Kumarasingha et al.

Parasites & Vectors

DOI 10.1186/s13071-016-1458-9

RESEARCH Open Access

Anthelmintic activity of selected

ethno-medicinal plant extracts on
parasitic stages of Haemonchus contortus
Rasika Kumarasingha1, Sarah Preston2, Tiong-Chia Yeo3, Diana S. L. Lim3, Chu-Lee Tu3, Enzo A. Palombo4,
Jillian M. Shaw5, Robin B. Gasser2* and Peter R. Boag1,2*

Abstract
Background: Parasitic roundworms (nematodes) cause substantial morbidity and mortality in livestock animals
globally, and considerable productivity losses to farmers. The control of these nematodes has relied largely on
the use of a limited number of anthelmintics. However, resistance to many of these these anthelmintics is now
widespread, and, therefore, there is a need to find new drugs to ensure sustained and effective treatment and
control into the future.
Methods: Recently, we developed a screening assay to test natural, plant extracts with known inhibitory effects
against the free-living worm Caenorhabditis elegans. Using this assay, we assessed here the effects of the extracts on
motility and development of parasitic larval stages of Haemonchus contortus, one of the most important nematodes
of small ruminants worldwide.
Results: The study showed that two of five extracts from Picria fel-terrae Lour. have a significant inhibitory effect
(at concentrations of 3–5 mg/ml) on the motility and development of H. contortus larvae. Although the two
extracts originated from the same plant, they displayed different levels of inhibition on motility and development,
which might relate to the presence of various active constituents in these extracts, or the same constituents at
different concentrations in distinct parts of the plant.
Conclusions: These results suggest that extracts from P. fel-terrae Lour. have promising anthelmintic activity and
that more broadly, plant extracts are a potential rich source of anthelmintics to combat helminthic diseases.
Keywords: Medicinal plant extracts, Anthelmintic activity, Developmental assay, In vitro-assay

Background pole worm; Nematoda: Strongylida), feeds on blood in the

Parasitic diseases cause major morbidity and mortality in stomach (abomasum) and causes gastritis, anaemia and
animals globally, and considerable losses to food produc- associated complications, leading to production losses and
tion. For instance, haemonchosis is one of the most signifi- death in severely affected animals. This nematode is trans-
cant parasitic diseases of livestock worldwide, affecting mitted orally from contaminated pasture to the host
hundreds of millions of small ruminants (including sheep through a complex life-cycle [3]: eggs are excreted in the
and goats) and causing substantial losses to the livestock host faeces and hatch into first-stage larva (L1) usually
industry estimated at tens of billions of dollars per annum within 1 day and then develop through to the second (L2)
[1, 2]. The causative agent, Haemonchus contortus (barber’s and third (L3) larval stages in about one week. Infective L3s
are then ingested by the host, exsheath (xL3) and, after a
* Correspondence: robinbg@unimelb.edu.au; peter.boag@monash.edu histotrophic phase, develop through fourth-stage larvae
1
Development and Stem Cells Program, Monash Biomedicine Discovery (L4) to dioecious adults (within 3 weeks) in the abomasum.
Institute and Department of Biochemistry and Molecular Biology, Monash
University, Melbourne, VIC 3800, Australia
Although a vaccine (Barbervax®) was recently released
2
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, in Australia to support anthelmintic treatment programs
Parkville, VIC 3010, Australia against haemonchosis, the control of H. contortus and
Full list of author information is available at the end of the article

© 2016 Kumarasingha et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kumarasingha et al. Parasites & Vectors Page 2 of 7

