Benzimidazole Resistance In Haemonchus Contortus Research Articles

Prevalence of gastrointestinal parasites and molecular identification of beta-tubulin mutations associated with benzimidazole resistance in Haemonchus contortus in goats from selected districts of Uganda.

Gastrointestinal parasites are among the most economically important pathogens of small ruminants causing serious economic losses and animal welfare problems for the livestock industry worldwide. The emergence of anthelmintic resistant H. contortus in small ruminants is a serious problem because it undermines effective helminth control and results in reduced productivity. Little is known about resistance to benzimidazoles (BZ) in Haemonchus in goats and sheep in Uganda. The objective of this study was to determine the prevalence of gastrointestinal parasites and to identify the presence of benzimidazole resistance associated mutations in the β-tubulin isotype 1 gene of Haemonchus contortus in goats from selected districts of Uganda. A total of 200 goats from 10 districts of Uganda slaughtered at Kalerwe abattoir in Kampala were sampled for H. contortus adult worms. Faecal samples were also collected to detect other intestinal parasites. Faecal microscopy and analysis were performed using flotation and sedimentation techniques. DNA was extracted from adult worms and PCR and sequencing of the ITS- 2 region and β-tubulin isotype 1 gene performed to identify H. contortus species and to determine the presence of mutations associated with anthelmintic resistance respectively. Faecal microscopy showed that the most prevalent intestinal parasites were coccidia (98%), strongyles (97.5%), Strongyloides (82%), Paramphistomum (74.5%), Moniezia (46%), Fasciola (1.5%) and Trichuris (1%). Most goats had a high intestinal burden of coccidia (≥ 5000 oocyst per gram) and strongyles (≥ 1000 egg per gram), 65% and 67.5%, respectively. The prevalence of H. contortus adult worms was 63% (126/200). Sequencing of the partial β-tubulin isotype 1 gene of 54 Haemonchus contortus adult male isolates revealed the presence of mutations associated with anthelmintic resistance. The F200Y mutation was the most common mutation (13% of samples with good beta-tubulin sequences) followed by the E198A and E198K mutations, both found in 9% of sequenced samples. Mutation F167Y was not identified in any of the samples and there were no heterozygous individuals for any of the SNPS associated with BZ resistance that were identified in this study. These findings highlight the need for controlled use of anthelmintics especially benzimidazoles, to enable sustainable control of H. contortus in Uganda, and a need for further investigation to understand the resistance of other parasites identified in this study.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL−1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

Population replacement of benzimidazole-resistant Haemonchus contortus with susceptible strains: evidence of changes in the resistance status.

The spread of anthelmintic resistance (AR) in nematode populations threatens the viability of sheep production systems worldwide, and warrants the adoption of sensitive, practical, and standardized tests to detect AR. The aim of this study was to characterize the replacement of an Haemonchus contortus population resistant to benzimidazoles (BZDs) by a susceptible one, by means of both phenotypic and genotypic techniques. Phenotypic methods to assess BZD resistance included in vivo tests, such as the fecal egg count reduction test (FECRT), and in vitro tests, such as the egg hatch assay (EHA). Additionally, genotypification of polymorphisms associated with BZD resistance by sequencing a fragment of the isotype 1 β-tubulin gene was carried out. The initial, BZD-resistant population (initial Balcarce population) exhibited an egg count reduction (ECR) of 59.3%. Following refugium replacement, the final population (final Balcarce population) exhibited an ECR of 95.2%. For the initial Balcarce population, the median effective dose (ED50) for the EHA was 0.607 μg thiabendazole (TBZ)/mL, with a rate of eclosion at a discriminating dose (EDD) of 0.1 μg TBZ/mL of 76.73%. For the final Balcarce population, ED50 was 0.02 μg TBZ/mL, and EDD was 1.97%. In the initial population, 93% of the analyzed individuals exhibited genotypic combinations associated with BZD resistance (53% Phe/Phe167-Tyr/Tyr200, 37% Phe/Tyr167-Phe/Tyr200, and 3% Phe/Tyr167-Glu/Leu198). Conversely, no combination associated with resistance was found in individuals from the final population. All of the tests were useful for detecting AR to BZDs. The results from the genetic and phenotypical studies were consistent, and the resulting information greatly aided in interpreting the outcomes of the population replacement and the potential impact of this strategy on management of AR.

The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand.

Background and Aim:Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene.Materials and Methods:A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol.Results:The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001).Conclusion:This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the β-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.

Characterization of benzimidazole resistance in Haemonchus contortus: integration of phenotypic, genotypic and proteomic approaches.

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 β-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.

