Haemonchus Contortus Isolates Research Articles

Susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of polyphenol-rich plants. Part 2: Infective L3 larvae

This study explored the variation in susceptibility to acetone:water plant extracts between infective larvae (L3) of ten Haemonchus contortus isolates from different geographical origin. The L3 of 10 different isolates were exposed either to the acetone:water extract of a temperate plant (Onobrychis viciifolia) or a tropical plant (Acacia pennatula) and were evaluated with the larval exsheathment inhibition assay (LEIA). The L3 of each isolate were incubated with different concentrations of each extract (0, 25, 50, 100, 200, 400, 600, 800, 1000 and 1200μg/mL of phosphate buffered saline (PBS)). After incubation, the exsheathment process of L3 was induced using a solution with sodium hypochlorite (2%) and sodium chloride (16.5%). The proportion of exsheathed L3 was determined for each concentration at 0, 20, 40 and 60min. Effective concentrations 50% (EC50) and the corresponding 95% confidence intervals (95% CI) were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated for each extract to compare isolates, using the most susceptible isolate as the respective reference for each extract. To determine the role of polyphenols on the reported effect, a second set of incubations was made for each isolate and each extract, using the extracts at a concentration of 1200μg/mL PBS with or without polyvinylpolypyrrolidone (PVPP), a polyphenol blocking agent, and controls without extract. The ten different H. contortus isolates showed variation in susceptibility for each of the 2 extracts tested (P<0.05). The EC50 values for A. pennatula extract ranged from 36 to 501μg/mL (RR: 2.11–13.68). Meanwhile, the EC50 values for O. viciifolia extract ranged from 128 to 1003μg/mL (RR: 1.25–7.82). The use of PVPP revealed that polyphenols were responsible for the anthelmintic activity recorded for both extracts. However, tested H. contortus isolates suggested that susceptibility to one polyphenol-rich extract did not determine the susceptibility to the other polyphenol rich extract. The latter result indicated that the different H. contortus isolates varied in their susceptibility to the polyphenols present in each extract evaluated.

Genotypic and phenotypic evaluation for benzimidazole resistance or susceptibility in Haemonchus contortus isolates.

Haemonchus contortus isolates were evaluated for benzimidazole (BZ) resistance or susceptibility by allele-specific PCR based on β-tubulin isotype 1 gene polymorphisms at the F167Y, E198A, and F200Y sites. Two isolates, one presumed susceptible from wild pronghorn antelope (PH) and one known to be resistant from goats (VM), were also assayed phenotypically for BZ resistance or susceptibility in the larval development assay (Drenchrite®). The BZ EC50 was 0.198μM (intermediate between susceptible and weak resistant) for PH with critical well 5 (intermediate between susceptible and weak resistant) and 1.456μM (intermediate weak resistant and resistant) for VM with critical well 8.5 (resistant). Genotypically, DNA extracted from pooled VM L3 larvae in the Drenchrite® wells with the highest BZ concentration was homozygous susceptible (SS) at the F167Y and E198A sites and homozygous resistant (RR) at the F200Y site by PCR, and sequence analysis bore this out. PH L3 larvae DNA from a control well (no BZ) was SS at all three sites by PCR, confirmed by sequence analysis. All single adult worm samples (N=21) from PH, VM, Egypt goat (EG), and a Texas llama were SS at F167Y and E198A by PCR; however, only 3 PH worms and 1 EG worm were SS at F200Y. Three additional PH worms were RS and upon cloning two clones were identified as resistant by sequencing and two as susceptible. Clones from single adult worms VM, llama, and EG samples that were RR by PCR at F200Y were sequence verified as resistant. In this study, F200Y was the most frequently found genotypic marker for BZ resistance or susceptibility in the different Haemonchus isolates.

Piper aduncum against Haemonchus contortus isolates: cross resistance and the research of natural bioactive compounds.