related nematodes relies largely on the use of anthelmintic this progress, these plant extracts had not been tested on
drugs. The excessive use of such drugs has led to wide- any parasitic nematodes.
spread resistance in these nematodes to most classes of The recent development of a new and inexpensive
anthelmintics [4–10], seriously compromising the control whole-organism assay for the rapid screening of chemical
of parasites in many countries. Although the development compounds against parasitic stages of H. contortus [29, 30]
of the compounds monepantel [11, 12] and derquantel has provided a unique prospect to screen plant extracts for
(2-deoxy-paraherquamide) [13] have provided fresh hope activity against one of the most important parasitic nema-
for the development of new classes of nematocides, todes of small ruminants, as a starting point for future as-
success in discovering new drugs has been limited. sessments on other nematodes. This screening assay,
Natural compounds from plants provide a unique oppor- which relies on video-capture to measure the inhibitory
tunity in the search for new, effective and safe anthelmintics properties of compounds on the motility of parasitic larval
[14, 15]. In China, for example, plant-derived medicines stages of H. contortus and subsequent, morphological as-
have been used (for centuries) to treat many disease condi- sessment of larval development, was employed specifically
tions in humans [16, 17] and other animals, including para- to screen extracts PE1, PE2, PE4, PE5 and PE7 against this
sitic diseases [18–20]. It is likely that many of these natural parasitic nematode.
medicines may be acting on pathways in worms that differ
from targets of currently used anthelmintic drugs [21, 22]
and, therefore, might be able to kill nematodes that are Methods
resistant to one or more anthelmintics. However, for Preparation of plant extracts for screening and assay
the vast majority of such natural compounds, there has plate preparation
been limited systematic, scientific evaluation of efficacy, Plant extracts (Table 1) were prepared by the Sarawak
mode of action and identity of their active compo- Biodiversity Centre (SBC), Kuching, Malaysia, as described
nent(s) [23–25], and no plant-based anthelmintic is yet previously [26]. Briefly, whole plants, or parts thereof, were
commercially available. dried, ground into a powder, extracted into 1:1 v/v dichlor-
Recently, we tested eight extracts (PE1 to PE8) from the omethane:methanol and then concentrated using a rotary
plants Picria fel-terrae Lour., Linariantha bicolor, Lansium evaporator. Dried plant extracts were stored at SBC and
domesticum and Tetracera akara for nematocidal activity sent to Australia upon request. Before their use, the dried
against seven strains of the free-living nematode Caenor- plant extracts were dissolved in absolute ethanol (Merck,
habditis elegans (one wild-type and six strains with GFP- Australia). Serial dilutions (1–5 mg/ml) of these extracts
tagged stress response pathways), and characterised the (PE1, PE2, PE4, PE5 and PE7) were prepared in Luria
stress responses caused by these extracts [26]. These plants Bertani medium (LB) [10 g tryptone (cat no. LP0042; Oxoid
are widely distributed throughout Asia and have been used England), 5 g yeast extract (cat no. LP0042; Oxoid), 5 g
by indigenous Malaysian healers to treat worm infections NaCl (cat no. K43208004210; Merck, Denmark) in 1 l of
and gastrointestinal disorders in humans [26–28]. Five of sterile water]. LB was autoclaved and supplemented with
the eight plant extracts (designated PE1, PE2, PE4, PE5 final concentrations of 2.5 μg/ml of amphotericin, 100 IU/
and PE7; Table 1), had significant nematocidal activity ml of penicillin and 100 μg/ml of streptomycin (Fungizone®,
against both larval and adult stages of C. elegans [26]. The antibiotic-antimycotic; cat no. 15240–062; Gibco); this
most effective extracts were from P. fel-terrae [26], and supplemented LB was designated LB*. Compounds were
triggered stress response pathways that were distinct from then dispensed in 50 μl volumes in triplicate into the wells
commercially available anthelmintics (doramectin and of sterile 96-well, flat-bottom microplates (cat no. 3635;
levamisole). This study showed that using traditional Corning 3650, Life Sciences). The anthelmintic monepantel
knowledge of plant medicinal properties, in combination (Zolvix®, Novartis Animal Health, Switzerland) was used at
with a C. elegans in vitro-screen, provided a practical and 20 μM as the positive-control compound, and LB* contain-
economical approach to search for nematocides. Despite ing 1 % ethanol (Merck) was used as the negative-control.

