signs consistent with the disease have been observed in snakes from many areas of Louisiana in the last few years. They observed a juvenile Nerodia fasciata confluens (Blanchard) (Broad-banded Watersnake) coiled and basking alongside the trail on the southwestern part of the lake at Cypress Island Preserve. When captured, the snake was lethargic (despite warm temperatures near 25 °C), emaciated, and had numerous areas of ulceration, crusting, and firm swelling on the skin of the body and head . They collected the snake and brought it to the laboratory, where its health continued to decline. The snake was moribund when checked on 30 March 2015 (extremely unresponsive and unable to right itself), and 98 was euthanized with an intracoelomic injection of MS-222. On microscopic examination, fungi consistent with O. ophiodiicola were observed in the skin lesions, and O. ophiodiicola was isolated in culture from multiple skin lesions. Fungal identification was confirmed by sequencing the entire internal transcribed spacer region of the ribosomal RNA gene as described in Bohuski et al. (2015). A juvenile Broad-banded Watersnake (Nerodia fasciata confluens) collected during a survey at Cypress Island Preserve, St. Martin Parish, LA, which tested positive for Ophidiomyces ophiodiicola. The snake exhibited ulceration of the skin on the head (A), several crusty ventral scales (B), and numerous nodules overlaid by areas of roughened skin on the dorsal surface (C, D). Guthrie et al. (2016) clinically examined 30 free-ranging snakes on public lands from April to October 2014 to confirm the presence of SFD and O. ophiodiicola in snakes of eastern Virginia, US,. Skin biopsy samples were collected from nine snakes that had gross lesions suggestive of SFD; seven of these biopsies were suitable for histologic interpretation, and eight were suitable for culture and PCR detection of O. ophiodiicola. Seven snakes had histologic features consistent with SFD and eight were positive for O. ophiodiicola by PCR or fungal culture. Humphrey et al. (2016) described the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis. Skin biopsy samples from two adult male tuatara submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results. After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR. Last et al. (2016) presented a free-ranging mud snake (Farancia abacura) from Bulloch County, Georgia with facial swelling and emaciation. Extensive ulceration of the skin, which was especially severe on the head, and retained shed were noted on 99 external examination. Microscopic examination revealed severe heterophilic dermatitis with intralesional fungal hyphae and arthroconidia consistent with O. ophiodiicola A skin sample incubated on Sabouraud dextrose agar yielded a white-totan powdery fungal culture that was confirmed to be O. ophiodiicola by polymerase chain reaction and sequence analysis. Heavy infestation with adult tapeworms (Ophiotaenia faranciae) was present within the intestine. Various bacterial and fungal species, interpreted to either be secondary invaders or postmortem contaminants, were associated with oral lesions. Although the role of these other organisms in the overall health of this individual is not known, factors such as concurrent infections or immunosuppression should be considered in order to better understand the overall manifestation of snake fungal disease, which remains poorly characterized in its host range and geographic distribution. Masters et al. (2016) reported 6 cases of dermatomycosis which were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five Masters. Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA. All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates.These were the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Ravesi et al. (2016) documented an alarming number of cases of Snake Fungal Disease (SFD), a condition frequently resulting in morbidity and mortality in snakes, in numerous species across much of the eastern US. showing a skin lesion on the face of a free-ranging Lampropeltis triangulum (Eastern Milksnake) from the northern Lower Peninsula of Michigan. The lesion tested positive for Ophidiomyces ophiodiicola, the causative agent of SFD. Our results document the second species from Michigan known to be infected with SFD. This case adds to the growing body of literature detailing the distribution of snake species affected, and further indicates that this pathogen is widespread in the eastern US. We stress the continued need for increased, systematic sampling efforts to determine the species affected by SFD and the potentially deleterious impacts it has on snake populations. 100 Schmidt-Ukaj et al. (2016) identified chronic dermatomycosis in 3 central bearded dragons (Pogona vitticeps), held as companion animals by the same owner. Clinical signs of dermatomycosis included subcutaneous masses as well as crusty, erosive, and ulcerative skin lesions. The facial region was affected in 2 of the 3 cases. Masses were surgically excised, and histology confirmed necrotizing and granulomatous inflammatory processes associated with fungal hyphae. Two of the bearded dragons were euthanized because of their deteriorating condition. In both cases, postmortem histology confirmed systemic fungal infections despite treatment of 1 animal with itraconazole. In the third bearded dragon, therapy with voriconazole at 10 mg/kg was initially effective, but mycotic lesions reappeared 15 months later. Nannizziopsis chlamydospora was identified by PCR and subsequent DNA sequencing in 2 of these cases. References: 1. Abarca, M. L., J. Martorell, G. Castella, A. Ramis, and F. J. Cabanes. 2008. Cutaneous hyalohyphomycosis caused by a Chrysosporium species related to Nannizziopsis vriesii in two green iguanas (Iguana iguana). Med. Mycol. 46349354. 2. Abarca ML, Martorell J, Castellá G, Ramis A, Cabañes FJ. 2009. 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Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand. New Zealand veterinary journal 64(5):1-16 · April 2016 24. McBride ,Michael P.., Kimberlee B. Wojick, Timothy A. Georoff., Jason Kimbro, Michael M. Garner, Xiaoling Wang, April L. Childress, and James F. X. Wellehan, Jr., Ophidiomyces ophiodiicola dermatitis in eight freeranging Timber rattlesnakes (crotalus horridus) from Massachusetts 25. Pare JA, Sigler L, Rypien KL, Gibas C-F. Cutaneous mycobiota of captive squamate reptiles with notes on the scarcity of Chrysosporium anamorph Nannizziopsis vriesii. J Herpetological Med Surg. 2003;13:10–5 26. Paré JA, Sigler L, Hunter DB, Summerbell RC, Smith DA, Machin KL. 1997. Cutaneous mycoses in chameleons caused by the Chrysosporium anamorph of Nannizziopsis vriesii (Apinis) Currah. J. Zoo Wildl. Med. 28:443–453 27. Paré A, Coyle KA, Sigler L, Maas AK 3rd, Mitchell RL. Pathogenicity of the Chrysosporium anamorph of Nannizziopsis vriesii for veiled chameleons (Chamaeleo calyptratus). Med Mycol. 2006 Feb;44(1):25-31. 28. Nichols, D.K., R.S. Weyant, E.W. Lamirande, L. Sigler, and R.T. Mason. 1999. Fatal mycotic dermatitis in captive Brown Tree Snakes (Boiga irregularis). Journal of Zoo and Wildlife Medicine 30(1):111–118. 29. Rajeev, S., D.A. Sutton, B.L. Wickes, D.L. Miller, D. Giri, M. Van Meter, E.H. Thompson, M.G. Rinaldi, A.M. Romanelli, and J.F. Cano. 2009. Isolation and characterization of a new fungal species, Chrysosporium ophiodiicola, from a mycotic granuloma of a Black Rat Snake (Elaphe obsolete obsoleta). Journal of Clinical Microbiology 47:1264–1268. 30. Ravesi M.J., S.J. Tetzlaff, M.C. Allender, and B.A. Kingsbury Dolinski, A.C., M.C. Allender, V. Hsiao, and C.W. Maddox. 2014. Systemic Ophidiomyces ophiodiicola infection in a free-ranging Plains Garter Snake (Thamnophis radix). Journal of Herpetological Medicine and Surgery 24:7–10. 31. Schmidt-Ukaj, S. , Igor Loncaric , Joachim Spergser , Barbara Richter , Manfred Hochleithner. Dermatomycosis in Three Central Bearded Dragons (Pogona Vitticeps) Associated With Nannizziopsis Chlamydospora J Vet Diagn Invest 28 (3), 319322. 2016 Mar 07. 32. Seifert K, Morgan-Jones G, Gams W, Kendrick B (2011) The genera of Hyphomycetes. Utrecht, The Netherlands: Centraalbureau voor Schimmelcultures. 33. Pare´ JA, Sigler L, Rosenthal KL, Mader DR (2006) Microbiology: fungal and bacterial diseases of reptiles. In: Mader DR, editor. Reptile medicine and surgery. St.Louis, USA: Saunders Elsevier. pp. 217–238. 34. Sigler, L., S. Hambleton, and J.A. Paré. 2013. Molecular characterization of reptile pathogens currently known as members of the Chrysosporium anamorph of Nannizziopsis vriesii complex and relationship with some human-associated isolates. Journal of Clinical Microbiology 51:3338–3357. 35. Sigler L, Hambleton S, Pare JA (2013) Molecular characterization of reptile pathogens currently known as members of the Chrysosporium anamorph of Nannizziopsis vriesii complex and relationship with some human-associated isolates. J Clin Microbiol 51: 3338–3357. 103 36. Stchigel AM, Sutton DA, Cano-Lira JF, Caban es FJ, Abarca ML, et al. (2013). Phylogeny of chrysosporia infecting reptiles: proposal of the new family Nannizziopsiaceae and five new species. Persoonia 31: 86–100. 37. Tetzlaff, S.J., M. Allender, M. Ravesi, J. Smith, and B. Kingsbury. 2015. First report of snake fungal disease from Michigan, USA, involving Massasaugas, Sistrurus catenatus (Rafinesque 1818). Herpetology Notes 8:31–33. 38. Thomas, A. D., L. Sigler, S. Peucker, J. H. Norton, and A. Neilan. 2002. Chrysosporium anamorph of Nannizziopsis vriesii associated with fatal cutaneous mycoses in the salt water crocodile (Crocodylus porosus). Med. Mycol. 40143-151. 39. Vidal P, Vinuesa MA, Sa´nchez-Puelles JM, Guarro J (2000) Phylogeny of the anamorphic genus Chrysosporium and related taxa based on rDNA internal transcribed spacer sequences. In: Kushwaha RKS, Guarro J, editors. Biology of dermatophytes and other keratinophilic fungi. Bilbao, Spain: Revista Iberoamericana de Micologı´a. pp. 22–29. 40. Vissiennon, T., K. F. Schuppel, E. Ullrich, and A. F. Kuijpers. 1999. Case report. A disseminated infection due to Chrysosporium queenslandicum in a garter snake (Thamnophis). Mycoses 42, 107-110. 104 4. Snake fungal disease (SFD) Snake Fungal Disease (SFD) is an emerging disease that has garnered increased awareness for freeranging snakes. Clinical signs of SFD include facial swelling and disfiguration, scale discolouration, lesions, granulomas and dysecdysis. The fungus Ophidiomyces ophiodiicola (formerly Chrysosporium ophiodiicola) is frequently correlated with snakes presenting symptoms of SFD.           An outbreak of fungal mycosis with clinical signs consistent with SFD was seen in 42 pigmy rattlesnakes (S. miliarius), one garter snake (Thamnophis sp.), and a ribbon snake (Thamnophis sauritus) within a 2 yr period, and retrospectively the authors identified an additional 59 pigmy rattlesnakes with signs consistent with mycotic disease, but neither Ophidiomyces nor CANV were identified in that case (Cheatwood et al., 2003). In 2006, a population of timber rattlesnakes (Crotalus horridus) in New Hampshire was observed with lesions on the head, neck, and body consistent with SFD (Clark et al., 2011). Since 2008, Eastern Massasaugas from the Carlyle Lake population in Illinois have been diagnosed with SFD (Allender et al., 2011, 2015). A case of O. ophiodiicola was observed in a captive black rat snake (Elaphe obsoleta obsoleta) with a subcutaneous nodule (Rajeev et al., 2009). Snakes with skin lesions and fungal dermatitis have been reported from several areas largely within the eastern half of the United States in colubrid and viperid species. (Rajeev et al., 2009, Allender et al., 2011; Clark et al., 2011; Smith, Edwards and Lorch, 2013) The manifestation of SFD in North American colubrids snakes included pneumonia, ocular infections, and subcutaneous nodules (Rajeev et al., 2009; Sleeman, 2013; Dolinski et al., 2014). Mycosis was observed in the skin as well as a more systemic invasion involving the lungs and eye (one case) and lungs and liver (one case) of garter snakes (Dolinski et al., 2014) Prior to 2014, lesions were thought to only affect the head and neck of snakes, but lesions have now been observed in the skin of the entire body in massasaugas in Michigan (Tetzlaff et al., 2015). Infections have been observed with great frequency in pitvipers (Cheatwood et al., 2003; Allender et al., 2011; Clark et al., 2011; Smith et al., 2013; Tetzlaff et al., 2015), The genus Ophidiomyces currently contains only one species, O. ophiodiicola, and it is known to infect only snakes leading to the syndrome Snake Fungal Disease (SFD) which causes widespread morbidity and mortality across the eastern United States (Allender et al., 2013; Sigler et al., 2013; Sleeman, 2013). 105 Aetiology: Ophidiomyces ophiodiicola (Guarro, Deanna A. Sutton, Wickes & Rajeev) Sigler, Hambl. & Paré, Index Fungorum 19: 1 (2013) =Chrysosporium ophiodiicola Guarro, Deanna A. Sutton, Wickes & Rajeev, Journal of Clinical Microbiology 47 (4): 1268 (2009) Cultures of O. ophiodiicola are powdery with whitish mycelium that becomes light yellowish with age. The cultures emit a pungent, skunk like odour. Optimal growth for O. ophiodiicola occurs at a temperature of 25 °C. Most isolates fail to grow at 35 °C. O. ophiodiicola is able to grow over pH range of 5-11 with optimal growth observed at pH of 9. O. ophiodiicola is able tolerate matric induced water stress below -5 MPa. The fungus exhibits strong urease activity and produces robust growth on ammonium sulfate, sulfite and thiosulfate Colonial and microscopic morphology of Chrysosporium ophiodiicola R-3923. (A) PYE, front and reverse; (B) potato dextrose agar, front and reverse; (C) PCA, front and reverse; (D) fertile hyphae and conidia; (E) conidia showing remnants of wall following rhexolytic dehiscence; (F) fertile hyphae with arthroconidia and terminal and lateral conidia Rajeev et al. (2009) 106 a, b. Fertile hyphae bearing sessile conidia, intercalary conidia (black arrows) and intercalary chains of arthroconidia (white arrows); c, d. arthroconidia; e. sessile conidia. — Scale bars = 10 µm (a, b, d, differential interference contrast; c, e, phase contrast). Stchigel et al., 2013 Reports: Rajeev et al. (2009) reported isolation and characterization of the new species Chrysosporium ophiodiicola from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta). Analysis of the sequences of different fragments of the ribosomal genes demonstrated that this species belongs to the Onygenales and that this species is genetically different from other morphologically similar species of Chrysosporium. This new species is unique in having both narrow and cylindricalto-slightly clavate conidia and a strong, pung ent odor. Cutaneous masses; (B) hematoxylin and eosin-stained section of the lesion; (C) Grocott-Gomori methenamine silver-stained section of the lesion. 107 Allender et al. (2011), in a note to the editor of the Emerg Infect Dis., mentioned that during 2008, the ninth year of a long-term biologic monitoring program, 3 eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) with severe facial swelling and disfiguration died within 3 weeks after discovery near Carlyle, Illinois, USA. In spring 2010, a similar syndrome was diagnosed in a fourth massasauga; this snake continued to be treated with thermal and nutritional support and antifungal therapy. A keratinophilic fungal infection caused by Chrysosporium sp. was diagnosed after physical examination, histopathologic analysis, and PCR in all 4 snakes. The prevalence of clinical signs consistent with Chrysosporium sp. infection during 2000– 2007 was 0.0%, and prevalence of Chrysosporium sp.–associated disease was 4.4% (95% confidence interval [CI] 1.1%–13.2%) for 2008 and 1.8% (95% CI 0.0%– 11.1%) for 2010. Clinical and gross necropsy abnormalities were limited to the heads of affected animals. In each case, a unilateral subcutaneous swelling completely obstructed the nasolabial pits. In the most severely affected snake, swelling extended to the cranial aspect of the orbit and maxillary fang. Notable histologic lesions were restricted to skin, gingiva, and deeper tissues of the head and cervical region and consisted of cutaneous ulcers with granulomas in deeper tissues. Ulcers had thick adherent serocellular crusts and were delineated by small dermal accumulations of heterophils and fewer macrophages. Crusts contained numerous 4–6-µm diameter right-angle branching fungal hyphae with terminal structures consistent with spores. In 1 snake, infection was associated with retained devitalized layers of epidermis consistent with dysecdysis. In the same snake, the eye and ventral periocular tissues were effaced by inflammation, but the spectacle and a small fragment of cornea remained; the corneal remnant contained few fungal hyphae. In all snakes, in addition to deep cutaneous ulceration, the dermis, hypodermis and skeletal muscle of the maxillary and or mandibular region contained multiple granulomas, centered on variable numbers of fungal hyphae. In 1 snake, similar granulomas were also observed in maxillary gingival submucosa and subjacent maxillary bone. Chrysosporium sp. fungal infection in eastern massasagauga rattlesnake (Sistrurus catenatus catenatus). A) Facial dermatitis and cellulitis caused by Chrysosporium sp. infection in rattlesnake from Carlyle, Illinois, USA; B) close-up 108 showing maxillary fang destruction. C) Maxillary dermal and subcutaneous fungal granuloma (circled area). Hematoxylin and eosin stain, original magnification ×2, scale bar = 5 μ . D Gra ulo a e ter ith large u ers of fu gal h phae. Gro ott ethe a i e sil er stai , origi al ag ifi atio × , s ale ar = μ . Sleeman (2013) mentioned that Snake Fungal Disease (SFD) is an emerging disease in certain populations of wild snakes in the eastern and midwestern United States. While fungal infections were occasionally reported in wild snakes prior to 2006, recently the number of free-ranging snakes with fungal dermatitis submitted to the USGS National Wildlife Health Center (NWHC) and other diagnostic laboratories has been increasing. Laboratory analyses have demonstrated that the fungus Ophidiomyces (formerly Chrysosporium) ophiodiicola is consistently associated with SFD, but often, additional fungi are isolated from affected snakes. At this time, definitive evidence that O. ophiodiicola causes SFD is inconclusive. As its name implies, SFD is only known to afflict snakes. To date, the NWHC has confirmed fungal dermatitis (or the suspected fungal pathogen in association with skin lesions) in wild snakes from nine states, including Illinois, Florida, Massachusetts, Minnesota, New Jersey, New York, Ohio, Tennessee, and Wisconsin. However, it is suspected that SFD is more widespread in the United States than is currently documented. Multiple species of snakes have been diagnosed with SFD at the NWHC including northern water snake (Nerodia sipedon), eastern racer (Coluber constrictor), rat snake (Pantherophis obsoletus species complex), timber rattlesnake (Crotalus horridus), massasauga (Sistrurus catenatus), pygmy rattlesnake (Sistrurus miliarius), and milk snake (Lampropeltis triangulum). The most consistent clinical signs of SFD include scabs or crusty scales, subcutaneous nodules, premature separation of the outermost layer of the skin (stratum corneum) from the underlying skin (or abnormal molting), white opaque cloudiness of the eyes (not associated with molting), or localized thickening or crusting of the skin (hyperkeratosis). Skin ulcers, swelling of the face, and nodules in the deeper tissues of the head have also been documented. Clinical signs of SFD and disease severity may vary by snake species. Eastern racer (Coluber constrictor) showing signs of fungal skin infection. Obvious external abnormalities are an opaque infected eye (spectacle), roughened crusty scales on the chin, and several discolored roughened scales on the side of neck. Snake captured in Volusia County, Florida, in January 2013 (case 24266). Photograph by D.E. Green, USGS National Wildlife Health Center. Eastern rat snake (Pantherophis alleghaniensis) showing signs of fungal infection. Obvious external abnormalities are an opaque infected eye (spectacle) and roughened, crusty scales on the snout. Snake captured in New Jersey in March 2012 (case 23906). Photograph by D.E. Green, USGS National Wildlife Health Center. 109 Northern water snake (Nerodia sipedon) with crusty and thickened scales overlaying raised blisters as a result of a fungal skin infection, captured from island in western Lake Erie, Ohio, in August 2009 (case 22747). Photograph by D.E. Green, USGS National Wildlife Health Center Northern water snake (Nerodia sipedon) with crusty and thickened scales overlaying raised blisters as a result of a fungal skin infection, captured from island in western Lake Erie, Ohio, in August 2009 (case 22747). Photograph by D.E. Green, USGS National Wildlife Health Center. Pygmy rattlesnake (Sistrurus miliarius) with multiple raised lumps (nodules) in the dorsal skin of the lower body and tail covered with roughened and crusty scales, captured in Volusia County, Florida, in October 2012. Photograph by D.E. Green, USGS National Wildlife Health Center. Milk snake (Lampropeltis triangulum) showing signs of fungal and bacterial infections, captured in Westchester County, New York, February 2013 (case 24281). Photograph by D.E. Green, USGS National Wildlife Health Center. Eastern racer (Coluber constrictor) showing signs of fungal infection, numerous white spots on belly scales, captured in Dutchess County, New York, in July 2012 (case 24042). Photograph by D.E. Green, USGS National Wildlife Health Center. 110 Eastern rat snake (Pantherophis alleghaniensis) showing signs of fungal infection with crusting on lateral scales, captured in Passaic County, New Jersey, in March 2012 (case 23906). Photograph by D.E. Green, USGS National Wildlife Health Center. Dolinski et al. (2014) reported a free-ranging plains garter snake (Thamnophis radix) with a brown, encrusted, cutaneous facial lesion presented depressed, emaciated, and dehydrated. It developed respiratory distress and was later euthanized after it failed to respond to antibiotics and supportive treatment. Disseminated granulomatous disease with prominent lung involvement was diagnosed at necropsy. Intralesional hyaline fungal elements, consisting of septate hyphae, were detected histologically among the necrotic core of granulomas and aggregates of epithelioid macrophages. Ophidiomyces ophiodiicola was cultured from tissue specimens and identification was confirmed using 18S rRNA ITS DNA sequencing. Isolation of O. ophiodiicola from lesions that contain morphologically consistent fungal elements strongly incriminates this fungus as the cause of disease in this garter snake. Plains garter snake, systemic mycosis. There is a 1cm round, non-proliferative, brown, dry, encrusted lesion caudal to the left eye. Plains garter snake, systemic mycosis, histologic section of the left eye. There is diffuse granulomatous panophthalmitis with corneal edema, cataract, retina effacement, and orbital cellulitis. At higher magnification (inserted box), the granulomas (*) consist primarily of coalescing aggregates of epitheloid and foamy macrophages. H&E. 111 Plains garter snake, systemic mycosis, histologic section of lung. There are interspersed granulomas (*) and a coagulum of inflammatory exudate in a bronchus (arrow). At higher magnification (right panel), the granuloma (*) comprises a central coagulum of necrotic cellular debris surrounded by layers of epithelioid and foamy macrophages. H&E. Allender et al. (2015a) provided a detailed literature review, introduce new ecological and biological information and consider aspects of O. ophiodiicola that need further investigation. The current biological evidence suggests that this fungus can persist as an environmental saprobe in soil, as well as colonizing living hosts. Not unlike other emerging fungal pathogens, many fundamental questions such as the origin of O. ophiodiicola, mode of transmission, environmental influences, and effective treatment options still need to be investigated. 112 Morphology and carbon utilization of O. ophiodiicola. (A) Ophidiomyces ophiodiicola isolate on PDA at 25 d; left is the upper surface of the colony, right is the bottom of the plate. (B) Ophidiomyces ophiodiicola asexual reproduction via arthroconidia demonstrating schizolytic dehiscence and (C) stalked aleurioconidia. Ophidiomyces ophiodiicola growth at after 25 d on (D) autoclaved Carassius sp., (E) autoclaved Locusta migratoria and (F) autoclaved Lentinula edodes Growth of O. ophiodiicola after 20 d (G) demineralized Pleoticus muelleri exoskeletons and (H) demineralized and deproteinated exoskeletons. All size bars [ 5 mm. 113 In vitro growth of Ophidiomyces ophiodiicola. (A) O. ophiodiicola demonstrating gelatinase activity. (B) Carbon enzymatic assays: PDA (control, bottom), Mn-dependent peroxidase (negative, left), chitinase (negative, top) and b-glucosidase (positive, right). (C) Carbon enzymatic assays: PDA (control, bottom), lipase using olive oil (left, positive), lipase using lard (top, positive) and lipase/esterase using Tween 80 (right, positive). (D) Keratinase assay: control left (negative) and inoculated right (positive). (E) Sole nitrogen source assays: left to right, columns 1e2 [ nitrate, columns 3e4 [ nitrite, columns 5e6 [ ammonium, columns 7e8 [ L-asparagine, columns 9e10 [ control with no nitrogen source, columns 11e12 [ uric acid. Rows A-D (12-34933), Rows E-H (12-33400), rows A, E (pH 5), rows B, F (pH 6), rows C, G (pH 7), rows D, H (pH 8). (F) Colony diameter of O. ophiodiicola isolates over the pH range of 5e11. Isolate colony diameters vary but growth pattern was consistent. Solid line represents mean values; dotted lines represent standard error of the mean. Ophidiomyces ophiodiicola in vipers. (A) Active infection in the ocular region in a cottonmouth (Agkistrodon piscivorous). (B) Crusts within the scales in a massasauga (Sistrurus catenatus). (C, D) More advanced infection of the face and tail in a massasauga. Arrows indicate location of infection. Allender et al. (2015b) conducted an experiment to understand the role of O. ophiodiicola in SFD. A cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted. 114 Summary of survival, DNA detection from swabs, and presence of microscopic lesions consistent with SFD in cottonmouth snakes (Agkistrodon piscivorous) experimentally challenged with Ophidiomyces ophiodiicola. Facial swelling in Cottonmouths with Snake Fungal Disease. Facial swelling observed in cottonmouths (Agkistrodon piscivorous) experimentally challenged with Ophidiomyces ophiodiicola. Severity ranged from severe (A, B), moderate (C), and mild (D) Skin lesions in cottonmouths with Snake Fungal Disease. Non-facial skin lesions observed in a cottonmouth (Agkistrodon piscivorous) experimentally challenged with Ophidiomyces ophiodiicola Microscopic lesions in cottonmouth snakes inoculated with Ophidiomyces ophiodiicola. A. Normal nasolabial pit. HE stain. B. Nasolabial pit with epithelial thickening and crusts (arrowheads), erosion, ulceration, and dermatitis. Deeper in the head, there is osteomyelitis (asterisk). HE stain. C. Nasolabial pit with crusts and heterophilic dermatitis (arrowheads). HE stain. D. Granuloma deep to the nasolabial pit that contains intralesional fungal hyphae (black linear branching). GMS stain 115 Bohuski et al. (2015) developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targeted the internal transcribed spacer region (ITS) of the fungal genome while the other targeted the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. Lorch et al. (2015) experimentally infected captive-bred corn snakes (Pantherophis guttatus) in the laboratory with pure cultures of O. ophiodiicola. All snakes in the infected group (n = 8) developed gross and microscopic lesions identical to those observed in wild snakes with SFD; snakes in the control group (n = 7) did not develop skin infections. Furthermore, the same strain of O. ophiodiicola used to inoculate snakes was recovered from lesions of all animals in the infected group, but no fungi were isolated from individuals in the control group. Monitoring progression of lesions throughout the experiment captured a range of presentations of SFD that have been described in wild snakes. The host response to the infection included marked recruitment of granulocytes to sites of fungal invasion, increased frequency of molting, and abnormal behaviors, such as anorexia and resting in conspicuous areas of enclosures. While these responses may help snakes to fight infection, they could also impact host fitness and may contribute to mortality in wild snakes with chronic O. ophiodiicola infection. This work provided a basis for understanding the pathogenicity of O. ophiodiicola and the ecology of SFD by using a model system that incorporates a host species that is easy to procure and maintain in the laboratory. 116 Clinical signs of SFD in snakes experimentally challenged with Ophidiomyces ophiodiicola. (A to C) Shaminoculated sites of snakes in the control group did not develop gross lesions characteristic of SFD. However, subtle damage to the scales (arrow) caused by the abrasion process was visible at the dorsal midbody site. In contrast, snakes exposed to O. ophiodiicola developed a range of clinical signs as the disease progressed. (D) Initially, individually infected scales were swollen and whitened (arrow). (E and F) Infected scales later became thickened and turned yellow to brown (E), eventually forming crusts of necrotic skin (F). (G) Infected skin on the snout became similarly thickened and yellow-brown. (H and I) Immediately prior to shedding, fluid accumulated between the old and new layers of skin, causing distortion of the head (H) and vesicle formation at inoculation sites on the body (I). (J to L) The presentations observed in experimentally infected snakes were consistent with those observed in wild snakes diagnosed with SFD at the U.S. Geological Survey National Wildlife Health Center, which often included thickened, yellow-brown areas of skin on the head (J) and ventral scales (K) and edematous scales (arrow) and crusting (asterisk) of the skin (L) 117 Microscopic lesions of SFD in snakes experimentally challenged with Ophidiomyces ophiodiicola. (A and B) Skin samples from sham-inoculated snakes were within normal limits (PAS stain). Bar, 500 µm (A) or 100 µm (B). (C) Skin samples from sham-inoculated snakes exhibited focal breaks in the stratum corneum attributed to mechanical damage from abrasion; the underlying epidermis was generally within normal limits (PAS stain). Bar, 100 µm. (D and E) Skin samples from snakes exposed to O. ophiodiicola developed multifocal superficial epidermal necrosis with extensive epidermal edema (D) and heterophil infiltration and mononuclear to granulocytic dermal inflammation (E) (PAS stain). Bar, 500 µm (D) or 100 µm (E). (F) Breaks in the stratum corneum in infected snakes were most common over areas of epidermal necrosis and granulocytic inflammation, suggesting that infection may be facilitated by preexisting damage to the skin surface (PAS stain). Bar, 100 µm. (G) Some infected snakes developed granulomas consisting of fungal hyphae (arrow) and epithelioid macrophages surrounded by lymphocytes and plasma cells (PAS stain; bar, 50 μm). (H) Areas of epidermal necrosis in snakes exposed to O. ophiodiicola often contained 2- to 5-µm-diameter, parallel-walled, septate, branching, fungal hyphae and ~2- by 5-µm superficial rectangular arthroconidia (GMS stain). Bar, 50 µm. (I) In a snake preparing to undergo ecdysis, the new stratum corneum can be seen beneath the necrotic epidermis (asterisk) of a lesion. Most fungal hyphae (stained black) are within the older epidermis that will be shed; however, hyphae that have invaded the new epidermis (arrow) may persist after the molt (GMS stain.) Bar, 100 µm. (J to L) Skin samples from free-ranging wild snakes diagnosed with SFD at the U.S. Geological Survey National Wildlife Health Center exhibited lesions similar to those that developed in experimentally infected snakes, including multifocal epidermal necrosis, granulocytic inflammation, and edema (PAS; bar, 500 µm) (J), mixed dermal inflammation (PAS; bar, 100 μm) (K), and fungal hyphae and arthroconidia morphologically consistent with O. ophiodiicola (GMS; bar, 50 µm) (L). McBride et al. (2015) observed 8 free-ranging timber rattlesnakes (Crotalus horridus) from two geographically isolated Massachusetts populations with skin lesions located primarily on the head but occasionally also on the lateral and ventral surfaces of the body. The snakes underwent health assessments that included physical examination, clinical pathology, full body radiographs, and full thickness biopsies of skin lesions. Each snake had fungal elements present histologically in tissue sections from skin 118 lesions. Ophidiomyces ophiodiicola was identified polymerase chain reaction in all eight snakes. from skin lesions using Tetzlaff et al. (2015) reported on cases of SFD involving S. catenatus in Michigan, USA, which is several hundred kilometres north of Carlyle Lake. In June 2013, two adult male S. catenatus (herein ―Snake One‖ and ―Snake Two‖, snout-to-vent length = 57.8 cm and 62.0 cm; tail length = 6.7 cm and 6.3 cm, mass = 253.2 g and 258.2 g, respectively) were captured for a radio-telemetry study in Grayling, Michigan, USA. Radio transmitters were implanted in both snakes on 19 June 2013. They were released at their points of capture on 27 June 2013 and radio-tracked three times per week. Upon relocating Snake One for the first time in the field post-surgery on 28 June 2013, slight misalignment of the mandibles was observed. The snake continued to be monitored in the field. It also showed signs of dysecdysis for several weeks, beginning on 29 June 2013. From the release date until 15 July 2013, Snake One remained in the same general area without large movements compared to several other asymptomatic telemetered males in the study area concurrently being radiotracked. The snake continued to have problems shedding, and the facial misalignment progressively worsened. On 04 August 2013 it was found to be very lethargic, with the mandibles appearing out of place and the trachea slightly everted from the oral cavity. The right side of the face had crusty lesions, especially on the labial area. In order to perform additional diagnostics to better understand the cause of the lesions, the snake was removed from the field on 05 August 2013. Snake Two (captured within 50 m of Snake One) was not immediately symptomatic for SFD after several weeks of relocations, though it, too, did not make large movements. On 31 August 2013, what appeared to be a protruding scale on the left side of the snake‘s face was observed. After several weeks, a noticeable lesion had formed in this area that was crusted and concave. Additionally, lesions on the neck, dorsal region and tail formed. Because the number of lesions increased and they resembled those of Snake One, Snake Two was removed from the field on 21 September 2013 to perform additional diagnostics. The presence of O. ophiodiicola was confirmed by testing biopsied tissue of lesions from both snakes using qPCR and genetic sequencing. The snakes were brought into captivity in attempts to treat them, but to no avail. Snake One and Snake Two died just over two months and within two weeks of treatment, respectively, and necropsy of the snakes confirmed fungal dermatitis and myositis. These are the first confirmed cases of O. ophiodiicola infection in free-ranging snakes in Michigan and the second population of S. catenatus for which individuals have tested positive. . Snake One with crusty lesions on face 119 Snake Two with face (A) and neck (B) lesions circled in yellow Allender et al. (2016) assayed 112 swabs from 102 individual eastern massasaugas (Sistrurus catenatus) at three locations in Michigan in 2014 for Ophidiomyces using quantitative PCR (qPCR). They observed a 12.7% qPCR prevalence of skin lesions. Individuals at each site had lesions, and occurrence of skin lesions was not significantly different between sites. We detected Ophidiomyces DNA at each of the three sites in five individuals (4.9%). no difference was found in detection probabilities between sites; however, snakes with dermatitis had higher Ophidiomyces DNA detection probabilities (P=0.15±0.08 SE) than snakes without dermatitis (P=0.02±0.01 SE, P=0.026). The emergence of SFD mortalities has potentially serious consequences for the viability of the eastern massasauga in Michigan. Future work should track temporal patterns in vital rates and health parameters, link health data to body condition indices for individual snakes, and conduct a "hotspot" analysis to examine health on a landscape scale. Last et al. (2016) autopsied a free-ranging mud snake (Farancia abacura) from Bulloch County, Georgia, because of facial swelling and emaciation. Extensive ulceration of the skin, which was especially severe on the head, and retained shed were noted on external examination. Microscopic examination revealed severe heterophilic dermatitis with intralesional fungal hyphae and arthroconidia consistent with O. ophiodiicola A skin sample incubated on Sabouraud dextrose agar yielded a white-to-tan powdery fungal culture that was confirmed to be O. ophiodiicola by polymerase chain reaction and sequence analysis. Heavy infestation with adult tapeworms (Ophiotaenia faranciae) was present within the intestine. Various bacterial and fungal species, interpreted to either be secondary invaders or postmortem contaminants, were associated with oral lesions. Although the role of these other organisms in the overall health of this individual is not known, factors such as concurrent infections or immunosuppression should be considered in order to better understand the overall manifestation of snake fungaldisease, which remains poorly characterized in its host range and geographic distribution. Lorch et al. (2016) reviewed the current state of knowledge about O. ophiodiicola and SFD. The original findings demonstrated that O. ophiodiicola is widely distributed in eastern North America, has a broad host range, is the predominant cause of fungal skin infections in wild snakes and often causes mild infections in snakes emerging from hibernation. This new information, together with what is already 120 available in the scientific literature, advances our knowledge of the cause, pathogenesis and ecology of SFD. However, additional research is necessary to elucidate the factors driving the emergence of this disease and develop strategies to mitigate its impacts. This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience. Parke (2016) mentioned that SFD has become a point of discussion and concern among the scientific community, especially after significant declines in localized snake populations across the Midwest and Eastern United States, had been discovered as a result of infection(s) confirmed to be associated with this fungus. In New Jersey several snake species, including Timber Rattlesnake, Corn Snake, Pine Snake, Black Rat Snake, and Black Racer, have been confirmed with SFD. According to the USGS National Wildlife Health Center, ―The most consistent clinical signs of SFD include scabs or crusty scales, subcutaneous nodules, premature separation of the outermost layer of the skin (stratum corneum) from the underlying skin (or abnormal molting), white opaque cloudiness of the eyes (not associated with molting), or localized thickening or crusting of the skin (hyperkeratosis). Skin ulcers, swelling of the face, and nodules in the deeper tissues of the head have also been documented. Clinical signs of SFD and disease severity may vary by snake species.‖ In some cases it has been documented to affect the snake‘s ability to obtain prey and can lead to malnutrition and die of starvation. Additionally SFD can lead a snake to exhibit behaviors that, in the wild, could cause the snake to spend more time in open areas to bask and thus become more exposed to predation. Photos by John Parke McCoy et al. (2017) monitored the severity of clinical signs of SFD, surface air temperature, reproductive status, body condition and serum complement activity (plasma bactericidal ability) in free-ranging pigmy rattlesnakes, Sistrurus miliarius, over the course of 18 months. Seasonal increases in the severity of clinical signs of SFD were correlated negatively with monthly air surface temperature and the mean 121 body condition of the population. Bactericidal ability varied seasonally, but pigmy rattlesnakes suffering from active SFD infections did not exhibit deficits in innate immune function. Infected snakes were in significantly lower body condition when compared with the general population, but seasonal patterns in the mean body condition of the population were not driven by seasonal patterns of infection severity. Our results highlight the potential importance of the thermal environment and energetic status in determining infection severity and outcomes and the need for managers and researchers to consider seasonality of symptom presentation when the goal is to identify the prevalence or incidence of SFD in populations. Example images of typical lesions on ventral surface (A), lesions on the dorsal surface (B) and a snake that was scored as a 3 (high) based on multiple body lesions in addition to lesions on the face (C and D). Lesions are indicated by arrows in A and B. References: 1. Allender MC, Dreslik M, Wylie S, et al. Chrysosporium sp. Infection in Eastern Massasauga Rattlesnakes. Emerging Infectious Diseases. 2011;17(12):2383-2384. 2. Allender MC, Hileman ET, Moore J, Tetzlaff S. Detection of Ophidiomyces, the Causative Agent of Snake Fungal Disease, in the Eastern Massasauga ( Sistrurus catenatus ) in Michigan, USA, 2014. J Wildl Dis. 2016 Jul;52(3):694-8. 3. Allender MC, Baker S, Wylie D, Loper D, Dreslik MJ, Phillips CA, et al. (2015) Development of Snake Fungal Disease after Experimental Challenge with Ophidiomyces ophiodiicola in Cottonmouths (Agkistrodon piscivorous). PLoS ONE 10(10): e0140193. doi:10.1371/journal.pone.0140193 4. Allender MC, Raudabaugh DB, Gleason FH, Miller AN. The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential impact on freeranging snake populations. Fungal Ecol 2015; Available: doi: 10.1016/j.funeco.2015.05.003. 122 5. Allender MC, Bunick D, Dzhaman E, Burrus L, Maddox C. Development and use of real-time polymerase chain reaction assay for detection of Ophidiomyces ophiodiicola in snakes. J Vet Diagn Invest 2015; 27: 217–220. doi: 10.1177/1040638715573983. pmid:25776546 6. Bohuski E, Lorch JM, Griffin KM, Blehert DS. TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease. BMC Vet Res 2015; 11: pmid:25889462 7. Dolinski AC, Allender MC, Hsiao V, Maddox CW. Systemic Ophidiomyces ophiodiicola infection in a free-ranging plains garter snake (Thamnophis radix). J Herp Med Surg 2014; 24: 7–10. 8. Last LA, Fenton H, Gonyor-McGuire J1, Moore M1, Yabsley MJ1. Snake fungal disease caused by Ophidiomyces ophiodiicola in a free-ranging mud snake (Farancia abacura). J Vet Diagn Invest. 2016 Nov;28(6):709-713. 3. 9. Lorch JM, Lankton J, Werner K, Falendysz EA, McCurley K, Blehert DS. Experimental Infection of Snakes with Ophidiomyces ophiodiicola Causes Pathological Changes That Typify Snake Fungal Disease. MBio. 2015 Nov 17;6(6):e01534-15 10. Lorch JM, Knowles S2, Lankton JS2, Michell K3, Edwards JL4, Kapfer JM5, Staffen RA, Wild ER7, Schmidt KZ2, Ballmann AE2, Blodgett D8, Farrell TM9, Glorioso BM1, Last LA11, Price SJ12, Schuler KL13, Smith CE4, Wellehan JF Jr14, Blehert DS. Snake fungal disease: an emerging threat to wild snakes. Philos Trans R Soc Lond B Biol Sci. 2016 Dec 5;371(1709). pii: 20150457. 11. McBride MP, Wojick KB, Georoff TA, Kimbro J, Garner MM, Wang X, et al. Ophidiomyces ophiodiicola dermatitis in eight free-ranging timber rattlesnakes (Crotalus horridus) from Massachusetts. J Zoo Wildl Med 2015; 46:86–94. 12. McCoy CM, Lind CM, Farrell TM. Environmental and physiological correlates of the severity of clinical signs of snake fungaldisease in a population of pigmy rattlesnakes, Sistrurus miliarius. Conserv Physiol. 2017 Jan 27;5(1):cow077. doi: 10.1093/conphys/cow077. eCollection 2017. 13. Rajeev S, Sutton DA, Wickes BL, Miller DL, Giri D, Van Meter M, et al. Isolation and characterization of a new fungal species Chyrsosporium ophiodiicola, from a mycotic granuloma of a black rat snake (Elaphe obsolete obsolete). J Clin Microbiol 2009; 47:1264–1268. doi: 10.1128/JCM.01751-08. pmid:19109465 14. Sleeman JM. 2013. Snake fungal disease in the United States. National Wildlife Health Center Wildlife Health Bulletin 2013–02. USGS, Madison, WI. 15. Tetzlaff SJ, Allender MC, Ravesi M, Smith J, Kingsbury B. 2015. First report of snake fungal disease from Michigan, USA involving Massasaugas, Sistrurus catenatus (Rafinesque 1818). Herp Review 44: 31–32. 123 5. White-nose syndrome (WNS) White-nose syndrome (WNS) is a recently emerged wildlife disease in North America, which in 4 years has resulted in unprecedented deaths of hibernating bats in the northeastern United States and is a widespread epizootic disease among bats (Blehert et al., 2009, Reichard and Kunz, 2009, Turner GR, Reeder,2009),.      White-nose syndrome (WNS) in North America is known to affect 6 species of bats that use hibernation as their winter survival strategy: the big brown bat (Eptesicus fuscus), the eastern small-footed bat (Myotis leibii), the little brown bat (M. lucifugus), the northern long-eared bat (M. septentrionalis), the tricolored bat (Perimyotis subfl avus), and the Indiana bat (M. sodalis) (Blehert et al., 2009, Turner GR, Reeder,2009, Courtin et al., 2010). o White-nose syndrome (WNS) has spread >1,300 km into Connecticut, Massachusetts, New Hampshire, New Jersey, Pennsylvania, Tennessee, Vermont, Virginia, and West Virginia in the United States and the provinces of Ontario and Quebec in Canada in a pattern suggesting the spread of an infectious agent (Blehert et al., 2009, Turner GR, Reeder,2009, USGS, 2010). o In response to WNS in North America, researchers in Europe initiated a surveillance effort during the winter of 2008–09 for WNS-like fungal infections among hibernating populations of bats in Europe. Blehert et al. (2008) described the fungus associated with white-nose syndrome as a member of the genus Geomyces. o Gargas et al. (2009) named the fungus Geomyces destructans o Minnis &. Lindner (2013) changed the name to Pseudogymnoascus destructans, based on the phylogenetic relationship that indicated that this fungus was more closely related to the genus Pseudogymnoascus than to the genus Geomyces Lorch et al. (2013a) performed a culture survey of fungi associated with bat hibernacula and determined that there were more P. species in bat hibernacula of eastern North America than have been described in the genus. Lorch et al. (2013a) also noted that there were about 17 named species of Geomyces, not including heterotypic synonyms, under a broadly defined generic concept based on their ITS phylogeny of Geomyces and allies under a one name per fungus system of classification. Muller et al. (2013) used the cultures to generate additional molecular data and a new intergenic spacer (IGS)-based qPCR test for P. destructans that is both sensitive and accurate. Bat species identified with diagnostic symptoms of WNS: In North America (Blehert et al. 2009) 1. Gray bat (Myotis grisescens) 2. Big brown bat (Eptesicus fuscus) 3. Eastern small-footed bat (Myotis leibii) 4. Indiana bat (Myotis sodalis) 5. Little brown bat (Myotis lucifugus) 6. Northern long-eared bat (Myotis septentrionalis) 124 7. Tricolored bat (Perimyotis subflavus) Gray bat - Wikipedia Kaieteur News Big Brown Bat (Eptesicus fuscus) Wikipedia Little brown bat Tri-colored Bat (Eastern Pipistrelle), | Flickr biology.eku.edu Small-footed myotis Northern Long-eared Bat (Myotis septentrionalis)...Reddit Indiana bat - Wikipedia In Europe (Pikula et al. 2012, Zukal et al., 2014, Zukal et al., 2016)  Greater mouse-eared bat (Myotis myotis)  Daubenton's bat (Myotis daubentonii)  Bechstein's bat (Myotis bechsteinii) 125  Natterer's bat (Myotis nattereri)  Brandt's bat (Myotis brandtii)  Geoffroy's bat (Myotis emarginatus)  Pond bat (Myotis dasycneme)  Northern bat (Eptesicus nilssonii)  Barbastelle (Barbastellus barbastellus)  Brown long-eared bat (Plecotus auritus)  Mediterranean horseshoe bat (Rhinolophus euryale)  Common bent-wing bat (Miniopterus schreibersii)  Lesser horseshoe bat (Rhinolophus hipposideros) 123RF.com Common bent wing bat miniopterus schreibersii, Sam Dyer Ecology Greater Mouse-eared Bat (Myotis myotis) NaturePhoto-CZ.com Daubenton's Bat. (Myotis daubentonii) Natterer's bat - Wikipedia Bechstein's bat (Myotis bechsteinii) · iNaturalist.org Myotis brandtii (Brandt's bat) UniProt 126 Geoffroy's Bat (Myotis emarginatus) BioPix Pond Bat (Myotis dasycneme) BioLib Eptesicus nilssonii - Northern Bat Bigstock The Barbastelle Bat (barbastella Barbastellus) Brown Long-Eared Bat (Plecotus auritus) - Ireland's Wildlife NaturePhoto-CZ.com Mediterranean Horseshoe Bat Alamy Lesser horseshoe bat (Rhinolophus hipposideros) 127 In Asia 1. Eastern water bat (Myotis petax) (Hoyt et al. 2016) zmmu.msu.ru В ая в дя ая и а (Myotis petax peta Aetiology : Pseudogymnoascus destructans  Pseudogymnoascus destructans is a species genus Pseudogymnoascus (previously Geomyces) of the o Traaen (1914) erected the genus Geomyces to accommodate four species of anamorphic hyphomycetes: namely Geomyces auratus, Geomyces cretaceus, Geomyces sulphureus, and Geomyces vulgaris. o Carmichael (1962) synonymized these four species and numerous others under the name Chrysosporium pannorum. o Sigler & Carmichael (1976) recognized Geomyces as a genus distinct from Chrysosporium and typified the genus with G. auratus o Van Oorschot (1980) supported Sigler and Carmichael‘s (1976) assertion that Geomyces is distinct from Chrysosporium. o The genus Geomyces is widespread and has been reported from a multitude of substrates, but it is especially common in soil in colder regions (Carmichael 1962; van Oorschot 1980; Rice & Currah 2006; Domsch et al. 2007; Loque et al. 2009; Gonc¸alves et al. 2012). Classification: NCBI Taxonomy view in classification  Cellular organisms + o Eukaryota +  Opisthokonta +  Fungi +  Dikarya +  Ascomycota +  Saccharomyceta +  Pezizomycotina +  Leotiomyceta +  Sordariomyceta + o Leotiomycetes +  Leotiomycetes incertae sedis +  Pseudeurotiaceae + 128        Pseudogymnoascus + Pseudogymnoascus destructans Pseudogymnoascus appendiculatus Pseudogymnoascus bhattii Pseudogymnoascus pannorum + Pseudogymnoascus roseus 40 more... Description: Pseudogymnoascus destructans (Blehert & Gargas) Minnis & D.L. Lindner, Fungal Biology: 646 (2013) ≡Geomyces destructans Blehert & Gargas, Mycotaxon 108: 151 (2009) Morphology (Chaturvedi e t al., 2010): On potato dextrose agar, incubated at 4°C and 15°C for 28 days, the initial colony appearance is white, velvety, glabrous turning grayish green, powdery in texture. Reverse with no pigmentation initially, later on revealing diffusible dark brown pigment. Older colony also exhibite exudates on surface. No growth in cultures incubated at −10°C or at 25°C. Microscopically, abundant curved conidia are borne in whorls directly on septate hyphae hyphae without any fruiting bodies. Chaturvedi V, Springer DJ, Behr MJ, Ramani R, Li X, Peck MK, et al. (2010) Morphological and Molecular Characterizations of Psychrophilic Fungus Geomyces destructans from New York Bats with White Nose Syndrome (WNS). PLoS ONE 5(5): e10783. doi:10.1371/journal.pone.0010783 Reports: Blehert et al. (2009) reported that, White-nose syndrome (WNS) is a condition associated with an unprecedented bat mortality event in the northeastern United States. Since the winter of 2006*2007, bat declines exceeding 75% have been observed at surveyed hibernacula. Affected bats often present with visually striking white fungal growth on their muzzles, ears, and/or wing membranes. Direct microscopy and culture analyses demonstrated that the skin of WNS-affected bats is colonized by a psychrophilic fungus that is phylogenetically related to Geomyces spp. 129 but with a conidial morphology distinct from characterized members of this genus. This report characterizes the cutaneous fungal infection associated with WNS. Chaturvedi et al. (2010) have investigated 100 bat and environmental samples from eight affected sites in 2008. The findings provided strong evidence for an etiologic role of G. destructans in bat WNS. Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. G. destructans DNA was directly amplified from infected bat tissues, Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. In vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans was identified. Microscopic and histopathological evidence of G. destructans in bats with WNS. (A) Direct lactophenol cotton blue mount prepared from skin scrape taken from the muzzle of a little brown bat from Graphite Mine on April 6, 2008 revealed fungal hyphae and curved conidia, bar 10 µm. (B) Control, [Bi] and infected muzzle tissue section [Bii] stained with PAS revealed epidermal colonization by fungal hyphae and spores; the sample was from a little brown bat from Williams Hotel Mine on March 27, 2008. Notably, a few neutrophils are present in the underlying dermis (arrows), bar 10 µm. Bacteria are also seen in this sample (C). SEM photomicrograph of muzzle sample from bat from Williams Hotel Mine showing characteristic curved conidia and septate hyphae spread over bat skin tissues. Note heavy fungal growth with profuse curved conidia covering the skin and hair shaft (Ci, muzzle, bar 100 µm; Cii, higher magnification of a portion of muzzle, bar 10 µm; Ciii & Cvi, higher magnifications, bar 10 µm). http://dx.doi.org/10.1371/journal.pone.0010783.g001 130 G. destructans in culture from bat tissues.(A). Original culture tubes of Sabouraud agar supplemented with nine antibiotics and incubated at 4°C for six- or eight-weeks; notice the profuse growth of G. destructans strains. (B) Some fungal contamination on individual isolates was visible as depicted in the close-up of a culture tube. (C) Enrichment and recovery of pure fungal colonies by treating a culture contaminated with bacteria with hydrochloric acid.http://dx.doi.org/10.1371/journal.pone.0010783.g002 G. destructans in bat tissues and culture are similar.(A) SEM of photomicrograph prepared from bat tissues samples, examined from Fig. 1C at high magnification, showed fungal hyphae and spores on the surface. (B) SEM photomicrograph prepared from G. destructans culture isolated from bat tissue samples collected from Williams Hotel Mine; note curved conidia borne in whorls on septate hyphae; this pattern is similar to SEM image in Fig. 3A, bar is 2 µm. All images are pseudo-colored in Adobe Photoshop 9.0.http://dx.doi.org/10.1371/journal.pone.0010783.g003 Molecular analysis of bat tissues and fungal cultures. (A) ITS PCR analysis of bat tissues and fungal cultures from DNA extracted from bat tissues and from pure G. destructans isolates. PCR amplification was carried out with primer set V47/V50. PCR amplicons were electrophoresed on 2% agarose gel, stained with ethidium bromide and photographed with a imaging software. Four bat tissues and respective fungal isolates showed perfect matches (blue connectors); one tissue DNA amplicon did not match with G. destructans amplicon obtained from pure culture (green connector). Also shown are amplicons from two additional G. destructans isolates (MYC80280, MYC80282) where corresponding tissues 131 samples were not processed. (B) ITS PCR analysis of bat tissue samples positive for G. destructans. Ten bat tissues including five untreated samples and five paraffin-fixed samples were positive for G. destructans DNA (details in Table 1). (C-D) Molecular typing of G. destructans was performed with RAPD primers. (C) Results shown were obtained by PCR of fungal genomic DNA with M-13 and (GACA)4 primers, amplicons were run on 2% agarose gels and band patterns were used to construct dendrograms with Applied Math software. Geomyces pannorum (UAMH 1062 and UAMH 2586) were used as outgroup. (D) Results shown were obtained by PCR of genomic DNA with Operon Technology 10-mer primers OPA1, OPA2 and OPA3; outgroup strains are similar to panel in C. Genotyping with five different primers showed that all six G. destructans culture isolates obtained from two sites, approximately 200-km apart, had indistinguishable band patterns. These preliminary results raised the possibility of involvement of a single strain of G. destructans in the outbreak of WNS in bats in upstate NY. http://dx.doi.org/10.1371/journal.pone.0010783.g004 G. destructans proteolytic activities. Results from a representative strain, G. destructans MYC80-0251, showed secretory proteases after 28-days growth on albumin agar (Ai-ii, 4°C front & reverse; Aiii-iv, 15°C front & reverse), Casein agar(Bi-ii, 4°C front & reverse; Biii-iv, 15°C front & reverse), Geleatin agar(Ci-ii, 4°C front & reverse; Ciii-iv, 15°C front & reverse) or Urea agar at 4°C and 15° (Di-ii, 4°C front & reverse; Diii-iv, 15°C front & reverse), marker is 10 mm. Similar patterns of secretory proteases were seen with remaining four G. destructans strains. http://dx.doi.org/10.1371/journal.pone.0010783.g007 Puechmaille et al. (2010) made intensive monitoring of bat hibernation in France. They found only one bat (Myotis myotis) on March 12, 2009, near Périgueux (45°8′N, 0°44′E), showed a powdery, white fungal growth on its nose, which is characteristic of WNS. Sterile dry cotton swabs were used to collect fungus material from the nose of the bat. The bat was then weighed, measured, and released. Swabs were moistened with 50 μL of sterile water and streaked onto plates containing potato dextrose agar supplemented with 0.1% mycologic peptone. Plates (9 cm in diameter) were sealed with parafilm and incubated inverted at 10°C. A dense fungus growth developed within 14 days. Cultures were established by transferring inoculum to other mycologic media, including malt extract agar and Sabouraud agar. Colonies on malt extract agar were initially white but after spore production and aging they quickly darkened from the center to a dull gray, often showing a faint green hue. Spores were hyaline, irregularly curved, broadly crescent-shaped (typically 6–8 μm long and 3–4 μm wide), and narrowed at each end, one of which was broadly truncate, often showing an annular frill. Microscopic examination of the original swab samples showed numerous spores with the above-mentioned features. The psychrophilic nature of the fungus and its species-specific morphologic features led to the conclusion that this fungus was G. destructans 132 A) Myotis myotis bat found in a cave on March 12, 2009, in France, showing white fungal growth on its nose (arrow). B) Fungus colony on malt extract medium after incubation for 3 weeks at 10°C. Scale bar = 1 cm. C) Clusters of unstained spores of Geomyces destructans. Spores in the inset were stained with lactophenol cotton blue, which shows the truncate spore base (arrows) and surface granulation. Scale bars = 10 µm. Wibbelt et al. (2010) sampled hibernating bats in Germany, Switzerland, and Hungary to determine whether G. destructans is present in Europe. Microscopic observations, fungal culture, and genetic analyses of 43 samples from 23 bats indicated that 21 bats of 5 species in 3 countries were colonized by G. destructans. It was hypothesized that G. destructans is present throughout Europe and that bats in Europe may be more immunologically or behaviorally resistant to G. destructans than their congeners in North America because they potentially coevolved with the fungus. A) Greater mouse-eared bat (Myotis myotis) with white fungal growth around its muzzle, ears, and wing membranes (photograph provided by Tamás Görföl). B) Scanning electron micrograph of a bat hair colonized by Geomyces destructans. Scale bar = 10 μm. 133 Lorch et al. (2011) demonstrated that exposure of healthy little brown bats (Myotis lucifugus) to pure cultures of G. destructans caused WNS. Live G. destructans was subsequently cultured from diseased bats, successfully fulfilling established criteria for the determination of G. destructans as a primary pathogen. We also confirmed that WNS can be transmitted from infected bats to healthy bats through direct contact. Our results provide the first direct evidence that G. destructans is the causal agent of WNS and that the recent emergence of WNS in North America may represent translocation of the fungus to a region with a naive population of animals. Demonstration of causality is an instrumental step in elucidating the pathogenesis and epidemiology of WNS and in guiding management actions to preserve bat populations against the novel threat posed by this devastating infectious disease. Histological sections of representative wing membranes (periodic acid-Schiff stain). a, Normal wing membrane of a healthy bat from the negative control group showing no signs of fungal growth. b, c, WNS lesions, including 134 invasion of the underlying connective tissue by fungal hyphae (arrows), are visible in sections from a bat with WNS from the positive control group (b) and a bat from the treated group that developed WNS after experimental exposure to G. destructans (c). Insets are higher magnification images and scale bars indicate 20 mm | Survival curves. a, Survival curves for the treated (n 5 29), contact exposure (n 5 18), airborne exposure (n 5 36), negative control (n 5 34) and positive control (n 5 25) groups. Bats in the positive control group, which consisted of animals naturally infected with WNS at the time they were collected, exhibited significantly decreased survival (asterisk) relative to the other groups (P , 0.001). Survival among bats of the remaining groups did not differ significantly from one another (P 5 0.72). b, Percentage of bats submitted by month (January 2008 to June 2011) to the USGS–National Wildlife Health Center that tested positive for WNS (n 5 54 submission events). The blue bars represent submissions that were not associated with major mortality events; the red bars depict submissions associated with high mortality. Annually, WNSassociated mortality events are first observed in January; the number of submissions involving mortality events for a given month peaks in March. Assuming the positive control bats were first exposed to G. destructans in late September, mortality due to WNS did not occur in the laboratory until approximately 120 days after exposure, consistent with what is observed in freeranging wild bats (the dotted line represents the exposure period in the wild before the animals were collected for this study). The duration of this infection trial (102 days) was insufficient to observe WNS-associated mortality in the treated and contact exposure groups (the treated group mortality curve is shifted such that duration of exposure corresponds to that of the positive control group; contact and airborne exposure group mortality curves are not shown). Puechmaille et al. (2011) collected data on the presence of bats with white fungal growth in 12 countries in Europe between 2003 and 2010 and conducted morphological and genetic analysis to confirm the identity of the fungus as Geomyces destructans. The results demonstrated the presence of the fungus in eight countries spanning over 2000 km from West to East and provide compelling photographic evidence for its presence in another four countries including Romania, and Turkey. Furthermore, matching prevalence data of a hibernaculum monitored over two consecutive years with data from across Europe show that the temporal occurrence of the fungus, which first becomes visible around February, peaks in March but can still be seen in some torpid bats in May or June, is strikingly similar throughout Europe. Finally, they isolated and cultured G. destructans from a cave wall adjacent to a bat with fungal growth. It was concluded that, G. destructans is widely found over large areas of the European continent without associated mass mortalities in bats, suggesting that the fungus is native to Europe. The characterisation of the temporal variation in G. destructans growth on bats provides reference data for studying the spatio-temporal dynamic of the fungus. Finally, the presence of G. destructans spores on cave walls suggests that hibernacula could act as passive vectors and/or reservoirs for G. destructans and therefore, might play an important role in the transmission process. Distribution of confirmed and suspected records of G. destructans on hibernating bats in Europe.Data are presented for genetically confirmed records of G. destructans in red (circles, this study; triangles, published records), photographic evidence in yellow, visual reports in green. Dead bats from Northern France which culture and genetic analysis did not reveal the presence of G. destructans are depicted as black dots. Countries abbreviated names are as follows: AUT: Austria, BEL: Belgium, CHE: Switzerland, 135 CZE: Czech Republic, DEU: Germany, DNK: Denmark, EST: Estonia, FRA: France, HUN: Hungary, NLD: Netherlands, POL: Poland, ROM: Romania, SVK: Slovakia, TUR: Turkey, UKR: Ukraine. Photographic evidence showing bats with confirmed or suspected growth of G. destructans. Photographs of cases confirmed by genetic analysis, from (A) Estonia (M. brandtii, May 23rd 2010, © L. Lutsar), (B) Poland (M. myotis, th th March 7 2010, © A. Wojtaszewski), (C) Germany (M. myotis, March 10 2010, © C. Jungmann), (D) France (M. myotis, March 4th 2010, © Y. Le Bris), (E) Netherlands (M. daubentonii, March 9th 2010, © T. Bosch), (F) Germany (M. myotis, March 23rd 2010, © K. th rd Passior) (G) Belgium (M. mystacinus, March 18 2010, © B. Mulkens), (H) Germany (M. mystacinus, March 23 2010, © K. Passior) th or bats with white-fungal growth suspected as G. destructans from (I) Denmark (M. dasycneme, March 14 2010, © B. Ohlendorf), (J) nd th Austria (M. myotis, February 2 2007, © O. Gebhardt), (K) Hungary (M. myotis, February 19 2010, © T. Görföl), (L) Belgium (M. th th myotis, March 7 2010, © F. Forget), (M) France (M. myotis, February 13 2010, © J. Vittier), (N) Ukraine (M. myotis, February th th 13 2010, © A.-T. Bashta), (O) France (M. escalerai/sp. A, June 25 2010, © F. Blanc), (P) Turkey (M. myotis/blythii, March nd th 22 2009, © M. Doker), and (Q) Romania (M. blythii, March 29 2008, © B. Szilárd). 136 Confirmed records of Geomyces destructans on hibernating bats in Europe and details of the culture and genetic analyses.http://dx.doi.org/10.1371/journal.pone.0019167.t001 Lorch (2012) tested the hypothesis that G. destructans is the primary cause of WNS by using a combination of classical and molecular microbiology techniques. He first examined the ability of G. destructans to serve as a primary pathogen in healthy little brown bats (Myotis lucifugus), in fulfillment of Koch‘s postulates, by conducting an infection trial. He found that 100% of healthy bats inoculated with pure cultures of G. destructans exhibited signs of WNS by 102 days post-exposure, while no animals in the negative control group contracted the disease. Geomyces destructans was reisolated from the ii experimentally-infected bats, conclusively implicating the fungus as the cause of WNS. He also demonstrated that WNS can be transmitted from infected to healthy bats through direct contact. He additionally investigated whether the distribution of G. destructans in the United States correlated with the observed distribution of WNS. To do this, He first isolated over 332 viable fungi from sediment samples collected from bat hibernacula across the eastern U.S. He found that closely related Geomyces species were the most abundant and diverse group cultured, representing approximately 33% of all isolates (many of which likely represent undescribed taxa). He sequenced and compared partial intergenic spacer regions of the rRNA gene complex from many of these isolates (n = 145), and used the data to design a molecular assay (real-time TaqMan PCR) to specifically detect G. destructans in environmental samples. Next, he screened sediment samples collected from 55 caves and mines in the eastern U.S. for the presence of G. destructans. We found that G. destructans is limited to hibernacula within the known range of WNS, further implicating it as the cause, rather than a manifestation, of WNS. This finding, 137 in combination with the ability of G. destructans to cause disease in healthy animals, provided conclusive evidence that G. destructans is the causative agent of WNS Meteyer et al. (2012) reported that, White nose syndrome, caused by Geomyces destructans, has killed more than 5 million cave hibernating bats in eastern North America. During hibernation, the lack of inflammatory cell recruitment at the site of fungal infection and erosion is consistent with a temperature-induced inhibition of immune cell trafficking. This immune suppression allows G. destructans to colonize and erode the skin of wings, ears and muzzle of bat hosts unchecked. Yet, paradoxically, within weeks of emergence from hibernation an intense neutrophilic inflammatory response to G. destructans is generated, causing severe pathology that can contribute to death. It was hypothesized that the sudden reversal of immune suppression in bats upon the return to euthermia leads to a form of immune reconstitution inflammatory syndrome (IRIS). IRIS was first described in HIVinfected humans with low helper T lymphocyte counts and bacterial or fungal opportunistic infections. IRIS is a paradoxical and rapid worsening of symptoms in immune compromised humans upon restoration of immunity in the face of an ongoing infectious process. One of 30 Little Brown Bats with white-nose syndrome collected from hibernation April 13, 2010 and taken into rehabilitation. Warmth, food, and water were provided and this bat was photographed over time. Photographs were used with permission from Gregory Turner, Mick Valent and Jackie Kashmer. (A) Photograph taken with top lighting in hibernacula at collection April 13, 2010. No gross lesions can be seen, but the dusting of white material on the wing surface is evidence of fungal infection. (B) Photograph of bat (A) taken on April 29, 2010 after 16 d of rehabilitation. The transilluminated wing was photographed outstretched over a light box and shows a reticular pattern of wing damage. (C) Photograph of bat (A) taken May 11, 2010 shows progressively worsening of wing damage 29 d after being taken into rehabilitation. Loss of tissue is evident and the wing membrane is fragile. In the wild, without provision of food, water, and protection, this bat would be unlikely to survive. (D) Photograph of bat (A) taken May 20, 2010, 138 Little Brown Bat found February 8, 2009 frozen outside of the small opening of a copper mine. The transilluminated wing was photographed outstretched over a light box and shows no evidence of wing damage. (B) Periodic acid Schiff stained section of wing membrane from bat (2A) shows characteristic dense aggregates of robust hyphae forming a defined interface with the skin, erosion along the broad zone of skin contact (arrows) and no visible inflammatory response. (C) One of nine Little Brown. Nine bats were found on the ground and unable to fly between April 4 and May 7, 2012. This bat was collected April 4, taken into rehabilitation, ate and drank, but died within 18 h of arrival. The wing was photographed outstretched over a light box and visible damage can be seen with dark areas of contraction and loss of elasticity. (D) Periodic acid Schiff stained section of wing membrane from the bat in Figure 2C. Severe neutrophilic inflammation and edema (bracket) in response to fungal hyphae (arrow). (E) Different field from same slide as in Figure 2D shows a thick layer of degenerating neutrophils (brackets) at the margins of a dense aggregate of fungal hyphae eroding epidermis (arrow). (F) Little Brown Bat in Figure 2C. Degenerating neutrophils (arrowheads) surround the dense aggregate of fungal hyphae (arrows).. Verant et al. (2012) described temperature-dependent growth performance and morphology for six independent isolates of G. destructans from North America and Europe. Thermal performance curves for all isolates displayed an intermediate peak with rapid decline in performance above the peak. Optimal temperatures for growth were between 12.5 and 15.8°C, and the upper critical temperature for growth was between 19.0 and 19.8°C. Growth rates varied across isolates, irrespective of geographic origin, and above 12°C all isolates displayed atypical morphology that may have implications for proliferation of the fungus. This study demonstrates that small variations in temperature, consistent with those inherent of bat hibernacula, affect growth performance and physiology of G. destructans, which may influence temperature-dependent progression and severity of WNS in wild bats. 139 Weekly growth curves for two isolates of Geomyces destructans. In an initial experiment, two isolates of G. destructans (one from New York and one from Germany) exhibited differences in growth performance but had similar thermal optima and upper critical temperatures for growth. Topt and upper critical temperatures (CLu) for growth at week 5 are marked on the graphs with arrows. For this figure, each curve is represented using a Brière2 function, although in some cases, other functions were equally parsimonious (Table S1). Topt and CLu in this figure represent the values specific to the Brière2 function shown in the graph; therefore T opt does not match the weighted averages presented in Table 1. The isolates were grown on Sabouraud dextros Five-week growth curves for four isolates of Geomyces destructans. In a follow-up experiment, differences in growth performance were confirmed among four additional isolates of G. destructans, two from North America and two from Europe. A consistent intercontinental trend in growth performance was not observed among the isolates. The isolates were grown on Sabouraud dextrose agar. Twenty-one replicate colonies of each isolate (Pennsylvania, Virginia, Hungary, and Switzerland) were incubated across a range of five temperatures from 1.9 to 17.7°C. The area of each expanding colony was measured after five weeks, and a growth curve was fit to each dataset. Each curve is represented using the best-fit function. For comparison to the weekly growth curve analysis (Fig. 1), 21 replicate co 140 Morphology of Geomyces destructans varies with incubation temperature. (a) A characteristically branched conidiophore following growth at approximately 7°C. (b) Curved conidia typical of those produced following incubation at approximately 7°C. (c) Hyphae were thickened, fragmented into arthrospores (arrows), and produced chlamydospore-like structures (arrowhead) following incubation at approximately 12°C. (d) Conidia were primarily pyriform to globoid in shape and frequently formed short chains (arrow) following incubation at approximately 12°C. (e) At elevated temperatures (above 15°C), thickened, deformed hyphae showed evidence of degeneration, and hyphal tips exhibited branched antler-like morphology (arrow). Chlamydospore-like structures were also common (arrowhead). (f) Thick irregular hyphal fragments were produced by colonies grown at approximately 18°C; conidia were not observed. Scale bars, 10 µm. http://dx.doi.org/10.1371/journal.pone.0046280.g003 Warneckea et al. (2012) mentioned that White-nose syndrome (WNS) is an emerging disease of hibernating bats associated with cutaneous infection by the fungus Geomyces destructans (Gd), and responsible for devastating declines of bat populations in eastern North America. Affected bats appear emaciated and one hypothesis is that they spend too much time out of torpor during hibernation, depleting vital fat reserves required to survive the winter. The fungus has also been found at low levels on bats throughout Europe but without mass mortality. This finding suggests that Gd is either native to both continents but has been rendered more pathogenic in North America by mutation or environmental change, or that it recently arrived in North America as an invader from Europe. Thus, a causal link between Gd and mortality has not been established and the reason for its high pathogenicity in North America is unknown. It was shown that experimental inoculation with either North American or European isolates of Gd causes WNS and mortality in the North American bat, Myotis lucifugus. In contrast to control bats, individuals inoculated with either isolate of Gd developed cutaneous infections diagnostic of WNS, exhibited a progressive increase in the frequency of arousals from torpor during hibernation, and were emaciated after 3–4 mo. The results demonstrated that altered torpor-arousal cycles underlie mortality from WNS and provide direct evidence that Gd is a novel pathogen to North America from Europe. 