Ian J. Lenane

Characterisation of an avermectin resistant strain of Australian Haemonchus contortus

A strain of Haemonchus contortus (CAVR) isolated in Australia was found to be resistant to ivermectin (IVM) with 0.4 mg kg-1 of the anthelmintic failing to significantly reduce worm burdens. Resistance to IVM was sex-influenced in the CAVR strain with adult males showing a greater sensitivity to IVM. Cross resistance to moxidectin was evident with approximately 15% of the population surviving a dose of 0.1 mg kg-1. The free-living stages of the CAVR isolate had a reduced sensitivity to avermectin (AVM) inhibition of development and motility. Similar structure-activity patterns and resistance factors were obtained for a series of related AVMs as inhibitors of larval development and L3 motility in CAVR and White River II, an IVM-resistant H. contortus isolate from South Africa. Further, both isolates were found to be 3 times more sensitive to paraherquamide than a susceptible H. contortus isolate. This suggest that the same resistance mechanism is operating in both isolates. The CAVR strain is susceptible to the benzimidazoles, levamisole and closantel.

Selection for anthelmintic resistance by macrocyclic lactones in Haemonchus contortus

Two morphologically marked strains of Haemonchus contortus, CAVRS (smooth-macrocyclic lactone resistant) and McMaster (linguiform-macrocyclic lactone susceptible), were used to investigate the selection for anthelmintic resistance following exposure to ivermectin (IVM), a non-persistent anthelmintic. and a more persistent anthelmintic, oral moxidectin (MOX). Three types of selection were investigated: (1) selection of resident worms at the time of treatment (Head selection); (2) selection of incoming-larvae post-treatment (Tail selection); and (3) selection of both resident population and incoming larvae (Head + Tail selection). The experimental animals were adult sheep and lambs. In the controls where there was no anthelmintic selection, the proportion of CAVRS in the adult worm population was the same as the proportion in larvae given to both adults and lambs indicating that CAVRS and McMaster H. contortus were equally infective. There was a significant effect of anthelmintic on total worm numbers in adult sheep with MOX treated adults having less worms, but selection type was non-significant. Anthelmintic type had a significant effect on numbers of resistant worms in adult sheep with less resistant worms in the MOX treated groups, but selection type had no effect. Analysis of variance of arcsine-transformed proportions of resistant worms found that the type of anthelmintic had a highly significant effect, with MOX treated adults having a higher proportion of resistant worms, while type of selection was not significant. In the lambs, nil treated controls and IVM Head + Tail and Tail selected groups had similar geometric mean total worm burdens while Head selected had less total worms. In the MOX treated lamb groups the worm burdens were similar within selection type but less than the IVM treated groups. In the lambs, the types of selection that resulted in more resistant worms were IVM Tail, MOX Head + Tail and MOX Tail. Resistant worm numbers were similar in both adult and lamb groups with Head selection by either MOX or IVM. Moxidectin selected out higher proportions of resistant worms than did IVM in the lambs, with Tail and Head + Tail being stronger selectors than Head. Computer simulations were used to estimate the rate at which resistance developed in the field using the information generated in the present study. The anthelmintic treatments used in the simulation followed a strategic parasite control program for H. contortus in which all sheep receive three Closantel (CLS) treatments in summer. all sheep receive a broad-spectrum (BS) drench or capsule at weaning and lambs receive an additional two BS drenches insummer or no further treatment in the case of the capsule. Moxidectin, IVM-capsule and IVM were the broad spectrum anthelmintics simulated. All simulations were run four times assuming high or low efficacy against resident resistant worms and in the presence or absence of CLS resistance. The simulations indicated that the presence of CLS resistance hastened selection for macrocyclic lactone (ML) resistance. While the IVM-capsule will select most rapidly for ML resistance, IVM oral is expected to be least selective. Moxidectin treatment is intermediate, except in simulations with no CLS resistance and when MOX is assumed to be highly effective against resident ML-resistant worms, in which case MOX can be expected to select more slowly than IVM oral treatments.

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 malesxVRSG females=CXV, and VRSG malesxCAVRS 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 200microgkg(-1) ivermectin. This dose rate (200microgkg(-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.

A hybridisation technique to identify anthelmintic resistance genes in Haemonchus.

The identification of genes associated with anthelmintic resistance can be facilitated in Haemonchus contortus by the ability of this species to hybridise with Haemonchus placei. Although the hybrid males are sterile, the lines can be rescued by backcrossing the females to either parental species. Resistance genes can be retained in Haemonchus hybrids, while the unwanted contortus background is removed through backcrossing to H. placei and anthelmintic selection of the progeny. Under this selection, genes involved in resistance would retain the H. contortus nucleotide sequence, while those that are not would either be H. placei or a random mixture of both, depending on the amount of backcrossing that had occurred. The first candidate gene to be tested in this system was a Haemonchus P-glycoprotein, hcpgp-1. hcpgp-1 was amplified, cloned and sequenced from H. contortus and H. placei. Two restriction sites were then identified in the sequenced product; one specific to H. contortus hcpgp-1 and the other found only in the H. placei gene. These genes were identified from macrocyclic lactone selected and non-selected worms by restricting PCR products from individual worms. Fitted occurrence of the H. contortus allele was 49% of unselected worms and 69% of macrocyclic lactone selected worms. The probability of this percentage occurring by chance was P = 0.006. Thus macrocyclic lactone selection was acting to increase the percentage of hcpgp-1 from macrocyclic-lactone-resistant CAVRS.

Selection of different genotype larvae and adult worms for anthelmintic resistance by persistent and short-acting avermectin/milbemycins.

To understand the factors that influence selection for anthelmintic resistance, it is necessary to examine the impact of drug treatment, particularly persistent drugs, on all phases of the worm life cycle. The efficacy of various avermectin/milbemycin anthelmintics was determined against resident worms, incoming larvae (L3) and development of eggs in faecal culture. Homozygote-resistant and maternal and paternal F1-heterozygote genotypes of Haemonchus contortus were used to infect sheep before or after treatment with ivermectin (IVM) oral, IVM capsule, moxidectin (MOX) oral or MOX injectable. Total worm count and quantitative larval culture were used to determine efficacy against parasitic and free-living stages, respectively. Selection for resistance by IVM capsules occurred at the adult and L3 stages because of poor efficacy against these stages for all resistant genotypes. However, the selective advantage of these surviving worms was reduced due to the low development of their eggs to L3 in faecal culture. For MOX, selection for resistance predominantly occurred after treatment because of high efficacy against resident adult worms of all resistant genotypes but poor efficacy against resistant L3 ingested after drug administration. The results indicated no evidence of sex-linked inheritance for IVM resistance. Mean IVM efficacies against homozygous and heterozygous resistant adult worms were not different, and IVM capsule efficacy against incoming L3 was approximately 70% for all resistant genotypes, consistent with a dominant trait. MOX was highly effective against adults of all resistant genotypes and approximately 76% effective against incoming L3 regardless of resistance genotype, also consistent with a dominant trait. These results will enable the impact of persistent drugs on worm control and anthelmintic resistance to be estimated. The results indicate that IVM capsules should not be used in populations where avermectin/milbemycin resistance is present.