The anthelminthic activity of the essential oil (EO) of Piper aduncum L. was tested in vitro on eggs and larvae of resistant (Embrapa2010) and susceptible (McMaster) isolates of Haemonchus contortus. The EO was obtained by steam distillation and its components identified by chromatography. EO concentrations of 12.5 to 0.02 mg/mL were used in the egg hatch test (EHT) and concentrations of 3.12 to 0.01 mg/mL in the larval development test (LDT). Inhibition concentrations (IC) were determined by the SAS Probit procedure, and significant differences assessed by ANOVA followed by Tukey's test. In the EHT, the IC50 for the susceptible isolate was 5.72 mg/mL. In the LDT, the IC50 and IC90 were, respectively, 0.10 mg/mL and 0.34 mg/mL for the susceptible isolate, and 0.22 mg/mL and 0.51 mg/mL for the resistant isolate. The EO (dillapiole 76.2%) was highly efficacious on phase L1. Due to the higher ICs obtained for the resistant isolate, it was raised the hypothesis that dillapiole may have a mechanism of action that resembles those of other anthelmintic compounds. We further review and discuss studies, especially those conducted in Brazil, that quantified the major constituents of P. aduncum-derived EO.

Increased expression of ATP binding cassette transporter genes following exposure of Haemonchus contortus larvae to a high concentration of monepantel in vitro.

BackgroundThere is some evidence that ATP binding cassette (ABC) transporters play a role in resistance to anthelmintics, particularly against macrocyclic lactones. Some anthelmintics, including ivermectin (IVM), have been shown to induce transcription of multiple ABC transporters in nematodes; however, the effects of monepantel (MPL) on transcription of these transporter genes has not been studied.MethodsLarvae of two MPL-susceptible isolates of Haemonchus contortus were exposed to MPL at two concentrations (2.5 and 250 μg/ml) for periods of 3, 6 and 24 h. Transcription levels of sixteen ABC transporter genes were measured at the end of the incubation periods. The consequences of MPL exposure were examined by measuring rhodamine-123 efflux from the larvae, and their sensitivity to subsequent treatment with IVM or levamisole.ResultsMultiple ABC transporter genes showed significantly higher transcription in both worm isolates following exposure to MPL at 250 μg/ml for 3, 6 or 24 h, particularly the P-glycoprotein (P-gp) genes pgp-11, pgp-12 and pgp-14. Of these, only pgp-11 maintained the elevated levels 24 h after the end of the drug exposure period. In contrast, there was only a single instance of low-level upregulation as a result of exposure to MPL at 2.5 μg/ml. Larvae exposed to MPL at 250 μg/ml showed an increased efflux of rhodamine-123 and a proportion of the larval population showed an ability to subsequently tolerate higher concentrations of IVM in migration assays. There was no increased tolerance to IVM following pre-exposure to MPL at 2.5 μg/ml.ConclusionsExposure of H. contortus larvae to 250 μg/ml MPL results in increased transcription of multiple transporter genes and increased R-123 efflux. The subsequent ability of a proportion of the larvae to tolerate IVM suggests a protective role of ABC transporters across different chemical entities. However, these observations were only made at a concentration of MPL well above that experienced by parasitic life stages in vivo, and hence their significance remains unclear.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1806-9) contains supplementary material, which is available to authorized users.

Genotyping of benzimidazole resistant and susceptible isolates of Haemonchus contortus from sheep by allele specific PCR.

Extensive and indiscriminate use of the benzimidazole class of drugs has led to the onset of anthelmintic resistance. In tropical countries like India, Haemonchus contortus is the most pathogenic parasite infecting sheep and goats. The widespread presence of resistant helminths (especially H. contortus) threatens the livestock farming. The use of various drugs has led to single nucleotide polymorphism that causes specific amino acid substitutions in β-tubulin protein of H. contortus to confer resistance. This emphasizes the need for a survey on the present status of resistance in India. In this study, allele specific PCR was employed to screen the presence of a SNP, a thymine-to-adenine transversion which leads to substitution of amino acid in codon 200 of β-tubulin gene that is correlated specifically with BZ resistance. Third stage larvae (L3) from pooled faecal cultures of four organized sheep farms served as a source of genomic DNA for identification of H. contortus and further genotype analysis. A total of 1000 larvae was screened, out of which 673 larvae were identified as H. contortus. Among 673 H. contortus larvae, 539 larvae (80%) were genotyped as homozygous resistant (rr) and remaining 134 (20%) were heterozygous susceptible (Sr) by allele specific PCR. The concluded resistance status reasons out the failure of anthelmintic drug in treating ruminants. Immediate steps are needed to avoid further aggravation of the problem. Target selective treatment by reviewing the resistance status of individual drugs, appropriate use of anthelmintic drugs and other control strategies will provide a pragmatic option for delaying the further spread of anthelmintic resistance.

