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

Ultrastructural characteristics and small subunit ribosomal DNA sequence of Vairimorpha cheracis sp. nov., (Microspora: Burenellidae), a parasite of the Australian yabby, Cherax destructor (Decapoda: Parastacidae)

This is the first record of a species of Vairimorpha infecting a crustacean host. Vairimorpha cheracis sp. nov. was found in a highland population of the Australian freshwater crayfish, Cherax destructor. The majority of spores and earlier developmental stages of V. cheracis sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, each of which gave rise to four sporoblasts in a rosette-shaped plasmodium, so that eight uninucleate spores were produced within the persistent ovoid SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and averaged 3.4×1.9 µm in dimensions. The anterior polaroplast was lamellar in structure, and the posterior polaroplast vesicular. The polar filament was coiled 10-12 times, lateral to the posterior vacuole. The small subunit ribosomal DNA (SSU rDNA) of V. cheracis sp. nov. was sequenced and compared with other microsporidia. V. cheracis sp. nov. showed over 97% sequence identity with Vairimorpha imperfecta and five species of Nosema, and only 86% sequence identity with V. necatrix. The need for a taxonomic revision of the Nosema/Vairimorpha group of species is discussed

Inheritance of avermectin resistance in Haemonchus contortus

A larval development assay was used to compare the responses of the Chiswick Avermectin Resistant (CAVRS) isolate of Haemonchus contortus, an avermectin-susceptible isolate (VRSG) and their crosses to avermectins, The F-1 and F-2 generations of reciprocal crosses between CAVRS and VRSG were denoted as CAVRS males x VRSG females = CXV, and VRSG males x CAVRS females = VXC. The levels of avermectin resistance in the developing larvae of the F-1 of both CXV and VXC were indistinguishable from that in the avermectin-resistant parent, indicating that the resistance trait is completely dominant. Avermectin dose-response curves for the CXV F-1 did not show a 50% mortality rate at low concentrations, indicating that avermectin resistance is not sex-linked. This conclusion was confirmed when adult male worms of the F-1 of the CXV mating were found to have survived treatment of the host with 200 mu g kg(-1) ivermectin. This dose rate (200 mu g kg(-1) ivermectin) caused a 50% reduction in the number of adult males in the F-1 from both CXV and VXC crosses, but only a non-significant reduction in the number of adult females in the F-1. Dose-response curves obtained for the F-2 generations in the larval development assay indicated the presence of 25% of avermectin-susceptible individuals, suggesting that a single major gene largely controls the avermectin-resistance trait. This genetic analysis of avermectin resistance in an Australian H. contortus isolate indicates that the expression of the gene for avermectin resistance is an autosomal complete dominant in the larvae; however, in adults its expression is sex-influenced, with males having a lower resistance to avermectin than females. (C) 2000 Australian Society for Parasitology Inc, Published by Elsevier Science Ltd. All rights reserved

Susceptibility assay of Haemonchus contortus to commonly used anthelmintics in Jimma, southwest Ethiopia

An experimental study to investigate the susceptibility pattern of Haemonchus contortus to commonly used anthelmintics (albendazole, tetramisole, ivermectin, and triclobendazole) was conducted between June and September 2009. Adult H. contortus parasites were collected from a total of 30 sheep slaughtered at Jimma municipal abattoir. The anthelmintics were assessed for egg hatch inhibition ability against H. contortus eggs. The eggs (500 eggs/ml) were incubated with different concentrations (0.25, 0.125, 0.0625, 0.03125, and 0.015625 mu g/ml) at 26A degrees C for 48 h. Egg hatching inhibition of different anthelmintics at different concentrations was recorded. The overall mean percent inhibition showed that there was a significant difference (p < 0.05) among the different concentrations. Mean egg hatch inhibition of ivermectin showed the highest result (84.90 %) at 0.25 mu g/ml, followed by albendazole (78.77 %), triclobendazole (76.66 %), and tetramisole (78.98 %), respectively. The overall mean percent inhibition showed a significant difference (p < 0.05) among the different anthelmintics. Further evaluation of these anthelmintics was recommended