Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals.

Diagnosis of gastrointestinal nematodes in livestock and companion animals has been neglected for years and there has been an historical underinvestment in the development and improvement of diagnostic tools, undermining the undoubted utility of surveillance and control programmes. However, a new impetus by the scientific community and the quickening pace of technological innovations, are promoting a renaissance of interest in developing diagnostic capacity for nematode infections in veterinary parasitology. A cross-cutting priority for diagnostic tools is the development of pen-side tests and associated decision support tools that rapidly inform on the levels of infection and morbidity. This includes development of scalable, parasite detection using artificial intelligence for automated counting of parasitic elements and research towards establishing biomarkers using innovative molecular and proteomic methods. The aim of this review is to assess the state-of-the-art in the diagnosis of helminth infections in livestock and companion animals and presents the current advances of diagnostic methods for intestinal parasites harnessing (i) automated methods for copromicroscopy based on artificial intelligence, (ii) immunodiagnosis, and (iii) molecular- and proteome-based approaches. Regardless of the method used, multiple factors need to be considered before diagnostics test results can be interpreted in terms of control decisions. Guidelines on how to apply diagnostics and how to interpret test results in different animal species are increasingly requested and some were recently made available in veterinary parasitology for the different domestic species.

Similarities and differences in the biotransformation and transcriptomic responses of Caenorhabditis elegans and Haemonchus contortus to five different benzimidazole drugs.

We have undertaken a detailed analysis of the biotransformation of five of the most therapeutically important benzimidazole anthelmintics - albendazole (ABZ), mebendazole (MBZ), thiabendazole (TBZ), oxfendazole (OxBZ) and fenbendazole (FBZ) - in Caenorhabditis elegans and the ruminant parasite Haemonchus contortus. Drug metabolites were detected by LC-MS/MS analysis in supernatants of C. elegans cultures with a hexose conjugate, most likely glucose, dominating for all five drugs. This work adds to a growing body of evidence that glucose conjugation is a major pathway of xenobiotic metabolism in nematodes and may be a target for enhancement of anthelmintic potency. Consistent with this, we found that biotransformation of albendazole by C. elegans reduced drug potency. Glucose metabolite production by C. elegans was reduced in the presence of the pharmacological inhibitor chrysin suggesting that UDP-glucuronosyl/glucosyl transferase (UGT) enzymes may catalyze benzimidazole glucosidation. Similar glucoside metabolites were detected following ex vivo culture of adult Haemonchus contortus. As a step towards identifying nematode enzymes potentially responsible for benzimidazole biotransformation, we characterised the transcriptomic response to each of the benzimidazole drugs using the C. elegans resistant strain CB3474 ben-1(e1880)III. In the case of albendazole, mebendazole, thiabendazole, and oxfendazole the shared transcriptomic response was dominated by the up-regulation of classical xenobiotic response genes including a shared group of UGT enzymes (ugt-14/25/33/34/37/41/8/9). In the case of fenbendazole, a much greater number of genes were up-regulated, as well as developmental and brood size effects suggesting the presence of secondary drug targets in addition to BEN-1. The transcriptional xenobiotic response of a multiply resistant H. contortus strain UGA/2004 was essentially undetectable in the adult stage but present in the L3 infective stage, albeit more muted than C. elegans. This suggests that xenobiotic responses may be less efficient in stages of parasitic nematodes that reside in the host compared with the free-living stages.

Prevalence, fecal egg counts, and species identification of gastrointestinal nematodes in replacement dairy heifers in Canada.

Information is scarce regarding the epidemiology of gastrointestinal nematodes in Canadian dairy heifers. The objectives of this study were to estimate the prevalence and fecal egg counts of gastrointestinal nematodes in dairy heifers, and using a novel deep-amplicon sequencing approach, to identify the predominant gastrointestinal nematode species in Canadian dairy replacement heifers. Fresh environmental fecal samples (n = 2,369) were collected from replacement heifers on 306 dairy farms across western Canada, Ontario, Québec, and Atlantic Canada. Eggs per gram of feces (EPG) were determined using a modified Wisconsin double-centrifugation sugar flotation technique. Predominant nematode species at the farm level were identified by deep-amplicon nemabiome sequencing of the internal transcribed spacer-2 rDNA locus of nematode third-stage larvae. Generalized estimating equations were used to estimate predicted parasite prevalence and mean EPG in all heifers and by province, allowing for clustering within herds. Individual heifer egg counts ranged from 0 to 141 EPG (median: 0 EPG; interquartile range: 0 to 71 EPG). Gastrointestinal nematodes were detected in 20.9% (95% confidence interval: 17.2 to 24.6%) of heifers, and the predicted mean strongylid EPG accounting for clustering on farms was 1.1 (95% confidence interval: 0.6 to 1.6). The predominant parasite species were Cooperia oncophora and Ostertagia ostertagi. This is the first study in Canada to use a combination of deep-amplicon nemabiome sequencing and a traditional egg count method to describe the epidemiology of gastrointestinal nematodes in dairy heifers.