New codon 198 \u03b2-tubulin polymorphisms in highly benzimidazole resistant Haemonchus contortus from goats in three different states in Sudan

BackgroundBenzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms.MethodsFaecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 β-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur.ResultsAlbendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5–90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples.ConclusionsTo our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.

Molecular detection of benzimidazole resistance in Haemonchus contortus of sheep in Punjab

Single nucleotide polymorphism (SNP) at codon 167, 198 and 200 of β-tubulin isotype 1 gene accounts for benzimidazole resistance in Haemonchus contortus. To assess the anthelmintic resistance to benzimidazole group, allele specific polymerase chain reaction (AS-PCR) was employed on 50 adults of local abattoir of Ludhiana and 138 harvested larvae of H. contortus from faecal samples collected from field flocks of 6 districts of western zone of Punjab. The adults were found to be homozygous resistant (rr) showing 100% prevalence of resistant allele (r). AS-PCR on 138 larvae collected from different districts of Punjab showed 49.28% (69) homozygous resistant (rr), 3.62% (5) homozygous susceptible (SS) and 46.37% (64) heterozygous (rS) population. Species was confirmed as H. contortus by PCR-RFLP from the three fragments of size 462 bp, 211 bp and 147 bp. The pioneer study on the molecular diagnosis of benzimidazole resistance in H. contortus showed an overall allele frequency for resistant and susceptible population to be 0.72 (TTC) and 0.28 (TAC), espectively indicating development of high level of resistance in sheep.

Genetic polymorphism of p- Tubulin isotype 1 gene conferring benzimidazole resistance in Haemonchus contortus in sheep of Andhra Pradesh

Genetic polymorphism of p- Tubulin isotype 1 gene conferring benzimidazole resistance in Haemonchus contortus in sheep of Andhra Pradesh

Efficacy of closantel against benzimidazole resistant Haemonchus contortus infection in sheep

Benzimidazoles are widely used and readily available ovine anthelminthics across the country. However, widespread resistance to this drug class has been documented, primarily in Haemonchus spp. The present study was conducted to determine the efficacy of closantel against benzimidazole resistant Haemonchus contortus infection in sheep. Naturally infected sheep (n=34) were divided into four groups on the basis of fecal egg counts (FEC) using modified McMaster technique with a lower detection limit of 8.3 eggs per gram of faeces (EPG) viz. Group-A (500-10000), Group- B (>10,000-20,000), Group-C (>20,000-30,000) and Group-D (>30,000). Generic differentiation of larvae was carried out by coproculture performed on pooled faeces which showed the presence of Haemonchus, Trichostrongylus, Oesophagostomum and Strongyloides larvae. To ascertain the evidence of benzimidazole resistance, Egg Hatch Assay (EHA) was performed. All animals were treated with Closantel @ 10mg\kg body weight and EPG was determined on 7th and 11th day post treatment. Animals of different groups had mean EPG range of 400 -760 at 11th day of treatment. In group A, B, C and D mean per cent efficacy of closantel was 91.24±3.49, 95.15±1.72, 97.73±0.72, 98.23±0.86 respectively. Efficacy of closantel against Haemonchus and other gastrointestinal nematodes was further confirmed by performing coproculture 11th day post treatment. Post treatment coproculture revealed presence of Trichostrongylus, Oesophagostomum and Strongyloides larvae and were devoid of Haemonchus larvae. To clear the left out infection of Trichostrongylus, Oesophagostomum and Strongyloides animals were further treated with Fenbendazole @5mg/kg body weight and EPG 14 days post treatment became zero. The results of the study suggested that closantel can be used for Targeted Selective Treatment (TST) in sheep primarily infected with Haemonchus. Since closantel is highly efficacious against Haemonchus its use as an alternative to benzimidazoles group may be helpful to decrease pasture contamination. Overall control of gastrointestinal nematodiasis may therefore be possible by use of closantel along with benzimidazoles.

Epidemiology of strongyle nematode infections and first report of benzimidazole resistance in Haemonchus contortus in goats in South Darfur State, Sudan