Comparison of two in vitro methods for the detection of ivermectin resistance in Haemonchus contortus in sheep

Summary Gastrointestinal parasitic nematodes in sheep cause severe economic losses. Anthelmintics are the most commonly used drugs for prophylaxis and therapy against parasitic helminths. The problem of drug resistance has developed for all commercially available anthelmintics in several genera and classes of helminths. In vitro and in vivo tests are used to detect anthelmintic resistance. Two in vitro methods (larval migration inhibition test and micromotility test) for the detection of ivermectin (IVM) resistance were compared using IVM-resistant and IVM-susceptible isolates of Haemonchus contortus. The degree of resistance for each test was expressed as a resistance factor (RF). The micromotility test was more sensitive for quantitatively measuring the degree of resistance between susceptible and resistant isolates. The RFs for this test for IVM and eprinomectin ranged from 1.00 to 108.05 and from 3.87 to 32.32, respectively.

Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

In vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins towards acetone:water extracts of two tannin rich plants

The aim of the study was to examine the variation in the in vitro susceptibility of ten Haemonchus contortus isolates from different geographical origins using respective egg hatch assays (EHA) with acetone:water extracts of two tannin containing plants, chimay (Acacia pennatula) and sainfoin (Onobrychis viciifolia). Fresh eggs were incubated in PBS with different concentrations of each extract (0, 600, 1200, 2400, 3600, 5000 and 8000μg/ml PBS). Additional concentrations were tested for O. viciifolia (75, 100, 200 and 400μg/ml PBS). Effective concentrations 50% (EC50), with the corresponding 95% confidence interval (95% CI), were calculated for every isolate with both extracts. Moreover, a resistance ratio (RR) was calculated to compare the isolates, using the most susceptible isolate for each extract as the respective reference. A second set of incubations were made using polyvinylpolypyrrolidone (PVPP) (0, 5000μg/ml, 5000μg/ml+PVPP) to determine the influence of polyphenols on the AH effect. The proportion of morulated eggs, eggs with L1 larvae failing eclosion (%LFE), and emerged larvae were estimated at different extract concentrations. Data of each isolate was used to calculate the effective concentration 50% (EC50) for each extract. The EC50 of each isolate was used to determine resistance ratio (RR) for the different isolates. For the 2 extracts, a susceptibility variation in egg hatching was observed for the different H. contortus isolates. The EC50 values for A. pennatula ranged from 2203 to 14106μg (RR from 2.01 to 6.40). The O. viciifolia extract showed higher variability with EC50 values ranging from 104 to 4783μg (RR from 3.66 to 45.74). The main AH effects of the two extracts tested on the ten isolates consisted in blocking the emergence of L1 larvae (higher% LFE). Additional observations on emerged larvae showed that extract exposure caused alterations in the internal structure, separating the cuticle from the pharynx, bulb and intestinal cells. The use of PVPP revealed that (a) condensed tannins were not the sole plant secondary metabolites responsible for the AH effects, and (b) different H. contortus isolates showed variability in the role of tannins either on the ovicidal effect or the %LFE.

Comparative Response of the West African Dwarf Goats to Experimental Infections with Red Sokoto and West African Dwarf Goat Isolates of Haemonchus contortus.

Response of the West African Dwarf (WAD) goats to two different isolates of Haemonchus contortus, the Red Sokoto (RS) goat isolate (RSHc) and the WAD goat isolate (WADHc) (isolated from WAD goats), was studied by experimental infections of 4–6-month-old male WAD goat kids. Group 1 and Group 2 goats were each infected with 4500 infective larvae (L3) of RSHc and WADHc, respectively. Group 3 animals served as uninfected control. Prepatent period (PPP), faecal egg counts (FEC), worm burden (WB), body weight (BW), packed cell volume (PCV), and body condition score (BCS) were determined. WAD goats infected with RSHc isolate and the ones infected with WADHc isolate had mean PPP of 19.63 ± 0.26 and 19.50 ± 0.19, respectively. Goats infected with WADHc isolate had significantly higher FEC (P = 0.004) and WB (P = 0.001). BW were significantly higher (P = 0.004) both in the controls and in Group 2 goats infected with WADHc isolate than in Group 1 goats infected with the RSHc isolate. BCS of animals in both infected groups dropped significantly (P = 0.001). There was a significant drop in PCV (P = 0.004) of both infected groups in comparison. Both isolates of H. contortus were pathogenic to the host.

