Strains Of Haemonchus Contortus Research Articles

Analysis of lncRNA-related studies of ivermectin-sensitive and -resistant strains of Haemonchus contortus.

In this study, 858 novel long non-coding RNAs (lncRNAs) were predicted as sensitive and resistant strains of Haemonchus contortus to ivermectin. These lncRNAs underwent bioinformatic analysis. In total, 205 lncRNAs significantly differed using log2 (difference multiplicity) > 1 or log2 (difference multiplicity) < - 1 and FDR < 0.05 as the threshold for significant difference analysis. We selected five lncRNAs based on significant differences in expression, cis-regulation, and their association with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. These expressions of lncRNAs, namely MSTRG.12610.1, MSTRG.8169.1, MSTRG.6355.1, MSTRG.980.1, and MSTRG.9045.1, were significantly downregulated. These findings were consistent with the results of transcriptomic sequencing. We further investigated the relative expression of target gene mRNAs and the regulation of mRNA and miRNA, starting with lncRNA cis-regulation of mRNA, and constructed a lncRNA-mRNA-miRNA network regulation. After a series of statistical analyses, we finally screened out UGT8, Unc-116, Fer-related kinase-1, GGPP synthase 1, and sart3, which may be involved in developing drug resistance under the regulation of their corresponding lncRNAs. The findings of this study provide a novel direction for future studies on drug resistance targets.

Comparative analysis on transcriptomics of ivermectin resistant and susceptible strains of Haemonchus contortus.

BackgroundIvermectin (IVM) is one of the most important and widely used anthelmintics in veterinary medicine. However, its efficacy is increasingly compromised by widespread resistance, and the exact mechanism of IVM resistance remains unclear for most parasitic nematodes, including Haemonchus contortus, a blood-sucking parasitic nematode of small ruminants.MethodsIn this study, an H. contortus IVM-resistant strain from Zhaosu, Xinjiang, China, was isolated and assessed by the control test, faecal egg count reduction test (FECRT) and the larval development assay (LDA). Subsequently, comparative analyses on the transcriptomics of IVM-susceptible and IVM-resistant adult worms of this parasite were carried out using RNA sequencing (RNA-seq) and bioinformatics.ResultsIn total, 543 (416 known, 127 novel) and 359 (309 known, 50 novel) differentially expressed genes (DEGs) were identified in male and female adult worms of the resistant strain compared with those of the susceptible strain, respectively. In addition to several previously known candidate genes which were supposed to be associated with IVM resistance and whose functions were involved in receptor activity, transport, and detoxification, we found some new potential target genes, including those related to lipid metabolism, structural constituent of cuticle, and important pathways such as antigen processing and presentation, lysosome, autophagy, apoptosis, and NOD1-like receptor signalling pathways. Finally, the results of quantitative real-time polymerase chain reaction confirmed that the transcriptional profiles of selected DEGs (male: 8 genes, female: 10 genes) were consistent with those obtained by the RNA-seq.ConclusionsOur results indicate that IVM has multiple effects, including both neuromuscular and non-neuromuscular targets, and provide valuable information for further studies on the IVM resistance mechanism in H. contortus.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-022-05274-y.

Transcriptome sequencing and analysis of lncRNA related to albendazole resistant strain of Haemonchus contortus