BackgroundSince pastoralists in South Darfur, Sudan, had complained about lack of albendazole (ABZ) efficacy to control nematodes in goats, the frequency of infection with gastrointestinal helminths was studied before in vivo faecal egg count reduction tests (FECRT) were conducted using ABZ orally either at the dose recommended for sheep, 5 mg/kg body weight (bw) or at 10 mg/kg bw. Experiments included goats naturally infected with gastrointestinal nematodes or experimentally infected with local Haemonchus contortus isolates. Three study areas (Nyala, Beleil and Kass) were visited in autumn or winter.ResultsOut of 478 screened goats, 82.4% were infected with gastrointestinal helminths and 82% were shedding eggs of strongyle nematodes with 90% of the strongyle larvae representing Haemonchus spp. A FECRT using naturally infected goats (n = 225: 71 untreated, 104 and 50 treated with 5 and 10 mg ABZ/kg bw, respectively) detected reduced ABZ efficacy in Nyala and Kass. Paired and unpaired FECRT calculations detected reductions of 72–92% with samples taken at 8 days post treatment with 5 mg ABZ/kg bw and of 85–94% with 10 mg ABZ/kg bw. The FECRT based on day 14 post treatment samples showed reductions of 69–77% with 5 mg/kg and of 75–87% with 10 mg ABZ/kg bw. In Beleil, ABZ efficacy was 95%. In the egg hatch test EC50 values for Nyala and Kass ranged from 0.12–0.24 μg thiabendazole/ml, corresponding to benzimidazole resistant phenotypes. Only Haemonchus spp. larvae were present after treatments in coprocultures. When the efficacy was evaluated experimentally using isolates of H. contortus from Nyala and Kass, the 5 mg ABZ/kg dose revealed reductions of 76–78% on day 8 and of 62–70% on day 14 with the unpaired method. Using 10 mg ABZ/kg, the FECR was still only 77–82%.ConclusionsBoth, in vivo and in vitro methods detected resistant H. contortus populations in goats from South Darfur State. The time point 14 days post treatment was more sensitive for detection of ABZ resistance than 8 days post treatment. This is the first report on the occurrence of anthelmintic resistance in Sudan confirming that anthelmintic resistance selection is occurring in African subsistence farming systems.

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR

Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the β tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.

Population genetics of benzimidazole-resistant Haemonchus contortus and Haemonchus placei from buffalo and cattle: implications for the emergence and spread of resistance mutations

The population genetics of nematode parasites are poorly understood with practical reference to the selection and spread of anthelmintic resistance mutations. Haemonchus species are important to study the nematode population genetics due to their clinical importance in ruminant livestock, and the availability of genomic resources. In the present study, it has been examined that Haemonchus contortus and Haemonchus placei populations from three buffalo and nine cattle hosts. Seventy-three individual adult worms of H. contortus and 148 of H. placei were analysed using a panel of seven microsatellite markers. The number of alleles per locus in H. contortus and H. placei indicated that all populations were polymorphic for the microsatellites used in the present study. Genetic diversity parameters included high levels of allelic richness and heterozygosity, indicating effective population sizes, high mutation rates and high transmission frequencies in the area. Genetic structure parameters revealed low genetic differentiation between and high levels of genetic variation within H. contortus and H. placei populations. Population dynamic analyses showed an absence of heterozygosity excess in both species, suggesting that there was no deviation from genetic drift equilibrium. Our results provide a proof of concept for better understanding of the consequences of specific control strategies, climatic change or management strategies on the population genetics of anthelmintic resistance alleles in Haemonchus spp. infecting co-managed buffalo and cattle.

Variation in the β-tubulin isotype 1 gene at codon 167 and 200 responsible for benzimidazole resistance in Haemonchus contortus in sheep of Krishna District, AP, India

Variation in the β-tubulin isotype 1 gene at codon 167 and 200 responsible for benzimidazole resistance in Haemonchus contortus in sheep of Krishna District, AP, India

Benzimidazole resistance in Haemonchus contortus recovered from farmed red deer.

Thirty Haemonchus contortus male worms were collected from farmed red deer yearlings in order to determine whether routine administration of albendazole for a long-term period (17years) could select anthelmintic resistance. PCR-RFLP method based on single-nucleotide polymorphism of codon 200 in isotype 1 ß-tubulin gene (Phe200Tyr) was applied. The results showed a significant frequency of either the resistant allele (85%) or the homozygous resistant genotype (70%). By chi-square test, Hardy-Weinberg equilibrium of the population was accepted (p = 0.334, power of test 0.01). True prevalence of the resistant genotype (RR) was estimated to be 46.5-87.2% (confidence interval 95%) calculated by Sterne's exact method. These results confirmed that long-term use of benzimidazoles could change the relative allele frequency of genes associated with drug resistance and may cause a large-scale spread of the resistant allele. To our knowledge, this study supported benzimidazole resistance in Haemonchus contortus in red deer for the first time.

Status of benzimidazole resistance in Haemonchus contortus of goats from different geographic regions of Uttar Pradesh, India

Present study was designed to survey the status of benzimidazole (BZ) resistance in goat flocks of different agro climatic regions of Uttar Pradesh (U.P.), India by faecal egg count reduction test (FECRT) and allele specific PCR (AS-PCR). Faecal egg counts and larval culture were made from representative samples of all regions. The results of the faecal culture and PCR-RFLP on beta tubulin isotype-1 gene showed Haemonchus contortus was predominant species. FECRT results showed BZ resistance prevalent in all regions. However, six farms of twenty screened, harboured susceptible populations of strongyles. Three hundred H. contortus infective larvae from all regions of the state were genotyped for BZ resistance. AS-PCR results revealed 55–85% of H. contortus homozygous resistant (rr), 10–21% homozygous susceptible (SS) and 5–24% heterozygous (rS) among different regions of U.P. The allele frequencies were 67–87.5% for resistant (TTC) and 12.5–33% for susceptible (TAC). The survey indicated that the status of BZ resistance is in alarming conditions in all the parts of the state.