A new enabling proteomics methodology to investigate membrane associated proteins from parasitic nematodes: Case study using ivermectin resistant and ivermectin susceptible isolates of Caenorhabditis elegans and Haemonchus contortus

The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of AR management is essential for effective and sustainable control of parasitic helminth worms in livestock. Current tests to measure AR are time consuming and can be technically problematic, gold standard diagnostics are therefore urgently required to assist in combatting the threat from drug resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop and be measurable in the target associated proteins present in the parasite membrane. The model nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein expression differences, between IVM resistant and IVM susceptible isolates in the presence and absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton ×100 and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a pathogenic blood feeding parasitic nematode which is of global importance in livestock health, welfare and productivity. We have demonstrated the successful resolution of membrane associated proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR.

Development of an in vitro screen for compound bioaccumulation in Haemonchus contortus.

The objective of the current study was to establish an in vitro screen and a highly sensitive analytical assay to delineate key physicochemical properties that favor compound bioaccumulation in the L3 life stage of a Haemonchus contortus isolate. Time-dependent studies revealed that absorption and elimination kinetics during the first 6 hr of exposure were sufficient to achieve maximum bioaccumulation for the majority of compounds tested. In subsequent studies, the larvae were incubated for 6 hr in a medium containing 146 compounds (5 μM initial concentration), including both human and veterinary medicines, characterized by a broad range of physicochemical properties. Bioaccumulation of the compounds by the nematodes was determined, and multiple physicochemical descriptors were selected for correlation. Data analysis using Bayes classification model and partial least-square regression revealed that clogD7.4, rotatable bond, E-state, and hydrogen bond donor each correlated with compound bioaccumulation in H. contortus L3. The finding that lipophilicity was critical for transcuticle compound permeation was consistent with previous studies in other parasitic species and in adult H. contortus . The finding of additional physicochemical properties that contribute to compound conformational flexibility, polarity, and electrotopological state shed light on the mechanisms governing transcuticle permeation. The relatively poor correlation between transcuticle and transmembrane permeation indicated the distinct mechanisms of compound permeation, likely due to the different constituents, and their contributions to overall transport function, of the lipid membranes and the porous collagen barrier of the nematode cuticle. Our study, for the first time, establishes a high-throughput screen for compound bioaccumulation in a parasitic nematode and further elucidates physicochemical factors governing transcuticular permeation of compounds. Application of this methodology will help explain the basis for discrepancies observed in receptor binding and whole organism potency assays and facilitate incorporation of drug delivery principles in the design of candidate anthelmintics.

Reduction of benzimidazole resistance in established Haemonchus contortus populations in goats using a single infection with a benzimidazole-susceptible isolate.

An in vivo study in goats evaluated the effect of superimposing a single artificial infection with a benzimidazole (BZ)-susceptible Haemonchus contortus isolate upon established H. contortus populations of known BZ resistance by measuring the phenotypic BZ resistance of eggs collected from faeces before and after re-infection. Two H. contortus isolates, one benzimidazole resistant (BZR) and the other susceptible (BZS), were used to infect worm-free goats. Eight goats were initially infected with 2000 third-stage larvae (L3). In each case the inoculum contained a pre-determined proportion of the two isolates: 100% BZS (one goat), 75% BZS/25% BZR (two goats), 50% BZS/50% BZR (two goats), 25%BZS/75% BZR (two goats) and, finally, 100% BZR (one goat). The phenotypic BZ susceptibility of the H. contortus population formed in each goat after the first infection was determined on days 30 and 36 post-infection using an egg-hatch assay (EHA) that estimated the concentration of thiabendazole (TBZ) required for 95% inhibition of larval hatching (EC(95)) with a 95% confidence interval (95% CI). On day 49 post-infection, goats were re-infected with 2000 L3 of the BZS isolate alone. A second set of EHA bioassays was performed 28 days and 34 days after re-infection. The first infection protocol produced three populations classified as BZS (EC(95) 0.055-0.065 μg TBZ/ml) while four were categorized as BZR (EC(95) 0.122-0.344 μg TBZ/ml). The status of one other population could not be determined. After re-infection with BZS L3, the number of susceptible populations increased to six (EC(95) 0.043-0.074 μg TBZ/ml) while the remaining two were deemed resistant (EC(95) 0.114-119 μg TBZ/ml). Re-infection with BZS L3 thereby reduced the resistance status of most H. contortus populations.