141 Lorch et al (2013) used culture-based techniques to investigate the diversity of fungi in soil samples collected from 24 bat hibernacula in the eastern United States. Ribosomal RNA regions (internal transcribed spacer and partial intergenic spacer) were sequenced to preliminarily characterize isolates. Geomyces species were one of the most abundant and diverse groups cultured, representing approximately 33% of all isolates. Geomyces destructans was isolated from soil samples from three hibernacula in states where WNS is known to occur, and many of the other cultured Geomyces isolates likely represent undescribed taxa. Minnis and Lindner (2013) generated DNA sequence data for the internal transcribed spacer (ITS) region, nuclear large subunit (LSU) rDNA, MCM7, RPB2, and TEF1 from a diverse array of Geomyces and allies that included isolates recovered from bat hibernacula as well as those that represent important type species. Phylogenetic analyses indicated Geomyces and allies should be classified in the family Pseudeurotiaceae, and the genera Geomyces, Gymnostellatospora, and Pseudogymnoascus should be recognized as distinct. True Geomyces are restricted to a basal lineage based on phylogenetic placement of the type species, Geomyces auratus. Thus, G. destructans is placed in genus Pseudogymnoascus. The closest relatives of Pseudogymnoascus destructans are members of the Pseudogymnoascus roseus species complex, however, the isolated and long branch of P. destructans indicated that none of the species included in this study were closely related, thus providing further support to the hypothesis that this pathogen is non-native and invasive in eastern North America. Several conidia-producing isolates from bat hibernacula previously identified as members of Pseudeurotium were determined to belong to the genus Leuconeurospora, which is widespread, especially in colder regions. Teberdinia hygrophila was transferred to Pseudeurotium as Pseudeurotium hygrophilum, comb. nov., in accordance with the one name per fungus system of classification, and two additional combinations were made in Pseudogymnoascus including Pseudogymnoascus carnis and Pseudogymnoascus pannorum. 142 Coleman and Reichard (2014) made a brief assessment for seven years after discovery of a virulent fungal pathogen in North America. They mentioned that, White-nose syndrome (WNS) was unknown to science before it was discovered in New York in 2007 and took all by surprise. Since then the conservation and scientific communities have come together to mount a coordinated international effort to address research and management needs to respond to this growing disaster. Among many accomplishments, great progress was made in understanding the disease, the biology of the causative fungus, and the biology and physiology of hibernating bats; guidance focusing on containing the fungus was developed to slow its spread; multiple novel approaches were explored to treat bats and affected environments in ways that reduce the impacts of the disease; jn addition to field testing a standardized and robust monitoring strategy to assess population trends for all bat species across North America. Zukal et al. (2014) investigated Geomyces destructans infection, the cause of whitenose syndrome (WNS), in relation to chiropteran ecology, behaviour and phylogenetics. While this fungus has caused devastating declines in North American bat populations, there have been no apparent population changes attributable to the disease in Europe. 276 bats of 15 species from the Czech Republic were screened over 2012 and 2013, and provided histopathological evidence for 11 European species positive for WNS. With the exception of Myotis myotis, the other ten species were all new reports for WNS in Europe. Of these, M. emarginatus, Eptesicus nilssonii, Rhinolophus hipposideros, Barbastella barbastellus and Plecotus auritus were new to the list of P. destructans-infected bat species. While the infected species were all statistically phylogenetically related, WNS affected bats from two suborders. These are ecologically diverse and adopt a wide range of hibernating strategies. Occurrence of WNS in distantly related bat species with diverse ecology suggests that the pathogen may be a generalist and that all bats hibernating within the distribution range of P. destructans may be at risk of infection. 143 Bats examined for white-nose syndrome and Pseudogymnoascus destructans infection in Czech hibernacula (Europe). http://dx.doi.org/10.1371/journal.pone.0097224.t00 Histopathological skin lesions consistent with white-nose syndrome in ten European bat species. (A) Myotis emarginatus, (B) Eptesicus nilssonii, (C) Rhinolophus hipposideros, (D) Plecotus auritus, (E) Barbastella barbastellus, (F) M. dasycneme, (G) M. nattereri, (H) M. daubentonii, (I) M. bechsteinii, (J) M. brandtii. The photographs illustrate i) extensive infection of the wing membrane and cup-shaped epidermal erosions (A, E, H, J; long black arrow); ii) cup-like epidermal erosions in the pinna (B; long black arrow), iii) Pseudogymnoascus destructans hyphae obscuring the basement membrane and invading the dermis (A, B, C, E, H; black arrow); iv) a single cupping erosion packed with fungal hyphae in the wing membrane (C, D, G, I; long black arrow); v) colonisation of a hair follicle by P. destructans, fungal hyphae present in the associated sebaceous gland and regional connective tissue (F; black arrow); vi) marked signs of inflammation (B, F, J); and vii) a cellular inflammatory crust that sequesters fungal hyphae (A, J). White arrows within each photograph indicate the interface between epidermis and dermis. Periodic acid-Schiff stain; scale bar = 50 µm. http://dx.doi.org/10.1371/journal.pone.0097224.g001 Bernard et al. (2015) collected epidermal swabs from bats captured during winters 2012-13 and 2013-14 in mist nets set outside of hibernacula in Tennessee. Epidermal swab samples were collected from eight Rafinesque's big-eared bats (Corynorhinus rafinesquii), six eastern red bats (Lasiurus borealis), and three silver-hair bats (Lasionycteris noctivagans). Using real-time PCR methods, we identified DNA sequences of P. destructans from skin swabs of two Rafinesque's big-eared bats, two eastern red bats, and one silver-haired bat. This was the first detection of the WNS fungus on Rafinesque's big-eared bats and eastern red bats and the second record of the presence of the fungus on silver-haired bats. 144 Janicki et al. (2015) determined the efficacy of visual detection of P. destructans by examining visual signs and molecular detection of P. destructans on 928 bats of six species at 27 sites during surveys conducted from January through March in 20122014 in the southeastern USA on the leading edge of the disease invasion. Cryptic infections were widespread with 77% of bats that tested positive by qPCR showing no visible signs of infection. The probability of exhibiting visual signs of infection increased with sampling date and pathogen load, the latter of which was substantially higher in three species (Myotis lucifugus, M. septentrionalis, and Perimyotis subflavus). In addition, M. lucifugus was more likely to show visual signs of infection than other species given the same pathogen load. Nearly all infections were cryptic in three species (Eptesicus fuscus, M. grisescens, and M. sodalis), which had much lower fungal loads. The presence of M. lucifugus or M. septentrionalis at a site increased the probability that P. destructans was visually detected on bats. Our results suggest that cryptic infections of P. destructans are common in all bat species, and visible infections rarely occur in some species. However, due to very high infection prevalence and loads in some species, it was estimated that visual surveys examining at least 17 individuals of M. lucifugus and M. septentrionalis, or 29 individuals of P. subflavus are still effective to determine whether a site has bats infected with P. destructans. In addition, because the probability of visually detecting the fungus was higher later in winter, surveys should be done as close to the end of the hibernation period as possible. Langwig et al. (2015) examined patterns and drivers of seasonal transmission of P. destructans by measuring infection prevalence and pathogen loads in six bat species at 30 sites across the eastern United States. Bats became transiently infected in autumn, and transmission spiked in early winter when bats began hibernating. Nearly all bats in six species became infected by late winter when infection intensity peaked. In summer, despite high contact rates and a birth pulse, most bats cleared infections and prevalence dropped to zero. These data suggested that the dominant driver of seasonal transmission dynamics was a change in host physiology, specifically hibernation. The timing of infection and fungal growth resulted in maximal population impacts, but only moderate rates of spatial spread. 145 (a) Boxplot of colony sizes, on a log scale, of M. lucifugus at winter hibernacula and summer maternity colonies. (b) Photos of M. lucifugus roosting in groups in winter (top) and summer (bottom). (Online version in colour.) Leopardi et al. (2015) presented the first informative molecular comparison between isolates from North America and Europe and provided strong evidence for the longterm presence of the fungus in Europe and a recent introduction into North America. The results further demonstrated great genetic similarity between the North American and some European fungal populations, indicating the likely source population for this introduction from Europe. Lorch et al. (2015) mentioned that, before the discovery of white-nose syndrome (WNS), a fungal disease caused by Pseudogymnoascus destructans, there were no reports of fungal skin infections in bats during hibernation. In 2011, bats with grossly visible fungal skin infections similar in appearance to WNS were reported from multiple sites in Wisconsin, US, a state outside the known range of P. destructans and WNS at that time. Tape impressions or swab samples were collected from affected areas of skin from bats with these fungal infections in 2012 and analyzed by microscopy, culture, or direct DNA amplification and sequencing of the fungal internal transcribed spacer region (ITS). A psychrophilic species of Trichophyton was isolated in culture, detected by direct DNA amplification and sequencing, and observed on tape impressions. Deoxyribonucleic acid indicative of the same fungus was also detected on three of five bat carcasses collected in 2011 and 2012 from Wisconsin, Indiana, and Texas, US. Superficial fungal skin infections caused by Trichophyton sp. were observed in histopathology for all three bats. Sequencing of the ITS of Trichophyton sp., along with its inability to grow at 25 C, indicated that it represented a previously unknown species, described herein as Trichophyton redellii sp. nov. Genetic diversity present within T. redellii suggested it is native to North America but that it had been overlooked before enhanced efforts to study fungi associated with bats in response to the emergence of WNS. 146 .Comparison between Trichophyton redellii infection (top panels) and Pseudogymnoascus destructans infection (i.e., white-nose syndrome [WNS]; bottom panels) in bats. Bats infected with T. redellii have visible white fungal growth on the ears, legs, wings, tail, or uropatagium (A); lesions may manifest as a distinct ring (arrow); the muzzle often lacks clinical signs of infection. Bats with WNS generally have visible fungus on the muzzle in addition to other areas of unfurred skin (B). Fungal tape impressions collected from bats with clinical signs of T. redellii infection display radially symmetric obovate to pyriform microconidia (C), as opposed to the asymmetrical curved conidia typical of P. destructans (D). In histologic sections (prepared with periodic acid–Schiff staining), the wing skin of bats with T. redellii infections (E) generally have superficial colonization and invasion of the keratin layers by the dermatophyte (arrow); aerial hyphae and fertile structures in the form of conidiophores and microconidia may also be present (arrowhead). In contrast, histologic cross sections of wing skin from bats with WNS (F) typically display cuplike aggregations of fungal hyphae (arrows), erosion of the epidermis, and occasional curved conidia (arrowheads). Scale bars = 20 µm. Trichophyton redellii (ex-type living culture): colony surface on Sabouraud dextrose agar (SDA) after 37 d incubated at 10 C in the dark (A); colony reverse on SDA after 37 d incubated at 10 C in the dark (B); conidia (C). Bar = 20 µm. Hoyt et al. (2016a) collected epidermal swabs from bats captured during winters 2012-13 and 2013-14 in mist nets set outside of hibernacula in Tennessee. Epidermal swab samples were collected from eight Rafinesque's big-eared bats (Corynorhinus rafinesquii), six eastern red bats (Lasiurus borealis), and three silver-hair bats (Lasionycteris noctivagans). Using real-time PCR methods, we identified DNA sequences of P. destructans from skin swabs of two Rafinesque's big-eared bats, two eastern red bats, and one silver-haired bat. This was the first detection of the WNS fungus on Rafinesque's big-eared bats and eastern red bats and the second record of the presence of the fungus on silver-haired bats. 147 Hoyt et al. (2016b) presented a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, was capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method proved to be highly sensitive, highthroughput for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi. Powers et al. (2016) did not find any marked changes in BMI across years after WNS for Gray Bats. This finding suggested that surviving bats are either not negatively impacted by WNS or have recovered sufficiently by late summer as to not document obvious differences across years. After limiting our analyses of juvenile recruitment to only the individuals that we had definitively aged via backlit photos (2010–2014), we found a non-significant declining trend in juvenile recruitment; a trend that merits continued monitoring in the years to come. As Gray Bats have only recently shown to be susceptible to WNS infection, it is possible that observable population declines are forthcoming. Zukal et al. (2016) showed high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. The data provided evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established. 148 Prevalence of white-nose syndrome and Pseudogymnoascus destructans infection in bat species from the Holarctic region. Screened = number of bats captured and examined by PCR (to detect the pathogen), UV light trans-illumination or histopathology (to detect WNS lesions). + = percentage of positive bats from the number screened by the method. NA = not available. Samples subjected for histopathology examination were suspect lesions selected under field conditions based on UV trans-illumination and thus prevalence on histopathology is not based on a randomized sample. It rather reflects qualitative informationthat WNS was confirmed in the species with histopathology. Fungal growth on hibernating bats from Russia. (a) A hibernating cluster of pond bats Myotis dasycneme in a cave near Yekaterinburg, Russia, in May 2014. Black and white arrows indicate fungal growth on the muzzle and forearm, respectively. (b) A pond bat from the same hibernaculum showing visible fungal growths on the uropatagium, pelvic limb toes, plagio- and pro-patagium and the ears and muzzle (white arrows). Photo: Jiri Pikula 149 White-nose syndrome on a pond bat Myotis dasycnemenear Yekaterinburg, Russia, in May 2014.(A) Microscopic identification of the characteristic curved conidia of Pseudogymnoascus destructans (black arrow). (B) Invasive fungal growth penetrating the full-thickness of the wing membrane (black arrows), with several inflammatory cells (neutrophils) situated at both margins of the lesion (white arrows). (C) Packed fungal hyphae of cupping erosions (black arrows) sequestered by neutrophils (white arrows). (D) Histopathological finding from a WNS-positive Nearctic Myotis lucifugus, identical to that found in Palearctic Asia. Skin sections stained with periodic acid-Schiff stain. References: 1. Bernard, R. F., Foster, J. T., Willcox, E. V., Parise, K. L. & McCracken, G. F. Molecular detection of the causative agent of white-nose syndrome on Rafinesque’s big-eared bats (Corynorhinus rafinesquii) and two species of migratory bats in the southeastern USA. J. Wildl. Dis. 51, 519–522 (2015). 2. Blehert DS, Hicks AC, Behr M, Meteyer CU, Berlowski-Zier BM, Buckles EL, et al. Bat white-nose syndrome: an emerging fungal pathogen? Science. 2009;323:227 3. Boyles JG, Willis CKR (2009) Could localized warm areas inside cold caves reduce mortality of hibernating bats affected by white-nose syndrome? Front Ecol Environ 18(2):92–98. 4. Burton RS, Reichman OJ (1999) Does immune challenge affect torpor duration? Funct Ecol 13:232–237. 5. Chabasse D, Guiguen C, Couatarmanac‘h A, Launay H, Reecht V, de Bièvre C Keratinophilic fungal flora isolated from small wild mammals and rabbit-warren in France. Discussion on the fungal species found[in French] Ann Parasitol Hum Comp. 1987;62:357–68 6. Chaturvedi V, Springer DJ, Behr MJ, Ramani R, Li X, Peck MK, et al. (2010) Morphological and Molecular Characterizations of Psychrophilic Fungus Geomyces 150 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. destructans from New York Bats with White Nose Syndrome (WNS). PLoS ONE 5(5): e10783. doi:10.1371/journal.pone.001078 Coleman, J. T. H. & Reichard, J. D. Bat white-nose syndrome in 2014: A brief assessment seven years after discovery of a virulent fungal pathogen in North America. Outlooks in Pest Management25, 374–377 (2014). Consensus statement. Austin (TX): Second WNS Emergency Science Strategy Meeting; 2009. May 27–28 Courtin F, Stone W, Risatti G, Gilbert K, Van Kruiningen H Pathologic findings and liver elements in hibernating bats with white-nose syndrome. Vet Pathol. 2010;47:214–9 de Bellis T, Kernaghan G, Widden P Plant community influences on soil microfungal assemblages in boreal mixed-wood forests. Mycologia. 2007;99:356–67 Feldmann R Teichfledermaus–Myotis dasycneme (Boie, 1825). Die Säugetiere Westfalens. Münster: Westfälisches Museum für Naturkunde; 1984. p. 107–11. Frick WF, et al. (2010) An emerging disease causes regional population collapse of a common North American bat species. Science 329:679–682. Hoyt, J. R. et al. Widespread bat white-nose syndrome fungus, Northeastern China. Emerg. Infect. Dis. 22, 10.3201/eid2201.151314 (2016b). Gargas A, Trest MT, Christensen M, Volk TJ, Blehert DS Geomyces destructans sp. nov. associated with bat white-nose syndrome. Mycotaxon. 2009;108:147–54 Geiser F (2004) Metabolic rate and body temperature reduction during hibernation and daily torpor. Annu Rev Physiol 66:239–274. Gianni C, Caretta G, Romano C Skin infection due to Geomyces pannorum var. pannorum. Mycoses. 2003;46:430–2 Janicki AF, Frick WF, Kilpatrick AM, Parise KL, Foster JT, McCracken GF. Efficacy of Visual Surveys for White-Nose Syndrome at Bat Hibernacula. Russo D, ed. PLoS ONE. 2015;10(7):e0133390. doi:10.1371/journal.pone.0133390. Kochkina GA, Ivanushkina NE, Akimov VN, Gilichinskii DA, Ozerskaia SM Haloand psychrotolerant Geomyces fungi from arctic cryopegs and marine deposits Mikrobiologiia. 2007;76:39–44 Langwig, K. E. et al. Host and pathogen ecology drive the seasonal dynamics of a fungal disease, white-nose syndrome. Proc. R. Soc. B Biol. Sci. 282, 20142335 (2015). 20. Leopardi, S., Blake, D. & Puechmaille, S. J. White-nose syndrome fungus introduced from Europe to North America. Curr. Biol. 25, R217–219 (2015) 21. Lorch, J.M., C.U. Meteyer, M.J. et al. 2011. Experimental infection of bats with Geomyces destructans causes white-nose syndrome. Nature 480:376–378. 22. Lorch, J. M. Role of the Fungus Geomyces destructans in Bat White-Nose Syndrome A dissertation in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular & Environmental Toxicology) at the UNIVERSITY OF WISCONSIN-MADISON 2012 23. Lorch, J. M. et al. A culture-based survey of fungi in soil from bat hibernacula in the eastern United States and its implications for detection of Geomyces destructans, the causal agent of bat white-nose syndrome. Mycologia 105, 237–252 (2013). 24. Lorch, J. M. et al. Distribution and environmental persistence of the causative agent of white-nose syndrome, Geomyces destructans, in bat hibernacula of the Eastern United States. Appl. Environ. Microbiol. 79, 1293–1301 (2013) 25. Lorch, J. M. et al. The fungus Trichophyton redellii sp. nov. causes skin infections that resemble white-nose syndrome of hibernating bats. J. Wildl. Dis. 51, 36–47 (2015). 26. Mercantini R, Marsella R, Prignano G et al. Isolation of keratinophilic fungi from the dust of ferry boats and trains in Italy. Mycoses. 1989;32:590–4 151 27. Mercantini R, Marsella R, Cervellati M Keratinophilic fungi isolated from Antarctic soil.Mycopathologia. 1989;106:47–52 10.1007/BF00436926 28. Meteyer CU, Buckles EL, Blehert DS, Hicks AC, Green DE, Shearn-Bochsler V, et al. Histopathologic criteria to confirm white-nose syndrome in bats. J Vet Diagn 2009;21:411–4 29. Meteyer, C. U., Barber, D. & Mandl, J. N. Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome. Virulence 3, 583–588 (2012). 30. Minnis AM, Lindner DL. Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern North America. Fungal Biol. 2013 Sep;117(9):638-49. 31. Puechmaille SJ, Verdeyroux P, Fuller H, Ar Gouilh M, Bekaert M, Teeling EC White-nose syndrome fungus (Geomyces destructans) in bat, France. Emerg Infect Dis. 2010;16:290–3 32. Puechmaille SJ, Wibbelt G, Korn V, Fuller H, Forget F, Mühldorfer K, et al. (2011) Pan-European Distribution of White-Nose Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality. PLoS ONE 6(4): e19167. 33. Rachowicz LJ, et al. (2005) The novel and endemic pathogen hypotheses: Competing explanations for the origin of emerging infectious diseases of wildlife. Conserv Biol 19:1441–1448. 34. Reichard JD, Kunz TH White-nose syndrome inflicts lasting injuries to the wings of little brown myotis (Myotis lucifugus). Acta Chiropt. 2009;11:457–64 35. Simmons NB Order Chiroptera. In: Wilson DE, Reeder DM, editors. Mammal species of the world: a taxonomic and geographic reference, 3rd ed. Baltimore: Johns Hopkins University Press; 2005. p. 312–529. 36. Thomas DW, Dorais M, Bergeron J-M (1990) Winter energy budgets and cost of arousals for hibernating little brown bats Myotis lucifugus. J Mammal 71:475–479. 37. Turner GG, Reeder DM Update of white nose syndrome in bats, Bat Research News. 2009;50:47–53 38. Turner GG, Reeder DM, Coleman JTH (2011) A five-year assessment of mortality and geographic spread of white-nose syndrome in North American bats and a look to the future. Bat Research News 52(3):13–27. 39. United States Geological Survey. Update on white-nose syndrome: Tennessee finding. USGS wildlife health bulletin. Reston (VA): The Survey; 2010. http://www.nwhc.usgs.gov/disease_information/white-nose_syndrome/ 40. Verant ML, Boyles JG, Waldrep W Jr, Wibbelt G, Blehert DS (2012) TemperatureDependent Growth of Geomyces destructans, the Fungus That Causes Bat WhiteNose Syndrome. PLoS ONE 7(9): e46280. doi:10.1371/journal.pone.0046280 41. Warneckea Lisa, James M. Turnera, et al., Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of whitenose syndrome PNAS | May 1, 2012 | vol. 109 | no. 18 | 6999–7003 42. Wibbelt G, Kurth A, Hellmann D, et al. White-Nose Syndrome Fungus (Geomyces destructans) in Bats, Europe. Emerging Infectious Diseases. 2010;16(8):1237-1243. 43. WNS Science Strategy Group (2008) Questions, observations, hypotheses, predictions, and research needs for addressing effects of white-nose syndrome (WNS) in hibernating bats. http://batcon.org/pdfs/WNSMtgRptFinal2.pdf 44. Zelenková H Geomyces pannorum as a possible causative agent of dermatomycosis and onychomycosis in two patients. Acta Dermatovenerol Croat. 2006;14:21–5 45. Zukal J, Bandouchova H, Bartonicka T, Berkova H, Brack V, Brichta J, et al. (2014) White-Nose Syndrome Fungus: A Generalist Pathogen of Hibernating Bats. PLoS ONE 9(5): e97224. doi:10.1371/journal.pone.0097224 46. Zukal, J , Hana Bandouchova, Jiri Brichta, et al.. White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America, Scientific Reports 6, No. 19829 (2016) 152 6. Chytridiomycosis Chytridiomycosis is an emerging infectious disease of amphibians caused by the aquatic fungal pathogens, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal)  Chytridiomycosis has been documented in numerous frog species, some salamander species, and a single caecilian species (Typhlonectes sp.) in captivity.  Chytridiomycosis was reported in Central, and South America, Australia, Africa, and Europe;  Chytridiomycosis has caused, over just the past 30 years, the catastrophic decline or extinction of at many species of frogs around the world (e.g., Costa Rica, Panama, Brazil, Australia etc.  Chytridiomycosis is an infection of the superficial, keratin-containing layers of amphibian skin. In frog tadpoles, only the mouthparts are keratinized leading to mouthpart depigmentation and sometimes defects.  Chytridiomycosis causes drop electrolyte blood levels, leading to death from cardiac arrest.  Batrachochytrium dendrobatidis (―Bd‖) is an emerging infectious amphibian chytrid fungus. o Bd was discovered in 1997 (Berger et al. 1998) o Bd continues to be a major factor in the recent declines and extinctions of amphibian populations worldwide (Lips et al. 2005; Pounds et al. 2006; Skerratt et al. 2007; Catenazzi et al. 2014). o Bd was listed as a World Organisation for Animal Health (OIE) notifiable disease in 2008 (OIE 2008; Schloegel et al. 2010). o Bd has infected >500 species and caused extinctions or declines in >200 species worldwide (Yap et al. , 2016) o Bd causes chytridiomycosis which may be responsible for the most spectacular loss of vertebrate biodiversity due to disease in recorded history. o Bd is found on every continent where amphibians exist, infecting close to 700 amphibian species globally and is implicated in worldwide amphibian population declines.  Batrachochytrium salamandrivorans (Bsal) is a virulent fungal pathogen that infects salamanders. o Bsal is implicated in the recent collapse of several populations of fire salamanders in Europe. o Bsal seems much like that of its sister species, Batrachochytriumdendrobatidis (Bd), the agent responsible for anuran extinctions and extirpations worldwide o Bsal is considered to be an emerging global threat to salamander communities. Classification Cellular organisms +  Eukaryota + 153 o  Opisthokonta + Fungi +  Chytridiomycota +  Chytridiomycetes +  Rhizophydiales +  Rhizophydiales incertae sedis +  Batrachochytrium +  Batrachochytrium dendrobatidis +  Batrachochytrium salamandrivorans+ Aetiology: Batrachochytrium dendrobatidis Longcore, Pessier & D.K. Nichols, Mycologia 91 (2): 220 (1999) Batrachochytrium salamandrivorans sp. nov. Martel, A.; Spitzen-van der Sluijs, A.; Blooi, M.; Bert, W.; Ducatelle, R.; Fisher, M. C.; Woeltjes, A.; Bosman, W.; Chiers, K.; Bossuyt, F.; Pasmans, F. (2013). Thalli single or aggregated in amphibian epidermis. One to several rhizoidal axes; rhizoids thread-like. Each segment of the thallus forms a zoosporangium. Zoosporangia flask-shaped, up to 40 ?m diam, with one of several inoperculate discharge papillae. Zoospores spherical or slightly ovoidal, with a single flagella about 20 ?m in length. Ribosomes aggregated. Numerous lipid globules present. Kinetosomal root consisting of a group of microtubules. Rumposome and transition zone plug absent. In vitro, B. dendrobatidis thrives best in tryptone-gelatin hydrolysate-lactose (TGhL) broth or 1% tryptone broth. 154 Morphology of Batrachochytrium species in culture. a Culture of B. dendrobatidis on tryptone/gelatinhydrolysate/lactose (TGhL)-broth, showing abundant mature zoosporangia (black arrow) containing zoospores and empty, discharged sporangia (white arrow); b In culture (TGhL-broth) B. salamandrivorans is characterized by predominant monocentric thalli (black arrow), few colonial thalli (white arrow) and zoospore cysts with germ tubes (asterisk); scale bars 100 µm Lifecycle The lifecycle of B. dendrobatidis and B. salamandrivorans in culture o The zoospore first encysts by developing a cell wall and absorbing its flagellum, to finally form a germling with fine tread-like rhizoids. o The maturing germling develops into a zoosporangium in which the cytoplasm cleaves mitotically to form new zoospores. o Zoosporangia are predominantly monocentric (a thallus containing a single sporangium) and rarely colonial (a thallus containing more than one sporangium). o Discharge papillae or tubes, blocked inside by a plug, are formed during the growth of the sporangium. o At maturity the plug dissolves and the zoospores are released into the environment to continue their lifecycle. o Distinctive features of B. salamandrivorans in culture are its lower thermal preference, the presence of tubular extension or germ tubes arising from the encysted zoospores and from which new sporangia arise and the more abundant colonial thalli . The lifecycle of Batrachochytrium species in culture. In culture Batrachochytrium dendrobatidis continues the life cycle stages A–E, while in Batrachochytrium salamandrivorans additional life cycle stages B1-B2 are observed: (A) flagellated motile zoospores; (B) encysted zoospore; (B1) germling with germtube; (B2) transfer of the cell contents into a newly formed thallus; (C) zoospore cyst with rhizoids; (D) immature sporangium; (E) mature 155 monocentric zoosporangium with discharge tube (at the right), colonial thallus containing several sporangia, each with their own discharge tube (at the left). Modified from Berger et al. The lifecycle of B. dendrobatidis in amphibian skin o Upon colonization of the host epidermis, the zoospores encyst; the flagellum is absorbed and a cell wall is formed o The zoospore cyst germinates and develops a germ tube that invades the host epidermis. o At the tip of the germtube a new sporangium arises. o The fungus proliferates intracellularly, within the cells of the stratum corneum and the stratum granulosum. o Immature sporangia are carried from the deeper skin layers to the skin surface by differentiating epidermal cells. o Developed sporangia discharge tubes and contain mature zoospores, o Mature zoospores finally occur in stratum corneum o zoospores are released in the environment. o The lifecycle of B. salamandrivorans in amphibian skin has not yet been illustrated in great detail, but is assumed to be similar. Growth and survival of Batrachochytrium species Growth and survival of Batrachochytrium species are strongly temperature dependent.  For Batrachochytrium dendrobatidis o Optimal growth of B. dendrobatidis is observed between 17 and 25 °C and pH 6–7. o At 10 °C or lower, B. dendrobatidis grows slowly. At 28 °C or higher B. dendrobatidis ceases growth, while its zoospores are killed within 4 h at 37 °C. o Desiccation is poorly tolerated o 5% NaCl solutions are lethal  For Batrachochytrium salamandrivorans o Optimum temperatures for growth are between 10 and 15 °C. o It can still grow at 5 °C o Temperatures of 25 °C and higher are lethal.  Under laboratory conditions, B. dendrobatidis grows on a variety of keratin containing substrates such as o autoclaved snake skin, o 1% keratin agar, o frog skin agar, o feathers and geese paws Epidemiology   B. dendrobatidis has a broad host range and infects at least 520 species of o Anurans (frogs and toads), o Urodeles (salamanders and newts) and o Caecilians. B. salamandrivorans seems to be restricted to salamanders and newts. 156 Origin of Batrachochytrium species      Africa, North-America, Asia, the Atlantic Forest of Brazil have been suggested as sites of origin. o At the moment the Atlantic Forest of Brazil is suggested as cradle of B. dendrobatidis. o The Brazilian lineage or genotype of B. dendrobatidis (BdBz) has been enzootic in local amphibian assemblages for at least 100 year and has diverged earliest in the phylogenetic history of the pathogen. o In Asia, B. dendrobatidis was present more than 100 years ago. o B. dendrobatidis could be trace in Korea back to 1911 by analysis of museum specimens. o B. salamandrivorans is thought to have its origins in Asia. o B. salamandrivorans occurs historically at low levels on salamanders throughout at least Japan, Thailand and Vietnam. o B. salamandrivorans was found in a museum specimen of Cynops ensicauda (sword-tailed newt) dating back from 1861. B. salamandrivorans was most likely introduced to Europe through amphibian trade. o B. salamandrivorans was found in imported Asian salamanders. o At least five Asian salamander species (Cynops pyrrhogaster, Cynops cyanurus and Paramesotriton deloustali and Salamandrella keyserlingii) could be suitable reservoirs of the disease and are able to shed zoospores for at least 5 months without necessarily developing clinical disease. o The risk for indroduction of B. salamandrivorans in other regions than northern-Europe is thus quite realistic. The international trade in live amphibians has paved the way to the dispersal of the pathogen between continents. o more than 94% of the amphibian trade in the US is restricted to L. catesbeianus, with more than 20 million of specimens traded over an 8 years‘ period. o X. laevis is widely traded for scientific research o L. catesbeianus is imported at large scale to mainly the US, SouthAmerica, China and Europe for consumption. Both species are highly invasive when introduced into new environments. X. laevis and L. catesbeianus are subclinical carriers of B. dendrobatidis infection and act as reservoir, transmitting the infection to naïve native amphibians species. Transmission:  Transmission among hosts is mainly established by o the motile waterborne zoospores or o through direct contact with infected amphibians (e.g. during mating). o Infected amphibians may shed considerable loads of zoospores into waterbodies, making them potential environmental reservoirs. o under sterile conditions, B. dendrobatidis can survive in water and moist soil for weeks up to several months. 157 o B. dendrobatidis is able to saprobically grow on e.g. sterile bird feathers, arthropod exoskeletons, keratinous paw scales of waterfowl and to survive in the gastrointestinal tract of crayfish o Both waterfowl and crayfish have been suggested as potential nonamphibian vectors for B. dendrobatidis contributing to the dissemination of B. dendrobatidis. Clinical signs   In anuran larvae, clinical signs of chytridiomycosis due to B. dendrobatidis are generally limited to depigmentation of the mouthparts, without morbidity and mortality. o B. dendrobatidis may cause sub-lethal effects, including lethargy or poor swimming abilities, leading to low foraging efficiencies which is reflected in reduction in body size. In metamorphosed amphibians clinical signs are variable and range from sudden death without obvious disease to significant skin disorder but infection may nonetheless elapse asymptomatically. Most common signs of chytridiomycosis are o excessive shedding of the skin, o erythema (redness) or discoloration of the skin.  In frogs and toads, the skin of the ventral abdomen, especially the pelvic patch (a highly vascularized skin area on the ventral side of the body), feet and toes are predilection sites of infection  in salamanders the pelvic region, fore and hind limbs and the ventral side of the tail seem more prone to infection. o Other clinical signs include o lethargy, anorexia, o abnormal posture (abduction of the hind legs), o neurological signs such as loss of righting reflex and flight Clinical signs and pathology associated with infection due to Batrachochytrium dendrobatidis. a Naturally infected moribund common midwife toad (Alytes obstetricans) showing abnormal posture (abduction hind legs) and loose sloughed skin; b section through the ventral skin (drink patch) of the same infected toad; infection is characterized by diffuse epidermal hyperkeratosis and hyperplasia combined with the presence of numerous zoosporangia at various stages of maturation; HE; scale bar 50 µm; c detail of intracellular septate zoosporangia; HE; scale bar 10 µm 158 Clinical signs and pathology associated with infection due to Batrachochytrium salamandrivorans. a a naturally infected fire salamander (Salamandra salamandra) found during a B. salamandrivorans-outbreak (Robertville, Belgium) showing several ulcers (white arrows) and excessive skin shedding; b extensive ulceration (white arrows) at the ventral side of an infected fire salamander; c skin section through an ulcer evidences abundant intracellular colonial thalli in all epidermal skin layers; immunohistochemical stain with polyclonal antibodies to B. dendrobatidis; scale bar 10 µm; d magnification of the intracellular colonial thalli from micrograph c; immunohistochemical stain; scale bar 10 µm Pathology  Infection with Batrachochytrium is mainly associated with o a mild to severe irregular thickening (hyperkeratosis) of the outermost keratinized layers of the epidermis (the stratum corneum and stratum granulosum), o erosion of the stratum corneum, o increased tissue growth (hyperplasia) of the stratum spinosum which lies beneath the keratinized superficial skin layers. o ulceration of the skin may occur. o Other pathological changes in the epidermis adjacent to the foci of infection include  mild focal necrosis,  intercellular edema (spongiosis),  cytoplasmic degeneration with minimal to mild inflammation  vacuolation of the deeper cell layers. o Dissemination to the deeper layers of the skin or the internal organs does not occur. . Diagnosis:    Bd infection is suspected when dead or dying frogs are found (though other pathogens can also cause mortality: Clinical signs of severe chytridiomycosis in post-metamorphic frogs (juveniles and adults) include lack of appetite, lethargy, abnormal posture with hind legs extended, and lack of righting reflex (Berger et al. 2005); o In tadpoles chytrid infection can result in loss of dark coloration in tadpole mouthparts o In salamanders, chytrid infection may be visible as small dark spots on the ventral surface (belly), and result in skin sloughing Sample collection:  A sample is collected by swabbing the underside of the adult or juvenile animal 159 o For larval amphibians, the tadpole mouthparts should be swabbed o The swabbing protocol for adults and juveniles involves wearing gloves and carefully swabbing the underside of the animal (particularly the "drink patch" on the belly, the underside of the thighs, and the underside of the toe webbing)   Bd infection is usually then confirmed in two ways: o histology (sectioning skin and looking for the presence of chytrid zoosporangia within the skin o swabbing amphibians thought to be infected and testing for the presence of chytrid DNA using :  Nested PCR has also been used to detect Bd presence (with the first round of PCR using fungal-specific primers and the second round using Bd-specific primers  Quantitative real-time PCR (qPCR) is preferable as it allows an estimate of infection load.  Immunohistochemistry  Electron microscopy. A new rapid protocol has also been developed to distinguish viable, motile Batrachochytrium dendrobatidis zoospores from dead ones, using a two-color fluorescence assay comprised of the fluorescent stains SYBR 14 and propidium iodide (Stockwell et al. 2010). o SYBR 14 passes through intact cell membranes and fluoresces green when it binds to nucleic acids. o Propidium iodide passes only through compromised cell membranes (of dead/dying cells) and fluoresces red when it binds to nucleic acids. o Thus live, motile zoospores stain green while dead or dying zoospores stain red. Histology of a skin section from a White's treefrog (Litoria caerulea), showing heavy chytrid infection. 160 Scanning electron microscopy of toe skin surface from Litoria lesueuri, showing heavy chytrid infection. Wild animals mentioned in the available reports to be infected with chytridiomycosis 1. Anolis humilis lizards Kilburn et al. (2011) 2. Anolis lionotus lizards Kilburn et al. (2011) 3. Pliocercus euryzonus Kilburn et al. (2011) 4. Imantodes cenchoa Kilburn et al. (2011) 5. Nothopsis rugosus Kilburn et al. (2011) 6. red-legged frog (Rana aurora) Hamilton et al. (2012) 7. Pacific chorus frog (Pseudacris regilla) Hamilton et al. (2012) 8. bullfrog (Lithobates catesbeianus) Greenspan et al. (2012) 9. wood frogs (L. sylvaticus) Greenspan et al. (2012) 10. the stream frog Rana italica Zampiglia et al. (2013) 11. the fire salamander Salamandra salamandra gigliolii Zampiglia et al. (2013) 12. the alpine newt Mesotriton alpestris apuanus Zampiglia et al. (2013) 13. Xenopus laevis Vredenburg et al. (2013) 14. Xenopus borealis Vredenburg et al. (2013) 15. Agalychnis callidryas Rebollar et al. (2014) 16. Dendropsophus ebraccatus Rebollar et al. (2014) 17. Craugastor fitzingeri Rebollar et al. (2014) 18. Kihansi spray toad (Nectophrynoides asperginis) Makange et al. (2014) 19. Eastern hellbender (Cryptobranchus alleganiensis alleganiensis Bales et al. (2015) 20. Rugosa emeljanovi Fong et al. (2015) 21. American bullfrogs (Lithobates catesbeianus) Eskew et al. (2015) 22. Green-eyed tree frogs Litoria serrata Hagman and Alford (2015) 23. Wood frogs (Lithobates sylvaticus) Bradley et al. (2015) 24. eastern red-backed salamanders Plethodon cinereus Hess et al. (2015) 25. Brazilian Dendropsophus minutes Bovo et al. (2016) 26. Ischnocnema parva Bovo et al. (2016) 27. Brachycephalus pitanga Bovo et al. (2016) 161 28. Lake Titicaca frog (Telmatobius culeus) Berenguel et al. (2016) 29. Hellbenders Seeley et al. (2016) 30. red-legged salamander Plethodon shermani Fonner et al. (2017) 31. American toads (Anaxyrus americanus) Jones et al. (2017) 32. Western toads (A. boreas) Jones et al. (2017) 33. Spring peepers (Pseudacris crucifer) Jones et al. (2017) 34. Pacific treefrogs (P. regilla) Jones et al. (2017) 35. leopard frogs (Lithobates pipiens) Jones et al. (2017) 36. Cascades frogs (Rana cascadae) Jones et al. (2017) California H Red-legged Salamander . acadskidmore. Red-backed Salamander. Salamandra salamandra gigliolii American Toad | SREL .. Western Toad · iNaturalist.org Spring Peeper (Pseudacris crucifer) Pacific tree frog - Wikipedia Northern Leopard Frog (Lithobates pipiens)-. Cascades Frog - Rana cascadae California Telmatobius culeus (Titicaca Water Frog) Eastern Hellbender (Cryptobranchus alleganiensis) .flickr. Brachycephalus pitanga Alamy small frog (Dendropsophus elegans New Hampshire Public Tv Ischnocnema henselii EurekAlert! A Rugosa emeljanovi 162 . green eyed tree frog - Litoria serrata Wood Frog (Lithobates sylvaticus) Bullfrog (Lithobates catesbeianus) Italian Stream Frog (rana Italica),.Alamy Alpine newt (Mesotriton alpestris apuana (Nectophrynoides asperginis) Agalychnis callidryas - Wikipedia Dendropsophus ebraccatus UniProt iNaturalist (Craugastor fitzingeri) Eastern hellbender (Cryptobranchus alleganiensis Frog (Lithobates sylvaticus bullfrog (Lithobates catesbeianus) African Clawed Frog (Xenopus laevis CalPhotos: Anolis humilis CalPhotos: Xenopus borealis Wikim Tree Frog (Pseudacris regilla). Panama Birds & Wildlife blogger Anole (Anolis lionotus) CalPhotos: Rana aurora 163 Pliocercus euryzonus | Flickr Imantodes cenchoa | Tom's Blog Reports: Skerratt et al. (2007) mentioned that, the global emergence and spread of the pathogenic, virulent, and highly transmissible fungus Batrachochytrium dendrobatidis, resulting in the disease chytridiomycosis, has caused the decline or extinction of up to about 200 species of frogs. Key postulates for this theory have been completely or partially fulfilled. In the absence of supportive evidence for alternative theories despite decades of research, it is important for the scientific community and conservation agencies to recognize and manage the threat of chytridiomycosis to remaining species of frogs, especially those that are naive to the pathogen. The impact of chytridiomycosis on frogs is the most spectacular loss of vertebrate biodiversity due to disease in recorded history. Forzán et al. (2010) collected skin swabs from 115 frogs from 18 separate sites across the province during the summer of 2009. The swabs were tested through single round end-point PCR for the presence of Bd DNA. Thirty-one frogs were positive, including 25/93 (27%) green frogs Lithobates (Rana) clamitans, 5/20 (25%) northern leopard frogs L. (R.) pipiens, and 1/2 (50%) wood frogs L. sylvaticus (formerly R. sylvatica); 12 of the 18 (67%) sites had at least 1 positive frog. The overall prevalence of Bd infection was estimated at 26.9% (7.2-46.7%, 95% CI). Prevalence amongst green frogs and leopard frogs was similar, but green frogs had a stronger PCR signal when compared to leopard frogs, regardless of age (p < 0.001) and body length (p = 0.476). Amongst green frogs, juveniles were more frequently positive than adults (p = 0.001). Green frogs may be the most reliable species to sample when looking for Bd in eastern North America. The 1 wood frog positive for Bd was found dead from chytridiomycosis; none of the other frogs that were positive for Bd by PCR showed any obvious signs of illness. Further monitoring will be required to determine what effect Bd infection has on amphibian population health on PEI. Bodinof et al. (2011) carried out a study to determine whether Bd occurred historically in Missouri hellbender populations or is a relatively novel occurrence. Epidermal tissue was removed from 216 archived hellbenders collected from 7 Missouri streams between 1896 and 1994. Histological techniques and an immunoperoxidase stain were used to confirm historic occurrence of Bd infection in hellbenders from the North Fork of the White (1969, 1973, 1975), Meramec (1975, 1986), Big Piney (1986), and Current rivers (1988). Bd was not detected in hellbenders from the Niangua, Gasconade or Eleven Point rivers. The study detected no evidence for endemism of Bd in Missouri hellbender populations prior to 1969, despite the fact that nearly one third of the hellbenders sampled were collected earlier. 164 Our findings are consistent with the hypothesis that Bd is a non-endemic pathogen in North America that was introduced in the second half of the twentieth centur. Kilburn et al. (2011) surveyed for the presence of B. dendrobatidis DNA among 211 lizards and 8 snakes at 8 sites at varying elevations in Panama where the syntopic amphibians were at pre-epizootic, epizootic or post-epizootic stages of chytridiomycosis. Detection of B. dendrobatidis DNA was done using qPCR analysis. Evidence of the amphibian pathogen was present at varying intensities in 29 of 79 examined Anolis humilis lizards (32%) and 9 of 101 A. lionotus lizards (9%), and in one individual each of the snakes Pliocercus euryzonus, Imantodes cenchoa, and Nothopsis rugosus. In general, B. dendrobatidis DNA prevalence among reptiles was positively correlated with the infection prevalence among co-occurring anuran amphibians at any particular site (r = 0.88, p = 0.004). These reptiles, therefore, may likely be vectors or reservoir hosts for B. dendrobatidis and could serve as disease transmission agents. Although there is no evidence of B. dendrobatidis diseaseinduced declines in reptiles, cases of coincidence of reptile and amphibian declines suggest this potentiality. The study is the first to provide evidence of non-amphibian carriers for B. dendrobatidis in a natural Neotropical environment. Savage et al. (2011) reported the first survey for Bd in Peninsular Malaysia. They swabbed 127 individuals from the six amphibian families that occur on Peninsular Malaysia, including two orders, 27 genera, and 47 species. They detected Bd on 10 out of 127 individuals from four of five states and five of 11 localities, placing the 95% confidence interval for overall prevalence at 4-14%. No variation was detected in Bd prevalence among regions, elevations, or taxonomic groups. The infection intensity ranged from 1 to 157,000 genome equivalents. The presence of Bd infections in native species without clinical signs of disease suggests that Bd may be endemic to the region. Alternately, Bd may have been introduced from non-native amphibians because of the substantial amphibian food trade in Peninsular Malaysia. Under both scenarios, management efforts should be implemented to limit the spread of nonnative Bd and protect the tremendous amphibian diversity in Peninsular Malaysia. Greenspan et al. (2012) conducted an ex-situ experiment between May and July 2010 to determine whether B. dendrobatidis-infected bullfrogs could transmit the fungus to wood frog tadpoles when the two species shared a body of water. They tested for B. dendrobatidis infections with quantitative polymerase chain reactions (qPCR) in a subsample of the wood frog tadpoles and in all metamorphosed wood frogs and compared risk of death of froglets exposed and unexposed to infected bullfrogs. They detected B. dendrobatidis sporadically in subsampled treatment tadpoles (nine of 90, 10%) and frequently in treatment froglets (112 of 113, 99.1%). Pooled risk of froglet death was higher (P<0.001) in treatment enclosures than in control enclosures. Our results indicate that, at the low infection loads bullfrogs tend to carry, swabbing for PCR analyses may underestimate prevalence of B. dendrobatidis in this species. Hamilton et al. (2012) tested the response of red-legged frog (Rana aurora) tadpoles to increased variation in temperature, a component of climate linked to amphibian declines, and Bd exposure. Included were tadpoles of a sympatric competitor species, Pacific chorus frog (Pseudacris regilla), in a fully factorial design to test the effects of Bd and temperature on interspecific interactions. It was found that higher variation in temperature had numerous effects in mesocosms, including interacting with Bd presence to decrease the condition of R. aurora, shifting the relative performance of 165 competing P. regilla and R. aurora, and accelerating the development of P. regilla relative to R. aurora. The results demonstrated that increased variation in temperature can affect amphibians in multiple ways that will be contingent on ecological context, including the presence of Bd and competing species. Hauselberger and Alford (2012) examined samples from a total of 595 individuals of 9 species of direct-developing Australian frogs in the family Microhylidae for the presence of infection by Batrachochytrium dendrobatidis (Bd). Between 1995 and 2004, 336 samples were collected; 102 of these were analysed histologically and 234 were tissues stored in alcohol, which were examined using diagnostic quantitative PCR (qPCR). Swab samples were collected from 259 frogs from 2005 to 2008 and were examined using qPCR. None of the 595 samples showed evidence of infection by Bd. The data suggested that Australian microhylids have a very low prevalence of infection by Bd in nature, and thus are either not susceptible, or are only slightly susceptible, to chytridiomycosis. This could be due solely to, or in combination with, low rates of transmission and to factors that promote resistance to infection, including ecological or behavioural characteristics, innate immune functions such as antimicrobial skin peptides, or antimicrobial symbionts in skin flora. Doherty-Bone et al. (2013) presented survey data from 12 localities across 6 regions of Cameroon from anurans (n = 1052) and caecilians (n = 85) of ca. 108 species. Bd was detected in 124 amphibian hosts at 7 localities, including Mt. Oku, Mt. Cameroon, Mt. Manengouba and lowland localities in the centre and west of the country. None of the hosts were observed dead or dying. Infected amphibian hosts were not detected in other localities in the south and eastern rainforest belt. Infection occurred in both anurans and caecilians, making this the first reported case of infection in the latter order (Gymnophiona) of amphibians. There was no significant difference between prevalence and infection intensity in frogs and caecilians. We highlight the importance of taking into account the inhibition of diagnostic qPCR in studies on Bd, based on all Bd-positive hosts being undetected when screened without bovine serum albumin in the qPCR mix. The status of Bd as an indigenous, cosmopolitan amphibian parasite in Africa, including Cameroon, is supported by this work. Isolating and sequencing strains of Bd from Cameroon should now be a priority. Longitudinal host population monitoring will be required to determine the effects, if any, of the infection on amphibians in Cameroon. Gold et al. (2013) tested the efficacy of 5 disinfectants and 1 anti-fungal treatment, at 1 and 5 min contact durations, in inactivating Batrachochytrium dendrobatidis (Bd) grown on tryptone media. The study focused on concentrations of disinfectants known to inactivate ranaviruses, which can be found at the same sites as Bd and can concurrently infect amphibians. Disinfectants tested were chlorhexidine gluconate (0.25, 0.75, and 2%), Pro-San (0.19, 0.35, and 0.47%), Virkon S (1%), household bleach (0.2, 1, and 3%), and Xtreme Mic (5%). The anti-fungal was terbinafine HCl at 0.005, 0.05, 0.1, and 1 mg ml-1. Inactivation of Bd was determined by microscopic evaluation of zoospore motility and growth of colony mass after 14 d. All disinfectants were effective at inactivating zoospore motility and colony growth of Bd at all concentrations and both contact times; however, terbinafine HCl inactivated Bd at only the highest concentration tested (1 mg ml-1) and 5 min duration. Thus, a minimum of 0.25% chlorhexidine gluconate, 0.19% Pro-San, 1% Virkon, 0.2% bleach, and 5% Xtreme Mic with 1 min contact was sufficient to inactivate Bd. Also, terbinafine HCl (1 mg ml-1) with a 5 min contact time might be effective in treating amphibians infected with Bd. Based on this study and previously published findings, 166 0.75% Nolvasan, 1% Virkon S, and 3% bleach with 1 min contact are sufficient to inactivate both Bd and ranaviruses. Vredenburg et al. (2013) conducted a survey on 178 archived specimens of 6 species of Xenopus collected in Africa from 1871-2000 and on 23 archived specimens (all wild-caught Xenopus laevis) collected in California, USA between 2001 and 2010. The overall prevalence rate of Bd in the tested Xenopus was 2.8%. The earliest positive specimen was X. borealis collected in Kenya in 1934. The overall prevalence of Bd in the X. laevis collected in California was 13% with 2 positive specimens from 2001 and one positive specimen from 2003. The positive Xenopus (3/23) collected in California were collected in 2001 (2/3) and 2003 (1/3). These data documented the presence of Bd-infected wild Xenopus laevis in California. The findings reported here support the prevailing hypothesis that Bd was present as a stable, endemic infection in Xenopus populations in Africa prior to their worldwide distribution likely via international live-amphibian trade. Zampiglia et al. (2013) investigated the presence and distribution of Bd among populations of 3 mid- to high-altitude species spanning the entire Italian peninsula (486 individuals from 39 sites overall): the stream frog Rana italica, the fire salamander Salamandra salamandra gigliolii, and the alpine newt Mesotriton alpestris apuanus. They found Bd in all of the analyzed species. Despite the widespread distribution of the pathogen, its overall prevalence (6, 9 and 19%, respectively) was lower than previously reported for the endangered Apennine yellow-bellied toad Bombina pachypus (62.5%). Moreover, several populations of the species studied here were not infected, even at sites where Bd has been detected in other host species. When coupled with the lack of evidence for Bd-related mortalities in these species in peninsular Italy, these results suggested that mechanisms of resistance and/or tolerance are protecting populations of these species from the pathogenic activity of Bd. In light of the dynamic pattern of Bd-host interactions reported in other studies, of Bd-related mortalities in at least 1 study species (S. s. salamandra) in other areas, and the ongoing climate changes in montane environments, it was suggested that the occurrence of Bd should be considered a potential threat to the long-term persistence of these species, and urge the implementation of monitoring and conservation plans. Makange et al. (2014) observed mass mortalities of Kihansi spray toads Nectophrynoides asperginis at the Kihansi captive breeding facility, located in the Udzungwa Mountains, Tanzania. Mortalities increased rapidly, and dead toads showed typical clinical signs of chytridiomycosis, including reddening of the skin that was especially evident on the toe pads. Treatment of toads with itraconazole rapidly reduced mortalities. Dead toads (n = 49) were collected and used to perform Bdspecific polymerase chain reaction and subsequent nucleotide sequencing. All toads collected at the facility were positive for Bd. The obtained Bd 5.8S rRNA gene and flanking internal transcribed spacer regions (ITS1 and ITS2) were not 100% identical to any other Bd sequences in GenBank, but closely resembled isolates from Ecuador, Japan, USA, Brazil, Korea, and South Africa. To our knowledge, this is the first study reporting molecular characteristics of Bd isolated from the Udzungwa Mountains. Strict biosecurity measures at the breeding facility and in Kihansi spray wetlands where toads have been reintroduced have been implemented. Further studies on Bd epidemiology in the Udzungwa Mountains awere recommended in order to understand its origin, prevalence, and molecular characteristics in wild amphibian populations. This will be important for conservation of several endemic amphibian 167 species in the Udzungwa Mountains, which are part of the Eastern Arc Mountains, a global biodiversity hotspot. Rebollar et al. (2014) determined the prevalence and intensity of Bd infection in three species of frogs in one highland and four lowland tropical forests, including two lowland regions in eastern Panamá in which the pathogen had not been detected previously. Bd was present in all the sites sampled with a prevalence ranging from 1534%, similar to other Neotropical lowland sites. The intensity of Bd infection on individual frogs was low, ranging from average values of 0.11-24 zoospore equivalents per site. This work indicated that Bd is present in anuran communities in lowland Panamá, including the Darién province, and that the intensity of the infection may vary among species from different habitats and with different life histories. The population-level consequences of Bd infection in amphibian communities from the lowlands remain to be determined. Detailed studies of amphibian species from the lowlands will be essential to determine the reason why these species are persisting despite the presence of the pathogen. Tamukai et al. (2014) surveyed amphibians imported into Japan and those held in captivity for a long period or bred in Japan to clarify the Bd infection status. Samples were taken from 820 individuals of 109 amphibian species between 2008 and 2011 and were analyzed by a nested-PCR assay. Bd prevalence in imported amphibians was 10.3% (58/561), while it was 6.9% (18/259) in those in private collections and commercially bred amphibians in Japan. The genotypes of this fungus were identified using partial DNA sequences of the internal transcribed spacer (ITS) region. Sequencing of PCR products of all 76 Bd-positive samples revealed 11 haplotypes of the Bd ITS region. Haplotype A (DNA Data Bank of Japan accession number AB435211) was found in 90% (52/58) of imported amphibians. The results showed that Bd is currently entering Japan via the international trade in exotic amphibians as pets, suggesting that the trade has indeed played a major role in the spread of Bd. Bales et al. (2015) surveyed populations of an aquatic salamander that is declining in the United States, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis), for the presence of Bs and Bd. Skin swabs were collected from a total of 91 individuals in New York, Pennsylvania, Ohio, and Virginia, and tested for both pathogens using duplex qPCR. Bs was not detected in any samples, suggesting it was not present in these hellbender populations (0% prevalence, 95% confidence intervals of 0.0-0.04). Bd was found on 22 hellbenders (24% prevalence, 95% confidence intervals of 0.16 ≤ 0.24 ≤ 0.34), representing all four states. All positive samples had low loads of Bd zoospores (12.7 ± 4.9 S.E.M. genome equivalents) compared to other Bd susceptible species. Bletz et al. (2015) documented surveys conducted across the country between 2005 and 2014, showing Bd's first record in 2010. Subsequently, Bd was detected in multiple areas, with prevalence reaching up to 100%. Detection of Bd appears to be associated with mid to high elevation sites and to have a seasonal pattern, with greater detectability during the dry season. Lineage-based PCR was performed on a subset of samples. While some did not amplify with any lineage probe, when a positive signal was observed, samples were most similar to the Global Panzootic Lineage (BdGPL). These results may suggest that Bd arrived recently, but do not exclude the existence of a previously undetected endemic Bd genotype. Representatives of all native anuran families have tested Bd-positive, and exposure trials confirmed infection by Bd is 168 possible. Bd's presence could pose significant threats to Madagascar's unique "megadiverse" amphibians. Bradley et al. (2015) experimentally investigated intraspecific differences in host sensitivity to Bd across 10 populations of wood frogs (Lithobates sylvaticus) raised from eggs to metamorphosis. The post-metamorphic wood frogs was exposed to Bd and survival was monitored for 30 days under controlled laboratory conditions. Populations differed in overall survival and mortality rate. Infection load also differed among populations but was not correlated with population differences in risk of mortality. Such population-level variation in sensitivity to Bd may result in reservoir populations that may be a source for the transmission of Bd to other sensitive populations or species. Alternatively, remnant populations that are less sensitive to Bd could serve as sources for recolonization after epidemic events. Bresciano et al. (2015) conducted a study to determine for the first time if specific anti-Bd bacteria are present on amphibians in the Andes of Ecuador, to monitor antiBd bacteria across developmental stages in a focal amphibian, the Andean marsupial tree frog, Gastrotheca riobambae, that deposits larvae in aquatic habitats, and to compare the Bd presence associated with host assemblages including 10 species at sites ranging in biogeography from Amazonian rainforest (450 masl) to Andes montane rainforest (3200 masl). They sampled and identified skin-associated bacteria of frogs in the field using swabs and a novel methodology of aerobic counting plates, and a combination of morphological, biochemical, and molecular identification techniques. The following anti-Bd bacteria were identified and found to be shared among several hosts at high-elevation sites where Bd was present at a prevalence of 32.5%: Janthinobacterium lividum, Pseudomonas fluorescens, and Serratia sp. Bd were detected in Gastrotheca spp. and not detected in the lowlands (sites below 1000 masl). In G. riobambae, recognized Bd-resistant bacteria start to be present at the metamorphic stage. Overall bacterial abundance was significantly higher postmetamorphosis and on species sampled at lower elevations. Further metagenomic studies are needed to evaluate the roles of host identity, life-history stage, and biogeography of the microbiota and their function in disease resistance. Coutinho et al. (2015) compared singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. They concluded that the nested-PCR technique, due to its ease of execution and 169 reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations. Eskew et al. (2015) reported on experimental exposures of American bullfrogs (Lithobates catesbeianus) to three different isolates of Batrachochytrium dendrobatidis (Bd), including one implicated in causing mass mortality of wild American bullfrogs. Exposed frogs showed low infection prevalence, relatively low infection load, and lack of clinical disease. The results suggested that environmental co-factors are likely important contributors to Bdassociated American bullfrog mortality and that this species both resists and tolerates Bd infection. Fong et al. (2015) used quantitative PCR to screen 244 museum specimens from the Korean Peninsula, collected between 1911 and 2004, for the presence of Bd to gain insight into its history in Asia. Three specimens of Rugosa emeljanovi (previously Rana or Glandirana rugosa), collected in 1911 from Wonsan, North Korea, tested positive for Bd. Histology of these positive specimens revealed mild hyperkeratosis a non-specific host response commonly found in Bd-infected frogs - but no Bd zoospores or zoosporangia. The results indicated that Bd was present in Korea more than 100 years ago, consistent with hypotheses suggesting that Korean amphibians may be infected by endemic Asian Bd strains. Hagman and Alford (2015) conducted an experiment using larval green-eyed tree frogs Litoria serrata in semi-natural streamside channels to test the hypotheses that (1) the fungus can be transmitted downstream in stream habitats and (2) infection affects tadpole growth and mouthpart loss. The results showed that transmission can occur downstream in flowing water with no contact between individuals, that newly infected tadpoles suffered increased mouthpart loss in comparison with controls that were never infected and that infected tadpoles grew at reduced rates. Although recently infected tadpoles showed substantial loss of mouthparts, individuals with longstanding infections did not, suggesting that mouthparts may re-grow following initial loss. The study suggested that any management efforts that can reduce the prevalence of infections in tadpoles may be particularly effective if applied in headwater areas, as their effects are likely to be felt downstream. Hess et al. (2015) explored topic of immune function using eastern red-backed salamanders Plethodon cinereus and Batrachochytrium dendrobatidis (Bd). They conducted an experiment in which we repeatedly observed the feeding activity of Bdinfected and non-infected salamanders. It was found that Bd-infected salamanders generally increased their feeding activity compared to non-infected salamanders. Kolby et al. (2015) explored the possible spread of Bd from an aquatic reservoir to terrestrial substrates by the emergence of recently metamorphosed infected amphibians and potential deposition of Bd-positive residue on riparian vegetation in Cusuco National Park, Honduras (CNP). Amphibians and their respective leaf perches were both sampled for Bd presence and the pathogen was detected on 76.1% (35/46) of leaves where a Bd-positive frog had rested. Although the viability of Bd detected on these leaves cannot be discerned from the quantitative PCR results, the cool air temperature, closed canopy, and high humidity of this cloud forest environment in CNP is expected to encourage pathogen persistence. High prevalence of infection (88.5%) detected in the recently metamorphosed amphibians and frequent shedding of Bd-positive residue on foliage demonstrates a pathway of Bd dispersal between aquatic and terrestrial habitats. This pathway provides the opportunity for 170 environmental transmission of Bd among and between amphibian species without direct physical contact or exposure to an aquatic habitat. Rollins-Smith et al. (2015) described the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. It was hypothesized that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen. Berenguel et al. (2016) found moderate levels of chytrid infection using quantitative PCR. The results enhanced the understanding of chytrid tolerance to high pH and low water temperature. Bovo et al. (2016) experimentally tested whether an enzootic strain of Bd caused significant mortality and altered host water balance (evaporative water loss, EWL; skin resistance, R(s); and water uptake, WU) in individuals of 3 Brazilian amphibian species (Dendropsophus minutus, n = 19; Ischnocnema parva, n = 17; Brachycephalus pitanga, n = 15). Infections with enzootic Bd caused no significant mortality, but an increase was found in R(s) in 1 host species concomitant with a reduction in EWL. These results suggested that enzootic Bd infections can indeed cause sub-lethal effects that could lead to reduction of host fitness in Brazilian frogs and that these effects vary among species. Thus, the findings underscored the need for further assessment of physiological responses to Bd infections in different host species, even in cases of sub-clinical chytridiomycosis and long-term enzootic infections in natural populations. Byrne et al. (2016) focused on the application of genomics to understanding the biology of the fungal pathogen Batrachochytrium dendrobatidis (Bd), a novel and deadly pathogen of amphibians. They provided a brief history of the system, then focused on key insights into Bd variation garnered from genomics approaches, and finally, highlighted new frontiers for future discoveries. Genomic tools have revealed unexpected complexity and variation in the Bd system suggesting that the history and biology of emerging pathogens may not be as simple as they initially seem. Clare et al. (2016) detected significantly higher fungal burdens from moribund metamorphs compared to visually healthy individuals; however, the ability of these swab data to provide an accurate indication of the true fungal burden was not reliable. These data suggest that fungal load dynamics played an important role in diseaseinduced mortality in A. obstetricans at these sites, but that using swab data to infer an exact threshold for Bd-associated mortality might be inappropriate and misleading. Fernández-Beaskoetxea et al. (2016) used experimental approaches in captive and wild populations to determine the effect of common midwife toad larvae on infection of other amphibian species found in the Peñalara Massif, Spain. It was observed that the most widely and heavily infected species, the common midwife toad, may be amplifying the infection loads in other species, all of which have different degrees of susceptibility to Bd infection. The results have important implications for performing 171 mitigation actions focused on potential 'amplifier' hosts and for better understanding the mechanisms of Bd transmission. Julian et al. (2016) identified 4 natural populations of northern green frogs Lithobates clamitans melanota in Pennsylvania (USA) that contained Bd-infected tadpoles during post-wintering collections in May and June, after hibernating tadpoles had overwintered in wetlands. However, they failed to detect infected tadpoles at those wetlands when pre-wintering collections were made in late July through early September. They observed 2 cohorts of tadpoles that appeared to lack Bd-infected individuals in pre-wintering collections, yet contained Bd-infected individuals the following spring. They also observed 4 cohorts of pre-wintering tadpoles that were Bd-free, even though post-wintering tadpoles collected earlier in the year were infected with Bd. The results suggested that tadpoles either reduce Bd infections during the summer months, and/or infections proliferate sometime prior to (or shortly after) tadpoles emerge from hibernation. It is unlikely that pre-wintering tadpoles were too small to detect Bd zoospores because (1) there was no correlation between Bd zoospore levels and tadpole size or stage, and (2) size was not a significant predictor of infection status. These results suggest that, while sampling larvae can be an effective means of collecting large sample sizes, investigators in our Mid-Atlantic region should conduct sampling by early summer to maximize the chances of detecting Bd. Further research was recommended to determine whether wetland topography and warm, shallow microhabitats within wetlands contribute to a population's ability to drastically reduce Bd prevalence prior to overwintering at ponds. Petersen et al. (2016) reported on prevalence and intensity of Bd in the United States amphibian populations across three longitudinally separated north-to-south transects conducted at 15 Department of Defense installations during two sampling periods (late-spring/early summer and mid to late summer). Such a standardized approach minimized the effects of sampling and analytical bias, as well as human disturbance (by sampling restricted military bases), and therefore permited a cleaner interpretation of environmental variables known to affect chytrid dynamics such as season, temperature, rainfall, latitude, and longitude. The prevalence of positive samples was 20.4% (137/670), and the mean intensity was 3.21 zoospore equivalents (SE = 1.03; range 0.001-103.59). Of the 28 amphibian species sampled, 15 tested positive. Three sites had no evidence of Bd infection; across the remaining 12 Bd-positive sites, neither infection prevalence nor intensity varied systematically. A more complicated pattern of Bd prevalence than anticipated was foud. Early season samples showed no trend associated with increasing temperature and precipitation and decreasing (more southerly) latitudes; while in late season samples, the proportion of infected individuals decreased with increasing temperature and precipitation and decreasing latitudes. A similar pattern held for the east-west gradient, with the highest prevalence associated with more easterly/recently warmer sites in the early season then shifting to more westerly/recently cooler sites in the later season. Bd intensity across bases and sampling periods was comparatively low. Some of the trends in the data have been seen in previous studies, and our results offer further continental-level Bd sampling over which more concentrated local sampling efforts can be overlaid. Seeley et al. (2016) evaluated the prevalence of Bd in 42 Eastern Hellbender (Cryptobranchus alleganiensis) at four sites in West Virginia, US, from June to September 2013, using standard swab protocols and real-time PCR. Overall prevalence of Bd was 52% (22/42; 37.7-66.6%; 95% confidence interval). Prevalence 172 was highest in individuals with body weight ≥695 g (χ(2)=7.2487, df=1, P=0.007), and was higher in montane sampling sites than lowland sites (t=-2.4599, df=44, P=0.02). While increased prevalence in montane sampling sites was expected, increased prevalence in larger hellbenders was unexpected and hypothesized to be associated with greater surface area for infection or prolonged periods of exposure in older, larger hellbenders. Wild hellbenders have not been reported to display clinical disease associated with Bd; however, prevalence in the population is important information for evaluating reservoir status and risk to other species, and as a baseline for investigation in the face of an outbreak of clinical disease. Sun et al. (2016) detected 19 bacterial genes transferred to Bd, including metallobeta-lactamase and arsenate reductase that play important roles in the resistance to antibiotics and arsenates. Moreover, three probable HGT gene families in Bd are from plants and one gene family coding the ankyrin repeat-containing protein appears to come from oomycetes. The observed multi-copy gene families associated with HGT are probably due to the independent transfer events or gene duplications. Five HGT genes with extracellular locations may relate to infection, and some other genes may participate in a variety of metabolic pathways, and in doing so add important metabolic traits to the recipient. The evolutionary analysis indicates that all the transferred genes evolved under purifying selection, suggesting that their functions in Bd are similar to those of the donors. Collectively, the results indicated that HGT from diverse donors may be an important evolutionary driver of Bd, and improve its adaptations for infecting and colonizing host amphibians. Wombwell et al. (2016) mentioned that there is increasing evidence that the global spread of the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been facilitated by the international trade in amphibians. Bd was first detected in the UK in 2004, and has since been detected in multiple wild amphibian populations. Most amphibians imported into the UK for the pet trade from outside the European Union enter the country via Heathrow Animal Reception Centre (HARC), where Bd-positive animals have been previously detected. Data on the volume, diversity and origin of imported amphibians were collected for 59 consignments arriving at HARC between November 2009 and June 2012, along with a surveillance study to investigate the prevalence of Bd in these animals. Forty-three amphibian genera were recorded, originating from 12 countries. It was estimated that 5000-7000 amphibians are imported through HARC into the UK annually for the pet trade. Bd was detected in consignments from the USA and Tanzania, in six genera, resulting in an overall prevalence of 3.6%. This suggests that imported amphibians are a source of Bd within the international pet trade. Xie et al. (2016) examined a broad set of climate metrics to model the Bd-climate niche globally and regionally, then projected how climate change may influence Bd distributions. Previous research showed that Bd distribution is dependent on climatic variables, in particular temperature. They trained a machine-learning model (random forest) with the most comprehensive global compilation of Bd sampling records (~5,000 site-level records, mid-2014 summary), including 13 climatic variables. They projected future Bd environmental suitability under IPCC scenarios. The learning model was trained with combined worldwide data (non-region specific) and also separately per region (region-specific). One goal of this study was to estimate of how Bd spatial risks may change under climate change based on the best available data. Our models supported differences in Bd-climate relationships among geographic regions. It was projected that Bd ranges will shift into higher latitudes and altitudes 173 due to increased environmental suitability in those regions under predicted climate change. Specifically, this model showed a broad expansion of areas environmentally suitable for establishment of Bd on amphibian hosts in the temperate zones of the Northern Hemisphere. These projections are useful for the development of monitoring designs in these areas, especially for sensitive species and those vulnerable to multiple threats. Yap et al. (2016) conducted a museum specimen survey (1910-1997) of Bd in amphibians on 11 California islands and found a pattern consistent with the emergence of Bd epizootics on the mainland, suggesting that geographic isolation did not prevent Bd invasion. It was proposed that suitable habitat, host diversity, and human visitation overcome isolation from the mainland and play a role in Bd invasion. Zevallos et al. (2016) screened frogs confiscated by the Administration of Forestry and Wildlife in Lima, Peru, for Bd. They used real-time PCR to diagnose Bd at the Laboratory of Wildlife, Faculty of Veterinary Medicine and Zootecnics, Universidad Peruana Cayetano Heredia, in Lima and Pisces Molecular Laboratory in Boulder, Colorado, US. Of 62 samples collected during this study, 60% (37) were PCR positive for Bd, confirming that illegal trade of amphibians can pose a risk for disseminating Bd. 174 Zhu et al. (2016) collected a total of 1284 non-invasive skin swabs from wild and captive anurans and caudates, including free-ranging, farmed, ornamental, and museum-preserved amphibians. Bd was detected at low prevalence (1.1%, 12 of 1073) in live wild amphibians, representing the first report of Bd infecting anurans from remote areas of northwestern China. There was no evidence of the historical presence of Bd from museum amphibians (n = 72). Alarmingly, Bd was not detected in wild amphibians from the provinces of northeastern China (>700 individuals tested), but was widely present (15.1%, 21 of 139) in amphibians traded in this region. Urgent implementation of measures is required to reduce the possibility of further spread or inadvertent introduction of Bd to China. It is unknown whether Bd in northern China belongs to endemic and/or exotic genotypes, and this should be the focus of future research. Burrowes and De la Riva (2017) reported the presence of B. dendrobatidis in the feet of preserved aquatic birds in the Bolivian high Andes during the time of drastic amphibian declines in the country. 