Regional heterogeneity and unexpectedly high abundance of Cooperia punctata in beef cattle at a northern latitude revealed by ITS-2 rDNA nemabiome metabarcoding.

BACKGROUND: The species composition of cattle gastrointestinal nematode (GIN) communities can vary greatly between regions. Despite this, there is remarkably little large-scale surveillance data for cattle GIN species which is due, at least in part, to a lack of scalable diagnostic tools. This lack of regional GIN species-level data represents a major knowledge gap for evidence-based parasite management and assessing the status and impact of factors such as climate change and anthelmintic drug resistance. METHODS: This paper presents a large-scale survey of GIN in beef herds across western Canada using ITS-2 rDNA nemabiome metabarcoding. Individual fecal samples were collected from 6 to 20 randomly selected heifers (n = 1665) from each of 85 herds between September 2016 and February 2017 and 10-25 first season calves (n = 824) from each of 42 herds between November 2016 and February 2017. RESULTS: Gastrointestinal nematode communities in heifers and calves were similar in Alberta and Saskatchewan, with Ostertagia ostertagi and Cooperia oncophora being the predominant GIN species in all herds consistent with previous studies. However, in Manitoba, Cooperia punctata was the predominant species overall and the most abundant GIN species in calves from 4/8 beef herds. CONCLUSIONS: This study revealed a marked regional heterogeneity of GIN species in grazing beef herds in western Canada. The predominance of C. punctata in Manitoba is unexpected, as although this parasite is often the predominant cattle GIN species in more southerly latitudes, it is generally only a minor component of cattle GIN communities in northern temperate regions. We hypothesize that the unexpected predominance of C. punctata at such a northerly latitude represents a range expansion, likely associated with changes in climate, anthelmintic use, management, and/or animal movement. Whatever the cause, these results are of practical concern since C. punctata is more pathogenic than C. oncophora, the Cooperia species that typically predominates in cooler temperate regions. Finally, this study illustrates the value of ITS-2 rDNA nemabiome metabarcoding as a surveillance tool for ruminant GIN parasites.

Validation of ITS-2 rDNA nemabiome sequencing for ovine gastrointestinal nematodes and its application to a large scale survey of UK sheep farms.

We have validated ITS-2 rDNA nemabiome next-generation amplicon sequencing to determine relative species abundance of gastrointestinal nematode species in ovine fecal samples. In order to determine species representation biases, ITS-2 rDNA amplicon sequencing was applied to mock communities or field populations with known proportions of L3 for eight of the major ovine gastrointestinal nematode species: Teladorsagia circumcincta, Trichostrongylus vitrinus, Haemonchus contortus, Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, Chabertia ovina and Oesophagostumum venulosum. Correction factors, calculated from this data, were shown to reduce species representation biases when applied to an independent set of field samples of known composition. We compared ITS-2 rDNA amplicon sequencing data that was generated from harvested eggs, freshly hatched L1 or L3 larvae following fecal culture and no statistically significant differences were found for the more abundant parasite species. We then applied the validated ITS-2 rDNA nemabiome amplicon sequencing assay to a set of archived L1 gastrointestinal nematode populations, collected in 2008 from fecal samples from 93 groups of 20 ewes and 61 groups of 20 lambs derived from 99 UK sheep farms. The presence of the major gastrointestinal nematode species had previously been determined on this large sample set by species-specific PCR. We show how the ITS-2rDNA amplicon sequencing data provided much more detailed information on species abundance than the previous species-specific PCR. This new data represents the most comprehensive overview of the relative abundance of the major gastrointestinal nematode species across UK sheep farms to date. Substantial variance in the relative abundance of both T. circumcincta and T. vitrinus between farms was revealed with the former species being of statistically significantly higher abundance in all three regions sampled (England, Scotland and Wales). The data also revealed that the relative abundance of T. circumcinta was statistically significantly higher in ewes than in lambs with the opposite pattern being the case for T. vitrinus. The nemabiome sequencing data also clearly illustrated the sporadic nature and skewed distribution of H. controtus across UK sheep farms as well as a higher relative abundance on farms from England compared to Wales and Scotland. The nemabiome survey also provides the first widescale data on the relative abundance of the two major large intestinal nematodes C. ovina and O. venulosum. This work validates ITS-2 rDNA nemabiome sequencing for use in sheep and illustrates the power of the approach for large scale surveillance of ovine gastrointestinal nematodes.

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.

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

Parasitic nematode ß-tubulin genes are of particular interest because they are the targets of benzimidazole drugs. However, in spite of this, the full ß-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 ß-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 ß-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 ß-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 ß-tubulin genes in mechanisms of benzimidazole resistance.

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.

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.

