Elizabeth Redman

The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery

BackgroundThe small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.ResultsHere we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates.ConclusionsThe H. contortus genome and transcriptome provide an essential platform for postgenomic research in this and other important strongylid parasites.

Caenorhabditis elegans is a useful model for anthelmintic discovery

Parasitic nematodes infect one quarter of the world’s population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery. Screening for new anthelmintic compounds that are active against parasitic nematodes is costly and labour intensive. Here, the authors use the non-parasitic nematode Caenorhabditis elegansto identify 30 anthelmintic lead compounds in an effective and cost-efficient manner.

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.

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.

Density-related variation in vertical transmission of a virus in the African armyworm

Larvae of the African armyworm, Spodoptera exempta, are darker and more resistant to baculovirus infection when reared in groups (gregarious form) compared to being reared singly (solitary form). Lepidoptera that survive virus challenge as larvae could potentially retain a sublethal virus infection which is then transmitted vertically to the next generation. Here we examine whether gregarious and solitary forms of the armyworm differ in the costs of surviving virus infection and in their capacity to transmit an active baculovirus infection to their offspring. Pupae of larvae reared gregariously that survived virus challenge weighed significantly less than uninfected individuals, but this was not so for those reared solitarily. This did not, however, translate into differences in fecundity, at least under laboratory conditions. As found in previous studies, pre-oviposition period was shorter for solitary than gregarious insects, and it was also shorter for females that had been challenged with virus as larvae. Both the prevalence of egg batches containing larvae that died from nucleopolyhedrovirus (NPV) infection and the proportion of infected larvae within each egg batch were significantly increased (approximately doubled) when parental moths were previously challenged with the virus during their larval state. This demonstrates that horizontal transmission in one generation can elevate vertical transmission to the next generation. Moreover, prevalence of overt infection in the offspring generation was two to three times greater when parental moths were reared solitarily as larvae than when reared gregariously. Disease prevalence and proportional infection were both independent of the sex of the infected parent and whether or not the egg batch was surface-sterilized to remove potential contaminants. This suggests that the eggs are infected internally (transovarial) rather than externally (transovum). These results help to shed light on the observed temporal pattern of virus epizootics in eastern Africa.

Genetic evidence for hybridisation between Haemonchus contortus and Haemonchus placei in natural field populations and its implications for interspecies transmission of anthelmintic resistance.

Genetic hybridisation between parasitic nematode species has potentially important consequences. It could lead to the introgression of genes between species including those involved in pathogenicity, host specificity, transmission and drug resistance. It could also complicate diagnosis and control. However, there are few compelling examples of its occurrence in parasites in the field. Haemonchus contortus and Haemonchus placei are two closely related parasitic nematode species that predominantly infect small ruminants and cattle, respectively. They are capable of experimental hybridisation when adult worms of each species are transplanted into the same individual host. Given that co-infection occurs in both small ruminants and cattle, there is potential for hybridisation in the field. However, this has not been definitively demonstrated and its extent is unknown. We investigated the occurrence of co-infection and interspecies hybridisation in H. contortus and H. placei in field populations from small ruminants from Pakistan and southern India using a number of independent genetic markers. Haemonchus contortus and H. placei co-infections were common in Pakistan but not in southern India where H. placei appeared to be absent in small ruminant hosts. In the former region, a number of worms were identified that were heterozygous for fixed, species-specific rDNA internal transcribed spacer 2 (ITS-2) single nucleotide polymorphisms. Genotyping of these ITS-2 heterozygotes with an additional four nuclear markers conclusively demonstrated them to be F1 interspecies hybrids. Mitochondrial NADH dehydrogenase subunit 4 haplotype analysis demonstrated that four of the hybrid worms had a H. placei maternal parent and one had a H. contortus maternal parent showing that hybridisation could occur in either direction. Interestingly, one of these hybrids contained an H. contortus isotype-1 β-tubulin benzimidazole resistance allele, suggesting there is a potential for interspecies introgression of drug resistance loci. We believe this is the first definitive genetic evidence of hybridisation between H. contortus and H. placei in the field and represents the most comprehensive genetic evidence of F1 hybrids between any human or livestock parasitic nematode species to date. Further, it suggests that interspecies transmission of anthelmintic resistance mutations warrants further investigation.

Genetic evidence for the spread of a benzimidazole resistance mutation across southern India from a single origin in the parasitic nematode Haemonchus contortus

It is important to understand how anthelmintic drug resistance mutations arise and spread in order to determine appropriate mitigation strategies. We hypothesised that a molecular genetic study of Haemonchus contortus in southern India, a region where resistance may be less advanced than in western Europe and North America, might provide some important insights into the origin and spread of anthelmintic resistance. The F200Y (TAC) isotype-1 β-tubulin benzimidazole resistance mutation is common in H. contortus throughout the world and the F167Y (TAC) and E198A (GCA) mutations, although less common, have been reported in a number of different countries. We have investigated the haplotypic diversity and phylogenetic relationship of isotype-1 β-tubulin benzimidazole resistance alleles for 23 H. contortus populations from small ruminants across southern India. The F200Y (TAC) mutation was most common, being detected in 18/23 populations at frequencies between 9% and 84% and the E198A (GCA) mutation was also detected in 8/23 populations at frequencies between 8% and 18%. The F167Y (TAC) mutation was not detected in any of the 23 populations. Phylogenetic haplotype network analysis suggested that the F200Y (TAC) mutation has arisen multiple independent times in the region with at least three independent origins of resistance alleles across the populations surveyed. In contrast, the E198A (GCA) mutation was present on a single haplotype which, given the high level of haplotypic diversity of the susceptible alleles in the region, suggests this particular mutation has spread from a single origin, likely by anthropogenic animal movement. Population genetic analysis of 12 of the H. contortus populations, using a panel of eight microsatellite markers, revealed extremely low genetic differentiation between populations, consistent with the hypothesis of high gene flow among sites. Additionally, there was no significant genetic differentiation between H. contortus taken from sheep and goats which is consistent with H. contortus populations being freely shared between these two different hosts. Overall, we believe these results provide the first clear genetic evidence for the spread of an anthelmintic resistance mutation to multiple different locations from a single origin.

High levels of genetic diversity in Spodoptera exempta NPV from Tanzania.

The African armyworm, Spodoptera exempta, is a major pest in sub-Saharan Africa. A nucleopolyhedrovirus (NPV) is often recorded in later population outbreaks and can cause very high levels of mortality. Research has been addressing whether this NPV can be developed into a strategic biological control agent. As part of this study, the variation in natural populations of NPV is being studied. An isolate of S. exempta NPV was cloned in vivo and found to contain at least 17 genetically-distinct genotypes. These genotypes varied in size from approximately 115 to 153 kb.

Exploring the Gastrointestinal "Nemabiome": Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities.

Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions during co-infection, parasite epidemiology, parasite ecology and the response of parasite populations to both drug treatments and control programs.