48 aquatic birds were collected from the Bolivian Andes that were preserved in museum collections. Birds were sampled for the presence of B. dendrobatidis DNA by swabbing, taking small pieces of tissue from toe webbing, or both. We detected B. dendrobatidis by DNA using quantitative PCR in 42% of the birds sampled via toe tissue pieces. This method was significantly better than swabbing at detecting B. dendrobatidis from bird feet. They confirmed B. dendrobatidis presence by sequencing B. dendrobatidis -positive samples and found 91-98% homology with B. dendrobatidis sequences from GenBank. The study confirmed that aquatic birds can carry B. dendrobatidis and thus may serve as potential vectors of this pathogen across large distances and complex landscapes. In addition, they recommend using DNA from preserved birds as a novel source of data to test hypotheses on the spread of chytridiomycosis in amphibians. Dillon et al. (2017) described the generation of an IgM monoclonal antibody (mAb), 5C4, specific to Bd as well as the related salamander and new pathogen Batrachochytrium salamandrivorans (Bsal). The mAb, which binds to a glycoprotein antigen present on the surface of zoospores, sporangia and zoosporangia, was used to develop a lateral-flow assay (LFA) for rapid (15 min) detection of the pathogens. The LFA detected known lineages of Bd and also Bsal, as well as the closely related fungus Homolaphlyctis polyrhiza, but did not detect a wide range of related and unrelated fungi and oomycetes likely to be present in amphibian habitats. When combined with a simple swabbing procedure, the LFA was 100% accurate in detecting the water-soluble 5C4 antigen present in skin, foot and pelvic samples from frogs, newts and salamanders naturally infected with Bd or Bsal. The results demonstrated the potential of the portable LFA as a rapid qualitative assay for tracking these amphibian pathogens and as an adjunct test to nucleic acid-based detection methods. Ellison et al. (2017) characterized the transcriptomic profile of Bd in vivo, using laser-capture microdissection. Comparison of Bd transcriptomes (strain JEL423) in culture and in two hosts (Atelopus zeteki and Hylomantis lemur), reveals >2000 differentially expressed genes that likely include key Bd defense and host exploitation mechanisms. Variation in Bd transcriptomes from different amphibian hosts demonstrates shifts in pathogen resource allocation. Furthermore, expressed genotype variant frequencies of Bd populations differ between culture and amphibian skin, and among host species, revealing potential mechanisms underlying rapid changes in virulence and the possibility that amphibian community composition shapes Bd 175 evolutionary trajectories. Our results provide new insights into how changes in gene expression and infecting population genotypes can be key to the success of a generalist fungal pathogen. Fonner et al. (2017) tested whether elevation of corticosterone (CORT). would reduce resistance to Batrachochytrium dendrobatidis (Bd) and chytridiomycosis development in the red-legged salamander Plethodon shermani. Plasma CORT was elevated daily in animals for 9 d, after which animals were inoculated with Bd and subsequently tested for infection loads and clinical signs of disease. On average, Bdinoculated animals treated with CORT had higher infection abundance compared to Bd-inoculated animals not treated with CORT. However, salamanders that received CORT prior to Bd did not experience any increase in clinical signs of chytridiomycosis compared to salamanders not treated with CORT. The lack of congruence between CORT effects on infection abundance versus disease may be due to threshold effects. Nonetheless, our results show that elevation of plasma CORT prior to Bd inoculation decreases resistance to infection by Bd. More studies are needed to better understand the effects of CORT on animals exposed to Bd and whether CORT variation contributes to differential responses to Bd observed across amphibian species and populations. Jones et al. (2017) simultaneously exposed postmetamorphic American toads (Anaxyrus americanus), western toads (A. boreas), spring peepers (Pseudacris crucifer), Pacific treefrogs (P. regilla), leopard frogs (Lithobates pipiens), and Cascades frogs (Rana cascadae) to a factorial combination of two pathogen treatments (Bd+, Bd-) and four pesticide treatments (control, ethanol vehicle, herbicide mixture, and insecticide mixture) for 14 d to quantify survival and infection load. They found no interactive effects of pesticides and Bd on anuran survival and no effects of pesticides on infection load. Mortality following Bd exposure increased in spring peepers and American toads and was dependent upon snout-vent length in western toads, American toads, and Pacific treefrogs. Previous studies reported effects of early sublethal pesticide exposure on amphibian Bd sensitivity and infection load at later life stages, but they found simultaneous exposure to sublethal pesticide concentrations and Bd had no such effect on postmetamorphic juvenile anurans. Future research investigating complex interactions between pesticides and Bd should employ a variety of pesticide formulations and Bd strains and follow the effects of exposure throughout ontogeny. Parrott et al. (2017) screened specimens of salamanders representing 17 species inhabiting mountain ranges in three continents: The Smoky Mountains, the Swiss Alps, and the Peruvian Andes. We screened 509 salamanders, with 192 representing New World salamanders that were never tested for Bsal previously. Bsal was not detected, and Bd was mostly present at low prevalence except for one site in the Andes. Piovia-Scott et al. (2017) investigated the ability of host-associated bacteria to inhibit the proliferation of Bd when grown in experimentally assembled biofilm communities that differ in species number and composition. Six bacterial species isolated from the skin of Cascades frogs (Rana cascadae) were used to assemble bacterial biofilm communities containing 1, 2, 3, or all 6 bacterial species. Biofilm communities were grown with Bd for 7 days following inoculation. More speciose bacterial communities reduced Bd abundance more effectively. This relationship between bacterial species 176 richness and Bd suppression appeared to be driven by dominance effects-the bacterial species that were most effective at inhibiting Bd dominated multi-species communities-and complementarity: multi-species communities inhibited Bd growth more than monocultures of constituent species. 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Julian JT, Gould VA, Glenney GW, Brooks RP. Seasonal infection rates of Batrachochytrium dendrobatidis in populations of northern green frog Lithobates clamitans melanota tadpoles. Dis Aquat Organ. 2016 Sep 26;121(2):97-104. 34. Kilburn VL, Ibáñez R, Green DM. Reptiles as potential vectors and hosts of the amphibian pathogen Batrachochytriumdendrobatidis in Panama. Dis Aquat Organ. 2011 Dec 6;97(2):127-34. 35. Kilpatrick AM, Briggs CJ, Daszak P (2010) The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends in Ecology and Evolution 25(2):109–118. doi: 10.1016/j.tree.2009.07.011. pmid:19836101 36. Kolby JE, Ramirez SD, Berger L, Richards-Hrdlicka KL, Jocque M, Skerratt LF. Terrestrial Dispersal and Potential Environmental Transmission of the Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis). PLoS One. 2015 Apr 30;10(4):e0125386. 37. La Marca, E. et al. 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Vredenburg VT, Felt SA, Morgan EC, McNally SV, Wilson S, Green SL. Prevalence of Batrachochytrium dendrobatidis in Xenopus collected in Africa (1871-2000) and in California (2001-2010). PLoS One. 2013 May 15;8(5):e63791. Wake, D. B. & Vredenburg, V. T. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proc. Natl. Acad. Sci. U. S. A. 105, 11466– 11473 (2008). Weldon, C., du Preez, L. & Vences, M. Lack of detection of the amphibian chytrid fungus (Bactrachochytrium dendrobatidis) in Madagascar in. A Conservation strategy for the amphibians of Madagascar (ed Andreone, F.) 95–106 (Monographie del Museo Regionale di Scienze Naturali di Torino, 2008). Whittaker, K. and Vance Vredenburg. An Overview of Chytridiomycosis May 17, 2011. Wombwell EL, Garner TW, Cunningham AA, Quest R, Pritchard S, Rowcliffe JM, Griffiths RA. Detection of Batrachochytrium dendrobatidis in Amphibians Imported into the UK for the Pet Trade. Ecohealth. 2016 Sep;13(3):456-466. 180 63. Yap TA, Gillespie L, Ellison S, Flechas SV, Koo MS, Martinez AE, Vredenburg VT. Invasion of the Fungal Pathogen Batrachochytrium dendrobatidis on California Islands. Ecohealth. 2016 Mar;13(1):145-50. 64. Xie GY, Olson DH, Blaustein AR. Projecting the Global Distribution of the Emerging Amphibian Fungal Pathogen, Batrachochytrium dendrobatidis, Based on IPCC Climate Futures. PLoS One. 2016 Aug 11;11(8):e0160746. doi: 10.1371/journal.pone.0160746. eCollection 2016. 65. Zampiglia M, Canestrelli D, Chiocchio A, Nascetti G. Geographic distribution of the chytrid pathogen Batrachochytrium dendrobatidis among mountain amphibians along the Italian peninsula. Dis Aquat Organ. 2013 Nov 25;107(1):61-8. 66. Zevallos S, Elías RK, Berenguel RA, Weaver TJ, Reading RP. Batrachochytrium dendrobatidis in Confiscated Telmatobius in Lima, Peru. J Wildl Dis. 2016 Oct;52(4):949-952. 67. Zhu W, Fan L, Soto-Azat C, Yan S, Gao X, Liu X, Wang S, Liu C, Yang X, Li Y. Filling a gap in the distribution of Batrachochytrium dendrobatidis: evidence in amphibians from northern China. Dis Aquat Organ. 2016 Mar 30;118(3):259-65. 181 7. Aspergillosis in wild animals Aspergillus is a filamentous, cosmopolitan and ubiquitous fungus found in nature. It is commonly isolated from soil, plant debris, and indoor air environment. While a teleomorphic state has been described only for some of the Aspergillus species, others are accepted to be mitosporic, without any known sexual spore production. The genus Aspergillus includes over 185 species. Around 20 species have so far been reported as causative agents of opportunistic infections in man and animals. Among these, Aspergillus fumigatus is the most commonly isolated species, followed by Aspergillus flavus and Aspergillus niger. Aspergillus clavatus, Aspergillus glaucus group, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor are among the other species less commonly isolated as opportunistic pathogens. Aspergilloses in animals Aspergillus infection is found worldwide and in almost all domestic animals and birds as well as in many wild species.   Aspergilloses is primarily a respiratory infection that may become generalized; however, tissue predilection varies among species. The most common forms are pulmonary infections in poultry and other birds; mycotic abortion in cattle; guttural pouch mycosis in horses; infections of the nasal and paranasal tissues, intervertebral sites, and kidneys of dogs; and sinonasal, sino-orbital, and pulmonary infection in domestic cats. The most common forms of wild animal aspergillosis are: o Pulmonary aspergillosis (Rewell et al., 1947, Frick et al., 1968, Jasmin et al., 1968, Joseph et al., 1986 Brian et al., 1986, de los Monteros et al., Jensenet al., 1989, Niki et al., 1991, Reidarson et al., 1998, 1999, Muntz, 1999,Vítovec et al.,1072,Woods et al., 1999) o Invasive aspergillosis (Severo et al., 1989, Ahmad et al., Chandrasekar et al., 2000, Jurczynski et al., 2012, 2014) o Systemic aspergillosis (Kim et al., 2015) o Pulmonary and hepatic aspergillosis(Vítovec et al.,1078) o Pulmonary aspergillosis and disseminated cutaneous involvement (Boyer et al., 2004) o Pulmonary aspergillosis and meningoencephalitis (Peden et al., 1985) o Mycotic encephalitis with the gastrointestinal involvement (Dagleish et al.,2006, Barley et al., 2007 and Dagleish et al.,2008) o Chronic Obstructive Pulmonary Disease (Delaney et al., 2013,Wu et al., 2016) o Pulmonary granulomas (Hall et al.,2011, Tamam and Refai, 2013) o Renal Oxalosis (Wyandk et al. , 1971) o Edematous and necrotic lesions (Tappe et al., 1984). o Fungal keratitis (Myers et al., 2009). o Otitis media and interna (Prah et al., 2011). 182 Aspergillus species reported in wild animals A. Aspergillus fumigatus 1. 2. 3. 4. 5. 6. 7. San Esteban chuckawallas Tappe et al. (1984) Garter snakes (Austwick and Keymer 1981), Japanese rat snakes (Austwick and Keymer 1981), Corn snakes (Girling 2002), Girling and Fraser (2009) American alligator Jasmin et al. (1968) Hares Thjostta, 1933; Hölphers & Lilleengen, 1947) Roe-deer Krembs, 1937; Burgisser, 1955, Vítovec et al. (1972), Vítovec and Fragner (1978) 8. Red deer (Cervus elaphus) Jensen et al. (1989) 9. Bison Rewell and Ainsworth (1947) 10. Guinea-pigs Ainsworth & Austwick (1955), Chandrasekar et al. (2000) 11. Dolphins Delaney et al. (2013) 12. Fisher Martes Pennanti Pinterest Williamson et al. (1963) 13. Blesbok (Damaliscus albifrons) Williamson et al. (1963) 14. Rats Niki et al. (1991) , Becker et al. (2006), Ahmad et al. (2014) 15. Green Iguanas Girling and Fraser (2009) 16. Bearded Dragon Girling and Fraser (2009) 17. Putty-nosed-monkey Jurczynski et al. (2012) 18. Siamese crocodile KIM et al. (2016) 19. Northern bottlenose whale Barley et al. (2007), Dagleish et al. (2008) 20. Harbour porpoise (Phocoena phocoena) Dagleish et al. (2008) B. Aspergillus amstelodami 1. Sitatunga 2. Galapagos testudo Williamson et al. (1963) Williamson et al. (1963) C. Aspergillus terreus 1. neonatal snow leopard Peden et al. (1985) Aspergillus candidus 1. Blind mole rats Tamam and Refai (2013) D. Aspergillus flavus 1. albino Wister rats Udeani (2013) E. Aspergillus niger 1. 2. 3. 4. Stag Burgeon (1929) Alpaca Muntz (1999) Western diamondback rattlesnake Boyer and Garner (2004) Garter snakes (Thamnophis sirtalis) Girling and Fraser (2009) 183 F. Aspergillus species 1. white-tailed deer (Odocoileus virgimiianus) Wyandk et al. (1971) 2. giant tortoise (Testudo gigantea Anderson and Ericksen (1968) 3. Alpaca (Lama pacos) Severo et al. (1989) 4. Dolphines Brian et al. (1986), Reidarson et al. (1998) 5. Geochelone gigante Anderson and Ericksen (1968) 6. American Bison de los Monteros et al. (1999) 7. Black rhinoceros Woods et al. (1999) 8. Gopher tortoise Myers et al., 2009) 9. Rhesus macaques Ahasan et al. (2013) 10. Samber deer Ahasan et al. (2013) 11. Nilgai Ahasan et al. (2013) 12. Stripped hyena Ahasan et al. (2013) 13. Gayal Ahasan et al. (2013) 14. Beisa oryx Ahasan et al. (2013) 15. Water buck Ahasan et al. (2013) 16. Greater kudu Ahasan et al. (2013) Guinea pig Hare - Wikipedia Albino Laboratory Wistar Rat Hub Sprague Dawley® Rat | Charles River Hardaker's Greater Red Musk Shrew . ARKive Egyptian blind mole rat 184 Alpaca - Lama pacos Rhesus macaque Fisher Martes Pennanti on Pinterest Wikipedia Male plains bison of Oklahoma Putty-nosed Monkey Archive Snow Leopard - ZooBorns Black Rhinoceros - Diceros bicornis -Tom Brakefield / Getty Images. Bos gaurus frontalis UniProt Boselaphus tragocamelus (Nilgai) UniProt Striped Hyena « Creepy Animals 185 Hunting in Bulgaria Roe Deer Dream of animals Stag Blesbok (Damaliscus albifrons) Sitatunga - Wikipedia White-tailed Deer New Hampshire Televi Red Deer (Cervus elaphus) Sambar Deer (Cervus unicolor) Flickr Beisa oryx photo - ARKive Waterbuck - Wikipedia Greater kudu - Wikiwand Phys.org Atlantic bottlenose dolphins 186 Harbour porpoise Northern Bottlenose Whale Crocodylus siamensis UniProt Female Killer whale (Orcinus orca) American Alligator (Alligator mississippiensis) Texas Parks San Esteban chuckwalla - Wikipedia Gopher tortoise - Wikiwand Galápagos tortoise - Wikiwand Giant Tortoise (testudo Gigantea,. Alamy Geochelone gigantea Wikimedia 187 Green Iguanas, Animals - National Geographic Valley Gartersnake - California Herps Bearded Dragon Reptiles Magazine 888 Reptiles Japanese Rat Snake Elaphe climacophora Sunshine Serpents Western diamondback rattlesnake - Wikipedia Description of Aspergillus species reported in wild animals Aspergillus amstelodami (L. Mangin) Thom & Church, The Aspergilli: 113 (1926) ≡Eurotium amstelodami L. Mangin, Ann. Sci. Nat., Bot.: 360 (1909) ≡Eurotium repens var. amstelodami (L. Mangin) Vuill., Bulletin de la Société Mycologique de France 36: 131 (1920) Colonies on Czapek's solution agar restricted, 2.5 to 3.0 cm. in diameter in 2 to 3 weeks at room temperature (24-26°C), plane or closely wrinkled, yellow to dull yellow-gray in color from cleistothecia admixed with sterile hyphae and developing conidial heads; reverse uncolored to yellowish, be-coming tawny in age. 188 A Colonies on MEA + 20% sucrose after two weeks; B ascomata, x 40; C conidia and conidiophore, x 920; D ascospores and conidia x2330; E portion of ascoma with asci x920 B.Flannigan, R Samson & JD Miller Aspergillus candidus Link, (1809) Synonyms: Aspergillus albus K. Wilh. Aspergillus okazakii Saito, (1907) Aspergillus albus var. thermophilus Nakaz., Takeda & Suematsu, (1932) Aspergillus tritici B.S. Mehrotra & M. Basu, (1976) Aspergillus triticus B.S. Mehrotra & M. Basu (1976) Morphology ( http://www.bcrc.firdi.org.tw/fungi/fungal Colony diameters on Czapek‘s Agar 1.5-1.7 cm in 14 days at 25°C, dense, plane; conidial heads radiate, white to ivory yellow; mycelium white; reverse white to cream color or warm buff to light ochraceous-buff, stipes 64-800 × 4.0-8.7 μm, hyaline, smooth; vesicles subglobose, globose, ellipsoidal or obovoid, 5.6-26.0 μm wide. Aspergilla biseriate, occasionally unseriate; metulae 4.4-11.1 × 2.1-3.8 μm, usually swollen, covering the whole surface of the vesicle; phialides 5.8-10.6 × 2.5-3.6 μm. Conidia subglobose or globose to ellipsoidal, smooth, 2.2-3.7 μm wide. Colony diameters on Malt Extract Agar 1.8-2.2 cm in 14 days at 25°C, dense, velutinous; conidial heads radiate, white to pale ivory; mycelium white; reverse ivory yellow to cream color. 189 Aspergillus flavus Link, 1809 Synonyms: Monilia flava (Link) Pers., (1822) Sterigmatocystis lutea Tiegh., (1877) Aspergillus flavus var. proliferans Anguli, Rajam, Thirum., Rangiah & Ramamurthi, (1965) Morphology A. flavus is known as a velvety, yellow to green or brown mould with a goldish to red-brown reverse. On Czapek dox agar, colonies are granular, flat, often with radial grooves, yellow at first but quickly becoming bright to dark yellow-green with age. Conidial heads are typically radiate, mostly 300-400 um in diameter, later splitting to form loose columns .The conidiophores are variable in length, rough, pitted and spiny. They may be either uniseriate or biseriate. They cover the entire vesicle, and phialides point out in all directions. Conidia are globose to subglobose, conspicuously echinulate, varying from 3.5 to 4.5 mm in diameter. Based on the characteristics of the sclerotia produced, A. flavus isolates can be divided into two phenotypic types. The S strain produces numerous small sclerotia (average diameter ,400 mm). The L strain produces fewer, larger sclerotia (Cotty, 1989 190 Aspergillus fumigatus Fresenius, 1863. Synonyms: Aspergillus fumigatus var. acolumnaris Rai, Agarwal & Tewari (1971) Aspergillus fumigatus var. albus Rai, Tewari & Agarwal (1974) Aspergillus fumigatus var. cellulosae Sartory, Sartory & Mey. (1935) Aspergillus fumigatus var. coeruleus Malchevsk. (1939) Aspergillus fumigatus var. ellipticus Raper & Fennell ( 1965) Aspergillus fumigatus var. fulviruber Rai, Tewari & Agarwal (1974) Aspergillus fumigatus var. fumigatus Fresen. (1863) Aspergillus fumigatus griseibrunneus var. Rai & Singh (1974) Aspergillus fumigatus var. helvolus Yuill (1937) Aspergillus fumigatus var. lunzinense Svilv. (1941) Aspergillus fumigatus var. minimus Sartory (1919) Aspergillus neoellipticus Kozak. (1989) Aspergillus phialoseptus Kwon-Chung (1975) Aspergillus bronchialis Blumentritt (1901) Aspergillus septatus Sartory & Sartory (1943) Aspergillus arvii Aho, Horie, Nishimura & Miyaji (1994) Description Colony diam (7 d): CYA25: 21-67 mm; MEA25: 25-69 mm; YES25: 48-74 mm; OA25: 34-62 mm, CYA37: 60-75 mm, CREA: poor growth, no or very weak acid production Colour: greyish turquoise or dark turquoise to dark green to dull green. 191 Reverse colour (CYA): creamy, yellow to orange. Colony texture: velutinous, st. floccose .Conidial head: columnar. Conidiation: abundant, rarely less abundant. Stipe: 50-350 × 3.5-10 μm. Vesicle diam, shape: 10-26 μm, pyriform to subclavate, sometimes subglobose, but rarely globose. Conidia length, shape, surface texture: 23.5(-6) μm, globose to ellipsoidal, smooth to finely rough Aspergillus niger van Tieghem 1867 Synonyms: Sterigmatocystis nigra (Tiegh.) Tieghem, (1877) Aspergillopsis nigra (Tiegh.) Speg., (1910) Rhopalocystis nigra (Tiegh.) Grove (1911) Aspergillus pyri W.H. English Aspergillus fuliginosus Peck, (1873) Aspergillus cinnamomeus E. Schiemann (1912) Aspergillus fuscus E. Schiemann (1912) Aspergillus niger var. altipes E. Schiemann, (1912) Aspergillus schiemanni Thom (1916) Morphology: On Czapek dox agar, colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads. Conidial heads are large (up to 3 mm x 15-20 um in diameter), globose, dark brown, becoming radiate and tending to split into several loose columns with age. Conidiophores are smooth-walled, hyaline or turning dark towards the vesicle. Conidial heads are biseriate with the phialides borne on brown, often septate metulae. Conidia are globose to subglobose (3.5-5.0 um in diameter), dark brown to black and rough-walled. 192 Aspergillus terreus Thom, (1918) Synonym: Aspergillus terrestris Colonies on potato dextrose agar at 25°C are beige to buff to cinnamon. Reverse is yellow and yellow soluble pigments are frequently present. Moderate to rapid growth rate. Colonies become finely granular with conidial production. Hyphae are septate and hyaline. Conidial heads are biseriate (containing metula that support phialides) and columnar (conidia form in long columns from the upper portion of the vesicle). Conidiophores are smooth-walled and hyaline, 70 to 300μm long, terminating in mostly globose vesicles. Conidia are small (2-2.5 μm), globose, and smooth. Globose, sessile, hyaline accessory conidia (2-6 μm) frequently produced on submerged hyphae. On Malt-Agar growth medium (MA) (initial pH 5) – Moderately fast growing colonies (reaching 78 cm in 21 days), velvet-like, white at first and then becoming cinnamon to brown-orange. The reverse is cream to slightly orangey. Emission of a yellowish pigment in the medium. The species slightly acidifies the medium (final pH 4). This fungus is readily distinguished from the other species of Aspergillus by its cinnamon-brown colony colouration and its production of aleurioconidia. Aspergillus terreus is a thermotolerant species since it has optimal growth in temperatures between 35–40 °C, and maximum growth within 45–48 °C. 193 Reports: Burgeon (1929) reported A.niger from nodular lesions in the lungs of a bull, a heifer and a stag in Indo-China. Scott (1930) gave details of nine cases of mycosis (aspergillosis?) and a further nine associated with tuberculosis in captive monkeys encountered at the London zoo. The lungs were the chief organs involved but miliary nodules were occasionally found throughout the viscera. Rewell and Ainsworth (1947) reported Aspergillus fumigatus in a case of fatal and apparently uncomplicated mycosis in a bison. Ainsworth & Austwick (1955) have recorded the disease in guinea-pigs. Recently, guinea-pigs are used for assessment of Aspergillus fumigatus burden in pulmonary tissue of antimycotics. Williamson et al. (1963) presented listings of mycotic infections occurring in vertebrates at the Chicago Zoological Park from September, 1954 to December, 1962. Most of the identifications were made by Dr. Tilden and Mrs. Getty from cultures of the fungi involved. Except for a few cases noted among the mammals, the findings were made from necropsy material. It is interesting to note the wide variety and numbers of birds with mycotic infections in contrast to the few findings in mammals 194 Anderson and Ericksen (1968) reported aspergillosis in a giant tortoise (Testudo gigantea, Geochelone gigante 195 Wyandk et al. (1971) reported an adult wild male white-tailed deer (Odocoileus virgimiianus) with severe skin lacerations of the left rear leg and rump. Other than exhaustion there were no other clinical problems and any previous history was unknown. The skin wounds were sutured and one intramuscular injection of penicillin was given. The deer was transferred to a holding facility and kept with two other deer. A diet of grain concentrate, timothy hay, maple and ash branches, and free choice 1: 10 phenothiazine-salt mixture was fed. Clinical improvement was rapid and within a week the deer appeared normal. Two days before death signs of depression, anorexia, and diarrhea appeared. Death occurred ten days after arrival at the laboratory. Necropsy Fimidimigs. Gross examination of the lungs revealed miliary 3 to 5 mm pale firm nodules, many of which had surrounding zones of hemorrhage or congestion. The significant microscopic lesions were limited to the lungs, brain, and kidneys. In the lungs there were disseminated focal necrotizing granulomas which contained dichotomous branching septate hyphae, neutrophils, and pyknotic nuclear debris . Within the lesions complete necrosis of alveolar walls occurred. There was alveolar hemorrhage, congestion, and edema in the adjacent lung tissue. In some areas hyphae were penetrating the pleura from contigu- ous necrotic foci resulting in small raised colonies of branching hyphae. Involvement of the central nervous system was principally hyphae within blood vessels and the adjacent. Focal lung lesions were streaked on blood agar plates and incubated at 37 C. A wet mount of the aerial mycelium which developed was prepared using lactophenol cotton blue stain. brain. Many were seen in the meninges underlying the basal ganglia. These hyphae were located free in the meningeal spaces and particularly in perivascular areas accompanied by infiltrates of neutrophils, eosinophils, and macrophages. Many vessels in the gray matter contained hyphae with smaller blood vessels showing only acute inflammatory cellular infiltrates and necrosis of the vascular walls. Hyphae with no inflammatory reaction were common in the adjacent brain parenchyma. The cerebral cortical gray matter had similar morphologic findings except that vascular necrosis and cellular infiltrates were more severe. The spinal cord was not examined. The septate hyphae with dichotomous branching found in the lung and brain were morphologically consistent with Aspergillus species. Septate bramic/iimig /ii’phiae surroumided bı’ necrotic imifla’nmatory cells imi the cemiter of a pulmnomiarv gramiulo,na. II & E X 480. Focal miecrotizing gramiu/ommia and adjacent pulmnonarv edemna amid comigestion. H & E X 12. 196 Mature conidial head of Aspergillus fumigatus. Note tue parallel rows of conidia w/iicbi arise fromn a simigle FOW of sterigmnata which are on tile termnimial portiomi of a flask-shaped vesiclc. Lactophiemiol cottomi blue X 480. Vítovec et al. (1972) described 2 cases of pulmonary aspergillosis in roe-deer. In one case there was a scattered necrotizing hemorrhagic aspergillic bronchopneumonia complicated by Listeria infection. In the second case, there was a solitary aspergilloma in the left upper lobe, similar to pulmonary aspergillomas in man. In the first case cultures yielded Aspergillus fumigatus FRESENIUS and Listeria monocytogenes, in the second case only Aspergillus fumigates FRESENIUS was found. 197 Vítovec and Fragner (1978) described 4 solitary aspergillomas in a deer. Two were found in the lungs and two in the liver. Morphologically, they were bulky, local lesions filled with homogenous, fragile, necrotic tissue of conspicuously green color. Pulmonary aspergillomas communicated with the conducting bronchus having affected fibers. The necrotic tissue of aspergillomas was interwoven by diffuse abundant fibers of Aspergillus fumigatus. Peden et al. (1985) reported mycotic pneumonia and meningitis due to Aspergillus terreus in a neonatal snow leopard. 198 Brian et al. (1986) described and discussed the clinical history, laboratory data, and gross and histological findings in three cases of fatal pulmonary aspergillosis in three species of dolphins. Jensen et al. (1989) diagnosed during 1988, pulmonary mycosis in four of 116 farmed deer examined on suspicion of tuberculosis. The histopathology showed allergic bronchopulmonary mycosis in a red deer (Cervus elaphus) and the agent was identified as a zygomycete, probably Absidia corymbifera, by immunofluorescence staining. Three fallow deer (Dama dama) had invasive necrotizing mycotic pneumonia and progressive exudative mycotic alveolitis caused by Aspergillus fumigatus. In the red deer, weakness due to paratuberculosis had probably promoted the mycotic infection. The three fallow deer were bred on another farm, where predisposing factors included mouldy straw and incorrect management. 199 Severo et al. (1989) described an invasive form of aspergillosis in an alpaca (Lama pacos), with dissemination causing small abscesses and multifocal areas of necrosis in the lung, heart, spleen and kidneys. Histological sections showed hyphae morphologically compatible with an Aspergillus species. Direct immunofluorescent testing confirmed the diagnosis of aspergillosis. Niki et al. (1991) treated male Sprague-Dawley rats with cortisone acetate and fed a low-protein diet for 3 weeks. At the end of week 2, animals were infected intratracheally with 105 conidia of Aspergilus fumigatus H11-20. Despite discontinuation of steroids and the low-protein diet 1 week after the infection, 94% of controls died of invasive pulmonary aspergillosis within 3 weeks postinfection. When rats were treated with a single dose of 1.6 mg of aerosolized amphotericin B per kg of body weight 48 1I prior to the infection, mortality was reduced to 11% within 3 weeks postinfection. Despite apparent good health and rapid weight gain, all survivors showed multiple lesions in histopathological sections of the lungs, and 103 to 104 CFU of aspergilli was recovered from cultures of their lungs. With discontinuation of immunosuppression, the infection was slowly cleared; however, when cortisone acetate was restarted during week 5, reactivation of progressive invasive pulmonary aspergillosis was observed. On the basis of these results, it was conclude that a single low dose of aerosolized amphotericin B prophylaxis was effective in preventing an exogenous aspergillus infection of the lung. Additional therapy was needed to prevent recurrent infection caused by endogenous aspergilli when immunosuppression was resumed. Reidarson et al. (1998) reported a 4-yr-old male bottlenose dolphin (Tursiops truncatus) that developed an Aspergillus fumigatus pneumonia. Fungal elements were identified by cytology and microbiology from endoscopic bronchoalveolar lavage and brushings of a raised yellow endobronchial lesion. The results of qualitative immunodiffusion serology, a technique that identifies specific circulating antibodies to Aspergillus fumigatus, were suggestive of an active infection. The dolphin was treated with itraconazole for over 2 yr, which resulted in remission of clinical signs. Pneumonia caused by Aspergillus sp. accounts for the large majority of pulmonary mycoses in marine mammals. Bronchoscopy facilitated an early definitive diagnosis, accurate treatment, and remission. de los Monteros et al. (1999) diagnosed concomitant nasal zygomycosis and pulmonary aspergillosis in a 3-mo-old female American bison calf (Bison bison) in Pennsylvania (USA). Etiologic diagnosis was made by immunohistochemistry using a panel of monoclonal antibodies and heterologously absorbed polyclonal antibodies. In the lungs fungal infection was accompanied by hemorrhage, fibrin exudation, and infiltration with neutrophils. Fungi were observed to penetrate apparently normal epithelial lining of the nasal turbinates, and there was hemorrhage, edema, and invasion of blood vessels in the submucosa. In vessels fungi were typically associated with thrombosis. The calf may have been infected due to a high level of exposure to mouldy feed and litter in the environment in combination with a collapse of it's natural defence mechanisms. Muntz (1999) presented an aging lactating alpaca in sternal recumbency. Although bright and alert, she did not respond to symptomatic treatment and was euthanized 5 weeks after initial presentation. Gross postmortem examination revealed purulent material in the pulmonary airways. Histologic examination of the lungs revealed an 200 extensive pyogranulomatous pneumonia with bronchiectasis. There were abundant fungal hyphae and high numbers of associated oxalate crystals, which were presumed to have been produced by the fungus. Low numbers of yeast cells were also present. Microbiological culture of tissues on horse blood agar and Sabouraud's agar identified the fungus to be Aspergillus niger. There was also moderate growth of Candida albicans. Calcium oxalate crystals in cytologic and histologic preparations can suggest an underlying Aspergillus infection. This is the first reported veterinary case of pneumonia due to Aspergillus niger infection and the associated production of oxalate crystals. Woods et al. (1999) treated captive black rhinoceros (Diceros bicornis) with a hoof abscess with long-term antibiotic therapy. After 9 months of treatment, there was rapid deterioration, marked weight loss and reluctance to stand. Profuse, bilateral epistaxis developed accompanied by collapse and the animal was euthanased. Necropsy revealed pulmonary aspergillosis with concurrent Pseudomonas aeruginosa infection. Though a well-recognized disease of black rhinoceros, fungal pneumonia has not been reported in this species in Australia. The cost and efficacy of treatment have been questioned, however, prophylactic antifungal drug administration will be considered in any further cases of chronic, debilitating illness in black rhinoceros at Western Plains Zoo. Chandrasekar et al. (2000) compared the efficacies of amphotericin B and voriconazole against invasive pulmonary aspergillosis in a guinea-pig model. A susceptible isolate of Aspergillus fumigatus was used to produce the infection. Voriconazole-treated animals had significantly better survival and decreased fungal burden in the lungs as compared with controls. Although no statistical difference was seen between the efficacies of voriconazole and amphotericin B, a trend favouring voriconazole was noted. Thus, voriconazole, with its cidal activity, may be an attractive alternative to potentially toxic amphotericin B in the treatment of invasive pulmonary aspergillosis. Boyer and Garner (2004) reported a western diamondback rattlesnake (Crotalus atrox) with dermatitis. Biopsies revealed severe granulomatous dermatitis with intralesional fungal elements, probably the result of visceral fungal infection. The snake died about 4 wk after presentation, while on ketoconazole therapy. Multiple white fungal granulomas were found in the lung at necropsy. Histopathology detected granulomatous and nonsuppurative myocarditis and bronchointerstitial pneumonia, both with intralesional fungal elements. Aspergillus niger was cultured from the pulmonary granuloma. A baby chicken died in this snake‘s hide box 10 days prior to 201 presentation, and was overgrown with fungus when the owner found it. It is suspected that this was the source of the pulmonary fungal granuloma. The fungus probably spread hematogenously to the skin. Becker et al. (2006) examined changes in pulmonary and general physiology during aspergillosis in an animal model. In a model of fatal left-sided IPA, 19 persistently neutropenic rats were monitored for clinical signs including body temperature, body weight and respiratory distress. A separate group of nine rats with IPA was used for measurements of arterial blood pressure, arterial O2 and CO2 pressure, lung compliance and surfactant function. Body temperature and body weight decreased, whereas respiratory distress increased during progression of the disease. Compared to uninfected controls, in rats with IPA arterial blood pressure and lung compliance were significantly lower, and left lung minimal surface tension was significantly higher. Right lung surfactant function was not affected. Arterial O2 and CO2 pressures were not different between rats with IPA and uninfected controls. Infection with Aspergillus fumigatus in neutropenic rats resulted in hypothermia, body weight loss and respiratory distress. Loss of left lung function was probably compensated by the uninfected right lung, even in a late stage of the disease. Circulatory failure was a major feature in the terminal phase of the infection. Dagleish et al. (2006) reported intracranial granuloma caused by Aspergillus fumigates in a harbor propoise. 202 Barley et al. (2007) reported a case of encephalitis in a northern bottlenose whale caused by Aspergillus fumigates in a letter to the editor of Vet. Rec. Dagleish et al. (2008) reported fatal mycotic encephalitis caused by Aspergillus fumigatus in a northern bottlenose whale (Hyperoodon ampullatus). 203 Girling and Fraser (2009) described the successful treatment of seven cases of cutaneous and deep subcutaneous aspergillosis infections with itraconazole at metabolically scaled oral doses specifically for reptiles. Details of the reptile species, presentation and Aspergillus species isolated are shown the following Table . In each case, a diagnosis was made by histopathology after biopsy of affected tissue and staining with periodic acid-Schiff stain to demonstrate fungal hyphae in the centre of the granulomas. In addition, the species of Aspergillus were identified after culture of affected tissue samples collected aseptically and inoculated on to Sabouraud‘s media, which was incubated at 28°C for four to seven days. The biopsies were performed with the animals under general anaesthesia; they were induced with 8 to 10 mg/kg propofol, administered intravenously, intubated and maintained with 1·5 to 2 per cent isoflurane in 100 per cent oxygen, using intermittent positive pres- 204 Myers et al. (2009) reported a free-ranging gopher tortoise (Gopherus polyphemus) with trauma and blindness. Fibrinous exudate obscured visualization of the globes. This exudative crust extended from the conjunctival fornices through the palpebral fissure and was manually removed. Ophthalmic examination revealed bilateral corneal ulcerations and scarring and phthisis bulbi of the left globe. Histology of the crust revealed a necrotic conjunctivitis with intralesional fungal hyphae. Culture of the corneal ulcer of the left eye isolated moderate growth of a mixed fungal flora consisting of Curvularia sp. and Aspergillus sp. Miconazole ophthalmic solution was administered and the ulcers in both eyes healed, but corneal edema continued. After 2 mo of treatment with miconazole, tramadol, acetylcysteine, hypertonic saline ointment, artificial tears, and hypertonic saline flushes, the right eye was normal with only a small scar. The left eye remained phthisical. Prahl et al. (2011) reported a case of severe mycotic otitis media in a cetacean, a juvenile female harbour porpoise (Phocoena phocoena) from British waters that stranded alive. Gross examinations were followed by histological and microbiological investigations of the auditory apparatus. Both tympanic cavities and periotic sinuses displayed copious greenish-yellow purulent and caseous material. Severe fungal infestation by Aspergillus terreus was documented in the otic region but not in any other site of the body. Adjacent to the promontorium, massive accumulation of fibrinous secretion and infiltration of clusters of inflammatory cells were present. Newly formed cysts and vessels replaced the round window membrane location, reminiscent of granulation tissue. Inflammatory cells and a severe fibrin net were noted within the perilymphatic spaces of scala tympani and scala vestibuli, indicative of an acute fibrinous otitis. Inflammatory reactions have probably been caused by this fungal organism. The basilar membrane was solely covered by a simple cuboidal epithelium. Complete absence of sensory cells of the Organ of Corti characterised a further severe phenomenon, which possibly led to the animal's poor nutritional status and stranding. Yamauchi et al. (2011) reported a male cynomolgus macaque at the age of 3 years and 11 months suffered sudden cardiac arrest during a surgical operation. This animal had been clinically asymptomatic for 6 months from the acclimatization period to death. At necropsy, a white mass approximately 5 cm in diameter was found at the base of the heart. Histopathologically, the mass consisted of a granuloma with a number of multinucleated giant cells and multiple necrotic foci. Fungal hyphae characterized by parallel cell walls, distinct septa, and branching were observed in the lesion. The granuloma extended into the thoracic lymph nodes and the subepicardium of the left atrium, compressed the bronchioli, and was separated from the pulmonary parenchyma by a thick fibrous layer. The hyphal morphology and results of polymerase chain reaction assays demonstrated that the pathogen was Aspergillus sp. Abdo et al. (2012) described hematological findings in a female killer whale (Orcinus orca) undergoing rehabilitation after sudden severe anorexia revealed continuing increases in serum lactate dehydrogenase and aspartate aminotransferase activities as well as fibrinogen concentration. Serologic evidence of herpesvirus infection and skin vesicles 2 weeks into the treatment regimen of antibiotics and corticosteroids. The whale showed signs of improvement after treatment with anti-herpesvirus drugs, but sudden severe anorexia reappeared, along with marked elevation of fibrinogen concentration that continued until the death. Postmortem examination revealed multiple light tan foci of necrosis in the skeletal and cardiac muscles, and lung 205 consolidation. Microscopic findings indicated disseminated fungal granulomas in the skeletal and cardiac muscles, as well as myocarditis, mycotic embolic thromboarteritis of cardiac blood vessels, and bronchopneumonia with numerous typical Aspergilluslike fungi. Mucor-like structures in granulomas in the heart and skeletal muscle and Aspergillus-like fungi in the lungs were identified using periodic acid-Schiff, Gomori methenamine silver stain, and immunohistochemistry. The present case involved dual infection with Mucor and Aspergillus species in a killer whale with concurrent herpesvirus Jurczynski et al. (2012) reported an 18-year-old captive female putty-nosedmonkey (Cercopithecus nictitans) with a history of long-term infertility and hyperglucocorticism. The animal was euthanized because of perforating thoracic trauma induced by group members and subsequent development of neurological signs. Complete necropsy and histopathological examination of formalin-fixed tissue samples was carried out. The monkey showed invasive pulmonary and cerebral infection with Aspergillus fumigatus together with adrenocortical neoplasia and signs of Cushing‘s syndrome, such as alopecia with atrophic skin changes, evidence for diabetes mellitus and marked immunosuppression. (A, B) Necropsy findings of the female putty-nosed monkey (Cercopithecus nictitans) showing marked invasive aspergillosis: (A) Lung with right-sided severe compression atelectasis and marked diffuse fibrino-suppurative pleuropnemonia (asterisk). Left lung shows marked congestion and multifocal abscesses of different size (arrows). (B) Brain with diffuse meningeal hyperemia, multifocal suppurative meningitis, and multiple necro-hemorrhagic foci on the cerebral and cerebellar cortex (arrows). (A–D) Representative histopathological findings of the female putty-nosed monkey (Cercopithecus nictitans) with marked invasive aspergillosis and hyperglucocorticism: (A) Lung parenchyma showing extensive necrosis admixed with abundant fungal hyphae surrounded by numerous degenerate inflammatory cells, intraalveolar edema and fibrin (HE stain, scale bar 200 μm). Inlet: morphologic detail of Aspergillus fumigatushyphae [periodic acid-Schiff (PAS)]. (B) Brain section with multifocal thrombosis and necrotizing vasculitis throughout the neuropil associated with moderate hemorrhage and gliosis (HE stain, scale bar 200 μm). Inlet: Microthrombus with angioinvasive fungal hyphae (Grocott silver stain, scale bar 20 μm). (C) Skin with severe dermal and epidermal thinning, follicular atrophy, and comedone formation (arrow) indicative for hyperglucocorticism (HE stain, scale 206 bar 200 μm). (D) Spleen with scarce, inactive lymphoid tissue indicating immunosuppression (HE stain, scale bar 1 mm). Delaney et al. (2013) reported 4 captive adult bottlenose dolphins succumbed to chronic, progressive respiratory disease with atypical recurrent upper respiratory signs. All dolphins had severe, segmental to circumferential fibrosing tracheitis that decreased luminal diameter. Histologically, tracheal cartilage, submucosa, and mucosa were distorted and replaced by extensive fibrosis and pyogranulomatous inflammation centered on fungal hyphae. In 3 of 4 cases, hyphae were morphologically compatible with Aspergillus spp and confirmed by culture in 2 cases. Amplification of fungal DNA from tracheal tissue was successful in one case, and sequences had approximately 98% homology to Aspergillus fumigatus. The remaining case had fungi compatible with zygomycetes; however, culture and polymerase chain reaction were unsuccessful. Lesions were evaluated immunohistochemically using antibodies specific to Aspergillus spp. Aspergillus-like hyphae labeled positively, while presumed zygomycetes did not. These cases represent a novel manifestation of respiratory mycoses in bottlenose dolphins. Ahasan et al. (2013) conducted a study to investigate aspergillosis in animals at Dhaka Zoo to ascertain animal health, welfare and public health safety standard. One hundred and two necropsied tissue samples preserved in 10% neutral buffered formalin at necropsy from 36 animals of 25 different species were collected from Dhaka Zoo. Twenty five out of 36 study animals were suffering from granulomatous diseases. Among them 13 animals were suffering from Aspergillosis. Nodular lesions from necropsy findings, granulomatous reactions along with fungal spores and characteristic radiating club on histopathology; dichotomously branching septate hyphae and mycelial conidiophore on special staining revealed Aspergillosis in 13 animals of nine species that included four rhesus macaques (Macaca mulatta), two samber deer (Cervus unicolor) and one of each species were nilgai (Boselaphus tragocamelus), horse (Equus caballus), stripped hyena (Hyena hyena), gayal (Bos frontalis), beisa oryx (Oryx beisa beisa), water buck (Kobus L. leche) and greater kudu (Tragelaphus strepsiceros). Present study provided evidence of existing aspergillosis and similar long standing zoonotic diseases in majority of rest of the animals with health risk that shades health safety standard at Dhaka Zoo. 207 Tamam and Refai (2013) established the cause of death in seven blind mole rats that died naturally in the wild. All the animals had large pulmonary lesions that on microscopic, microbiological, and ultrastructural analysis were shown to contain mixed infections with Alternaria alternata and Aspergillus candidus. Some of the lesions were circumscribed with fibroblastic proliferation and inflammatory response. The lungs had haemorrhage and chronic inflammatory response to the organisms, which is likely to have been the cause of death. This is the first report of some pathogenic organisms resulting in death of the blind mole rat. 208 Macroscopic features. (A) Thoracic cavity of Spalax leucodon showing large, tumour-like lesions replacing the apical left lung lobe and large, firm brown lesions in the caudal and right lobes (yellow arrows). (B) Thoracic cavity containing brown, consolidating focal lesions in the centre of the left lung lobe, emphysematous change (red arrows), and red hepatisation of the lower right lobe (violet arrow). Microscopic and ultrastructural features. (A) Histological features of the lesions demonstrating fungal organisms within foamy macrophages (H&E x40). (B) Alveolar macrophages engulfing hemosiderin pigment (H&E x40). (C) Aspergilloma wall showing fibroblastic proliferation with slightly pleomorphic nuclei, vesicular chromatin, and grooved and folded nuclei (H&E x100). (D) TEM photomicrograph demonstrating fungal hyphae being engulfed by a foamy macrophage|(X1000). 209 Colonies (1) and microscopic morphology of Aspergillus candidus (white) possessing white, globose conidial heads producing thin-walled conidia measuring 2.5–3.5 μm in diameter (2), and Alternaria alternate (black colonies) showing branched acropetal chains and multicelled, obclavate to obpyriform conidia with short conical beaks (3-8). Udeani (2013) determined the association between antiretroviral (ARV) drugs and Aspergillus flavus in experimental rats. Inbred albino Wister rats, aged18-21weeks; weighing 200- 250g were used. The experiment was carried out in two models, excluding antifungal drugs. In model 1; the rats were infected with Aspergillus flavus and treated with antiretroviral drugs; grouped into four of six rats each, based on their weights. Group A: Neat control while Groups B-D were administered with varying concentrations of Nevirapine, Zidovudine and Lamivudine, Zidovudine, Lamivudine and Nevirapine. In model 2, the animals were immunosuppressed with cyclophosphamide and then administered with ARV and A. flavus. In both models; pre and post inoculation blood samples were collected to assay for liver enzymes and the liver was harvested for histological assessment and recovery of A. flavus. Results: In model1, pre-inoculation liver enzyme values showed no statistical increase between control group and experimental groups (P> 0.05) while the post-inoculation liver enzyme values were statistically significant. Ahmad et al. (2014) evaluated an experimental inhalational model of IPA in rats and the efficacy of three biomarkers, namely β-D-glucan (BDG), a panfungal marker, galactomannan (GM), a genus-specific marker, and A. fumigatus DNA, a speciesspecific marker in serum and bronchoalveolar lavage (BAL) specimens at different time points postinfection for early diagnosis of IPA. BDG and GM were detected by using commercial Fungitell and Platelia Aspergillus EIA kits, respectively. A. fumigatus DNA was detected by developing a sensitive, single-step PCR assay. IPA was successfully developed in immunosuppressed rats and all animals until 5 days post-infection were positive for A. fumigatus by culture and KOH-calcofluor microscopy also showed A. fumigatus in 19 of 24 (79%) lung tissue samples. Fourteen of 30 (47%) and 27 of 30 (90%) serum and BAL specimens, respectively, were positive for all three biomarkers with 100% specificity (none of sera or BAL specimens of 12 control rats was positive for biomarkers). The data show that BAL is a superior specimen than serum and combined detection of BDG, GM and A. fumigatus DNA provided a sensitive diagnosis of IPA in an experimental animal model. Moreover, combined detection of GM and DNA in BAL and detection of 210 either GM or DNA in serum was also positive in 27 of 30 (90%) animals. For economic reasons and considering that the positive predictive value of BDG is low, the detection of GM and/or DNA in serum and BAL samples has the potential to serve as an integral component of the diagnostic-driven strategy in high-risk patients suspected for IPA KOH-calcoflour mounts and histopathology of lung tissue sections.The KOH-calcoflour mounts (a and b), and histopathology (c and d) of lung tissue sections obtained from 4 immunosuppressed rats sacrificed on Day 3 postinfection showing abundant growth of A. fumigatus. Positivity for the detection of 1,3 β-D glucan (BDG), galactomannan (GM) and A. fumigatus DNA in serum and bronchoalveolar lavage (BAL) samples and culture positivity in BAL of experimentally infected rats sacrificed on different days postinfection. 211 Tamam and Refai (2014) undertook a detailed and systematic histopathological, parasitological, and ultrastructural examination of thirty-five Greater Red Musk Shrews that had died mainly of natural causes in Egypt between March 2008 and April 2014. Parasitic enteritis caused by Hilmylepis spp. was observed most frequently (n = 20), followed by parasitic pneumonia (n = 5), gastric giardiasis (n = 5), intestinal coccidiosis (n = 4), testicular degeneration (n = 4), intestinal amebiasis (n = 3), mycotic pneumonia (n = 3), Streptococcus pneumonia (n = 3), and blood protozoal infection (n = 2). There was a causative agent in 20/32 (63%) cases and several etiological agents 12/32 (37%) cases. In three cases, the lungs were invaded with thick, branched, PAS-positive -hyphae of Aspergillus fumigatus. and surrounded by massive tissue damage and neutrophils Lung invaded with Aspergillus fumigatus . and appearing as thick, branching hyphae surrounded by neutrophils and tissue debris (H&E x400). (D) Lung invaded with Aspergillus fumigatus . and appearing as thick, branching hyphae (PAS stain; red arrow) and associated with Gram-positive bacteria (blue arrow) (background H&E x10). Seyedmousavi et al. (2015) stated that the importance of aspergillosis in humans and various animal species has increased over the last decades. Aspergillus species are found worldwide in humans and in almost all domestic animals and birds as well as in many wild species, causing a wide range of diseases from localized infections to fatal disseminated diseases, as well as allergic responses to inhaled conidia. Some prevalent forms of animal aspergillosis are invasive fatal infections in sea fan corals, stonebrood mummification in honey bees, pulmonary and air sac infection in birds, mycotic abortion and mammary gland infections in cattle, guttural pouch mycoses in horses, sinonasal infections in dogs and cats, and invasive pulmonary and cerebral infections in marine mammals and nonhuman primates. This article represented a comprehensive overview of the most common infections reported by Aspergillus species and the Aspergillus species are saprophytic filamentous fungi 212 that are commonly found in soil, where they thrive as saprophytes, with an occasional potential to infect living hosts including plants, insects, birds, and mammals. Muscle and heart of a killer whale ( Orcinus orca) with disseminated mycoses caused by dual infection with Mucor and Aspergillus species. Adopted from Abdo et al. (REF 50) with permission of the publisher. Top lef t: Multifocal well-demarcated , light tan, and soft foci were present in various muscles in the caudal thorax and abdominal areas. Bottom left: Two tan nodules (arrow), 1 cm × 4 cm, bulged slightly from the surface in the left ventricle 7 cm from the apex. Top right: Frequent mycotic embolisms in small blood vessels. Hematoxylin and eosin stain. Scale bar = 40 μ m. Bottom right: Necrotizing thromboarteritis due to fungal invasion of the arterial wall. Periodic acid–Schiff reaction. Scale bar = 20 μ m. Brain of a northern bottlenose whale ( Hyperoodon ampullatus) with encephalitis due to Aspergillus fumigatus. Adopted from Dagleish et al. (REF 240) with permission of the publisher. Top: There are roughly circular, poorly circumscribed areas of hemorrhage (black) in coronal sections of the cerebrum (top left) and midbrain (bottom), both anterior views, and a sagittal section of the cerebellar vermis (top right). Scale bar = 1 cm. Bottom: Histological preparation of the cerebral cortex: Thrombosed blood vessel and surrounding thick cuff composed of poly- morphonuclear neutrophils, hemorrhage and fibrin (arrow) and fungal hyphae in the neuropil (arrowhead) are visible. Haematoxylin and eosin. Scale bar = 100 μ m. Inset: Histological preparation of cerebral cortex showing the presence of septate dichotomously branching fungal hyphae, typical of Aspergillus fumigatus, in the neuropil. GrocottGomori methenamine silver. Scale bar = 50 μ m Kim et al. (2016) reported a 4-year-old female Siamese crocodile (Crocodylus siamensis) housed at a zoo died without any prior clinical signs. During necropsy, numerous scattered, well-demarcated, yellowish- white, firm nodules were observed throughout the liver and lungs. Microscopic examination with periodic acid-Schiff staining revealed granulomatous inflammation in the liver and lungs. Liver granulomas were characterized by the presence of a connective tissue barrier and hyphae, and the centers of the granulomas showed signs of necrosis. Lung samples showed characteristics similar to those observed in the liver samples. The fungus was identified as Aspergillus fumigatus based on its appearance on Sabouraud dextrose agar, microscopic examination with lactophenol cotton blue staining and genetic sequencing. 213 Gross appearance of the lungs and liver in the diseased Siamese crocodile at necropsy and samples of the lungs and liver stained with periodic acid-Schiff. (A) Multiple scattered nodules in the lung lobes. (B) Many white nodules (approximately 1 cm in diameter) in the parenchyma of the dissected liver. (C) Well-demarcated granuloma in the liver (×40). Bar = 200 µm. (D) Hyphae and 198 inflammatory cells in the liver parenchyma (×400). Bar = 50 µm. Species identification of Aspergillus fumigatus cultured from the lesions was based on the appearance of colonies, microscopic observation and nucleotide sequencing. (A) Colonies cultured on Sabouraud dextrose agar appear velvet-like and bluish-green. (B) Dome-shaped colonies stained with lactophenol cotton blue show a columnar conidial head and phialidae on the upper half, which are typical of A. fumigatus (×200). (C) Electrophoresis gel showing the 492-bp amplicon of an A. fumigatus β-tubulin gene fragment. Lanes: M, 100-bp DNA ladder; 1, A. fumigatus in this study; 2, 207 a negative sample. Wu et al. (2016) used cigarette smoke exposure to generate COPD rat model. colonyforming units (CFU) count assessment and phagocytosis were applied to evaluate the defense function of COPD rats against Aspergillus challenge. ELISA, western blotting, and GST-Rac1 pull-down assays were conducted to determine the expressions of cytokines and TLR2-associated signaling pathway. Our data showed that Aspergillus burdens increased, phagocytosis of Aspergillus as well as the expressions of inflammatory cytokines from alveolar macrophages (AMs) were impaired in COPD rats compared with normal rats. Though TLR2 signaling-related proteins were induced in response to the stimulation of Aspergillus or Pam3csk4 (TLR2 agonist), the activation of TLR2-associated signaling pathway was apparently 214 interfered in rats with COPD, compared to that in normal rats. Taken together, our study demonstrated that COPD caused the deficiency of AMs function and impaired the activation of TLR2/PI3K/Rac 1 signaling pathway, leading to invasion of Aspergillus infection, which also provides a future basis for the infection control in COPD patients. BCS staining of AMs infected with biotinylated. Afumigatus conidia. Differential staining of extra and intracellular conidia. a Extracellular conidia stained in red using Cy3-labeled streptavidin; b calcofluor white staining (blue) of extra and intracellular conidia; c macrophages visualized by Concanavalin AFITC are shown in green (Concanavalin A-FITC also recognizes the surface of extracellular conidia.) d an overlay of all micrographs. Positions of extracellular conidia are indicated by arrows Cytokine levels in supernatant before and after conidia stimulation. The analysis of variance with factorial design showed differences between control and COPD groups. There was interaction between grouping and conidia 215 stimulation, suggesting that the alteration trend of TNF-α, MIP-2, IL-1β, and IL-10 in the culture supernatant after stimulation was different between the groups. In control group, cytokine levels increased more significantly. Number sign signifies the analysis of single factor effect which showed that before conidia stimulation, levels of TNF-α and MIP-2 in COPD group were significantly higher than those in control group (P < 0.001). Asterisk signifies after spore stimulation, although cytokine concentrations in both groups increased significantly, the final cytokine levels were significantly higher in control group than in COPD group (P < 0.001). Expressions of TLR2 on AMs after intratracheal instillation of Aspergillus spores in each group. The analysis of variance with factorial design showed significant difference between normal and COPD groups (P = 0.049), and there were significant differences between different day points (P < 0.001). There was interaction between grouping and day points, suggesting that the alteration trends of TLR2 receptor cells in two groups were different. A. fumigatus stimulates TLR2/Akt activity through a Rac1-dependent mechanism. a Representative image of the western blot results, which showed that the TLR2 protein expression level was downregulated in the COPD rats comparing to the control after A. fumigatus infected. These results have also been gotten from the protein expression levels of p-AKT and GTP-Rac1. There were no significant differences found in the AKT and totalRac1 protein expression levels between the COPD and control groups. b–d Statistical analysis of the western blot results (*P < 0.05, #P < 0.01). References: 1. Abdo W, Kawachi T, Sakai H, Fukushi H, Kano R, Shibahara T, Shirouzu H, Kakizoe Y, Tuji H, Yanai T. Disseminated mycosis in a killer whale (Orcinus orca). J Vet Diagn Invest. 2012 Jan;24(1):211-8. doi: 10.1177/1040638711416969. Epub 2011 Oct 10. 2. Ahasan S. A, E. H. Chowdhury , M. M. Rahman and M. A. Rahman J. Bangladesh Agril. Univ. 11(2): 265–270, 2013 3. Ahmad S, Al-Shaikh AA, Khan Z (2014) Development of a Novel Inhalational Model of Invasive Pulmonary Aspergillosis in Rats and Comparative Evaluation of 216 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Three Biomarkers for Its Diagnosis. PLoS ONE 9(6): e100524. doi:10.1371/journal.pone.0100524 Anderson, S., and E. Ericksen. 1968. Aspergillose bei einer elephanten-schildkrote (Testudo gigantean elephantine). Proc. Tenth Intl Symp Erkr Zootiere (Salzberg): Adamademie Verlag, Berlin, Democratic Republic of Germany: 65. Barley J, Foster G, Reid B, Dagleish M, Howie F. Encephalitis in a northern bottlenose whale. Vet Rec. 2007 Mar 31;160(13):452. Becker , Martin J. , Siem De Marie, Marcel H. A. M. Fens Jack J. Haitsma Henri A. Verbrugh Burkhard Lachmann Irma A. J. M. Bakker-Woudenberg. Pathophysiology of unilateral pulmonary aspergillosis in an experimental rat model Med Mycol (2006) 44 (2): 133-139 Boyer , Thomas H., and Michael M. Garner. 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Joseph BE, Cornell LH, Simpson JG, Migaki G, Griner L. 1986. Pulmonary aspergillosis in three species of dolphin. Zoo Biol 5:301–308. Jasmin A. M., Carroll J. M., Baucom J. N. 1968. Pulmonary aspergillosis of the American alligator (Alligator mississippiensis). Am. J. Vet. Clin. Path. 2: 93. Jurczynski K., Gruber-Dujardin E., Widmer D., Kaup F. J., Mätz-Rensing K. 2012. Invasive aspergillosis in a putty-nosed monkey (Cercopithecus nictitans) with adrenocortical Cushing‘s syndrome. J. Med. Primatol. 41: 172–175 KIM K-T, LEE S-H, KWAK D. Sudden death of a Siamese crocodile (Crocodylus siamensis) due to systemic aspergillosis. The Journal of Veterinary Medical Science. 2016;78(11):1723-1726. doi:10.1292/jvms.16-0260. Debbie A. Myers, , Ramiro Isaza, , Gil Ben-Shlomo , Jeffrey Abbott,, and Caryn E. Plummer, Fungal Keratitis in a Gopher Tortoise (Gopherus polyphemus). Journal of Zoo and Wildlife Medicine 40(3):579-582. 2009 doi: http://dx.doi.org/10.1638/20080219.1 217 23. Muntz FH. Oxalate-producing pulmonary aspergillosis in an alpaca. Vet Pathol. 1999 Nov;36(6):631-2. 24. Niki , Yoshihito, Edward M. Bernard, Fitzroy F. Edwards, Heinz J. Schmitt, Bessie Yu, And Donald Armstrong. Model of Recurrent Pulmonary Aspergillosis in Rats. JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1317-1322 25. Peden W. M., Richard J. L., Trampel D. W., Brannian R. E. 1985. Mycotic pneumonia and meningoencephalitis due to Aspergillus terreus in a neonatal snow leopard (Panthera uncia). J. Wildl. Dis.21: 301–305. doi: 10.7589/0090-355821.3.301 26. Prahl S, Jepson PD, Sanchez-Hanke M, Deaville R, Siebert U. Aspergillosis in the middle ear of a harbour porpoise (Phocoena phocoena): a case report. Mycoses. 2011 Jul;54(4):e260-4. 27. Reidarson TH, Harrell JH, Rinaldi MG, McBain J. Bronchoscopic and serologic diagnosis of Aspergillus fumigatus pulmonary infection in a bottlenose dolphin (Tursiops truncatus). J Zoo Wildl Med. 1998 Dec;29(4):451-5. 28. Rewell R. E. & G. C. Ainsworth. Occurrence of Aspergillus fumigatus Fresen. in the Lung of an American Bison. Letters to Editor. Nature 160, 362-363 (13 September 1947) 29. Seyedmojtaba Seyedmousavi , Jacques Guillot Pascal Arné G. Sybren de Hoog Johan W. Mouton Willem J. G. Melchers Paul E. Verweij . Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease Med Mycol (2015) 53 (8): 765-797. 30. Severo LC, Bohrer JC, Geyer GR, Ferreiro L. Invasive aspergillosis in an alpaca (Lama pacos). J Med Vet Mycol. 1989;27(3):193-5. 31. Tamam Omar A.S., And Mohamed Refai Dual mycotic pulmonary granulomas caused by alternaria alternata and aspergillus candidus in the wild egyptian mole rat (Spalax Leucodon Egyptiacus). Assiut Vet. Med. J. Vol. 59 No. 139 October 2013 32. Tamam Omar A.S. and Mohamed Refai. A retrospective analysis of mortalities in Greater Red Musk Shrews (Crocidura flavescens). Egypt. J. Comp. Path &Clinic Path. Vol.27 No.1, 2014 ; 73- 87 33. Tappe JP, Chandler FW, Liu SK, Dolensek EP. Aspergillosis in two San Esteban chuckwallas. J Am Vet Med Assoc. 1984 Dec 1;185(11):1425-8. 34. Udeani, Theophilus K, Chukwugozie N Okwuosa, Ezinne U Adiele. Effect of Aspergillus flavus on the Liver of Experimental Rats Administered with Antiretroviral Drugs . AJPCT1[6][2013]443-456 35. Vítovec J, Fragner P. [Pulmonary and hepatic aspergillomas in the deer]. Vet Med (Praha). 1978 Jan;23(1):49-54. 36. Vítovec, J., Vladík, P. and Frágner, P. (1972), Morphologie der Lungenveränderungen bei Aspergillose des Rehwildes. Mycoses, 15: 289–294. doi: 10.1111/j.1439-0507.1972.tb02518.x 37. Williamson , W.M., E.B. Tilden, ANDR.E. Getty B.S. Mycotic Infections Occurring during an eight Year Period at the Chicago Zoological Park, Brookfield, Illinois. Bijdragen tot de Dierkunde, Vol. 33, No. 1, p.83-85. ISSN 0067-8546 38. Woods R, Blyde DJ, Seaman JT, Thorne AH. Fungal pneumonia in a captive black rhinoceros. Aust Vet J. 1999 Nov;77(11):717-9. 39. Wu, Y., Xu, H., Li, L. et al. Inflammation (2016) 39: 1310. Susceptibility to Aspergillus Infections in Rats with Chronic Obstructive Pulmonary Disease via Deficiency Function of Alveolar Macrophages and Impaired Activation of TLR2. August 2016, Volume 39, Issue 4, pp 1310–1318 40. Wyandk D. S.. Langheinrichc. F. Helmboldt . Aspergillosis and Renal Oxalosis in a White-tailed Deer Journal of Wildlife Diseases, 7(1):52-56., 1971 41. Yamauchi H, Takai Y, Yamasaki H, Fukuda R. Thoracic mass in a cynomolgus macaque (Macaca fascicularis). Vet Pathol. 2011 Jul;48(4):E1-5. 218 8. Penicilliosis in wild animals Ecology and source of infection     The fungus may have a natural habitat in soil in areas of southern China and South-East Asia where it is endemic . Talaromyces marneffei was originally isolated from the bamboo rat, Rhizomys sinensis, in 1956. Since then, additional studies demonstrated that three other bamboo rat species may act as reservoirs: Rhizomys pruinosus, Rhizomys sumatrensis and the reddish-brown subspecies of Cannomys badius. Within these rodent species, the prevalence of infection varies widely across South-East Asia . Disease in animals      Penicilliosis is a rare infection in domestic animals such as dogs and cats. Clinical signs of infection include skin dermatitis, rhinitis and otitis externa. Symptomatic animals have nasal discharge and ulceration of external nares and epistaxis Disseminated infections have been reported in dogs, with peripheral lymphadenopathy and bronchopneumonia. No clinical signs have been reported in naturally infected bamboo rats . The prevalence of infection varies widely across South-East Asia, suggesting that there are regional variations in the endemicity of infection or there are geographical variations in the predisposition to infection within different species of bamboo rats. Mode of transmission     Bamboo rats are a natural reservoir, they are generally not in close contact with humans and there is no evidence of direct transmission from rats to humans . Bamboo rats and HIV-positive patients have been found to share genetically similar strains of T. marneffei, suggesting that rat-to-human transmission might be possible or co-infection from a common but still unidentified source . Infected rats appear healthy . To date T. marneffei has never been recovered from environments other than those that are intimately associated with bamboo rats. Penicillium marneffei is the only known dimorphic and pathogenic species in the genus of Penicillium.  Penicillium marneffei is an emerging pathogen that can cause lethal penicilliosis marneffei.  Penicillium marneffei was discovered in 1956 from bamboo rats, it has been isolated from the internal organs of four species of rodents (Rhizomys 219      sinensis, Rhizomys pruinosus, Rhizomys sumatrensis and Cannomys badius) (Chariyalertsak et al., 1996a; Fisher et al., 2004a; Gugnani et al., 2004; 2007; Vanittanakom et al., 2006). Penicilliosis marneffei infection is endemic in Southeast Asia, including Thailand, Vietnam, Hong Kong, Southern China, Taiwan, India and Laos (Hu et al., 2013). Penicilliosis marneffei, in these endemic areas, has been proven to be the third commonest AIDS-indicating systemic opportunistic infection among HIV-positive patients (Wong and Wong, 2011). The distribution of bamboo rat species generally followed the distribution of P. marneffei (Vanittanakom et al., 2006; Cao et al., 2011; Li et al., 2011), The multilocus microsatellite typing system showed little difference in allele frequencies between P. marneffei isolates from bamboo rats and human, which means these two host-associated populations of P. marneffei shared high similarity (Fisher et al., 2004b; Cao et al., 2011 Soil exposure, especially during the rainy season, was found to be a risk factor associated with the infection caused by P. marneffei (Chariyalertsak et al., 1996b; 1997). Penicillium griseofulvum is a species of the genus of Penicillium which produces patulin, penifulvin A, cyclopiazonic acid, roquefortine C, shikimic acid and griseofulvin. Penicillium griseofulvum occurs on cereals and nuts.  Penicillium griseofulvum was reported to cause sudden fatal illness in a group of New World toucanets held captive in Finland (Aho et al., 1990).  