John S. Gilleard

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.

RNA interference in parasitic helminths : current situation, potential pitfalls and future prospects

RNA interference (RNAi) has become an invaluable tool for the functional analysis of genes in a wide variety of organisms including the free-living nematode Caenorhabditis elegans. Recently, attempts have been made to apply this technology to parasitic helminths of animals and plants with variable success. Gene knockdown has been reported for Schistosoma mansoni by soaking or electroporating different life-stages in dsRNA. Similar approaches have been tested on parasitic nematodes which clearly showed that, under certain conditions, it was possible to interfere with gene expression. However, despite these successes, the current utility of this technology in parasite research is questionable. First, problems have arisen with the specificity of RNAi. Treatment of the parasites with dsRNA resulted, in many cases, in non-specific effects. Second, the current RNAi methods have a limited efficiency and effects are sometimes difficult to reproduce. This was especially the case in strongylid parasites where only a small number of genes were susceptible to RNAi-mediated gene knockdown. The future application of RNAi in parasite functional genomics will greatly depend on how we can overcome these difficulties. Optimization of the dsRNA delivery methods and in vitro culture conditions will be the major challenges.

Acaricide resistance in cattle ticks and approaches to its management: The state of play

Cattle ticks are an important constraint on livestock production, particularly in tropical and subtropical areas. Use of synthetic acaricides is the primary method of tick control; therefore, it would be imperative to develop strategies to preserve the efficacy of existing acaricides. This paper summarizes the status of acaricide resistance in cattle ticks from different parts of the world and reviews modes of action of currently used acaricides, mechanism of resistance development, contributory factors for the development and spread of resistance, management of resistant strains and strategies to prolong the effect of the available acaricides. Use of vaccines, synthetic and botanical acaricides and educating farmers about recommended tick control practices are discussed, along with the integration of currently available options for the management of drug resistance and, ultimately, the control of cattle ticks.

Activation of hypodermal differentiation in the Caenorhabditis elegans embryo by GATA transcription factors ELT-1 and ELT-3.

ABSTRACT The Caenorhabditis elegans GATA transcription factor genes elt-1 and elt-3 are expressed in the embryonic hypodermis (also called the epidermis). elt-1 is expressed in precursor cells and is essential for the production of most hypodermal cells (22). elt-3 is expressed in all of the major hypodermal cells except the lateral seam cells, and expression is initiated immediately after the terminal division of precursor lineages (13). Although this expression pattern suggests a role for ELT-3 in hypodermal development, no functional studies have yet been performed. In the present paper, we show that either elt-3 or elt-1 is sufficient, when force expressed in early embryonic blastomeres, to activate a program of hypodermal differentiation even in blastomeres that are not hypodermal precursors in wild-type embryos. We have deleted the elt-3gene and shown that ELT-3 is not essential for either hypodermal cell differentiation or the viability of the organism. We showed that ELT-3 can activate hypodermal gene expression in the absence of ELT-1 and that, conversely, ELT-1 can activate hypodermal gene expression in the absence of ELT-3. Overall, the combined results of the mutant phenotypes, initial expression times, and our forced-expression experiments suggest that ELT-3 acts downstream of ELT-1 in a redundant pathway controlling hypodermal cell differentiation.

Population genetics of anthelmintic resistance in parasitic nematodes

A key aim of anthelmintic resistance research is to identify molecular markers that could form the basis of sensitive and accurate diagnostic tests. These would provide powerful tools to study the origin and spread of anthelmintic resistance in the field and to monitor strategies aimed at preventing and managing resistance. Molecular markers could also form the basis of routine diagnostic tests for use in surveillance and clinical veterinary practice. Much of the research conducted to date has focused on the investigation of possible associations of particular candidate genes with the resistance phenotype. In the future, as full parasite genome sequences become available, there will be an opportunity to apply genome-wide approaches to identify the genetic loci that underlie anthelmintic resistance. Both the interpretation of candidate gene studies and the application of genome-wide approaches require a good understanding of the genetics and population biology of the relevant parasites as well as knowledge of how resistance mutations arise and are selected in populations. Unfortunately, much of this information is lacking for parasitic nematodes. This review deals with a number of aspects of genetics and population biology that are pertinent to these issues. We discuss the possible origins of resistance mutations and the likely effects of subsequent selection on the genetic variation at the resistance-conferring locus. We also review some of the experimental approaches that have been used to test associations between candidate genes and anthelmintic resistance phenotypes and highlight implications for future genome-wide studies.