Table 1 Sources of the plant extracts used in this study

Extract/origin SBC specimen reference number Plant species Plant family
PE1/whole plant A000423010301 Picria fel-terrae Lour. Scrophulariaceae
PE2/leaves A000423020301 Picria fel-terrae Lour. Scrophulariaceae
PE4/roots A001293020103 Linariantha bicolor Acanthaceae
PE5/leaves A001293030103 Linariantha bicolor Acanthaceae
PE7/whole plant A002698010103 Lansium domesticum Meliaceae
Kumarasingha et al. Parasites & Vectors Page 3 of 7

Production of H. contortus and storage Evaluation of the effects of extracts on the development
Haemonchus contortus (Haecon-5 strain) was maintained of xL3 to L4
in experimental sheep as described previously [31], and in Following the measurement of larval motility, larvae in
accordance with the institutional animal ethics guidelines 5 mg/ml of plant extracts were re-incubated for four more
(permit no. 1111938; The University of Melbourne). In days at 38 °C and 10 % v/v CO2 in a humidified environ-
brief, helminth-free Merino sheep (eight weeks of age) ment. Subsequently, worms were fixed through the addition
were inoculated intra-ruminally with 5,000 third-stage of 50 μl of 1 % iodine, and 30 worms from each well were
larvae (L3s) of H. contortus. Four weeks after infection, examined at 20x magnification to assess the development
faecal samples were collected each day. L3s were produced of H. contortus L4s (based on the presence/absence of a
from eggs by incubating faeces at 27 °C for one week. Then, well-developed pharynx/mouth; cf. [29, 33]). The number
L3s were sieved through two layers of nylon mesh (pore of L4s was expressed as a percentage of the total worm
size: 20 μM; Rowe Scientific, Australia) to remove debris or number (n = 30). All compounds were tested in triplicate
dead larvae, and stored at 10 °C for up to three months. on three different days. To establish whether the inhibitory
effect of individual plant extracts was reversible, a separate
(‘recovery’) assay was conducted: xL3s incubated at 5 mg/
Exsheathment of L3s
ml plant extracts for 72 h (same conditions as above) were
L3s were exsheathed and sterilised by incubation in
washed four times with 200 μl LB* to remove extracts from
0.15 % v/v sodium hypochlorite (NaClO) at 37 °C for
wells, fresh LB* (100 μl) added, and plates incubated for
20 min [32]. Following this incubation, exsheathed L3s
four more days under the same conditions. Then, worms
(xL3s) were washed five times in sterile physiological saline
were fixed in 1 % iodine and development to the L4 stage
by centrifugation at 600 x g (5 min) at room temperature
assessed. Differences in development between treated and
(22–24 °C). After the last wash, xL3s were suspended in
untreated worms (negative-controls) were assessed by
LB* at a density of 300 xL3 per 50 μl.
statistical analysis using one-way ANOVA.

Screening xL3s for reduced motility upon exposure to Results

plant extracts To test the effects of the five plant extracts on the motil-
We assessed the effect of plant extracts on xL3 motility, ity of xL3 stage of H. contortus, PE1, PE2, PE4, PE5 and
essentially as described recently [29, 30]. In brief, on 96- PE7 were each assessed at five concentrations (1, 2, 3, 4
well plates, test compounds (at concentrations ranging and 5 mg/ml). Both PE1 and PE4 significantly reduced
from 1–5 mg/ml), the positive-control compound (mone- xL3 motility in a dose-dependent manner with reference
pantel) and the ethanol-control in LB* were arrayed in to the negative-control. Although PE1 did not reduce
triplicate; six wells were used for the negative-control (LB* motility significantly at 1 and 2 mg/ml, it did at 3, 4 and
+ 1 % ethanol). Then, 300 xL3s in 50 μl of LB* were trans- 5 mg/ml (Fig. 1). PE4 significantly reduced xL3 motility
ferred to each well of each plate (with the exception of at 4 mg/ml and 5 mg/ml, but did not affect motility at
perimeter wells) using a multi-channel pipette. The final lower concentrations (Fig. 1). PE2 had a significant,
concentration of the positive-control anthelmintic (mone- adverse effect on xL3 motility at all five concentrations
pantel) was 20 μM, and plant extracts (Table 1) were indi- tested, but this effect was not dose dependent (Fig. 1).
vidually tested at 1, 2, 3, 4 and 5 mg/ml. Plates were Neither PE5 nor PE7 significantly reduced xL3 motility
incubated at 38 °C and 10 % v/v CO2. After 48 and 72 h, with reference to negative-controls (Fig. 1). Although
plates were agitated (126 rotations per min) using an PE1, PE2 and PE4 significantly inhibited xL3 motility
orbital shaker for 30 min at 38 °C. To capture the motility after 48 h (PE1: F(5, 48) = 28.77; PE2: F(5, 48) = 27.24; PE4:
of xL3s, a 10 s video recording of each well on each plate F(5, 48) = 9.802; all P ≤ 0.001), these extracts did not
was captured using an eyepiece camera (Dino-eye, ANMO demonstrate statistically significant, time-dependent ef-
Electronic Corporation, Taiwan) attached to a stereo fects on these larvae, with no increased inhibition of
dissecting microscope (Olympus, Japan). After 3 min of motility at 72 h (Fig. 2). Negative-controls showed a
imaging, plates were re-agitated for 5 min. The motility of constant motility index (Mi) throughout all experiments.
xL3s was recorded in each well on each plate. Each 10 s After seven days, the effects of individual extracts on L4
video was processed using a custom macro in the program development were assessed (Fig. 3). After this time, 76.7 %
Image J (1.47v, imagej.nih.gov/ij) to measure larval motil- of untreated xL3s developed to L4s in negative-control
ity, represented by the motility index (= Mi), in each well wells. By contrast, 17.3 % and 5.9 % of xL3s exposed to
[30]. All extracts were tested individually in triplicate on 5 mg/ml of PE1 and PE4, respectively, developed to L4s
three different days. Differences in motility between after 7 days. Although PE2, PE5, PE7 and monepantel re-
treated and untreated worms (negative-controls) were duced the development of xL3 to L4, this reduction was
assessed by statistical analysis using one-way ANOVA. not significant (Fig. 3a). Approximately 80 % of untreated
Kumarasingha et al. Parasites & Vectors Page 4 of 7