The only reported infection in wild animals caused by Penicillium griseofulvum concerned a Seychelles Giant Tortoise (Oros et al.,1996) Animals reported to be infected with Penicillium marneffei Rhizomys pruinosus Wikipédia Wikimedia Commons Rhizomys sinensis Hardwicke. Omar Ariff - PhotoShelter Bamboo Rat (Rhizomys sumatrensis). ADW: Cannomys badius: Description 220 Penicillium marneffei Segretain, Capponi & Sureau, Bulletin de la Société Mycologique de France 75: 416 =Talaromyces marneffei (Segretain, Capponi & Sureau) Samson, Yilmaz, Frisvad & Seifert, Studies in Mycology 70: 176 (2011) Colonies (CzA, 30°C) flat, sparse, compact, greenish to purplish, on MEA exuding an orange or red pigment into the medium; primary cultures often canary yellow due to sterile aerial mycelium. At 37°C colonies restricted, whitish, yeast-like. Microscopy. Hyphae in part spirally twisted. Conidiophores creeping or fasciculate, 70-150 x 2.5-3.0 µm; penicilli generally biverticillate but also irregularly monoverticillate or more complex. Metulae 7-11 µm long, in whorls of 3-5. Phialides in whorls of 4-7, ampulliform to acerose, 6-10 x 2.5-3.0 µm. Conidia smooth-walled, ellipsoidal, 2.5-4.0 x 2-3 µm, often with prominent scars, borne in short, disordered chains Hindawi Granular colony of P. marneffei with a characteristic red diffusible pigment on Sabouraud's dextrose agar after 7 days incubation at 25°C. Penicillium marneffei - Wikipedia Mycobank Reports: Capponi et al. (1956) made the Initial isolation of P. marneffei from a captive bamboo rat (Rhizomys sinensis) used for laboratory experiments in South Vietnam. The native bamboo rat had been experimentally inoculated with the scrub typhus 221 bacterium Rickettsia orientalis (now designated Rickettsia tsutsugamushi). At autopsy, the rodent was found to have an enlarged liver and spleen, viscous ascitic fluid, and epiploic nodules. Cultures from all the organs yielded a Penicillium species, which proved pathogenic to hamsters (Mesocricetus auratus). The fungus was subsequently described as a new species by Segretain (25), who named the fungus Penicillium marneffei in honor of Hubert Marneffei, then-director of the Pasteur Institute in French Indochina. Deng et al. (1986) surveyed the wild rats in in the Guangxi region of the People's Republic of China. The rats captured were of bamboo rat (R. pruinosus). Of the 19 rats captured, 18 yielded cultures of P. marneffei from one or more of their internal organs. Li et al (1989) studied the natural carrier of P. marneffei by conducting experiments in 16 Rhizomys pruinous senex, a species of bamboo rats trapped in various districts of Guangxi. The results of mycological cultivation demonstrated that the natural positive carrier rate of bamboo rats was 93.1%. Among the internal organs taken for culture, the lung had the highest positive rate, 87.5%; next in decreasing order was the liver (56.3%), spleen (56.3%) and mesentery lymph node (50%). Thus, it is reasonable to consider that bamboo rats are a reservoir host of P. marneffei. In this paper, the colony shape and microstructure of P. marneffei, isolated from bamboo rats, are described. A preliminary discussion is given on the epidemiological relations between bamboo rats and human Penicilliosis marneffei. Ajello et al. (1995) carried out a survey of bamboo rats for natural infections by Penicillium marneffei in the central plains of Thailand during June-September, 1987. Thirty-one lesser bamboo rats (Cannomys badius) and eight hoary bamboo rats (Rhizomys pruinosus) were trapped. Portions of their internal organs were cultured to determine if they had been infected by P. marneffei. Six each of C. badius (19.4%) and R. pruinosus (75%) yielded cultures of this unique, dimorphic Penicillium species. All of the isolates were readily converted to their unicellular form that multiplies by the process of schizogony by incubating them at 37 degrees C on plates of brain heart infusion agar. Their identity was further confirmed by a specific immunological test. Among the internal organs of the positive rats, the lungs had the highest positivity (83.3%), next in decreased order of frequency were the liver (33.3%) and the pancreas (33.3%). The use and value of domestic and wild animals in locating and demarcating endemic areas of geophilic fungal pathogens are discussed. Penicilliosis marneffei is considered to be a zooanthroponosis--a disease that occurs in lower animals, as well as, humans. Gugnani et al. (2004) surveyed wild rodents and their associated environment in order to identify the natural populations of this fungus. Surveys recovered P. marneffei from the internal organs of 10 (9.1%) of 110 bamboo rats (Cannomys badius) examined from Manipur state, India, an area endemic for Penicilliosis marneffei. All the P. marneffei-positive bamboo rats appeared healthy. At autopsy, no gross lesions were observed on any of the internal organs of the rats. Two of the isolates were recovered from rats trapped in the Senapati district, and the remaining eight isolates were from rats trapped in the Tamenglong district in Manipur state. In 2 of the 10 positive rats, P. marneffei was cultured from the liver and spleen. Cultures of the liver, spleen, and pancreas from three rats yielded P. marneffei; cultures of the 222 fungus from lungs, liver, and spleen of another two animals also produced the fungus. For the remaining three P. marneffei-positive animals, only spleen tissue cultures yielded the fungus. Histopathological examination of lungs, liver, and spleen of 15 C. badius rats (including 5 of the P. marneffei-positive animals) did not reveal any fungal elements P. marneffei was not recovered from any of the 25 samples of soil from the burrows of C. badius or from 10 samples (each) of bamboo shoots and leaves from the surrounding areas. Further sampling of soil samples from the immediate areas surrounding the burrows of bamboo rats and from the burrows of other rodent species (120 soil samples in total) were also negative for P. marneffei. None of the 72 rodents of the other five species (B. bengalensis, R. norvegicus, R. rattus, R. niditus, and M. musculus) trapped on bamboo plantations from other areas of northeast India were found to harbor P. marneffei. . (A) Lactophenol blue mount of 7 days‘ growth of P. marneffei (isolate VPCI 214) showing characteristic biverticillate and monverticillate penicilli. Magnification, _425. (B) Lactophenol blue mount of 3 days‘ growth of P. marneffei isolate (VPCI 214), showing fission yeast form. Magnification, _425. Cao et al. (2011) trapped bamboo rats during June 2004–July 2005 across Guangxi Province, China, and demonstrated 100% prevalence of infection. Multilocus genotypes showed that P. marneffei isolates from humans were similar to those infecting rats and were in some cases identical. Humans and bamboo rats are sampling an as-yet undiscovered common reservoir of infection, or bamboo rats are a vector for human infections by acting as amplifiers of infectious dispersal stages. Huang et al. (2015) carried out a study to explore the distribution of P. marneffei in bamboo rats, their associated environment and non-ratassociated environments. Totally, 270 samples were collected in Guangdong province of China in 2012; the prevalence of P. marneffei was much higher in samples collected from surrounding areas of burrows (8.2%) than in the samples obtained from non-rat-associated sites (2%) or artificial farms of bamboo rats (0%). There was no difference in P. marneffei isolated rate from different areas of Guangdong province. The infection was prevalent in all rats, and this fungus could be frequently seen in the rats‘ lungs. This study confirmed that bamboo rat is the ecological niche of P. marneffei and hypothesizes that bamboo rats become infected by inhaling aerosolized conidia originating from 223 environmental sources, rather than by the fecal–oral route or transplacental crossing. According to the result of no detection of P. marneffei in the artificial farm, the activity of bamboo rats might be more relevant to the distribution and dissemination of P. marneffei in natural environment. The bamboo rat (R. pruinosus) we captured and surrounding environmental samples. (A) Rat’s cave and stool of the rat; (B) bamboo root in the burrow; (C) the soil and debris of food; (D) petiole; (E) bamboo leaves; (F) bamboo rat. The isolation of P. marneffei recovered from different samples. Black arrows point to the suspected colonies. (A-C) Growth of P. marneffei recovered from soil, bamboo root and stool of bamboo rats at 25°C. (D-K) The grow th of P. marneffei recovered from different organs of bamboo rats at 25°C and 37°C. 224 The organs of bamboo rat we captured. (A) Lung; (B) liver; (C) spleen; (D) intestine; (E) uterus; (F) embryo. Penicillium griseofulvum  The only reported infection in wild animals caused by Penicillium griseofulvum concerned a Seychelles Giant Tortoise (Oros et al.,1996) Seychelles Giant Tortoise, Aldabran Giant Tortoise,..Alamy Description: Penicillium griseofulvum Dierckx, Annales de la Société Scientifique de Bruxelles 25 (1): 88 (1901) Colonies (CzA) slowly growing, fasciculate to synnematal, greyish-green; soluble pigment reddish-brown. Conidiophore stipes of very variable length, smooth-walled, brownish; penicilli terverticillate to quaterverticillate. Metulae 7-10 µm long, sometimes apically inflated. Phialides closely packed, very short, ampulliform, 4.56.0 µm. Conidia ellipsoidal, smooth-walled, 3.0-3.5 µm long.Intolerant to benomyl. No growth at 37°C. (Pitt, 1979). 225 Mycobank Report: Oros et al. (1996) reported a 50-year-old, 40 kg, female Seychelles giant tortoise belonging to the collection at Gran Canaria's Reptilandia Park with systemic mycosis. The tortoise had been maintained with three others of the same species for five years in an outdoor enclosure with a shelter from bad weather, and was the only survivor of a fire which took place in the enclosure eight months before its death. Having sustained shell bums of varying severity, it had been treated by the park's veterinarians and had apparently recovered. The tortoise was treated for suspected salmonellosis, receiving 20 mg/kg trimethoprim-sulphadiazine intramuscularly once daily with parenteral fluid supplementation; despite this the animal died. At post mortem examination the most significant lesions were observed in the cardiac region. The parietal pericardium was highly distended and numerous randomly-scattered whitishyellow nodules (4 to 5 mm in diameter) protruded from the surface. The pericardial cavity contained an abundant, dense, greenish-yellow fluid. The visceral pericardium was distended. with various sized necrotic foci and numerous scattered irregularly-shaped nodules (I to 1.5 cm in diameter), which were soft and whitish in colour. Whitish yellow nodules (2 to 4 mm in diameter) were seen in the kidneys, the liver, the omentum, in the serosae of the stomach, small and large intestine, and in the mucosa of the pyloric antrum. Samples from all the organs stained with haematoxylin and periodic acid-Schiff, Gram and Grocott's revealed methenamine silver nitrate stains multifocal granulomata composed of necrotic tissue, with numerous branching septate hyphae. Samples from tissues showing gross lesions were cultured on blood agar plates, MacConkey's agar, Sabouraud's dextrose agar and mycobiotic agar and were incubated at 25°C. Fungal colonies were isolated four days later but bacteria were not isolated from any of the cultures. The fungus was identified as P griseofulvum on the basis of its growth pattern and the morphological characteristics. 226 Visceral pericardium showing various-sized whitish necrotic foci. Inset: Branching septate hyphae within the caseated core of a pericardial granuloma. Grocott's methenamine silver nitrate stain x 190 References: 1. Aho R, Westerling B, Ajello L, Padhye AA, Samson RA. Avian penicilliosis caused by Penicillium griseofulvum in a captive toucanet. J Med Vet Mycol. 1990;28(5):349-54. 2. Ajello, L., A. A. Padhye, S. Sukroongreung, C. H. Nilakul, and S. Tantimavanic. 1995. Occurrence of Penicillium marneffei infections among wild bamboo rats in Thailand. Mycopathologia 131:1–8. 3. Cao C, Liang L, Wang W, et al. Common Reservoirs for Penicillium marneffei Infection in Humans and Rodents, China. Emerging Infectious Diseases. 2011;17(2):209-214. doi:10.3201/eid1702.100718. 4. Capponi M, Sureau P, Segretain G. Pénicilliose de Rhizomys sinensis. Bull Soc Pathol Exot 1956;49:418-421 5. Chariyalertsak, S., P. Vanittanakom, K. E. Nelson, T. Sirisanthana, and N. Vanittanakom. 1996. Rhizomys sumatrensis and Cannomys badius, new natural animal hosts of Penicillium marneffei. J. Med. Vet. Mycol. 34:105–110. 6. Deng, Z., M. Yun, and L. Ajello. 1986. Human penicilliosis and its relation to the bamboo rat (Rhizomys pruinosus) J. Med. Vet. Mycol. 24:383–389. Wei, X. G., Y.. Ling, C. Li, and F. S. Zhang. 1989. Study of 179 bamboo rats carrying Penicillium marneffei. Chin. J. Zoonoses 3:34–35. (In Chinese.) 7. Gugnani H, Fisher MC, Paliwal-Johsi A, Vanittanakom N, Singh I, Yadav PS. Role of Cannomys badius as a natural animal host of Penicillium marneffei in India. J Clin Microbiol. 2004 Nov;42(11):5070-5 8. Huang , Xiaowen, Guohua He, Sha Lu, Yuheng Liang and Liyan Xi. Role of Rhizomys pruinosus as a natural animal host of Penicillium marneffei in Guangdong, China. Microbial Biotechnology (2015) 8(4), 659–664 9. Li JC, Pan LQ, Wu SX. Mycologic investigation on Rhizomys pruinous senex in Guangxi as natural carrier with Penicillium marneffei. Chin Med J (Engl). 1989 Jun;102(6):477-85. 10. Oros J, Ramirez AS, Poveda JB, et al: Systemic mycosis caused by Penicillium griseofulvum in a Seychelles giant tortoise (Megalochelys gigantea). Vet Rec 139:295-296,1996 227 9. Zygomycosis in wild animals           The term zygomycosis denotes infection by one of several genera of fungi of the class Zygomycetes. The frequency of reports of zygomycosis in humans and animals has increased recently. Several other terms have been applied to infections with these fungi. For example, the term mucormycosis has been used to designate infections caused by various species within the order Mucorales, in the class Zygomycetes. The term phycomycosis had been widely used since 1959 to describe infections caused by fungi previously grouped in the class Phycomycetes.26 in 1976 it was proposed that the archaic term be replaced with zygomycosis. The class Zygomycetes is subdivided into the orders of Mucorales and Entomophthorales. o Members of the order Mucorales tend to cause disseminated disease characterized by angioinvasion. o Members of the Entomophthorales tend to cause localized subcutaneous granulomas. Most zygomycotic infections in animals are caused by one of several genera of the family Mucoraceae, within the order Mucorales. Zygomycetes are ubiquitous and regularly isolated from soil, foodstuff, and air in many environments where cattle are raised. Zygomycosis generally manifests as subcutaneous, systemic or rhinocerebral infections. Zygomycosis is caused most frequently by members of the family Mucoraceae, including Rhizopus, Absidia, Rhizomucor, Mucor, and Apophysomyces. mucormycosis has been reported in juvenile Florida softshell turtles (Apaloneferox) that had necrotizing shell and skin lesions. Mucor was isolated from the lesions. Mucor sp was also isolated from infected skin of wood turtles (Clemmys insculpta) Zygomycetes were reported in 1. 2. 3. 4. 5. 6. 7. 8. Crocodilians Silberman et al. (1977) Red milksnake (Lampropeltis triangulum syspila) Sindler et al. (1978) Eastern Indigo Snake Werner et al. (1978) western massasaugua rattlesnake (Sistrurus catenatus) Williams et al. (1979) Softshell turtles Jacobson et al. (1980) Gopher snake (Pituophis melanoleucos) Jessup and Seely (1981) Copperhead (Agkistrodon contortrix) Jessup and Seely (1981) Farmed deer Jensen et al. (1989) 9. Female American bison calf (Bison bison) de los Monteros et al. (1999) 10. Harbor porpoise (Phocoena phocoena) Wünschmann et al. (1999) 11. killer whale (Orcinus orca) Robeck and Dalton (2002), Abdo et al. (2012) 12. White-sided dolphins (Lagenorhynchus obliquidens) Robeck and Dalton (2002) 13. Bottlenose dolphins (Tursiops truncatus) Robeck and Dalton (2002), IsidoroAyza et al. (2014) 228 14. Grey seal (Halichoerus grypus) Barnett et al. (2014) Red Deer (Cervus elaphus) » Focusi American Bison bison | ARKive Florida softshell turtle - Wikipedia aboutanimals Western Massasauga Rattlesnake Alamy California gopher snake Pituophis melanoleucus Shutterstock Red Milk Snake Southern Copperhead-Agkistrodon contortrix SWCHR 229 ARKive Harbour porpoise (Phocoena phocoena). Harbour porpoise r Killer whale - Wikipedia The Atlantic Grey Seal Halichoerus grypus...Focusing on Wildlife University of Florida Crocodilians Dreamstime.com Bottle-nose Dolphin (Tursiops truncatus) www.oceanwideimages.com Pacific White-sided Dolphin Description: Apophysomyces elegans P.C. Misra, K.J. Srivast. & Lata, Mycotaxon 8 (2): 378 (1979) Colony characteristics. Colonies (MEA, 30°C) growing rapidly, brownish-grey. Microscopy. Sporangiophores generally singly, emerging from aerial hyphae, straight or curved, unbranched, slightly tapering towards the apex, light greyish-brown, up to 540 ?m long, 3.4-5.7 ?m wide. Sporangia produced terminally and singly, pyriform, with distinct apophyses and columellae, 20-58 ?m diam; apophyses vase- or bellshaped, 10-46 x 11-40 ?m; columellae hemispherical, 18-28 ?m diam. Sporangiospores subspherical to cylindrical, subhyaline, smooth-walled, 5.4-8.0 x 4.0-5.7 ?m. 230 Mycobank Cunninghamella bertholletiae Stadel, Über einen neuen Pilz, Cunninghamella bertholletiae: 1 (1911) Colony characteristics. Colonies (MEA) expanding, with abundant floccose mycelium, tannish-grey. Sporangiophores erect, in the apical region with a whorl of short lateral branches, each branch ending in a swollen vesicle up to 40 ?m diam, with 1-spored sporangiola (each becoming a sporangiospore) covering the entire surface. Sporangiospores spherical to ovoidal, 7-11 ?m diam, smooth-walled, sometimes finely echinulate. [ Mycobank MB#230361] 231 Rhizomucor pusillus (Lindt) Schipper, Studies in Mycology 17: 54 (1978) [MB#322484] ≡ Mucor pusillus Lindt, Arch. Exp. Path. Pharmacol.: 272 (1886) =Mucor septatus Bezold, Schimmelmyc. memschl. Ohres: 97 (1889) [MB#182694] =Mucor parasiticus Lucet & Costantin, Compt. Rend. Hebd. Séances Acad. Sci., Sér. D: 1033 (1899) =Mucor buntingii Lendn., Bulletin de la Société Botanique de Genève 21: 260 (1930) Morphology Colonies on PDA and synthetic Mucor agar (SMA) about 2 mm. High, at first white with unbranched sporangiophores, later brown, slightly smoky with strongly branched brown sporangiophores 5-18 µm diam., always with a septum below the sporangium. Sporangia globose, 50-80 µm diam., bright grey to brown with more or less quickly diffluent margin. Columellae oval or pyriform, bluish-brown, up to 50 x 60 µm, often with a collarette. Sporangiospores globose to subglobose, occasionally oval, 2,5-4 µm, often mixed with crystalline pieces of the sporangial wall. Zygospores homothallic, globose to slightly flattened at the sides, black, 55-75 µm diam., covered with conical warts. Suspensors approximately equal, elongate and conical. Gemmae unknown. Mycobank Rhizopus rhizopodiformis (Cohn) Zopf, Handbuch der Botanik 4: 587 (1890) =Mucor rhizopodiformis Cohn, Z. klin. Med.: 140 (1884) =Rhizopus cohnii Berl. & De Toni, Sylloge Fungorum 7: 213 (1888) =Rhizopus microsporus var. rhizopodiformis (Cohn) Schipper & Stalpers, Studies in Mycology 25: 30 (1984) =Rhizopus equinus Costantin & Lucet, Bulletin de la Société Mycologique de France 19: 200 (1903) =Rhizopus pusillus Naumov, Opredelitel Mukorovykh (Mucorales): 74 (1935) 232 Morphology Colony dark greyish brown. Rhizoids simple. Sporangiophores on stolons up to 500 µm in length, 8 µm in width, brownish, with 1-4 together. Sporangia blueish to greyish black, powdery in appearance, up to 100 µm diam. Larger columellae pyriform; mouse grey; columella/sporangia: 4/5. Sporangiospores (sub-) globose, up to 5(-6) µm diam, rather homogeneous, minutely spinulose. Good growth at 50°C. Saksenaea vasiformis S.B. Saksena, Mycologia 45: 434 (1953 Colonies (OA, 30°C) expanding, grey. Sporangiophores single, unbranched, 25-60 x 6-9 ?m, with dichotomously branched, darkly pigmented rhizoids. Sporangia single, terminal, flask-shaped, up to 50 ?m long, with basis up to 20 ?m wide, multi-spored; columella hemispherical, 11-15 ?m diam. Sporangiospores smooth-walled, ellipsoidal to cylindrical, 3-4 x 1.5-2.0 ?m. Physiology. Maximum growth temperature 44°C. Microscopic characteristics of the isolate of Saksenaea vasiformis cultured on Czapek agar. A) Typical flask-shaped spora gia s ale ar = 5 μ o tai i g B s ooth- alled, re ta gular spora giospores s ale ar = μ . Blanchet D, Dannaoui E, Fior A, et al. Saksenaea vasiformis Infection, French Guiana. Emerging Infectious Diseases. 2008;14(2):342-344. doi:10.3201/eid1402.071079. 233 Reports: Silberman et al. (1977) described a case of phycomycoses that resulted in the death of Crocodilians in a common Pool. Sindler et al. (1978) reported phycomycosis in a red milksnake (Lampropeltis triangulum syspila). Werner et al. (1978) described a case of phycomycotic dermatitis in an Eastern Indigo Snake Williams et al. (1979) reported Phycomycosis in a western massasaugua rattlesnake (Sistrurus catenatus) with infection of the telencephalon, orbit, and facial structures. Jacobson et al. (1980) submitted 6 hatchling softshell turtles selected from a group of approximately 400 with circular gray integumentary lesions for clinical evaluation. The turtles died within 24 hours. Histologic examination of the carapace revealed ulcerative epidermitis, with bacterial colonies and tightly packed groups of fungi. A Mucor sp was isolated from the lesions. 234 1 Jessup and Seely (1981) reported zygomycete fungus infection in two captive snakes: Gopher snake (Pituophis melanoleucos);Copperhead (Agkistrodon contortrix). Jensen et al. (1989) diagnosed during 1988, pulmonary mycosis in four of 116 farmed deer examined on suspicion of tuberculosis. The histopathology showed allergic bronchopulmonary mycosis in a red deer (Cervus elaphus) and the agent was identified as a zygomycete, probably Absidia corymbifera, by immunofluorescence staining. de los Monteros et al. (1999) diagnosed concomitant nasal zygomycosis and pulmonary aspergillosis in a 3-mo-old female American bison calf (Bison bison) in Pennsylvania (USA). Etiologic diagnosis was made by immunohistochemistry using a panel of monoclonal antibodies and heterologously absorbed polyclonal antibodies. In the lungs fungal infection was accompanied by hemorrhage, fibrin exudation, and infiltration with neutrophils. Fungi were observed to penetrate apparently normal epithelial lining of the nasal turbinates, and there was hemorrhage, edema, and invasion of blood vessels in the submucosa. In vessels fungi were typically associated with thrombosis. The calf may have been infected due to a high level of exposure to mouldy feed and litter in the environment in combination with a collapse of it's natural defence mechanisms. Wünschmann et al. (1999) described a case of systemic mycosis due to a Rhizopus sp. infection in a dead-stranded, 10-yr-old, male harbor porpoise (Phocoena phocoena) found on the beach of Neustadt, Schleswig-Holstein on the Baltic Sea (Germany). At necropsy, granulomatous mycotic lesions in brain, lung, kidneys, testis, and draining lymph nodes were found. In addition, a focal ulcerative gastritis of 235 the first stomach, due to a nematode infection, was present and is suspected to be the portal of entry for the fungus. Robeck and Dalton (2002) mentioned that during a 10-yr period, a killer whale (Orcinus orca), two Pacific white-sided dolphins (Lagenorhynchus obliquidens), and two bottlenose dolphins (Tursiops truncatus), all housed at SeaWorld of Texas from 1991 to 2001, were infected with fungi from the class Zygomycetes. In four out of five cases, the fungi were identified as either Saksenaea vasiformis or Apophysomyces elegans. All fungi in the class Zygomycetes aggressively invade the vascular system. Death occurred within 23 days after the initial clinical signs. The primary site of infection involved the s.c. tissue and skeletal musculature. In one case, infection originated in the placenta and uterus of a periparturient animal. All cases exhibited systemic spread of the organisms, including two to the central nervous system. The fifth and most recent case, a bottlenose dolphin, was treated with liposomal nystatin, an antifungal formulation with reduced nephrotoxicity. This animal initially responded to therapy; however, 14 days after cessation of therapy, fungal growth reoccurred. Thus, the animal was euthanatized 39 days after the initial clinical signs. This drug represents a promising treatment option if combined with early disease detection and aggressive tissue resection. Abdo et al. (2012) reprted an adult female killer whale (Orcinus orca) which was transported to the Port of Nagoya public aquarium in June 2010. While the animal was being maintained in the aquarium there was a gradual decrease in body weight. On October 1st, 2010 the whale exhibited signs of gastrointestinal disease and died on January 14th, 2011. At necropsy examination the gastric compartments were filled with a large number of variably-sized rocks (total weight 81.4 kg) and there was marked ulceration in the third compartment. There were multifocal tubercle-like nodules within the lungs and on sectioning there were numerous abscesses and pulmonary cavities. Microscopically, there was severe suppurative pneumonia associated with fungal hyphae that were infrequently septate and often branched. Numerous bacterial colonies were also present. The hyphae demonstrated immunohistochemical cross-reactivity with Rhizomucor spp. and Cunninghamella bertholletiae was cultured. Bacteriological culture revealed the presence of Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas oryzihabitans. This case represents the first documentation of zygomycosis associated with C. bertholletiae in a marine mammal. Barnett et al. (2014) presented a three-month-old grey seal (Halichoerus grypus), admitted to a rehabilitation facility with severe gingivitis and minor trauma, that developed neurological signs 36 hours after arrival, including loss of swallowing reflex, muscle tremors and a poor response to stimuli. The animal was euthanased due to the poor prognosis. Initial gross postmortem findings were unremarkable, but 236 coronal slicing of the brain found a focal extensive area of red/brown discolouration within the right thalamic region. On histological examination, subacute, neutrophilic and histiocytic, leucocytoclastic, necrotising cerebral vasculitis, encephalitis and meningitis was present, with intralesional fungal hyphae with morphology suggestive of a zygomycete. PCR sequences of fungal DNA purified from digested paraffin sections of the brain matched best those for Rhizomucor pusillus (99 per cent homology) demonstrating the value of this technique in speciating fungi. This is believed to be the first published case of CNS infection in a pinniped with a zygomycete fungus. Isidoro-Ayza et al. (2014) reported an adult, male bottlenose dolphin (Tursiops truncatus) found stranded and dead on the Spanish Mediterranean coast. At necropsy, several areas of malacia were macroscopically observed in the periventricular parenchyma of the cerebrum. Microscopically a severe, diffuse, pyogranulomatous, and necrotizing meningoencephalomyelitis was associated with numerous intralesional highly pleomorphic fungal structures. After culture, the fungus, Cunninghamella bertholletiae, was identified by culture and PCR. To our knowledge, this is the first reported case of central nervous system mucormycosis due to Cunninghamella bertholletiae in a cetacean. Bottlenose dolphin (Tursiops truncatus) with Cunninghamella bertholletiae infection in the central nervous section. (a) Rostral section of the cerebrum; malacia was observed in the nucleus caudatus, beneath the lateral ventricle of the left cerebral hemisphere. (b) Cerebrum (nucleus caudatus); inflammatory infiltration composed of groups of macrophages and large multinucleated giant cells containing large and pleomorphic fungal structures (arrowheads). Numerous neutrophils surround the giant cells. H&E stain. (c) Medulla oblongata (leptomeninges); highly pleomorphic, pauciseptate, thin-walled, and irregularly branching hyphae that often appear collapsed, associated with the same vessel shown in Fig. 1d. Grocott's silver stain. (d) Medulla oblongata (leptomeninges); medium-size arteriole showing a severe hyalinization of the tunica media (fibrinoid necrosis) with inflammatory cells and leukocytoclastic changes (leukocytoclastic vasculitis). Few fungal structures similar to those shown in Fig. 1b are identified within the vascular wall (arrowheads). H&E. (e) Gross microscopic view of medulla oblongata, showing severe 237 multifocal vasculitis and periarteriolar edema in the ventral leptomeningeal arteriolar network (arrows). More external arterioles are spared. H&E. (f) Fungal culture microphotography; Terminal portion of a sporangiophore of C. bertholletiae showing a vesicle bearing sporangioles on short stalks. Lactophenol cotton blue stain. References: 1. Abdo W, Kakizoe Y, Ryono M, Dover SR, Fukushi H, Okuda H, Kano R, Shibahara T, Okada E, Sakai H, Yanai T. 2012. Pulmonary zygomycosis with Cunninghamella bertholletiae in a killer whale (Orcinus orca). J Comp Pathol 147:94–99. 2. Barnett, J., Riley, P., Cooper, T., Linton, C., Wessels, M. (2014) Mycotic encephalitis in a grey seal (Halichoerus grypus) pup associated with Rhizomucor pusillus infection Veterinary Record Case Reports 2: e000115. doi: 10.1136/vetreccr2014-00011 3. Isidoro-Ayza Marcos , Lola Pérez, F. Javier Cabañes, Gemma Castellà, Marina Andrés, Enric Vidal, and Mariano Domingo. Central Nervous System Mucormycosis Caused by Cunninghamella Bertholletiae in a Bottlenose Dolphin (Tursiops truncatus). Journal of Wildlife Diseases: July 2014, Vol. 50, No. 3, pp. 634-638. 4. Jacobson ER, Calderwood MB, Clubb SL: Mucormycosis in hatchling Florida softshell turtles.J Am Vet Med Assoc177:835-837, 1980 5. Jessup DA, SeelyJC: Zygomycete fungus infection in two captive snakes: Gopher snake (Pituophis melanoleucos);Copperhead (Agkistrodon contortrix). J Zoo Anim Med. 12:54-59, 1981 6. Robeck TR, Dalton LM. 2002. Saksenaea vasiformis and Apophysomyces elegans zygomycotic infections in bottlenose dolphins (Tursiops truncatus), a killer whale (Orcinus orca), and pacific white-sided dolphins (Lagenorhynchus obliquidens). J Zoo Wildl Med 33:356–366 7. Silberman, MS, Blue, J, Mahaffey, E. Phycomycoses Resulting in the Death of Crocodilians in a Common Pool. in: Annual Proceedings of the American Association of Zoo Veterinarians. Hill's,Topeka, KS; 1977:100–101 8. Sindler RB, Plue RE, Herman DW: Phycomycosis in a red milksnake (Lampropeltis triangulum syspila). Vet Med Small Anim Clin 73:64-65, 1978 9. Werner R, Balady MA, Kolaja GJ: Phycomycotic dermatitis in an Eastern Indigo Snake. Vet Med Small Anita Clin 73:362-363, 1978 10. Williams LW, Jacobson ER, Gelatt KN, et ah Phycomycosis in a western massasaugua rattlesnake (Sistrurus catenatus) with infection of the telencephalon, orbit, and facial structures. Vet Med Small Anim Clin 74:1181-1184, 1979 11. Wunschmann A, Siebert U, Weiss R. 1999. Rhizopus mycosis in a harbor porpoise from the Baltic Sea. J Wildl Dis 35:569–573. 238 10. Beauveria bassiana infection in wild animals   Beauueria bassiana is a ubiquitous soil saprophyte and has been recognized as pathogenic agent in insects since 1835, when Agostino Bassi proved mts role in muscardine in the silk-worm. Nowadays, it is considered not only entomopathogenic, but it has also been reported to be a pathogenic agent in reptiles, such as giant tortoises, crocodiles, and American alligators Classification: Species 2000 & ITIS Catalogue of Life: April 2013  Fungi + o  Ascomycota + Sordariomycetes +  Hypocreales +  Cordycipitaceae +  Beauveria +  Beauveria bassiana (Bals. -Criv.) Vuill. 1912  Beauveria amorpha (Höhn.) Samson & H. C. Evans 1982  Beauveria arenaria (Petch) Arx 1986  Beauveria brongniartii (Sacc.) Petch 1926  Beauveria caledonica Bissett & Widden 1988  Beauveria epigaea (Brunaud) Langeron 1936  Beauveria felina (DC.) J. W. Carmich. 1980  Beauveria velata Samson & H. C. Evans 1982  Beauveria vermiconia de Hoog & V. Rao 1975 Wild animals reported to be infected with Beauvaria bassiana 1. 2. 3. 4. tortoise Galapagos Georg et al. (1962) tortoise Aldabra Georg et al. (1962) tortoise (Tachemys scripta) González Cabo et al. (1995) American alligator (Alligator mississipiensis) Fromding et al. (1979) 239 Reports: Georg et al. (1962) reported fatal pulmonary infections in 4 giant land tortoises (Galapagos and Aldabra tortoises) that had been in captivity. In 2 of the tortoises, cultural and histological evidence for infection by Beauvaria bassiana was obtained. The tissues of the abdominal cavity appeared to be normal, the peritoneal walls and organs had smooth, glistening surfaces. areas of brown discoloration as well as areas covered with a white, cottony growth were observed in the lungs (Fig. 1, a). Direct examination of the lung tissue from these areas revealed it to be tough in consistency. In the NaO H mounts, large quantities of septate mycelium were observed. Culture media were inoculated and incubated both at room temperature and at 37° G. Culture at room temperature developed white,, fluffy aerial mycelial growth after 3 days. The colonies were well developed after 6 days. No growth was obtained on media incubated at 37° C. Cultures and fresh tissue, as well as preserved lung tissue were forwarded to the Mycology Unit of the Communicable Disease Center for further study. Cultures made at the Mycology Unit from the fresh tissue were identical to those isolated at the Chicago Zoological Park Animal Hospital. The plate culture grew rapidly and developed a flat, white to cream-colored colony that was coarsely powdery at the center, but fluffy at the edge. In slide culture, delicate oval conidia borne on flask-shaped sporophores or "phialides" developed. These sporophores were borne either singly along the hyphae, in pairs opposite each other, in verticils, or most commonly in clusters with their bases arranged in a whorl-like pattern. Th e phialides developed long delicate filaments at their tips. Th e spores were borne upon these filaments in a zigzag fashion. The fungus was forwarded to Dr. W. Bridge Cooke of the Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio. Dr. Cooke kindly identified the fungus as Beauvarìa bassìana. a. Tortoise # 2. Gross lesion on lung surface. b. Beauvarìa bassiana. Colony on Sabouraud dextrose agar. c. Conidiophores of B. bassiana. 850 X. ci. Conidiophores of B. bassiana. 850 X. e. Tortoise # 2. Mycelium in lung tissue, Gomori, 340 X. f. Tortoise # 2. Mycotic abscess in lung tissue, Gomori, 85 X. a. Tortoise # 3. Lung tissue showing masses of mycelium in bronchioles. Gomori, 50 X. b. Tortoise # 3. Granulomatous abscesses in lung, H. & E., 50 X. c. Tortoise # 4. Mycelium and conidiophores in lung tissue. Gomori, 850 X. d. Tortoise # 4. Mycelium and conidiophores in lung tissue. Gomori, 850 240 X. e. Tortoise # 4. Lung tissue showing exudative reaction. H. & E., 100 X. f. Tortoise # 4. Lung tissue showing organization of exudate. H. & E., 100 X. Fromding et al. (1979a) isolated the entomopathogenic fungus, Beauveria bassiana, from pulmonary lesions of a dead American alligator (Alligator mississipiensis) at the Oklahoma City Zoo. Colonies of the fungus, which had sporulated in vivo, were found in the thoracic air spaces. Septate, branching hyphae and fungal spores were seen in stained histologic sections of pleura and lung. Dissemination to other viscera had not occurred. This case indicated that B bassiana, a rare vertebrate pathogen, may be a fatal mycotic agent in captive reptiles. Fromding et al. (1979b) reported fatal pulmonary disease in two captive American alligators. The entomopathogenic fungus, Beauveria bassiana, was isolated from pulmonary lesions in both alligators. An extended hibernation period because of a severe winter and a failure of the zoo heating system may have predisposed the alligators to infection. González Cabo et al. (1995) reported a case of fatal pulmonary infection in a female tortoise (Tachemys scripta) imported into Spain from Cuba. Necropsy revealed general pulmonary congestion with pleuritis and a large number of yellowish nodules of the granulomatous type, similar to aspergillomata. Histological examination showed some infiltration of round cells, surrounding a small mass of fungal hyphae. Culturing on Sabouraud glucose agar, demonstrated the presence of a fungus whose macroscopic and microscopic characteristics corresponded to those of Beauveria bassiana. Pulmonary lesions due to Beauvh barsiana. General pulmonary congestion with pleuritis and a large number of yellowish nodules Granulomatous lesions in the lung due to Beauveria basszana. Infiltrate of round cells (bar), focus of necropsy (asterisk) and a large amount of fungal hyphae (arrows) (PAS stain, x 100). Aetiology: Beauveria bassiana (Bals.-Criv.) Vuill., Bulletin de la Société Botanique de France 59: 40 (1912) =Botrytis bassiana Bals.-Criv., Linnaea 10: 611 (1835) =Spicaria bassiana (Bals.-Criv.) Vuill. (1910) =Penicillium bassianum (Bals.-Criv.) Biourge, La Cellule 33: 101 (1923) =Sporotrichum densum Link, Magazin der Gesellschaft Naturforschenden Freunde Berlin 3 (1): 13 (1809) =Sporotrichum larvatum Peck, Annual Report on the New York State Museum of Natural History 32: 44 (1879) =Sporotrichum globuliferum Speg., Anales Soc. Ci. Argent.: 278 (1880) [MB#201601] 241 =Sporotrichum minimum Speg., Anales de la Sociedad Científica Argentina 13 (1): 24 (1882) [MB#194578] =Botrytis bassiana subsp. tenella Sacc., Michelia 2 (8): 544 (1882) [MB#453299] =Botrytis brongniartii subsp. delacroixii Sacc., Sylloge Fungorum 10: 540 (1892) [MB#139039] =Isaria vexans R.H. Pettit, Bull. Cornell Univ. agric. Exp. Stn: 399 (1895) [MB#206438] =Isaria citrinula Speg., Anales del Museo Nacional de Historia Natural Buenos Aires 20 (13): 449 (1910) =Beauveria doryphorae R. Poiss. & Patay, Soc. Scient. Bretagne: 1 (1935) [MB#263487] =Trichoderma minima (Speg.) Gunth. Müller (1965) References: 1. Fromding RA, Kosanke SD, Jensen JM, et al: Fatal Beauveria bassiana infection in'a captive American alligator. J Am Vet Med Assoc 175:934-936, 1979a 2. Fromtling RA, Jensen JM, Robinson BE, Bulmer GS. Fatal mycotic pulmonary disease of captive American alligators. Vet Pathol. 1979b Jul;16(4):428-31. 3. Georg L. K., Williamson W. M., Tilden E. B., Getty R. E.: Mycotic pulmonary disease of captive giant tortoises due to Beauveria bassiana and Paecilomyces fumoso-roseus. Sabouraudia 2: 80–86, 1962 242 4. J. F. González Cabo, J. Espejo Serrano, M. C. Bárcena Asensio. Mycotic pulmonary disease by Beauveria bassiana in a captive tortoise. Mycoses. Volume 38, Issue 3-4 March 1995 Pages 167–169 Metarhizium anisopliae infection in wild animals Hall et al. (2011) reported an 18-yr-old, male, albino, American alligator (Alligator mississippiensis) evaluated for decreased appetite and abnormal buoyancy. Computed tomography (CT) of the coelomic cavity showed multifocal mineral and soft tissue attenuating pulmonary masses consistent with pulmonary fungal granulomas. Additionally, multifocal areas of generalized, severe emphysema and pulmonary and pleural thickening were identified. The alligator was euthanized and necropsy revealed severe fungal pneumonia associated with oxalosis. Metarhizium anisopliae var. anisopliae was cultured from lung tissue and exhibited oxalate crystal formation in vitro. Crystals were identified as calcium oxalate monohydrate by X-ray powder defractometry. Fungal identification was based on morphology, including tissue sporulation, and DNA sequence analysis. This organism is typically thought of as an entomopathogen. Clinical signs of fungal pneumonia in nonavian reptiles are often inapparent until the disease is at an advanced stage, making antemortem diagnosis challenging. This case demonstrates the value of CT for pulmonary assessment and diagnosis of fungal pneumonia in the American alligator. Fungal infection with associated oxalosis should not be presumed to be aspergillosis.. Right lateral scout computed tomography image (upper left) and three dorsal-plane reconstructed images of the thorax. The alligator’s head is to the left and dorsal is to the top of scout image. For each of the dorsal-plane images, the right side of the alligator is the viewer’s left and the alligator’s head is at the top of the image. A. Right lung lobe contains large cavitated masses and severe emphysematous changes from cranial to midzone region with a small focal pneumocoelom along the right lateral and caudal aspect of the thoracic coelom and thickening of the adjacent portion of right lung lobe. A single, large, curvilinear septation is noted to divide these severe changes from the caudal medial lung segment, where less severe emphysematous changes are present. B. Lung image centered on the most severe right-sided pulmonary pathologic changes. Similar changes as seen in Figure 1A are noted. C. Left lung has multifocal emphysematous changes (absence of visible lung septation), focal pleural and pulmonary thickening, and mild thickening of the pulmonary septation. All images were reconstructed from 5-mm transverse images and are 5 mm in thickness. A bone algorithm was used for reconstruction. In addition, all images are presented for review with a window width (WW)¼3,500 and a window level (WL)¼_350. Dorsoventral scout computed tomography image (upper left) and three sagittal plane reconstructed images of the thorax. The alligator’s head is at the top, and the alligator’s right side is to the viewer’s left as marked with the RT (right) in the scout image. The alligator’s head is to the left, and dorsal is at the top for each of the sagittal reconstructions. A. Right lung lobe with large cavitated mass, ventral plate-like areas of soft tissue thickening of the cranial lung lobe, severe dorsal and cranial emphysema, and marked visceral pleural thickening.B. Several right lung lobe cavitated masses with similar changes as seen in Figure 2A. C. Left lung with multifocal emphysema (absence of visible lung septation) and focal pleural and pulmonary thickening. All images were reconstructed from 5-mm transverse images and are 5 mm in thickness. A bone algorithm was used forreconstruction. In addition, all images are presented for review with a WW¼ 3,500 and a WL¼_350 243 Microscopic features of M. anisopliae. A. View of the right lung tissue showing adventitious sporulation with characteristic chains of cylindrical conidia. Hematoxylin and eosin (H&E),340. Inset is the same tissue at 3100 magnification. B. View of the fungal mat within the right lung demonstrating oxalosis with characteristic centrally radiating crystal structure. H&E, 3100. Inset is 3400 magnification of wet mount from cultured isolate exhibiting crystal production and conidiation in vitro.. Richini-Pereira et al. (2010) mentioned that Road-killed wild animals have been for years used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for ecoepidemiological studies. In the current study, fungal infection was evaluated by PCR and nested-PCR in tissue samples collected from 19 road-killed wild animals. The necropsies were carried out and samples were collected for DNA extraction. Using PCR with a panfungal primer and nested PCR with specific primers, indicated that some animals are naturally infected with Amauroascus aureus, Metarhizium anisopliae, Aspergillus flavus, Aspergillus oryzae, Emmonsia parva, Paracoccidioides brasiliensis or Pichia stipitis. The approach employed herein proved useful for detecting the environmental occurrence of several fungi, as well as determining natural reservoirs in wild animals and facilitating the understanding of host-pathogen relationships. References: 1. Hall, N. H., Kenneth Conley, Clifford Berry,Lisa Farina, Lynne Sigler, James F. X. Wellehan, Michael H. A. Roehrl, andDarryl Heard,. Oxalosis in an American Alligator (Alligator mississippiensis)Associated with Metarhizium anisopliae var anisopliae: Journal of Zoo and Wildlife Medicine, 42(4):700-708. 2011 244 2. Richini-Pereira VB, Bosco SMG, Theodoro RC, Barrozo L, Bagagli E. Road-killed wild animals: a preservation problem useful for eco-epidemiological studies of pathogens. J. Venom. Anim. Toxins incl. Trop. Dis [Internet]. 2010 ; 16( 4 ): 607613 11. Fusarium infections in wild animals Frelier et al. (1985) reported fatal pulmonary infection in a captive alligator (Alligator mississippiensis). At necropsy, the animal appeared to be in excellent nutritional condition, but a severe necrotizing bronchitis with bronchiectasis was present. Histological examination revealed numerous branched, septate, hyaline hyphae within the necrotic debris lining the bronchi and rarely infiltrating into the adjacent stroma. The fungus cultured from the lung was identified as F. moniliforme. Smith et al. (1989) reported the occurrence of fatal fusariosis in baby bonnethead sharks (Sphyrna tiburo) born at the National Aquarium, Baltimore, Maryland. An atypical strain of Fusarium solani was cultured from the tissues of two of the infected sharks following postmortem examination. Histopathology revealed an apparent predilection of the fungus for hyaline cartilage. Invasion of the cartilage resulted in hyphae with a distorted morphology. In slide culture the fungus displayed the unusual characteristic of terminal chlamydoconidium generation on macroconidia; this may be of some taxonomic significance. Cabanes et al. (1997) found in September 1996, a subadult (26.3-kg) loggerhead sea turtle (Caretta caretta L.) floating off the coast of Barcelona, Spain (Mediterranean Sea). The turtle had a fishing hook anchored in the proximal esophagus and a traumatic injury with important loss of shell tissues. The hook was removed by surgical procedures, and debridement of the necrotic shell tissues was carried out. Treatment consisted of supportive therapy and control of secondary infections (amoxicillin, 22 mg/kg of body weight, intramuscularly, once a day, for 8 days, doxycycline, 250 mg, orally once a day for 30 days). During the second month of rehabilitation, the turtle developed several white-scaled skin lesions that were 10 to 35 mm in diameter over the dorsal region of the neck and head (Fig. 1). Samples of skin scrapings of the skin lesions for routine microbial culturing and biopsy specimens were obtained. Histologic sections of biopsy material were stained with hematoxylin and eosin and periodic acid-Schiff stain (PAS). KOH-lactophenol-, hematoxylin and eosin-, and PAS-stained preparations revealed the presence of numerous hyaline septate hyphae in the keratin layers of the stratum corneum (Fig. 2). Samples were inoculated on Sabouraud glucose agar supplemented with chloramphenicol, blood agar, and MacConkey agar. Cultures on Sabouraud glucose agar supplemented with chloramphenicol yielded numerous vinaceous fungal colonies in pure culture consistent with Fusarium sp. Culture of the fungal isolate on potato dextrose agar (14) and synthetic nutrient-poor agar (15) for identification generated bluish-green colonies which presented characteristic conidial structures. The micromorphology showed elongate monophialides bearing oval to kidney-shaped microconidia (Fig. 3). Macroconidia were abundant, stout, thick walled, and generally cylindrical, with dorsal and ventral surfaces parallel for most of their length (Fig. 4). The fungus was identified as F. solani according to the description by Nelson et al. (14). Bacteriological cultures yielded Pseudomonas fluorescens with the API 20E identification system (API, bioMe´rieux, Barcelona, Spain). The turtle was first 245 treated with a topical 10% solution of iodine in alcohol at accessible skin lesions and, afterwards, when the fungal infection was diagnosed, was treated simultaneously with a topical 10% solution of iodine in alcohol and topical ketoconazole. Subsequent susceptibility tests of the strain isolated were performed with antifungal tablets (NeoFusarium solani was reported as the agent of a cutaneous infection in an injured sea turtle collected in the Mediterranean Sea. The turtle was treated with both a topical 10% solution of iodine in alcohol and ketoconazole. The source of the causal agent was traced to the sand in the tank in which the turtle was maintained. Lesions on the dorsal regions of the neck and head. PAS-stained section of a portion of the tissue biopsy sample (stratum corneum) showing hyphal elements. Bar, 10 mm.Characteristic conidiogenous cell and microconidia in false heads of F. solani. Bar, 8 mm. Characteristic macroconidia of F. solani. Bar, 8 mm. Orós et al. (2004) conducted a study to describe the microscopic and immunohistochemical findings in a case of pulmonary hyalohyphomycosis in a Kemp's ridley sea turtle (Lepidochelys kempi). Samples of lung, liver and kidney from a stranded, dead Kemp's ridley sea turtle were routinely processed for histopathological studies. Two monoclonal antibodies that reacted specifically with antigens of Aspergillus spp and the Mucorales (Zygomycetes) group, and a panel of polyclonal antibodies raised against Aspergillus fumigatus, Candida albicans, Geotrichum candidum, Fusarium solani, and Scedosporium apiospermum were used for immunohistochemical or immunofluorescence staining. Histologically, a severe multifocal granulomatous pneumonia associated with fungal infection was diagnosed. All hyphae were identified as Fusarium spp because a strong and uniform reactivity was obtained only with a heterologously-absorbed polyclonal antibody raised against somatic antigens of Fusarium solani. Fusarium spp should be included in the differential diagnosis of mycotic pneumonia in Kemp's ridley sea turtles. Williams et al. (2012) reported a cold-stunned Kemp's ridley sea turtle, Lepidochelys kempii, which developed an abscess associated with Fusarium solani, Vibrio 246 alginolyticus, and a Shewenalla species after receiving a bite wound to the front flipper during rehabilitation. The lesion failed to respond to medical therapy and was treated successfully with surgery. Histopathology of the excised tissue demonstrated septic heterophilic inflammation with necrosis and granulation tissue, fungal elements, and bacteria, despite appropriate antimicrobial therapy. Variably thick bands of dense collagenous tissue partially surrounded affected areas which might have limited drug penetration into the tissue. Postoperative healing and eventual releases were uneventful. This is the first report of surgical treatment of cutaneous Fusarium infection in a sea turtle and supports surgery as an effective treatment for a fungal abscess in a reptil References: 1. Cabanes FJ, Alonso JM, Castella G, et al: Cutaneous hyalohyphomycosis caused by Fusarium sdani in a loggerhead sea turtle (Caretta caretta L). J Clin Microbiol 35:3343-3345, 1997 2. Frelier PF, Sigler L, Nelson PE. Mycotic pneumonia caused by Fusarium moniliforme in an alligator. Sabouraudia. 1985 Dec;23(6):399–402 3. Orós J , C Delgado , L Fernández & HE Jensen. Pulmonary hyalohyphomycosis caused by Fusarium spp in a Kemp's ridley sea turtle (Lepidochelys kempi): an immunohistochemical study. N Z Vet J. 2004 Jun;52(3):150-2. 4. Smith AG, Muhvich AG, Muhvich KH, Wood C. Fatal Fusarium solani infections in baby sharks. J Med Vet Mycol. 1989;27(2):83–91. 5. Williams,S. R., Michele A. Sims, Lois Roth-Johnson, Brian Wickes, (2012) Surgical removal of an abscess associated with fusarium solani from a kemp's ridley sea turtle (LEPIDOCHELYS KEMPII). Journal of Zoo and Wildlife Medicine: June 2012, Vol. 43, No. 2, pp. 402-406. 247 12. Paecilomyces lilacinus infection in wild animals Gordon (1984) recovered Paecilomyces lilacinus in culture from pulmonary lesions and other internal organs of a captive armadillo. Heard et al. (1986) reported hyalohyphomycosis caused by Paecilomyces lilacinus in an Aldabra tortoise 248 249 Maslen et al. (1988) reported sudden death of a captive Estuarine crocodile hatchling (Crocodylus porosus). On autopsy, granuloma-like lesions were seen in the liver, left lung and spleen, and branching, septate fungal hyphae were observed in sections of liver and spleen. The fungus isolated from the liver showed characteristics of both Paecilomyces lilacinus and Paecilomyces marquandii but was closer to the former species. This is apparently the first report of the isolation of this fungus from a reptile in Australia Marin et al. (2005) reported 11 wild-caught Fly River turtle hatchlings, Carettochelys insculpta, presented for anorexia and circular shell lesions. One animal died shortly after arrival, and three others within the next month. Necropsy of two animals and one shell biopsy revealed systemic and shell mycoses. A biopsy culture demonstrated infection due to Paecilomyces lilacinus, a ubiquitous fungal pathogen rarely affecting mammals, fish, and reptiles. Malachite green, formaldehyde, and parenteral itraconazole were used to effectively treat the shell lesions. In addition, changes in husbandry were made, and included frequent water changes, increasing the salinity to 7 ppt, increasing the water temperature to 32 degrees C (90 degrees F), and provision of enteral support. 250 Shell lesions on a fly river turtle, Carettochelys insculpta, caused by Paecilomyces lilacinus Numerous dark staining septate branching fungal hyphae are visible within a granuloma, Grocott’s Methenamine Silver (GMS) stain, Bar = 50 μm Li et al. (2008) reported for the first time in China that the white-spot disease of Chinese soft-shelled turtles in a culture farm was caused by Paecilomyces lilacinus according to its morphological characters of the isolate, challenge studies and sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). Paecilomyces lilacinus isolate from a diseased turtle after 7 d of incubation at 25 °C on Czapek agar (CA). (a) Visual colony morphology; (b) Colony morphology viewed under stereo microscope; (c) Conidiophore from the culture; (d) Conidia from the culture 251 References: 1. Bowater RO, Thomas A, Shivas RG, Humphrey JD. Deuteromycotic fungi infecting barramundi cod, Cromileptes altivelis (Valenciennes), from Australia. Journal of Fish Diseases. 2003;26(11-12):681–686. doi: 10.1046/j.1365-2761.2003.00503 2. Gordon MA. 1984. Paecilomyces lilacinus (Thom) Samson, from systemic infection in an armadillo (Dasypus novemcinctus). Sabouraudia, 22:109-116. 3. Heard DJ, Cantor GH,Jacobson ER, et al: Hyalohyphomycosis caused by Paecilomyces lilacinus in an Aldabra tortoise. J Am Vet Med Assoc 189:1143-1145, 1986 4. Li X, Zhang C, Fang W, Lin F. White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus . Journal of Zhejiang University Science B. 2008;9(7):578-581. doi:10.1631/jzus.B0720009 5. Marin, M L F , James Wellehan, Terrell SP, Kimbrough JW. Shell and systemic hyalohyphomycosis in Fly River turtles (Carettochelys insculpta) caused by Paecilomyces lilacinus. Journal of Herpetological Medicine and SurgeryVolume 15, No. 2, 2005, 1 6. Maslen M, Whitehead J, Forsyth WM, McCraken H, Hocking AD. 1988. Systemic mycotic disease of captive crocodile hatchling (Crocodylus porosus) caused by Paecilomyces lilacinus. J Med Vet Mycology, 26:219-225. 252 13. Dematiaceous fungal infections in wild animals Wild animals reported to be infected with dematiaceous fungi 1. 2. 3. 4. 5. 6. Toads (Bufo marinus) Velasquez and Restrepo (1975) Wyoming toads (Bufo baxteri) Taylor et al. (1996) Spadefoot toad (Scaphiopus holbrooki)Juopperi et al. (2002) Galapagos tortoise Manharth et al. (2005) Eastern box turtle (Terrapene carolina carolina) Joyner et al. (2006) Aldabra tortoise (Geochelone gigantea) Stringer et al. (2009) 7. Green Iguana (Iguana iguana) Olias et al. (2010) 8. Risso ‘ s dolphin ( Grampus griseus ) Elad et al. (2011) 9. Toads (Rhinella icterica) Brito-Gitirana and Silva-Soares (2012) 10. Sea turtles (Chelonia mydas) Donnelly et al. (2015) 11. Bufo japonicas formosus Hosoya et al. (2015) 12. Dysscophus guineti Hosoya et al. (2015) 13. Litoria caerula Hosoya et al. (2015 Bufo/ Cane Toad /Bufo marinus Galápagos Tortoises, A N G Wyoming toad - Wikipedia Alamy Eastern spadefoot toad, Scap Turtle Terrapene carolina -WPClipart 123RF.com Aldabra Giant Tortoise Green Iguana (Iguana iguana) - J Weigner . Rhinella icterica adult male.. MBio.org Chelonia mydas. Sea Turtles Bufo japonicus formosus (Hasumi) Tomato Frog (Dyscophus guineti ) Tol 253 Litoria caerulea Tracie Marine Toad - Bufo Marinus FreshMarine. Aldabra Tortoise - Geochelone gigantean STW - Risso's Dolphin Dematiaceous fungi reported in wild 1. Basidiobolus ranarum 2. Cladophialophora devriesii 3. Exophiala salmonis 4. Exophiala jeanselmei 5. Exophiala oligosperma 6. Exophiala angulospora 7. Lecythophora mutabilis 8. Neoscytalidium dimidiatum 9. Oidiodendron spp 10. Scolecobasidium humicola 11. Wangiella dermatitidis Reports: Cicmanec et al. (1973) reported spontaneous neurological disorders in marine toads (Bufo marinus). Agents were frequently identified as Fonsecaea pedrosoi; the analyses suggest that, in recent taxonomy, the causal isolates would be more likely to have been Cladophialophora species close to C. devriesii (G.S. de Hoog, unpubl. data). Elkan & Philpot (1973) described an Exophiala species (as ‗Phialophora‘) with septate conidia, thus strongly resembling E. salmonis or E. pisciphila, from a systemic infection in a frog (Phyllobates trinitatis). Velasquez and Restrepo (1975) found 2 of 75 toads (Bufo marinus) infected by black molds. The internal organs of these animals had granulomatous lesions containing brown fungi identical to those found in human chromomycosis. Cultures gave rise to slow-growing black molds but all attempts to induce sporulation failed. The fungi did not grow at 36 degrees C or above and failed to hydrolyse gelatin or casein. Immunodiffusion and immunoelectrophoresis revealed that both isolates were identical and shared common antigens with the recognized human pathogens P. pedrosoi, P. verrucosa and C. carrioni. The findings are compared with other reports of black mold infections in amphibians. Bube et al. (1992) performed post-mortem examinations on two marine toads, one animal showing neurological disorders and the other multifocal dermatitis. In one 254 case, lesions consisted of a severe granulomatous encephalomyelitis and in the other of multiple granulomas in the nasal cavity, lungs, heart, bone marrow, ovaries and skin. Histologically, the lesions revealed varying amounts of dark brown fungal elements, predominantly sclerotic bodies indicative of a mycotic infection due to a pigmented fungus. Muotoe-Okafor and Gugnani (1993) was isolated Lecythophora mutabilis from the lungs of 3 and from the liver of 2 bats, Eidolon helvum a fruit eating species. Wangiella dermatitidis was recovered from the liver of 2 bats of the same species. The isolates were pathogenic for laboratory mice when injected by subcutaneous, intraperitoneal and intravenous routes. W. dermatitidis was neurotropic in the mice injected intravenously. L. mutabilis: Fasciculate hyphae with cylindrical to curved conidia aggregating along the sides. Adelophialides and ellipsoid to clavate chlamydoconidia are also seen. ×525 (Lactophenol blue mount of a 12 day old culture). W. dermatitidis. Toruloid hyphae bearing phialides with sulglobose to ovoid phialoconidia accumulating in clusters (arrowed) Yeast like cells are also observed. ×1050 (Lactophenol blue mount of a 2 weeks o Section of the liver of mouse infected with L. mutabilis showing a granuloma containing a few hyphal fragments. Section of the brain of a mouse infected with W. dermatitidis showing mycelium, hyphal fragments and yeast cells, x875 (Grocott Stain). (arrowed) yeast like cells, x 625 (PAS Stain).ld culture). Taylor et al. (1996) reported Wyoming toads (Bufo baxteri) that died from January 1989 to June 1996. These consisted of 108 free-ranging toads and 170 animals from six captive populations. Ninety seven (90%) of 108 free-ranging toad carcasses were submitted during September and October. From 1989 to 1992, 27 (77%) of 35 mortalities in the captive populations occurred in October, November, and December. From 1993 to 1996, mortality in captive toads occurred without a seasonal pattern and coincided with changes in hibernation protocols that no longer mimicked natural cycles. Cause of mortality was determined in 147 (53%) of the 278 cases. Mycotic dermatitis with secondary bacterial septicemia was the most frequent diagnosis in 104 (71%) of 147 toads. Basidiobolus ranarum was found by microscopic examination of skin sections in 100 (96%) of 104 of these mortalities. This fungus was isolated from 255 30 (56%) of 54 free ranging and 24 (48%) of 50 captive toads. This research documents the causes of mortality for both free-ranging and captive endangered Wyoming toads over a 7 yr period. Juopperi et al. (2002) reported an 8-month-old spadefoot toad (Scaphiopus holbrooki) presented to the North Carolina State University (NCSU) for evaluation of a proliferative to ulcerative dermatitis of 1-month duration. On physical examination, the toad appeared to be in good body condition. Examination of the skin revealed multiple distinct to coalescing white, raised foci of various sizes, from 1 mm to 5 mm in diameter. Several of the lesions were ulcerated. Though the skin was diffusely affected, the lesions appeared to be concentrated on the lateral surface of the animal and in the inguinal and axillary regions. A biopsy of the skin lesions was obtained, and touch imprints were submitted for cytologic examination. Cytologic examination of 2 slides that were moderately cellular and mildly hemodiluted revealed a mixed cell population consisting primarily of equal numbers of nondegenerate neutrophils and macrophages. A moderate number of eosinophils and lower numbers of small lymphocytes were noted. Multinucleated giant cells were observed frequently. Several dark blue-green pigmented septate fungal hyphae, approximately 4 μm in width, were scattered throughout the specimen. A moderate number of sclerotic bodies also were observed, both alone and in small clusters. These structures were spherical, approximately 10–15 μm in diameter, and had a thick dark wall with a blue-green color. The pyogranulomatous inflammation was compatible with a fungal infection. The primary differential for the fungal hyphae and sclerotic bodies was chromomycosis. Histologic examination of the skin biopsy revealed findings similar to those observed in cytologic specimens. The hematoxylin and eosin-stained skin sections were characterized by multilobular to coalescing dermal masses composed primarily of macrophages and multi-nucleated giant cells. A moderate number of eosinophils was scattered throughout the granuloma. Lower numbers of neutrophils and lymphocytes were also observed. Scattered within the granulomas were moderate numbers of spherical, thick-walled septate sclerotic bodies (Figure 2). The sclerotic bodies were seen individually, in small clusters, and occasionally within multinucleated giant cells. Brown-pigmented septate hyphae were also observed infrequently throughout the specimen. Both tissue forms of the fungus were present within the epidermis, along with mild mononuclear inflammation. The histologic diagnosis was granulomatous dermatitis with an intralesional fungus, consistent with chromomycosis. Weitzman et al. (2003) reported Scolecobasidium humicola to cause cutaneous lesions in a tortoise, Terrapine carolina var. carolina. S. humicola was isolated from lesions on the foot and dematiaceous hyphae were observed in KOH preparations of the biopsy and in stained preparations. This isolate and others were compared morphologically and physiologically with isolates of Dactylaria gallopava which it resembles. As a result of this investigation, It was concluded that D. gallopava may be differentiated from S. humicola macroscopically, by the production in D. gallopava of an extensive diffusible purplish-red to reddish-brown pigment when cultured on Sabouraud dextrose agar; microscopically, by the presence and usually predominance of conidia, whose apical cell is markedly wider than the basal cell, and usually constricted at the septum; and physiologically, by the ability to grow on media containing cycloheximide and by the ability to grow well at 36-45 degrees C. In contrast, S. humicola does not usually produce a diffusible pigment on Sabouraud's 256 dextrose agar or if present, is not extensive; it lacks the wider upper cell; is less constricted or non-constricted at the central septum; grows on media containing cycloheximide, although some inhibition may occur and lastly, does not grow at 36 degrees C or higher. Both species were urease positive, hydrolysed tyrosine but not casein, xanthine, or gelatin. Manharth et al. (2005) described a disseminated infection in a Galapagos tortoise (Geochelone nigra), caused by an Exophiala species, Joyner et al. (2006) described a subcutaneous inflammatory mass in an eastern box turtle (Terrapene carolina carolina). The turtle (Terrapene carolina carolina) was referred to the Wildlife Center of Virginia with a three month history of marked swelling of the right hind limb initially diagnosed as chromomycosis by histopathology. Hematology revealed severe anemia (9%), leukocytosis (12.8 cells3103 /ml), heterophilia (6.14 cells3103 /ml), and monocytosis (0.51 cells3103 /ml). Gross necropsy revealed a firm, encapsulated 331 cm subcutaneous mass filled with dark brown-black, friable necrotic material of the distal right hind limb. Microscopically, the mass was characterized by a granulomatous inflammatory process with numerous multinucleated histiocytic giant cells. Fungal elements were present within necrotic centers and associated with multinucleated cells. Special stains revealed numerous phaeoid hyphae and yeast; Exophiala jeanselmei was isolated by routine mycologic culture. Phaeohyphomycosis was diagnosed based on the histologic appearance of the fungal elements within the mass and culture results. There was no histopathological evidence of systemic infection. This is the first report of phaeohyphomycosis caused by fungi of the genus Exophiala in free-living reptiles Necropsy specimen of the right hind limb of an eastern box turtle (Terrapene Carolina carolina). The skin is reflected distally to expose a subcutaneous mass. Note the small opening and pigmentation in the caudodistal portion of the capsule, as well as edema. Photomicrograph of the subcutaneous lesion on the right hind limb of an eastern box turtle (Terrapene carolina carolina). Note the granuloma with peripheral multinucleated histiocytic giant cells and a necrotic center with brown pigmented fungal hyphae and conidia (arrow). H&E. Bar510 mm Stringer et al. (2009) described an adult male Aldabra tortoise (Geochelone gigantea) presented with a deep flaking area of the carapace, and histologic examination of biopsies from this area revealed phaeohyphomycosis of the superficial keratinized layers. The disease progressed rapidly and spread to numerous sites on the carapace. After several weeks of regular debridement, deep bone involvement was evident and was confirmed through histologic examination. Fungal culture was attempted but was unsuccessful at isolating the infectious agent. Polymerase chain reaction analysis of extracted DNA from the fixed tissue block identified the fungus as Exophiala oligosperma. Initial treatment included weekly debridement and oral and topical 257 antifungal agents. A nuclear scintigraphy bone scan was performed to determine the extent and status of the infection. Multiple foci of uptake of the radiopharmaceutical marker were present within the carapace, indicating active lesions. The tortoise was maintained on oral antifungal treatment, and lesions resolved over several months. A repeat bone scan performed 1 yr after initial presentation showed reduction in marker uptake, indicating a response to treatment in the deeper lesions. Phaeohyphomycosis should be considered as a differential diagnosis for cases of shell. Olias et al. (2010) diagnosed cerebral phaeohyphomycosis in a 4-year-old green iguana (Iguana iguana) with paroxysmal spastic paralysis of all limbs and circling motion. Formalin-fixed tissues were collected at necropsy examination and submitted for evaluation. The left cerebrum and the left ventricle were replaced by a solid brown coloured mass. Microscopical examination revealed the presence of necrotizing and granulomatous encephalitis affecting the cerebrum, cerebellum and brainstem, with severe vasculitis and intralesional dematiaceous fungal hyphae. No other lesions or fungi were found in other organs. Fungi were identified as Oidiodendron spp. by sequence analysis of the internal transcribed spacer (ITS) region 1 extracted from formalin-fixed and paraffin wax-embedded brain tissue. This case represents the first report of phaeohyphomycosis with tropism for the central nervous system in a reptile. In the absence of fresh tissue, the diagnosis in such cases may be assisted by molecular analysis of fixed tissue specimens. De Hoog et al. (2011) mentioned that the majority of mesophilic waterborne species of the black yeast genus Exophiala (Chaetothyriales) belong to a single clade judging from SSU rDNA data. Most taxa are also found to cause cutaneous or disseminated infections in cold-blooded, water animals, occasionally reaching epidemic proportions. Hosts are mainly fish, frogs, toads, turtles or crabs, all sharing smooth, moist or mucous skins and waterborne or amphibian lifestyles; occasionally superficial infections in humans are noted. Cold-blooded animals with strictly terrestrial life styles, such as reptiles and birds are missing. It is concluded that animals with moist skins, i.e. those being waterborne and those possessing sweat glands, are more susceptible to black yeast infection. Melanin and the ability to assimilate alkylbenzenes are purported general virulence factors. Thermotolerance influences the choice of host. Exophiala species in ocean water mostly have maximum growth temperatures below 30 °C, whereas those able to grow until 33(-36) °C are found in shallow waters and occasionally on humans. Tissue responses vary with the phylogenetic position of the host, the lower animals showing poor granulome formation. Species circumscriptions have been determined by multilocus analyses involving partial ITS, TEF1, BT2 and ACT1. Gjessing et al. (2011) observed abnormal swimming behaviour and skin pigmentation and increased mortality in cod kept in an indoor tank. Necropsy revealed foci of different sizes with a greyish to brownish colour in internal organs of diseased fish. The foci consisted of ramifying darkly pigmented fungal hyphae surrounded by 258 distinct layers of inflammatory cells, including macrophage-like cells. In the inner layer with many hyphae, the macrophage-like cells were dead. No apparent restriction of fungal growth was observed by the inflammatory response. A darkly pigmented fungus was repeatedly isolated in pure culture from foci of diseased fish and identified as Exophiala angulospora using morphological and molecular characters. This species has not been previously reported to cause disease in cod, but has been reported as an opportunistic pathogen of both marine and freshwater fish. Based on the morphology and sequence analysis presented here, we conclude that E. angulospora caused the observed chronic multifocal inflammation in internal organs of cod, leading to severe disease and mortality. Elad et al. (2011) isolated Neoscytalidium dimidiatum from two 12 – 18 cm abscesses in the lung and the mediastinal lymph nodes of a stranded Risso‘ s dolphin ( Grampus griseus ). Histopathologic examination of samples of these organs revealed the presence of hyphaeand sclerotic body-like fungal elements. Photobacterium damselae subsp. Damselae was recovered from the dolphin ‘ s organs which also were found to contain numerous Monorygma grimaldii cysts. No histopathological signs of morbillivirus infection were seen. Neoscytalidium dimidiatum – microscopic appearance Histopathology: hyphae and sclerotic body-like fungal elements in (a) the lung – Periodic acid Schiff staining and (b) the mediastinal lymph nodes – hematoxylin and eosin staining. 259 Brito-Gitirana and Silva-Soares (2012) analyzed the integuments of five toads (Rhinella icterica) histologically and two of them exhibited brownish encapsulated subcutaneous mass of the integument. Fungal elements involved by multinucleated histiocytic giant cells were restrict to the hypodermis. This granulomatous inflammatory process showed an extracellular matrix rich in a hyaluronic acid. Moreover, these areas were surrounded by fibrous connective tissue, where collagenous fibers predominate. Since chromomycosis was previously reported in a bufonid from Amazon region and this work was first in Southeast from Brazil, it is possible that this disease may be spread to other Brazilian regions. Light micrograph of the integument of the dorsal region of ) in the hypodermis,Rhinella icterica. Note granulomatous area ( ). SD = spongiousshowing multinucleated histiocytic giant cells ( dermis; CD = compact dermis; GG = granular gland. HE-staining. Detail of the granuloma. Fungal elements ( within multinucleated phagocytic giant cells (*). HE-staining. Light micrograph of the integument staining with Alcian blue at pH 2.5 with hyaluronidase pre-treatment. In the granuloma region, the alcianophilic staining disappeared when compared to the hypodermis (*), indicating that the extracellular matrix of the granuloma is rich in hyaluronan 260 In the granuloma, several collagenous fibers ( intermingled polymorphic cells and giant cells with fungal elements Under polarized light microscope, thick collagenous fibers ) are visualized in red intermingled polymorphic cells, but they( are absent around giant cells containing fungal elements (*). Picrosirius red polarization method Tamam and Refai (2013) established the cause of death in seven blind mole rats that died naturally in the wild. All the animals had large pulmonary lesions that on microscopic, microbiological, and ultrastructural analysis were shown to contain mixed infections with Alternaria alternata and Aspergillus candidus. Some of the lesions were circumscribed with fibroblastic proliferation and inflammatory response. The lungs had haemorrhage and chronic inflammatory response to the organisms, which is likely to have been the cause of death. This is the first report of some pathogenic organisms resulting in death of the blind mole rat. Macroscopic features. (A) Thoracic cavity of Spalax leucodon showing large, tumour-like lesions replacing the apical left lung lobe and large, firm brown lesions in the caudal and right lobes (yellow arrows). (B) Thoracic cavity containing brown, consolidating focal lesions in the centre of the left lung lobe, emphysematous change (red arrows), and red hepatisation of the lower right lobe (violet arrow). 261 Microscopic and ultrastructural features. (A) Histological features of the lesions demonstrating fungal organisms within foamy macrophages (H&E x40). (B) Alveolar macrophages engulfing hemosiderin pigment (H&E x40). (C) Aspergilloma wall showing fibroblastic proliferation with slightly pleomorphic nuclei, vesicular chromatin, and grooved and folded nuclei (H&E x100). (D) TEM photomicrograph demonstrating fungal hyphae being engulfed by a foamy macrophage|(X1000 Donnelly et al. (2015) found 3 wild immature green sea turtles Chelonia mydas alive but lethargic on the shores of the Indian River Lagoon and Gulf of Mexico in Florida, USA, and subsequently died. Necropsy findings in all 3 turtles included partial occlusion of the trachea by a mass comprised of granulomatous inflammation. Pigmented fungal hyphae were observed within the lesion by histology and were characterized by culture and sequencing of the internal transcribed spacer 2 domain of the rRNA gene and D1/D2 region of the fungal 28s gene. The dematiaceous fungus species Veronaea botryosa was isolated from the tracheal mass in 2 cases, and genetic sequence of V. botryosa was detected by polymerase chain reaction in all 3 cases. Genetic sequencing and fungal cultures also detected other dematiaceous fungi, including a Cladosporium sp., an Ochroconis sp., and a Cochliobolus sp. These cases are the first report of phaeohyphomycosis caused by V. botryosa in wild marine animals. Hosoya et al. (2015) determined a dematiaceous hyphomycete, isolated from frogs, as the possible etiologic agent of a case of systemic chromomycosis in a cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran. 262 263 Pathological findings of chromomycosis in frogs. A. Polypoid skin nodule in Bufo japonicus formosus, no. 5. B. The kidneys are highly enlarged and discolored in Dyscophus guineti, no. 6. C. Multiple white nodules are observed in the liver of Litoria caerulea, no. 8. D. Cutaneous granuloma formation with necrosis of the epidermis and skeletal muscle in B. japonicus formosus, no. 5, HE. E. Granulomatous inflammation is observed in the dermis along with brown fungal hyphae, in B. japonicus formosus, no. 5, HE. F. Mycotic granuloma in the liver and brown fungal hyphae, in D. guineti, no. 6, HE. G. Brown fungal hyphae in granulomas in B. japonicus formosus, no. 5, HE. HE sta ds for he ato li a d eosi . Bar, μ . This Figure is reproduced in color in the online version of Medical Mycology 264 3. Veronaea botryosa NBRC 109680. A. Colony on potato dextrose agar (PDA). B. Colony on brain heat infusion (BHI) agar. C. Brown-colored hyphae with septa, constricted at the septa. D–K. Light microscopy of the conidiaproducing structure on PDA. D–F. The same conidia-producing structure focused at different optical sections, showing the three-dimensional production of conidia. G. Conidia produced sympodially on the conidiogenous cells. H. Three septate conidia rarely observed on conidiogenous cells. I. Geniculate conidiogenous cells with scars. J. Curved conidiogenous cells with a prominent detachment scar. K. Conidiogenous cell with a detachment scar and pointed apex. References: 1. Brito-Gitirana, L. de, and T. Silva-Soares). Chromomycosis in Rhinella icterica. The Open Zoology Journal, 2012, 5, 38-41 2. Bube A, Burkhardt E, Weiss R. Spontaneous chromomycosis in the marine toad (Bufo marinus). J Comp Pathol. 1992 Jan;106(1):73-7. 3. De Hoog GS, Vicente VA, Najafzadeh MJ, Harrak MJ, Badali H, Seyedmousavi S. Waterborne Exophiala species causing disease in cold-blooded animals. Persoonia : Molecular Phylogeny and Evolution of Fungi. 2011;27:46-72. doi:10.3767/003158511X614258. 4. Donnelly , Kyle Donnelly, Thomas B. Waltzek1, James F. X. Wellehan Jr.2, Deanna A. Sutton3, Nathan P. Wiederhold3, Brian A. Stacy. Phaeohyphomycosis resulting in obstructive tracheitis in three green sea turtles Chelonia mydas stranded along the Florida coast. Inter-Research ,113 , 3 , 257-262, 2015 5. Elad, D. Danny Morick, Dan David, Aviad Scheinin,, Gilad Yamin , Shlomo Blum OZ GOFFMAN. Pulmonary fungal infection caused by Neoscytalidium dimidiatum in a Risso‘s dolphin (Grampus griseus). Medical Mycology May 2011, 49, 424–426 6. Gjessing, M. C., Marie Davey, Agnar Kvellestad , Trude Vrålstad. Exophiala angulospora causes systemic inflammation in Atlantic cod Gadus morhua.Dis Aquat. Org. Vol. 96: 209–219, 2011 7. Hosoya,T., Yasuko Hanafusa, Tomoo Kudo, Kenichi Tamukai, Yumi Une. First report of Veronaea botryosa as a causal agent of chromomycosis in frogs . Med Mycol (2015) 53 (4): 369-377. 8. Juopperi T, Karli K, De Voe R, Grindem CB. Granulomatous dermatitis in a spadefoot toad (Scaphiopus holbrooki). Vet Clin Pathol. 2002;31(3):137-9. 9. Miller EA, Montali RJ, Ramsay EC, Rideout BA. 1992. Disseminated chromoblastomycosis in a colony of ornate-horned frogs (Ceratophrys ornata). J. Zoo Wildl. Med. 23:433–438 10. Muotoe-Okafor FA, Gugnani HC. Isolation of Lecythophora mutabilis and Wangiella dermatitidis from the fruit eating bat, Eidolon helvum. Mycopathologia. 1993 May;122(2):95-100 11. Olias, P., M. Hammer†, R. Klopfleisch. Cerebral Phaeohyphomycosis in a Green Iguana (Iguana iguana). Journal of Comparative Pathology Volume 143, Issue 1, July 2010, Pages 61–64 265 12. Stringer, E. M., Michael M Garner, Jeffry Proudfoot, Daniel S Bradway. Phaeohyphomycosis of the Carapace in an Aldabra Tortoise (Geochelone gigantea). Journal of Zoo and Wildlife Medicine 40(1):160-7 · April 2009 13. Taylor, S. K., Elizabeth S. Williams, E. Tom Thorne, Ken W. Mills, David I. Withers, and A. C. Pier Causes of mortality of the wyoming toad. Journal of Wildlife Diseases, 35(1), 1999, pp. 49–57 14. Velasquez, L.F. V. and A. N. Restrepo. Chromomycosis in the toad (Bufo marinus) and a comparison of the etiologic agent with fungi causing human chromycosis. Sabouraudia 13 Pt 1(1):1-9 · April 1975 15. Weitzman, I., S. A. Rosenthal, and J. L. Shupack. 