Anthelmintic resistance: markers for resistance, or susceptibility?

The Consortium for Anthelmintic Resistance and Susceptibility (CARS) brings together researchers worldwide, with a focus of advancing knowledge of resistance and providing information on detection methods and treatment strategies. Advances in this field suggest mechanisms and features of resistance that are shared among different classes of anthelmintic. Benzimidazole resistance is characterized by specific amino acid substitutions in beta-tubulin. If present, these substitutions increase in frequency upon drug treatment and lead to treatment failure. In the laboratory, sequence substitutions in ion-channels can contribute to macrocyclic lactone resistance, but there is little evidence that they are significant in the field. Changes in gene expression are associated with resistance to several different classes of anthelmintic. Increased P-glycoprotein expression may prevent drug access to its site of action. Decreased expression of ion-channel subunits and the loss of specific receptors may remove the drug target. Tools for the identification and genetic analysis of parasitic nematodes and a new online database will help to coordinate research efforts in this area. Resistance may result from a loss of sensitivity as well as the appearance of resistance. A focus on the presence of anthelmintic susceptibility may be as important as the detection of resistance.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

Graphical abstract

Haemonchus contortus as a paradigm and model to study anthelmintic drug resistance.

Anthelmintic resistance is a major problem for the control livestock parasites and a potential threat to the sustainability of community-wide treatment programmes being used to control human parasites in the developing world. Anthelmintic resistance is essentially a complex quantitative trait in which multiple mutations contribute to the resistance phenotype in an additive manner. Consequently, a combination of forward genetic and genomic approaches are needed to identify the causal mutations and quantify their contribution to the resistance phenotype. Therefore, there is a need to develop genetic and genomic approaches for key parasite species identified as relevant models. Haemonchus contortus, a gastro-intestinal parasite of sheep, has shown a remarkable propensity to develop resistance to all the drugs used in its control. Partly because of this, and partly because of its experimental amenability, research on this parasite has contributed more than any other to our understanding of anthelmintic resistance. H. contortus offers a variety of advantages as an experimental system including the ability to undertake genetic crosses; a prerequisite for genetic mapping. This review will discuss the current progress on developing H. contortus as a model system in which to study anthelmintic resistance.

Botanicals: an alternative approach for the control of avian coccidiosis

Coccidiosis is recognized as the major parasitic disease of poultry and is caused by the apicomplexan protozoan Eimeria. In the past, conventional disease control strategies have depended mainly on anticoccidial drugs and, to a certain extent, live vaccines. Anticoccidial drugs have played a major role in the effective control of avian coccidiosis, but, their extensive use has resulted in the emergence of drug resistant coccidian strains. In such situations, new drugs should be available to replace the older ones against which resistance has developed, however it takes a long time to develop any new compounds. Because of the high cost of developing new drugs and vaccines, development of drug resistance and concerns over drug residues associated with the continuous use of these chemicals, there is a renewed interest in the use of botanicals for safe, effective and cheap control of avian coccidiosis. Several poultry scientists all over the world are now actively engaged in research into the use of plants and plant derived products to fight and reduce the heavy economic losses in poultry industry caused by coccidiosis. This paper reviews the research on botanicals, herbal complexes and commercially available botanical products having anticoccidial properties against avian coccidiosis. Information regarding active compounds, doses and mechanism of action of plants provided in this paper may serve as a guideline for the use of botanical anticoccidial agents as a part of integrated control strategies for the effective control of resistant coccidian parasites.

Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel

BackgroundMicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question.ResultsThe small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (pi)RNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes.ConclusionsThis in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention.