Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep
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Keywords:
Teladorsagia circumcincta
Benzimidazole
Trichostrongylus
Pyrosequencing
SUMMARY Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in β-tubulin genes have been associated with BZ-resistance. In the present study, we investigated β-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000–10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus . The molecular methods were more sensitive than the ‘egg hatch test’, and less time-consuming than current in vivo - or in vitro -anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.
Pyrosequencing
Parasitology
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Flock
Moxidectin
Teladorsagia circumcincta
Levamisole
Benzimidazole
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Weekly tests, over a period of eighteen months, of the sera of a flock of grazing sheep showed a relationship between the serum titres and the faecal egg counts. This relationship was particularly obvious during the summer months. When egg counts fell, the titres rose, and as the titres dropped, the egg counts usually rose again. This relationship between egg counts and serum titres was more striking in individual sheep than in the whole flock because all the sheep did not behave similarly at the same time. The antibody levels rose in individual sheep irrespective of whether the sheep were heavily or lightly infested at the time. There was no evidence that a permanent resistance to Haemonchus contortus was acquired under field conditions. Seven periods of "self-cure" occurred in the grazing flock during the period of observation. On each occasion, the majority of sheep which showed a drop in egg count also showed a rise in serum titre. This behaviour was strikingly similar to the result when infective larvae of H. contortus were superimposed upon an existing infestation of H. contortus or Trichostrongylus spp. in penned sheep. 'Self-cure' was produced in the field by giving naturally infested sheep large doses of infective larvae of H. contortus by mouth. A statistically significant fall in the faecal egg count of H. contortus and Trichostrongylus spp., but not of Oesophagostomum columbianum was produced by this means. The fact that 'self-cure' takes place in naturally grazing flocks during the summer months after rain indicated that the intake of large doses of infective larvae of H. contortus was the exciting cause of the phenomenon.
Flock
Trichostrongylus
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Abstract Background Haemonchus contortus and Trichostrongylus spp. are reported to be the most prevalent and highly pathogenic parasites in livestock, particularly in small ruminants. However, the routine conventional tool used in Malaysia could not differentiate the species accurately and therefore limiting the understanding of the co-infections between these two genera among livestock in Malaysia. This study is the first attempt to identify the strongylids of veterinary importance in Malaysia (i.e., H. contortus and Trichostrongylus spp.) by amplification and sequencing of the Internal Transcribed Spacer II DNA region. Results Overall, 118 (cattle: 11 of 98 or 11.2%; deer: 4 of 70 or 5.7%; goats: 99 of 157 or 63.1%; swine: 4 of 91 or 4.4%) out of the 416 collected fecal samples were microscopy positive with strongylid infection. The PCR and sequencing results demonstrated that 93 samples (1 or 25.0% of deer; 92 or 92.9% of goats) contained H. contortus . In addition, Trichostrongylus colubriformis was observed in 75 (75.8% of 99) of strongylid infected goats and Trichostrongylus axei in 4 (4.0%) of 99 goats and 2 (50.0%) of 4 deer. Based on the molecular results, co-infection of H. contortus and Trichostrongylus spp. ( H. contortus + T. colubriformis denoted as HTC; H. contortus + T. axei denoted as HTA) were only found in goats. Specifically, HTC co-infections have higher rate (71 or 45.2% of 157) compared to HTA co-infections (3 or 1.9% of 157). Conclusions The present study is the first molecular identification of strongylid species among livestock in Malaysia which is essential towards a better knowledge of the epidemiology of gastro-intestinal parasitic infection among livestock in the country. Furthermore, a more comprehensive or nationwide molecular-based study on gastro-intestinal parasites in livestock should be carried out in the future, given that molecular tools could assist in improving diagnosis of veterinary parasitology in Malaysia due to its high sensitivity and accuracy.