1985. A comparison between Dactylaria gallopava and Scolecobasidium humicola: First report of an infection in a tortoise caused by S. humicola. Sabouraudia 23: 287–293. J Wildl Dis. 2003 Apr;39(2):329-37. 14.Scedosporium apiospermum infection in wild animals Haulena et al. (2002) reported a recently weaned, stranded, male northern elephant seal (Mirounga angustirostris) pup that had been undergoing rehabilitation that was found severely obtunded with hyponatremia, hypokalemia, hypochloremia, and hypophosphatemia after a history of intermittent regurgitation. The animal was euthanatized, and gross postmortem findings included multifocal abscessation affecting brain, spleen, kidney, muscle, and subcutaneous tissue. Scedosporium apiospermum and mixed bacteria were cultured from brain, kidney, and subcutaneous tissue. Histopathologic examination revealed multiple fungal granulomas of variable size in the kidneys, brain, liver, and skeletal muscle. This is the first report of S. apiospermum infection associated with lesions in a marine mammal Reference: Haulena M, Buckles E, Gulland FMD, Lawrence JA, Wong A, Jang S, Christopher M, Lowenstine LJ. 2002. Systemic mycosis caused by Scedosporium apiospermum in a stranded northern elephant seal (Mirounga angustirostris) undergoing rehabilitation. J Zoo Wildl Med 33:166–171 266 15. Sporobolomyces infection in wild animals Sporobolomyces koalae is a species of fungus in the order Sporidiobolales. It is an anamorphic yeast. Strains of the yeast were isolated from nasal swabs from three of five captive Queensland koalas (Phascolarctos cinereus) kept at the Kobe Oji Zoo in Kobe, Japan. Swabs from three zoo keepers were examined as well, but tested negative for the presence of the yeast. It is not suspected to be pathogenic, as the koalas from which it was isolated were healthy. Classification  Sp. recognized by Index Fungorum: Fungi + o Basidiomycota +  Microbotryomycetes +  Sporidiobolales +  Incertae sedis +  Sporobolomyces Kluyver & C.B. Niel 1924 +  Sporobolomyces koalae Satoh & Makiura 2008  Sporobolomyces albidus C. Ramírez 1957  Sporobolomyces alborubescens Derx 1930  Sporobolomyces albus W. F. Hanna 1929  Sporobolomyces antarcticus Goto, Sugiy. & Iizuka 1969  Sporobolomyces bannaensis F.Y. Bai & J.H. Zhao 2003  Sporobolomyces beijingensis F.Y. Bai & Q.M. Wang 2004  Sporobolomyces bischofiae Hamam., Thanh & Nakase 2002  Sporobolomyces blumeae M. Takash. & Nakase 2000  Sporobolomyces boleticola C. Ramírez 1957  90 more... show full tree... Description Sporobolomyces koalae Satoh and Makimura sp. nov. 2008 After 3 days in malt extract at 20 oC, cells are ovoid, ellipsoidal to elongate, 2.5– 5.065.0–15.0 mm, single, in pairs or groups of four. After 1 month on malt extract agar at 20 uC, the streak culture is butyrous to viscous, pink to orange–red, smooth and glistening with an entire margin. Pseudohyphae are formed in YM broth on overnight cultivation at 28 oC. Pseudohyphae are not formed in plate culture on malt extract agar. On cornmeal agar, ballistoconidia are formed on short sterigmata and are asymmetrical, ellipsoidal to reniform, 2.0–5.063.0–7.0 mm. Fermentation of glucose is negative. The following carbon compounds are assimilated: glucose, sucrose, maltose, cellobiose, trehalose (weak), raffinose, melezitose (weak), inulin (weak), soluble starch, L-arabinose (weak), glycerine (weak), D-mannitol, D-sorbitol (weak), salicin (weak), D-gluconate (weak) and succinic acid. The following are not assimilated: D-galactose, L-sorbose, lactose, melibiose, D-xylose, D-arabinose, Dribose, Lrhamnose, D-glucosamine, N-acetyl-D-glucosamine, methanol, ethanol, erythritol, adonitol, galactitol, methyl a-Dglucoside, DL-lactic acid, citric acid, inositol, hexadecane, 2-keto-D-gluconate and xylitol. Ammonium sulphate and potassium nitrate are utilized as sole sources of nitrogen; sodium nitrite, L-lysine, ethylamine and cadaverine are not utilized. Growth in vitamin-free medium is 267 positive. Optimum growth temperature is 28–30 oC; growth is negative at 32 uC. Growth does not occur in 50 % (w/w) glucose/yeast extract broth. No starch-like substrate is produced. Urease activity is positive. Diazonium blue B reaction is positive. . Sporobolomyces koalae JCM 15063T . (a) Vegetative cells and pseudohyphae grown in YM broth overnight at 28 6C. (b) Ballistoconidia-producing cell and vegetative cells. (c) Ballistoconidia produced on cornmeal agar after 3 days at 17 6C. Bars, 10 mm. Satoh et al. (2008) Report: Satoh et al. (2008) isolated 3 strains (JCM 15063T, JCM 15098 and JCM 15099) of a novel basidiomycetous yeast species belonging to the genus Sporobolomyces from nasal smears of Queensland koalas kept in a Japanese zoological park. Analyses of sequences of the nuclear rDNA internal transcribed spacer region and the 26S rDNA D1/D2 domain and morphological studies indicated that these strains represent a novel species with a close phylogenetic relationship to Sporobolomyces carnicolor and Sporobolomyces japonicus in the Sporidiobolus lineage, for which the name Sporobolomyces koalae sp. nov. is proposed (type strain JCM 15063T =CBS 10914T=DSM 19992T). Reference: Satoh K, Makimura K (2008) Sporobolomyces koalae sp. nov., a novel basidiomycetous yeast isolated from nasal smears of Queensland koalas kept in a Japanese zoological park. Int J Syst Evol Microbiol 58:2983–2986 268 16. Pneumocystosis infection in wild animals Historical  One of the first reports described lesions in male and female F344 rats on multiple prechronic toxicity studies performed over several years in different facilities in the United States (Elwell et al.,1997).  The lesions consisted of prominent increases in perivascular lymphocytes throughout the lung, infiltrates of macrophages, neutrophils, and lymphocytes within alveolar spaces, focal hyperplasia of pneumocytes, and a variable increase in peribronchiolar lymphoid tissue.  Similar lesions developed in male and female Wistar Hsd/Cpb:Wu rats used in toxicologic research (Slaoui et al., 1998).  In 2009, the transmission of idiopathic lung lesions from rats in a colony endemic for lung lesions to Wistar Han rats (Crl:WI[Han]) originating from a colony negative for lung lesions was demonstrated (Albers et al., 2009).  All these reports provide were suggestive evidence that the etiology of idiopathic lung lesions in rats is a transmissible infectious agent (Riley et al.,1997 and 1999, Besselsen et al., 2008. Albers et al., 2009).  Cytopathic effect was seen in cell lines incubated with lung homogenates from diseased rats, and lung lesions were reproduced in rats inoculated with the cultured cells (Riley et al., 1999, Besselsen et al., 2008). o In conjunction with the perivascular and interstitial lesion distribution and lack of an identifiable etiologic agent, these data led researchers to suspect a viral etiology. o The term ‗rat respiratory virus’ (RRV) was adopted to confer a putative viral etiology to the idiopathic lung lesions and has been used over the past decade in reference to this disease.  Attempts to identify the etiology of idiopathic lung lesions in rats have been unsuccessful (Riley et al.,1997 and 1999, Besselsen et al., 2008).  Livingston et al. (2011) demonstrated that Pneumocystis DNA was present in the lungs of several immunocompetent rats with RRV(rat respiratory virus)type lesions.  Henderson et al. (2012) confirmed a causative association between P. carinii infections and the time course and severity of interstitial pneumonia in immunocompetent laboratory rats. They proposed this prevalent, once idiopathic disease, to be called P. carinii pneumonia (PCP).  P. carinii is a single-celled fungal respiratory pathogen of mammals that is transmitted via an airborne route (Walzer et al., 1988, Hughes et al., 1982).  P. carinii is known to cause a severe and often lethal pneumonia in immunocompromised hosts (Walzer et al., 1976, Pohlmeyer and Deerberg, 1993).  Several studies have found PCP in immunocompetent hosts, including rats and mice naturally or experimentally infected with Pneumocystis spp. (Icenhour et al.2001, An et al., 2003, Chabé et al., 2004).  Recently, inflammatory lesions have been observed in the lungs of immunocompetent laboratory rats, very similar to those previously described (Albers et al., 2009, Henderson et al., 2012). 269      The latest evidence indicates that P. carinii causes the infectious interstitial pneumonia that was previously attributed to RRV in laboratory rats (Livingston et al., 2011, Henderson et al., 2012). o The taxonomy of P carinii was a matter of discussion:either a protozoan or a fungus The life cycle of the agent is in favour of being a protozoan: o The structural forms of P carinii that have been recognized are the cyst, which is thick-walled; o the sporozoite, an intracystic structure; and the thin-walled trophozoite. o The cyst is a spherical to ovoid structure 4 to 6 μm in diameter. It contains up to eight pleomorphic sporozoites. o The trophozoite is a thin-walled extracystic cell representing an excysted sporozoite. o The organism can be briefly propagated in embryonic thick epithelial lung cells, Vero cells, and WI-38 cells. o The organism does not enter the host cell, but instead attaches to its surface during a phase in the replicative cycle. o There is no evidence of toxin production. .. Pneumocystis carinii life cycle | by Arieviln Recent studies showed rRNA sequences, thymidylate synthase, dihydrofolate reductase, beta tubulin, mitochondrial DNA and chitin in the cell wall of P carinii more closely resemble fungi than protozoa. Eriksson's treatise places P carinii in a new family, Pneumocystidaceae, and in a new order, Pneumocystidales (Ascomycota).  Taxon recognized by NCBI Taxonomy: Cellular organisms + o Eukaryota +  Opisthokonta +  Fungi +  Dikarya +  Ascomycota +  Taphrinomycotina +  Pneumocystidomycetes +  Pneumocystidales +  Pneumocystidaceae + o Pneumocystis +  Pneumocystis carinii + 270  Pneumocystis carinii f. sp. macacae  Pneumocystis carinii f. sp. mustelae  Pneumocystis carinii f. sp. rattus-quarti  Pneumocystis carinii f. sp. rattus-secundi  Pneumocystis carinii f. sp. rattus-tertii  Pneumocystis carinii f. sp. suis o Pneumocystis jirovecii Etiology:   Pneumocystis carinii is the cause of diffuse pneumonia in immunocompromised hosts. o Even in fatal cases, the organism and the disease remain localized to the lung. o The pneumonia rarely, if ever, occurs in healthy individuals. Pneumocystis is a single-celled fungal (Ascomycota) respiratory pathogen of mammals. P. carinii and P. wakefieldiae are the two Pneumocystis species that have been identified in laboratory rats, with P. carinii being most commonly found. Transmission:  Transmission occurs via direct contact and airborne routes.  Experimental infection studies have shown that Pneumocystis isolates are host species-specific.  Interspecies transmission does not occur even in immunodeficient hosts. Clinical Signs:  Immunodeficient rats with pneumocystosis present with weight loss, ruffled fur or dry skin and a hunched posture.  The animals may suffer from severe, often lethal pneumonia.  Neonatal rats which acquire infection within hours after birth and can harbor the organism without evidence of clinical disease unless subjected to chronic immune suppression.  P. carinii has been found in the lungs of clinically healthy commercially produced immunocompetent rats. Pathology: Gross pulmonary lesions include 1-4 mm grey, flat to raised foci randomly distributed throughout all lung lobes at the peak of disease expression Lesion severity is greatest at 5 wk after exposure and consists of moderate to severe multifocal perivascular lymphohistiocytic infiltrates and moderate to severe multifocal lymphohistiocytic interstitial pneumonia. In areas of interstitial pneumonia, there is occasional to marked type 2 pneumocyte hyperplasia, and many alveolar spaces are filled with macrophages. Livingston, et al., 2011. 271  Classification and Antigenic Types Antigenic differences have been demonstrated between organisms obtained from humans and from lower mammals such as the rat, rabbit, and ferret. Pathogenesis  The portal of entry for P carinii is a likely inhalation.  Airborne transmission has been demonstrated in animals.  In most individuals, the organism is dormant and sparsely dispersed in the lung, with no apparent host response (latent infection). I  n susceptible (immunocompromised) hosts, the organism occurs in massive numbers, filling the alveolar spaces and eliciting an active response of the alveolar macrophages and phagocytosis.  In debilitated infants with Pneumocystis pneumonia, the alveolar septum is thickened and there is an interstitial plasma cell and lymphocyte infiltration.  The infection results in impaired ventilation and severe hypoxia. Host defenses     With rare exceptions, P carinii causes disease only when natural mechanisms of host defense are compromised. Pneumonitis tends to occur in patients with impaired cell-mediated immunity. Both IgG and IgM antibody may appear in response to infection or experimental immunization, but humoral antibody does not protect against the disease. Alveolar macrophages actively engulf and digest the parasite in the presence of specific antibody. Infected infants show extensive plasma cell infiltration of the alveolar septae, but immunosuppressed children and adults do not. Epidemiology    Pneumocystis carinii has been found in the lungs of rats, rabbits, mice, dogs, sheep, goats, ferrets, chimpanzees, guinea pigs, horses, and monkeys. The organism has been reported in lower animals and humans from all continents. Animal-to-animal transmission by the airborne route has been demonstrated. Diagnosis    Diagnosis requires the identification of P carinii in pulmonary tissue or lower airway fluids. The Gomori, Giemsa, fluorescence-labelled antibody, or toluidine blue O stains may be used to identify the organism. Serologic studies for antibodies and antigen are not helpful in establishing a specific diagnosis. Control 272      Experimental studies show that immunization with P carinii does not protect the animal from pneumonia The disease can be prevented by prophylactic administration of trimethoprimsulfamethoxazole, aerosolized pentamidine or dapsone. Four drugs currently available for therapy of P carinii pneumonitis are pentamidine isethionate, trimethoprim-sulfamethoxazole, atovaquone and trimetrevate. Trimethoprim-sulfamethoxazole is preferred because of its low toxicity and greater efficacy. Acute, fatal infections with this parasite are also recorded in a number of captive "coatimundis", Nasua narica (Carnivora: Procyonidae) and a sloth, Bradypus tridactylus (Edentata). Pneumocystis was also encountered in lung smears from a newly captured and apparently healthy sloth, Choloepus didactylus. Zoonotic aspect   Pneumocystis carinii is a well known cause of fatal, interstitial plasma-cell pneumonia in human infants and sometimes the weakened adult: The keeping of exotic pets such as the coatimundi is, therefore, not without some hazard in this respect. Reports: Lainson and Shaw (1975) detected Pneumocystis carinii in lung smears from a newly captured Oryzomys capito (Cricetidae). Laakkonen (1995) mentioned that Pneumocystis carinii is a pulmonary pathogen causing a fatal pneumonia (PCP) in immunocompromised hosts. Notable interspecific differences in the prevalence of P. carinii have been observed in small mammals in Finland. Although seasonal peaks (up to 30 %) occur in some species, prevalences usually were low (< 10 %). On the contrary, 40 % to 70 % of the common shrews, Sorer araneus, had the cyst form of P. carinii. The prevalence was high in all sex and age groups, in all seasons and throughout the country. Despite the high prevalence, no signs of PCP have been detected, and the infected shrews are apparently healthy. The occurrence of cysts has no significant relation to the histological changes detected in the lungs of the shrews. The number of cysts found per gram of lung tissue in wild S. araneus was low compared to those of clinically ill rats, but seems on the average to be higher than those found in Microtus agrestis caught in the same area. The interspecific differences in the prevalence of P. carinii infection observed in shrews and other wild mammals are most likely due to the strong species specificity of P. carinii. 273 common shrews, Sorer araneus. ARKive Wu Deming (1996) induced pneeumocystis carinii pneumonia (PCP) in Wistar rats injected subcuteneously with cortisone acetate twice a week for 12 weeks. From the 6th to 1 2th week, two rats were necropsied weekly. A development of PCP was found in experimental rats, Pathological changes showed:(1) the PC cysts and trophozoites were identified in impression smears,(2) histopathologic changes in HE staining sections were: from the 6th to 8th week,interstitial pneumonitis with moderate infiltration of lymphocytes and the absence of foamy intra-alveolar exudate; from the 9th to 12th week, the identification of foamy intraalveolar exudate.With PAS staining, the exudate showed positive reaction. Black cysts were found in GMS staining sections. (3) After 4～6weeks of stopping injection with cortisone acetate,the PCP mended. It showed that the suffered rats may cure without any treatments. Electronic microscopy showed ultrastructure of PC cysts and trophozoites and injured alveolar epithelial cells. Laakkonen (1998) summarised the present state of knowledge on the occurrence of P. carinii in wild mammals in their natural habitats, and briefly discussed various characteristics of P. carinii infection important for understanding the distribution and abundance of this organism. Some aspects of P. carinii infection in wild hosts of particular interest for future research in this field were discussed. Icenhour et al. (2001) conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of pneumocystis in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke Pneumocystis growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of Pneumocystis by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of Pneumocystis carinii f. sp. carinii. Upon receipt, 64% of rats were positive for P. carinii f. sp. carinii-specific antibodies, while P. carinii f. sp. carinii DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed P. carinii f. sp. carinii DNA in all rat lungs, illustrating widespread occurrence of Pneumocystis in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of Pneumocystis exposure. 274 Laakkonen et al. (2001) detected cyst forms of the opportunistic fungal parasite Pneumocystis carinii in the lungs of 34% of the desert shrew, Notiosorex crawfordi (n 5 59), 13% of the ornate shrew, Sorex ornatus (n 5 55), 6% of the dusky-footed wood rat, Neotoma fuscipes (n 5 16), 2.5% of the California meadow vole, Microtus californicus (n 5 40), and 50% of the California pocket mouse, Chaetodipus californicus (n 5 2) caught from southern California between February 1998 and February 2000. Cysts were not found in any of the harvest mouse, Reithrodontomys megalotis (n 5 21), California mouse, Peromyscus californicus (n 5 20), brush mouse, Peromyscus boylii (n 5 7) or deer mouse, Peromyscus maniculatus (n 5 4) examined. All infections were mild; extrapulmonary infections were not observed. Notiosorex crawfordi www.wtamu.edu Ornate Shrew, Sorex ornatus Natural History of Orange County, California dusky-footed wood rat, Neotoma fuscipes, UniProt California vole - Wikipedia Chaetodipus californicus Sharif et al. (2006) stated that Pneumocystis carinii (P. carinii) organisms constitute a large group of heterogenous atypical microscopic fungi that are able to infect immunocompromised mammals and proliferate in their lungs, inducing Pneumocystis carinii pneumonia. Despite intensive investigation in human and animal hosts, information on the occurrence and nature of infections in wild animals is scarce, although characterization of infections in wild-animal populations may help to elucidate the life-cycle and transmission of this elusive organism. Due to the interspecific with P. carinii in differences in prevalence and intensity of P. carinii infection and to the antigenic and genetic diversity of P. carinii organisms originating 275 from various host species, which may affect the infectivity and pathogenicity of these organisms, one should be cautious when making generalizations about the nature of P. carinii infection. This study conducted to find out the prevalence of infection in wild rats in their natural habits and also in immunosuppressed rats in Sari, Mazandaran Province of Iran. Albers et al. (2009) introduced 104 Wistar Han rats, free of known pathogens and of RRV-associated lesions, into a rat production colony positive for RRV-type lesions, but free of other histologic, serologic, or microbiologic evidence of infectious disease. Lungs of 8 of the naïve rats were examined grossly and microscopically each week, weeks 0-13. Irregular gray-white lesions suggestive of interstitial pneumonia were grossly evident from weeks 6 through 13. Primary histopathologic evaluation of all lungs by one pathologist found multifocal, lymphohistiocytic interstitial pneumonia or prominent perivascular lymphoid cuffing from weeks 5 through 13. Based on results of the initial evaluation, diagnostic criteria for RRV infection (i.e., changes seen only after exposure to the RRV-positive colony) were tentatively selected and used by 2 other pathologists to classify each lung as RRV positive, RRV equivocal, or RRV negative. The secondary evaluation found 95% concordance in RRV diagnosis between pathologists, and correlated well with the initial evaluation, thus confirming the consistency of the criteria. These data showed that RRV-naïve rats introduced into an RRV-endemic colony develop equivocal microscopic lesions of RRV by 5 weeks of exposure, and positive diagnostic lesions by 7 weeks. Interstitial pneumonia becomes grossly evident after 6 weeks of exposure. Cavallini Sanches et al. (2009) detected Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10RRI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%) and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil. 276 Tadarida brasiliensi Smithsonian National Museum of Natural History, Vampire Bat Desmodus Rotundus Portrait by Michael & Patricia Fogden Nyctinomops laticaudatus | by Setsuo Tahara 277 Livingston et al. (2011) conducted a study to determine whether Pneumocystis carinii infection in immunocompetent rats can cause idiopathic lung lesions previously attributed to RRV. In archived paraffin-embedded lungs (n = 43), a significant association was seen between idiopathic lung lesions and Pneumocystis DNA detected by PCR. In experimental studies, lung lesions of RRV developed in 9 of 10 CD rats 5 wk after intratracheal inoculation with P. carinii. No lung lesions developed in CD rats (n = 10) dosed with a 0.22-µm filtrate of the P. carinii inoculum, thus ruling out viral etiologies, or in sham-inoculated rats (n = 6). Moreover, 13 of 16 CD rats cohoused with immunosuppressed rats inoculated with P. carinii developed characteristic lung lesions from 3 to 7 wk after cohousing, whereas 278 no lesions developed in rats cohoused with immunosuppressed sham-inoculated rats (n = 7). Both experimental infection studies revealed a statistically significant association between lung lesion development and exposure to P. carinii. These data strongly support the conclusion that P. carinii infection in rats causes lung lesions that previously have been attributed to RRV. Photomicrographs of lungs from (A, B) immunocompetent F344 rat with naturally occurring idiopathic interstitial pneumonia consistent with so-called RRV and (C, D) immunocompetent CD rat 5 wk after intratracheal inoculation with P. carinii. Note that lesions induced from P. carinii inoculation are indistinguishable from those of naturally acquired lesions previously attributed to RRV infection, presented in panels A and B. (E, F) Lungs from immunocompetent CD rat 5 wk after intratracheal inoculation with P. carinii inoculum passed through a 0.22-µm filter. Note the absence of lesions in these rats inoculated with a preparation that excludes P. carinii but not viruses. Hematoxylin and eosin stain. Bar, 200 µm (A, C, E); 50 µm (B, D, F). Panels B, D, and F are higher-magnification images of panels A, C, and E, respectively. (A through F) Photomicrographs of lungs from immunocompetent CD rats cohoused with P. carinii-inoculated immunosuppressed rats at 3, 5 and 7 wk after cohousing. Note the progression of lesions over time. (A, B) 3 wk after cohousing: mild perivascular lymphohistiocytic infiltrates with eosinophils and minimal lymphohistiocytic interstitial pneumonia. (C, D) 5 wk after cohousing: marked multifocal perivascular lymphohistiocytic infiltrates and severe locally extensive lymphohistiocytic interstitial pneumonia. (E, F) 7 wk after cohousing: mild perivascular lymphohistiocytic infiltrates and minimal lymphohistiocytic interstitial pneumonia. (G through L) Photomicrographs of lungs from immunocompetent CD rats cohoused with sham-inoculated rats at 3, 5 and 7 wk after cohousing. Note 279 the absence of lesions at all time points. Hematoxylin and eosin stain. Bar, 200 µm (A, C, E, G, I, K); 50 µm (B, D, F, H, J, L). Panels B, D, F, H, J, and L are higher-magnification images of panels A, C, E, G, I, and K, respectively. Kim et al. (2014) investigated whether the pulmonary lesions observed were caused by P. carinii infection. Male Sprague?Dawley rats, free of known pathogens, were introduced into a rat colony positive for RRV?type lesions. Routine histopathological examinations were performed on the rat lung tissues following exposure. The presence of Pneumocystis organisms was confirmed using Grocott's methenamine silver (GMS) staining. At week 3 following introduction, a few small lymphoid aggregates were located adjacent to the edematous vascular sheath. By week 5, foci of dense perivascular lymphoid cuffing were observed. Multifocal lymphohistiocytic interstitial pneumonia and prominent lymphoid perivascular cuffs were observed between week 7 and 10. GMS staining confirmed the presence of Pneumocystis cysts. Thus, the results of the present study demonstrated that P. carinii caused lymphohistiocytic interstitial pneumonia in a group of laboratory rats. The observations strongly support the conclusion that P. carinii infection in immunocompetent laboratory rats causes the lung lesions that were previously attributed to RRV. Histopathology of PCP in immunocompetent laboratory rats. (A and B) At week 3 following introduction, the earliest change was the appearance of edema with a few small lymphoid aggregates located adjacent to the edematous vascular sheath (single arrows). (C and D) At week 5, dense lymphoid cuffs surrounded the blood vessels and the adjacent alveolar septa were minimally infiltrated by lymphocytes. (E) At week 7, areas of lymphohistiocytic interstitial pneumonia showing alveoli infiltrated by lymphocytes and macrophages and thickened alveolar septa were observed. Dense perivascular lymphoid cuffs were more frequently identified (arrowheads). (F) At week 10, thick bands of lymphocytes and macrophages encircled the blood vessels adjacent to the areas of alveolar septal thickening and leukocytic infiltrates. (G) Lymphohistiocytic interstitial inflammation led to septal thickening with extensive alveolar leukocytic infiltrates and the formation of dense cuffs and aggregates of lymphocytes around the blood vessels. Foamy eosinophilic exudates were also observed (double arrows) and a number of GMS-positive Pneumocystis cysts (inset), characteristic of PCP, were identified in the same areas as the interstitial pneumonia. (A–G) Hematoxylin and eosin staining and (G; inset) GMS staining. (A and B) magnification, ×40; (C–G) magnification, ×100; and (G; inset) magnification, ×400. PCP, Pneumocystis carinii pneumonia; GMS, Grocott‘s methenamine silver. 280 References: 1. Albers TM, Simon MA, Clifford CB. 2009. Histopathology of naturally transmitted ‗rat respiratory virus‘: progression of lesions and proposed diagnostic criteria. 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Genetic stability and diversity of Pneumocystis carinii infecting rat colonies. Infect Immun. 1993;61:4801–4813. 7. Deerberg F, Pohlmeyer G, Wullenweber M, Hedrich HJ. History and pathology of an enzootic Pneumocystis carinii pneumonia in athymic Han:RNU and Han:NZNU rats. J Exp Anim Sci. 1993;36:1–11.[ 8. Elwell MR, Mahler JF, Rao GN. 1997. Have you seen this? Inflammatory lesions in the lungs of rats. Toxicol Pathol 25:529–531 9. Farrar P, LaRegina M. 1997. Diagnostic exercise: interstitial pneumonia in viral and mycoplasmal antibody-free Sprague Dawley rats. ACLAD News 18:5–9. 10. Gigliotti F, Harmsen AG, Wright TW. Characterization of transmission of Pneumocystis carinii f. sp muris through immunocompetent BALB/c mice. Infect Immun. 2003;71:3852–3856. 11. Gilbert BE, Black MB, Waldrep JC, Bennick JB, Montgomery C, Knight V. 1997. Cyclosporin A liposome aerosol: lack of acute toxicity in rats with a high incidence of underlying pneumonitis. Inhal Toxicol9:717–730. 12. González-González AE, Aliouat-Denis CM, Ramírez-Bárcenas JA, Demanche C, Pottier M, Carreto-Binaghi LE, Akbar H, Derouiche S, Chabé M, Aliouat el M, Dei-Cas E, Taylor ML. Histoplasma capsulatum and Pneumocystis spp. coinfection in wild bats from Argentina, French Guyana, and Mexico. BMC Microbiol. 2014 Feb 5;14:23. 13. Icenhour CR, Rebholz SL, Collins MS, Cushion MT. Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs. J Clin Microbiol. 2001 Oct;39(10):3437-41. 14. Henderson KS, Dole V, Parker NJ, et al. Pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to ‗rat respiratory virus‘ Vet Pathol. 2012;49:440–452. 15. Hughes WT. Natural mode of acquisition for de novo infection with Pneumocystis carinii. J Infect Dis. 1982;145:842–848. 16. Icenhour CR, Rebholz SL, Collins MS, Cushion MT. Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs. J Clin Microbiol. 2001;39:3437–3441. 17. Kim H-S, Do S-I, Kim Yw. Histopathology of Pneumocystis carinii pneumonia in immunocompetent laboratory rats. Experimental and Therapeutic Medicine. 2014;8(2):442-446. doi:10.3892/etm.2014.1732. 18. Laakkonen. J. Characterization of Pneumocystis carinii infection in Sorex araneus – A review Mammalia 59(4):623-628 · January 1995 19. Laakkonen J Pneumocystis carinii in wildlife. International Journal for Parasitology [1998, 28(2):241-252] 281 20. Laakkonen, Juha, Robert N. Fisher, and Ted J. Pneumocystosis in Wild Small Mammals from California Author(s): Journal of Wildlife Diseases, 37(2):408-408, 2001. 21. Lainson R, Shaw JJ. Pneumocystis and Histoplasma infections in wild animals from the Amazon region of Brazil. Trans R Soc Trop Med Hyg. 1975;69(5-6):505-8. 22. Livingston RS, Besch-Williford CL, Myles MH, et al. Pneumocystis carinii infection causes lung lesions historically attributed to rat respiratory virus. Comp Med. 2011;61:45–59. 23. Pohlmeyer G, Deerberg F. Nude rats as a model of natural Pneumocystis carinii pneumonia: sequential morphological study of lung lesions. J Comp Pathol. 1993;109:217–230. [PubMed] 24. Pritchett-Corning KR, Cosentino J, Clifford CB. Contemporary prevalence of infectious agents in laboratory mice and rats. Lab Anim. 2009;43:165–173. 25. Riley LK, Purdy G, Dodds J, Franklin C, Besch-Williford C, Hook RR, Wagner JE. 1997. Idiopathic lung lesions in rats: search for an etiologic agent. Contemp Top Lab Anim Sci 36:46 26. Riley LK, Simmons JH, Purdy G, Livingston R, Franklin C, Besch-Williford C. 1999. Research update: idiopathic lung lesions in rats. ACLAD Newsl 20:9–11 27. Sharif , M., H. Ziaei , A. Daryani , M. Nasrolahei and B. Lackterashi , 2006. Epidemiological Survey of Pneumocystis Carinii in Rodents of Sari, Northern Iran. Journal of Animal and Veterinary Advances, 5: 390-394. 28. Slaoui M, Dreef HC, van Esch E. 1998. Inflammatory lesions in the lungs of Wistar rats. Toxicol Pathol26:712–713. 29. Soulez B, Dei-Cas E, Charet P, et al. The young rabbit: a nonimmunosuppressed model for Pneumocystis carinii pneumonia. J Infect Dis. 1989;160:355–356. 30. Vestereng VH, Bishop LR, Hernandez B, et al. Quantitative real-time polymerase chain-reaction assay allows characterization of Pneumocystis infection in immunocompetent mice. J Infect Dis. 2004;189:1540–1544. 31. Walzer PD, Schnelle V, Armstrong D, Rosen PP. Nude mouse: a new experimental model for Pneumocystis carinii infection. Science. 1977;197:177–179. 32. Walzer PD, Schultz MG, Western KA, Robbins JF. Pneumocystis carinii pneumonia and primary immune deficiency diseases. Natl Cancer Inst Monogr. 1976;43:65–74. 33. Weisbroth SH, Geistfeld J, Weisbroth SP, et al. Latent Pneumocystis carinii infection in commercial rat colonies: comparison of inductive immunosuppressants plus histopathology, PCR, and serology as detection methods. J Clin Microbiol. 1999;37:1441–1446. 34. Wu Deming . Experimental study on pathology of pneeumocystis carinii pneumonia (PCP). Chinese Journal Of Clinical And Experimental Patholog 1996-02 282 17. Adiaspiromycosis infection in wild animals     Adiaspiromycosis is a rare chronic pulmonary infection caused by dimorphic fungi from the genus Emmonsia within the family Ajellomycetaceae. The infection is characterized by the presence of very large adiaspores in the lungs. Inhaled conidia of Emmonsia produced from the mycelial phase growing at ambient temperatures fail to germinate in the lung, and instead simply increase in volume to form thick-walled, non-replicating adiaspores. Ecology and epidemiology    Emmonsia species are commonly found in soil but also occur in mammalian species living in close association with soils such as rodents, insectivores, otters, stoats, weasels, moles and ground squirrels. o E. . parva var. crescens is widespread in continental Europe and UK o E. parva is found mainly in some xerothermic regions, including parts of the Americas, Central Asia and Africa. Emmonsia species have an extremely broad host range and infections have been reported in many species of small mammals, worldwide. The frequency of infected animals varies according to geographic area, altitude, habitat, and season. Several studies clearly indicated that almost 30% of all small wild mammals examined in the UK and parts of central and eastern Europe where it is endemic, had evidence of infection, raising the possibility that sporadic infections might also occur in domestic animals and humans. Mode of transmission Similar to other dimorphic onygenalean fungi, Emmonsia species are environmental pathogens, having a life cycle involving soil and vectoring by the animals. Histopathological examination of the infected animals generally reveals a multifocal extensive granulomatous reaction containing oval adiaspores scattered irregularly throughout the lungs. Adiaspiromycosis in man     Adiaspiromycosis is a pulmonary fungal infection caused by the dimorphic fungi, Emmonsia parva or Emmonsia crescens . Large globose, thick-walled, non-proliferating structures called adiaspore are seen in infected tissue. The term adiaspore was derived from the Greek verb speirein for scattering, with adia being a negative, so adiaspiromycosis describes an infection in which there is no multiplication or dissemination of the fungus from the original site. The infection's pathological effects range from asymptomatic infection to necrogranulomatous pneumonia and death, depending on the burden of adiaspore and host immunocompetence 283        Pulmonary adiaspiromycosis is associated with inhaled conidia of soil fungi Chrysosporium (Emmonsia) parvum or Chrysosporium parvum var. crescens, with resultant pulmonary granulomas. Inhalated E. crescens develops into large, thick-walled spherules called adiaspores, measuring as much as 700 µm, and originating from minute (2~4 µm) subglobose conidia. Infectious E. crescens cannot germinate at the elevated temperatures of the host, and instead increases in volume to form thick-walled, non-replicating adiaspores that elicit extensive granulomatous reaction. Expanding adiaspores cause collapse of the adjacent alveoli and respiratory distress or even failure. Adiapiromycosis patients have a chronic history of progressive dyspnea, nonproduction cough, fatigue, low-grade fever and, less frequently, with hemophysis, pain, chills, malaise, weight loss and auscultatory crackles In addition to the pulmonary organ, Emmonsia crescens can cause cutaneous adiaspiromycosis and associated acute conjunctivitis. In many of the reported cases, Emmonsia crescens infection was related to play in the surroundings of an animal burrow, which may have played the role of a reservoir, and other outdoor activities. Adiaspiromycosis in animals   Rare pulmonary infections associated with adiaspiromycosis have been reported in squirrels, armadillos, mice, rats, muskrats, beavers, rabbits, mink, martins, skunks, weasels, fox, Japanese pika, Australian hairy-nosed wombats, raccoons, goats, dogs, and humans. Clinical signs are not significant in rodents. Diagnosis of adiaspiromycosis    is difficult because the fungus is not easily cultured. Histological observation of characteristic adiaspores with light microscopy Emmonsia parva and Emmonsia crescens are morphologically indistinguishable in their mycelial phases, which makes differential diagnosis difficult. o E. crescens produces multinucleate adiaspores at temperatures above 30-37℃ (depending on the isolate) which routinely reach diameters in excess of 500 µm , o Emmonsia parva isolates produce adiaspores that are mononucleate and substantially smaller (20-40 µm in diameter) only at temperatures approaching or in excess of 40℃. Animals reported to be infected with adiaspiromycosis 1. 2. 3. 4. 5. ground squirrels Citellus richardsoni Leighton and Wobeser (1978) ground squirrels C. tridecemlineatus Leighton and Wobeser (1978) ground squirrels C. franklini Leighton and Wobeser (1978) fox (Vulpes vulpes) Otcenásek et al. (1975), Krivanec et al. (1976) badger (Meles meles) Krivanec et al. (1976) 284 6. the otter (Lutra lutra) Krivanec et al. (1976) 7. Tasmanian wombats Krivanec and Mason (1980) 8. Hairy-nosed wombats (Lasiorhinus latifrons) Mason and Gauhwin (1982) 9. Japanese pikas (Ochotona hyperborea yesoensis) Taniyama et al. (1985) 10. striped skunks (Mephitis mephitis) Mudher et al. (1986) 11. wallaby Hamir (1999) 12. Opossum Hamir (1999) 13. bullfrogs (Rana catesbeiana) Hill et al. (1996) 14. Clethrionomys glareolus Hubálek (1999) 15. Arvicola terrestris Hubálek (1999) 16. Apodemus flavicollis Hubálek (1999) 17. Apodemus sylvaticus Hubálek (1999) 18. Apodemus microps Hubálek (1999) 19. Microtus subterraneus Hubálek (1999) 20. Microtus arvalis Hubálek (1999) 21. Microtus agrestis Hubálek (1999) 22. Ondatra zibethicus Hubálek (1999) 23. Cricetus cricetus Hubálek (1999) 24. Crocidura suaveolens Hubálek (1999) 25. Neomys fodiens Hubálek (1999) 26. Sorex araneus Hubálek (1999) 27. European beaver (Castor fiber) Mörner et al. (1999) 28. immature otter Simpson et al. (2000) 29. European hedgehog Seixas et al. (2006) 30. Apodemus agrarius Kim et al. (2012) 31. crested porcupine (Hystrix cristata) Morandi et al. (2012) 32. Eurasian otter Malatesta et al. (2014) 33. Hokkaido sika deer Matsuda et al. (2015) Alamy Ground Squirrel (Citellus richardsoni) ground Squirrel (Citellus tridecemlineatus) Franklin's ground squirrel BioWeb Home Rana catesbeiana Gary 285 Hlasek Arvicola terrestris Nature-CZ.com Yellow-necked Field Mouse. Hlasek Apodemus agrariu BioLib Apodemus uralensis ASM Microtus subterraneus Microtus arvalis Saxifraga Microtus agrestis | NaturePhoto Dreamstime.com (Ondatra Zibethicus) Cricetus cricetus hamsterUniProt BioLib Crocidura suaveolens 123RF.com (Neomys fodiens) Sorex araneus NaturePhoto-CZ.com Eurasian Beaver, Alamy Otter - Animal Facts Apodemus agrarius by Dark-Raptor . ARKive North African crested porcupine Otter Facts Eurasian Otter 286 E uropean hedgehog - Wikiwand Fox (Vulpes vulpes YouTube European badger (Meles meles) wildaboutbritain.co.uk Otter Lutra Lutra Tasmanian wombats .natureworld. Ryan Lasiorhinus latifrons hairy-nosed wombat Pinterest Pika Ochotona hyperborea TOM CLARK: Striped Skunk wallaby - Wikipedia Sika deer - Wikipedia 287 Opossum - Wikipedia Aetiology: o Emmonsia crescens o Emmonsia parva. o Emmonsia pasteuriana Description: Emmonsia parva var. crescens (C.W. Emmons & Jellison) Oorschot, Studies in Mycology 20: 59 (1980) Synonyms =Chrysosporium parvum var. crescens (C.W. Emmons & Jellison) J.W. Carmich., Canadian Journal of Botany 40 (8): 1164 (1962) =Emmonsia parva var. crescens (C.W. Emmons & Jellison) Oorschot, Studies in Mycology 20: 59 (1980) Colonies (SGA, 24°C) with moderate growth, white to pale brown, often with yellowish or reddish tinge, glabrous, later cottony; reverse pale greyish-brown brown to reddish-brown. At 37°C on BHI colonies butryous, cerebriform. Microscopy. Conidia at 24°C sessile or on slender stalks or terminal inflations, subhyaline, verrucose or smooth-walled, thin-walled, 1-celled, (sub)spherical, 2-5 — 2-4 ?m, with narrow basal scars. At 37°C on BHI the blastic conidia swell and become spherical, thick-walled, 20-140 ?m diam (adiaspores). Adiaspores produced in vivo multinucleate, 200-700 Mycobank Emmonsia parva CIFERRI et MONTEMARTINI, 1959 Synonyms: Chrysosporium parvum EMMONS et ASHBURN 1942 288 Haplosporangium parvum EMMONS et ASHBURN 1942 Emmonsia crescens EMMONS et JELLISON, 1960 Emmonsia parva grows moderately rapidly and produces glabrous to velvety white to buff to pale brown colonies. Microscopically, septate hyaline hyphae, conidiophores, and aleurioconidia are seen. Conidiophores are simple or may occasionally branch at right angles. Conidia are sessile or are located on slender stalks. They are unicellular, thin-walled, (sub) spherical, and 2-5 x 2-4 µm in size. These conidia are found solitary or may form twoto three-celled chains. At 37-40°C, the conidia tend to swell and give rise to thick-walled, big, liberated conidia known as adiaspores. Adiaspores are formed only at 37-40°C and on blood or brain heart infusion agar in vitro Emergomyces pasteurianus (Drouhet, Guého & Gori) Dukik, Sigler & de Hoog, comb. nov Basionym: Emmonsia pasteuriana Drouhet, Guého & Gori, in Drouhet, Guého, Gori, Huerre, Provost, Borgers & Dupont − J. Mycol. Méd. 8: 90, 1998 Colonies at 24°C on MEA whitish, composed of hyaline hyphae. Conidiophores short, slender, unbranched, arising at right angles from narrow hyphae and slightly swollen at the tip; bearing 1-3 (up to 8) conidia on short thin pedicels or sometimes sessile alongside hyphae. Conidia subhyaline, slightly verruculose, thin-walled, onecelled, subspherical, 2-3 × 3-4 μm. At 37°C, budding cells present which are ellipsoidal, 2-4 μm in length, budding at a narrow base, in addition to broad-based budding cells. Physiology: minimum growth temperature 6°C, optimum 24°C, reaching 35 mm diameter, maximum 40°C. Reports: Otcenásek et al. (1975) reported on adiaspiromycosis of a fox (Vulpes vulpes) caused by Emmonsia parva. Species diagnosis was made on the basis of dimensions of the fungus found in the lungs of the animal, the histopathological picture of tissue changes and the properties of the isolated culture. The identification of the fungus was also confirmed by experimental infection. Krivanec et al. (1976) diagnosed adiaspiromycosis in 6 animals in the examination of the lungs of 90 large carnivores. Emmonsia crescens (Chrysosporium parvum var. crescens) was demonstrated as the causative agent in 5 cases of disease-in the badger 289 (Meles meles), the otter (Lutra lutra) and the fox (Vulpes vulpes). E. parva was demonstrated in the remaining case of disease in a fox. The badger is a new, up to the present unknown host of E. crescens. The sporadic occurrence of adiaspiromycosis in the fox and the otter classifies this disease among rare diseases of this animals. Leighton and Wobeser (1978) examined lungs from three species of ground squirrels collected in south central Saskatchewan by histopathology and a digestion technique for adiaspores of Emmonsia crescens. Two of 81 (2.5%) Citellus richardsoni, 3 of 17 (17.6%) C. tridecemlineatus and 35 of 44 (79.5%) C. franklini were infected. Infection was more common in adults than in young-of-the-year. Tissue digestion was the more sensitive method for detecting adiaspores. Possible reasons for the difference in prevalence among the species are discussed. Krivanec and Mason (1980) described the finding of small spherules in the lungs of two species of wombats from Tasmania. A histological examination of lung tissue caused adiaspiromycosis to be suspected and the etiological agent was thought to Chrysosporium parvum. Mason and Gauhwin (1982) detected pherical organisms, with an average diameter of about 22 microns, in the lungs of adult and pouched young hairy-nosed wombats (Lasiorhinus latifrons). Although infections of up to 640 X 10(3) organisms per cubic centimeter were detected, their presence produced only limited pathological change. In-vitro growth was obtained at 30 C but not at 37 C or 40 C. However, at the higher temperatures, typical chlamydospore spherules were produced by colonies initially grown at 30 c. This report presents the first record of adiaspiromycosis in Australia and in wombats. Taniyama et al. (1985) described the morphology of pulmonary adiaspiromycosis caused by Chryso-sporium parvum var. crescens in two Japanese pikas (Ochotona hyperborea yesoensis Kishida). Mudher et al. (1986) diagnosed pulmonary adiaspiromycosis in seven of 25 striped skunks (Mephitis mephitis) in east-central Alberta. The infection varied from mild, where only microscopic lesions were seen, to severe, where gross lesions of grayishwhite nodules were observed in the lung parenchyma. Mild lesions were restricted to the lung, while severe lesions extended to the tracheobronchial and mediastinal lymph nodes. Histologically, the lesions were characterized by a centrally located fungal spherule, surrounded by granulomatous inflammation. The morphology of the fungal spherules was consistent with that of Emmonsiu crescens. By electron microscopy, the fungal cells had an outer thick fibrillar wall and an inner cytoplasm filled with large lipid vacuoles with relatively few mitochondria, ribosomes or glycogen inclusions. The absence of endosporulation and budding suggested that each fungal cell in the lung represented a separate inhaled spore. Infection was by inhalation, nevertheless adiaspores may disseminate to the regional lymph nodes. 290 Formalin-fixed lung of mature striped skunk heavily infected with adiaspores. Note multiple nodules distributed throughout the lung. Histological section of three fungal spherules in the lung of a striped skunk. Note the thick fungal cell wall with globular central mass and variable inflammatory reaction around fungal cells. H&E, ~109 Histological section of a fungal spherule in the lung of a striped skunk. Note focal thinning and destruction of the wall with marked intlammatory cell infiltration. H&E, x435. Histological section of a fungal spherule in the mediastinal lymph node of a striped skunk. Note the mild granulomatous inflammatory reaction around the spherule. H&E, x 109. Hill et al. (1996) described adiaspores of Chrysoporium sp. found in skeletal muscles of bullfrogs (Rana catesbeiana). Bullfrogs were obtained from a small farm pond, and rear legs were skinned in preparation for cooking. Discoloration of leg muscles was observed, and affected tissues were presented to the Clemson University Animal Diagnostic Laboratory for examination. Muscles of 6 rear legs contained randomly scattered flattened dark red to blue irregular lesions up to 1 cm in diameter. Affected muscle was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 µm, stained with hematoxylin and eosin (HE), and examined by light microscopy. Multiple thick-walled oval organisms up to 240 µm in diameter were randomly scattered throughout the leg muscles. Cell walls of the organisms stained palely eosinophilic with periodic acid-Schiff (PAS) reaction and dark black with Grocott‘s methenamine silver (GMS). Hamir (1999) examined 63 adult raccoons found dead on highways (roadkills), submitted as rabies suspects, or obtained from local wildlife control organizations. Representative tissues of all major organs were immersion fixed in 10% buffered formalin. Only findings relevant to the presence of spherules of Chrysosporium parvum in lungs were reported here. A large thick-walled spherule of Chrysosporium parvum was present within a granuloma. The fungal spherule did not contain 291 endospores and was surrounded by epithelioid macrophages and multinucleate giant cells. Selected sections containing fungal spherules were also stained with periodic acid–Schiff (PAS), Go¨mo¨ri‘s methenamine silver (GMS), and mucicarmine to differentiate C. parvum spherules from the fungal spherules of Coccidioides immitis and Rhinosporidium seeberi. 3 Histologic evidence of Chrysosporium parvum was seen in lung tissue sections of 5 raccoons (3 males and 2 females). Gross lesions of the infection were not seen in any of the affected lungs. Microscopic lesions were present in the alveolar tissue and consisted of various numbers (1–5 per tissue section) of multifocal, randomly distributed granulomas containing epithelioid macrophages and lesser numbers of multinucleate giant cells. Within the centers of these inflammatory foci was usually 1 large (up to 300 mm) fungal spherule (adiaconida) composed of fine granular eosinophilic material and surrounded by a thick laminated capsule. The capsule stained positive with PAS and GMS and was negative by mucicarmine. Lung, raccoon. A large thick-walled spherule of Chrysosporium parvum is present within a granuloma. HE. 100X. Lung, raccoon. Higher magnification of Fig. 1. The fungal spherule does not contain endospores and is surrounded by epithelioid macrophages and multinucleate giant cells. HE. 240X . Hubálek (1999) detected adiaspores of the fungus Emmonsia crescens microscopically in the lung tissue of 13% of 10.081 small mammals belonging to 24 species examined in 14 areas of the Czech Republic between 1986 and 1997; 441/1.934 (23%) Clethrionomys glareolus, 1/6 (17%) Arvicola terrestris, 357/2.172 (16%) Apodemus flavicollis, 220/1.981 (11%). A sylvaticus, 23/265 (9%) A. microps, 11/81 (14%) Microtus subterraneus, 93/1.275 (7%) M. arvalis, 98/1.439 (7%) M. agrestis, 1/3 (33%) Ondatra zibethicus, 1/1 Cricetus cricetus, 1/20 (5%) Crocidura suaveolens, 2/40 (5%) Neomys fodiens, and 13/529 (2%) Sorex araneus were infected. Emmonsiosis was not recorded among the species of rodents that do not build their nests in the soil (Muscardinus avellanarius, Micromys minutus, Mus musculus, Rattus norvegicus). The overall prevalence of emmonsiosis was significantly higher in adult (19%) than in juvenile (7%) mammals, and in rodents (13%, and 20% in adults) than in insectivores (2%, and 4% in adults). The frequency of infected mammals also varied according to geographic area, altitude, habitat, and season. Mörner et al. (1999) diagnosed an infection with the rare mycosis Chrysosporium parvum in a European beaver (Castor fiber) shot in northern Sweden. The animal was in normal body condition and no signs of disease were observed. In the lungs a large number of nodules, up to 5 mm diameter, were 292 observed. A large number of adiaspores were observed in the interstitium of the lungs and in the mediastinal lymph node. A chronic inflammatory reaction dominated by mononuclear leukocytes and giant cells was observed around the spores. This is the first report of adiaspiromycosis (Chrysosporium parvum) in the European beaver. Simpson et al. (2000) described the postmortem examination of an immature otter which died in the wild which showed that large areas of the lungs were swollen and firm, with emphysema and haemorrhage in the remaining areas. Histopathological examination revealed large numbers of fungal adiaspores and an unusually severe inflammatory response. It was considered that respiratory impairment was the primary cause of the otter's death. Seixas et al. (2006) reported pulmonary adiaspiromycosis in a European hedgehog (Erinaceus europaeus) in Portugal. Borman et al. (2009) reported the results of histopathological examination of lungs of free-living wild mammals from the south-west UK coupled with digestion of lung material in potassium hydroxide followed by centrifugation and microscopic examination for the presence of adiaspores. The combined results showed that almost one-third (27/94, 28.7%) of animals examined had evidence of infection with E. crescens. Attempts to culture E. crescens from infected lungs were unsuccessful. However, E. crescens could be confirmed as the causative agent by PCR amplification and sequencing of DNA from adiaspores micro-dissected from animal lungs. The prevalence of adiaspiromycosis was largely independent of animal species or precise geography. Adiaspore burdens in most animals were low, consistent with transient exposure to E. crescens. However, burdens in several animals suggested heavy or repeated exposures to E. crescens, and were considered sufficient to have significantly impaired respiratory function. Finally, since E. crescens is apparently widespread in UK mammals and the first UK human case of adiaspiromycosis was reported recently, we present data obtained using a previous isolate of E. crescens demonstrating that both the mycelial and adiaspore phases of the organism are susceptible to amphotericin B, voriconazole, itraconazole and caspofungin Kim et al. (2012) detected multiple circular spore-like materials surrounded by granulomatous inflammation following histopathological examination of a female Apodemus agrarius captured on Jeju Island (N 33°20'33.8", E126°20'12.1") in August 2010. The body weight was 37.3 g and the body length was 107.84 mm. On that day necropsy was performed on the wild rodent. In the necropsy, specific gross lesions were not detected; brain, heart, lung, liver, spleen, stomach, intestine, and kidney were collected and fixed in 10% buffered formalin for 48 hours. After alcoholxylene processing and embedding with paraffin, 4 µm thick sections were stained by haematoxylin and eosin and observed with a light microscope. Round structures were randomly scattered throughout the entire lung field. These structures were located in alveolar space, had thick trilaminar walls consisting of a basophilic outer-layer (3.4 µm in diameter 227 µm structure), an eosinophilic mid-layer (11 µm in diameter 227 µm structure) and a pale colored inner-layer (43 µm in diameter 227µm structure) and a basophilic granular retiform part in the center (approximately 119 µm in diameter 227 µm structure). The diameter of the structures was 195-500 µm (mean 263 µm). The structures mildly compressed the surrounding tissues that were encapsulated by 293 multinucleated giant cells, macrophages (epitheloid cells) and lymphocytes Adiaspores of Emmonsia sp. in the lung parenchyma of an Apodemus agrarius. (A) Round structures (black arrows) scattered in the lung. (B) Adiaspore was located in alveolar space encapsulated by granulomatous inflammatory lesion. Macrophages and Langerhans giant cells infiltrated in the surrounding granulomatous tissue. The spores have three layers in their walls and basophilc granular structures in their inner. Morandi et al. (2012) reported a young crested porcupine (Hystrix cristata) found dead showed multiple fractures, chronic pleuritis, and granulomatous pneumonia. Microscopically, cystic structures were consistent with adiaspiromycosis by Emmonsia crescens. The diagnosis was confirmed using molecular methods. Lungs from a crested porcupine (Hystrix cristata) from Italy consistent with adiaspiromycosis by Emmonsia crescens. Note the presence of multiple disseminated grayish nodules (arrowheads ) on the surface that are indicative of granulomas by Emmonsia crescens. Bar53 cm. FIGURE 2. Lung tissue from a crested porcupine (Hystrix cristata) showing granuloma containing adiaconidia (Grocott's technique). Bar550 mm. Malatesta et al. (2014) reported a road-killed male Eurasian otter submitted for the post-mortem examination on 21st December 2009 at the Veterinary Pathology Unit of the Faculty of Veterinary Medicine of Teramo. Histologically, multifocal round structures with a PAS-positive thick tri-laminar wall and a central basophilic granular mass were observed within the alveoli. The adiaspores were surrounded by a severe 294 granulomatous reaction with high number of macrophages, multinucleated giant cells, eosinophils, neutrophils and fibroblasts. Numerous multifocal cholesterol granulomas were observed close to those fungal-induced. Lung of a male Eurasian otter found near the National Park of Cilento, Vallo di Diano and Alburni (Salerno, Italy; 21.12.2009): multiple variably sized adiaspores randomly scattered in the pulmonary parenchyma (Bar = 110 μm) (H&E). Lung of a male Eurasian otter found near the National Park of Cilento, Vallo di Diano and Alburni (Salerno, Italy; 21.12.2009): adiaspores surrounded by a severe granulomatous inflammation (Bar = 55 μm) (H&E). Insets: strongly PAS- (a) and GMS- (b) positive adiaspores (Bar = 85 μm). Lung of a male Eurasian otter found near the National Park of Cilento, Vallo di Diano and Alburni (Salerno, Italy; 21 st December 2009): a focal granuloma containing cholesterol clefts, associated with adiaspore-induced granulomatous inflammation (Bar = 110 μm) (H&E). Matsuda et al. (2015) detected a granuloma containing fungal spherules, which were morphologically consistent with the adiaspores of E. crescens in the lungs of a female Hokkaido sika deer. This is the first reported case of adiaspiromycosis involving a cervid in the world. Two adiaspores are surrounded by fused lymphoid follicles, forming a nodular lesion in the lung. Hematoxylin and eosin. Bar=200 µm. An adiaspore with fine granular contents and a thick bilaminar wall. The outer third of the adiaspore‘s wall is eosinophilic, and the inner two-thirds are unstained. Hematoxylin and eosin. Bar=100 µm. References: 1. Borman, A. M , Vic R Simpson, Michael D Palmer, Elizabeth Johnson. Adiaspiromycosis Due to Emmonsia crescens is Widespread in Native British Mammals. Mycopathologia 168(4):153-63 · July 2009 2. Hamir, A. N. Pulmonary adiaspiromycosis in raccoons (Procyon lotor) from Oregon. J Vet Diagn Invest 11:565–567 (1999) 295 3. Hill Joseph E., Pamela G. Parnell. Adiaspiromycosis in bullfrogs (Rana catesbeiana) J Vet Diagn Invest 8:496-497 (1996) 4. Hubálek Z. Emmonsiosis of wild rodents and insectivores in Czechland. J Wildl Dis. 1999 Apr;35(2):243-9. 5. Kim T-H, Han J-H, Chang S-N, et al. Adiaspiromycosis of an Apodemus agrarius captured wild rodent in Korea. Laboratory Animal Research. 2012;28(1):6769. doi:10.5625/lar.2012.28.1.67. 6. Krivanec K, Otcenásek M, Slais J. Adiaspiromycosis in large free living carnivores. Mycopathologia. 1976 Jun 4;58(1):21-5. 7. Krivanec K, Mason RW. Adiaspiromycosis in Tasmanian wombats? Mycopathologia. 1980 Jul 1;71(2):125-6. 8. Leighton FA, Wobeser G. The prevalence of adiaspiromycosis in three sympatric species of ground squirrels. J Wildl Dis. 1978 Jul;14(3):362-5. 9. Malatesta D, Simpson VR, Fontanesi L, Fusillo R, Marcelli M, Bongiovanni L, Romanucci M, Palmieri C, Della Salda L. First description of adiaspiromycosis in an Eurasian otter (Lutra lutra) in Italy. Vet Ital. 2014 Jul-Sep;50(3):199-202. 10. Mason RW, Gauhwin M. Adiaspiromycosis in south Australian hairy-nosed wombats (Lasiorhinus latifrons). J Wildl Dis. 1982 Jan;18(1):3-8. 11. Morandi, F., Roberta Galuppi, Maria Jose Buitrago, Giuseppe Sarli. Disseminated Pulmonary Adiaspiromycosis in a Crested Porcupine (Hystrix cristata Linnaeus, 1758). Journal of wildlife diseases 48(2):523-5 · April 2012 12. Mörner T, Avenäs A, Mattsson R. Adiaspiromycosis in a European beaver from Sweden. J Wildl Dis. 1999 Apr;35(2):367-70. 13. Matsuda K, Niki H, Yukawa A, et al. First detection of adiaspiromycosis in the lungs of a deer. The Journal of Veterinary Medical Science. 2015;77(8):981-983. 14. Mudher A. Albassam, Rakesh Bhatnagar, Leonard E. Lillie, and Laurence Roy. Adiaspiromycosis In Striped Skunks In Alberta, Canada. lournul of Wfldlife D(seases. =(I). 1986. pp 13-18 Cc Wildlife Disease Auocintion 1986 15. Otcenásek M, Krivanec K, Slais J. Emmonsia parva as causal agent of adiaspiromycosis in a fox. Sabouraudia. 1975 Mar;13 Pt 1:52-7. 16. Seixas F, Travassos P, Pinto ML, Pires I, Pires MA Pulmonary adiaspiromycosis in a European hedgehog (Erinaceus europaeus) in Portugal. Vet Rec. 2006 Feb 25;158(8):274-5. 17. Simpson VR, Gavier-Widen D. Fatal adiaspiromycosis in a wild Eurasian otter (Lutra lutra). Vet Rec. 2000 Aug 26;147(9):239-41. 18. Stebbins WG, Krishtul A, Bottone EJ, Phelps R, Cohen S. Cutaneous adiaspiromycosis: a distinct dermatologic entity associated with Chrysosporium species. J Am Acad Dermatol. 2004 Nov;51(5 Suppl):S185-9. 19. Taniyama , H. , Hidefumi Furuoka, Takane Matsui, Takeshi Ono. Two Cases of Adiaspiromycosis in the Japanese Pika (Ochotona hyperborea yesoensis Kishida)The Japanese Journal of Veterinary Science 47 (1985), 1, 139-142 296 18. Lobomycosis in wild animals Lobomycosis, a disease caused by the uncultivable dimorphic onygenale fungi Lacazia loboi, remains to date as an enigmatic illness, both due to the impossibility of its etiological agent to be cultured and grown in vitro, as well as because of its unresponsiveness to specific antifungal treatments. Lobomycosis was first described by Lobo (1931) at Recife, in northeast Brazil. For many years it was considered an exclusively human pathology, limited to Latin America, but this view has changed with the first reports of the disease in bottlenose dolphins, Tursiops truncatus, derived from the Florida coast (Migaki et al. 1971, Woodard 1972, Poelma et al. 1974, Caldwell et al. 1975, Dudok van Heel 1977, Bossart 1984, Dailey 1985).         Lobomycosis (lacaziosis) is a chronic fungal disease of the skin and subcutaneous tissues that occurs only in dolphins and humans under natural conditions. Dermal lesions seen in bottlenose dolphins (Tursiops truncatus) are similar to those described in humans but may be more extensive, covering large areas of the body. Lesions are typically found on the leading edges of the dorsal and pectoral fins, the head, fluke, and caudal peduncle where they form multiple aggregations of firm, white raised nodules that may extend >30 cm in the broadest dimension (Reif et al., 2006). Lesions may be associated with sites of apparent previous trauma, such as shark bites. The histologic response to the organism in dolphins and humans consists of multifocal, subepidermal granulomas with inflammatory infiltrates containing histiocytes, multinucleated giant cells, and large numbers of yeast-like cells, 6–12 lm in diameter, arranged singly or in chains connected by tube-like bridges (Migaki et al., 1971; Bossart et al., 1984). Lobomycosis was first identified in dolphins along the Gulf coast of Florida (Migaki et al., 1971), near Vero Beach, FL along the Atlantic coast (Caldwell et al., 1975) and in a dolphin from Marineland, FL (Woodard, 1972) in the 1970s. Subsequently, cases were reported from o the Surinam River estuary (De Vries et al., 1973), o the Spanish–French coast (Symmers et al., 1983), o the south Brazilian coast (Lopes et al., 1993), o the Texas coast of the Gulf of Mexico (Cowan et al., 1993). o The Indian River Lagoon (IRL)(Bossart et al., 2003). Between 2003 and 2005, lobomycosis was identified in 9 of 89 (10.1%) dolphins examined (Reif et al., 2008). 297 Diagnosis: Diagnosis of Lobo's disease is made by taking a sample of the infected skin (a skin biopsy) and examining it under the microscope. Lacazia loboi is characterized by long chains of spherical cells interconnected by tubules. The cells appear to be yeast-like with a diameter of 5 to 12 μm. Attempts to culture L. loboi have so far been unsuccessful.The disease is often misdiagnosed as Blastomyces dermatitidis or Paracoccidiodes brasiliensis due to its similar morphology. Aetiology: Lacazia loboi CIFERRI et al., 1956 Synonyms: Glenosprella loboi FONSECA FILHO et AREA LEAO 1940 Gelenosporopsis amazonica FONSECA FILHO 1943 Paracoccidiodes loboi ALMEIDA et LACAS 1949 Blastomyces loboi VANBREUSEGHEM 1952 Lobomyces loboi (Fonseca & Leão) Borelli 1968 Lacazia loboi is predominantly an intracellular pathogen. Organisms, singly or in chains, reside predominantly in macrophage vacuoles. They probably reproduce by budding; linear or radiating chains of as many as 20 organisms linked by tubules have been observed. The fungus is abundant in lobomycotic skin lesions. It is a spherical intracellular yeast 6-12 μm diameter. The fungus is remarkably homogeneous, with an average diameter of 9 μm. Reports: Migaki et al. (1971) diagnosed Lobo's disease histologically in the skin of the tail stock and flukes of a feral Atlantic bottle-nosed dolphin (Tursiops truncatus) found in a bay on the west coast of Florida. The cutaneous lesions appeared as extensive white crusts. There were large, discrete, histiocytic granulomas in the dermis, resulting in severe acanthosis. The causative mycotic or-ganism, Loboa loboi, was readily demonstrated in the granulomas 298 Vries and Laarman (1973) reported the occurrence of this mycosis in Sotalia guianensis (= Sotulia fluviatilis Gervais) based on a specimen captured in the estuary of the Surinam River. Caldwell (1975) investigated skin lesions on an Atlantic bottlenosed dolphin, captured off the coast of Florida, and found to be histologically and microbiologically 299 indistinguishable from those caused in humans by Loboa loboi. All attempts to isolate the etiologic agent or to transmit the infection to mice and monkeys ended in failure. Sight records of other suspected dolphin cases of lobomycosis in Florida waters were described. Symmers (1983) described a granuloma of the skin in a dolphin (Tursiops truncatus). caught in the Bay of Biscay. Yeast-like organisms, morphologically indistinguishable from Loboa loboi, were found in a biopsy specimen. Identical organisms were present in skin lesion of one hand, accompanied by supratrochlear lymphadenitis, about 3 months after the patient, an aquarium attendant, had had occupational contact, in Europe, with the bottle-nosed dolphin. The Society for Marine Mammalogy (1993) reported lobomycosis in a wild bottlenose dolphin which was the first record for the southwestern South Atlantic. A fresh adult female T. trucatus was recovered on 22 February 1990 in the jetties of Laguna (28‖3O‘S, 48‖45‘W), Santa Catarina State, southern Brazil. Its skull was preserved (UFSC 1089) in the Laboratorio de Mamiferos Aquaticos collection of the Universidade Federal de Santa Catarina. It was an exceptionally large animal (320 cm long) with several light-colored, verrucoid cutaneous lesions. These lesions were concentrated mainly on the flanks and ventral portion of the body, affecting the bottom and sides of the lower jaw, throat, both sides of the flippers extending through the axillary region and part of the thorax, in addition to single patches on the sides of the tailstock. Gross and microscopic findings were typical for this disease, Yellowish nodules (1 cm in diameter) with firm consistency could be observed into the reticular dermis. Histologic analysis of the cutaneous lesion revealed a chronic granulomatous inflammation with severe acanthosis. This tissue presented agglomerates of fungal lemon-shaped elements 15 pm in diameter with double walls and unipolar branching. The diagnosis was based on the cytologic examination of skin lesions, considering that it was not possible to isolate the etiological agent. The fungal elements were identified as Loboa loboi by the use of light microscopy. Cowan (1993) diagnosed Lobo's disease in a free-ranging male bottlenose dolphin Tursiops truncatus examined as part of a live-capture and release program conducted in Matagorda Bay, Texas, USA, during July 1992. Diagnosis was based on typical presentation of a raised skin lesion, with sub-epidermal granuloma and demonstration of typical features of the organism by light microscopy, using hematoxylin and eosin, and methenamine silver stains. Bossart et al. (2003) found lobomycosis in 3 of 17 (17.6%) animals examined in a series of necropsies of stranded dolphins from the IRL,. In a cross-sectional health assessment of dolphins conducted in the Indian River Lagoon in Florida. Mazzoil et al. (2003) mentioned that preliminary analyses of images of IRL dolphins taken from 1996 to 2003 documented a high prevalence of dermal lesions consistent with lobomycosis. Mazzoil et al. (2004) described field and photo-identification protocols for identification of individual dolphins using digital images of dorsal fins previously. Reif et al. (2006) carried out a cross-sectional study to determine the prevalence of lobomycosis, a mycotic infection of dolphins and humans caused by a yeast-like organism (Lacazia loboi), among dolphins in the Indian River Lagoon in Florida.146 Atlantic bottlenose dolphins were subjected to comprehensive health assessments of 300 bottlenose dolphins in the Indian River Lagoon of Florida (n = 75) and in estuarine waters near Charleston, SC (71), were conducted during 2003 and 2004. Bottlenose dolphins were captured, examined, and released. Skin lesions were photographed and then biopsied. Tissue sections were stained with H&E and Gomori methenamine silver stains for identification of L. loboi. 9 of 30 (30%) dolphins captured in the southern portion of the Indian River Lagoon had lobomycosis, whereas none of the 45 dolphins captured in the northern portion of the lagoon or of the 71 dolphins captured near Charleston, SC, did. Affected dolphins had low serum alkaline phosphatase activities and high acute-phase protein concentrations. Results suggested that lobomycosis may be occurring in epidemic proportions among dolphins in the Indian River Lagoon. Localization of the disease to the southern portion of the lagoon, an area characterized by freshwater intrusion and lower salinity, suggests that exposure to environmental stressors may be contributing to the high prevalence of the disease, but specific factors are unknown. Because only dolphins and humans are naturally susceptible to infection, dolphins may represent a sentinel species for an emerging infectious disease. Murdoch et al. (2008) studied the occurrence and distribution of lobomycosis in the IRL using photo-identification survey data collected between 1996 and 2006. The objectives were to (1) determine the sensitivity and specificity of photo-identification for diagnosis of lobomycosis in free-ranging dolphins; (2) determine the spatial distribution of lobomycosis in the IRL; and (3) assess temporal patterns of occurrence. Photographs from 704 distinctly marked dolphins were reviewed for skin lesions compatible with lobomycosis. The presumptive diagnosis was validated by comparing the results of photographic analysis with physical examination and histologic examination of lesion biopsies in 102 dolphins captured and released during a health assessment and 3 stranded dolphins. Twelve of 16 confirmed cases were identified previously by photography, a sensitivity of 75%. Among 89 dolphins without disease, all 89 were considered negative, a specificity of 100%. The prevalence of lobomycosis estimated from photographic data was 6.8% (48/704). Spatial distribution was determined by dividing the IRL into six segments based on hydrodynamics and geographic features. The prevalence ranged from <1% in the Mosquito Lagoon to 16.9% in the south Indian River. The incidence of the disease did not increase during the study period, indicating that the disease is endemic, rather than emerging. In summary, photo-identification is a useful tool to monitor the course of individual and population health for this enigmatic disease. 301 Bermudez et al. (2009) reported one case of lobomycosis caused by Lacazia loboi in a fisherman and one case of lobomycosis-like disease in a bottlenose dolphin (Tursiops truncatus) along the coast of Venezuela. These findings suggested that the marine environmentis a likely habitat for L. loboi and a reservoir for infection. A) Multiple, confluent, keloid-like, hyperchromic nodules with flat shiny surfaces involving the entire free border, posterior aspect, and lobule of the left ear of a fisherman, Venezuela. B) Numerous Lacazia loboi tissue-phase organisms within the stroma. Note the typical chain pattern showing simple gemation budding (Gomori-Grocott stain, magnification ×100). C) Yeast cells showing typical double refraction of the membrane and protoplasmic bodies within cells (periodic acid–Schiff stain, magnification ×600). Extensive lobomycosis-like disease on the beak (A) and dorsal fin (B) of a bottlenose dolphin (Tursiops truncatus) stranded on Margarita Island, Venezuela. 302 Kiszka et al. (2009) reported on Lobomycosis-like disease (LLD) in Indo-Pacific bottlenose dolphins Tursiops aduncus from the tropical lagoon of Mayotte, between Mozambique and Madagascar. From July 2004 to June 2008, boat surveys were conducted in Mayotte waters. At least 71 adult dolphins were photo-identified. Six (5 males, 1 female) tailstock that suggested LLD. The lesions were extensive in some cases. The calf of the positive female was also affected. LLD has been present in this community since at least 1999. As sampling was not possible, the aetiology of the disease could not be explored. The emergence of LLD in Mayotte may be related to degradation of the coastal environment associated with rapid urbanization, expanding agriculture and increased release of untreated freshwater runoffs. Other skin lesions included scars, healing wounds, whitish lesions and lumps. Tursiops aduncus. Lobomycosis-like disease in dolphins around Mayotte. Photo credits: N. Bertrand, J. Kiszka and J. Wickel Tursiops aduncus. Development of lobomycosis-like disease in male MY16 from December 2004 to February 2008 Paniz-Mondolfi et al. (2009) mentioned that lobomycosis has been confirmed in 2 species of inshore and estuarine Delphinidae: 1) the common bottlenose dolphin (Tursiops truncatus) from Brazil, the Atlantic coast of the United States, and Europe and 2) the Guiana dolphin (Sotalia guianensis) from the Surinam River estuary. 303 The fact that lobomycosis is endemic in humans in the Amazon basin could logically raise the suspicion that other animal species in this area may act as reservoirs or even be affected by the disease. However, the infection has never been reported in botos (Inia geoffrensis) or tucuxis (Sotalia fluviatilis) from the Amazon and Orinoco Rivers. Grocott methamine silver–stained section from a skin biopsy specimen of a bottlenose dolphin (Tursiops truncatus) showing abundant Lacazia loboi yeast cells individually and in chains connected by thin tubular bridges. Magnification ×400. Rotstein et al. (2009) reported 2 cases of lobomycosis in stranded bottlenose dolphins (Tursiops truncatus) and 1 case of lobomycosis-like disease in 1 free-swimming, pelagic, offshore bottlenose dolphin from North Carolina, where no cases have previously been observed. A) Serpiginous dermal nodules covering the dorsum of an offshore bottlenose dolphin (KLC020). B) Gomori methenamine silver–stained sections of dermis showing yeast-like structures connected by neck and arranged at various angles (magnification ×400). Scale bar = 100 μm 304 Free-swimming bottlenose dolphin (offshore ecotype) sighted off the Outer Banks of North Carolina with raised gray to white nodules over the dorsal surface, consistent with those of lobomycosis seen in other Atlantic bottlenose dolphins. Xenobalanus sp., a barnacle, is adhered to the tip of the dorsal fin. Image provided by Ari Friedlander, Duke University Marine Laboratory, Beaufort, NC, USA. Daura-Jorge et al. (2011) evaluated the occurrence of lobomycosis-like disease (LLD) throughout this population. Of 47 adult dolphins and 10 calves identified, 7 (12%) presented some form of epidermal lesion and 5 (9%) had evidence of LLD. The lesions were stable in all but 2 cases, in which a progressive development was recorded in a presumed adult female and her calf (referred to here as the LLD pair). During the first few months of observation, the lesion grew slowly and at a constant rate on the adult. However, in the fourteenth month, the growth rate increased rapidly and the first lesions appeared on the calf. Compared to the rest of the population, the LLD pair also presented a different spatial ranging pattern, suggesting a possible social or geographic factor. Current and previous records of LLD or lobomycosis indicate that the disease is endemic in this population. These findings highlight the importance of monitoring both the health of these cetaceans and the quality of their habitat. Esperon et al. (2012) reported the diagnosis and molecular characterization of lobomycosis-like lesions in a captive bottlenose dolphin. The clinical picture and the absence of growth in conventional media resembled the features associated with Lacazia loboi. However sequencing of ribosomal DNA and further phylogenetic analyses showed a novel sequence more related to Paracoccidioides brasilensis than to L. loboi. Moreover, the morphology of the yeast cells differed from those L. loboi causing infections humans. These facts suggest that the dolphin lobomycosislike lesions might have been be caused by different a different fungus clustered inside the order Onygenales. A successful treatment protocol based on topic and systemic terbinafine is also detailed. 