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.
Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the ?-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length ?-tubulin isotype 1 and 2 and ?-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora ?-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be ?-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans ?-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like ?-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in ?-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.
Two different classes of small nematode specific lipid-binding proteins, the nematode polyprotein allergens/antigens (NPAs) and the fatty acid- and retinol-binding (FAR) proteins, are secreted by helminth parasites. Until now, there was no evidence of the expression or secretion of these two families of proteins in Haemonchus contortus. In this study, we applied proteomic and bioinformatic tools in an iterative manner to reveal the expression and complexity of these proteins in the excretory/secretory products (ESP) of adult H. contortus at the protein and gene levels. Initial examination of the mass spectra of ESP fractions against standard databases returned nine peptides mapping to Ostertagia ostertagi NPA and FAR sequences. Searches of the H. contortus EST and genomic contig databases with the O. ostertagi and Caenorhabditis elegans homologues retrieved diverse sequences encoding H. contortus NPA and FAR proteins. H. contortus sequences were then integrated into a customized database and a new search of the mass spectra achieved a 10-fold improvement in coverage of the predicted H. contortus NPAs. The final analyses of the mass spectra achieved 49-60% coverage of H. contortus NPAs and 7-47% coverage of H. contortus FARs. Moreover, the diversity in structures of the encoding genes was revealed by assembling the genomic sequence data with predicted protein sequences confirmed by the peptide evidence. We predict there are at least one Hc-NPA gene and six Hc-FAR genes in H. contortus, and life stage gene expression of Hc-FAR-1 to -6 revealed unique transcription patterns for each of these genes.
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