Genetic Diversity and Population Structure of Haemonchus contortus.

Haemonchus contortus is one of the most successful and problematic livestock parasites worldwide. From its apparent evolutionary origins in sub-Saharan Africa, it is now found in small ruminants in almost all regions of the globe, and can infect a range of different domestic and wildlife artiodactyl hosts. It has a remarkably high propensity to develop resistance to anthelmintic drugs, making control increasingly difficult. The success of this parasite is, at least in part, due to its extremely high levels of genetic diversity that, in turn, provide a high adaptive capacity. Understanding this genetic diversity is important for many areas of research including anthelmintic resistance, epidemiology, control, drug/vaccine development and molecular diagnostics. In this article, we review the current knowledge of H. contortus genetic diversity and population structure for both field isolates and laboratory strains. We highlight the practical relevance of this knowledge with a particular emphasis on anthelmintic resistance research.

Haemonchus contortus: Genome Structure, Organization and Comparative Genomics.

One of the first genome sequencing projects for a parasitic nematode was that for Haemonchus contortus. The open access data from the Wellcome Trust Sanger Institute provided a valuable early resource for the research community, particularly for the identification of specific genes and genetic markers. Later, a second sequencing project was initiated by the University of Melbourne, and the two draft genome sequences for H. contortus were published back-to-back in 2013. There is a pressing need for long-range genomic information for genetic mapping, population genetics and functional genomic studies, so we are continuing to improve the Wellcome Trust Sanger Institute assembly to provide a finished reference genome for H. contortus. This review describes this process, compares the H. contortus genome assemblies with draft genomes from other members of the strongylid group and discusses future directions for parasite genomics using the H. contortus model.

Molecular evidence shows that the liver fluke Fasciola gigantica is the predominant Fasciola species in ruminants from Pakistan.

Fascioliasis is an important disease affecting livestock, with great costs to producers worldwide. It has also become a serious issue for human populations in some endemic areas as an emerging zoonotic infection. There are two Fasciola species of liver fluke responsible for this disease, which occur worldwide, Fasciola hepatica and Fasciola gigantica. Identifying these two species on the basis of adult or egg morphology requires specialist knowledge due to the similarity of characters, and may misidentify putative intermediate or hybrid forms. In this study we sequenced the internal transcribed spacer 2 (ITS-2) rDNA of liver flukes collected from multiple species of hosts from seven localities in the Punjab and Baluchistan provinces of Pakistan, to determine the distribution of these two species. All 46 flukes processed in this study, collected from seven sites, showed the rDNA ITS-2 genotype corresponding to F. gigantica, contradicting previous reports, based on adult and egg morphology, that both species are present in Pakistan, with F. hepatica being the more common.

Hepatogenous photosensitisation in Scottish sheep casued by Dicrocoelium dendriticum.

Dicrocoeliosis was identified as the probable predisposing cause of weight loss and hepatogenous photosensitisation affecting half of a group of 14 month-old ewe lambs on a farm on the Inner Hebridean island of Coll. Oral dosing of the ewe lambs with 15 mg/kg albendazole only achieved a 79.2% reduction in Dicrocoelium dendriticum egg count, 21 day post treatment. Thus, this report of dicrocoeliosis differs from the disease that occurs elsewhere, both in the severity and nature of its clinical signs, and in the response of the parasite to anthelmintic drug treatment. These differences could indicate the existence of a genetically divergent D. dendriticum population, which may have evolved within the unique and isolated biotope afforded by the machair on the island of Coll. Better understanding of these factors is a prerequisite for effective and sustainable disease control.

Use of diagnostic markers to monitor fasciolosis and gastrointestinal nematodes on an organic dairy farm.