The objective of this study was to explore the link between long non-coding RNAs (lncRNAs) and resistance mechanism of albendazole in Haemonchus contortus by analyzing the gene expression profile of lncRNA in sensitive and resistant strains. This study provided the base for the resistance mechanism of Haemonchus contortus. In this experiment, the cDNA sequencing libraries were collected constructed for the sensitive and resistant strains of Haemonchus contortus, and the sequencing was performed using Illumina HiSeq 4000 platform, and differentially expressed lncRNAs (DEIncRNAs) were screened. Then the cis-targeted and trans-targeted genes of DElncRNAs were predicted, and the GO enrichment, KEGG pathway enrichment analysis were also made. The results displayed that 6 377 and 6 356 candidate lncRNA transcripts of sensitive and resistive strains were respectively obtained, 168 DElncRNAs of which were selected. Compared with resistive strains, there were 92 up-regulated and 76 down-regulated lncRNAs in sensitive strains. Meanwhile, 416 candidate target genes of DElncRNAs were obtained. The analytical results indicated that these genes participated in 641 GO terms and 92 pathways. The pathways involved in drug resistance are drug metabolism-other enzymes, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450 and so on. In conclusion, these findings inferred that some lncRNA-mediated target genes were associated with the resistance of Haemonchus contortus, and lncRNA may play an important role in the resistance of Haemonchus contortus. This study explored the expression profile of lncRNA in the Haemonchus contortus of sensitive and resistant strains. It was found that DElncRNAs in the sensitive and resistant strains, which helped to find out the Haemonchus contortus resistance mechanism of albendazole and provided a scientific basis for exploring the mechanism of resistance to Haemonchus contortus.

Haemonchus contortus resistance to monepantel in sheep: fecal egg count reduction tests and randomized controlled trials

Worm infections are a major cause of economic losses in sheep farming, and their control depends essentially on anthelmintic drugs. After decades without the discovery of new drug types, a new class of anthelmintics, called aminoacetonitrile derivatives, represented by monepantel, was released on the market. In order to evaluate a report of low efficacy of monepantel on a sheep farm in the municipality of Taiúva, state of São Paulo, Brazil, fecal egg count reduction and critical tests were performed on a Haemonchus contortus strain collected from this farm. To this end, ten animals were experimentally infected with 5000 H. contortus third-stage larvae, and these animals were divided into two groups, control and treated, according to the treatment with monepantel. The parameters evaluated were reduction in fecal egg counts on post-treatment days (PTD) 0, 2, 4, 7 and 14, and the therapeutic efficacy at the end of this period. Egg count reduction was observed starting on PTD 2, with a maximum reduction of 32.89% recorded on PTD 14. The critical test revealed 24.65% efficacy, indicating resistance to the active ingredient monepantel. The ineffectiveness of monepantel may be associated with the excessive use of the anthelmintic, which the farmer administered at intervals of less than one month, without technical guidance or a defined criterion. Given that the anthelmintic was used on this farm for less than one year, according to the farmer, it can be inferred that H. contortus develops resistance to monepantel in a few generations if the product is used incorrectly.

Biotransformation of albendazole and activities of selected detoxification enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics

The increased activity of drug-metabolizing enzymes can protect helminths against the toxic effect of anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug albendazole (ABZ) and the activities of selected biotransformation and antioxidant enzymes in three different strains of Haemonchus contortus: the ISE strain (susceptible to common anthelmintics), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (multi-resistant).H. contortus adults were collected from the abomasum of experimentally infected lambs. In vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (living nematodes cultivated in flasks with medium) experiments were performed. HPLC with spectrofluorimetric and mass-spectrometric detection was used in the analysis of ABZ metabolites. The in vitro activities of oxidation/antioxidation and conjugation enzymes toward model substrates were also assayed.The in vitro data showed significant differences between the susceptible (ISE) and resistant (BR, WR) strains regarding the activities of peroxidases, catalase and UDP-glucosyltransferases. S-oxidation of ABZ was significantly lower in BR than in the ISE strain. Ex vivo, four ABZ metabolites were identified: ABZ sulphoxide and three ABZ glucosides. In the resistant strains BR and WR, the ex vivo formation of all ABZ glucosides was significantly higher than in the susceptible ISE strain. The altered activities of certain detoxifying enzymes might partly protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.

Sensitivity of two in vitro assays for evaluating plant activity against the infective stage of Haemonchus contortus strains

The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P < 0.05) in the sensitivity of the LPA and the MTT-formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.