Trichostrongylus
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Infections with trichostrongylid worms are common and a major cause of production loss in small ruminants worldwide. In Australia, infections with Haemonchus contortus and Trichostrongylus spp cost the Australian sheep industry up to AUD$369 million annually. Currently, the control of these two parasites relies heavily on the use of anthelmintics and grazing management. However, the frequent use of drugs resulted in widespread development of anthelmintics resistance in many trychostrongylid species including H. contortus and Trichostrongylus spp. Monepantel (Zolvix®, Novartis, Australia), an amino-acetonitrile derivate, is one of the most recent anthelmintics released in Australia and it proved to be active against strains of H. contortus and Trichostrongylus spp that are resistant to broad-spectrum anthelmintics. However, lack of efficacy of monepantel against Teladorsagia circumcincta and T. colubriformis was recently reported in New Zealand and Australia. In this trial, the efficacy of monepantel in sheep and goats naturally infected with H. contortus and Trichostrongylus spp was assessed by faecal egg count reduction tests (FECRT) and total worm counts (WC).
Trichostrongylus
Teladorsagia circumcincta
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Teladorsagia circumcincta
Trichostrongylus
Levamisole
Pyrantel
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Resistance to benzimidazoles (BZs) in trichostrongyloid nematodes is a worldwide problem for livestock production, particularly regarding small ruminants. Sensitive and reliable methods are required to assess anthelmintic resistance status. Currently available methods for BZ resistance detection can be divided into three main groups, in vivo (e.g. faecal egg count reduction test), in vitro (e.g. egg hatch assay) and molecular tests. Three single nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene of various nematode species correlate with BZ resistance. While PCR-based methods have been reported for the three most economically important nematodes of sheep, namely, Trichostrongylus, Haemonchus and Teladorsagia, pyrosequencing assays are so far only available for the latter two. Here, the design and evaluation of pyrosequencing assays for isotype-1 and isotype-2 β-tubulin genes of Trichostrongylus colubriformis are described. PCR fragments carrying the susceptible and corresponding resistant genotype were combined in defined ratios to evaluate assay sensitivity and linearity. The correlation between the given and the measured allele frequencies of the respective SNPs (codons F167Y, E198A and F200Y) was very high. Pyrosequencing assays for Haemonchus, Teladorsagia and Trichostrongylus were subsequently used for a BZ resistance survey, carried out in the three European countries, namely Ireland, Italy and Switzerland. Larval cultures obtained from field survey samples in 2012 and 2013 were used for pyrosequencing. The test was applied when the target species represented at least 10% of the sample. Trichostrongylus and Teladorsagia were detected in all countries' samples whereas Haemonchus was not detected in samples from Ireland. SNPs in isotype-1 associated with resistance were detected for all three species, with frequencies at codon F200Y far exceeding those at codons F167Y and E198A. Elevated SNP frequencies in isotype-2 of Trichostrongylus were only rarely detected. Farms with BZ resistance-associated SNP frequencies above 10% were most often found in Switzerland followed by Ireland and Italy.
Pyrosequencing
Teladorsagia circumcincta
Trichostrongylus
Isotype
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Teladorsagia circumcincta
Flock
Domestic sheep reproduction
Nematode infection
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The effects of tannins on adult populations of Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta
in goats are characterised mainly by a decrease in egg excretion without any significant changes in worm number. In contrast,
the impact of tannins on T. colubriformis or T. circumcincta third-stage larvae (L3) is associated with a significant reduction in worm
establishment. The objective of the present study was to examine the effects of quebracho extract tannins on H. contortus L3. The
consequences of consumption of sainfoin hay were also examined.
Twenty-one nai¨ve kids were divided into three experimental groups. Group Q received quebracho extract and group S received
sainfoin hay from days D3 to D5. Group C remained as an infected control group. All kids received 1500 L3 H. contortus on D0, D1
and D2. On D18, post-infection, the kids were slaughtered and the worm populations compared in the different groups. Compared
to the control values, the worm counts decreased, respectively, by 33% and 38% in groups Q and S but the differences were not significant.
No differences were found in pathophysiological measurements between the three groups. The results confirm differences in
tannin effect according to nematode species but not parasitic stage.
Teladorsagia circumcincta
Condensed tannin
Tannin
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