305 (A) Lobomycosis-like disease on the dorsal fi n of a bottlenose dolphin ( Tursiops truncatus ). (B) Presence of yeastlike organisms forming chains (magnifi cation _ 400). Scale bar: 30 m. Neighbor-joining phylogram of the sequence obatined in this study (Dolphin isolate GenBank accession number: HQ413323), nine different sequences obtained by BLAST search, and an Aspergillus fl avus sequence as an outgroup member. E, Emmonsia; P, Paracoccidioides; L, Lacazia; A, Aspegrillus. The bootstrap consensus tree inferred from 5,000 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Paniz-Mondolfi et al. (2012) mentioned that lobomycosis, a disease caused by the uncultivable dimorphic onygenale fungi Lacazia loboi, remains to date as an enigmatic illness, both due to the impossibility of its aetiological agent to be cultured and grown in vitro, as well as because of its unresponsiveness to specific antifungal treatments. It was first described in the 1930s by Brazilian dermatologist Jorge Lobo and is known to cause cutaneous and subcutaneous localised and widespread infections in humans and dolphins. Soil and vegetation are believed to be the chief habitat of the fungus, however, increasing reports in marine mammals has shifted the attention to the aquatic environment. Infection in humans has also been associated with proximity to water, raising the hypothesis that L. loboi may be a hydrophilic microorganism that penetrates the skin by trauma. Although its occurrence was once thought to be restricted to New World tropical countries, its recent description in African patients has wrecked this belief. Antifungals noted to be effective in the empirical management of other cutaneous ⁄ subcutaneous mycoses have proven unsuccessful and unfortunately, no satisfactory therapeutic approach for this cutaneous infection currently exists. 306 (a) Keloid-like lesions over the upper limb of a patient with lobomycosis. (b and c) Multiple confluent nodular lesions admixed with small verrucous areas (c). Lobomycosis-like disease (LLD) in a bottlenose dolphin (Tursiops truncatus) stranded on Venezuela. Note the numerous white greyish proliferating lesions with keloidal and verrucous aspect forming rosettes on the dorsal fin (a) and flanks (b). Lobomycosis in a bottlenose dolphin (Tursiops truncatus) from Brasil. Note the numerous proliferating lesions with keloidal aspect on the head (a) and dorsal fin (b). Microscopy (high magnification) depicting typical lemon-shaped budding organisms. Note the variation in size and lesser overall surface of the organisms (Bar: 5 lm). Exfoliative smear (a-b). Uniform round and lemonshaped yeast presenting with characteristic thick and birefrigent walls (Direct examination ・ 400). 307 (a). Stretched, atrophic epidermis with an area of central acanthosis. The dermis is filled by a granulomatous infiltrate composed mainly of histiocytes, giant cells and an overwhelming number of fungal cells. Skin appendages are replaced by the infiltrate. (b). Chains of rounded fungal cells connected by thin tubular structures (arrowheads), Gomori_s methamine silver stain, ・100. (c). A giant cell containing fungal cells within the cytoplasm. Lying free in the stroma are cells revealing protoplasmic structures and a double refractile body (arrow) as well as other cells that appear empty (arrowhead), Trichrome stain, ・100 (Bar: 10 lm). (d). Fungal cells capsular material (arrowhead), Gridley stain ・100 (Bar: 10 lm). (e). Asteroid body within the cytoplasm of a multinucleated giant cell. Adjacent is another giant cell containing fungal cells (Haematoxylin and eosin, ・100). (f). Viable and empty fungal cells within the cytoplasm of a giant cell (Periodicacid-Schiff–PAS-stain, ・100). surrounded by a dense red Bessesen et al. (2014) used Photo-ID records to identify LLDaffected bottlenose dolphins and to assess their lesions. Findings showed between 13.2 and 16.1% of the identified dolphins exhibited lesions grossly resembling lacaziosis. By combining efforts and cross-referencing photographic data, the teams explored the presence of LLD in Golfo Dulce over a time gap of approximately 20 yr. These findings expand the geographical range of the disease and offer insight into its longevity within a given population of dolphins. 308 Bossart et al. (2015) biopsied mucocutaneous lesions from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and estuarine waters of Charleston (CHS), South Carolina, USA, between 2003 and 2013. A total of 78 incisional biopsies from 58 dolphins (n=43 IRL, n=15 CHS) were examined. Thirteen dolphins had 2 lesions biopsied at the same examination, and 6 dolphins were re-examined and re-biopsied at time intervals varying from 1 to 8 yr. Biopsy sites included the skin (n=47), tongue (n=2), and genital mucosa (n=29). Pathologic diagnoses were: orogenital sessile papilloma (39.7%), cutaneous lobomycosis (16.7%), tattoo skin disease (TSD; 15.4%), nonspecific chronic to chronic-active dermatitis (15.4%), and epidermal hyperplasia (12.8%). Pathologic diagnoses from dolphins with 2 lesions were predominately orogenital sessile papillomas (n=9) with nonspecific chronic to chronic-active dermatitis (n=4), TSD (n=3), lobomycosis (n=1), and epidermal hyperplasia (n=1). Persistent pathologic diagnoses from the same dolphins re-examined and re-biopsied at different times included genital sessile papillomas (n=3), lobomycosis (n=2), and nonspecific dermatitis (n=2). This is the first study documenting the various types, combined prevalence, and progression of mucocutaneous lesions in dolphins from the southeastern USA. The data support other published findings describing the health patterns in dolphins from these geographic regions. Potential health impacts related to the observed suite of lesions are important for the IRL and CHS dolphin populations, since previous studies have indicated that both populations are affected by complex infectious diseases often associated with immunologic disturbances and anthropogenic contaminants. Tajima et al. (2015) described an Indo-Pacific bottlenose dolphin (Tursiops aduncus) stranded in Kagoshima, Japan, with severe skin lesions characterized by granulomatous reactions and hyperkeratosis that were similar to those of the lobomycosis, but no fungal organism was observed in the skin lesion. Left side of Indo-Pacific bottlenose dolphin. Male and 270 cm in body length. The skin includes numerous serpiginous and coalescing, raised, ulcerated to papillary nodules on the top of the melon to the blowhole/ External appearance of the flipper is the same as that of the blowhole. 309 a: Hyperkeratosis in the skin. Bar is 500 µm. b: Dermal and subcutaneous granulomas composed of numerous phagocytic cells, lymphocytes and plasma cells were present. Bar is 20 µm. Van Bressem et al. (2015) reported on the epidemiology of lobomycosis-like disease (LLD), a cutaneous disorder evoking lobomycosis, in 658 common bottlenose dolphins Tursiops truncatus from South America and 94 IndoPacific bottlenose dolphins T. aduncus from southern Africa. Photographs and stranding records of 387 inshore residents, 60 inshore non-residents and 305 specimens of undetermined origin (inshore and offshore) were examined for the presence of LLD lesions from 2004 to 2015. Seventeen residents, 3 non-residents and 1 inshore dolphin of unknown residence status were positive. LLD lesions appeared as single or multiple, light grey to whitish nodules and plaques that may ulcerate and increase in size over time. Among resident dolphins, prevalence varied significantly among 4 communities, being low in Posorja (2.35%, n = 85), Ecuador, and high in Salinas, Ecuador (16.7%, n = 18), and Laguna, Brazil (14.3%, n = 42). LLD prevalence increased in 36 T. truncatus from Laguna from 5.6% in 2007-2009 to 13.9% in 2013-2014, albeit not significantly. The disease has persisted for years in dolphins from Mayotte, Laguna, Salinas, the Sanquianga National Park and Bahía Málaga (Colombia) but vanished from the Tramandaí Estuary and the Mampituba River (Brazil). The geographical range of LLD has expanded in Brazil, South Africa and Ecuador, in areas that have been regularly surveyed for 10 to 35 yr. Two of the 21 LLD-affected dolphins were found dead with extensive lesions in southern Brazil, and 2 others disappeared, and presumably died, in Ecuador. These observations stress the need for targeted epidemiological, histological and molecular studies of LLD in dolphins, especially in the Southern Hemisphere. Minakawa et al. (2016) diagnosed a case of Lacaziosis in a Pacific whitesided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin. 310 Schaefer et al. (2016) utilized both classical and novel microbiological methods in order to stimulate growth of Lacazia cells collected from dolphin lesions. This included the experimental inoculation of novel media, cell culture, and the use of artificial skin matrices. Although unsuccessful, the methods and results of this study provide important insight into new approaches that could be utilized in future investigations of this elusive organism. Characteristic nodular lesions from a bottlenose dolphin with lobomycosis. Photomicrograph of a granuloma from an affected dolphin showing multiple round yeasts arranged containing a thick double wall and birefringent capsule with a folded basophilic nucleus. Hematoxylin and eosin X600. Photomicrograph of a granuloma from an affected dolphin showing multiple round yeasts arranged individually and in short chains connected by tube-like bridges characteristic of Lacazia loboi. Gomori methenamine silver X600 References: 1. Bermudez Luis, Marie-Françoise Van Bressem, Oscar Reyes-Jaimes, Alejandro J. Sayegh, and Alberto E. Paniz-Mondolfi Lobomycosis in Man and Lobomycosis-like Disease in Bottlenose Dolphin, Venezuela. Emerg Infect Dis. 15(8):1301-1303. 2. Bessesen BL, Oviedo L, Burdett Hart L, Herra-Miranda D, Pacheco-Polanco JD, Baker L, Saborío-Rodriguez G, Bermúdez-Villapol L, Acevedo-Gutiérrez A. Lacaziosis-like disease among bottlenose dolphins Tursiops truncatus photographed in Golfo Dulce, Costa Rica. Dis Aquat Organ. 2014 Jan 16;107(3):173-80 3. Bossart GD, Schaefer AM, McCulloch S, Goldstein J, Fair PA, Reif JS. Mucocutaneous lesions in free-ranging Atlantic bottlenose dolphins Tursiops truncatus from the southeastern USA. Dis Aquat Organ. 2015 Aug 20;115(3):175-84. 4. Caldwell DK, Caldwell MC, Woodard JC, Woodard JC, Ajello L, Kaplan M, et al. (1975) Lobomycosis as a disease of the Atlantic bottlenose dolphin (Tursiops truncatus Montagu, 1821). American Journal of Tropical Medicine and Hygiene 24:105–114\ 5. Cowan DF (1993) Lobo‘s disease in a bottlenose dolphin (Tursiops truncatus) from Matagorda Bay, Texas. Journal of Wildlife Diseases 29:488–489 6. Daura-Jorge FG1, Simões-Lopes PC. Lobomycosis-like disease in wild bottlenose dolphins Tursiops truncatus of Laguna, southern Brazil: monitoring of a progressive case. Dis Aquat Organ. 2011 Jan 21;93(2):163-70. = 7. De Vries GA, Laarman JJ (1973) A case of Lobo‘s disease in the dolphin Sotalia guianensis. Aquatic Mammals 1:26–33 8. Esperon, F., D. Garcia-Parraga, E.N. Belliere and J.M. Sanchez-Vizcaino, 2012. Molecular diagnosis of lobomycosis-like disease in a bottlenose dolphin in captivity. Med. Mycol., 50: 106-109. 9. Kiszka Jeremy , Marie-Françoise Van Bressem3, Claire Pusineri. Lobomycosis-like disease and other skin conditions in Indo-Pacific bottlenose dolphins Tursiops aduncus from the Indian Ocean. Dis Aquat Org. 84: 151–157, 2009 311 10. Migaki, G, MG Valerio, B Irvine, FM Garner -Lobo's disease in an Atlantic bottle-nosed dolphin J Am Vet Med, 1971 sarasotadolphin.org 11. Minakawa T, Ueda K, Tanaka M, Tanaka N, Kuwamura M, Izawa T, Konno T, Yamate J, Itano EN, Sano A, Wada S. Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific WhiteSided Dolphin (Lagenorhynchus obliquidens). Mycopathologia. 2016 Aug;181(78):523-9. 12. Murdoch ME, Reif JS, Mazzoil M, McCulloch SD, Fair PA, Bossart GD. Lobomycos is in bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon, Florida: estimation of prevalence, temporal trends, and spatial distribution. EcoHealth. 2008;5:289–97. 13. Paniz-Mondolfi AE, Sander-Hoffmann L. Lobomycosis in Inshore and Estuarine Dolphins. Emerging Infectious Diseases. 2009;15(4):672-673. doi:10.3201/eid1504.080955 14. Paniz-Mondolfi, C. Talhari, L. Sander Hoffmann, D. L. Connor,5 S. Talhari, L. Bermudez-Villapol, M. Hernandez-Perez7 and M. F. Van Bressem. Lobomycosis: an emerging disease in humans and delphinidae. Mycoses, 2012, 55, 298–309 15. Reif JS, Mazzoil MS, McCulloch SD, Varela RA, Goldstein JD, Fair PA, Bossart GD. Lobomycosis in Atlantic bottlenose dolphins from the Indian River Lagoon, Florida. J Am Vet Med Assoc. 2006 Jan 1;228(1):104-8. 16. Rotstein DS, Burdett LG, McLellan W, Schwacke L, Rowles T, Terio KA, et al. Lobomycosis in Offshore Bottlenose Dolphins (Tursiops truncatus), North Carolina. Emerg Infect Dis. 2009;15(4):588-590. https://dx.doi.org/10.3201/eid1504.081358 17. Schaefer AM, Reif JS, Guzmán EA, Bossart GD, Ottuso P, Snyder J, Medalie N, Rosato R, Han S, Fair PA, McCarthy PJ. Toward the identification, characterization and experimental culture of Lacazia loboi from Atlantic bottlenose dolphin (Tursiops truncatus). Med Mycol. 2016 Aug 1;54(6):65965. 18. Simões-Lopes MF, PC, Félix F, Kiszka JJ, Daura-Jorge FG, Avila IC, Secchi ER, Flach L, Fruet PF, du Toit K, Ott PH, Elwen S, Di Giacomo AB, Wagner J, Banks A, Van Waerebeek K. Epidemiology of lobomycosis-like disease in bottlenose dolphins Tursiops spp. from South America and southern Africa. Dis Aquat Organ. 2015 Nov 17;117(1):59-75. 19. Society for Marine Mammalogy (1993) First case of lobomycosis in a bottlenose dolphin from southern Brazil. MARINE MAMMAL SCIENCE, 9(3):329-33 1 (July 1993) 0 1993 20. Symmers WS. A possible case of Lôbo‘s disease acquired in Europe from a bottlenosed dolphin (Tursiops truncatus). Bull Soc Pathol Exot Filiales. 1983;76:777–84 Bull Soc Pathol Exot Filiales. 1983 Dec;76(5 Pt 2):777-84. 21. Tajima Y, Sasaki K, Kashiwagi N, Yamada TK. A case of stranded IndoPacific bottlenose dolphin (Tursiops aduncus) with lobomycosis-like skin lesions in Kinko-wan, Kagoshima, Japan. J Vet Med Sci. 2015 Aug;77(8):989-92. 312 19. Cryptococcosis in wild animals           Cryptococcosis is a systemic fungal disease that may affect the respiratory tract (especially the nasal cavity), CNS, eyes, and skin. Cryptococcosis is caused by Cryptococcus neoformans or C gattii. Cryptococcosis occurs worldwide. Cryptococcosis route of infection is similar amongst host species, with Cryptococcus spp. acquired from the environment primarily through the inhalation of basidiospores, ingestion of desiccated yeast cells or more rarely,direct cutaneous inoculation. Cryptococcosis in animals is usually sporadic in occurrence but outbreaks are also documented Cryptococcosis is not zoonotic, infection does not spread between animals and humans. Both are exposed to the environmental source of infection. Cryptococcosis symptoms vary depending on the organ systems affected by the fungus. Often, symptoms are systemic and nonspecific, such as diminished appetite, weight loss, or lethargy. o The primary pulmonary lesion is often clinically silent. o If the fungus has invaded the central nervous system, there can be head tilting, nystagmus (a strange, abnormal back and forth eye movement), the inability to blink due to paralysis of the facial nerves, or loss of coordination, including circling and seizures. o Eye problems are also very common and can include hemorrhage in the retina, as well as inflammatory conditions of the eye like chorioretinitis and anterior uveitis. Cryptococcosis occurs in different domestic. o Dogs and cats become infected by inhalation of spores through the nasal cavity, and the infection spreads throughout the respiratory system and often reaches the nervous system. o In horses, sheep, and goats, the lesions are restricted to the respiratory system o In cattle, lesions are usually located in the mammary glands. Cryptococcosis occurs in different wild animals such as koalas, snakes, ferrets, porpoises, monkeys, cheetah etc o In ferrets, Cryptococcus spp. can affect the gastrointestinal and respiratory systems such nasal cavity and eyes. o The koala often have primary infection of the upper or lower respiratory tract as the first disease feature. o Marine mammals have also been infected. Lesions associated with cryptococcosis vary from a gelatinous mass, consisting of numerous organisms with minimal inflammation, to granuloma formation. o The lesion is usually composed of aggregates of encapsulated organisms within a connective tissue reticulum. 313 o The cellular response is primarily macrophages and giant cells with a few plasma cells and lymphocytes. o Epithelioid giant cells and areas of caseous necrosis are less common than with the other systemic mycoses. Diagnosis:  Direct detection of the organism in smears from nasal exudate, skin exudate, CSF etc. o Gram stain is most useful; the organism retains the crystal violet, whereas the capsule stains lightly red with safranin. o India ink is also used to visualize the organism, which appears unstained and silhouetted against a black background. o Wright stain has been used, but this stain can cause the organism to shrink and the capsule to become distorted. o New methylene blue and periodic acid-Schiff (PAS) stains are better than Wright stain for this reason. o The organism can be stained with H&E, but the capsule does not stain. o The organism is more easily visualized with PAS and Gomori methenamine silver stains, but the capsule does not stain with these, either. o The best stain for Cryptococcus is Mayer mucicarmine because of its ability to stain the capsule. o Immunofluorescent staining can also be used.  Detection of cryptococcal capsular antigen in serum, urine, or CSF is a useful, rapid method of diagnosis in those suspected cases in which the organism is not identified. o A latex agglutination test is commercially available in kit form.  Isolation of the organism can be cultured from exudate, CSF, urine, joint fluid, and tissue samples if a large enough sample volume is available. o Sabouraud agar with antibiotics is used if bacterial contamination is likely. Wild animals reported to be infected with Cryptococcosis Marmoset monkey, Leontocebus geoffroyi Takoms and Eltonn (1953) Rhesus monkey (Macca mulatta ) Pal et al. (1984) Squirrel monkey (Saimiri sciureus) Roussilhon et al. (1987) Short-eared elephant shrews (Macroscelides proboscides) Telll et al. (1997) Large tree shrews (Tupaia tana) Telll et al. (1997) Lesser tree shrews (Tupaia minor) Telll et al. (1997) Common marmoset (Callithrix jacchus) Juan‐Salles et al. (1998) Eastern gray squirrels (Sciurus carolinensis) Duncan et al. (2006 Bandicota indica Singh et al. (2007) Cheetah (Acinonyx jubatus) Polo Leal et al. (2010) Spinner dolphin (Stenella longirostris). Rotstein et al. (2010) Koala Alshahni et al. (2011), Kido et al. (2012) , Satoh et al. (2013) Ferret (Mustela putorius furo) Morera et al. (2011), Ropstad et al. (2011), Morera et al. (2014) 14. California sea lion (Zalophus californianus) Mcleland et al. (2012) 15. Gorilla gorilla Mischnik et al. (2014) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 314 16. Common Toad (bufo Bufo) 17. Harbor Porpoise 18. Wild rabbit ARKive Geoffroy's marmoset Seixas et al. (2008) Norman et al. (2011) Shin et al. (2006) Rhesus macaque - Wikipedia Pinterest Squirrel Monkey (Saimiri sciureus Gorilla Female Photogram Common marmoset - Wikipedia North American WildlifeEastern Gray Squirrel Short-eared Elephant Shrew..Alamy Tupaia tana Large tree shrew. Ryan CalPhotosTupaia minor;Lesser Tree Shrew European domestic ferret) (Mustela furo)UniProt Bandicota indica UniProt Koala | San Diego Zoo Animals ARKive Spinner dolphin Harbor Porpoise. - Wikimed California Sea Lion . Zalophus californianus 315 Common Toad (bufo Bufo) Freshwater Habitats  wild rabbit | Mark's Mural | Pinterest Cheetah Acinonyx Jubatus Winfried Wisniewski Aetiology:      The causative agent is Cryptococcus spp., which is considered infectious only as a desiccated yeast cell or basidiospore as found in the environment. The genus Cryptococcus includes over 37 species; Only C. neoformans is commonly considered to be pathogenic. Conventional nomenclature includes 3 recognized varieties of Cryptococcus neoformans: o Cryptococcus neoformans var. grubii (serotype A), o Cryptococcus neoformans var. neoformans (serotype D), and o Cryptococcus neoformans var. gattii (serotypes B and C), o A hybrid of C. neoformans var. grubii and C. neoformans var. neoformans (serotype AD). Recently, proposed changes to the taxonomy suggest that C. neoformans should be divided into 2 distinct species, o Cryptococcus neoformans  Cryptococcus neoformans var. grubii (serotype A),  Cryptococcus neoformans var neoformans (serotypes D and AD) o Cryptococcus gattii  Cryptococcus gattii serotypes B  Cryptococcus gattii serotypes C Historically, the organism responsible for clinical disease has been thought to be determined by environmental and ecological factors. o Cryptococcus gattii had been restricted to the tropics and subtropics, o Cryptococcus neoformans has a global distribution Current and proposed species of the C. gattii/C. neoformans species complex (F. Hagen et al. / Fungal Genetics and Biology 78 (2015) 16–48) ________________________________________________________________________________ 316 Diagram showing results of identification of test set of all species and interspecies hybrids by MALDITOF MS. (F. Hagen et al. / Fungal Genetics and Biology 78 (2015) 16–48) Description of species reported in wild animals: Cryptococcus neoformans (Sanfelice) Vuillemin1901 Confirmed synonyms:  Saccharomyces neoformans Sanfelice, 1894  Debaryomyces neoformans (Sanfelice) Redaelli, Ciferri & Giordano, 1937.  Cryptococcus hominis Vuillemin, 1901  Saccharomyces hominis Constantin, 1901.  Atelosaccharomyces hominis (Vuillemin) Todd & Herrman, 1936.  Torulopsis hominis (Vuillemin) Redaelli, 1931.  Candida psicrophylicus Nino, 1934.  Cryptococcus neoformans (Sanfelice) Vuillemin variety grubii Franzot, Salkin & Casadevall., 1999,  Cryptococcus neoformans var. grubii Description After 3 days at 25 oC in 3% glucose medium, sediment is present; yeast cells are globose, usually with a single bud, but occasionally some cells may adhere, 4.0–7.0 o 4.0–7.0 lm. Bigger cells measuring up to 9 lm diameter are present, On YMoA, streak colonies are 5–6.5 mm width, smooth, moist to mucoid, shiny, creamish white, with an entire margin; cells are subglobose to globose, 4.0–6.0 lm in diameter, usually with one bud, but occasionally with two buds. On Dalmau plates only yeast cells are 317 present. On MEA colonies measure 8–11 mm, and are pale yellowish beige (pale isabella), and becoming highly mucoid. C. neoformans capsule images. Top row: negative stain with India ink; quick freeze deep-etch electron micrograph of a portion of the cell wall with capsule fibers extending to the left); thin section electron micrograph of three cells. Bottom row: immunoelectron micrograph of a portion of a cell (capsule fibers extending upwards) stained with gold-conjugated anticapsule antibody; differential interference contrast micrograph of a budding cell; confocal immunofluorescence micrograph with the capsule stained blue and the cell wall stained green. Views of C. neoformans. A. Colonies grown on medium containing 0.1% L-DOPA to demonstrate melanization. Top, wild type; bottom, laccase mutant. B. Brightfield microscopy. C. Transmission electron microscopy (pseudocolored). D. Mating filaments. Cryptococcus gattii (Vanbreuseghem & Takashio) Kwon-Chung and Boekhout, 2002 (nomen conservandum, McNeil et al., 2006) Confirmed synonyms 318          Saccharomyces subcutaneous tumefaciens Curtis, 1896. serotype B (Boekhout et al., 2001). Cryptococcus hominis Vuillemin variety tumefaciens (Curtis) Benham, 1935. Cryptococcus hondurianus Castellani, 1933. Torulopsis hominis Vuillemin variety honduriana Castellani, 1933. Torulopsis neoformans variety sheppei Giordani, 1935. Cryptococcus neoformans variety gattii Vanbreuseghem and Takashio, 1970. Cryptococcus neoformans var. gattii var. nov. Ann. Soc. Belg. Med. Trop. 50: 695–702. Cryptococcus neoformans variety shanghaiensis Liao, Shao, Zhang & Li, 1983 Description After 3 days at 25 _C in 3% glucose medium, sediment is present; cells are globose, subglobose, ellipsoid, fusoid to ovoid, 4.0–7.0 _ 3.5–6.0 lm, usually with a single bud, but occasionally some cells may adhere. On YMoA, streak colonies are 5.0–6.0 mm width, smooth, moist to mucoid, shiny, creamish white, with an entire margin; cells are broadly ellipsoid, subglobose to globose, 4.0–6.0 lm in diameter. On Dalmau plates only yeast cells present. On MEA colonies measure approximately up to 8 mm, pale yellowish beige (pale isabella), and are highly mucoid. Cryptococcus albidus (Saito) C.E. Skinner, 43: 249 (1950) Synonyms: 1. Torulopsis albida var. albida 319 2. Torula albida Saito, Journal of Japanese Botany 1: 43 (1922) 3. Torulopsis albida (Saito) Lodder, Verhandelingen Koninklijke Nederlandse Akademie van Wetenschappen Afdeling Natuurkunde 32: 163 (1934) 4. Torulopsis neoformans var. albida (Saito) W. Kaufman, Zentralblatt für Bakteriologie und Parasitenkunde Abteilung 2 106: 442 (1944) 5. Cryptococcus albidus (Saito) C.E. Skinner, The American Midland Naturalist 43: 249 (1950) 6. Rhodotorula albida (Saito) Galgoczy & E.K. Novák, Acta Microbiol. Acad. Sci. Hung. 12 (2): 155 (1965) Morphology The colonies are cream-colour to pale pink, with the majority of colonies being smooth with a mucoid appearance. Some of the colonies may be rough and wrinkled, but this is a rare occurrence. This species is very similar to Cryptococcus neoformans, but can be differentiated because it is phenol oxidase-negative, and, when grown on Niger or birdseed agar, C. neoformans produces melanin, causing the cells to take on a brown color while the C. albidus cells stay cream-color. Microscopically, C. albidus has an ovoid shape, and when viewed with India ink it is apparent that a capsule is present. This species also reproduces through budding. The formation of pseudohyphae has not been seen. C. albidus is able to use glucose, citric acid, maltose, sucrose, trehalose, salicin, cellobiose, and inositol, as well as many other compounds, as sole carbon sources. This species is also able to use potassium nitrate as a nitrogen source. C. albidus produces urease, as is common for Cryptococcus species. Cryptococcus albidus is very easily mistaken for other Cryptococcus species, as well as species from other genus of yeast, and as such should be allowed to grow for a minimum of seven days before attempting to identify this species. Cryptococcus albidus var. albidus is a variety of C. albidus that has been considered unique. It differs from Cryptococcus neoformans because of its ability to assimilate lactose, but not galactose. This species is also considered unique because its strains have a maximum temperature range from between 25°C and 37°C. Cryptococcus yokohamensis Alshahni, Satoh & Makimura sp. nov., 2011 Description: After 3 days in 5% malt extract at 28 uC, cells are capsulated, globose to elongate (1.7–2.462.4–3.5 mm). Polar budding is observed and cells occur as parent bud pairs. After 10 days on YM agar at 28 uC, colonies are glistening, slimy, mucoid and tan with an entire margin. Pseudohyphae are not formed in Dalmau plate cultures on 320 cornmeal. On cornmeal agar, ballistoconidia are not formed. Fermentation is absent. The following carbon compounds are assimilated: glucose, galactose, L-sorbose, sucrose, maltose, cellobiose, trehalose, raffinose, melezitose, inulin (weak), soluble starch, xylose, L-arabinose, D-arabinose, ribose, L-rhamnose (weak), N-acetyl-Dglucosamine, ethanol, erythritol, ribitol, galactitol, mannitol, methyl a-D-glucoside, salicin, D-gluconate, succinate, citrate, inositol (weak), D-glucuronate and sorbitol. The following are not assimilated: lactose, melibiose, methanol, glycerol, DL-lactate, hexadecane and 2-keto-D-gluconate. LLysine, cadaverine, ethylamine and ammonium sulphate are utilized as nitrogen sources, but not sodium nitrite or potassium nitrate. Growth in vitamin-free medium is negative. Optimal growth temperature is 28 uC; delayed growth occurs at 37 uC but no growth is seen at 42 uC. Starch-like compounds are formed. Urease activity is positive. Diazonium blue B reaction is positive. Growth is observed in the presence of 0.1% (w/v) cycloheximide and in 10% sodium chloride/ 5% glucose media, but not in 50% glucose. Major ubiquinones are Q-9 and Q-10. Scanning electron micrograph of cells indicating polar budding of globose to elongated cells. Cells were grown on YM agar for 2 days before being processed for scanning electron microscopy. Bar, 1 mm. Cryptococcus lacticolor Satoh and Makimura sp. nov (2013) Description: After 3 days in yeast and mold (YM) broth at 28 C, cells globose, ovoid, ellipsoid, 2.0–2.5 9 2.0–3.0 lm, single or in parent–bud pairs. Sediment formation observed. Ballistospores not observed after 2 weeks on corn meal agar at 17 C. After 1 month on malt extract agar at 25 C, streak cultures butyrous to viscous, milky white, smooth, and glistening with entire margins. Pseudohyphae not produced on cornmeal agar slide cultures after 59 days at 17 C. Fermentation of glucose absent. Glucose, galactose, L-sorbose, sucrose, maltose, cellobiose, trehalose, lactose (weak), melibiose (weak), raffinose, melezitose, inulin (weak), soluble starch, D-xylose, L321 arabinose, D-arabinose, Dribose, L-rhamnose, D-glucosamine, N-acetyl-Dglucosamine, ethanol (weak), glycerol (weak), erythritol, ribitol, galactitol, Dmannitol, sorbitol, a-methyl-Dglucoside, salicin, D-gluconate, succinate (weak), citrate (weak), and myo-inositol assimilated. Methanol, DL-lactate, hexadecane, 2keto-D-gluconate, and xylitol not assimilated. Ammonium sulfate and L-lysine utilized as sole sources of nitrogen, but not sodium nitrite, potassium nitrate, ethylamine, or cadaverine. Growth in vitamin-free medium present. Growth in 50 % glucose medium absent. Optimum growth temperature 28–30 C; no growth at 35 C. Starch formation present. Urease activity present. Diazonium blue B reaction present. Cryptococcus mujuensis Shin & Park sp. nov. 2006 Description> After growth on YM agar for 3 days at 25 uC, cells are ovoid to ellipsoid and occur singly or in pairs. Budding is multilateral. In YM broth, after 1 month at 20 uC, sediment and a pellicle are present. Streak culture is pale, white to cream, smooth, glistening and butyrous, with an entire margin. D-Glucose, galactose, D-glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl a-D-glucoside, cellobiose, salicin, arbutin, melibiose (delayed), lactose, raffi- nose, melezitose, soluble starch, glycerol (delayed), erythritol, ribitol, xylitol, L-arabitol, D-glucitol, D-mannitol, galactitol, myo-inositol, glucono-dlactone, 2-ketogluconic acid, 5-ketogluconic acid, D-gluconate, D-glucuronate, Dgalacturonic acid, DL-lactate (delayed, weak) and succinic acid are assimilated. LSorbose, inulin, methanol, propane- 1,2-diol, butane-2,3-diol, quinic acid and saccharate are not assimilated. Ethylamine hydrochloride, L-lysine (delayed), cadaverine and glucosamine are assimilated. Potassium nitrate, sodium nitrite, creatine, creatinine, imidazole and D-tryptophan are not assimilated. Does not grow in vitamin-free medium. Grows in 0?01 % cycloheximide and 10 % NaCl/5 % glucose. Does not grow in 50 % glucose, 0?1 % cycloheximide and 16 % NaCl/5 % glucose. Positive for urease activity and diazonium blue B reaction. Produces starch-like compounds. Grows (very weakly) at 30 uC, but does not grow at 35 uC. The major ubiquinone is Q-10. Cryptococcus cuniculi Shin & Park sp. nov. 2006 Description: After growth on YM agar for 3 days at 25 uC, cells are spherical, ovoid to ellipsoidal, 2?1– 5?861?8–5?0 mm, single or in pairs. Budding is bipolar. Streak culture is cream to yellowishwhite, smooth, glistening and butyrous, with an entire margin. In YM broth after 1 month at 20 uC, sediment and a ring are formed. In Dalmau plate cultures on cornmeal agar after 2 weeks, no hyphae or pseudohyphae are formed. The following carbon compounds are assimilated: Dglucose, galactose, L-sorbose (delayed), D-ribose, D-xylose, L-arabinose, Darabinose, L-rhamnose, sucrose, maltose, trehalose, cellobiose, arbutin, lactose, melezitose, soluble starch, glycerol (delayed), xylitol, L-arabitol, D-glucitol, D-mannitol, galactitol, myoinositol, glucono-d-lactone, 2-ketogluconic acid, 5-ketogluconic acid, D-gluconate, Dglucuronate, D-galacturonic acid, succinic acid, ethanol and saccharate. No growth occurs on D-glucosamine, methyl a-D-glucoside, melibiose, raffinose, inulin, erythritol, DL-lactate, methanol, propane-1,2-diol, butane-2,3-diol or quinic acid. Does not assimilate potassium nitrate, sodium nitrite, creatine, creatinine, glucosamine or imidazole. Assimilates ethylamine hydrochloride, L-lysine, cadaverine and D-tryptophan (delayed). Does not grow 322 in vitamin-free medium. Growth in the presence of 10 % NaCl/5 % glucose is delayed and weak. Positive for the diazonium blue B reaction and urease activity. Growth in the presence of 0?01 % cycloheximide is delayed and positive. Does not grow on 50 % (w/w) glucose/yeast extract agar. Produces starch-like compounds. Grows at 30 uC (very weak), but not at 35 uC on YM agar. The ubiquinone type is Q-10. Reports: Takoms and Eltonn (1953) described 2 cases of spontaneous infection with Cryptococcus from the marmoset monkey, Leontocebus geoffroyi, of Panama. "This is the third species of lower animal to be reported as having acquired the infection spontaneously. Panama.". Backhouse and Bolliger (1960) reported 3 cases of infection by Cryptococcus neoformans (all in females) found at autopsy in a series of about 16 unselected koalas. Pal et al. (1984) diagnosed pulmonary cryptococcosis in a 5-year-old-male rhesus monkey from Delhi during 1979. The animal undergoing treatment for bacterial pneumonia was admitted to the indoor ward of Veterinary Hospital with a history of low grade fever, dyspnea and cough. Despite treatment with broad-spectrum antibiotics, hydrocostisones and other supportive drugs, the monkey died. Cryptococcus neoformans was cultured from the lungs of the autopsied animal on sunflower agar medium at 28OC. Histological section of the lung tissue showed large monuclear cells proliferation along with cryptocod cells. The isolate of C. neoformans proved pathogenic to albino mice as the yeast with budding and encapsulation could be demonstrated in the peritoneal exudate under India ink besides reisolation of the pathogen from the visceral organs. Sectionofalung tissueofa5-year-old male rhesus monkey (Macaca mulatta) showing large mononuclear cell proliferation resulting into consolidation of lung parenchyma. Cryptococcal cells were distributed throughout the lung. Peridoci acid - Schiff stain X 157.5. Roussilhon et al. (1987) reported an old female squirrel monkey (Saimiri sciureus) with a tumor-like growth of the lower jaw died in shock after 2 months of illness. Histological studies of different tissue samples demonstrated that the pathological agent was Cryptococcus. Multiple foci of fungus existed in the thoracic cavity with essentially pulmonary and glandular localizations. Bradley et al. 1997) described a subadult female koala (Phascolarctos cinereus) with an acute onset of dyspnoea, depression, and lethargy. The dam of this koala was euthanased due to lymphoma, but also had disseminated cryptococcosis. The koala 323 was thin and had generalised lymphadenopathy. The skeletal muscle was very pale. Approximately 25 mL of clear, straw coloured fluid was evident within the thoracic cavity. Both lungs were pale, wet and firm. The myocardium appeared pale. Clear, straw coloured fluid was evident within the abdominal cavity. The edges of the liver were pale, friable and rubbery. There are 1 mm firm, white nodules throughout the parenchyma of the spleen. No visible lesions: adrenal gland, pancreas, small intestine, skeletal muscle. Tell et al. (1997) reported fungal infections due to Cryptococcus neoformans in seven short-eared elephant shrews (Macroscelides proboscides), six large tree shrews (Tupaia tana), and five lesser tree shrews (Tupaia minor) at the National Zoological 324 Park during a 30-mo period in 1991-1993. Clinical signs were absent or included weight loss, shivering, dyspnea, and/or neurologic disease. Definitive antemortem diagnostic techniques included tracheal lavage and serum cryptococcal antigen latex agglutination titers. Thirteen cases were diagnosed solely by postmortem examination. The source of infection for these animals was uncertain, but C. neoformans is commonly found in soil and other organic material. Two lesser tree shrews and one large tree shrew received antifungal therapy and converted to a negative serum cryptococcal antigen titer. Juan‐Salles et al. (1998) mentioned that a five-year-old female common marmoset (Callithrix jacchus) died after a one-month clinical course of nonspecific signs. Pathologic findings were acute diffuse fibrinonecrotizing enteritis and granulomatous endolymphangitis of intestinal and mesenteric lymphatic vessels. Both lesions were associated with a marked proliferation of Mayer's mucicarmine-positive, 4 to 15 microm yeasts that were surrounded by a wide clear halo. The infection was probably acquired by oral route. Other findings included moderate multifocal granulomatous and necrotizing hepatitis and mesangial nephropathy. Although the immunological status of this marmoset was unknown, cryptococcosis might induce primary lethal intestinal infections in callitrichids. Intestine; common marmoset. Note full-thickness mucosal necrosis and extensive fibrin deposition (stars) associated with Cryptococcus yeasts. Inflammatory infiltrates are present in the submucosa (S), muscular layers (M), and serosa. Mayer‘s mucicarmine, bar = 80 pm. Intestine; common marmoset. The mucosa is effaced by proliferating Cryptococcus yeasts surrounded by a wide clear halo. There are numerous budding yeasts (arrows). Note inflammatory cells, particularly neutrophils around yeasts (arrowheads). Muscular layer (M). Mayer‘s mucicarmine, bar =40 pm. Mesentery; common marmoset. Note marked distension and partial occlusion of a lymphatic vessel (arrowheads) due to an intravascular granuloma. Hematoxylin and eosin, bar = 80 pm.Intestine; common marmoset. Note the inner (asterisk) and outer (stars) muscular layers. A lymphatic vessel (arrowheads) is occluded by mononuclear inflammatory cells and some spindle-shaped cells. Note intralesional yeasts with thinclear halos (arrows). Mayer‘s mucicarmine, bar = 40 pm. 325 Bolton et al. (1999) documented 2 cases of cryptococcosis with central nervous system involvement in captive cheetah (Acinonyx jubatus). In both cases the predominant post mortal lesions were pulmonary cryptococcomas and extensive meningoencephalomyelitis. Both cheetahs tested negative for feline immunodeficiency virus and feline leukaemia virus. The organism isolated in Case 2 was classified as Cryptococcus neoformans var. gattii, which is mainly associated with disease in immunocompetent hosts. Radiographs of the cheetah‘s skull in Case 1. The progression of cystic osteomyelitis in the right zygomatic arch from the 1st examination (on left) to 3 months later (on right), is noticeable. Parenchymal lesion in the brain in Case 2 Micrograph of the pulmonary lesion in Case 2. HE, 200 Micrograph of the lesion illustrated in Fig. 2 of the contiguous brain parenchyma withmarked perivascular cuffing. HE, 400. Connolly et al. (1999) obtained, over a 22-month period, sequential nasal and skin swabs from 52 healthy captive koalas (Phascolarctos cinereus) from the Sydney region. Cryptococcus neoformans was isolated in 17 koalas from 64 of 262 (24%) nasal swabs and from nine of 262 (3%) skin swabs. Prevalence of nasal colonization varied seasonally from 12% (3/25) to 38% (10/26). Cryptococcus neoformans var. gattii alone was cultured from 37, var. neoformans alone from 22 and both varieties from five nasal swabs. Of 33 koalas sampled on three or more occasions, organisms were isolated persistently from six, occasionally from eight and never from 19. Two koalas were persistently and heavily (]100 colonies/plate) colonized by C. neoformans var. gattii and two with var. neoformans. Isolation of C. neoformans var. gattii from the skin was low grade and sporadic. No koalas from which C. neoformans was persistently isolated showed clinical signs of cryptococcosis and all except one had a negative latex cryptococcal antigen test, therefore the nasal cavity was presumed to be colonized by, rather than infected with, C. neoformans. Preliminary observations of 326 koalas from Coffs Harbour indicated a much higher prevalence of colonization by C. neoformans, suggesting that environmental factors influenced the extent of carriage by C. neoformans Krockenberger et al. (2002a) mentioned that Cryptococcus neoformans var. gattii has been shown to have a strong association with eucalypts frequently used by koalas and, not surprisingly, it has been shown to colonize the nasal cavities of koalas. The progression from nasal colonization to tissue invasion is critical to understanding the pathogenesis of cryptococcosis in this species and provides a model for pathogenesis of cryptococcosis in other species. Cryptococcal antigenaemia was detected in twenty-eight healthy koalas from three different regions. This was interpreted as representing limited subclinical disease. One koala developed cryptococcal pneumonia 6 months after leaving the study, whereas another developed cryptococcal meningoencephalitis during the course of the study. Opportunistic necropsies on ten antigen-positive koalas resulted in discovery of small cryptococcal lesions in two (paranasal sinus and lung, respectively). Our data suggest that cryptococcal antigenaemia occurs commonly in koalas, especially in areas with a high environmental presence of C n. var. gattii. Subclinical disease appears most likely to manifest as a small focal lesion in the respiratory tract. Possible outcomes include elimination by an effective immune response, quiescence with possibility of later reactivation or direct progression to overt disease. Symptomatic and subclinical cases showed differences in levels of antigenaemia. The data presented have significant implications for koalas in captivity. Krockenberger et al. (2002b) studied the relationship among Cryptococcus neoformans var. gattii, koalas and the environment. The koala was used as a natural biological sampler in an attempt to understand the dynamics of C. neoformans var. gattii in Australian environments. Evidence of asymptomatic nasal and skin colonization for extended periods by large numbers of C. n. var. gattii was obtained and geographical factors assessed. The key finding was the ability of koalas to amplify numbers of C. n. var. gattii in certain environments. Koalas were not found to be obligatory for the survival of the organism in all environments. Geographical factors alone could not explain differing rates of nasal and skin colonization in koalas in different environments. A strong association between healthy koalas and C. n. var. gattii was confirmed and C n. var. gattii was isolated from novel sources, including the turpentine gum tree (Syncarpia glomulifera), tallowwood (Eucalyptus microcorys) and flooded gum (E. grandis). It seems likely that as yet undiscovered environmental sources of C. n. var. gattii exist in eastern Australia. Further investigation of host, environmental and organism factors integral to the hostpathogen relationship will assist an understanding of the progression from colonization to tissue invasion and cryptococcosis in all species. Malik et al. (2002) diagnosed cryptococcosis in seven ferrets (five from Australia; two from western Canada) displaying a wide range of clinical signs. Two of the ferrets lived together. One (5-years-old) had cryptococcal rhinitis and presented when the infection spread to the nasal bridge. Its sibling developed cryptococcal abscessation of the right retropharyngeal lymph node 12 months later, soon after developing a severe skin condition. DNA fingerprinting and microsatellite analysis demonstrated that the two strains isolated from these siblings were indistinguishable. Two ferrets (2- to 3-years-old) developed generalised cryptococcosis: one had primary lower respiratory tract disease with pneumonia, pleurisy and medi-astinal 327 lymph node involvement, while in the other a segment of intestine was the primary focus of infection with subsequent spread to mesenteric lymph nodes, liver and lung. The remaining three ferrets (1.75 to 4-years-old) had localised disease of a distal limb, in one case with spread to the regional lymph node. Cryptococcus bacillisporus(formerly C neoformansvar gattii) accounted for three of the four infections in Australian ferrets where the biotype could be determined. The Australian ferret with intestinal involvement and the two ferrets from Vancouver had C neoformansvar grubiiinfections. Miller et al. (2002) described the first case of cryptococcosis caused by Cryptococcus neoformans var. gattii in a male Atlantic bottlenose dolphin (Tursiops truncatus). The dolphin showed clinical signs of tachypnea, transient dyspnea, and mild tachycardia and developed multiple hyperechoic nodules, parenchymal consolidation, and thickening of pleura. A diagnosis of bronchopneumonia with pleuritis was made. Itraconazole therapy was implemented for 120 days, and trough levels in serum were within or above the suggested therapeutic range. Titers of cryptococcal antigen in serum increased eightfold during therapy, and the case had a fatal outcome. Necropsy examination findings included enlarged pulmonary lymph nodes and extensive coalescing granulomatous lesions throughout both lungs. Histologic examination revealed numerous, spherical to ellipsoidal, mucicarmine-positive, 3- to 14-μm, encapsulated, budding cells consistent with C. neoformans. Culture of the lung tissue yielded colonies of C. neoformans. The isolate was urease positive and nitrate negative and exhibited phenoloxidase activity. It was positive on canavanine-glycinebromothymol blue agar. When tested by the Iatron serodiagnostic reagent kit (Iatron Laboratories, Inc.), it was shown to belong to serotype B. Tissue section of lung of Atlantic bottlenose dolphin showing C. neoformans var. gattii encapsulated cells visualized with periodic acid-Schiff stain. Magnification, ×220 Ellipsoid, encapsulated cells of C. neoformans var. gattii, visualized with Mayer's mucicarmine. Magnification, ×750 Stephen et al. (2002) serotyped fungi isolated from 2 porpoises, 1 cat, and 1 dog and each organism was identified as C. neoformans gatti, the same as was isolated from the human cases. Serotyping was accomplished by culture on canavanine-glycinebromthymol blue agar and appropriate biochemical tests, followed by a slide agglutination test (Crypto Check; Iatron Laboratories, Tokyo, Japan). An immunohistochemical test was performed on 9 paraffin-embedded blocks from animals diagnosed with cryptococcosis in the years 2000–2001, including 2 of the porpoises that were serotyped as var gatti. The lack of var gatti specific antibodies for immunohistochemical testing made diagnoses by negation necessary. All paraffin blocks were more compatible with C. neoformans neoformans; however, inconsistent staining patterns made definitive identification difficult. The lack of subspecies 328 specific antibodies for var gatti likely accounts for the discrepancy between the immunoperoxidase and serotyping results. Krockenberger et al. (2003) analysed details of 11 previously reported cases and 32 new cases of cryptococcosis in captive and wild koalas. Cryptococcus neoformans var. gattii accounted for all 29 cases in which varietal status was determined. No age or sex predisposition was observed. The respiratory tract was the primary focus of disease in 77% of cases. Although the lower respiratory tract was affected most commonly (60% of cases), 30% of cases had upper respiratory tract lesions and 14% had both. Dissemination was common, especially to the central nervous system (37% cases). Local extension to surrounding tissues was a feature of upper respiratory tract disease. Other tissues showing cryptococcal invasion included lymph nodes (19%), gastrointestinal tract (12%), kidneys (12%), spleen (9%) and skin (7%). Only three cases (7%) had no respiratory tract or central nervous system involvement, two cases of primary skin inoculation and one case of primary lymphadenopathy. Late presentation was a likely factor in the high proportion of cases with disseminated disease (40%). The proportion of koala cases with involvement of the central nervous system, lower respiratory tract and skin, parallels what has been reported for immunocompetent people. Cryptococcosis in the koala appears to be an excellent naturally occurring model for examination of the cryptococcal host-parasite relationship in all species. Lester et al. (2004) determined clinical and pathologic findings associated with an outbreak of cryptococcosis in an unusual geographic location (British Columbia, Canada) in 1 pink-fronted cockatoo, 2 ferrets, 20 cats, and 15 dogs. A presumptive diagnosis of cryptococcosis was made on the basis of serologic, histopathologic, or cytologic findings, and a definitive diagnosis was made on the basis of culture or immunohistochemical staining. Microbiologic culture in 15 cases yielded 2 isolates of Cryptococcus neoformans var grubii (serotype A) and 13 isolates of C. neoformans var gattii (serotype B); all organisms were susceptible to amphotericin B and ketoconazole. Serologic testing had sensitivity of 92% and specificity of 98%. Duncan et al. (2006) employed opportunistic sampling methods to obtain nasal swabs for fungal culture from wild mammal species residing within the coastal Douglas fir biogeoclimatic zone on the southeast coast of the island. Samples were collected from 91 animals representing 14 species. Cryptococcus gattii was isolated from the nasal 329 swabs of two eastern gray squirrels (Sciurus carolinensis) trapped in Duncan, British Columbia. The relative proportion of nasal colonization in wild mammal species is consistent with findings in domestic animals, suggesting that animals may be good indicators of environmental organisms. Shin et al. (2006) identified 2 previously undescribed anamorphic yeasts, strains T11T and T-26T , recovered from wild rabbit faecal pellets collected in Muju, Korea, using phenotypic and molecular taxonomic methods. The isolates were characterized by the proliferation of budding cells, positive diazonium blue B and urease reactions, the presence of Q-10 as the major ubiquinone, the presence of xylose in whole-cell hydrolysates and the inability to ferment sugars. Phylogenetic analyses based on 26S rRNA gene partial sequences revealed that strain T-11T was located in the Bulleromyces clade and was related to Sirobasidium intermedium, Tremella exigua, Cryptococcus cellulolyticus and Bullera pseudoalba. Strain T-26T was located in the Mesenterica clade and was closely related to Cryptococcus sp. F6 and Cryptococcus heveanensis CBS 8976. Sequence divergence values of more than 4 % from other described Cryptococcus species, together with the phenotypic differences, showed that the isolated yeasts represent previously unrecognized members of this genus. Therefore, two novel yeast species are proposed: Cryptococcus mujuensis sp. nov., with strain T-11T (=KCTC 17231T =CBS 10308T ) as the type strain, and Cryptococcus cuniculi sp. nov., with strain T-26T (=KCTC 17232T =CBS 10309T ) as the type strain. Probiotics have been defined as live microbial. Singh et al. (2007) reported the first case of cryptococcosis caused by Cryptococcus neoformans var. grubii in a new species of bandicoot (Bandicota indica) is described. The animal was trapped in a bamboo thicket in a park located in the city of Jabalpur, India. On necropsy, pathological lesions were seen in the lungs and liver and C. neoformans var. grubii was isolated from the lungs, liver, kidneys, spleen and brain but not the heart or intestine. The soil of the animal's burrow and bamboo debris around it also revealed the presence of C. neoformans var. grubii. Histopathology of the lung: (a) showing inflammatory reaction and alveolar structure (_/60, HE), (b) presence of foci of blood cells incorporating yeast like cells of Cryptococcus neoformans (100, HE), (c) Cryptococcus neoformans invasion of the lung(_/400, GMS stained). Seixas et al. (2008) described pulmonary cryptococcosis in a free-living adult female common toad (Bufo bufo) that was killed by a vehicle. Both lungs had various eosinophilic, monomorphic, and spherical to elliptical organisms identified as Cryptoccocus spp. The yeasts were demonstrated by Grocott‘s silver method and the periodic acid-Schiff reaction and the capsule was positive for mucin with a mucicarmine stain. The agent was confirmed by immunohistochemistry, using the 330 monoclonal antibody anti-Cryptococcus neoformans, and by a polymerase chain reaction-based method using a C. neoformansspecific primer.. Lung. Fungal organisms in the respiratory space, with no inflammatory reaction (PAS). Bar5100 mm. FIGURE 2. Lung. Inflammatory reaction to the fungal organisms (arrows). H&E stain. Bar5100 mm. FIGURE 3. Lung. High-power magnification of Figure 2. Arrows show the fungal organisms. H&E stain. Bar550 mm. FIGURE 4. Lung. Immunoreactivity of the yeasts to antiCryptococcus antibody. Streptavidin-biotin-peroxidase, counterstained with hematoxylin. Bar550 mm. Agarose gel electrophoresis of polymerase chain reaction (PCR) products resulting from PCR amplification with three primers: two rDNA Universal delimiting primers led the amplification of a 600-base pair (bp) fragment, and a internal primer specific to Cryptococcus neoformans allowed an amplification of a species-specific fragment of 200 bp. M 5 Marker. 1 5 DNA from a pure culture of C. neoformans. 2 5 DNA from the free-living toad stained tissues. 3 5 DNA from blood from a human patient with cryptococcosis. 4 5 DNA from cerebrospinal fluid from a human patient with cryptococcosis. 5 5 DNA from a pure culture of Candida sp. Polo Leal et al. (2010) presented a case of a female cheetah (Acinonyx jubatus) kept in the Nacional Zoo of Havana. The animal came from South Africa. She began losing weight, and suffering asthenia, anorexia and breathing problems with abundant nasal secretion. Mycological testing of these secretions disclosed the presence of serotype B Cryptococcus gattii. Because of the origin and captive condition of the animal, it was believed that the infection had been latent for 16 months at least. They concluded that up to the present, in Cuba, all clinical Cryptococcus isolates were C. neoformans var. grubii, so it is considered that the infection was caught in the country of origin of the female cheetah. This is the first C. gattii isolate in Cuba from an animal coming from South Africa where this fungus is endemic. Rotstein et al. (2010) described cutaneous nodules and enlarged lymph nodes in a spinner dolphin (Stenella longirostris). Numerous Cryptococcus gattii VGI yeast were observed in multiple organs with minimal inflammation. This case represents the first reported infection of C. gattii in a dolphin from Hawaii. Alshahni et al. (2011) isolated 3 strains from the nostrils of a koala and the surrounding environment in a Japanese zoological park. Sequence analysis of the nuclear rDNA internal transcribed spacer (ITS) region and the large subunit rDNA 331 D1/D2 domains in addition to physiological and morphological studies indicated that the isolates represent a single novel species belonging to the basidiomycetous genus Cryptococcus (Tremellales, Tremellomycetes, Agaricomycotina). Phylogenetic analysis based on D1/D2 and ITS regions revealed that the novel species belongs to the Fuciformis clade. The name Cryptococcus yokohamensis sp. nov. is proposed to accommodate these isolates with strain JCM 16989T (=TIMM 10001T =CBS 11776T =DSM 23671T) as the type strain. DODVPR, Conference ( 2011) reported cryptococcosis in an elk. Brain: Disrupting the gray and white matter of the left aspect of the diencephalon, mesencephalon, and brain stem were multifocal to coalescing inflammatory nodules composed of a central areas of necrosis surrounded by epithelioid macrophages, fewer lymphocytes and plasma cells, and scattered multinucleated giant cells. Within these foci there were numerous 5–18 μm diameter, round extracellular yeasts with a 1 μm thin wall, narrow-based budding, and a 2–8 μm thick, amphophilic, mucicarminepositive mucinous capsule. There was spongiosis of the adjacent neuropil with few infiltrating lymphocytes and plasma cells. Occasionally, there was expansion of the Virchow-Robin space by small numbers of lymphocytes, plasma cells and occasional macrophages (perivascular cuffing). The leptomeninges overlying the cerebrum and brainstem were expanded by moderate numbers of lymphocytes, plasma cells, and macrophages associated with the same yeast bodies. Illnait-Zaragozı et al. (2011) reported a cheetah which was imported from South Africa to Cuba at the age of approximately1 year, where it was subsequently displayed at the carnivores_ area of the National Zoo. After 16 months in captivity, the cheetah showed signs of sudden loss of weight, fatigue, anorexia, shortness of breath and nasal discharge. A yeast-like fungus isolated from nasal discharge of a 332 cheetah was subsequently identified using macromorphological and micromorphological characteristics including growth at 37 _C, absence of germ tube formation, morphology on cornmeal agar with Tween 80, pigmentation on caffeic acid agar, urea hydrolysis, myo-inositol assimilation, growth on canavanineglycinebromothymol blue medium and serological agglutination using the Crypto Check system (Iatron Laboratories, Tokyo, Japan). The strain was deposited in the public culture collection of the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands) under accession number CBS11421. Using AFLP and MLST analysis the strain was classified as genotype AFLP4 ⁄ VGI. Additional matingtype analysis revealed that the STE12a allele was present and previous identification of the yeast as a C. gattii strain serotype B based on conventional techniques and serology was confirmed. Morera et al. (2011) reported a domestic ferret (Mustela putorius furo) with lymphadenopathy and acute bilateral blindness. Cytologic evaluation and biopsy of an affected lymph node revealed pyogranulomatous lymphadenitis with intralesional yeast consistent with Cryptococcus sp. Subsequent studies demonstrated Cryptococcus gattii serotype B VGI/AFLP4 as the causative agent. Postmortem examination revealed disseminated cryptococcosis with prominent neurologic involvement. Nasal swabs of other ferrets and humans from the same household revealed that two ferrets and two humans to be asymptomatic carriers of the same strain of cryptococcus as the necropsied ferret. Norman et al. (2011) reported a pregnant, dead, stranded, harbor porpoise (Phocoena phocoena) on western Whidbey Island, in Puget Sound, Washington State. The carcass was iced and necropsy was performed. Both lungs were exposed, extensively scavenged, firm, and nodular; a sectioned surface exuded clear to slightly opaque gelatinous to mucinous discharge. Mediastinal lymph nodes were grossly enlarged, multinodular, and firm with large numbers of yeasts visible by microscopy. The first stomach chamber contained two 3.5 cm × 2.5 cm raised, centrally umbilicated ulcers and several embedded anisakid nematodes. The uterus was gravid in the right horn with a mid-term fetus. Microscopically, the lung lesions correlated with granulomatous to pyogranulomatous infiltrates, often with a myriad of yeasts. The male fetus appeared grossly normal externally and was at a gestation of ≈5–6 months. Mediastinal lymph nodes had mild granulomatous inflammation and contained numerous yeasts morphologically consistent with Cryptococcus spp.. The lymph nodes were partially replaced with intracellular and extracellular mulilobulated yeast aggregates (length 8–20 μm) with pale eosinophilic central regions and a thin refractile wall peripherally bound by a 5-μm nonstaining capsule. Around the periphery of these aggregates, there were small numbers of macrophages and lymphocytes and fewer neutrophils. Specific staining showed a prominent mucicarminophilic capsule consistent with Cryptococcus spp. Yeasts were found in the amniotic fluid and interspersed within the chorioallantoic villi and submucosal vasculature of the placenta. Mild multifocal nonsuppurative myocarditis was detected. Maternal and fetal tissues were cultured for fungi, and diagnosis was based on Gram stain (budding yeast-like cells), India ink stain (positive for encapsulated cells), hydrolysis of urea (positive), and final confirmation by using API 20C Aux V3.0. Canavanine-glycine-bromthymol blue agar was used to differentiate between C. gattii and C. neoformans. Molecular typing by restriction fragment length polymorphism was used to definitively speciate and subtype C. gattii. Fungal culture 333 showed heavy growth of Cryptococcus spp. from the dam (lungs, mediastinal lymph nodes, and placenta) and fetus (mediastinal lymph nodes). Genotyping of primary isolates identified VGIIa C. gattii in both animals. A) Enlarged mediastinal lymph nodes of a stranded, pregnant, harbor porpoise (Phocoena phocoena) infected with Cryptococcus gattii that was transmitted to its fetus. B) Mucicarmine–stained sections of fetal mediastinal lymph. Ropstad et al. (2011) described a case of bilateral exudative chorioretinitis in an 18month-old male neutered ferret (Mustela putorius furo) with a generalized Cryptococcus gattii infection confirmed by PCR.. The postmortem histopathology confirmed the initial diagnosis of cryptococcosis and the presence of intraretinal Cryptococcus spp. Histological micrograph of a mandibular lymph node fine needle aspiration of the ferret, showing a cluster of encapsulated yeast organisms (arrow). (Wright‘s stain, ×400). Funduscopic photograph showing peripapillary subretinal edema and tapetal hyper-reflectivity in the ferret.. Cataract and subluxated lens of the right eye in the ferret. Histopathologic sample of the left eye of the ferret. A retinal detachment with loss of normal retinal architecture and subretinal scattered spherical microorganisms consistent with Cryptococcus spp. are observed (black arrow). The organisms are 10 to 40 μm in diameter, palely basophilic, with a central nucleus, thick capsule and a few narrow-based budding. (Hematoxylin-eosin, 40x) Kido et al. (2012) described the long-term surveillance and treatment of subclinical cryptococcosis and nasal colonization of koalas by Cryptococcus neoformans and C. gattii. Of the 15 animals investigated through the use of samples obtained by nasal swabs, antigen titer measurements, and pathologic examination, C. neoformans was found associated with nine koalas and C. gattii with one animal. Nine koalas showed 334 subclinical disease and one clinical infections and antigenemia. Treatment with fluconazole, itraconazole and amphotericin B upon detection of C. neoformans or C. gattii was not effective. The results of the present study showed that C. neoformans was the predominant species isolated from the nasal swab samples and the fungus might have naturally become associated with the koalas' nasal cavities at Kanazawa Zoological Gardens. The unclear treatment effectiveness might have been caused by a shorter treatment period that is routinely used and unstable itraconazole absorption. Mcleland et al. (2012) recovered Cryptococcus albidus, from a juvenile California sea lion (Zalophus californianus) rescued near San Francisco Bay, California. Yeast morphologically consistent with a Cryptococcus sp. was identified histologically in a lymph node and C. albidus was identified by an rDNA sequence from the lung. Infection with C. albidus was thought to have contributed to mortality in this sea lion, along with concurrent bacterial pneumonia.Cryptococcus albidus should be considered as a potential pathogen with a role in marine mammal morbidity and mortality. Satoh et al. (2013) isolated a total of 515 yeast strains from the nasal smears of Queensland koalas and their breeding environments in Japanese zoological parks between 2005 and 2012. The most frequent species in the basidiomycetous yeast biota isolated from koala nasal passages was Cryptococcus neoformans, followed by Rhodotorula minuta. R. minuta was the most frequent species in the breeding environments, while C. neoformans was rare. Seven strains representing two novel yeast species were identified. Analyses of the 26S rDNA (LSU) D1/D2 domain and nuclear ribosomal DNA internal transcribed spacer region sequences indicated that these strains represented new species with close phylogenetic relationships to Cryptococcus and Rhodotorula. A sexual state was not found for either of these two novel yeasts. Key phenotypic characters confirmed that these strains could be placed in Cryptococcus and Rhodotorula. The names Cryptococcus lacticolor sp. nov. (type strain TIMM 10013(T) = JCM 15449(T) = CBS 10915(T) = DSM 21093(T), DDBJ/EMBL/Genbank Accession No.; AB375774 (ITS) and AB375775 (26S rDNA D1/D2 region), MycoBank ID; MB 802688, Fungal Barcoding Database ID; 3174), and Rhodotorula oligophaga sp. nov. (type strain TIMM 10017(T) = JCM 18398(T) = CBS 12623(T) = DSM 25814(T), DDBJ/EMBL/Genbank Accession No.; AB702967 (ITS) and AB702967 (26S rDNA D1/D2 region), MycoBank ID; MB 802689, Fungal Barcoding Database ID; 3175) are proposed for these new species. 335 Cryptococcus lacticolor TIMM 10013T. Growth on 5 % malt extract agar after 7 days at 17 °C. Scale bar = 10 μm Rhodotorula oligophaga TIMM 10017T. Growth on 5 % malt extract agar after 5 days at 20 °C Mischnik et al. (2014) reported disseminated cryptococcosis in a simian immunodeficiency virus-negative 27-year-old female Gorilla gorilla presenting with lethargy, progressive weight loss and productive cough. The diagnosis was confirmed by positive lung biopsy culture, serum cryptococcal antigen, and cerebral histopathology demonstrating encapsulated yeasts. Molecular characterisation of lung culture isolate yielded Cryptococcus neoformans var. grubii. An immune-deficiency could not be demonstrated Morera et al. (2014) described two cases of cryptococcosis in ferrets in the Iberian Peninsula and Balearic Islands and documents a relationship of ferret cryptococcosis with environmental isolates in the same locations References: 1. Alshahni MM, Makimura K, Satoh K, Nishiyama Y, Kido N, Sawada T (2011) Cryptococcus yokohamensis sp. nov., a basidiomycetous yeast isolated from trees and Queensland koala kept in a Japanese zoological park. Int J Syst Evol Microbiol 61:3068–3071 2. Backhouse TC, Bolliger A. 1960. Cryptococcosis in the koala Phascolarctos cinereus. Aust. J. Sci. 23:86 –87. 3. BARRIMET , STADLECRK : Successful treatment of Cryptococcus neoformans infection in an Allen‘s swamp monkey (Allenopithecus nigroviridis) using fluconazole and flucytosine. J Zoo Wildl Med 26: 109-1 14, 1995 4. Bartlett, K., M. W. Fyfeb, And L. A. Macdougall. 2003. Environmental Cryptococcus neoformans var. gattii in British Columbia, Canada. American Journal of Respiratory and Critical Care Medicine 167: A499. 5. Boekhout, T., J. Fell, and M. Diaz. 2002. Proposal to conserve the name Cryptococcus gattii against C. hondurianus and C. bacillisporus (Basidiomycota, Hymenomycetes, Tremellomycetidae). Taxon 51: 804–806. 336 6. Bolton LA, Lobetti RG, Evezard DN, Picard JA, Nesbit JW, van Heerden J, Burroughs RE. Cryptococcosis in captive cheetah (Acinonyx jubatus): two cases. J S Afr Vet Assoc. 1999 Mar;70(1):35-9. 7. Bradley W. Bellenger C.R. Lamb W.A. Barrs V.R. Foster S. Hemsley S. Canfield P.J. Love D.N. (1997) Nasopharyngeal cryptococcosis. Australian Veterinary Journal. 75: 7, 483-488. 8. Connolly, J. H., M. B. Krockenberger, R. Malik, P. J. Canfield, D. I. Wigney, and D. B. Muir. 1999. Asymptomatic carriage of Cryptococcus neoformansin the nasal cavity of the koala (Phascolarctos cinereus). Medical Mycology 37: 331–338. 9. DODVPR, Conference: 14 - 2011 Case: 02 brain-Cryptococcosis in koala, Wednesday Slide Conference Online, 10. Duncan. C., H. Schwantje, C. Stephen, J. Campbell, and K. Bartlet. Cryptococcus gattii in Wildlife of Vancouver Island, British Columbia, Canada. Journal of Wildlife Diseases, 42(1), 2006, pp. 175–178 11. Hoang, L. M., J. A. Maguire, P. Doyle, M. Fyfe, And D. L. Roscoe. 2004. Cryptococcus neoformans infections at Vancouver Hospital and Health Sciences Centre (1997–2002): Epidemiology, microbiology and histopathology. Journal of Medical Microbiology 53: 935–940. 12. Juan‐Salles C, Marco A, Domingo M. Intestinal cryptococco‐sis in a common marmoset (Callithrix jacchus) J Med Primatol. 1998;27:298–302 13. Hough I: Cryptococcosis in an eastern water skink. Aust VetJ 76:471-472, 1998 14. Huckabone SE, Gulland FM, Johnson SM, Colegrove KM, Dodd EM, Pappagianis D, Dunkin RC, Casper D, Carlson EL, Sykes JE, Meyer W, Miller MA. Coccidioidomycosis and other systemic mycoses of marine mammals stranding along the central California, USA coast: 1998-2012. J Wildl Dis. 2015 Apr;51(2):295-308. 15. Illnait-Zaragozí MT, Hagen F, Fernández-Andreu CM, Martínez-Machín GF, PoloLeal JL, Boekhout T, Klaassen CH, Meis JF. Reactivation of a Cryptococcus gattii infection in a cheetah (Acinonyx jubatus) held in the National Zoo, Havana, Cuba. Mycoses. 2011 Nov;54(6):e889-92. 16. Kido N, Makimura K, Kamegaya C, Shindo I, Shibata E, Omiya T, Yamamoto Y. Long-term surveillance and treatment of subclinical cryptococcosis and nasal colonization by Cryptococcus neoformans and C. gattii species complex in captive koalas (Phascolarctes cinereus). Med Mycol. 2012 Apr;50(3):291-8. 17. Krockenberger MB, Canfield PJ, Barnes J, Vogelnest L, Connolly J, Ley C, Malik R (2002a) Cryptococcus neoformans var. gattii in the koala (Phascolarctos cinereus): serological evidence for subclinical cryptococcosis. Med Mycol 40:273–282 18. Krockenberger MB, Canfield PJ, Malik R (2002b) Cryptococcus neoformans in the koala (Phascolarctos cinereus): colonization by C. n. var. gattii and investigation of environmental sources. Med Mycol 40:263–272 19. Makimura K, Karasawa M, Hosoi H, Kobayashi T, Kamijo N, Kobayashi K, Hiramatsu H, Akikawa T, Yabe T, Yamaguchi A, Ishida O, Murakami A, Fujisaki R, Nishiyama Y, Uchida K, Yamaguchi H (2002) A Queensland koala kept in a Japanese zoological park was carrier of an imported fungal pathogen, Filobasidiella neoformans var. bacillispra (Cryptococcus neoformans var. gattii). Jpn J Infect Dis 55:31–32 20. Mcleland, Shannon , Colleen G Duncan, Terry Spraker, Frances Gulland. Cryptococcus albidus Infection in a California Sea Lion (Zalophus californianus). Journal of wildlife diseases 48(4):1030-4 · October 2012 21. Lester SJ, Kowalewich NJ, Bartlett KH, Krockenberger MB, Fairfax TM, Malik R. Clinicopathologic features of an unusual outbreak of cryptococcosis in dogs, cats, ferrets, and a bird: 38 cases (January to July 2003). J Am Vet Med Assoc. 2004 Dec 1;225(11):1716-22. 22. Pal, Mahendra, Sihin Tesfaye , Pratibha Dave. Cryptococcosis: An Enigmatic Mycosis of Humans and Animals. J Environ Occup Sci 2014; 3(1):53-60 337 23. Malik R, Alderton B, Finlaison D, Krockenberger MB, Karaoglu H, Meyer W, Martin P, France MP, McGill J, Lester SJ, O‘Brien CR, Love DN. 2002. Cryptococcosis in ferrets: a diverse spectrum of clinical disease. Aust. Vet. J. 80:749 –755. 24. Miller WG, Padhye AA, Van Bonn W, Jensen E, Brandt ME, Ridgway SH. 2002. Cryptococcosis in a bottlenose dolphin (Tursiops truncatus) by Cryptococcus neoformans var. gattii. J Clin Microbiol 40:721–724. 25. Norman SA, Raverty S, Zabek E, Etheridge S, Ford JK, Hoang LM,Morshed M. 2011 . Maternal-fetal transmission of Cryptococcus gattii in harbor porpoise. Emerg. Infect. Dis. 17:304–305. 10.3201/eid1702.101232. 26. Pal M, Dubey GD, Mehrotra BS. Pulmonary cryptococcosis in a rhesus monkey (Macca mulatta ). Mykosen 1984; 27: 309_ 312. 27. Polo-Leal, J. L., T. Boekhout,2C. H. Klaassen4 and J. F. Meis. Reactivation of a Cryptococcus gattii infection in a cheetah (Acinonyx jubatus) held in the National Zoo, Havana, Cuba. Mycoses 54, e889–e892 doi:10.1111/j.1439-0507.2011.02046.x 28. Rotstein DS, West K, Levine G, Lockhart SR, Raverty S, Morshed MG, Rowles T. 2010. Cryptococcus gattii VGI in a spinner dolphin (Stenella longirostris) from Hawaii. J Zoo Wildl Med 41:181–183 29. Roussilhon C, Postal JM, Ravisse P. Spontaneous cryptococcosis of a squirrel monkey (Saimiri sciureus) in French Guyana. J Med Primatol. 1987;16:39–47. 30. Satoh K, Maeda M, Umeda Y, Sugamata M, Makimura K. Cryptococcus lacticolor sp. nov. and Rhodotorula oligophaga sp. nov., novel yeasts isolated from the nasal smear microbiota of Queensland koalas kept in Japanese zoological parks. Antonie Van Leeuwenhoek. 2013 Jul;104(1):83-93. 31. Seixas, Fernanda, Maria da Luz Martins, Maria de Lurdes Pinto, Paulo Jorge Travassos, Manuela Miranda, and Maria dos Anjos Pires. A Case of Pulmonary Cryptococcosis in a Free-living Toad (Bufo bufo). Journal of Wildlife Diseases, 44(2):460-463. 32. Shin Kee-Sun, Hee-Mock Oh, Yong-Ha Park, Kang Hyun Lee, Haryoung Poo, GiSeok Kwon and O-Yu Kwon. Cryptococcus mujuensis sp. nov. and Cryptococcus cuniculi sp. nov., basidiomycetous yeasts isolated from wild rabbit faeces. Int J Syst Evol Microbiol. 2006 Sep;56(Pt 9):2241-4. 33. Singh SM, Naidu J, Sharma A, Nawange SR, Singh K. First case of cryptococcosis in a new species of bandicoot (Bandicota indica) caused by Cryptococcus neoformans var. grubii. Med Mycol. 2007 Feb;45(1):89-93. 34. Spencear, Leyc , Canfielp,D M Artipn, Perryr : Meningoencephalitis 35. in a koala (Phascolarctos cinereus) due to Cryptococcus neoformans var gattii infection. J Zoo Wildl Med 24519-522, 1993. 36. Srikanta D, Santiago-Tirado FH, Doering TL. Cryptococcus neoformans: Historical curiosity to modern pathogen. Yeast (Chichester, England). 2014;31(2):47-60. doi:10.1002/yea.2997. 37. Stephen C, Lester S, Black W, Fyfe M, Raverty S. British Columbia: Multispecies outbreak of cryptococcosis on southern Vancouver Island, British Columbia. The Canadian Veterinary Journal. 2002;43(10):792-794 38. Takoms J, Eltonn W: Spontaneous cryptococcosis of marmoset monkeys in Panama. AMA Arch Pathol55:403-407, 1953. 39. Tell, L. A., Nichols, D. K., Fleming, P. & Bush, M. 1997. Cryptococcosis in tree shrews (Tupaia tana and Tupaia minor) and elephant shrews (Macroscelides proboscideus). Journal of Zoo and Wildlife Medicine, 28, 175-181. 40. Tscharke, M. Huynh, K. H. Bartlett, M. Fyfe, L. Macdougall, T. Boekhout, K. J. Kwon-Chung, And W. Meyer. 2004. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proceedings of the National Academy of Sciences of the United States of America 101: 17258–17263. 338 20. Candidosis in wild animals         Candidosis is a fungal disease affecting the mucous membranes and the skin and may cause infection of any organ or system or systemic infections. Candida infection was the most frequently mentioned mycosis in the alimentary tract in wild animals. o The necropsy incidence of candidiasis (moniliasis, thrush) in the alimentary tract is about 0.1% to 0.4% (Mcclure, 1971, Mcclure et al., 1971, CDC, 1973) o Ulcers and whitish streaks and plaques on the tongue, oral cavity, and esophagus, and a thick pseudomembrane on the colon, were found in five rhesus monkeys (Macaca mulatta) (Wiks et al., 1970). o Esophageal lesion characterized by a yellowish adherent pseudomembrane was found in a rhesus monkey (M. mulatta) that had received antibiotic medication for diarrhea for about two months [16]. Candida was confined to the keratinized layer of the epithelium wit no invasion of the basal layer (Kaufmann and Quist, 1969) Candida spp. are common saprophytes of nonhuman primates as indicated by isolation surveys of asymptomatic animals. Reports of lesions in nonhuman primates relatable to infection with Candida spp. are few and most are associated with other disease processes or antibacterial prophylaxis. Investigation of experimentally induced candidiasis indicated that cellmediated immune response were more important than humoral antibody in resisting mucosal invasion by Candida. Candidosis is distributed worldwide in a variety of animals and is most commonly caused by Candida albicans. Candida albicans is a polymorphic fungus which grows in both yeast and filamentous forms and resides as a commensal in humans, particularly within the gastrointestinal (GI) tract (Brown et al., 2012). Candida albicans is also an opportunistic pathogen and one of the major aetiological agents of mucosal and systemic fungal infection (Brown et al., 2012). o In susceptible individuals, systemic C. albicans infections are thought to arise from organisms in the GI tract; a hypothesis supported by data from both patients and animal models (Koh et al., 2008; Miranda et al., 2009). o As filamentous forms predominate at sites of primary epithelial infection, morphogenic transition is thought to facilitate access of C. albicans to the bloodstream and subsequent systemic spread (Gow et al., 2012). o Morphogenetic transition is essential for virulence of this pathogen as mutants locked in either the yeast (Lo et al., 1997) or filamentous (Murad et al., 2001) forms are highly attenuated in animal models of systemic disease. Given the importance of mucosal barriers, considerable attention has been given to understanding the interactions between C. albicans and epithelial cells. 