Fig. 1 The effects of plant extracts on exsheathed third-stage larvae (xL3) of Haemonchus contortus after 48 h. Results were calculated from three
biological and technical replicates (cf. Table 1) (300 worms per well per replicate). Error bar indicates the standard error of the mean (SEM).
Negative-control (NC) represents LB* containing 1 % ethanol. ***P ≤ 0.001

xL3s as well as xL3s exposed to extract PE2, PE4, PE5 or or the same constituents at different concentrations in
PE7 (5 mg/ml each) or monepantel (20 μM) developed to different parts of the plant. Interestingly, none of the plant
L4s following the addition of fresh LB*, after 72 h of incu- extracts showed significant, time-dependent effects on the
bation. By contrast, only 41.8 % of xL3s exposed to extract larvae at the time points tested. This finding may be due to:
PE1 developed to L4s, even in the subsequent absence of (i) the active constituents in plant extracts having a max-
PE1, after incubation for 72 h (Fig. 3b). imum effect on H. contortus within 48 h; (ii) the degrad-
ation of active constituents in the extracts during testing in
Discussion the assay, such that they are no longer effective against
The present results show that extracts PE1 and PE2 from xL3s or L4s; (iii) xL3s may have rapidly developed an acute
P. fel-terrae Lour. have considerable activity against the “resistance” or used their defence mechanism to overcome
parasitic larval stages of H. contortus in vitro. Extract PE2 some of the effect(s) of the active constituent(s), for
was the most effective inhibitor of xL3 motility at all con- instance, via a complex mechanism on the cuticle of the
centrations tested, but with no observable dose-dependent worm [34–36].
effect. This latter extract reduced worm motility, even at In larval development and recovery assays, only PE1 had
the lowest concentration tested (1 mg/ml), which is consist- an adverse impact on development from xL3 to L4. Even
ent with its effect on C. elegans [26]. Although PE1 and though both PE1 and PE4 had a remarkable, adverse effect
PE2 originate from the same plant, they showed distinctive on the development of xL3s to L4s, most PE4-treated
inhibitory characteristics on both xL3 motility and L4 larvae recovered and developed to L4s after the removal of
development of H. contortus, which might relate to the the plant extract and replacement with LB*. This finding
presence of different active constituents in these extracts, suggests that the effect of extract PE4 is reversible, whereas
Kumarasingha et al. Parasites & Vectors Page 5 of 7

Fig. 2 The effects of plant extracts on exsheathed third-stage larvae (xL3) of Haemonchus contortus after 48 h and 72 h. Results were calculated from
three biological and technical replicates (cf. Table 1) (300 worms per replicate). Negative-control (NC) represents LB* containing 1 % ethanol. Error bar
indicates the standard error of the mean (SEM)

