Leo F. Le Jambre

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.

Resistance to antiparasitic drugs: the role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.

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.

Molecular variation in trichostrongylid nematodes from sheep and cattle

The application of molecular techniques such as restriction length polymorphisms, mitochondrial DNA analysis and sequence tagged microsatellite sites to determine the amount of genetic variation and population structure of trichostrongylid parasites of domestic ruminants is examined. Information already provided by these techniques indicates that the parasite populations have a large amount of genetic variation and that gene flow between populations is greater than previously expected. The implications are that parasites can rapidly respond to selection by either anthelmintics or genetically resistant hosts. Advance warning of the response to selection can be obtained, even when the trait under selection is unknown, by measuring changes in the DNA polymorphisms of the parasite populations. Resistance to the benzimidazoles in the trichostrongylids now appears to be dependent on changes at two beta-tubulin loci. The first stage in the development of benzimidazole (BZ) resistance involves selection for an allele encoding a resistant form of isotype 1 beta-tubulin. A higher level of resistance is achieved with the selection of the gene encoding isotype 2 beta-tubulin. Techniques that maintain BZs in the host for extended periods of time may select for the higher level of resistance. A DNA assay to test this hypothesis is proposed.

Thelohania montirivulorum sp. nov. (Microspora: Thelohaniidae), a parasite of the Australian freshwater crayfish, Cherax destructor (Decapoda: Parastacidae): fine ultrastructure, molecular characteristics and phylogenetic relationships

Thelohania montirivulorum sp. nov., a new species of microsporidian parasite, was found in a highland population of the Australian yabby, Cherax destructor. Data are presented on fine ultrastructure, developmental morphology and DNA sequence of the small subunit ribosomal DNA (SSU rDNA) and internal transcribed spacer region. The phylogenetic relationships of T. montrivulorum sp. nov. and other crayfish parasites in the genus Thelohania, based on the SSU rDNA sequence, are investigated. Fine ultrastructure, patterns of sporogony and SSU rDNA sequence similarities indicate T. montirivulorum sp. nov. is congeneric with T. parastaci, a parasite of lowland populations of C. destructor, and with T. contejeani, a parasite of European freshwater crayfish. SSU rDNA data suggests Thelohania species found in crustacean hosts are more closely related to the Vairimorpha/Nosema clade of species from insect and crustacean hosts than to the fire ant parasites, T. solenopsae and Thelohania sp.

Thelohania parastaci sp. nov. (Microspora: Thelohaniidae), a parasite of the Australian freshwater crayfish, Cherax destructor (Decapoda: Parastacidae).

Thelohania parastaci sp. nov. infects the Australian freshwater crayfish, Cherax destructor. Data on morphology, developmental patterns and sequences from the small subunit (SSU) and internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) of T. parastaci sp. nov. are described. The ultrastructural features of different life cycle stages are very similar to those of the European crayfish parasite Thelohania contejeani. T. parastaci sp. nov. exhibits simultaneous dimorphic sporogony in muscle tissue. Meronts, sporonts and spores are found in muscle tissue, within haemocytes in the hepatopancreas, and in the intestinal wall of infected crayfish. T. parastaci sp. nov. shows 92% sequence identity with T. contejeani and only 67% sequence identity with the fire ant pathogen T. solenopsae, when SSU rDNA sequences are compared. Analysis of SSU rDNA and ITS sequences of T. parastaci sp. nov. from crayfish from Victoria, Western Australia, and New South Wales indicate that the parasite has a wide geographical distribution in Australia.

Workshop on irradiated larval vaccines.

The development of anthelmintic resistance in gastrointestinal parasites of sheep and goats has meant that there is an urgent need for non-chemical control of these parasites. In order to investigate a potential alternative method of control, a workshop on irradiated larval vaccines (ILV) was held in conjunction with the Second International Conference on Novel Approaches to the Control of Helminth Parasites of Livestock. The objectives of the workshop were to: (1) Review knowledge of irradiated helminth vaccines through the presentation of invited and contributed papers. (2) Identify opportunities for an ILV and in which host/parasite/production systems. (3) Determine what are the gaps in knowledge required to produce and apply an ILV. The workshop concluded that ILVs could be useful as research tools in the further definition of immune responses to parasites and in stimulating the immune response in young sheep and goats to facilitate the estimating of heritability of faecal egg counts. Although the workshop identified some problems associated with a live attenuated vaccine, an ILV could be a useful control measure in the production systems identified by the workshop. Before an ILV could be used on farms, additional research is required into the period of protection afforded by an ILV especially under field conditions. The efficacy of an ILV could be improved by interaction between host genotype and nutritional status and is deserving of further investigation.