An Immunochemical Study of D. viviparus Infective Larvae

The bovine lungworm Dictyocaulus viviparus induces a highly effective immune response in infected cattle and a vaccine, consisting of radiation attenuated infective larvae, has been successfully used for over thirty years. In spite of this notable success, there is little understanding of natural or vaccine-induced immunity to this parasite. Since the infective larva is a potential source of important antigens and can be obtained in relatively large quantities, an immunochemical study of this stage formed the basis of the work presented in this thesis. An investigation of the mouse as a potential immunological model of D.vivipants infection revealed that larvae migrate to the lungs but are expelled without undergoing significant development. Although mice were capable of mounting an immune response to invading larvae, the results suggested the mouse was of limited value as an immunological model for this parasite. No polypeptides were detected by surface biotinylation of exsheathed L3 but several molecules were revealed by labelling sheathed L3. The generation of monoclonal antibodies and lectin binding studies on the L3 cuticular surface demonstrated the presence of phosphorylcholine and carbohydrate epitopes respectively. Lectin binding studies suggested that carbohydrate was not exposed on the external surface of the L3 sheath but was present on the internal surface. The generation of monoclonal antibodies revealed a 29-40kDa antigen on the externzil surface of the L3 sheath which appeared to be highly immunogenic and responsible for the marked antibody response produced to this surface by immunised cattle. These monoclonal antibodies also bound to the surface of the L3 sheath of numerous other nematodes from the order Strongylida, although the molecular weight of the detected antigen varied between some of the species. The antigen was located on a surface coat overlying the sheath epicuticle and was also found to be present in the somatic tissues of the L3. The stage specificity and immunochemical properties of this antigen were examined. In vitro culture of L3 revealed partial development to the L4 with the production of several antigens which were detected by immune bovine serum. An L3 cDNA expression library was produced but screening with immune bovine serum or the monoclonal antibodies failed to detect any positive recombinant clones

The emergence of resistance to the benzimidazole anthlemintics in parasitic nematodes of livestock is characterised by multiple independent hard and soft selective sweeps

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the “genetic signature” of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs

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

Understanding anthelmintic resistance: The need for genomics and genetics

Anthelmintic resistance is a major problem for the control of many parasitic nematode species and has become a major constraint to livestock production in many parts of the world. In spite of its increasing importance, there is still a poor understanding of the molecular and genetic basis of resistance. It is unclear which mutations contribute most to the resistance phenotype and how resistance alleles arise, are selected and spread in parasite populations. The main strategy used to identify mutations responsible for anthelmintic resistance has been to undertake experimental studies on candidate genes. These genes have been chosen predominantly on the basis of our knowledge of drug mode-of-action and the identification of mutations that can confer resistance in model organisms. The application of these approaches to the analysis of benzimidazole and ivermectin resistance is reviewed and the reasons for their relative success or failure are discussed. The inherent limitation of candidate gene studies is that they rely on very specific and narrow assumptions about the likely identity of resistance-associated genes. In contrast, forward genetic and functional genomic approaches do not make such assumptions, as illustrated by the successful application of these techniques in the study of insecticide resistance. Although there is an urgent need to apply these powerful approaches to anthelmintic resistance research, the basic methodologies and resources are still lacking. However, these are now being developed for the trichostrongylid nematode Haemonchus contortus and the current progress and research priorities in this area are discussed

Genetics of Mating and Sex Determination in the Parasitic Nematode Haemonchus contortus

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes

Characterization and comparative analysis of the complete Haemonchus contortus β-tubulin gene family and implications for benzimidazole resistance in strongylid nematodes