PE1 appears to irreversibly inhibit xL3 motility and larval the nematodes can develop resistance rapidly (sometimes
development. Interestingly, a similar pattern to PE4 was after as few as three generations; [37]), a similar situation
observed for monepantel at a dose of 20 μM. Given that might be the case for the active, natural compound PE4.
Kumarasingha et al. Parasites & Vectors Page 6 of 7

Fig. 3 The effects of plant extracts (cf. Table 1) on the development (panel a) and recovery (panel b) of exsheathed third-stage larvae (xL3) of
Haemonchus contortus. Monepantel (20 μM) was used as the positive-control (PC). The results were calculated from three biological replicates
(300 worms per replicate). Negative-control (NC) represents LB* containing 1 % ethanol. Error bar indicates the standard error of the mean (SEM)

Thus, resistance development in H. contortus should be inputs from other authors. All authors read and approved the final version of
evaluated. the manuscript.

The finding that three of the same five plant extracts Acknowledgements
shown previously to affect C. elegans [26] significantly re- Funding from the National Health and Medical Research Council (NHMRC) of
duced motility and development in xL3s of H. contortus in- Australia is gratefully acknowledged. R.K. was awarded an Australian Postgraduate
Award (APA) from the Australian Government via Monash University and
dicates variation between the free-living and parasitic Swinburne University of Technology.
nematodes in targets and/or the pathways, which might
relate to genomic differences and evolutionary distance Author details
1
Development and Stem Cells Program, Monash Biomedicine Discovery
between these worms [38–40]. However, it is also possible Institute and Department of Biochemistry and Molecular Biology, Monash
that the absorption of constituents from the plant extracts University, Melbourne, VIC 3800, Australia. 2Faculty of Veterinary and
differs between the two worms [41], or that, from a Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010,
Australia. 3Sarawak Biodiversity Centre (SBC), KM 20 Jalan Borneo Heights,
biological/evolutionary perspective, the parasitic nematode Semengoh, Locked Bag No. 3032, 93990 Kuching, Sarawak, Malaysia.
(H. contortus) is perhaps more adapted to a plant extract- 4
Department of Chemistry and Biotechnology, Faculty of Science,
rich environment in the abomasum of its ruminant host Engineering and Technology, Swinburne University of Technology, Victoria
3122, Australia. 5Department of Health and Medical Sciences, Faculty of
compared with the soil nematode, C. elegans. These aspects Health, Arts and Design, Swinburne University of Technology, Victoria 3122,
and differences in susceptibility or efficacy need to be taken Australia.
into consideration when the focus of screening is on
Received: 27 November 2015 Accepted: 16 March 2016
extracts or compounds expected to have an effect on a rela-
tively wide range of related nematodes
References
1. Roeber F, Jex AR, Gasser RB. Next-generation molecular-diagnostic tools for
Conclusion gastrointestinal nematodes of livestock, with an emphasis on small
Significant activities previously identified for PE1 and PE2 ruminants. A turning point? Adv Parasitol. 2013;832013:267–333.
2. Waller PJ, Chandrawathani P. Haemonchus contortus: parasite problem No. 1
in C. elegans in vitro [26] were also seen in H. contortus. from tropics - Polar Circle. Problems and prospects for control based on
These findings suggest that extracts from P. fel-terrae epidemiology. Trop Biomed. 2005;22(2):131–7.
Lour. have promising anthelmintic effects. We propose 3. Veglia F. The anatomy and life-history of the Haemonchus contortus (Rud.).
Rep Dir Vet Res. 1915;3–4:347–500.
that future work should focus on attempting to fractionate 4. Besier B. New anthelmintics for livestock: the time is right. Trends Parasitol.
extract PE1, in order to identify and characterise the con- 2007;23(1):21–4.
stituent(s) that are active against H. contortus, and then to 5. Jabbar A, Iqbal Z, Kerboeuf D, Muhammad G, Khan MN, Afaq M. Anthelmintic
resistance: The state of play revisited. Life Sci. 2006;79(26):2413–31.
explore which biological pathways are affected by these 6. Kaplan R, Vidyashankar A. An inconvenient truth: global worming and
components/fractions. anthelmintic resistance. Vet Parasitol. 2012;186:70–8.
7. Kaplan RM. Drug resistance in nematodes of veterinary importance: A status
report. Trends Parasitol. 2004;20(10):477–81.
Competing interests
8. Scott I, Pomroy WE, Kenyon PR, Smith G, Adlington B, Moss A. Lack of
The authors declare that they have no competing interests.
efficacy of monepantel against Teladorsagia circumcincta and
Trichostrongylus colubriformis. Vet Parasitol. 2013;198(1–2):166–71.
Authors’ contributions 9. Wolstenholme AJ, Fairweather I, Prichard R, Von Samson-Himmelstjerna G,
RA, RBG and PRB conceived the project with imput from EP and JS. RA carried Sangster NC. Drug resistance in veterinary helminths. Trends Parasitol. 2004;
out laboratory work and data analysis. SP supplied the parasites and assisted 20(10):469–76.
with the laboratory work. TY, DSLL and CT coordinated plant collection and 10. Wolstenholme AJ, Kaplan RM. Resistance to macrocyclic lactones. Curr
prepared plant extracts. RA, RBG and PRB wrote the manuscript with critical Pharm Biotechnol. 2012;13(6):873–87.
Kumarasingha et al. Parasites & Vectors Page 7 of 7