339 340 Aetiology: Candida albicans (Robin) Berkhout 1923 synonyms =Blastomyces albicans Brownlie: 425-431 (1920) =Candida biliaria Bat. & J.S. Silveira, Hospital Rio de Janeiro 56 (2): 295 (1959) =Candida claussenii Lodder & Kreger, The Yeasts: a taxonomic study: 578 (1952) =Candida intestinalis Bat. & J.S. Silveira, Hospital Rio de Janeiro 56 (2): 293 (1959) =Candida langeronii Dietrichson, Annales de Parasitologie Humaine Comparée 29: 479 (1954) =Candida mycotoruloidea Redaelli & Cif., Archiv für Mikrobiologie 6: 50 (1935) =Candida nouvelii Saëz, Bulletin de la Société Mycologique de France 89 (1): 82 (1973) =Candida truncata Vanbreus., Archives Belge de Dermatologie et Syphiligraphie 4: 307-313 (1948) =Endomyces albicans Okabe, Clblatt Bakt, Parasit Infektionskrankh, Erste Abt: 181-187 (1929) =Monilia alba Castell. & Chalm., Manual of Tropical Medicine: 1089 (1919) =Monilia albicans Plaut (1919) Morphology of Candida albicans On Sabouraud dextrose agar, C. albicans develops within 24-48 h raised, creamy, opaque colonies of 1-2 mm in diameter. After several days of incubation, the colonies show radiating outgrowths penetrating the medium. C. albicans is capable of producing yeast cells, pseudohyphae, true hyphae and chlamydospores. For germ tube testing a suspension of the suspected colony is made in 0.5 ml serum, incubated for 24 h at 370C and examined microscopically for the development of germ tubes, which extend from the cell without septum or constriction. After 72 h at 250C the inoculated plates are examined for pseudohyphae and chlamydoconidia. 341 Wild animals in which candidosis was reported 1. Kodiak bear Kaben and Essmann (1975) 2. Europa Beaver Saëz (1976) 3. Bottle-nosed dolphins Nakeeb et al. (1977), Morris et al. (2011) 4. Baboon (Papio papio) Saëz et al. (1978 a,b) 5. Cheetah (Acinonyx jubatus) La Perle et al. (1998) 6. Greek tortoise (Testudo graeca) Hernandez-Divers (2001) 7. guanaco (Lama guanicoe) Keck et al. (2009) 8. Gorilla, (Gorilla gorilla beringei) Fienners (1967) 9. Capuchin monkey (Cebus sp.) Fienners (1967), McCullough et al. (1977) 10. Woolly monkey (Lagothrix sp.) Fienners (1967) 11. Macaca irus monkeys Budtz-Jörgensen (1973), Wikse et al. (1970) chimpanzee Schmidt and Butler (1970) rhesus monkey (Macaca mulatto). Kauffman and Quist (1969) Owl monkey (Aotus trivirgatus) King (1976) Albino rats Nawange et al. (2009) 16. Wild Brazilian porcupine Castelo-Branco et al. (2013) 12. 13. 14. 15. Kodiak bear - Wikipedia Eurasian beaver Cheetah Acinonyx Jubatus Winfried Wisniewski Fine Art America ARKive Greek tortoise baboon (Papio papio) Chimpanzee - ZooBorns Capuchin monkey 342 macaque www.britannica.com gorilla Woolly Monkey (Lagothrix sp.) - Wiki Rhesus macaque - Wikipedia Owl monkey Bottlenose Dolphin National Geographic Kids Albino rat 123RF.com Brazilian Porcupine (Coendou prehensilis) Lama guanicoe (Guanaco) UniProt Reports: Fienners (1967) reported lesions containing Candida in animals in a gorilla, (Gorilla gorilla beringei), a capuchin monkey (Cebus sp.) and a woolly monkey (Lagothrix sp.) in London Zoo. The infection was restricted to the tongue in the gorilla and woolly monkey, whereas the capuchin monkey had lesions in the tongue, oral mucosa, intestine, liver, and lungs. Baboons (Papio papio) at the Zoological Park in Paris had buccal lesions caused by Candida Kauffman and Quist (1969) reported a case of thrush in a male rhesus monkey (Macaca mulatto). The illness was characterized by progressive inanition, anorexia and diarrhoea over a 2-month period. At necropsy a rough, yellow adherent 343 pseudomembrane histologically consistent oesophagus. Candida albicans was present. with thrush lined the entire Schmidt and Butler (1970) reported 1-yr-old chimpanzee that developed anorexia, diarrhoea and dehydration. It did not respond to treatment, including antibiotics, and died 7 days after the appearance of symptoms. At post-mortem the fat at the base of the heart was found to be yellow and gelatinous, and a yellowish-white material adhered closely to the oesophagus. Candida albicans was cultured from this material. The oesophageal epithelium was necrotic and numerous blastospores and pseudohyphae were found adjacent to the normal epithelium. No abnormalities were seen in the remainder of the gastrointestinal tract. Wikse et al. (1970) diagnosed candidosis in six monkeys over a 10-month period. Most cases had been on antibiotic therapy for enterocolitis. Fungal invasions were seen in epithelium of the tongue, oral cavity, oesophagus, colon, and hard keratin of the nails. Gross lesions of the anterior alimentary tract were either white patches or ulcers of the mucosa. Lesions of the colon consisted of a thick pseudomembrane that contained numerous Candida organisms. The nails exhibited typical Candida onychomycosis. C. albicans was isolated from the two cases that were cultured. Tissue invasion by Candida blastospores and hyphae was histologically demonstrated in all cases. Budtz-Jörgensen (1973) produced an experimental Candida infection of the palatal mucosa in 14 Macaca irus monkeys by inoculating C. albicans under an acrylic plate. Half of the animals were subjected to immunosuppressive therapy (azathioprine) for 2 weeks before and 2 weeks after infection. The course of development of the cellular and humoral immune response to C. albicans was assessed using the leukocyte migration test and the agglutination reaction, respectively. Normal animals:migration inhibition was significant from 1 week to 5 months after infection. Cellular hypersensitivity developed concomitantly with clearing of the infection, while antibody was not yet detectable. As the antibody titer rose, cellular hypersensitivity declined. Azathioprine-treated animals: the infection persisted and cellular hypersensitivity did not develop until 1–3 weeks after the treatment with azathioprine was discontinued. On the other hand, the humoral antibody response was early and strong. Clinically and histologically, inflammation was reduced as compared with normal animals and intraepithelial invasion by hyphae was seen. It was concluded that cellular hypersensitivity to Candidais of primary importance in host resistance against experimentally induced superficial candidiasis, as compared with serum antibody. Patterson et al. (1974) described enterocolitis caused by Candida in three spider monkeys (Ateles paniscus, A. geoffroyi, A. belzebruth) that had been treated with antibiotics and corticosteroids for diarrhea for several weeks before they died. BUDTZ-Jörgensen (1975) infected 13 adult monkeys (Macaca irus) with Candida albicans by inoculating the microorganisms under an acrylic plate covering the palatal mucosa. Six of the monkeys were treated with the steroid triamcinolone acetonide intramuscularly for 2 weeks before and 2 weeks after inoculation. The palatal mucosa was studied clinically and histologically at weekly or biweekly intervals for up to 5 months after inoculation. The cellular immune response was studied using the direct 344 leukocyte migration test. In the group of seven non-steroid-treated monkeys an acute atrophic candidiasis developed that healed spontaneously in 2—3 weeks. No tissue invasion by Candida was seen in tissue sections, but the inflammation was pronounced. Migration inhibition was significant up to 5 months after infection. In the group of six steroid-treated monkeys an acute pseudomembranous candidiasis was induced that showed retarded healing, tissue invasion by Candida, and enhanced yeast proliferation. Inflammation was only slight and the peripheral blood leukocytes were not inhibited in their migration by Candida antigen. The study has shown that systemic treatment with the steroid, triamcinolone acetonide, potentiate oral Candida infections, probably by suppressing both non-specific inflammatory responses and cellular immunity. Kaben and Essmann (1975) reported an 11 month old kodiak bear in the zoological garden in Rostock with round, partly confluent, sharply demarcated skin lesions scattered over the whole body and covered with dense scales. Lesions on the nasal cones had developed conical to 15 mm high skin tumors. The horny layer of the paws were almost completely detached, so that the epidermal relief was freed. The claws had on their underside, easily peelable hyperkeratoses. Results obtained by mycological and histological investigations revealed a cutaneous candidosis caused by Candida albicans Skin granuloma Yeasts isolated from KOH wet preparation Yeasts isolated post mortem 345 Saez (1975) reported a young Primate - Hylobates concotlor leucogenys – that died some hours after its birth in captivity at the Paris‘s zoological Park. He found a mycosis of the stomach due to Candida albicans. That candidosis, at its beginning, developed on a tumoral mass of the stomach formed during the intra-uterine life. King (1976) reported esophageal candidiasis in a severely debilitated owl monkey (Aotus trivirgatus); no other information was provided. 346 Saëz (1976) diagnosed a candidiasis due to Candida albicans on the thigh of an Europa Beaver, Castor fiber L., dead after 8 years of captivity. In the epiderma parasited of the Beaver, C. albicans has developed in a yeast-form in the superficial strates of the skin and in the filamentous-form in the deeper. McCullough et al. (1977) reported a case of spontaneous candidosis in a capuchin monkey (Cebus apella) involving nasal, pharyngeal, and intestinal mucosal surfaces and a pharyngeal lymph node. The animal had been experimentally infected with Schistosoma haematobium (Iran strain). Persistent nasal exudation and weight loss characterized the clinical disease preceding the animal's death (autopsy). Histology showed granulomatous inflammation, involving the organs mentioned above; blastospores and pseudohypnae could be demonstrated. Nakeeb et al. (1977) reported, in a 20-month period, generalized chronic cutaneous candidiasis developed in 3 performing bottle-nosed dolphins kept in an indoor pool. Extensive esophagogastric ulcerations were observed in 2 of the dolphins, each of which died, presumably because of these lesions. The 3rd dolphin died during a surgical procedure and did not have any esophagogastric ulcerations. Candida albicans was the only organism isolated from skin lesions but was not isolated from adjacent normal skin of dolphins. Treatment with antifungal drugs was unsuccessful. Subsequently, immunopotentiating treatment with levamisole phosphate resulted in formation of granulation tissue and healing of the skin lesions. Olsen and Haanaes (1977) inserted maxillary acrylic plates, inoculated with Candida albicans for 3 weeks in 10 monkeys (Cercopithecus aethiops) (Series I), and reinserted in five of the animals 8 weeks after removal (Series II). To suppress saliva flow oxyphencyclimine was injected intramuscularly (0.125 mg/kg) thrice daily for 3 weeks in six monkeys of Series I, while four controls received no drug. In Series II the oxyphencyclimine dose was doubled in three animals, and two controls were sham-treated with sodium chloride. Mean saliva flow was reduced to 58 % after 1 week and to 63 % after 3 weeks with the low dose of oxyphencyclimine. The values with the high dose were 56 % and 64 %, respectively. After 1 week thrush had developed beneath the plates of all monkeys. The patches were more extensive and regressed slower with oxyphencyclimine. Enlarged lesions were seen with the double dose, In Series I intraepithelial invasion by hyphae was detected more frequently and longer after inoculation in the oxyphencyclimine group. Such invasion was not found in biopsies from Series II. It is likely that saliva offers some protection against yeasts colonizing the fitting side of a denture. Olsen and Bondevik (1978) sat up an experimental model of yeast-induced denture stomatitis has been in the rat by inoculating Candida albicans on the fitting side of a maxillary acrylic plate retained by an orthodontic band around the incisors. Thirtyeight Wistar rats were used in two series of experiments with an observation period of 2 weeks. In each of the series there were one control and three experimental groups. Control rats were left untreated, while rats of the experimental groups wore either uninoculated or inoculated plates, or had their palatal mucosa smeared with the yeast. For cytologic examination the palate was scraped in Series I and the fitting side of the plate in Series II. After 1 week a generalized simple inflammation had developed in the palate of most animals of the experimental groups. It was most severe and persistent in rats with inoculated plates. Histologic signs of inflammation and hyphal formation were also most pronounced in this group. Hyphae did not invade the 347 epithelium. Except for an initial loss of body weight, which was restored by day 10 or 12, the rats tolerated their plates. The Wistar rat seems to be well suited for experimental studies on denture stomatitis. Saëz et al. (1978a) described PM findings in a zoo baboon (Papio papio) that died at 15 years of age, included buccal Candida infection and intestinal invagination. Of 128 animals of this species examined in recent years nine (7%) had buccal Candida infection and three (2.3%) intestinal invagination. Both conditions occurred in two animals. Glossitis, the most common form of Candida infection, appearedusually in winter (78% of cases) and most frequently in older baboons with trichuriasis or tuberculosis. Saëz et al. (1978b) reported a case of candidosis of the digestive tract associated to an obstructive epithelial tumor, with great invasive ability, in a captive Baboon, Papio papio (Desm.). Included is a discussion of the incidence of some intestinal disturbances involving candidiasis of subhuman Primates. Migaki et al. (1982) mentioned that ulcers and necrosis of the mucosa of the alimentary tract are the principal gross lesions. A granulomatous inflammatory process occurs in which the fungi are visible histologically on hematoxylin and eosin (HE)-stained sections, but they are seen and characterized better when stained with periodic acid-Schiff (PAS) or Gomori methenamine silver (GMS) techniques. Cultural or immunofluorescence studies, or both, are necessary for specific identification of the fungi. Immunosuppression is suggested as a predisposing factor in certain mycotic diseases. Candidiasis. Ulcerative glossitis in chimpanzee. Many colonies of blastospores and pseudohyphae of Candida (inset: higher magnification. PAS) on superficial portion of epithelium. HE. Courtesy of J. H. Vickers 348 Candidiasis. a. Thick pseudomembrane, composed of many pseudohyphae, onsuperficial portion of epithelium of esophagus in rhesus monkey. HE. AFIP Neg. 79-4710. b. Higher magnification of a. PAS. AFIP Neg. 79-47 12. Candidiasis. Ulcerative colitis in marmoset. PAS. Courtesy of L. V. Chalifoux. La Perle et al. (1998) diagnosed systemic candidiasis, with involvement of the spleen, liver, kidneys, and lymph nodes in a geriatric captive cheetah (Acinonyx jubatus). The animal had a long clinical history of intermittent chronic gastritis associated with Helicobacter acinonyx and chronic renal failure, both of which were repeatedly treated with broad-spectrum antimicrobial therapy. Following euthanasia, a postmortem examination showed numerous microabscesses and granulomas composed of degenerate eosinophils and containing asteroids or Splendore-Hoeppli material throughout the body. Yeast, pseudohyphae, and infrequently branching septate hyphae, demonstrated with special stains, were identified as a Candida sp. by fluorescent antibody testing. Low genetic variation in cheetahs may increase their susceptibility to infectious agents. Additional factors contributing to the overgrowth and dissemination of Candida sp. in this case may have included changes in the bacterial flora of the alimentary tract as a result of repeated antimicrobial therapy and alterations in the topography of the alimentary mucosa caused by chronic gastritis. Hernandez-Divers (2001) presented an adult female Greek tortoise (Testudo graeca) with dyspnea, lethargy, and anorexia. Severe unilateral pulmonary candidiasis was diagnosed and confirmed by histologic and microbiologic evaluations. Initial treatment with ketoconazole resulted in plasma elevations of aspartate aminotransferase, lactate dehydrogenase, and bile acids consistent with imidazole-induced hepatotoxicity. Plasma chemistry abnormalities resolved upon withdrawal of the drug. Temporary osteotomy permitted access to the diseased lung and facilitated intrapulmonary catheterization. Intrapulmonary amphotericin B therapy at 0.1 mg/kg s.i.d. for 34 days proved to be both safe and effective in this case. 349 Keck et al. (2009) reported systemic candidosis in a guanaco (Lama guanicoe) Nawange et al. (2009) observed naturally acquired disseminated dual infection caused by Candida famata and Candida catenulata in a group of albino rats bred in an animal house for sale at Jabalpur, India. Out of 200 rats examined, 40 (20%) revealed disseminated infection from which 10 (5%) exhibited infection of the brain. Mixed colonies of C. famata and C. catenulata were isolated in culture from brain, heart, lungs, liver, kidneys, spleen and stomach of the diseased animals. Histopathology revealed the presence of necrotic lesions containing yeast cells. Epidemiological studies showed the presence of the pathogens in the soil of the animal_s breeding place. It is suggested that the rats may have acquired infection from the soil either through contaminated food, drinking water or aerosol. This is the first report of the 350 naturally acquired dual infection in albino rats caused by C. famata (Debaryomyces hansenii) and C. catenulate Photograph of a diseased albino rat caused by Candida famata and Candida catenulate Histopathology of the rat_s lungs showing numerous yeast-like cells of Candida spp. in the tissues. GMS stained ·400. Morris et al. (2011) cultured bacteria and fungi from the upper respiratory tract (blowhole), gastric fluid and anus of 180 wild bottlenose dolphins (Tursiops truncatus) from two estuarine locations along the southeastern Atlantic Coast of the United States. A total of 339 and 491 isolates from Charleston, SC (CHS) and Indian River Lagoon, FL (IRL) dolphins, respectively, were cultured from gastric (70 CHS/82 IRL), fecal (141 CHS/184 IRL), and blowhole (128 CHS/225 IRL) swabs on selective media used for routine clinical microorganisms of human concern. The most frequently cultured Gram-negative bacteria from all sample and study types were Plesiomonas shigelloides, Aeromonas hydrophila, Escherichia coli, and Pseudomonas fluorescens. Among the Gram-positive bacteria, Clostridium perfringens, Bacillus sp., and Staphylococcus Coag. Neg were the predominant organisms. For fungi, the most abundant species were Candida glabrata, budding yeasts, and Candida tropicalis. Of concern, the MRSA strain of Staphylococcus aureus was detected in the blowhole and gastric swabs from CHS dolphins. In general, a greater prevalence of bacteria and fungi (four-fold increase) were cultured from IRL than CHS animals. Together, these culture-dependent studies, coupled to on-going culture-independent approaches, should help establish a baseline of microorganisms associated with bottlenose dolphins and aid in the identification of organisms responsible for infectious diseases(s) in these animals. Castelo-Branco et al. (2013) evaluated the in vitro antifungal susceptibility of Candida albicans isolates obtained during necropsy of a wild Brazilian porcupine and the mechanism of azole resistance. Initially, we investigated the in vitro susceptibility of the three isolates to amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole. Afterwards, three sub-inhibitory concentrations (47, 21 and 12 mg/l) of promethazine, an efflux pump inhibitor, were tested in combination with the antifungal drugs in order to evaluate the role of these pumps in the development of antifungal resistance. In addition, the three isolates were submitted to RAPD-PCR and M13-fingerprinting analyses. The minimum inhibitory concentrations (MICs) obtained with the isolates were 1, 0.03125, 250, 125, 8 and 250 mg/l for amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole, respectively, and the isolates were found to be resistant to all tested azoles. The addition of the three subinhibitory concentrations of promethazine resulted in statistically significant (P < 0.05) reductions in the MICs for all tested drugs, with decreases to azoles being statistically greater than those for amphotericin 351 B and caspofungin (P < 0.05). The molecular analyses showed a genetic similarity among the three tested isolates, suggesting the occurrence of candidemia in the studied animal. These findings highlight the importance of monitoring antifungal susceptibility of Candida spp. from veterinary sources, especially as they may indicate the occurrence of primary azole resistance even in wild animals. Vautier et al. (2015) made use of wild-type and morphological mutants of C. albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C. albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C. albicans, and have implications for our understanding of human disease References: 1. Budtz-Jörgensen, E. Effects of triamcinolone acetonide on experimental oral candidiasis in monkeys , J. Oral Sci 83, 3 June 1975 , 171–178 2. Budtz-Jörgensen, F. Immune response to C. albicans in monkeys with experimental candidiasis in the palate. Europ. J. Oral Sci. 81, 1973 ,360–371 3. Castelo-Branco, D. S. C. M. Castelo-Branco, R. S. N. Brilhante, M. A. N. Paiva, C. E. C. Teixeira, E. P. Caetano, J. F. Ribeiro, R. A. Cordeiro, J. J. C. Sidrim, A. J. Monteiro ,M. F. G. Rocha. Azole-resistant Candida albicans from a wild Brazilian porcupine (Coendou prehensilis): a sign of an environmental imbalance? Med Mycol (2013) 51 (5): 555-560. 4. CENTER for DISEASE CONTROL: Primate Zoonoses Surveillance, Annual Summary 1973. Atlanta, 1975 5. Fienners.:, Zoonoses of Primates, pp. 77-92. Cornell University Press, Ithaca, 1967 6. Hernandez-Divers SJ. Pulmonary candidiasis caused by Candida albicans in a Greek tortoise (Testudo graeca) and treatment with intrapulmonary amphotericin B. J Zoo Wildl Med. 2001 Sep;32(3):352-9. 7. Kaben U, Tessmann K. [Granulomatous candida mycosis in a Kodiak bear (Ursus arctos middendorfi); mycological and histological studies]. Mykosen. 1975 Jun;18(6):277-84. 8. Kauffman, A. F.; Quist, K. D. Thrush in a rhesus monkey: report of a case. 9. Laboratory Animal Care 1969 Vol.19 No.4 pp.526-527 10. Keck N, Libert C, Rispail P, Albaric O. Systemic candidosis in a guanaco (Lama guanicoe). Vet Rec. 2009 Feb 21;164(8):245. 11. King, N.W. JR.: Synopsis of the pathology of New World monkeys. In: First Inter12. American Conference on Conservation and Utilization of American Nonhuman Primates, pp. 169-178. Pan American Health Organization, Washington, 1976 13. La Perle KM, Wack R, Kaufman L, Blomme EA. Systemic candidiasis in a cheetah (Acinonyx jubatus). J Zoo Wildl Med. 1998 Dec;29(4):479-83. 14. MCCLURE, H.M.: Postmortem observation and classification of 946 nonhuman primate deaths at the Yerkes Primate Center colony. In: Center for Disease Control: Primate Zoonoses Surveillance, April-June 197 1, pp. 8-16. Atlanta, 1971 15. MCCLURE, H.M.; GUILLOUD, N.B.: Comparative pathology of the chimpanzee. In: The Chimpanzee, vol. 4, ed. Bourne, pp. 104-244. University Park Press, Baltimore, 1971 352 16. Migaki, G., R. E. Schmidt, J. D. Toft 11, And A. F. Kaufmann. Mycotic Infections of the Alimentary Tract of Nonhuman Primates: A Review. Vet. Pathol. 19(Supp. 7): 93-103, 1982 17. Morris PJ, Johnson WR, Pisani J, Bossart GD, Adams J, Reif JS, Fair PA. Isolation of culturable microorganisms from free-ranging bottlenose dolphins (Tursiops truncatus) from the southeastern United States. Vet Microbiol. 2011 Mar 24;148(24):440-7. 18. McCullough B, Moore J, Kuntz RE. Multifocal candidiasis in a capuchin monkey (Cebus apella). J Med Primatol. 1977;6(3):186-91. 19. Nakeeb S, Targowski SP, Spotte S. Chronic cutaneous candidiasis in bottle-nosed dolphins. J Am Vet Med Assoc. 1977 Nov 1;171(9):961-5. 20. Nawange, SR, Singh, K, Naidu, J, Singh, SM. Naturally acquired systemic dual infection caused by Candida famata (Debaryomyces hansenii) and Candida catenulata in albino rats bred for sale in the market at Jabalpur (Madhya Pradesh), India, Mycoses , 2009, vol. 53,173-175 21. Nelson, B.; Cosgrove, G.E.; Gengozian,.: Diseases of an imported primate Tamarinus nigricollis. Lab Anim Care 16255-275, 1966 22. Olsen I., O. Bondevik. Experimental Candida-induced denture stomatitis in the Wistar rat. J. Oral Sci. 86, 5 October 1978 , 392–398 23. Olsen I, H. R. Haanaes. Experimental palatal candidosis and saliva flow in monkeys. J. Oral Sci. 85, 2 April 1977, 135–141 24. Patterson DR, Wagner JE, Owens DR, Ronald NC, Frisk CS. Candida albicans infections associated with antibiotic and corticosteroid therapy in spider monkeys. J Am Vet Med Assoc. 1974 Apr 1;164(7):721-2. 25. Saëz H. [Cutaneous candidiosis in an European beaver, Castor fiber. Epidimiological aspect and parasitic form of Candida albicans]. Acta Zool Pathol Antverp. 1976 Dec;(66):101-10. 26. Saëz H, Rinjard J. [Buccal candidiasis and intestinal invagination in captive baboons, Papio papio]. Rev Elev Med Vet Pays Trop. 1978a;31(1):39-43. 27. Saëz H, Coudert J, Rinjard J. [Candidiasis and obstructive intestinal tumor in a captive baboon 'Papio papio' (Desm.)]. Mycopathologia. 1978b Nov 10;64(3):173-7. 28. Schmidt, R. E.; Butler, T. M.Esophageal candidiasis in a chimpanzee. Journal of the American Veterinary Medical Association 1970 Vol.157 No.5 pp.722-723 29. Wikse, S. E.; Fox, J. G.; Kovatch, R. M. Candidiasis in simian primates. Laboratory Animal Care 1970 Vol.20 pp.957-963 21. Histoplasmosis in wild animals   Histoplasmosis is the most common endemic mycosis and a major cause of morbidity in patients who live in endemic areas. It has emerged as an important opportunistic infection in immunocompromised patients including those with AIDS or who are taking medications that impair cellular immunity. Histoplasmosis is caused by the dimorphic fungus Histoplasma species o The mould form of Histoplasma capsulatum grows in soil containing rotted bird or bat guano.  Microconidia are the infectious particles of the mould, o The yeast form of H. capsulatum grows above 35°C. 353        The yeast is the pathogenic form of the organism found in the tissues of infected individuals.. Based on phenotypic characteristics (host, morphology and pathogenicity), the genus Histoplasma was split into three varieties: o Histoplasma capsulatum var. capsulatum, o Histoplasma capsulatum var. duboisii and o Histoplasma capsulatum var. farciminosum. Outbreaks of histoplasmosis have been reported in the latitudes 54° North and 38° South. Outbreaks are often reported with Histoplasma-contaminated soils, commonly in the presence of bat or bird guano. Birds are infected with H. capsulatum sporadically, however they could play role in its dispersal. In endemic areas of histoplasmosis throughout the Americas, bats are often infected with this fungus [Taylor et al.,1999, Vite-Garin et al.,2014]. Histoplasma capsulatum has been detected in wild mammals such as; o non-human primates e.g., baboons) [Walker et al., 1960, Butler et al., 1988], o mustelids (e.g., badgers and northern sea otter) [Jensen et al., 1992, Eisenberg et al., 2013, Burek-Huntington et al.,2014,], o procyonids (e.g., raccoons) [Clothier et al., 2014], Histoplasma capsulatum is a cosmopolitan fungus, and epidemiological knowledge has been improved by serology, culturing and molecular-based diagnostic methods. o The endemicity of histoplasmosis is:  Low (Europe and Oceania)  moderate (Africa and South Asia)  high (Americas). o In the United States the Midwestern and Southeastern regions specifically the Ohio, St Lawrence and Mississippi river regions are considered highly endemic [Kauffman, 2007]. o In Latin America, the high prevalence areas range from Uruguay to Mexico, mostly in countries with a moderate climate and constant humidity [Negroni, 2011]. o In Africa,—southern Africa and western/central Africa [Loulergue et al., 2007]. o In southern Asia, in China, India and Thailand, Malaysia, Indonesia, Myanmar and the Philippines [Gopalakrishnan et al., 2012].  Population structure based on molecular genotyping suggested at least 8 clades o North America clade 1 (NAm Phylogenetic o North America clade 2 (NAm 2), o Latin America clade A (LAm A), o Latin America clade B (LAm B), o Australia clade o Netherlands clade 354 o Eurasia clade o Africa clade       Phylogenetic analysis of H. capsulatum sensu stricto revealed at least 17 cryptic phylogenetic species Latin America Histoplasma capsulatum isolates contributed to the highest genetic diversity compared with other regions of the globe. South and Central America contributes to the majority of cases of histoplasmosis in the world [Gomez , 2011, Negroni et al., 2011]. Histoplasma has a worldwide distribution [Untereiner et al., 2004]. Histoplasma is found on all continents, and therefore it is reasonable to hypothesize a mechanism of long-range dispersal [Bagagli et al., 2006]. Dispersion mechanisms of Histoplasma. o Histoplasma is found and grows in nitrogen/phosphate (bird and bat guano) enriched soils and environments, which selects against other microorganisms [Lockwood et al., 1968]. o Histoplasma can naturally infect avian and chiropteran species[Pollock et al., 2003, Quist et al., 2011]. o some avian and chiropteran species are long-range migratory reservoirs, aiding in the dispersal of this microorganism [Constantine Geographic, 2003]. o Histoplasma is recurrently isolated from bats in endemic areas of histoplasmosis [Hoff and Bigler, 1981, Taylor et al., 2005]. o The fungus was isolated from internal organs as well as in bat guanoenriched soils [Muniz et al., 2020]. o Bats are widely distributed across many continents and ecosystems. o Bats live in caves, abandoned or occupied buildings, mines, plant crowns, bark, or rocks, which in association with humidity and guano enriched microenvironment favors the development of Histoplasma [Hoff and Bigler, 1981]. Examples of Animals in which histoplasmosis was reported 1. Bats Zamora (1977), Taylor et al. (1999), Taylor et al. (2000), Lyon et al. (2004), Canteros et al. (2005) Galvão Dias et al. (2011) González-González et al. (2014) 2. Badgers Jensen et al. (1992), Bauder et al. (2000), Wohlsein et al. (2001), (Eisenberg et al., 2013) (Burek-Huntington et al.,2014) 3. G. pigs Correa and Pacheco (1967) 4. Proechimys guyanensis Lainson and Shaw (1975) 5. Didelphis marsupialis Naiff et al. (1985), Naiff et al. (1996) 6. Agouti paca. Naiff et al. (1985) 7. Callithricidae Costa et al. (1994) 8. Procyonidae Costa et al. (1994) 9. Felidae Costa et al. (1994) 10. bottlenose dolphin Jensen et al. (1998) 11. Raccoons (Clothier et al., 2014) 12. maras (Dolichotis patagonum) Rosas-Rosas et al. (2006) 13. snow leopards (Uncia uncia). Espinosa-Avilés et al. (2008) 355 14. African pygmy hedgehog (Atelerix albiventris) Snider et al. (2008) 15. Gazelle dorca (Gazella dorcas neglecta Fariñas et al. (2009) 16. European hedgehog (Erinaceus europaeus) Jacobsen et al. (2010) 17. Bengal tiger (Panthera tigris). Keller et al. (2011) 18. Cebidae monkey Costa et al. (1994) 19. Baboons [Walker et al., 1960, Butler et al., 1988] Molossus molossus bats Molossus rufus bats Nyctinomops macrotis bats Eumops glaucinus bats Eumops bonariensis bats Badger G. pigs Cebidae monkey Tadarida brasiliensis bats Raccoon Proechimys Baboon monkey opossum s mara 356 Agouti paca marmosets Snow leopard African hedgehog Wild felines European hedgehog Gazella bottlenose dolphin Description Histoplasma capsulatum Darling 1906 Synonyms: Cryptococcus capsulatus Castellani et Chalmers, 1910Posadasia capsulate Moore 1934Histoplasma pyriforma Dodge 1935 Perfect stage: Emmonsiella capsulate KWON-CHUNG 1972 On Sabouraud dextrose agar, at temperature below 35C, the fungus is slow growing, usually requiring 2-6 weeks. The growth initially appears moist and waxy, then aerial mycelium develops , which is gray to white in colour and turns to buff or dark with age. Microscopically, the hyphae are small, hyaline and septate. They bear both micro- and macroconidia. The microconidia are small, round, sessile or stalked, 2-6 microns in diameter. The macroconidia, which are diagnostic, are round to pear-shaped, 8-14 microns in diameter, tuberculate and born on narrow conidiophores. In tissues, the fungus exists in the form of small, round or oval yeast-like cells, 1-4 microns in diameter. They are intracellular, often filling the cytoplasm of mononuclear and occasionally polymorphonuclear cells. 357 Reports: Correa and Pacheco (1967) described histoplasmosis naturally occurring in laboratory guinea pigs in its clinical, necropsy, histological and mycological aspects.The animals if adult show a chronic disease with progressive emaciation and lameness of the hind legs. The young below three months of age died in 2 to 4 weeks presenting ruffled fur, great dorsal curvature and sometimes closed eyelids and catarrhal conjunctivitis. At necropsy the principal lesions were ulcerative gastritis, hemorrhagic and catarrhal enteritis, enlarged spleen and mesenteric lymph nodes. Sometimes the liver, lungs, mediastinal lymph nodes and other organs showed lesions. Histological and mycological demonstration of the fungus completed the diagnosis and the surviving animals were burned and sanitation measures instituted. Histological evidence of histoplasmosis in a cow's lung from the area from which the grass was obtained for the feeding of the guinea pigs suggests an epidemiological link. Efforts will be made to isolate and demonstrate H. capsulatum in wild animals on the same area. Lainson and Shaw (1975) isolated Histoplasma from 4 rodents, Proechimys guyanensis (Echimyidae), all from virgin forest along the newly opened Trans Amazon Highway, Pará State, and from a single sloth, Choloepus didactylus, from near Belém, Pará. All these animals showed no symptoms of infection: isolation of the parasite was made by the inoculation of laboratory hamsters with saline suspensions of triturated liver and spleen. Zamora (1977) isolated Histoplasma capsulatum from only one species of bats, Brachyphylla cavernarum. Bats were collected at the Aguas Buenas Caves in Puerto Rico. The other species are: Artibeusjamaicensis, Monophyllus redmani portoricensis, and Erophylla bibom- b([rons; these resulted negative for the fungus. Naiff et al. (1985), in a survey of 296 sylvatic animals captured from virgin forests in the north-eastern and south-western Amazon of Brazil, isolated Histoplasma capsulatum, via the indirect hamster inoculation method, from the liver and spleen of four common opossums Didelphis marsupialis and two pacas Agouti paca. The infected animals did not show any clinical symptoms or histopathology. The known Amazonian mammalian species with natural histoplasmosis now total five, the previously reported species being the spiny rat Proechimys guyannensis, the two-toed sloth Choloepus didactylus and the nine-banded armadillo Dasypus novemcinctus. Jensen et al. (1992) reported the first case of disseminated histoplasmosis in an animal in Scandinavia. Yeast cells compatible with those of Histoplasma capsulatum var. capsulatum were found in the skin, liver, spleen, a kidney, and a lymph node of a wild badger (Meles meles). The diagnosis was confirmed by electron microscopy and immunofluorescence staining of the yeast cells in tissue sections. Costa et al. (1994) performed an epidomiological study of sporotrichosis and histoplasmosis in captive Latin American wild mammals, São Paulo, Brazil. Mycopathologia.using delayed hypersensitivity tests (histoplasmin and sporotrichin) in Latin American wild mammals. This research was assayed using 96 healthy animals at Parque Zoológico de São Paulo, Brazil: Primates: 33 Cebus apella-weeping-capuchin and 16 Callithrix jacchus--marmoset; Procyonidae: 37 Nasua nasua--coatimundi and 10 Felidae (Panthera onca--jaguar; Felis pardalis--ocelot Felis wiedii--margay; Felis tigrina--wild cat). For intradermic tests, the following antigens 358 were used: Sporothrix schenkii cell suspension (sporotrichin, histoplasmin-filtrate), Histoplasma capsulatum cell suspension (histoplasmin), and Histoplasma capsulatum (polysaccharide). The positivity to histoplasmin was 44.79% (Cebidae 15.15%; Callithricidae 6.25%; Procyonidae 86.49% and Felidae 50.00%, respectively). With respect to sporotrichin, 30.21% (Cebidae 6.06%, Callithricidae 0.0%; Procyonidae 64.86% and Felidae 30.00% respectively). Naiff et al. (1996) recovered 28 isolates of Histoplasma capsulatum from eight species of forest mammals from the States of Amazonas, Pará and Rondônia in the Amazon Region of Brazil. Primary isolates were obtained by inoculating triturated liver and spleen tissue intradermally and intraperitoneally in hamsters. Mycological diagnosis in hamsters presenting lesions was confirmed by histopathology and culture on Sabouraud dextrose-agar. Infected hamsters developed signs of disease within two to nine months; all had disseminated visceral lesions and most also had skin lesions at the sites of inoculation. None of the hamsters inoculated with skin macerates of the original hosts developed histoplasmosis, and histopathological examination of the viscera of the wild hosts failed to reveal H. capsulatum. Prevalence of infection was considerably higher in females than in males both for the opossum Didelphis marsupialis and for total wild animals (479) examined. It is proposed that canopy-dwelling mammals may acquire the infection from conidia borne on convective currents in hollow trees with openings at ground-level. Jensen et al. (1998) reported an approximately 37-yr-old female Atlantic bottlenose dolphin (Tursiops truncatus) that died after a 4-mo illness characterized by intermittent anorexia, lethargy, mild neutrophilic leukocytosis, and mild nonregenerative anemia. At necropsy, the lungs were diffusely consolidated, and histopathology of the lungs revealed severe pneumonia with macrophages containing clusters of numerous yeast cells. Inflammatory lesions and yeast also were found in pulmonary, mediastinal, prescapular, and duodenal lymph nodes, spleen, liver, kidneys, urinary bladder, pancreas, right adrenal gland, and the pyloric stomach. Histomorphology, fungal culture, and polymerase chain reaction analysis indicated that the fungus was Histoplasma capsulatum var. capsulatum. This is the first report of histoplasmosis in a cetacean. Taylor et al. (1999) isolated Histoplasma capsulatum from gut, lung, liver, and spleen of 17 of 208 captured bats belonging to 6 different genera and species. Three of the 17 infected bats were from the State of Guerrero and 14 were from the State of Morelos. All were adult bats: 6 males (1 Pteronotus parnellii, 2 Natalus stramineus, 2 Artibeus hirsutus, and 1 Leptonycteris nivalis) and 11 females (1 Myotis californicus, 1 Mormoops megalophylla, 8 A. hirsutus, and 1 L. nivalis). High rates of bat infection with H. capsulatum were found in the monitored sites of the State of Morelos. Histoplasma infection of N. stramineus, A. hirsutus, and L. nivalis should be considered as the first records in the world. The fungus isolated from infected bats was identified by its typical mycelial-phase morphology and by its yeast-phase conversion. Exoantigen production confirmed the fungal identification by the presence of specific precipitation lines in double immunodiffusion assays using human immune serum. Histopathologic studies showed intracellular yeast-like cells compatible with H. capsulatum yeast-phase in tissues of several bats, especially in pulmonary (intra-alveolar and septal) macrophages, with none or minimal tissue reaction. In contrast to past reports, present data support a high risk of bat infection with H. capsulatum in Mexican cave environments. 359 Bauder et al. (2000) described the first case of histoplasmosis due to infection with Histoplasma capsulatum var. capsulatum in a wild badger (Meles meles) in Austria. Diagnosis was established by histopathological and immunohistochemical characterization of yeast forms in skin lesions and lymph nodes. Although Austria has yet to be regarded as an endemic region for H. capsulatum, infections of animals and humans exposed to contaminated soil cannot be excluded. Taylor et al. (2000) presented data on the genetic polymorphism of 13 Histoplasma capsulatum isolates recovered from infected bats randomly captured in the Mexican states of Morelos, Puebla, and Oaxaca. The polymorphic DNA patterns were analyzed by two-primer RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) method. To amplify the fungal genome by PCR, the following primer arrangements were used: 5'-AACGCGCAAC-3' and 5'-AAGAGCCCGT-3'; 5'AACGCGCAAC-3' and 5'-GTTTCCGCCC-3'; or 5'-AACGCGCAAC-3' and 5'GCGATCCCCA-3'. A common polymorphic DNA pattern of H. capsulatum was revealed in different assays. This pattern is shared by 7 H. capsulatum isolates recovered from different specimens of nonmigratory bats (Artibeus hirsutus) captured in a cave in Morelos, by 5 isolates recovered from infected migratory bats (Leptonycteris nivalis) captured in Morelos and Puebla, and by 1 isolate from another migratory bat (L. curasoae) captured in Oaxaca. This polymorphic DNA pattern of H. capsulatum could represent fungal markers for the geographic areas studied, and considering its distribution in three different states of the Mexican Republic, the role of bats as responsible for H. capsulatum spreading in nature, in relation to their movements and migrations besides their shelter habits, is suggested. Analyses of DNA patterns of H. capsulatum isolated from infected bats, from clinical cases, and from blackbird excreta, have shown a major relatedness between bats and clinical isolates, in contrast to those isolates from bird excreta. Wohlsein et al. (2001) diagnosed an infection with Histoplasma capsulatum in two wild badgers (Meles meles) in northern Germany, which was predominantly localized in the skin and the regional lymph nodes. The yeast-like fungi were identified in tissue sections using histological and immunohistological methods. Lyon et al. (2004) mentioned that between October 1998 and April 1999, 51 persons belonging to two separate groups developed acute pulmonary histoplasmosis after visiting a cave in Costa Rica. The first group consisted of 61 children and 14 adults from San Jose, Costa Rica; 44 (72%) were diagnosed with acute histoplasmosis. The second group comprised 14 tourists from the United States and Canada; 9 (64%) were diagnosed with histoplasmosis. After a median incubation time of 14 days, the most common symptoms were headache, fever, cough, and myalgias. Risk factors for developing histoplasmosis included crawling (odds ratio [OR] = 17.5, 95% confidence interval [CI] = 2.3-802) and visiting one specific room (OR = 3.4, 95% CI = 1.0-12.3) in the cave. Washing hands (OR = 0.1, 95% CI = 0.01-0.6) after exiting the cave was associated with a decreased risk of developing histoplasmosis. Histoplasma capsulatum was isolated from bat guano collected from inside the cave. Persons who explore caves, whether for recreation or science, should be aware of the risk bat-inhabited caves pose for developing histoplasmosis, especially if they are immunocompromised in any way. Canteros et al. (2005) reported the first isolation of Histoplasma capsulatum var. capsulatum from a male bat Eumops bonariensis captured in Buenos Aires city in 2003. The pathogen was recovered from spleen and liver specimens, and was 360 identified by its phenotypic characteristics. PCR with primers 1283, (GTG)5, (GACA)4 and M13 was used to compare both bat isolates with 17 human isolates, 12 from patients residing in Buenos Aires city, and 5 from other countries of the Americas. The profiles obtained with the four primers showed that both bat isolates were identical to each other and closer to Buenos Aires patients than to the other isolates (similarity percentage: 91-100% and 55-97%, respectively). The high genetic relationship between bat isolates and those from patients living in Buenos Aires suggests a common source of infection. This is the first record of E. bonariensis infected with H. capsulatum in the world, and the first isolation of the fungus in the Argentinean Chiroptera population. In the same way as these wild mammals act as reservoir and spread the fungus in the natural environment, infection in urban bats could well be associated with the increase in histoplasmosis clinical cases among immunosuppressed hosts in Buenos Aires city. Rosas-Rosas et al. (2006) described the causes of death of 54 maras (Dolichotis patagonum) in a captive colony in Mexico over a period of seven years. There were 35 adults, 11 juveniles, five neonates, two fetuses and one stillbirth--27 males, 21 females and six whose sex was not determined. Trauma was the cause of 25 deaths, and there were eight cases of fatal bacterial infection. Besnoitiosis was the only parasitic disease found frequently (13 cases), and was associated with fatal interstitial pneumonia in three juveniles. Right-sided hypertrophic cardiomyopathy attributed to high altitude was observed in 26 maras, and in three cases death was attributed to acute cardiac dysfunction. Two maras died of disseminated histoplasmosis and two of hyperthermia. Additional causes of death included one case each of uterine torsion, intestinal intussusception, aspiration pneumonia and hydranencephaly. Gastric erosions with luminal haemorrhage were found in 27 of the maras and splenic lymphoid depletion in 20, changes that were attributed to stress. Espinosa-Avilés et al. (2008) reported two cases of disseminated histoplasmosis in captive snow leopards (Uncia uncia). Histoplasmosis was diagnosed based on histopathology, immunohistochemistry, transmission electron microscopy, and molecular findings. Snider et al. (2008) examined a 2-year-old captive-bred sexually intact female African pygmy hedgehog (Atelerix albiventris) because of vague signs of illness including inappetence, weakness, lethargy, and weight loss over a 20-day period. Abnormalities detected via initial clinicopathologic analyses included anemia, thrombocytopenia, leukopenia, hypoproteinemia, and hypoglycemia. Results of a fecal flotation test were negative. Three weeks after the initial evaluation, splenomegaly was detected via palpation and ultrasonography. The hedgehog was treated with broad-spectrum antibacterial agents, resulting in an initially favorable response. Fenbendazole was also administered against possible occult parasitic infestation. After 3 weeks of illness, the hedgehog's condition had worsened and supportive care and administration of additional antibacterial agents were instituted. The hedgehog died, and pathologic examinations revealed severe splenomegaly; granulomatous infiltrates were evident in multiple organs, and Histoplasma capsulatum yeasts were detected intralesionally. Fariñas et al. (2009) reported a 17 month old female gazelle dorca (Gazella dorcas neglecta), kept in captivity in a Spanish zoo, showed several symptoms of illness including fever, lethargy and behavioural changes. (X)-ray revealed ruminal "foreign bodies" and pneumonia with a nodular pattern. After surgical intervention, the animal 361 died. At necropsy, histopathologic and microbiological findings were consistent with the diagnosis of disseminated histoplasmosis, with an inflammatory histological pattern associated with immunodepression in the animal, similar to those observed in patients with severe immunodeficiency (AIDS and others). Jacobsen et al. (2010) reported disseminated histoplasmosis in European hedgehog (Erinaceus europaeus) in northern Germany. 362 Galvão Dias et al. (2011) analysed 2427 bats in the São Paulo State region. Homogenates of the livers and spleens of the bats were plated on specific medium to identify animals infected with Histoplasma capsulatum. The fungus was isolated from 87 bats (3·6%). The infected bats were identified as Molossus molossus (74), Nyctinomops macrotis (10), Tadarida brasiliensis (1), Molossus rufus (1) and Eumops glaucinus (1), all insectivorous species Keller et al. (2011) diagnosed disseminated infection with Histoplasma capsulatum in a 7-yr-old female Bengal tiger (Panthera tigris). Clinical signs were nonspecific with the exception of brief periods of tachypnea for 5 days prior to death. H. capsulatum organisms were found in the lungs, tracheobronchial lymph nodes, and liver. Diagnosis was confirmed by tracheal wash, urine H. capsulatum enzyme immunoassay, and necropsy results. This report represents the first published account of disseminated histoplasmosis in a tiger. González-González et al. (2014) screened 21 bat lungs from Argentina (AR), 13 from French Guyana (FG), and 88 from Mexico (MX) using nested-PCR of the fragments, employing the Hcp100 locus for H. capsulatum and the mtLSUrRNA and mtSSUrRNA loci for Pneumocystis organisms. Of the 122 bats studied, 98 revealed H. capsulatum infections in which 55 of these bats exhibited this infection alone. In addition, 51 bats revealed Pneumocystis spp. infection of which eight bats exhibited a Pneumocystis infection alone. A total of 43 bats (eight from AR, one from FG, and 34 from MX) were found co-infected with both fungi, representing a co-infection rate of 35.2% (95% CI = 26.8-43.6%). 363 Percentages of H. capsulatum and Pneumocystis infection and their respective co-infection in bats randomly sampled in Argentina, French Guyana, and Mexico. Each percentage value was calculated based on the 122 bats captured. Bat infection was screened with specific molecular markers for each pathogen, as described in the Methods section. Teixeira et al. (2016) mentioned that Histoplasma capsulatum comprises a worldwide complex of saprobiotic fungi mainly found in nitrogen/phosphate (often bird guano) enriched soils. The microconidia of Histoplasma species may be inhaled by mammalian hosts, and is followed by a rapid conversion to yeast that can persist in host tissues causing histoplasmosis, a deep pulmonary/systemic mycosis. Histoplasma capsulatum sensu lato is a complex of at least eight clades geographically distributed as follows: Australia, Netherlands, Eurasia, North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B) and Africa. With the exception of the Eurasian cluster, those clades are considered phylogenetic species. Increased Histoplasma sampling (n = 234) resulted in the revision of the phylogenetic distribution and population structure using 1,563 aligned nucleotides from four protein-coding regions. The LAm B clade appears to be divided into at least two highly supported clades, which are geographically restricted to either Colombia/Argentina or Brazil respectively. Moreover, a complex population genetic structure was identified within LAm A clade supporting multiple monophylogenetic species, which could be driven by rapid host or environmental adaptation (~0.5 MYA). Two divergent clades were found, which include Latin American isolates (newly named as LAm A1 and LAm A2), harboring a cryptic cluster in association with bats. It is concluded that At least six new phylogenetic species are proposed in the Histoplasma species complex supported by different phylogenetic and population genetics methods, comprising LAm A1, LAm A2, LAm B1, LAm B2, RJ and BAC-1 phylogenetic species. The genetic isolation of Histoplasma could be a result of differential dispersion potential of naturally infected bats and other mammals. In addition, the present study guides isolate selection for future population genomics and genome wide association studies in this important pathogen complex. References: 1. 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[Disseminated histoplasmosis in a dorcas gazelle (Gazella dorcas neglecta) kept in captivity conditions in Spain]. Rev Iberoam Micol. 2009 Jun 30;26(2):152-4 16. Galvão Dias M. A. , R. M. Zancopé Oliveira M. C. Giudice , H. Montenegro Netto Isolation of Histoplasma capsulatum from bats in the urban area of São Paulo State, Brazil. Zoonoses. volume 139, Issue 10 October 2011, pp. 1642-1644 17. Gomez BL. Histoplasmosis: Epidemiology in Latin America. Current fungal infection reports. 2011; 5 (4):6. 18. González-González AE, Aliouat-Denis CM, Ramírez-Bárcenas JA, Demanche C, Pottier M, Carreto-Binaghi LE, Akbar H, Derouiche S, Chabé M, Aliouat el M, Dei-Cas E, Taylor ML. Histoplasma capsulatum and Pneumocystis spp. coinfection in wild bats from Argentina, French Guyana, and Mexico. BMC Microbiol. 2014 Feb 5;14:23. 19. Hoff, G.L., william J. Bigler . The role of bats in the propagation and spread of histoplasmosis: a review Journal of Wildlife Diseases, 17(2):191-196. 1981 20. Jacobsen et al. (2010) Disseminated histoplasmosis in European hedgehog (Erinaceus europaeus) in northern Germany. Mycoses 54, 538-541, 2010 21. Jensen HE, Bloch B, Henriksen P, Dietz HH, Schønheyder H, Kaufman L. Disseminated histoplasmosis in a badger (Meles meles) in Denmark. APMIS. 1992 Jul;100(7):586-92. 365 22. Jensen ED, Lipscomb T, Van Bonn B, Miller G, Fradkin JM, Ridgway SH. Disseminated histoplasmosis in an Atlantic bottlenose dolphin (Tursiops truncatus). J Zoo Wildl Med. 1998 Dec;29(4):456-60. 23. Kauffman CA. Histoplasmosis: a clinical and laboratory update. Clinical microbiology reviews. 2007; 20(1):115–32. 24. Keller DL, Steinberg H, Sladky KK. Disseminated histoplasmosis in a Bengal tiger (Panthera tigris). J Zoo Wildl Med. 2011 Dec;42(4):727-31. 25. Lainson R, Shaw JJ. Pneumocystis and Histoplasma infections in wild animals from the Amazon region of Brazil. Trans R Soc Trop Med Hyg. 1975;69(5-6):505-8. 26. Lockwood GF, Garrison RG. The possibel role of uric acid in the ecology of Histoplasma capsulatum. Mycopathologia et mycologia applicata. 1968;35(3):377– 88. 27. Lyon GM, Bravo AV, Espino A, Lindsley MD, Gutierrez RE, Rodriguez I, Corella A, Carrillo F, McNeil MM, Warnock DW, Hajjeh RA. Histoplasmosis associated with exploring a bat-inhabited cave in Costa Rica, 1998-1999. Am J Trop Med Hyg. 2004 Apr;70(4):438-42. 28. Muniz Mde M, Morais ESTP, Meyer W, Nosanchuk JD, Zancope-Oliveira RM. Comparison of different DNA-based methods for molecular typing of Histoplasma capsulatum. Applied and environmental microbiology. 2010;76(13):4438–47 29. Naiff RD, Barrett TV, Naiff Mde F, Ferreira LC, Arias JR. New records of Histoplasma capsulatum from wild animals in the Brazilian Amazon. Rev Inst Med Trop Sao Paulo. 1996 Jul-Aug;38(4):273-7. 30. Naiff RD, Mok WY, Naiff MF. Distribution of Histoplasma capsulatum in Amazonian wildlife. Mycopathologia. 1985 Mar;89(3):165-8. 31. Negroni R. [Histoplasmosis in Latin America]. Biomedica: revista del Instituto Nacional de Salud. 2011; 31(3):304. Epub 2012/08/02. 32. Pollock C. Fungal diseases of columbiformes and anseriformes. The veterinary clinics of North America Exotic animal practice. 2003;6(2):351–61. 33. Quist EM, Belcher C, Levine G, Johnson M, Heatley JJ, Kiupel M, et al. Disseminated histoplasmosis with concurrent oral candidiasis in an Eclectus parrot (Eclectus roratus). Avian pathology: journal of the WVPA. 2011;40(2):207–11. 34. Rosas-Rosas AG, Juan-Sallés C, Garner MM. Pathological findings in a captive colony of maras (Dolichotis patagonum). Vet Rec. 2006 May 27;158(21):727-31 35. Snider TA, Joyner PH, Clinkenbeard KD. Disseminated histoplasmosis in an African pygmy hedgehog. J Am Vet Med Assoc. 2008 Jan 1;232(1):74-6. doi: 10.2460/javma.232.1.74. 36. Taylor ML, Chavez-Tapia CB, Vargas-Yanez R, Rodriguez-Arellanes G, PenaSandoval GR, Toriello C, et al. Environmental conditions favoring bat infection with Histoplasma capsulatum in Mexican shelters. The American journal of tropical medicine and hygiene. 1999; 61(6):914–9. 37. Taylor ML, Chávez-Tapia CB, Reyes-Montes MR. Molecular typing of Histoplasma capsulatum isolated from infected bats, captured in Mexico. Fungal Genet Biol. 2000 Aug;30(3):207-12. 38. Teixeira MdM, Patané JSL, Taylor ML, Gómez BL, Theodoro RC, de Hoog S, et al. (2016) Worldwide Phylogenetic Distributions and Population Dynamics of the Genus Histoplasma. PLoS Negl Trop Dis 10(6): e0004732. doi:10.1371/journal. pntd.0004732 39. Untereiner WA, Scott JA, Naveau FA, Sigler L, Bachewich J, Angus A. The Ajellomycetaceae, a new family of vertebrate-associated Onygenales. Mycologia. 2004;96(4):812–21. 40. Vite-Garin T, Estrada-Barcenas DA, Cifuentes J, Taylor ML. The importance of molecular analyses for understanding the genetic diversity of Histoplasma capsulatum: an overview. Revista iberoamericana de micologia. 2014; 31(1):11–5. 366 41. Walker J, Spooner ET. Natural infection of the African baboon Papio papio with the large-cell form of Histoplasma. The Journal of pathology and bacteriology. 1960; 80:436–8. 42. Wohlsein P, Bauder B, Kuttin ES, Kaufman L, Seeliger F, von Keyserlingk M. [Histoplasmosis in two badgers (Meles meles) in northern Germany]. Dtsch Tierarztl Wochenschr. 2001 Jun;108(6):273-6. 43. Zamora,J.R.C.Isolation of histoplasma capsulatum from tissues of bats captured in the Aguas Buenas caves, Aguas Buenos, Puerto Rico. Mycopathologia Vol. 60, 3: pp. 167-169, 1977 22. Blastomycosis in wild animals Synonyms: "North American blastomycosis", "Blastomycetic dermatitis", "Gilchrist's disease"       Blastomycosis is a fungal infection caused by Blastomyces dermatitidis. Blastomycosis occurs in several endemic areas the most important of which is in eastern North America, particularly in the western and northern periphery of the Great Lakes Basin, extending eastward along the south shore of the St. Lawrence River Valley and southward in the territory spanned by the central Appalachian Mountains in the east, to the Mississippi River Valley in the west. Blastomycosis have been reported sporadically in continental Africa, the Arabian Peninsula and the Indian subcontinent.. Blastomycosis is most common in people, dogs, and cats. Blastomycosis has also been described in horses, ferrets, deer, wolves, African lions, bottlenosed dolphins, and sea lions. Infection occurs by inhalation of infectious spores (aleurioconidia). o In the pulmonary tract, the spores transform into yeasts, causing pyogranulomas. o Yeasts can be transported from the lungs via the bloodstream or lymphatics and disseminate to other organs, like the skin and the brain. Aetiology:  Blastomyces dermatitidis is a member of the phylum Ascomycota in the family Ajellomycetaceae.  Blastomyces dermatitidis has been recognised as the asexual state of Ajellomyces dermatitidis.. 367     Blastomyces dermatitidis, a spore-forming dimorphic saprophytic fungus that thrives in humid and acidic soil rich in decaying plant or animal waste. Blastomyces dermatitidis grows as a mould in the environment and becomes yeast in host tissues. Blastomyces dermatitidis is a primary pulmonary pathogen in humans and animals The states with areas of highest endemicity are Wisconsin, Minnesota, Pathogenesis and Clinical Findings    Blastomycosis is acquired by inhaling fungal spores, and causes a respiratory and/or disseminated infection. If the inoculum is small and the animal is not immunocompromised, the infection may be limited to the respiratory tract and may have few or no clinical signs. Inhaled conidia of B. dermatitidis are phagocytosed by neutrophils and macrophages in alveoli. o Some of these escape phagocytosis and transform into yeast phase rapidly. o Having thick walls, these are resistant to phagocytosis and express glycoprotein, BAD-1, which is a virulence factor as well as an epitope. o In lung tissue, they multiply and may disseminate through blood and lymphatics to other organs, including the skin, bone, genitourinary tract, and brain. o The incubation period is 30 to 100 days, although infection can be asymptomatic Clinical Findings:  The signs vary with organ involvement and are not specific. o Weight loss may be accompanied by coughing, anorexia, lymphadenopathy, dyspnea, ocular disease, lameness, skin lesions, and fever. o Severe pulmonary involvement results in hypoxemia, which indicates a poor prognosis. o Skin lesions may include proliferative granulomas and subcutaneous abscesses that ulcerate and drain a serosanguineous discharge. o The planum nasale, face, and nail beds are most often involved. o Signs of ocular blastomycosis include blindness, uveitis, glaucoma, and retinal detachment. o Lameness associated with fungal osteomyelitis or severe paronychia occurs in some animals. o CNS signs are uncommon. 368  Lesions: o Gross lesions consist of few to numerous, variable-sized, irregular, firm, gray to yellow areas of pulmonary consolidation and nodules in the lungs and thoracic lymph nodes. o Dissemination may result in nodular lesions in various organs but especially the skin, eyes, and bone. o Cutaneous lesions are single or multiple papules, or chronic, draining, nodular pyogranulomas. Diagnosis:    Radiographic findings in the lungs include noncalcified nodules or consolidation, with enlargement of the bronchial and mediastinal lymph nodes. o The predominant patterns on thoracic radiographs are those of diffuse nodular interstitial and peribronchial densities. o Commonly, the bronchial lymph nodes are greatly enlarged and appear in radiographs as dense masses. Diagnosis can be made from biopsy of tissue or aspirated specimens taken from cutaneous lesions or other involved organs by the presence of o Thick-walled yeast that often have daughter cells budding from a broad base. o These round to ovoid, pale pink (H&E) blastospores measure 8–25 μm and have a refractile, double-contoured wall. o They may be empty or contain basophilic nuclear material and have single, broad-based buds. Serological diagnosis: o An antibody response, detected by agar gel immunodiffusion, usually occurs, but this response is neither sensitive nor specific when attempting to make a definitive diagnosis. o An enzyme immunoassay for antibodies to rBAD-1 repeat has shown improved sensitivity. o A recently developed antigen enzyme immunoassay has been used in both serum and urine to detect cell-wall galactomannan. Wild animals in which blastomycosis was reported 1. Northern sea lion Williamson et al. (1959) 2. Hamsters Landay et al. (1968) 3. Deer Jarnagin and Thoen (1977) 4. Indian fruit bat Raymond et al. (1977) 5. African lion (Panthera leo) Stroud and Coles (1980) 6. Ferret Lenhard (1985) 7. The 'lesser rat-tailed bat Randhawa et al. (1985), Chaturvedi et al. (1986) 8. Bottlenose dolphin Cates et al. (1986) 9. Wild wolf Thiel et al. (1987) 10. Polar bear (Ursus maritimus) Morris et al. (1989) 11. Rhesus monkey (Macaca mulatta) Wilkinson et al. (1999) 369 12. California sea lions (Zalophus californianus) Zwick et al. (2000) 13. Asian lions (Panthera leo persicus ) Storms et al. (2003) 14. African lion (Panthera leo ) Storms et al. (2003) 15. North American ground-dwelling insectivores Baumgardner et al. (2005) 16. American black bear (Ursus americanus) Dykstra et al. (2012) 17. Coyote (Canis latrans) Rodríguez-Tovar et al. (2015) 18. Red ruffed lemur (Varecia rubra) Rosser et al. ( 2016) Marine Mammal Center Steller Sea Lion, California Sea Lion by edwin galeano Bottlenose dolphin - Wikipedia LitlePups Hamsters Deer, Agriland Pinterest Indian Fruit Bat TrekNature Lesser Rat - Tailed Bat 370 AllPosters.com African Lion (Panthera Leo) Asiatic lion - Wikipedia African lion (Panthera leo ) WoW Amazing Polar Bear Ursus Maritimus Photo ...Getty Images American black bear - Wikipedia Wild Wolf - Orrazz :Canis latrans (Yosemite, 371 \ Rhesus macaque - Wikipedia Red ruffed lemur (Varecia rubra)Pinterest Description Blastomyces dermatitidis GILCHRIST et STOCKES 1898 Synonyms: Oidium dermatitidis RICKETTS 1901Cryptococcus gilchrisii VUILLEMIN 1902Zymonema gilchrisii BEUREMANN et GOUGEGOT 1901 Glenospora gammeli POLACCI et NANNIZZI 1927Blastomycoides tulanensis CASTELLANI 1928Monosporium tulanensis AGOSTIN 1932 B. dermatitidis is a thermically dimorphic fungus which is assumed to be a soil saprophyte in nature. It grows at room temperature as mould, developing glabrous, tan, non-conidiating colonies, or colonies with fluffy white mycelium. The colonies mature in 2 weeks and may attain dark brown colour on age. Microscopically, the mycelium consists of hyaline and septate hyphae which bear delicate conidiophores that carry on their tips round, oval or pear-shaped conidia. The fungus readily converts to the yeast phase when plated on blood agar and incubated at 37 C. The yeast colonies are wrinkled and folded, glabrous and tan or creamy in colour. Microscopically, the yeast cells are characterized by broad-based buds. Reports: 372 Williamson et al. (1959) reported North American blastomycosis in a northern sea lion. Landay et al. (1968) reported disseminated blastomycosis in adult Williamson et al. (1959) after intramuscular, intraperitoneal or subcutaneous injection of the mycelial or yeast phases of Blastomyces dermatitidis. Following spontaneous death, gross examination revealed an abscess at the site of injection. In addition, cascous lesions were found in regional lymph nodes while in the lungs there were extensive caseated nodules, and pleural adhesions. Animals that survived the 63 day period of the experiment had less extensive disease, although lesions were often observed at the site of injection, in the regional lymph nodes and in the lungs. Jarnagin and Thoen (1977) Isolated Blastomyces dermatitidis from a deer. Raymond et al. (1977) described a case of pulmonary blastomycosis in an Indian fruit bat. 373 Stroud and Coles (1980) identified Blastomyces dermatitidis by fluorescent antibody techniques in pyogranulomatous lesions in the lungs, mediastinum, trachea, and cerebrum of an African lion (Panthera leo). Clinical signs included anorexia and dry inspiratory rales. Signs of nervous system dysfunction were not reported prior to death even though a large lesion was found in the cerebrum at necropsy. The lion was shipped from a geographic area where B dermatitidis infection is endemic. It was evident from histologic studies that organisms were being shed from lesions in the 374 trachea into the environment. It was not possible to culture the organism because freezing apparently destroyed its viability. Lenhard (1985) diagnosed an 18-month-old female ferret with an ulcerated metacarpal pad and signs of respiratory illness as having blastomycosis by visualization of organisms on a tissue imprint, an agarose gel immunodiffusion test, and thoracic radiography. The ferret was treated intravenously with amphotercin B at 0.4 to 0.8 mg/kg and orally with ketoconazole at 8 mg/kg for approximately 1 month, during which time clinical and radiographic improvement was noted. A change to SC amphoterocin B therapy resulted in relapse of clinical signs despite continuation of ketoconazole therapy, and necessitated euthanasia. Necropsy revealed granulomatous lesions typical of Blastomyces dermatitidis infection in the lungs, thoracic pleura, spleen, meninges, and brain. Comparisons between this case and canine blastomycosis cases are made and alternative treatment regimes for mustelid blastomycosis are suggested. Randhawa et al. (1985) reported Blastomyces dermatitidis for the first time from the liver of Rhinopoma hardwickei hardwickei Gray (the 'lesser rat-tailed bat'); it was cultured from one of 46 samples of the bat captured on December 10, 1982, from the basement of Safdar-Jang Tomb, a historical monument in New Delhi. The fungus was not found in 581 other bats representing R. hardwickei hardwickei, three more insectivorous and one frugivorous species investigated from several sites in Delhi and New Delhi metropolitan areas. The identity of the isolate was based upon its macroscopic and microscopic cultural morphology, dimorphic character and verification of pathogenicity for white mice. It was further confirmed by determining the capacity of the isolate to produce the 'A' exoantigen specific for B. dermatitidis. The infected bat did not manifest any obvious clinical signs and symptoms of illness. Its visceral organs were free from macroscopic lesions, and histopathologically none of them including the liver, revealed any fungal elements or tissue response. B. dermatitidis was not found in any of the 34 samples of bat guano investigated by direct culture or mouse-inoculation technique. The results reinforce the available evidence for the endemic occurrence of B. dermatitidis in India and focus on the possible role of R. hardwickei hardwickei as a natural host or vector for this pathogen. Cates et al. (1986) reported an Atlantic bottlenose dolphin, Tursiops truncatus, collected in the Gulf of Mexico and maintained in Kaneohe Bay, Hawaii for eleven months, which presented clinical signs of cellulitis and pneumonia prior to death. Response to antibiotic and antifungal therapy was unremarkable. A necropsy revealed granulomatous pneumonia and severe thoracic lymphadenitis. Fungal cells, morphologically and tinctorially compatible with Blastomyces dermatitidis, were found in the heart, lung, kidney, spleen, liver, lymph node, gastrointestinal tract, and skin. The diagnosis of systemic blastomycosis was established by specific immunofluorescent staining of the fungus in tissues and detection of specific serum precipitins by immunodiffusion tests. Chaturvedi et al. (1986) reported the survival of Blastomyces dermatitidis with markedly reduced population, in the gastrointestinal tract and faeces of Rhinopoma hardwickei hardwickei orally infected with 2·5×105 colony forming units of the fungus, is reported. B. dermatitidis was cultured from the stomach, intestine and faeces up to 16–24 h and from rectum up to 48 h post infection. The results demonstrate that orally infected R. hardwickei hardwickei bats transiently shed B. 375 dermatitidis through their faeces but the significance of this route of environmental dissemination requires further evaluation. Thiel et al. (1987) reported a fatal blastomycosis to a wild wolf in Minnesota, and serologic evidence of blastomycosis was found in a Wisconsin wolf. No unusual movements were detected in the Minnesota animal from October 1983 through October 1985. However, by early December 1985, this wolf was weak and debilitated, and it perished on 14 December after approaching a human residence. Morris et al. (1989) diagnosed systemic blastomycosis in a 5-yr-old male captive polar bear (Ursus maritimus). Initial signs of disease were intermittent anorexia, weight loss, malaise, and occasional weakness in the rear legs. Thoracic radiographs revealed pleural effusion and diffuse interstitial lung disease. The definitive diagnosis of blastomycosis was made by cytologic examination and culture of pleural fluid. The blastomycosis was cured by treatment with the investigational oral antifungal triazole drug, itraconazole, for 90 days. Complete blood count, serum chemistry, and radiologic evaluation were used at 30-day intervals to monitor treatment. At a dose of 4.3 mg/kg/day, serum and pleural fluid itraconazole levels of up to 1.65 μg/ml and 0.89 μg/ml were achieved. The polar bear did not show signs of toxicity to itraconazole. In vitro minimum inhibitory concentrations and minimum lethal concentrations of this isolate for ketoconazole and itraconazole were compared. Wilkinson et al. (1999) reported an 8-year-old male rhesus monkey (Macaca mulatta) that died following a 6-day illness consisting of progressive depression, anorexia, labored abdominal breathing, coughing, and tachypnea. Gross necropsy findings included severe multifocal (miliary) granulomatous pneumonia, granulomatous splenitis, and multifocal cerebral abscesses. Histologic examination revealed 10-15microm broad-based budding organisms within pyogranulomatous inflammatory lesions in the lung, tracheobronchial lymph node, brain, spleen, and liver. The distribution of extrapulmonary lesions was intermediate between that described for dogs and that described for humans. These findings were consistent with blastomycosis, which is previously unreported in nonhuman primates. 376 Zwick et al. (2000) diagnosed 2 captive California sea lions (Zalophus californianus) from different facilities with disseminated blastomycosis. The first, a 12-yr-old male, died after a 3-wk history of progressive anorexia and lethargy. Gross examination revealed acute jejunitis with focal perforation and associated peritonitis, along with severe purulent bronchopneumonia. The second, a 15-yr-old female, was euthanized after a 2-wk history of severe cutaneous ulceration and declining clinical condition. Gross examination revealed severe pyogranulomatous bronchopneumonia and ulcerative dermatitis. Histopathologic examination in both individuals revealed severe multifocal subacute to chronic pyogranulomatous pneumonia associated with massive numbers of fungal organisms morphologically compatible with Blastomyces sp. Fungal organisms were 8-20-μm-diameter broad-based budding yeasts with thick, refractile, double-contoured walls. The male sea lion had multifocal transmural Blastomyces-induced enteritis with subsequent rupture and peritonitis. The organism was also present in the liver, with minimal associated inflammation. The female had severe multifocal pyogranulomatous ulcerative dermatitis associated with large numbers of intralesional fungal organisms. Dissemination to the spleen had occurred in both animals. A serologic immunodiffusion test for Blastomyces dermatitidis was positive in the male. The presumptive primary pathogen in both cases was Blastomyces dermatitidis. Storms et al. (2003) diagnosed blastomycosis in six nondomestic felids from eastern Tennessee, including two Asian lions (Panthera leo persicus ), one African lion (Panthera leo ), one Siberian tiger (Panthera tigris ), one cheetah (Acinonyx jubatus), and one snow leopard (Panthera uncia ). Clinical signs included lethargy, anorexia, weight loss, dyspnea, sneezing, ataxia, and paresis. Variable nonspecific changes included leukocytosis, monocytosis, moderate left shift of neutrophils, moderate hypercalcemia, hyperproteinemia, and hyperglobulinemia. Thoracic radiographs revealed interstitial and alveolar changes, consolidation or collapse of a lung lobe, bullae formation, and a pulmonary mass. Agar gel immunodiffusion (AGID) serology for Blastomyces dermatitidis was performed in five felids and was positive in three. The tiger had cerebral blastomycosis and was positive for AGID serologic tests of both cerebrospinal fluid and serum. One percutaneous lung aspirate in the snow leopard and one bronchial aspirate in an Asian lion demonstrated B. dermatitidis organisms, whereas tracheal wash samples and a nasal discharge were nondiagnostic in others. Treatment with itraconazole was attempted in four cats. The tiger improved before euthanasia, whereas the others did not survive beyond initial treatments. In four felids, B. dermatitidis was found in the lungs and tracheobronchial lymph nodes, associated with a florid pyogranulomatous reaction; the tiger had a pyogranulomatous encephalomyelitis, and the cheetah had a single pulmonary granuloma. Thoracic radiography, cytologic examination of lung lesion aspirates, and B. dermatitidis AGID serology should be performed on clinically ill zoo felids in endemic areas to rule out blastomycosis 377 Left lateral radiographic projection (a) of the cranial thorax of a snow leopard with blastomycosis, showing consolidation of cranial and middle lobes and extensive air-bronchogram pattern; left lateral gross necropsy photograph (b) of the left lung, showing irregular contour to surface of lung lobes caused by nodules and diffuse consolidation. Cr 5 cranial lobe; M 5 middle lobe; Cd 5 caudal lobe 378 Percutaneous lung aspirate from a snow leopard, showing a cluster of numerous large, thick- and doublewalled, broad-based budding yeasts, consistent with Blastomyces dermatitidis. H&E. Bar 5 5 mm Baumgardner et al. (2005) attempted to isolate Blastomyces from shrews, North American ground-dwelling insectivores that have been shown to harbor Histoplasma capsulatum in endemic areas. Forty-seven masked shrews (Sorex cinereus) and 13 northern short-tailed shrews (Blarina brevicauda) were collected in endemic areas of northern Wisconsin and Michigan using pitfall traps. Specimens were collected between 1998 and summer 2002, stored frozen, then necropsied. Cultures of nasopharynx, lungs, liver, spleen and large and small bowel were placed on yeast extract phosphate agar with one or two drops of ammonium hydroxide. Cultures for Blastomyces were negative from all 60 shrews and two deer mice (Peromyscus maniculatus) and three southern red-backed voles (Clethrionomys gapperi), which were trapped inadvertently. Histological examination of 36 of these specimens revealed no Blastomyces yeast forms. Northern Wisconsin shrews do not appear to be carriers of B. dermatitidis. Dykstra et al. (2012) reporte an aged, free-ranging, female, radio-collared American black bear (Ursus americanus) that died after an approximately 5 month long period of weight loss. Gross necropsy findings included severe diffuse pyogranulomatous bronchopneumonia, marked granulomatous lymphadenitis of tracheobronchial lymph nodes and multiple intra-abdominal lymph nodes, chronic focal jejunal ulceration, and widespread alopecia. Histopathologic examination revealed abundant fungal organisms morphologically compatible with Blastomyces sp. within pyogranulomatous inflammatory lesions in the lungs, multiple lymph nodes, liver, kidneys, jejunum, and right adrenal gland. In addition, the haircoat had a mild infestation of chewing lice (Trichodectes pinguis euarctidos), and large numbers of rhabditid nematodes consistent with Pelodera sp. were histologically observed within hair follicles. 