Relative potency of macrocyclic lactones in in vitro assays with larvae of susceptible and drug-resistant Australian isolates of Haemonchus contortus and H. placei

The present study used in vitro assays to determine the relative potency of commercial macrocyclic lactone (ML) anthelmintics against larvae of drug-susceptible and drug-resistant Australian isolates of important parasites of sheep and cattle, Haemonchus contortus and Haemonchus placei, respectively. Cattle pour-on products containing abamectin, ivermectin, eprinomectin, doramectin or moxidectin were diluted in DMSO and used in larval development assays. Abamectin was the most potent chemical (lowest IC50 value) towards the drug-susceptible H. contortus Kirby isolate. The abamectin IC50 was approximately 2-fold lower than those for ivermectin, moxidectin, eprinomectin and doramectin. Moxidectin and abamectin were the most potent chemicals towards the resistant H. contortus Wallangra isolate. This isolate showed resistance ratios up to 70-fold towards eprinomectin. The resistance ratio for this species was lowest with moxidectin (ratio of 4.0-fold). Abamectin was also the most potent chemical towards both drug-susceptible (Bremner) and drug-resistant (Dayboro) H. placei isolates. The larval development assay only showed low levels of resistance for the drug-resistant H. placei, with resistance ratios ranging from 1.7 to 2.0 fold for moxidectin and abamectin, up to 3.3-fold for eprinomectin. This study examined the readily-accessible larval life stages of these parasites in in vitro assays, and, hence, the relationship between our findings and relative drug efficacies in vivo remains to be determined. Despite this, the study accords with some evidence from the use of these anthelmintics in the field in demonstrating the potency of moxidectin and abamectin against ML-resistant H. contortus. The study also highlights the usefulness of eprinomectin as a readily-available compound which is a more sensitive marker for ML resistance in in vitro larval development assays than the other commercial ML compounds examined.

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.

Genetic variability within and among Haemonchus contortus isolates from goats and sheep in China

BackgroundHaemonchus contortus (order Strongylida) is a common parasitic nematode infecting small ruminants and causing significant economic losses worldwide. Knowledge of genetic variation within and among H. contortus populations can provide a foundation for understanding transmission patterns, the spread of drug resistance alleles and might assist in the control of haemonchosis.Methods152 H. contortus individual adult worms were collected from seven different geographical regions in China. The second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA and mitochondrial nicotinamide dehydrogenase subunit 4 gene (nad4) were amplified by polymerase chain reaction (PCR) and sequenced directly. The sequence variations and population genetic diversities were determined.ResultsNucleotide sequence analyses revealed 18 genotypes (ITS-2) and 142 haplotypes (nad4) among the 152 worms, with nucleotide diversities of 2.6% and 0.027, respectively, consistent with previous reports from other countries, including Australia, Brazil, Germany, Italy, Malaysia, Sweden, the USA and Yemen. Population genetic analyses revealed that 92.4% of nucleotide variation was partitioned within populations; there was no genetic differentiation but a high gene flow among Chinese populations; some degree of genetic differentiation was inferred between some specimens from China and those from other countries.ConclusionsThis is the first study of genetic variation within H. contortus in China. The results revealed high within-population variations, low genetic differentiation and high gene flow among different populations of H. contortus in China. The present results could have implications for studying the epidemiology and ecology of H. contortus in China.

