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
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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
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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.
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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.
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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.
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(Cercopithecus lhoesti). Vet Rec. 2010 Nov 27;167(22):862-4.
22. Klingmüller G, Heymer T, Sobich E. [Trichophyton mentagrophytes var. erinacei
infection contracted from a hedgehog]. Hautarzt. 1979 Mar;30(3):140-3.
23. Klokke, A.H, and G.A, de Vries (1963): Tinea capitis in chimpanzees caused by
Microsporum canis Bodin 1902 resembling M, obesum Conant 1937. Sabouraudia, 2,
268-270.
24. Koch, HA, W Jänisch -Eine Mikrosporum‐gypseum‐Enzootie bei Rhesusaffen
(Macacus rhesus) Mycoses, 1964 - Wiley Online Library
25. Koroleva VP. Rasprostravennost vozbuditelei dermatomikozov zhivotnykh v raznykh
zonakh soyuza. Byulletin Vsesoyuznogo I nstituta Eksperimental'noi Veterinarii
1976; 25: 49-52.
26. Koroleva VP. Rasprostravennost vozbuditelei dermatomikozov zhivotnykh v raznykh
zonakh soyuza. Byulletin Vsesoyuznogo I nstituta Eksperimental'noi Veterinarii
1976; 25: 49-52.
27. Knudtson WU, Gates CE, Ruth GR, Haley LD. Trichophyton mentagrophytes
dermatophytosis in wild fox. J Wildl Dis. 1980 Oct;16(4):465-8.
28. Knudtson WU, Gates CE, Ruth GR, Haley LD. Trichophyton mentagrophytes
dermatophytosis in wild fox. J Wildl Dis. 1980 Oct;16(4):465-8.
29. Kuntze A, Gemeinhardt H, Bensch GJ. [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
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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
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38. Pombier EC, Kim JC. An epizootic outbreak of ringworm in a guinea-pig colony
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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.
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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).
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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)
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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.
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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
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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.
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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.
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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
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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.
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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)
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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
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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
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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
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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
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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.
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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. These results underscore the notion
that pathogen resistance is an emergent property of microbial communities, a
consideration that should be taken into account when designing probiotic treatments
to reduce the impacts of infectious disease.
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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.
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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.
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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.
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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
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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
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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.
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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).
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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-
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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
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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.
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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.
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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).
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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
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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.
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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
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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.
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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).
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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 = spongiousshowing 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
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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.
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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.
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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.
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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
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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
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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
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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
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species
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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.
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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
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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
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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.
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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.
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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.
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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
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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.
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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.
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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)
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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.
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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.
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Nishimura,M. Miyaji. Cerebriform colonies of Paracoccidioides brasiliensisisolated
from nine-banded armadillos (Dasypus novemcinctus) at room temperature. Mycoses.
44, 1-2, 2001, 9–12
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fo 3 bats (Artibeus lituratus) in Colombia S.A. Sabouraudia l965;4:124-125.
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Granulomas in Dolphins Caused by Novel Uncultivated Paracoccidioides brasiliensis.
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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.
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