A Common Beta Tubulin Isotype-1 Gene Single Nucleotide Polymorphism as a Tool for Detection and Quantitation of Anthelmintic Resistant Haemonchus contortus in Grazing Goats

Haemonchus contortus, a gastrointestinal parasite costs the global small ruminant industry massive production loss and drug costs per annum. In Haemonchus contortus , one of the single nucleotide polymorphisms (SNP) at codon 200 of the beta tubulin isotype-1 gene (TTC to TAC), renders this nematode resistant to benzimidazole based anthelmintics. Goats infected with such strains are resistant to benzimidazole based anthelmintics. We used this well established SNP as a diagnostic tool to detect and quantify resistant Haemonchus contortus in grazing goats. Spanish goats had 28% higher (P<0.05) amounts of both benzimidazole sensitive and resistant strains of Haemonchus contortus in stool than Myotonic goats. Bucks also had 25% higher (P<0.05) of both sensitive and resistant strains than Does. These data demonstrate that this SNP can be used as a diagnostic tool for detection and quantitation of Haemonchus contortus in the stool of grazing goats.

Immunoprotective effect of cysteine proteinase fractions from two Haemonchus contortus strains adapted to sheep and goats

A preliminary analysis of the significance of genetic diversity in cysteine proteinase genes has been performed simultaneously in sheep and goats, with regard to the immunological control using these enzymes against haemonchosis. For this purpose, we have studied the cross-immunoprotective effect of cysteine protease-enriched protein fractions (CPFs) in adult worms of two Haemonchus contortus strains from North America and Spain that are adapted to sheep and goats, respectively. Previous genetic analysis of cysteine proteinase genes in both strains has shown that some of loci are polymorphic and these differences are translated into changes in the amino acid sequences. However, our results show that CPFs from H. contortus adult worms have a protective effect against the parasite in both sheep and goats. These results are similar regardless of whether they were obtained from sheep or goat-adapted H. contortus strains, which could be very important in case H. contortus CPFs were commercially used in different countries, as vaccines to prevent the negative effects of this parasite.Interestingly, this experimental inoculation of both species with a heterologous strain of H. contortus contributes to the idea shown in previous studies about how difficult is the interpretation and the comparison of vaccination where strains not adapted to a specific host are used. Therefore, the challenger of using heterologous strains could provide similar results to those observed in immunised animals. This study suggests the possibility of exploring the mechanisms involved in natural protection against non-adapted strains, in order to develop strategies to control haemonchosis.

Import and efflux of flubendazole in Haemonchus contortus strains susceptible and resistant to anthelmintics

Drug entry into the body of a helminth is a key factor in the efficacy of anthelmintics. The present project was designed to study the ex vivo uptake and efflux of the benzimidazole anthelmintic flubendazole (FLU) in four strains of H. contortus: the ISE strain (fully susceptible to anthelmintics), the ISE-S strain (resistant to ivermectin), the BR strain (resistant to benzimidazoles) and the WR strain (multi-resistant). The transport of FLU between dead and living nematodes was also compared as well as the effect of verapamil, an inhibitor of the main efflux ABCB1 transporter (P-glycoprotein), on FLU accumulation in nematodes. The obtained results showed that FLU is able to effectively enter H. contortus adults due to high FLU lipophilicity. Passive diffusion is probably the only mechanism in both FLU import and efflux from nematodes. No differences in FLU transport were found among four H. contortus strains with different sensitivity to anthelmintics. No active FLU efflux from H. contortus and no effect of verapamil were observed, indicating that H. contortus cannot protect itself against FLU by the active removal of this anthelmintic from its body.

Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.

Evaluation of Cymbopogon schoenanthus essential oil in lambs experimentally infected with Haemonchus contortus