A 12-month study was conducted to assess and monitor gastrointestinal tract nematodes and liver fluke in cohorts of cattle on a Scottish organic dairy farm. Various diagnostic markers for helminth parasites of cattle from different age groups were assessed monthly from April 2007 to March 2008. First season grazing stock were subjected to significant challenge from Ostertagia ostertagi nematodes as reflected in serum pepsinogen concentrations, which rose markedly in the second half of the grazing season. In addition, plasma albumin concentrations decreased and faecal egg counts (FEC) increased moderately, indicating exposure to both O ostertagi and probably Cooperia oncophora. Second season grazing animals had a peak FEC early in the grazing period, suggestive of a potential carry-over of Ostertagia species infection ('Type 2') during housing. All classes of cattle showed evidence of fluke (Fasciola hepatica) infection. Adult cow exposure to O ostertagi and fluke was estimated via the use of ELISA testing to detect antibodies to O ostertagi and F hepatica and the high levels detected suggested a significant exposure response. Despite low stocking densities and sympathetic grazing management, there was a significant challenge to all grazing stock from gastrointestinal nematodes and liver fluke.

Effects of age and immune suppression of sheep on fecundity, hatching and larval feeding of different strains of Haemonchus contortus.

The effects of host age and immune suppression on abomasal parasitic infection in sheep were investigated following single experimental oral infections with MHco3 (ISE), MHco4 (WRS) and MHco10 (CAVR) strains of Haemonchus contortus in naïve 5-month-old crossbred lambs (n=1 per group) and 15-month-old Greyface sheep treated with methyl prednisolone acetate (n=2 per group) or without corticosteroid treatment (n=2 per group). Adult female H. contortus in 5-month-old lambs (n=1 per group) shed on average 6.5, 3.1 and 8.0 times more eggs than in 15-month-old sheep (n=4 per group) following infection with MHco3 (ISE), MHco4 (WRS) and MHco10 (CAVR) strains of H. contortus, respectively, over a period of 28 days following the commencement of patency. There was no obvious effect of age of sheep or corticosteroid treatment on the abomasal establishment of H. contortus or on in vitro assays for egg hatching or larval feeding at different concentrations of anthelmintics, although statistical analysis could not be performed due to the small group sizes.

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.

Characterisation of milbemycin-, avermectin-, imidazothiazole- and benzimidazole-resistant Teladorsagia circumcincta from a sheep flock.

Selection for milbemycin resistance in a population of Teladorsagia circumcincta was examined in a sheep flock in which a lack of persistence of an oral dose of 0.2 mg/kg moxidectin against T circumcincta had previously been identified. A faecal egg count reduction test also showed resistance to benzimidazole, levamisole and avermectin anthelmintic groups. Bioassays were used to compare the moxidectin-resistant T circumcincta with another previously characterised benzimidazole-, levamisole- and ivermectin-resistant (MTci5) strain that had been isolated from a sheep flock in the same region in south-east Scotland and with an anthelmintic-susceptible (MTci3) strain of T circumcincta. The mean ED(50) value (the concentration of drug required to prevent 50 per cent of eggs from hatching) obtained for thiabendazole in egg hatch assays was higher in the moxidectin-resistant T circumcincta than in the ivermectin-resistant MTci5 strain. The inclusion of the cytochrome p450 inhibitor piperonyl butoxide in larval feeding inhibition assays increased the level of ivermectin resistance in vitro in the ivermectin- and moxidectin-resistant populations, but not in the ivermectin-susceptible MTci3 strain of T circumcincta.

A case of canine Angiostrongylus vasorum in Scotland confirmed by PCR and sequence analysis.

This report describes the first recognised case of Angiostrongylus vasorum infection in a native Scottish dog and its diagnostic confirmation using polymerase chain reaction. The use of this technique to definitively diagnose canine angiostrongylosis confirms the presence of this epidemiologically significant case. The implications of the changing distribution of this parasite are discussed.

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.

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.

The use of Caenorhabditis elegans in parasitic nematode research.

There is increasing interest in the use of the free-living nematode Caenorhabditis elegans as a tool for parasitic nematode research and there are now a number of compelling examples of its successful application. C. elegans has the potential to become a standard tool for molecular helminthology researchers, just as yeast is routinely used by molecular biologists to study vertebrate biology. However, in order to exploit C. elegans in a meaningful manner, we need a detailed understanding of the extent to which different aspects of C. elegans biology have been conserved with particular groups of parasitic nematodes. This review first considers the current state of knowledge regarding the conservation of genome organisation across the nematode phylum and then discusses some recent evolutionary development studies in free-living nematodes. The aim is to provide some important concepts that are relevant to the extrapolation of information from C. elegans to parasitic nematodes and also to the interpretation of experiments that use C. elegans as a surrogate expression system. In general, examples have been specifically chosen because they highlight the importance of careful experimentation and interpretation of data. Consequently, the focus is on the differences that have been found between nematode species rather than the similarities. Finally, there is a detailed discussion of the current status of C. elegans as a heterologous expression system to study parasite gene function and regulation using successful examples from the literature.

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

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-3 gene 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.