11. Kaminsky R, Ducray P, Jung M, Clover R, Rufener L, Bouvier J, Weber SS, 36. Rudin W. Comparison of the cuticular structure of parasitic nematodes
Wenger A, Wieland-Berghausen S, Goebel T. A new class of anthelmintics recognized by immunocytochemical and lectin binding studies. Acta Trop.
effective against drug-resistant nematodes. Nature. 2008;452(7184):176–80. 1990;47(5–6):255–68.
12. Prichard RK, Geary TG. Drug discovery: Fresh hope to can the worms. 37. Sager H, Bapst B, Strehlau GA, Kaminsky R. Efficacy of monepantel,
Nature. 2008;452(7184):157–8. derquantel and abamectin against adult stages of a multi-resistant
13. Lee BH, Clothier MF, Johnson SS. Semi-synthesis of 2-deoxo- and 3-epi- Haemonchus contortus isolate. Parasitol Res. 2012;111(5):2205–7.
paraherquamide A. Bioorg Med Chem Lett. 2001;11(4):553–4. 38. Blaxter ML. Nematodes: The worm and its relatives. PloS Biol. 2011;9(4):e1001050.
14. Hammond JA, Fielding D, Bishop SC. Prospects for plant anthelmintics 39. Geary TG, Thompson DP. Caenorhabditis elegans: How good a model for
in tropical veterinary medicine. Vet Res Commun. 1997;21(3):213–28. veterinary parasites? Vet Parasitol. 2001;101(3–4):371–86.
15. Waller PJ, Bernes G, Thamsborg SM, Sukura A, Richter SH, Ingebrigtsen 40. Laing R, Kikuchi T, Martinelli A, Tsai IJ, Beech RN, Redman E, Holroyd N,
K, Höglund J.. Plants as de-worming agents of livestock in the Nordic Bartley DJ, Beasley H, Britton C et al. The genome and transcriptome of
countries: historical perspective, popular beliefs and prospects for the Haemonchus contortus, a key model parasite for drug and vaccine discovery.
future. Acta Vet Scand. 2001;42(1):31–44. Genome Biol. 2013; 14(8):R88.
16. Liu ZL, Liu JP, Zhang AL, Wu Q, Ruan Y, Lewith G, Visconte D. Chinese 41. Burns AR, Wallace IM, Wildenhain J, Tyers M, Giaever G, Bader GD, Nislow C,
herbal medicines for hypercholesterolemia. Cochrane Database Syst Rev. Cutler SR, Roy PJ. A predictive model for drug bioaccumulation and
2011;7:CD008305. bioactivity in Caenorhabditis elegans. Nature Chem Biol. 2010;6(7):549–57.
17. Xu HB, Jiang RH, Chen XZ, Li L. Chinese herbal medicine in treatment of
diabetic peripheral neuropathy: A systematic review and meta-analysis.
J Ethnopharmacol. 2012;143(2):701–8.
18. Hon KLE, Ma KC, Wong Y, Leung TF, Fok TF. A survey of traditional
Chinese medicine use in children with atopic dermatitis attending a
paediatric dermatology clinic. J Dermatol Treat. 2005;16(3):154–7.
19. Li ZH, Wan JY, Wang GQ, Zhao FG, Wen JH. Identification of compounds
from Paris polyphylla (ChongLou) active against Dactylogyrus intermedius.
Parasitology. 2013;140(8):952–8.
20. Zhu L, Dai JL, Yang L, Qiu J. In vitro ovicidal and larvicidal activity of the
essential oil of Artemisia lancea against Haemonchus contortus (Strongylida).
Vet Parasitol. 2013;195(1–2):112–7.
21. Hrckova G, Velebny S. Parasitic helminths of humans and animals: health impact
and control. In: Pharmacological Potential of Selected Natural Compounds in the