Microsatellite genotyping supports the hypothesis that Teladorsagia davtiani and Teladorsagia trifurcata are morphotypes of Teladorsagia circumcincta.

Traditionally nematode species designations have been based on morphological criteria. However, it is has long been recognised that valid species designations are critical for basic biological and epidemiological studies. The ever increasing use of molecular and genetic techniques has allowed traditional classifications to be more closely examined. The sub-family Ostertagiinae is of particular interest as many of the species within this group are of economic importance worldwide, with unresolved classification complicating epidemiology, management, control and genetic studies. This study examines genetic differences between three morphological variants (morphotypes) within the genus Teladorsagia (sub-family: Ostertagiinae) using a multi-locus population genetic analysis approach. Five microsatellites were used to genotype a total of 31 T. davtiani (ScKiTD), 30 T. trifurcata (ScKiTT), and 31 T. circumcincta (ScKiTC). Population genetic analysis detected no genetic differentiation between T. davtiani, T. trifurcata, and T. circumcincta supporting the hypothesis that these are morphotypes of the same species.

Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting.

Many of the Haemonchus contortus isolates currently used for experimental work were originally derived from different regions of the world and are commonly exchanged between laboratories. In most cases, these are largely genetically uncharacterised other than the analyses conducted on specific genes of interest. We have used a panel of eight microsatellite markers to genetically characterise five different commonly used H. contortus isolates including MHco3 (ISE), the isolate chosen for full genome sequencing as part of the H. contortus genome project. There is an extremely high level of genetic differentiation between each of the isolates except the two which have a common origin, MHco1 (MOSI) and MHco3 (ISE). We have investigated the amplification of microsatellite markers from pooled DNA as a potential method for fingerprinting different isolates. Good estimates of the true allele frequencies can be made by amplification from either pooled adult DNA or bulk L3 DNA for seven out of the eight markers tested. Both single worm genotyping and bulk DNA fingerprinting revealed no genetic differentiation between adult worms in the host and larvae derived from faecal culture. Furthermore, none of the eight markers showed genetic changes when isolates were passaged through different individual hosts. Hence the microsatellite genotyping of bulk larval DNA samples provides a simple and rapid method to genetically define and monitor laboratory isolates, and to determine their relationship with particular field populations.

Population genetic analysis of the ovine parasitic nematode Teladorsagia circumcincta and evidence for a cryptic species.

An understanding of genetic variation in parasite populations, and how it is partitioned, is required to underpin many areas of basic and applied research. Population genetic studies on parasitic nematode populations are still in their infancy and have been dominated by the use of single locus markers. We have used a panel of five microsatellite markers to undertake a genetic study of a number of field and laboratory populations of Teladorsagia circumcincta. High levels of polymorphism were seen in all the populations examined with the majority of diversity being within rather than between populations. There was no detectable genetic differentiation between the UK populations examined although they included both laboratory passaged and field isolates derived from different geographical regions and host species. This broadly supports previous mtDNA sequence diversity studies of this parasite in the UK and USA. However, some between-population genetic differentiation was apparent when several populations from French goats and a laboratory population from New Zealand were examined. Most notably, a population from a French goat farm, which has previously been suggested to contain a cryptic species, showed very high levels of genetic differentiation from all the other populations. Analysis of multi-locus genotypes suggested the presence of two sympatric parasite populations on this farm with little or no gene flow between them. This supports the hypothesis that parasites currently defined as T. circumcincta by routine morphological criteria comprise more than a single species.

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.

Characterisation of Teladorsagia circumcincta microsatellites and their development as population genetic markers.

There is a need to develop tools to study the genetics of parasitic nematodes. This is particularly urgent for those species in which anthelmintic resistance is common such as the sheep parasite Teladorsagia (Ostertagia) circumcincta. The lack of information on the population genetics of such parasites severely limits our ability to study the genetic basis of anthelmintic resistance. This paper presents the results of three approaches used to isolate microsatellite markers from T. circumcincta and the development of a panel of markers suitable for population genetic analysis. Hybridisation screening of small insert genomic libraries and interspecies PCR amplification of Haemonchus contortus microsatellites were used to identify CA/GT microsatellites. Many of these loci were associated with a 146bp tandem repeat, named TecRep, that is related to a repetitive element previously identified in other trichostrongylid nematode genomes but apparently absent from other nematode groups. A large proportion of the loci isolated were problematic for use as population genetic markers, predominantly due to a high frequency of null alleles or the association with the TecRep repeat. Bioinformatic screening of a T. circumcincta EST database identified both di- and tri-nucleotide microsatellite repeats and a greater proportion of these turned out to be more robust markers than those derived from genomic sequence. A panel of seven markers has been selected and characterised which are sufficiently robust and polymorphic to be valuable population genetic markers for this parasite.