Hematophagous gastrointestinal parasites cause significant economic losses in small ruminant grazing systems. The growing reports of multi-drug resistant parasites call for intensive research on alternative treatments for anthelmintics to help small ruminants cope with these parasites. Two-month-old lambs with mean body weight (BW) of 22.5kg were experimentally infected with a multidrug-resistant Haemonchus contortus strain. Infected animals were dosed orally with Cymbopogon schoenanthus essential oil to evaluate its anthelmintic potential. Eighteen animals were allocated into three groups of six animals, and each received one of the following treatments: Group 1 – control (10mL of water), Group 2 –C. schoenanthus essential oil (180mg/kg BW); and Group 3 –C. schoenanthus essential oil (360mg/kg BW). Animals received the oil once a day for 3 consecutive days. Lambs were evaluated clinically for blood biochemistry before, at 1, 5, 10, 15 and 20 days after treatment, and then were euthanized to assess the total worm burden. No statistically significant reduction in fecal egg count, packed cell volume or total worm count was observed after treatments. Also, no statistical difference among group means for blood levels of urea, creatinine, albumin, alkaline phosphatase, aspartate aminotransferase and gamma glutamyl transferase was found. Larval development assay (LDA) and egg hatch assay (EHA) were performed from feces of treated animals at 1, 5, 10 and 15 days after essential oil administration. An inhibition in LDA was observed 1 day after the 3-day treatment in larvae from feces of animals treated with 360mg/kg essential oil. In conclusion, the essential oil at the doses of 180mg/kg and 360mg/kg was safe to sheep, but failed as an anthelmintic treatment when applied to young sheep artificially infected with a multidrug-resistant H. contortus strain.

Pyrosequencing analysis of the beta-tubulin gene in Spanish Teladorsagia circumcincta field isolates

Benzimidazole (BZ) resistance in gastrointestinal nematodes has been associated with single nucleotide polymorphisms (SNPs) at codons 200, 167 and 198 in the beta-tubulin isotype 1 gene and, recently, these SNPs have also been found in macrocyclic lactone (ML) resistant strains of Haemonchus contortus. On this basis, we have studied the same putative SNPs in Spanish Teladorsagia circumcincta field isolates by pyrosequencing. Single L3 (infective 3rd stage larvae) from five sheep flocks were tested after confirming their BZ susceptibility and degree of ivermectin (IVM) resistance. According to the Faecal Egg Count Reduction Test (FECRT) one flock was classified as IVM susceptible, another one was resistant, and the rest had a suspicion of resistance to IVM. DNA extraction was carried out on 598 single L3 and 56% of these were identified as T. circumcincta after the amplification of a species-specific ITS2 fragment. The number of L3 analyzed for the SNPs 198/200 was 255 and for the SNP 167 was 187. Results clearly indicate no resistance-associated SNPs were present at any codon, before or after treatment. Therefore, all T. cicumcincta L3 were designated as susceptible homozygous genotypes for all SNPs. The absence of the mutations in these populations would argue against resistance haplotypes being present in the parasite population prior to drug treatment, at least in Spanish T. circumcincta.

Dose‐dependent systemic exposure of albendazole metabolites in lambs

The gastrointestinal absorption of most drugs follows a first-order kinetics, whereby a constant fraction of the total drug is absorbed in each equal time interval. Although this related absorption principle is applicable to the most of the therapeutically used drugs, it remains unclear for poorly water-soluble compounds such as the benzimidazole anthelmintics in ruminants. The goal of the current work was to characterize the albendazole (ABZ) metabolites plasma disposition kinetics after ABZ administration at different dosages to nematode-infected lambs. Eighteen Corriedale lambs artificially infected with a resistant Haemonchus contortus strain were allocated into three groups and intraruminally treated with ABZ at either 5 (ABZ(5)), 15 (ABZ(15)) or 45 (ABZ(45)) mg/kg. Blood samples were collected up to 120 h post-treatment, and the collected plasma was analysed by high-performance liquid chromatography. The estimated pharmacokinetic parameters were statistically compared using parametric and nonparametric tests. None of the animals involved in the current trial showed any adverse events during the study. While ABZ parent drug was not recovered in the bloodstream, the area under the concentration vs time curve (AUC) of the active ABZ-sulphoxide (ABZSO) metabolite increased significantly (P<0.05) from 21.0 (ABZ(5)) up to 158.6 (ABZ(15)) and 389.7 μg·h/mL (ABZ(45)), which indicates some type of nonproportionality in the relationship between dose level and drug systemic exposure. The overall kinetic disposition of the inactive sulphone metabolite did not change after treatment at threefold the therapeutic ABZ dosage. However, significantly (P<0.05) higher AUC, C(max) and mean residence time values were observed after the administration of the highest dosage level. The higher dosages accounted for a significantly (P<0.05) enhancement of the ABZSO peak plasma concentration, which were obtained at delayed times post-treatment. High correlations between AUC(0-LOQ) and C(max) and nematode counts were observed, with Spearman's coefficients of -0.83 and -0.84, respectively. The results obtained in the current experiment show that increasing the dose of ABZ in sheep is clearly associated with enhanced plasma ABZ metabolites exposure. The data showed a nonproportionality on the gastrointestinal absorption of ABZ in nematode-infected lambs.