Parasitic nematode β-tubulin genes are of particular interest because they are the targets of benzimidazole drugs. However, in spite of this, the full β-tubulin gene family has not been characterized for any parasitic nematode to date. Haemonchus contortus is the parasite species for which we understand benzimidazole resistance the best and its close phylogenetic relationship with Caenorhabditis elegans potentially allows inferences of gene function by comparative analysis. Consequently, we have characterized the full β-tubulin gene family in H. contortus. Further to the previously identified Hco-tbb-iso-1 and Hco-tbb-iso-2 genes, we have characterized two additional family members designated Hco-tbb-iso-3 and Hco-tbb-iso-4. We show that Hco-tbb-iso-1 is not a one-to-one orthologue with Cel-ben-1, the only β-tubulin gene in C. elegans that is a benzimidazole drug target. Instead, both Hco-tbb-iso-1 and Hco-tbb-iso-2 have a complex evolutionary relationship with three C. elegans β-tubulin genes: Cel-ben-1, Cel-tbb-1 and Cel-tbb-2. Furthermore, we show that both Hco-tbb-iso-1 and Hco-tbb-iso-2 are highly expressed in adult worms; in contrast, Hco-tbb-iso-3 and Hco-tbb-iso-4 are expressed only at very low levels and are orthologous to the Cel-mec-7 and Cel-tbb-4 genes, respectively, suggesting that they have specialized functional roles. Indeed, we have found that the expression pattern of Hco-tbb-iso-3 in H. contortus is identical to that of Cel-mec-7 in C. elegans, being expressed in just six “touch receptor” mechano-sensory neurons. These results suggest that further investigation is warranted into the potential involvement of strongylid isotype-2 β-tubulin genes in mechanisms of benzimidazole resistance

Prevalence of parasitic nematode species identified on the seven study farms and parasite population structure.

<p>(A) The relative frequencies of the eight major ovine gastro-intestinal nematode species based on species-specific PCR assays on 90 individual L₁ larvae on the seven study farms. (B) Pairwise F_ST values based on genotyping with ten microsatellite loci for <i>H</i>. <i>contortus</i> and <i>T</i>. <i>circumcincta</i> respectively. F_ST values estimated before correction for null alleles below the diagonal and after correction above. Values underlined and in bold show significant differences based on bootstrapping (10, 000 replicates) using the software program FreeNA. (C) SplitsTrees4 Networks based on populations of thirty worms on each farm genotyped using ten microsatellite markers for each species.</p

Network analysis of sequences of isotype-1 β-tubulin sequences.

<p>Split networks were generated with the neighbour-net method of SplitsTrees4 from (A) <i>H</i>. <i>contortus</i> sequences and (B) <i>T</i>. <i>circumcincta</i> sequences. The circles in Split networks represent the different haplotype and the size of the circles is proportional to the frequency in the population. The colours define the resistant haplotypes across positions P167, P198 and P200 (P167F:P198E:P200F, Susceptible = blue; P167F:P198E:P200Y, Resistant at P200 = Red; P167Y:P198E:P200F, Resistant at P167 = green; P167F:P198L:P200F, Resistant at P198 = orange).</p

Allelic diversity of microsatelllite loci and β-tubulin from <i>H</i>. <i>contortus</i> and <i>T</i>. <i>circumcincta</i> from each of the seven study farms and neutrality test statistics.

<p>H_E, Expected heterozygosity; A, mean number of alleles per locus; h_Sus, total number of susceptible haplotypes; h_Res, total number of resistant haplotypes;</p><p>h_Tot, total number of haplotypes; H_d, Gene diversity; S, number of segregating sites; k, mean number of pairwise differences; π, nucleotide diversity;</p><p>θ_S, Mutation parameter based on infinite site equilibrium model and the number of segregating sites; <i>D</i>, Tajima’s <i>D</i> test statistic; <i>H</i>, Fay and Wu’s <i>H</i> test statistic. Statistical significant departure from neutrality determined with the use of simulations of the coalescent at p<0.05 *, p<0.01 **, and p<0.005***.</p><p>Allelic diversity of microsatelllite loci and β-tubulin from <i>H</i>. <i>contortus</i> and <i>T</i>. <i>circumcincta</i> from each of the seven study farms and neutrality test statistics.</p

Relative frequency of individual resistant haplotypes in seven UK sheep parasite populations.

<p>A total number of five resistant haplotypes for (A) <i>H</i>. <i>contortus</i> and twenty eight resistant haplotypes for (B) <i>T</i>. <i>circumcincta</i> were identified. Susceptible haplotypes are not included this figure but are show in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003494#pntd.0003494.s003" target="_blank">S3 Fig</a>.</p