Control of Parasitic Diseases. Vienna: Springer; 2013. p. 29–99.
22. Roeber F, Kahn L. The specific diagnosis of gastrointestinal nematode
infections in livestock: Larval culture technique, its limitations and
alternative DNA-based approaches. Vet Parasitol. 2014;205:619–28.
23. Eguale T, Tadesse D, Giday M. In vitro anthelmintic activity of crude extracts
of five medicinal plants against egg-hatching and larval development of
Haemonchus contortus. J Ethnopharmacol. 2011;137(1):108–13.
24. Hoste H, Jackson F, Athanasiadou S, Thamsborg SM, Hoskin SO. The
effects of tannin-rich plants on parasitic nematodes in ruminants.
Trends Parasitol. 2006;22(6):253–61.
25. Kaewintajuk K, Cho PY, Kim SY, Lee ES, Lee HK, Choi EB, Park H.
Anthelmintic activity of KSI-4088 against Caenorhabditis elegans. Parasitol
Res. 2010;107(1):27–30.
26. Kumarasingha R, Palombo EA, Bhave M, Yeo TC, Lim DSL, Tu CL, Shaw JM,
Boag PR. Enhancing a search for traditional medicinal plants with
anthelmintic action by using wild type and stress reporter Caenorhabditis
elegans strains as screening tools. Int J Parasitol. 2014;44(5):291–8.
27. Chai PPK. Medicinal Plants of Sarawak: Paul Chai P.K.; 2006.
28. Christensen H. Ethnobotany of the Iban & the Kelabit: Nepcon. Malaysia;
University of Aarhus; Forest Department Sarawak; 2002.
29. Preston S, Jabbar A, Nowell C, Joachim A, Ruttkowski B, Baell J, Cardno T,
Korhonen PK, Piedrafita D, Ansell BRE. Low cost whole-organism screening
of compounds for anthelmintic activity. Int J Parasitol. 2015;45(5):333–43.
30. Preston S, Jabbar A, Nowell C, Joachim A, Ruttkowski B, Cardno T, Hofmann A,
Gasser RB. Practical and low cost whole-organism motility assay: A step-by-step
protocol. Mol Cell Probes. 2015. doi:10.1016/j.mcp.2015.08.005.
31. Schwarz E, Korhonen P, Campbell B, Young N, Jex A, Jabbar A, Hall R,
Mondal A, Howe A, Pell J et al. The genome and developmental Submit your next manuscript to BioMed Central
transcriptome of the strongylid nematode Haemonchus contortus. and we will help you at every step:
Genome Biol. 2013;14(8):R89.
32. Nikolaou S, Hartman D, Presidente P, Newton S, Gasser RB. HcSTK, a • We accept pre-submission inquiries
Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, • Our selector tool helps you to find the most relevant journal
Haemonchus contortus. Int J Parasitol. 2002;32:749–58.
• We provide round the clock customer support
33. Sommerville RI. The Development of Haemonchus contortus to the fourth
stage in vitro. J Parasitol. 1966;52(1):127–36. • Convenient online submission
34. Maizels RM, Blaxter ML, Selkirk ME. Forms and functions of nematode • Thorough peer review
surfaces. Exp Parasitol. 1993;77(3):380–4. • Inclusion in PubMed and all major indexing services
35. Ríos-de álvarez L, Jackson F, Greer A, Bartley Y, Bartley DJ, Grant G, Huntley
JF. In vitro screening of plant lectins and tropical plant extracts for • Maximum visibility for your research
anthelmintic properties. Vet Parasitol. 2012;186(3–4):390–8.
Submit your manuscript at
www.biomedcentral.com/submit