Molecular diagnosis of benzimidazole resistance in Haemonchus contortus in sheep from different geographic regions of North India

Molecular diagnosis of benzimidazole resistance in Haemonchus contortus in sheep from different geographic regions of North India

Frequency of benzimidazole resistance inHaemonchus contortus populations isolated from buffalo, goat and sheep herds

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: Comparison of detection methods for drug resistance

In 2011, a field study was conducted to assess drug resistance of gastro-intestinal nematodes in sheep flocks in Ontario, Canada. Benzimidazole resistance in Haemonchus contortus was assessed by genetic analysis of eggs; measurement of resistant allele percentages at codons 167, 198 and 200 in the β-tubulin gene was determined on pools of H. contortus eggs using pyrosequencing. Susceptibility to benzimidazoles in gastro-intestinal nematodes was also determined using a Faecal Egg Count Reduction Test (FECRT) and a Larval Development Assay (LDA). In total, 16 farms were assessed with the genetic test. Based on resistant allele frequencies, all of the farms (16/16) tested had benzimidazole resistance in H. contortus; the overall percentage of benzimidazole-resistant H. contortus (estimated prior to treatment using the Hardy–Weinberg formula) was 68.5%. The FECRT and LDA were performed on 11 and 13 farms, respectively. Resistance to fenbendazole was detected on 100% (11/11) of the farms where the FECRT was performed. The LDA revealed the presence of thiabendazole resistance in H. contortus in 92% (12/13) of the farms. Estimated percentages of resistant parasites in H. contortus populations obtained with the two biological tests and the genetic test were compared. The results of the genetic test were in agreement with the biological tests and confirmed that benzimidazole resistance in H. contortus is present in Ontario sheep flocks. Differences between the different methods of drug resistance detection are discussed in terms of cost, time and sampling.

Efficiency of a genetic test to detect benzimidazole resistant Haemonchus contortus nematodes in sheep farms in Quebec, Canada

Haemonchus contortus is a hemophilic nematode which infects sheep and causes anemia and death to lambs. Benzimidazole drugs are used to remove these parasites, but the phenomenon of resistance has arisen worldwide. A sensitive test to detect resistance before treatment would be a useful tool to enable farmers to anticipate the efficiency of the drug before drenching the flock. In this study, we compared a test for benzimidazole resistance based on detection of genetic markers in H. contortus before treatment with the common method of fecal egg count reduction test (FECRT). We recruited 11 farms from different regions of Quebec for this study. Fecal samples from animals were collected per rectum before and after treatment in control and treated groups (10 animals per group). The 10 sheep were treated with fenbendazole at the recommended dose rate. Among the 11 farms participating in the study, we found H. contortus in 8 of them and it was the most predominant nematode species detected by egg count. Using the genetic test, we found benzimidazole resistance in each of these 8 farms. In 5 of these 8 farms there were sufficient sheep with an egg count for H. contortus above 150 eggs per gram to allow the FECRT test to be conducted. Benzimidazole resistance was observed in each of these 5 farms by the FECRT. When we compared the results from the genetic test for samples off pasture and from individual sheep, with the results from the FECRT, we concluded that the genetic test can be applied to samples collected off pasture to estimate benzimidazole resistance levels before treatment for H. contortus infections.

First identification of benzimidazole resistant Haemonchus contortus in sheep in Greece

The present study investigated for the first time benzimidazole (BZ) resistance in Haemonchus contortus originating from sheep in the area of Thessaloniki, Northern Greece, using an allele-specific PCR. This method detects a polymorphism (Phe/Tyr) that affects the amino acid at position 200 of the β-tubulin protein, and is known to be linked to BZ resistance. Forty adult worms were collected from local abattoir from 8 flocks. From each flock a total of 5 sheep were sampled and from each sheep one worm was collected. It was found that all 40 worms were homozygous benzimidazole resistant, which demonstrates for the first time that resistant strains of H. contortus do exist in sheep farms in Northern Greece. These results have practical implications in sheep farmer consulting in Greece, because sensitive screening of important worms using molecular methods may offer in the future a valid estimation on the correct use of anthelmintics towards a more effective control of gastrointestinal nematode parasites of small ruminants.