Effects of age and immune suppression of sheep on fecundity, hatching and larval feeding of different strains of Haemonchus contortus

The effects of host age and immune suppression on abomasal parasitic infection in sheep were investigated following single experimental oral infections with MHco3 (ISE), MHco4 (WRS) and MHco10 (CAVR) strains of Haemonchus contortus in naïve 5-month-old crossbred lambs (n=1 per group) and 15-month-old Greyface sheep treated with methyl prednisolone acetate (n=2 per group) or without corticosteroid treatment (n=2 per group). Adult female H. contortus in 5-month-old lambs (n=1 per group) shed on average 6.5, 3.1 and 8.0 times more eggs than in 15-month-old sheep (n=4 per group) following infection with MHco3 (ISE), MHco4 (WRS) and MHco10 (CAVR) strains of H. contortus, respectively, over a period of 28 days following the commencement of patency. There was no obvious effect of age of sheep or corticosteroid treatment on the abomasal establishment of H. contortus or on in vitro assays for egg hatching or larval feeding at different concentrations of anthelmintics, although statistical analysis could not be performed due to the small group sizes.

Efficacy of combination anthelmintics against multiple resistant strains of sheep nematodes

Broad-spectrum anthelmintics were tested for their efficacy in either single active or combination formulations against multiple anthelmintic-resistant strains of Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Three efficacy trials based on post mortem worm counts showed that the triple and quadruple combinations of benzimidazoles, levamisole, macrocyclic lactones and closantel were effective against most strains that were resistant to each of the components when drenched individually. Consequently, use of any one of these classes of anthelmintics by itself against multiple-resistant strains would lead to a rapid increase in the level of resistance. When used in combinations, the anthelmintics reduced the survival rate of most strains to &lt;5%. However, because some strains of T. circumcincta and T. colubriformis were found to be resistant to all combinations currently marketed in Australia, it is clear that combinations should be used before resistance levels become too high.

Genomic analyses of Haemonchus contortus infection in sheep: Abomasal fistulation and two Haemonchus strains do not substantially confound host gene expression in microarrays

To determine whether fistulation and differing strains of Haemonchus contortus complicate genome analysis of the host response to infection, two pilot experiments examined parasite development and gene expression in the abomasal mucosa of parasitised sheep. No significant differentially-expressed genes were detected in a comparison between ivermectin-susceptible McMaster and ivermectin-resistant CAVR strains of H. contortus. This demonstrated that the sheep response was not significantly altered by the ivermectin-resistance status of the parasite. However, sheep infected with McMaster strain had a significantly lower proportion of larvae and a higher mean FEC at post-mortem than sheep infected with CAVR, suggesting that McMaster larvae advance to patency faster than CAVR larvae. Abomasal fistulation resulted in significant upregulation of three genes and significant downregulation of two genes. Fistulated sheep had significantly lower FEC than the other groups but the proportion of larvae at post-mortem was not significantly different to other groups infected with the same strain (CAVR). Hence fistulation does not alter establishment of the CAVR isolate, but may slow its progression to patency. The observation that different H. contortus strains and abomasal fistulation induced minimal changes in mucosal gene expression validated the design of a subsequent experiment (manuscript in preparation) where sequential biopsies taken during infection were analysed by microarray to describe the molecular responses which inhibit larval establishment.