379 380 381 Rodríguez-Tovar et al. (2015) injured a female coyote (Canis latrans) was fatally by a vehicle on a road in San Luis Potosi, Mexico. Because of deteriorating clinical signs, the animal was euthanized. Postmortem examination of the lungs showed numerous small multifocal white nodules (0.5–1 cm diameter) disseminated throughout. Histopathologic examination revealed multifocal coalescing granulomas with abundant macrophages, numerous neutrophils, fibroblasts, plasma cells, and lymphocytes. Abundant intracellular and extracellular thick-walled, refractile, spherical yeasts (10–15 μm) were observed within the granulomas. The yeasts were intensely PAS-positive, with granular protoplasm. Broad-based single budding yeasts were occasionally present. Based on the microscopic findings of the pulmonary lesions and the morphological features of the organism, a diagnosis of chronic 382 pyogranulomatous pneumonia caused by Blastomyces dermatitidis was made. To our knowledge, the case described herein is the first report of pulmonary blastomycosis in a wild coyote. Nemeth et al. (2016) mentioned that Blastomyces dermatitidis, a fungus that can cause fatal infection in humans and other mammals, is not readily recoverable from soil, its environmental reservoir. Because of the red fox‘s widespread distribution, susceptibility to B. dermatitidis, close association with soil, and well-defined home ranges, this animal has potential utility as a sentinel for this fungus. Rosser et al. ( 2016) reported a 5-yr-old, intact male red ruffed lemur (Varecia rubra) presented for evaluation as the result of a 1-wk history of lethargy and hyporexia. Physical examination findings included thin body condition, muffled heart sounds, harsh lung sounds, and liquid brown diarrhea. Complete blood count and serum biochemistry showed an inflammatory leukogram, mild hyponatremia, and mild hypochloremia. Orthogonal trunk radiographs revealed a severe alveolar pattern in the right cranial lung lobes with cardiac silhouette effacement. Thoracic ultrasound confirmed a large, hypoechoic mass in the right lung lobes. Fine-needle aspiration of the lung mass and cytology revealed fungal yeast organisms, consistent with Blastomyces dermatitidis. Blastomyces Quantitative EIA Test on urine was positive. Postmortem examination confirmed systemic blastomycosis involving the lung, tracheobronchial lymph nodes, spleen, kidney, liver, cerebrum, and eye. To the authors' knowledge, this is the first report of blastomycosis in a prosimian species. (A) The large soft tissue opacity mass is visible in the cranial to mid-thorax, causing effacement of the cardiac silhouette and caudal displacement of the carina. 383 (B) A severe alveolar pattern in the right cranial lung lobes, causing a mass effect with left lateral displacement of the trachea (arrows). Areas of increased soft tissue opacity were also visualized in the left caudal lung fields. Cytology of a lung aspirate of the same patient (31.000 magnification). Cytologic findings include a marked inflammatory infiltrate characterized by variably degenerate neutrophils and macrophages and a high number of extracellular yeast organisms. These yeasts measure 7–15 lm in diameter, with a double-contoured wall and frequent broad-based budding , consistent with B. dermatitidis. Inflammatory cells were in poor condition, which. Systemic blastomycosis primarily affecting the lungs of a captive red ruffed lemur. (A) The right cranial and middle lung lobes are noncollapsing and swollen and developed into a large mass-like structure (open arrows). The parenchyma of the entire right cranial and middle lung lobes is mostly tan and completely consolidated, with a multinodularmultilobulated appearance (insert). In contrast, the right caudal lung lobe is collapsed References: 1. Baumgardner, Dennis J., Richard Summerbell, Sigmund Krajden, Iakovina Alexopoulou, Bobby Agrawal, Mitch Bergeson, Milan Fuksa Christina Bemis & Mark A. Baumgardner. Attempted isolation of Blastomyces dermatitidis from native shrews in northern Wisconsin, USA. Medical Mycology August 2005, 43, 413/416 384 2. Cates, M. B., L. Kaufman, J. H. Grabau, J. M. Pletcher, and J. P. Schroeder, ―Blastomycosis in an Atlantic bottlenose dolphin,‖ Journal of the American Veterinary Medical Association, vol. 189, no. 9, pp. 1148–1150, 1986. 3. Chaturvedi UP, Randhawa HS, Kini S, .: 1986, Survivial of B. dermatiditis in the gastrointestinal tract of an orally infected insectivorous bat, Rhinopoma hardwickie hardwickie J Med Vet Mycol 24:349–35 4. Dykstra JA, Rogers LL, Mansfield SA, Wünschmann A. Fatal disseminated blastomycosis in a free-ranging American black bear (Ursus americanus). J Vet Diagn Invest. 2012 Nov;24(6):1125-8. 5. Jarnagin JL, Thoen CO. Isolation of dermatophilus congolensis and certain mycotic agents from animal tissues: A laboratory summary. Am J Vet Res 1977; 38(11): 1909-1910 6. Landay M.E. , E.P. Lowe, J. Mitten & F.X. Smith. Disseminated blastomycosis in hamsters after intramuscular, subcutaneous and intraperitoneal injection. Sabouraudia: Journal of Medical and Veterinary Mycology Volume 6, 1968 - Issue 4 Pages 318-323 | Published online: 09 Jul 2009 7. Lenhard L: Blastomycosis in a ferret. J Am Vet Med Assoc 186:70-72, 1985 8. Luis E. Rodríguez-Tovar, Alicia M. Nevárez-Garza, Ricardo Vladimir BarajasJuárez, Juan J. Zarate-Ramos, Rogelio A. Ledezma-Torres, and Armando TrejoChávez. Probable Pulmonary Blastomycosis in a Wild Coyote (Canis latrans). Case Reports in Veterinary Medicine Volume 2015 (2015), Article ID 564610, Case Report http://dx.doi.org/10.1155/2015/564610 9. Morris PJ, Legendre AM, Bowersock TL, et al: Diagnosis and treatment of systemic blastomycosis in a polar bear (Ursus maritimus). J Zoo Wildl Med 20:336-345, 1989 10. Nemeth NM, Campbell GD, Oesterle PT, Shirose L, McEwen B, Jardine CM. Red Fox as Sentinel for Blastomyces dermatitidis, Ontario, Canada. Emerging Infectious Diseases. 2016;22(7):1275-1277. doi:10.3201/eid2207.151789 11. Randhawa HS, Chaturvedi VP, Kini S, Khan ZU. Blastomyces dermatitidis in bats: first report of its isolation from the liver of Rhinopoma hardwickei hardwickei Gray. Sabouraudia. 1985 Feb;23(1):69-76. 12. Raymond, J.T., M. R. White, T.P. Kilbane and E. B. Janovitz. pulmonary blastomycosis in an Indian fruit bat (Pteropus giganteus). J. Vet. Diag. Invest. 9,8587, 1977 13. Rosser, M., Dana M. Lindemann, Anne M. Barger, Mark E. Howes. Systemic blastomycosis in a captive red ruffed lemur (Varecia rubra). Journal of Zoo and Wildlife Medicine 47(3):912-916 · September 2016 DOI: 10.1638/2016-0019.1 14. Storms,T. N., Victoria L. Clyde, Linda Munson, and Edward C. Ramsay. Blastomycosis in nondomestic felids. Journal of Zoo and Wildlife Medicine 34(3): 231–238, 2003 15. Stroud RK, Coles BM. Blastomycosis in an African lion. J Am Vet Med Assoc. 1980 Nov 1;177(9):842-4. 16. Sweeney JC, Migaki G, Vainik PM, et al: Systemic mycoses in marine mammals. J Am Vet Med Assoc 169:946-948, 1976 17. Thiel RP, Mech LD, Ruth GR, Archer JR, Kaufman L. Blastomycosis in wild wolves. J Wildl Dis. 1987 Apr;23(2):321-3. 18. Williamson WM, Lombard LS, Getty RE: North American blastomycosis in a northern sea lion. JAm Vet Med Assoc 135:513-515,1959 19. Wilkinson, L. M., J. M. Wallace, and J. M. Cline, ―Disseminated blastomycosis in a rhesus monkey (Macaca mulatta),‖ Veterinary Pathology, vol. 36, no. 5, pp. 460–462, 1999. 20. Zwick, L. S., M. B. Briggs, S. S. Tunev, C. A. Lichtensteiger, and R. D. Murnane, ―Disseminated blastomycosis in two California sea lions (Zalophus californianus),‖ Journal of Zoo and Wildlife Medicine, vol. 31, no. 2, pp. 211–214, 2000. View at Publisher · View at Google Scholar · View at Scopus 385 21. Storms, T. N., V. L. Clyde, L. Munson, and E. C. Ramsay, ―Blastomycosis in nondomestic felids,‖ Journal of Zoo and Wildlife Medicine, vol. 34, no. 3, pp. 231– 238, 2003. View at Publisher · View at Google Scholar ·View at Scopus 23. Coccidioidomycosis in wild animals Coccidioidomycosis is caused by Coccidioides immitis, a soil fungus native to the San Joaquin Valley of California (see the image below), and by C posadasii, which is endemic to certain arid-to-semiarid areas of the southwestern United States, northern portions of Mexico, and scattered areas in Central America and South America. Although genetically distinct, the 2 species are morphologically identical. Coccidioidomycosis is typically transmitted by inhalation of airborne spores of C immitis or C posadasii (see Etiology). Infection occurs in endemic areas and is most commonly acquired in the summer or the late fall during outdoor activities. Historical:         In 1892 Wernicke and Posadas first described a case of coccidioidomycosis in South America, in an Argentinean soldier with predominantly cutaneous manifestations. In 1894, a patient with disseminated coccidioidomycosis was first reported in California In 1896, Rixford and Gilchrist reported a few cases in which they identified the infecting agent as a protozoanlike organism and named it Coccidioides immitis. In 1905, Ophuls further described the fungal life cycle and pathology of C immitis In 1929, Harold Chope, a Stanford University medical student, accidentally inhaled a culture of Coccidioides and developed a nonfatal pulmonary illness accompanied by erythema nodosum. In the 1930s, Charles E. Smith subsequently developed coccidioidin skin test and serologic testing for coccidioidomycosis. Wildlife has also proven susceptible to the disease. In California, after the Jan. 17, 1994, Northridge earthquake triggered massive landslides in the Santa Rosa Mountains, the prevailing winds carried dust out to the Pacific coast where a population of sea lions, which had no previous exposure to the soil fungus and thus no immunity, was decimated by the disease. Residents of the desert areas of Arizona, California, Utah and New Mexico generally become aware that they could get a nasty fungal disease called coccidioidomycosis (kok-sid-e-oy-doh-my-KOH-sis). 99% of the cases 386       come from these 4 states. It is informally called Valley Fever, which is easier to pronounce. Coccidioidomycosis has been shown to affect non-human mammals, including domestic and non-domestic animals in the wild and in captivity.23 Coccidioidomycosis has been reported as the cause of death among a wide variety of animals. Examples include nondomestic canids, nondomestic felids, bats, wallabies and kangaroos, tapirs, and Przewalski's horses. (Pappagianis, 1988, Galgiani et al., 2005, Saubolle et al., 2007) Primates as a group appear particularly susceptible and many species, including lemurs, chimpanzees, gorillas, macaques, and baboons,13,15,16 have been reported to have died from disseminated coccidioidomycosis (Tamerius and Comrie , 2003, Saubolle et al., 2007, Talamantes et al., 2007) Coccidioidal infection has also been detected in free-ranging wildlife. There have been two reports of coccidioidomycosis in western cougars, in which it caused death (Elconin et al., 1964, Maddy, 1967) It has been reported as an incidental finding in three coyotes in southern Arizona (Egeberg and Ely , 1956) Necropsy revealed disseminated coccidioidomycosis as the cause of death in a bottlenose dolphin captured emaciated and ill off o southern California coast (Cordeiro et al., 2012) o sea otter in the same area (Eulalio et al., 2001) o ree-living California sea lions (Sharpton et al., 2009) Wild animals reported to be infected with Coccidoidomycosis 1. 2. 3. 4. 5. wild rodents Ashburn and Emmons, 1942. ring-tailed lemur Burton et al., 1986 monkeys Castleberry et al., 1963 western cougar (Felis concolor). Clyde et al., 1990 sea otter (Enhydra lutris) Cornell et al., 1979, Huckabone et al. (2015) 6. California sea lions Reed et al. (1976), (Zalophus californianus) Fauquier et al., 1996, (Zalophus californianus), Church (2010) Huckabone et al. (2015) 7. Bengal tigers Henrickson and Biberstein, 1972. 8. Gorilla (Gorilla beringeri) McKenney et al., 1944 9. Bottlenose dolphin Reidarson et al., 1998 10. Coyotes Straub et al., 1961 11. Sonoran gopher snake (Pituophis melanoleucus affinis) Timm et al., 1988 12. mandrill baboon (Mandrillus sphinx) Johnson et al. (1998) 13. Armadillos Eulalio et al. (2001) 14. Indochinese tiger (Panthera tigris corbetti) Helmick et al. (2006) 15. Black rhinoceros (Diceros bicornis) Wallace et al. (2009) 16. Black bear (Ursus americanus) Woods and Swift (2011),Burgdorf-Moisuk et al. (2012) 387 17. alpaca (Vicugna pacos) Diab et al. (2013) 18. gibbon (Nomascs gabriellae) Goe et al. (2013) 19. red coachwhip snake (Masticophis flagellum piceus) Churgin et al., 2013 20. Pacific walrus (Odobenus rosmarus divergens) Schmitt and Procter (2014) 21. harbor seal,Huckabone et al. (2015) 22. Carollia perspicillata bat Cordeiro et al. (2012) AllPosters.com (Felis Concolor), Coyote - Wikipedia Gorilla beringei (Eastern Gorilla) Western North America Armadillo Havahart Mandrill - Wikipedia 388 Hinterland Who's Who Black Bear Pinterest Ring-Tailed Lemur 123RF.com Yellow-cheeked gibbon sanctuarymonitoring.org California Sea Lion Pacific harbor sealPhillip Colla Flickr Adult Sea Otter (Enhydra lutris) Pacific Walrus...Photodune Black Rhinoceros The Animal Encyclopedia California Herps Sonoran Alpaca (Vicugna pacos) Flickr Gopher Snake Red Coachwhip Snake 389 Pinterest Bottlenose dolphin Carollia perspicillata bat Description Coccidioides immitis RIXFORD et GILCHRIST 1896 Synonyms: Posadasia esferiformis CANTON 1898 Blastomycoides immitis CASTELLANI 1928 Pseudococcidioides mazzai DA FONSECA 1928 Geotrichum immite AGOSTINI 1932 Coccidioides esferiformis MOORE 1932 Glenospora metaeuropea CASTELLANI 1933 Glenospora louisianoideum CASTELLANI 1933 Trichosporon proteolyticum NEGRONI et DE VILLAFANE 1938 C. immitis is a thermically dimorphic fungus that grows in nature, soil, or in the laboratory at room temperature as a mould and in tissues or in the laboratory at 37C as a yeast. The mould phase grows at first as moist, glabrous and grayish colonies that rapidly develop abundant, floccose, aerial mycelium. The mycelium is initially white, but usually becomes tan to brown with age. Microscopically, the fungus develops thin and septate hyphae that produce side branches that are much more thicker and have numerous septations. Thick-walled arthroconidia are produced in these side-branches. The arthroconidia alternate with thin-walled empty cells. The arthroconida are barrel-shaped, 2.5-4 by 3-6 um and are released by fragmentation of the mycelium. The arthroconidia are highly resistant to desiccation, temperature extremes and deprivation of nutrients and may remain viable for years. In the tissues the arthroconidium develops into spherules within a few hours or days. They become more rounded as they transform and enlarge. At maturity the spherules are 30-60 um in diameter, with a thick and prominent cell wall. Endospores are formed inside the spherules which are 2-5 um in diameter and may reach to hundreds in one spherule. At maturation the spherule ruptures and the endospores are released, which in turn develop into spherules. 390 Coccidioides posadasii M.C. Fisher, G.L. Koenig, T.J. White & J.W. Taylor, Mycologia 94 (1): 78 (2002) Reports: Reed et al. (1976) described coccidioidomycosis in an adult male California sea lion (Zalophus californianus). The animal was housed in a zoo in Tucson, Arizona, for 391 approximately 5 years. This is believed to be the first reported case of coccidioidomycosis in a marine mammal. Johnson et al. (1998) diagnosed a case of disseminated coccidioidomycosis caused by a dimorphic fungus Coccidioides immitis in a mandrill baboon (Mandrillus sphinx) following radiography, ultrasound-guided aspiration of thoracic lesions, and aspiration cytology of skeletal lesions of the left sixth rib. The diagnosis was confirmed by fungal culture and serum quantitative immunodiffusion for antibodies against C. immitis. Eulalio et al. (2001) studied natural infection of armadillos with Coccidioides immitis in the state of Piauí, northeast of Brazil, endemic for coccidioidomycosis. In 1998, 26 nine-banded armadillos (Dasypus novemcinctus) were captured in 4 different counties. The animals were sacrificed under deep anesthesia with ether. At necropsy fragments of spleen, liver, lungs and heart were homogenized and seeded onto Sabouraud dextrose agar with and without cycloheximide (BBL, USA). Part of each organ was also processed for histological examination. Suspected colonies of filamentous fungi observed after the second week of incubation at room temperature, exhibiting barrel-shaped arthroconidia alternating with empty spaces, were inoculated intraperitoneally into mice. Three armadillos proved to be infected with C. immitis. Mice inoculated with suspected colonies obtained from homogenized spleen of three and liver of two armadillos developed disseminated coccidioidomycosis and immature and mature spherules of C. immitis were disclosed in several organs. For the first time armadillos (D. novemcinctus) were found naturally infected with C. immitis, adding new data on the ecology and on a possible role of these ancestral mammals in the evolutionary life cycle of this fungus. 392 Helmick et al. (2006) reported a 19-yr-old, 78.2-kg captive female Indochinese tiger (Panthera tigris corbetti) from the El Paso Zoo (El Paso, Texas, USA) with chronic renal disease was euthanized after a 10-day course of anorexia, depression, progressive rear limb weakness, muscle fasciculations, and head tremors. Postmortem findings included pericardial effusion, generalized lymphadenopathy, glomerulosclerosis, glomerular atrophy with membranous glomerulonephropathy, and pancreatic adenocarcinoma. Pyogranulomatous pneumonia, pericarditis, and lymphadenitis were associated with fungal spherules histomorphologically consistent with Coccidioides immitis. Rising antibodies to C. immitis were detected on samples obtained perimortem and 2 mo before euthanasia. Retrospective serology was negative for two additional Indochinese tigers, two Iranian leopards (Panthera pardus saxicolor), two jaguars (Panthera onca), two bobcats (Lynx rufus texensis), two 393 ocelots (Leopardus pardalis), and three Amur leopards (Panthera pardus orientalis) housed at the zoo over an 8-yr period. Despite being located within the endemic region for C. immitis, this is only the second case of coccidioidomycosis reported from this institution. Wallace et al. (2009) reported a 5-yr-old female black rhinoceros (Diceros bicornis) which was euthanized 11 mo after arrival at the Milwaukee County Zoo (Milwaukee, Wisconsin, USA) from Glen Rose, Texas (USA) for a severe progressive rear leg lameness of 6-mo duration. Gross necropsy revealed complete rupture of the capital ligament of the left femur with synovitis and osteomyelitis. Multifocal lymphadenopathy with chronic suppurative lymphadenitis of the tracheobronchial, left supramammary, and iliac lymph nodes was present. Granulomatous pneumonia with a focal abscess was also noted. Histologically, fungal elements were seen in the lung, lymph nodes, and synovium, and Coccidioides immitis was isolated on fungal culture. Coccidioides immitis is not endemic to Wisconsin; therefore, the animal had to have been infected, although asymptomatic, at the time of arrival at the Milwaukee County Zoo. Whether the disease was active at the time of arrival or whether it was quiescent and then became active with the stress of shipment or injury is unknown. Church (2010) reported systemic coccidioidomycosis in a subadult male California sea lion (Zalophus californianus). Necropsy revealed severe atrophy of skeletal muscle and adipose tissue throughout the body. The peritoneal cavity contained 2 L of opaque, yellowish fluid. The omentum and mesentery were congested and thickened by multifocal, coalescing nodules measuring 1 – 5 mm in diameter that were also present on the serosal surface of the bladder, the peritoneal surface of the abdominal body wall, and the capsular surface of the liver and kidney. Gastric lymph nodes were 394 markedly enlarged. Histologically, the nodules corresponded to aggregates of intact and fragmented neutrophils with plump epithelioid macrophages and multinucleated giant cells. Lymphocytes, plasma cells and fibroblasts surrounded these aggregates. The centers of many of these nodules contained spherules characteristic of Coccidioides immitis (25 to 45 um diameter with a double contoured wall; containing granular basophilic material or round endospores approximately 5 um in diameter). Woods and Swift (2011) examined a biopsy of a pleural mass from an American Black bear (Ursus americanus) submitted to the California Animal Health and Food Safety Laboratory System at the University of California, Davis. Microscopically, the mass was diagnosed as a Coccidioides immitis pyogranuloma. The bear was a trophy bear that was harvested by a hunter in the San Joaquin Valley in the central valley of California in December 2010. After dressing out the bear, the hunter‘s friends and family ate the heart and liver. The hunter skinned the bear and submitted the skull and hide to the taxidermist and the remainder of the carcass to the meat processor. The taxidermist froze the skull upon receiving it. He scraped the hide, salted and pickled the hide for 14 days in water/citric acid (pH 1.5) and then froze it. After sawing the chest open, the meat processor observed large numbers of white, nodular, firm masses, 2-10 cm in diameter, on the parietal pleura of the thoracic wall. He contacted a biologist at the California Department of Fish and Game who sent the mass to the Wildlife Investigations Laboratory and advised the meat processor to discard the carcass. The mass was submitted to CAHFS in formalin a few months later at which time a diagnosis of coccidioidomycosis was made. Upon questioning, the hunter indicated he did not notice that any of the other tissues appeared to be affected when he eviscerated the carcass. No persons at risk of exposure, including the hunter, family, friends, taxidermist and meat processor reported any illness from the time of exposure until the diagnosis was made. Burgdorf-Moisuk et al. (2012) reported a 16-yr-old male koala (Phascolarctos cinereus) presented for nonspecific signs of illness and weight loss. Despite 2 mo of diagnostics and supportive care, the koala's health declined and euthanasia was elected. On histopathologic examination, lesions containing fungal organisms morphologically consistent with coccidioidomycosis were found in the lung, liver, spleen, kidney, lymph node, heart, eye, and bone marrow. Although disseminated infection was present, the koala was IgM and IgG seronegative for Coccidioides spp. 1 mo prior to euthanasia. Cordeiro et al. (2012) tested 83 bats for this fungus. Although H. capsulatum was not isolated, Coccidioides posadasii was recovered from Carollia perspicillata bat lungs. Immunologic studies detected coccidioidal antibodies and antigens in Glossophaga soricina and Desmodus rotundus bats. 395 Coccidioidal structures obtained from a naturally infected Carollia perspicillata bat (upper images) and experimentally infected mice (lower images). A) Macroscopic aspect of Coccidioides posadasii culture recovered from homogenate of bat lungs. B) Microscopic view of C. posadasii culture from bat lungs showing hyaline hyphae with arthroconidia and disjunctor cells (lactophenol cotton blue staining). C) Mature spherule filled with endospores in lung tissue (10% KOH) of bat. D) Bursting spherule with endospores in mouse lung tissue (10% KOH). E) Histopathologic features of mouse lungs revealing parasitic coccidioidal forms by periodic acid-Schiff staining. F) Coccidioidal forms on mouse lungs shown by Grocott-Gomori methenamine-silver staining. Scale bars = 20 μm. Diab et al. (2013) reported abortion and disseminated infection by Coccidioides posadasii in an alpaca (Vicugna pacos) fetus in Southern California. While the aborted dam never left California, other camelids on the premise had traveled briefly to shows in Arizona, New Mexico, Utah and Nevada. No other alpaca abortions were reported on the premise in recent past or in several weeks following this abortion. Prior cases of coccidioidomycosis occurring at the same farm included an adult alpaca, an 11-day old alpaca cria, and a guard dog. A full postmortem examination of the fetus, and gross examination of the placenta were performed and samples of heart, lungs, kidneys, liver, spleen, adrenal gland, gastrointestinal tract, brain, tongue, skin, skeletal muscle, eye and placenta were collected and fixed by immersion in 10% buffered formalin, pH 7.4, for approximately 24 h before preparing 4 µm thick histological sections. These were subsequently stained with hematoxylin and eosin, and a few select sections were additionally stained with GMS and PAS. The fetus was in a mild to moderate state of post-mortem decomposition. Roughly spherical, slightly raised, white-grey and firm, 0.2–1 cm diameter nodules were detected widely scattered throughout the lungs, spleen and intercostal skeletal muscles, less numerous on the diaphragm and skin, and rare in the liver and heart. The skin nodules were observed on the face, ventral abdomen, perianal region, and rear legs. The eyes showed marked external corneal opacity (edema) and a mid-sagittal section revealed anterior synechia and multiple white nodules expanding the iris, ciliary body and cornea. The placenta had many irregular, roughly round (~2–3 cm diameter) areas of hyperemia and hemorrhage covered by a fibrinous exudate on the chorionic and allantoic surfaces. 396 (a) Fetal lung depicting multifocal, variably sized, round, elevated, tan pyogranulomas. (b) Fetal spleen showing multifocal, variably sized, round, elevated, tan pyogranulomas. (c) Placenta showing a focal area of hyperemia and hemorrhage covered by a fibrinous exudate. (d) Microphotograph of the fetal lung showing a large, centrally mineralized pyogranuloma. Hematoxylin and eosin stain. (e) Higher magnification of Fig. 1d depicting numerous fungal spores compatible with Coccidioides spp. Hematoxylin and eosin stain.Dam's lung showing widespread, multifocal, granulomatous pneumonia caused by Coccidioides spp Dam's lung showing widespread, multifocal, granulomatous pneumonia caused by Coccidioides spp. Goe et al. (2013) reported an 8-yr-old male buff-cheeked gibbon (Nomascs gabriellae) acutely developed abnormal behavior, decreased appetite, and dull mentation. Mild generalized muscle wasting and weight loss were the only other abnormalities noted on examination. Routine immunodiffusion serology for Coccidioides spp. were IgG and IgM positive. Magnetic resonance imaging of the brain was suggestive of an infectious meningoencephalitis with secondary obstructive hydrocephalus. A ventriculoperitoneal shunt was placed in standard fashion to reduce the imminent risk of mortality from increased intracranial pressure. Postoperative treatment included oral fluconazole, a tapered course of prednisolone, and physical therapy. Clinical signs improved steadily and the gibbon was fit to return to exhibit 8 wk post-shunt placement. This case of coccidioidomycosis demonstrates the complications that can occur with dissemination to the central nervous system and its management. It is the first published report describing the use of ventriculoperitoneal shunt placement in this species. Churgin et al. (2013) reported coccidioidomycosis in an adult female, wild caught red coachwhip snake (Masticophis flagellum piceus). The snake was euthanized at the Phoenix Zoo due to severe neurologic signs. Necropsy and histopathology revealed an invasive liposarcoma of the vertebral column, which likely caused the neurologic signs. Histology of the small intestine revealed a granuloma with intralesional yeasts morphologically compatible with the genus Coccidioides. The diagnosis of 397 coccidioidomycosis was confirmed with immunohistochemistry staining. Coccidioides posadasii is endemic to Arizona and is an important cause of disseminated fungal infections in mammals in this region. This is the first known report of intestinal coccidioidomycosis in a veterinary species and the second report of coccidioidomycosis in a reptile. Schmitt and Procter (2014) reported an 11 yr-old female Pacific walrus (Odobenus rosmarus divergens) demonstrated decreased appetite and weight loss approximately 4 wk after truck transport from a northern California facility to a southern California facility. An initial blood analysis revealed a leukocytosis of 22,800 white blood cells (WBC)/microl, with a left shift, low iron (58 microg/dl), and mild hyperglobulinemia (4.3 g/dl). Empiric antibiotic therapy was started with amoxicillin and clavulanic acid (14 mg/kg p.o. b.i.d.). Clinical improvement was observed initially; however, followup blood analysis demonstrated a persistent leukocytosis (24,000 WBC/microl), with left shift and progressive hyperglobulinemia (6.7 mg/dl). As a result of the relapse of clinical signs on antibiotic therapy, aggressive antifungal therapy was initiated with voriconazole (1.8 mg/kg p.o. s.i.d.). Concurrent fungal immunodiffusion antibody assays and complement fixation were repetitively positive for coccidioidomycosis. The walrus improved clinically over the next 3 mo and is currently stable on antifungal therapy at its originating facility in northern California. Huckabone et al. (2015) analyzed stranding and necropsy data from three marine mammals to assess the prevalence, host demographics, and lesion distribution of systemic mycoses affecting locally endemic marine mammals. Between 1 January 1998 and 30 June 2012, >7,000 stranded marine mammals were necropsied at the three facilities. Necropsy and histopathology records were reviewed to identify cases of locally invasive or systemic mycoses and determine the nature and distribution of fungal lesions. Forty-one animals (0.6%) exhibited cytological, culture- or histologically confirmed locally invasive or systemic mycoses: 36 had coccidioidomycosis, two had zygomycosis, two had cryptococcosis, and one was systemically infected with Scedosporium apiospermum (an Ascomycota). Infected animals included 18 California sea lions (Zalophus californianus), 20 southern sea otters (Enhydra lutris nereis), two Pacific harbor seals (Phoca vitulina richardsi), one Dall's porpoise (Phocoenoides dalli), and one northern elephant seal (Mirounga angustirostris). Coccidioidomycosis was reported from 15 sea lions, 20 sea otters, and one harbor seal, confirming that Coccidioides spp. is the most common pathogen causing systemic mycosis in marine mammals stranding along the central California coast. They also reported the first confirmation of C. gattii infection in a wild marine mammal from California and the first report of coccidioidomycosis in a wild harbor seal. References: 1. Ashburn, L. L., and C. W. Emmons. 1942. Spontaneous coccidioidal granuloma in the lungs of wild rodents. Arch. Pathol. 34: 791–800. 2. Burgdorf-Moisuk A, Stalis IH, Pye GW. Disseminated coccidoidomycosis in a koala (Phascolarctos cinereus). J Zoo Wildl Med. 2012 Mar;43(1):197-9. 3. Burton, M., R. J. Morton, E. Ramsey, and E. L. Stair. 1986. Coccidioidomycosis in a ring-tailed lemur. J. Am. Vet. Med. Assoc. 9: 1209–1211. 4. Castleberry, M. W., J. L. Converse, and J. E. Del Favero. 1963. Coccidioidomycosis transmission to infant monkey from its mother. Arch. Pathol. 75: 459–461. 398 5. Church. M. (2010)Coccidioidomycosis in a California Sea Lion (Zalophus californianus) 6. Clyde, V. L., G. V. Kollias, M. E. Roelke, and M. R. Wells. 1990. Disseminated coccidioidomycosis in a western cougar (Felis concolor). J. Zoo Wildl. Med. 21: 200–205. 7. Cordeiro Rde A, e Silva KR, Brilhante RS, et al. Coccidioides posadasii infection in bats, Brazil. Emerg Infect Dis. 2012;18(4):668–670. 8. Cornell, L. H., K. G. Osborn, and J. E. Antrim. 1979. Coccidioidomycosis in a California sea otter (Enhydra lutris). J. Wildl. Dis. 15: 373–378. 9. Leslie Woods, Pam Swift.Coccidioidomycosis in an American Black Bear (Ursus americanus) Proceedings of the 55th Annual Coccidioidomycosis Study Group Meeting Number 55 April 2, 2011 University of California Davis Davis, California 10. Churgin, S. M., Michael M. Garner, Julie Swenson, Daniel S. Bradway, Stephanie French, Matti Kiupel, and Gary West (2013) INTESTINAL coccidioidomycosis in a red coachwhip snake (masticophis flagellum piceus). Journal of Zoo and Wildlife Medicine: December 2013, Vol. 44, No. 4, pp. 1094-1097. 11. Diab S, Johnson SM, Garcia J, Carlson EL, Pappagianis D, Smith J, Uzal FA. 2013. Case report: Abortion and disseminated infection by Coccidioides posadasii in an alpaca (Vicugna pacos) fetus in Southern California. Med Mycol Case Rep 2:159–162. 12. Egeberg RO, Ely AF. Coccidioides immitis in the soil of the southern San Joaquin Valley. Am J Med Sci. 1956;231(2):151–154. 13. Elconin AF, Egeberg RO, Egeberg MC. Significance of soil salinity on the ecology of coccidioides immitis. J Bacteriol. 1964;87:500–503. 14. Eulalio, K.D., de Macedo, R.L., Salmito Cavalcanti, M..A. et al. Coccidioides immitis isolated from armadillos (Dasypus novemcinctus) in the state of Piauí, northeast Brazil. Mycopathologia (2001) 149: 57. doi:10.1023/A:1007273019647 15. Fauquier, D. A., F. M. D. Gulland, J. G. Trupkiewicz, T. R. Spraker, and L. J. Lowenstine. 1996. Coccidioidomycosis in free-living California sea lions (Zalophus californianus) in central California. J. Wildl. Dis. 32: 707–710. 16. Galgiani JN, Ampel NM, Blair JE, et al. Coccidioidomycosis. Clin Infect Dis. 2005;41(9):1217–1223. 17. Goe A, Swenson J, West G, Evans J. Meningoencephalitis with secondary obstructive hydrocephalus caused by probable coccidioides species in a buff-cheeked gibbon (Nomascus gabriellae). J Zoo Wildl Med. 2013 Sep;44(3):781-5. 18. Helmick KE, Koplos P, Raymond J. Disseminated coccidioidomycosis in a captive Indochinese tiger (Panthera tigris corbetti) with chronic renal disease. J Zoo Wildl Med. 2006 Dec;37(4):542-4. 19. Henrickson, R. V., and E. L. Biberstein. 1972. Coccidioidomycosis accompanying hepatic disease in two Bengal tigers. J. Am. Vet. Med. Assoc. 161: 674– 677. 20. Huckabone SE, Gulland FM, Johnson SM, Colegrove KM, Dodd EM, Pappagianis D, Dunkin RC, Casper D, Carlson EL, Sykes JE, Meyer W, Miller MA. Coccidioidomycosis and other systemic mycoses of marine mammals stranding along the central California, USA coast: 1998-2012. J Wildl Dis. 2015 Apr;51(2):295-308. 21. Johnson JH, Wolf AM, Edwards JF, Walker MA, Homco L, Jensen JM, Simpson BR, Taliaferro L. Disseminated coccidioidomycosis in a mandrill baboon (Mandrillus sphinx): a case report. J Zoo Wildl Med. 1998 Jun;29(2):208-13. 22. Maddy KCH. Establishment of coccidioides immitis in negative soil following burial of infected animals and animal tissues. In: Ajello L, editor. Papers from the Second Symposium on Coccidioidomycosis. Tuscon, AZ: University of Arizona Press; 1967. pp. 309–312. 23. McKenney, F. D., J. Traum, and A. E. Bonestell. 1944. Acute coccidiomycosis in a mountain gorilla (Gorilla beringeri) with anatomical notes. J. Am. Vet. Med. Assoc. 104: 136–140. 399 24. Pappagianis D. Epidemiology of coccidioidomycosis. Curr Top Med Mycol. 1988;2:199–238. 25. Kolivras KN, Comrie AC. Modeling valley fever (coccidioidomycosis) incidence on the basis of climate conditions. Int J Biometeorol. 2003;47(2):87–101 26. Reed RE, Migaki G, Cummings JA. Coccidioidomycosis in a California sea lion (Zalophus californianus). J Wildl Dis. 1976 Jul;12(3):372-5. 27. Reidarson, T. H., L. A. Griner, D. Pappagianis, and J. McBain. 1998. Coccidioidomycosis in a bottlenose dolphin. J. Wildl. Dis. 34: 629–631. 28. Saubolle MA, McKellar PP, Sussland D. Epidemiologic, clinical, and diagnostic aspects of coccidioidomycosis. J Clin Microbiol. 2007;45(1):26–30. 29. Schmitt TL, Procter DG. Coccidioidomycosis in a Pacific walrus (Odobenus rosmarus divergens). J Zoo Wildl Med. 2014 Mar;45(1):173-5. 30. Sharpton TJ, Stajich JE, Rounsley SD, et al. Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives. Genome Res. 2009;19(10):1722–1731 31. Straub, M., R. J. Trautman, and J. W. Greene. 1961. Coccidioidomycosis in 3 coyotes. Am. J. Vet. Res. 22: 811–812. 32. Talamantes J, Behseta S, Zender CS. Fluctuations in climate and incidence of coccidioidomycosis in Kern County, California: a review. Ann N Y Acad Sci. 2007;1111:73–82. 33. Timm, K. I., R. J. Sonn, and B. D. Hultgren. 1988. Coccidioidomycosis in a Sonoran gopher snake (Pituophis melanoleucus affinis). J. Med. Vet. Mycol. 26: 101–104. 34. Wallace RS, Clyde VL, Steinberg H. Coccidioidomycosis in a black rhinoceros (Diceros bicornis). J Zoo Wildl Med. 2009 Jun;40(2):365-8. 24. Paracoccidioidomycosis in wild animals Paracoccidioidomycosis is a fungal infection endemic to South and Central America, most notably Brazil, Argentina, Colombia, and Venezuela (see the image below). Distribution of paracoccidioidomycosis in North, Central, and South America  Paracoccidioidomycosis infection is probably acquired by inhalating infective propagules present in the environment. 400      Paracoccidioidomycosis can be classified as PCM infection (infected individuals living in PCM endemic areas without symptoms of disease) and PCM disease (patients with clinical symptoms) Paracoccidioidomycosis is caused by the thermo-dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. Paracoccidioides brasiliensis, grows as mycelia when cultured at 25C or as yeast when cultured at 37C or in the host. The disease and particularly P. brasiliensis infection have been demonstrated in different species of domestic and wild animals. An armadillo species (Dasypus novemcinctus) is more associated with the eco-epidemiology of paracoccidioidomycosis, being frequently infected or showing histopathological changes suggestive of disease caused by P. brasiliensis. Armadillos dig and produce aerosols with soil particles and are probably infected by inhaling fungal conidia in suspension in the air, as is the case in humans. o The armadillos are amply distributed in Latin America where they occupy areas that coincide, at least partly, with the paracoccidioidomycosis endemic regions [Restrepo , 1994, Bagagli et al., 1998,]. o The first isolations from this mammal were those reported by Naiff et al., 1986., who in search for Leishmania reservoirs in the Brazilian region of the Amazonas river (State of Pará), surprisingly found four animals infected with the fungus as demonstrated by both histopathology and culture o Further studies by the same group, allowed recovery of the fungus from 18 new armadillos, captured in the same area although in a different locality [Naiff and Barreto, 1989]. o The fungus was cultured from various internal organs, including mesenteric lymph nodes, and also from the hamsters inoculated with the armadillos‘ tissues. Two new isolations were recently reported in Serra da Mesa, Brasil [Macedo et al., 1998]. Diagnosis:    Conclusive diagnosis of paracoccidioidomycosis has traditionally relied on the identication of P. brasiliensis from lesions found in patients, particularly upon the most characteristic feature of the yeast form, i.e., the pilot‘s wheel appearance of the mother cell surrounded by multiple peripheral daughter cells. Depending on the histopathological pattern, however, small forms of P. brasiliensis may be mistaken for other fungal infections The diagnosis of paracoccidioidomycosis by indirect serological methods that rely on antibody detection is highly valuable. Currently, rapid and efŽcient molecular methods to identify and distinguish fungal species are being applied for use as diagnostic tools. Wild animals reported to be infected with blastomyces: 1. Squirrel monkey (Saimiri sciureus) Johnson and Lang (1977) 401 2. Nine-banded armadillos (Dasypus novemcinctus) Bagagli et al. (1998), Corredor et al. (1999), Silva-Vergara et al. 2000), Tanaka et al. (2001). Hebeler-Barbosa et al. (2003), Nishikaku et al. (2008), Richini-Pereira et al. (2008) 3. wild New World monkeys (Cebus sp. and Alouatta caraya) Corte et al. (2007) 4. Cavia aperea (guinea pig) Richini-Pereira et al. (2008) 5. Cerdocyon thous (crab-eating-fox) Richini-Pereira et al. (2008) 6. Dasypus septemcinctus (seven-banded armadillo) Richini-Pereira et al. (2008) 7. Didelphis albiventris (white-eared opossum) Richini-Pereira et al. (2008) 8. Eira barbara (tayra) Richini-Pereira et al. (2008) 9. Gallictis vittata (grison) Richini-Pereira et al. (2008) 10. Procyon cancrivorus (raccoon) Richini-Pereira et al. (2008) 11. Sphiggurus spinosus (porcupine). Richini-Pereira et al. (2008) 12. captive bottlenose dolphin Esperón et al. (2012) 13. Akodon sp., Sbeghen et al. (2015) 14. Bats (Artibeus lituratus) Grose and Tamsitt (1965) 123RF.com Nine-banded Armadillo (Dasypus novemcinctus) 7-banded Armadillo (dasypus Septemcinctus), Alamy Wiki Village Common Squirrel Monkey (Saimiri sciureus sciureus) Pinterest Alouatta caraya monkey 402 Wikipedia New World monkeys (Cebus sp Pinterest Raccoon (Procyon cancrivorus) Cnaptured dolphi Fox - Cerdocyon thous Carnivora Forum Didelphis albiventris (opossum) UniProt The Online Zoo Tayra Eira Barbara Grison (Gallictis vittata) | Flickr ZooChat Porcupine (Sphiggurus spinosus) Guinea Pig Cavia Aperea ...Alamy Thaptomys nigrita, Euryoryzomys russatus, Oligoryzomys nigripes, Monodelphis sp., Sooretamys angouya, A greater fruit-eating bat, Artibeus lituratus 403 Abrawayaomys ruschii Description: Paracoccidioides brasiliensis (SPLENDORE) ALMEIDA 1930) Synonyms: Zymonema brasiliense SPLENDORE 1912 Zymonema histosporocellularis ALMEIDA 1914 Coccidioides brasiliensis ALMEIDA 1929 Coccidioides histosporocellularis FONSECA 1932 Paracoccidioides cerebriformis MOORE 1935 Paracoccidioides tenuis MOORE 1935 Lutziomyces histosporocellularis FONSECA FILHO 1939 Blastomyces brasiliensis CONANT et HOMMEL 1941 Aleurisma brasiliensis AROEIRA et BOGLIORI 1951 Perfect stage: unknown Paracoccidioides brasiliensis is the cause of paracoccidioidomycosis (South American blastomycosis). Paracoccidioides brasiliensis is a thermically dimorphic fungus. The mould phase grows slowly and matures within 3-4 weeks. The colonies vary from glabrous, leathery, brownish, flat ones with a tuft of aerial mycelium to wrinkled, folded, floccose to velvety, white, beige to pink forms. Microscopically, the hyphae are hyaline and septate and may be sterile or carry few terminal conidia. All cultures produce intercalary chlamydospores.. The mould phase is easily converted to yeast phase when subcultered and incubated at 37 C. The yeast phase grows slowly, producing wrinkled, folded, glabrous and whitish colonies. Microscopically, the yeast cells are 2-30 microns in diameter, thin-walled, oval or irregular in shape and produce multiple, thin-necked, round buds which develop from all areas of the mother cell. Reports: Grose and Tamsitt (1965) plated samples of faecal material from the intestines of 243 bats representing 6 spp. in the tropical zone of W.C. Colombia on Sabouraud's glucose medium and incubated at 25 and 37°C. P. brasiliensis was isolated from 3 of the bats, and was identified from lesions on guineapigs inoculated intratesticularly with cultures of the yeast-like phase. 404 Greer and Bolaños (1977) fed he fruit-eating bat, Artibeus lituratus, known quantities of viable yeast cells and mycelial particles of Paracoccidioides brasiliensis in an attempt to assess the role of this animal in the distribution of this agent in nature. Results of mycosal cultures of the stomach, upper intestine, lower intestine and rectum clearly showed that the fungal cells were unable to survive more than 8 hours in the digestive tract of the bat. The mycelial particles were more susceptible than the yeast and were killed before passing to the rectum. The fungus died rapidly in the voided fecal material. These findings indicate the improbability of isolating P. brasiliensis from the digestive tract of wild captured bats and show that A. lituratus probably plays no role in the distribution of this fungus in nature. Johnson and Lang (1977) reported a female squirrel monkey (Saimiri sciureus) that had granulomatous lesions in the liver and colon. There were many fungal organisms in sections of liver and many of these organisms had multiple buds on their surface. Although we did not prepare fungal cultures, the appearance of the organisms was sufficient to identify them as Paracoccidiodes brasiliensis, the cause of paracoccidioidomycosis (South American blastomycosis). Bagagli et al. (1998) reported on the high incidence of Paracoccidioides brasiliensis PCM infection in armadillos from a hyperendemic region of the disease. Four ninebanded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos are a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus. 405 406 407 Corredor et al. (1999) determinined the presence of infected armadillos in one of the paracoccidioidomycosis endemic areas of Colombia (Manizales, Department of Caldas). Based on the records of paracoccidioidomycosis patients available in the regional hospital, a locality corresponding to a permanent resident was selected, and found that it also had armadillo´s burrows. Counting with the proper authorization, two animals were captured, sacrificed under prolonged anaesthesia and various internal organs cultured in mycological media. PCR with specific P. brasiliensis´ primers was also done. The fungus was isolated from the mesenteric lymph node of one of the animals; fungal DNA amplification was positive in the same specimen as well as in the liver. The isolate from the Colombian armadillo indicated that these animals are regular hosts to P. brasiliensis in at least two endemic countries. Due to the restricted life pattern of these mammals they represent an important link with the natural habitat of the fungus. Consequently, a study of their movements and habits could prove rewarding in the search for this habitat. Silva-Vergara et al. 2000) stated that natural infection of armadillos (Dasypus novemcinctus) with Paracoccidioides brasiliensis in Northern Brazil was reported in 1986, raising great interest in the understanding of the role of this mammal in the epidemiological cycle of the fungus. Recently, P. brasiliensis was isolated from the soil of Ibiá, State of Minas Gerais, southeastern Brazil. Armadillos captured in this area were evaluated for the presence of P. brasiliensis in the viscera and infection was detected in 4/16 animals (25%). Fungal yeast phase cells were observed in three of the four infected armadillos by direct microscopic examination and by the indirect immunofluorescence test carried out on homogenized tissues. P. brasiliensis was isolated from three armadillos whose homogenized viscera had been injected into Swiss mice. The new strains (Ibiá-T1, Ibiá-T2 and Ibiá-T3) were identified as P. brasiliensis on the basis of macro- and micromorphology, thermodimorphism, production and serologic activity of exoantigens, and by polymerase chain reaction (PCR)-detection of the gp43 gene. The lethality and lesions caused to the mice from which the strains were recovered confirmed the virulence of the isolates. We conclude that P. brasiliensis infects armadillos in locations with different geoclimatic 408 characteristics and vegetation cover. The direct observation of yeast cells in tissues and the multiple visceral involvement, including the lungs, suggests the occurrence of paracoccidioidomycosis disease in these mammals and supports their role as wild hosts in the epidemiological cycle of the fungus. RESTREPO et al. (2001) mentioned that, when trying to understand the pathophysiology of any infectious agent, one key piece of information is the determination of its habitat. In the case of Paracoccidioides brasiliensis, the precise location of the fungus‘ environmental niche remains undeŽned despite the efforts of various research groups. This review summarizes recent studies on the ecology of P. brasiliensis and certain facets of paracoccidioidomycosis. Studies on the juvenile form of paracoccidioidomycosis in children less than 13 years of age, the characterization of the ecological factors in the ‗reservarea‘ where the infection is acquired and the presence of P. brasiliensis in the nine-banded armadillo (Dasypus novemcinctus), are all helping to pinpoint the microniche of this pathogen. The application of molecular biology techniques based on the ampliŽcation of nucleic acids will also hopefully help in establishing the precise habitat of P. brasiliensis. Tanaka et al. (2001) indicated that 12 isolates of Paracoccidioides brasiliensis generated cerebriform colonies at room temperature on potato glucose agar slants (PDA). These isolates contained abundant chlamydospores and yeast-like cells and were a subset of the 65 isolates obtained from nine-banded armadillos (Dasypus novemcinctus). They grew as a yeast form with typical multiple buddings at 37°C on brain heart infusion agar supplemented with 1% glucose. After replating on PDA and culturing at room temperature for 2 months, the mutants appeared as cottonous colonies, which indicated that the morphological characteristics were unstable. 409 Growth of cerebriform colonies from Paracoccidioides brasiliensis isolates Pb-267, PRT1, D4LIV1 and D4S9, respectively, at room temperature on PDA for 2 months.. (a) Paracoccidioides brasiliensis isolate PRT1 with large chlamydospores, multiple budding yeast-like cells and a few mycelia after growth at room temperature on PDA for 2 months. Cells were stained with lactophenol. Magnification: ×200. (b) Isolate Pb-265 with elongated yeast-like cells and multiple budding cells at room temperature on PDA for 2 months. Cells were stained with lactophenol. Magnification: ×200. Hebeler-Barbosa et al. (2003) tested virulence profiles of 10 P. brasiliensis isolates from different armadillos and of two clinical isolates in an experimental hamster model. Pathogenicity was evaluated by counting cfu and performing histopathological analysis in the testis, liver, spleen and lung. Circulating specific antibodies were measured using enzyme-linked immunosorbent assay (ELISA). All isolates from armadillos were virulent in the model, with dissemination to many organs. The clinical isolates, which had long been stored in cultured collections, were less virulent. The isolates were classified into four virulence categories according to number of cfu per gram of tissue: very high, high, intermediate and low. This study confirmed that armadillos harbor pathogenic genotypes of P. brasiliensis, probably the same ones that infect humans. Corte et al. (2007) evaluated the seroprevalence of Paracoccidioides brasiliensis infection in wild New World monkeys (Cebus sp. and Alouatta caraya). A total of 93 animals (Cebus sp., n = 68 and Alouatta caraya, n = 25) were captured in the Parana´ River basin, Parana´ State, Brazil and the serum samples were analyzed by ELISA and immunodiffusion using P. brasiliensis gp43 and exoantigen as antigens, respectively. The seropositivity observed by ELISA was 44.1% and 60% for Cebus sp. and A. caraya, respectively, while by immunodiffusion test Cebus sp. showed positivity of 2.9% only. No significant difference was observed in relation to age and sex. This is the first report of paracoccidioidomycosis in wild capuchin monkeys and in wild-black and golden-howler monkeys. The high positivity to P. brasiliensis infection in both species evaluated in our study and the positivity by immunodiffusion test in Cebus sp. suggest that natural disease may be occurring in wild monkeys living in paracoccidioidomycosis endemic areas. Nishikaku et al. (2008) compared the virulence profiles of Paracoccidioides brasiliensis isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates 410 induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirmed that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen. Richini-Pereira et al. (2008) evaluated the presence of Paracoccidioides brasiliensis infections by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic areas might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping 'hot spot' areas of PCM. Esperón et al. (2012) reported the diagnosis and molecular characterization of lobomycosis-like lesions in a captive bottlenose dolphin. The clinical picture and the absence of growth in conventional media resembled the features associated with Lacazia loboi. However sequencing of ribosomal DNA and further phylogenetic analyses showed a novel sequence more related to Paracoccidioides brasilensis than to L. loboi. Moreover, the morphology of the yeast cells differed from those L. loboi causing infections humans. These facts suggest that the dolphin lobomycosis-like lesions might have been be caused by different a different fungus clustered inside the order Onygenales. A successful treatment protocol based on topic and systemic terbinafine is also detailed. Albano et al. (2014) evaluated the prevalence of Paracoccidioides brasiliensis infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20%), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures. 411 Sbeghen et al. (2015) evaluated the infection of small wild mammals by P. brasiliensis in an endemic area for human PCM. Samples from different species such as Akodon sp., Thaptomys russatus, Oligoryzomys nigripes, Monodelphis sp., Abrawayaomys angouya, Abrawayaomys ruschii and 412 from 38 wild mammals nigrita, Euryoryzomys Sooretamys angouya, Akodontinae sp. were evaluated by ELISA, immunodiffusion, histopathology, nested PCR and culture. The overall positivity to gp43 observed in the ELISA was 23.7 %. Samples from heart and liver of one O. nigripes were PCR positive, and the animal was also seropositive to gp43 in ELISA. This study showed that wild animals living in endemic areas for PCM are infected with P. brasiliensis and can be valuable epidemiological markers of the fungus presence in the environment. This is the first evidence of PCM infection in Akodon sp., E. russatus, T. nigrita and O. nigripes. Minakawa et al. (2016) diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. Vilela et al. (2016) conducted phylogenetic analysis of fungi from 6 bottlenose dolphins (Tursiops truncatus) with cutaneous granulomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasiliensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidioidomycosis ceti. Infected tissues from 6 bottlenose dolphins (Tursiops truncatus) with paracoccidioidomycosis ceti, Indian River Lagoon, Florida, USA, showing typical branching chains of yeast-like cells of Paracoccidioides brasiliensis connected by small isthmuses. A) Strain FB-921; B) FB-938; C) FB-946; 413 D) FB-952; E) B92- 932; F) SW070458. Gomori‘s methenamine silver stained. Scale bars indicate 10 µm. References: 1. Albano AP, Klafke GB, Brandolt TM, Da Hora VP, Minello LF, Jorge S, Santos EO, Behling GM, Camargo ZP, Xavier MO, Meireles MC. Wild animals as sentinels of Paracoccidioides brasiliensis in the state of Rio Grande do Sul, Brazil. Mycopathologia. 2014 Apr;177(3-4):207-15. 2. Bagagli E, Sano A, Coelho KI, Alquati S, Miyaji M, de Camargo ZP, Gomes GM, Franco M, Montenegro MR. Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis. Am J Trop Med Hyg. 1998 Apr;58(4):505-12. 3. Corredor, G. G. , John H. Castaño , Luis A. Peralta , Soraya Díez , Myrtha Arango , Juan McEwen and Angela Restrepo. Original Isolation of Paracoccidioides brasiliensis from the nine-banded armadillo Dasypus novemcinctus, in an endemic area for paracoccidioidomycosis in Colombia. Rev Iberoam Micol 1999; 16: 216-220. 4. Corte , Andreia C. , Walfrido K. Svoboda , Italmar T. Navarro, Roberta L. Freire, Luciano S. Malanski, M. M. Shiozawa, Gabriela Ludwig, Lucas M. Aguiar, Fernando C. Passos, Angela Maron, Zoilo P. Camargo, Eiko N. Itano, Mario Augusto Ono. Paracoccidioidomycosis in wild monkeys from Parana´ State, Brazil. Mycopathologia (2007) 164:225–228 5. Costa EO, Diniz LS, Netto CF. The prevalence of positive intradermal reactions to paracoccidioidin in domestic and wild animals in São Paulo, Brazil. Vet Res Commun. 1995;19(2):127-30. 6. Esperón F, García-Párraga D, Bellière EN, Sánchez-Vizcaíno JM. Molecular diagnosis of lobomycosis-like disease in a bottlenose dolphin in captivity. Med Mycol. 2012 Jan;50(1):106-9. doi: 10.3109/13693786.2011.594100. Epub 2011 Aug 15. 7. Gezuele E. Aislamiento de Paracoccidioides sp. de heces de un pingüino de la Antártida. In Proc. IV International Symposium on Paracoccidioidomycosis. Caracas, Venezuela, l989: Abstract B-2. 8. Greer DL, Bolaños B. Role of bats in the ecology of Paracoccidioides brasiliensis: the survival of Paracoccidioides brasiliensis in the intestinal tract of frugivorous bat, Artibeus lituratus. Sabouraudia. 1977 Nov;15(3):273-82. 9. GROSE, ELIZABETH; TAMSITT, J. R. Paracoccidioides brasiliensis recovered from the intestinal tract of three bats (Artibeus lituratus) in Colombia, S.A Journal article : Sabouraudia 1965 Vol.4 No.2 pp.124-125 10. Hebeler-Barbosa , F., M. R. Montenegroy & E. Bagagli. Virulence profiles of ten Paracoccidioides brasiliensis isolates obtained from armadillos (Dasypus novemcinctus). Medical Mycology 2003, 41, 89–96 11. Johnson WD, Lang CM. Paracoccidioidomycosis (South American blastomycosis) in a squirrel monkey (Saimiri sciureus). Vet Pathol 1977;14:368–71. 12. Macedo R, Lacera M, Trilles Reis R, et al. Infeccão natural de tatus por Paracoccidioides brasiliensis em Serra da Mesa, Minais, Goiás: Estudo preliminar. II Congreso Brasileiro de Micologia. Rio de Janeiro, Brasil, 1998:182. 13. Minakawa T, Ueda K, Tanaka M, Tanaka N, Kuwamura M, Izawa T, et al. Detection of multiple budding yeast cells and a partial sequence of 43-kDa glycoprotein coding gene of Paracoccidioides brasiliensis from a case of lacaziosis in a female Pacific white-sided dolphin (Lagenorhynchus obliquidens). Mycopathologia. 2016;181:523– 9. http://dx.doi.org/10.1007/ s11046-016-998 14. Naiff R, Ferreira L, Barrett T, Naiff M, Arias J. Enzootic paracoccidioidomycosis in armadillos (Dasypus novemcinctus) in the State of Para. Rev Inst Med Trop Sao Paulo 1986;28:19-27. 414 15. Naiff R, Barreto T. Novos registros de Paracoccidioides brasiliensis em tatus (Dasypus novemcinctus ). Proc. Congreso Brasileiro Parasitol. Rio de Janeiro, 1989: 197. 16. NISHIKAKU, Angela Satie et al. Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates. Braz J Infect Dis [online]. 2008, vol.12, n.1, pp.57-62. <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S141386702008000100013 &lng=en&nrm=iso 17. RESTREPO , A., J. G. McEWEN & E. CASTANÄ EDA. The habitat of Paracoccidioides brasiliensis: how far from solving the riddle? Medical Mycology 2001, 39, 233±241 18. Richini-Pereira VB, Bosco Sde M, Griese J, Theodoro RC, Macoris SA, da Silva RJ, Barrozo L, Tavares PM, Zancopé-Oliveira RM, Bagagli E. Molecular detection of Paracoccidioides brasiliensis in road-killed wild animals. Med Mycol. 2008 Feb;46(1):35-40. 19. Sbeghen , Mônica Raquel, Thais Bastos Zanata, Rafaela Macagnan and Mario Augusto Ono. Paracoccidioides brasiliensis Infection in Small Wild MammalsMycopathologia 180(5-6) · August 2015 20. Silva-Vergara ML, Martinez R, Camargo ZP, Malta MH, Maffei CM, Chadu JB. Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus novemcinctus) in an area where the fungus was recently isolated from soil. Med Mycol. 2000 Jun;38(3):193-9. 21. Tanaka, R., A. Sano, M. Franco, E. Bagagli, M. Rubens Montenegro, K. Nishimura,M. Miyaji. Cerebriform colonies of Paracoccidioides brasiliensisisolated from nine-banded armadillos (Dasypus novemcinctus) at room temperature. Mycoses. 44, 1-2, 2001, 9–12 22. Grosse E, Tamsitt J. Paracoccidioides brasiliensis recovered from the intestinal tract fo 3 bats (Artibeus lituratus) in Colombia S.A. Sabouraudia l965;4:124-125. 23. Vilela Raquel, Gregory D. Bossart, Judy A. St. Leger, Leslie M. Dalton, John S. Reif, Adam M. Schaefer, Peter J. McCarthy, Patricia A. Fair, Leonel Mendoza. Cutaneous Granulomas in Dolphins Caused by Novel Uncultivated Paracoccidioides brasiliensis. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 12, December 2016, 2063-2069 25. Sporothricosis in wild animals       Sporotrichosis is a subcutaneous fungal infection that affects humans and other mammals and is caused by the thermally dimorphic fungus, Sporothrix schenckii. .Sporotrichosis has been reported in dogs, cats, horses, cows, camels, dolphins, goats, mules, birds, pigs, rats, armadillos, and people. Sporotrichosis is a sporadic, chronic, granulomatous disease . Sporotrichosis has a worldwide distribution, especially in tropical and subtropical areas. Sporotrichosis is endemic to Latin America and hyperendemic in areas including the rural highlands of Peru, Guatemala and Rio de Janeiro in BrazilSporotrichosis has been reported in the United States, South America (Brazil, Columbia, Guatemala, Mexico, Peru), Asia (China, India, Japan), Africa and Australia. 415           Sporotrichosis, in Uruguay, has a higher prevalence among armadillo hunters, following scratches received during armadillo hunting. In northeast India and Japan, there is a higher prevalence in females due to their greater engagement in agricultural activities Sporotrichosis usually results from direct inoculation of the organism into skin wounds via contact with plants or soil or penetrating foreign bodies. Sporothrix schenckii is dimorphic and forms mycelia on vegetation and in Sabouraud dextrose agar at 25°–30°C (77°–86°F) but is yeast-like in tissue and media at 37°C (98.6°F). Sporothrix schenckii is ubiquitous in soil, vegetation, and timber; is distributed worldwide. Currently, medically relevant Sporothrix spp. are o Sporothrix brasiliensis (Clade I), o Sporothrix schenckii sensu stricto (s. str.) (Clade II), o Sporothrix globosa (Clade III), o Sporothrix luriei (Clade VI) o Sporothrix mexicana (Clade IV) o Sporothrix pallida (Clade V) In nature, at 25 °C, Sporothrix spp. probably grows as mycelia and produce conidia, which are responsible for survival and dispersal in the environment. Propagules present in nature are the major source of contamination of patients, who are traumatically inoculated in tropical and subtropical zones. Soil is considered a great reservoir for major pathogenic and non-pathogenic fungal species. Sporothrix spp. could leave the saprophytic stage and infect a vertebrate host, causing disease when it undergoes a thermo dimorphic transition at 35–37 °C to a yeast-like phase, which is the parasitic form. Epidemiological studies using skin tests with sporotrichin, an antigenic preparation derived from Sporothrix spp., demonstrated that individuals are exposed naturally to Sporothrix spp. in nature. Clinical Findings and Lesions:  Sporotrichosis may be grouped into three forms: o The lymphocutaneous form is the most common. Small, firm dermal to subcutaneous nodules, 1–3 cm in diameter, develop at the site of inoculation. As infection ascends along the lymphatic vessels, cording and new nodules develop. Lesions ulcerate and discharge a serohemorrhagic exudates o The cutaneous form tends to remain localized to the site of inoculation, although lesions may be multicentric.  o Disseminated form is rare but potentially fatal and may develop with neglect of cutaneous and lymphocutaneous forms or if the animal is inappropriately treated with corticosteroids. Sporotrichosis develops via hematogenous or tissue spread from the initial site of inoculation to the bone, lungs, liver, spleen, testes, GI tract, or CNS. 416 Diagnosis:     Diagnosis can be made by culture (samples obtained from unopened lesions) or microscopic examination of the exudate or biopsy specimens. In tissues and exudate, the organism is present as few to numerous, cigarshaped, single cells within macrophages. The fungal cells are pleomorphic and small (2–10 × 1–3 μm); buds may be present and give the appearance of a ping-pong paddle. A fluorescent antibody technique has been used to identify the yeast-like cells in tissues. In cultures, a true mycelium is produced, with fine, branching, septate hyphae bearing pear-shaped conidia on slender conidiophores. Treatment:     Itraconazole is considered the treatment of choice for sporotrichosis. o Treatment should be continued 3–4 wk beyond apparent clinical cure. Terbinafine has also been used successfully. Alternatively, a supersaturated solution of potassium iodide, administered PO, has been used with some success; o therapy is continued 30 days beyond apparent clinical cure. During treatment, the animal should be monitored for signs of iodide toxicity: anorexia, vomiting, depression, muscle twitching, hypothermia, cardiomyopathy, cardiovascular collapse, and death. Zoonotic Risk:      Sporotrichosis is an important zoonosis, with animal-to-human transmission well documented. Sporotrichosis is distinct in the supposed high prevalence of animal-to-human transmission of the disease. However, it is not clear how the yeast phase transmits the infection through this route since it is generally accepted that conidia of the mycelial phase are the infectious propagules for humans. Infected nine-banded armadillos may be a source of zoonotic transmission, given their predilection for spontaneous systemic sporotrichosis , which has been extensively reported in Uruguay among armadillo hunters. Strict hygiene must be observed when handling animals (especially cats) with suspected or diagnosed sporotrichosis. People in contact with infected animals should be informed of the contagious nature of the disease when therapeutic options are discussed. Description of Sporothrix schenckii sensu stricto Sporothrix schenckii HEKTOEN et PERKINS 190 Synonyms : Sporotrichum schenckii MATRUCHOT1910 Sprotrichum beurmannii MATRUCHOT et RAMOND 1905 Sporotrichum asteroids SPLENDORE 1909 Perfect stage: Ceratocystis stenocera 417 S. schenckii is a thermically dimorphic fungus. On Sabouraud dextrose agar at 25 C colonies develop in 3-5 days, at first blackish and shiny but become fuzzy with age as aerial hyphae are produced. Initially, the colony is moist, glabrous and yeast-like , but becomes tough, wrinkled and folded in time. Microscopically, thin, branching, septate hyphae and small, 3-5 microns, conidia are seen. The conidia are delicately attached to the distal tapering ends of slender conidiophores. The conidia are arranged in flower-like clusters. At 37 oC, on media containing high concentration of sugars the organism grows in the yeast phase. Conversion to the yeast phase requires 3-5 days. The yeast colony is pasty and grayish Microscopically, the yeast cells are variable in shape, but often fusiform, 1-3 by 3-10 microns, with multiple buds. The elongated yeast cells, resembling cigars with buds, are characteristic. Reports: Kaplan et al. (1982) diagnosed two captive, adult, male, nine-banded armadillos (Dasypus novemcinctus) as having spontaneous systemic sporotrichosis. Cultures, histopathology, and immunofluorescence were used to diagnose the disease. Sporotrichosis has not been recorded previously in armadillos. 418 Conti Díaz (1989) applied the concept of 'epidemiological chain' and successively analyzed the etiologic agent, Sporothrix schenckii and its natural reservoirs (sources of infection); the different ways that infecting particles may reach man (mechanisms of infection); the susceptible population and the population at risk; the incidence and distribution by sex and age in countries of Latin America; the prevalence of the disease according to clinical cases in dermatological clinics and the variation of incidence rates in some countries with time; the influence of the environment mainly climatic conditions on the geographic distribution of the disease. Finally, according to Mackinnon's hypothesis, the climate could have a determining role on the predominance of a certain clinical form on another in different countries. He reported 138 cases of sporotrichosis over a span of 16 years in Uruguay, in which 81% were attributed to contact with armadillos. Vidal & Rodríguez (1993) reported fifteen cases of sporotrichosis within an endemic outbreak of the disease in the Northwest of the province of Santa Fe, Uruguay, among armadillo hunters (tatú mulita or Dasypus novemcinc-tus). Costa et al. (1994) performed an epidomiological study of sporotrichosis in captive Latin American wild mammals, São Paulo, Brazil. Mycopathologia.using delayed hypersensitivity tests (histoplasmin and sporotrichin) in Latin American wild mammals. This research was assayed using 96 healthy animals at Parque Zoológico de São Paulo, Brazil: Primates: 33 Cebus apella--weeping-capuchin and 16 Callithrix jacchus--marmoset; Procyonidae: 37 Nasua nasua--coatimundi and 10 Felidae (Panthera onca--jaguar; Felis pardalis--ocelot Felis wiedii--margay; Felis tigrina--wild cat). For intradermic tests, the following antigens were used: Sporothrix schenkii cell suspension (sporotrichin, histoplasmin-filtrate), Histoplasma capsulatum cell suspension (histoplasmin), and Histoplasma capsulatum (polysaccharide). With respect to sporotrichin, 30.21% (Cebidae 6.06%, Callithricidae 0.0%; Procyonidae 64.86% and Felidae 30.00% respectively). Wenker et al. (1998) reported an adult female nine-banded armadillo (Dasypus novemcinctus) died in the quarantine station of a private Swiss zoo. Multifocal ulcerative skin lesions and multiple hemorrhages in the lungs were found at necropsy. The spleen was enlarged and dark red. Histologically, there was diffuse granulomatous infiltration, including multinucleated giant cells, of the skin lesions, lungs, spleen, liver, heart, and kidneys. Abundant periodic acid-Schiff-positive yeastlike cells were demonstrated intracellularly in giant cells and extracellularly scattered throughout the tissues. Morphology of the cells varied, with some nonbudding cells resembling Cryptococcus neoformans and others resembling Sporothrix schenckii. A diagnosis of sporotrichosis was confirmed by immunofluorescence studies. This is the first report of sporotrichosis in an armadillo in a zoological garden and the third report of sporotrichosis in D. novemcinctus. Alves et al. (2010) reported ten cases of sporotrichosis evolving the armadillo's hunting diagnosed in some towns located in the central and west regions of Rio Grande do Sul State. The cases were established based on clinical and classic mycological laboratorial techniques. The susceptibility tests were conducted by microdilution technique according to M38-A2 CLSI documents. Ten cases of sporotrichosis associated with armadillo hunting detected in the State of Rio Grande do Sul were diagnosed by mycological methods. The susceptibility tests of Sporothrix 419 schenckii isolates to antifungal agents itraconazole, ketoconazole and terbinafine showed that all the isolates were susceptible. et al. (2014) reported a bottlenose dolphin Tursiops truncates, maintained in a natural sea water tank with 4 million liters water, which developed macroscopic skin lesions on the right flank and abdominal area. The case was initially diagnosed as lymphoplasmacytic perivascular dermatitis. The animal showed local suppurative ulcers and/or subcutaneous nodules without a specific microorganism or cause identified. Hematology and serum chemistry results were consistent with a nonspecific inflammatory response and included leukocytosis, neutrophilia, increased erythrocyte sedimentation rate, low serum iron and low alkaline phosphatase. No changes in behavior, appetite or energy were associated with the development of lesions. However and based on hematology and chemistry parameters, metrics of inflammation were linked to newly developing areas. Multiple diagnostic tests were performed on samples from the lesions including; PCR, cultures, histopathology, The samples collected from different areas and from different lesions were submitted for culture and histopathology and after 14 days on Sabouraud dextrose agar, PAS and blue cotton stains revealed a filamentous fungal growth. Histopathological sections showed severe pyogranulomatous inflammation of deep dermis and subcutaneous tissue, with intralesional oval to elongate-shaped budding yeast-like structures. These forms were compatible with Sporothrix schenckii and they were confirmed by phenotypic characterization and DNA sequencing. Different antifungal approaches and treatments were implemented, with no obvious resolution; however, during the last 7 months oral terbinafine (2 mg/kg SID) plus local hyperthermia had positive local and systemic effects. Bernal Bagagli et al. (2014) inoculated mice and hamsters with a samples collected at the interior (0.6–0.8 m depth) of an armadillo burrow in Manduri County. Colonies resembling Sporothrix spp. were present in cultures of the testis, spleen, liver, and mesenteric lymph node of 6 hamsters and the spleen and liver of 2 mice. The inoculated mice did not show any abnormalities during an external examination; while, the hamsters had very swollen testis and ulcerated lesions on the skin of the scrotum. It was also observed that all joints of the former and hind limbs were swollen with small ulcerated lesions of the skin. During the necropsies, lesions were not present in mice; although, there was discrete hepatosplenomegaly. Hamsters had ulcerated testes with orchitis containing caseous necrosis as well as hepatosplenomegaly. Histopathological sections showed great numbers of budding yeast cells, a characteristic of pathogenic species in the S. schenckii complex 420 Hamster 6 weeks after inoculation with a soil sample positive for S. schenckii. a An ulcerated skin lesion in the scrotum; b ulcerated skin lesions and swelling of the joint in the hind limb Histopathological section of an articular lesion from a hamster inoculated with a soil sample positive for S. schenckii showing a great number of yeast cells. a Gomori-Grocott, b PAS, X400 Chakrabarti et al. (2015) stated that sporotrichosis is an endemic mycosis caused by the dimorphic fungus Sporothrix schenckii sensu lato. It has gained importance in recent years due to its worldwide prevalence, recognition of multiple cryptic species within the originally described species, and its distinctive ecology, distribution, and epidemiology across the globe. In this review, they described the current knowledge of the taxonomy, ecology, prevalence, molecular epidemiology, and outbreaks due to S. schenckii sensu lato. Despite its omnipresence in the environment, this fungus has remarkably diverse modes of infection and distribution patterns across the world. They have delved into the nuances of how sporotrichosis is intimately linked to different forms of human activities, habitats, lifestyles, and environmental and zoonotic interactions. The purpose of this review is to stimulate discussion about the peculiarities of this unique fungal pathogen and increase the awareness of clinicians and microbiologists, especially in regions of high endemicity, to its emergence and evolving presentations and to kindle further research into understanding the unorthodox mechanisms by which this fungus afflicts different human populations. References: 421 1. Alves, Sydney Hartz, Boettcher, Cecília Schubert, Oliveira, Daniele Carvalho de, Tronco-Alves, Giordano Rafael, Sgaria, Maria Aparecida, Thadeu, Paulo, Oliveira, Loiva Therezinha, & Santurio, Janio Morais. (2010). Sporothrix schenckii associated with armadillo hunting in Southern Brazil: epidemiological and antifungal susceptibility profiles. Revista da Sociedade Brasileira de Medicina Tropical, 43(5), 523-525. 2. Chakrabarti, Arunaloke, Alexandro Bonifaz, Maria Clara Gutierrez-Galhardo, Takashi Mochizuki4 and Shanshan Li. Global epidemiology of sporotrichosis. Medical Mycology, 2015, 53, 3–14 3. Conti-Díaz IA. Epidemiology of sporotrichosis in Latin America. Mycopathologia 1989; 108:113-116. 4. Costa EO, Diniz LS, Netto CF, Arruda C, Dagli ML. Epidemiological study of sporotrichosis and histoplasmosis in captive Latin American wild mammals, São Paulo, Brazil. Mycopathologia. 1994 Jan;125(1):19-22. 5. Jaime A. Bernal1*; Robert T. Braun1; Martin Haulena4; Wayne F. Phillips1,2; Gloria M. González5; José A. Rueda3; Roberto A. Real1; Alejandro R. Garcia2; Ricardo C. Santos2; Marcos H. Cancino2; Hector R. Ramirez6; David A. Espinosa. Chronic Subcutaneous Sporotrichosis (S. schenckii) in T. truncatus Dolphin: Differentials, Diagnostics, Identification and Treatment. IAAAM 2014 6. Kaplan W, Broderson JR, Pacific JN. Spontaneous systemic sporotrichosis in ninebanded armadillos (Dasypus novemcinctus). Sabouraudia. 1982;20:289–94. 7. Rodrigues AM, Bagagli E, de Camargo ZP, de Moraes Gimenes Bosco S. Sporothrix schenckii sensu stricto isolated from soil in an armadillo‘s burrow. Mycopathologia 2014; 177: 199–206. 8. Vidal G, Rodriguez-de-Kopp N. Esporotricosis: enfoque epidemiológico, clínico y terapéutico. Arch Argent Dermatol 1993; 63:221-234. 9. Wenker CJ, Kaufman L, Bacciarini LN, Robert N. Sporotrichosis in a nine-banded armadillo (Dasypus novemcinctus). J Zoo Wildl Med. 1998 Dec;29(4):474-8. 422