Efficacy of monepantel against lower developmental stages of a multi-resistant and susceptible Haemonchus contortus isolates: an in vitro study

Abstract Monepantel (MOP) belongs to a new class of anthelmintic compounds, the amino-acetonitrile derivates, which have a different mode of action as the currently used anthelmintics. Many present studies confirmed the high efficacy of MOP against fourth larval and adult stages of Haemonchus contortus. The objective of this study was to determine in vitro efficacy of MOP against lower development stages (eggs, L1–L3 larvae) and to compare it between resistant and susceptible isolates of H. contortus. For this purpose, two in vitro tests - egg hatch test and micro-agar larval development test were used. Results were quantified as 50 % lethal concentration (LC50), 99 % lethal concentration (LC99) and resistance factor (RF). This study revealed the high efficacy against lower larval stages (L1–L3) of both resistant and susceptible strains of this parasite. Larval susceptibility was not dependent of the sensitivity status of the nematode isolate. On the other hand, ovicidal effect of MOP was very low.

Acetylcholine receptor subunit and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant Haemonchus contortus

The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50>600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to the averaging of gene expression data across the whole population.

F200Y polymorphism in the β-tubulin gene in field isolates of Haemonchus contortus and risk factors of sheep flock management practices related to anthelmintic resistance

Haemonchus contortus is the most prevalent and pathogenic nematode of sheep in tropical areas. The objectives of this study were to assess the frequency of the F200Y polymorphism on the β-tubulin gene in third-stage larvae of H. contortus from 33 sheep flocks in São Paulo state, Brazil, and to associate this frequency to risk factors based on farm management practices. The resistance allele frequency varied from 9 to 74%, and the resistance genotype frequency varied from 0 to 66.7%. Resistance genotype frequencies higher than 40% were associated with multiple risk factors – new sheep farming enterprises, the absence of farm records, the use of Dorper and Suffolk breeds, rotational grazing, the lack of wetlands on farms, pasture sharing with cattle or horses, frequent incorporation of animals into the flock, semi-intensive farming systems, whole-flock treatment, failure to use the FAMACHA method, lack of the dose-and-move practice, anthelmintic rotation after each application, visual estimation of animal weight for treatment, and lack of drug combination use. It can be concluded that genotyping the F200Y polymorphism can be used to monitor the resistance in sheep flocks and the knowledge of management strategies at the farm level is important to identify drug resistance related factors.

Is the micro-agar larval development test reliable enough to detect ivermectin resistance?

To aid the diagnosis of anthelmintic resistance, a range of in vivo and in vitro techniques have been developed. Amongst in vitro techniques, the larval development test is the most widely employed. Six lambs were infected with susceptible (three) and ivermectin-resistant (three) isolates of Haemonchus contortus. The micro-agar larval development test (MALDT) was able to easily distinguish between susceptible and resistant isolates. Different proportions of resistant and susceptible eggs were subsequently incubated, i.e. development to the third larval stage occurred only in the resistant isolate. The percentage of resistant eggs ranged from 2 to 20.0 % of all eggs in the wells. In all cases, the MALDT was able to detect the presence of a minimum of 10 % of resistant worms amongst a susceptible background population. The probability was approximately 87 % of positively diagnosing a proportion of resistant worms of only 2-4 % within the population.

Efficacy of monepantel, derquantel and abamectin against adult stages of a multi-resistant Haemonchus contortus isolate

Drug resistance in gastrointestinal nematodes is a severe problem for sheep farmers. With the recent introduction of monepantel (Zolvix®) and of derquantel plus abamectin (Startect®) in New Zealand, two new anthelmintic classes will be available to control gastrointestinal nematodes. While monepantel covers a broad spectrum of nematodes, the efficacy of derquantel is mid-spectrum and limited to a smaller number of species and stages. The combination of derquantel and abamectin allows to enlarge the spectrum and to cover most parasitic nematodes in sheep. However, the question remained open, if the efficacy of the new anthelmintics can be maintained in the presence of severe anthelmintic resistance. The present study investigated the efficacy against adult stages of a multi-resistant Haemonchus contortus isolate. While monepantel resulted in 100 % elimination, derquantel in combination with abamectin resulted in efficacies <95 % (faecal egg counts and worm counts).