Amphidial structure of ivermectin-resistant and susceptible laboratory and field strains of Haemonchus contortus.

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians, pet owners, and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides. As a result, resistance to ivermectin, moxidectin, nemadectin, and doramectin by Haemonchus contortus has been documented throughout the world. Sensory neurons located in the cephalic end of nematodes are in close contact with the external environment. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible strains of H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveal that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. These findings raise a number of questions regarding the relationship between amphidial structure and ivermectin resistance as well as the role of amphids as a means of entry for ivermectin. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to macrocyclic lactones.

The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep.

Resistance to the benzimidazole (BDZ) class of anthelmintics in nematodes of sheep has become a common and global phenomenon. The rate at which the selection process and development of resistance occurs is influenced by a number of factors. Of these, the effects of stage-specific exposures to anthelmintic were investigated with a BDZ-resistant strain of Haemonchus contortus (HCR) over five parasite generations. Sheep were infected at each generation with the HCR strain and were treated with thiabendazole (TBZ), either 5 days post-infection (p.i.) (larval line), 21 days p.i. (adult line), or left untreated (no selection line). Additionally eggs from each generation were exposed to TBZ (egg line). Geometric worm burdens were calculated from post-mortem worm counts, both at the start of the study, and after the final selection studies for each of the selection lines. Egg hatch assays (EHAs) were also conducted throughout the study. All data relating to worm burdens and EHAs for each generation were analysed by linear regression to produce dose titration curves and lethal dose(50) (LD(50)) values for each of the selection lines. Over the five generations, LD(50) values on dose-response were increased and worm survival occurred at higher dose rates of TBZ irrespective of the parasite stage exposed to treatment. A similar picture was seen with ED(50) values, which showed a fluctuating but generally upward trend for each of the three selection lines. In contrast, LD(50) and ED(50) values were decreased in the no selection line, indicating some degree of reversion albeit to levels still considered to be BDZ-resistant.

Selection for resistance to macrocyclic lactones by Haemonchus contortus in sheep

An anthelmintic-sensitive Haemonchus contortus strain was selected for moxidectin and ivermectin resistance concurrently for 22 generations. Treatment with 0.002 mg moxidectin/kg BW or 0.02 mg ivermectin/kg BW produced >99% efficacy against the susceptible parent strain passaged for 22 generations without any anthelmintic exposure. However, to obtain similar efficacy the moxidectin-selected and the ivermectin-selected strains of H. contortus required 0.05 mg moxidectin/kg BW or 0.4 mg ivermectin/kg BW. These results indicate that development of resistance to one macrocyclic lactone, simultaneously results in resistance to another macrocyclic lactone. However, rates of resistance development differ between compounds and occurs more slowly with moxidectin than with ivermectin.

Effect of multidrug resistance modulators on the activity of ivermectin and moxidectin against selected strains of Haemonchus contortus infective larvae

Nematode parasites have shown resistance to the anthelmintics, ivermectin and moxidectin, and there is evidence that the over-expression of parasite P-glycoprotein (P-gp) may account, at least in part, for resistance to ivermectin. The objective of this study was to evaluate whether the multidrug resistance (MDR) modulators, verapamil, CL 347.099 (an analog of verapamil) and cyclosporin A, would enhance the efficacy of ivermectin and moxidectin against selected strains of Haemonchus contortus using an in vitro larval migration assay. The modulators had no effects on the number of migrating larvae when used alone. Ivermectin and moxidectin showed a significant (P&lt;0.05) increase in its efficacy by 52.8 and 58.5% respectively, when used in association with verapamil against a moxidectin-selected strain. CL 347,099 also increased significantly (P&lt;0.05) the ivermectin and moxidectin efficacy by 24.2 and 40.0% respectively, against an ivermectin-selected strain and by 40.0 and 75.6% respectively, against an moxidectin-selected strain. At the concentrations tested cyclosporin A showed a variable effect on increasing the efficacy of the anthelmintics against the susceptible and resistant strains.