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

<b>BACKGROUND:</b> MicroRNAs (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. <b>RESULTS:</b> The 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. <b>CONCLUSIONS:</b> This 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

Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field

Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals

92 páginas, 3 tablas, 4 figuras.Diagnosis of gastrointestinal nematodes in livestock and companion animals has been neglected for years and there has been an historical underinvestment in the develop- ment and improvement of diagnostic tools, undermining the undoubted utility of surveillance and control programmes. However, a new impetus by the scientific com- munity and the quickening pace of technological innovations, are promoting a renais- sance 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 esta- blishing 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.This article is based in part upon work from COST Action COMBAR CA16230, supported by COST (European Cooperation in Science and Technology). It is also based upon work conducted by the STAR-IDAZ IRC Scientific sub-Committee “Diagnostic test roadmap” developed by the Livestock Helminth Research Alliance (LiHRA; June 2019) with major contributions of John Gilleard, Georg von Samson Himmelstjerna, Diana Williams, Laura Rinaldi, Edwin Claerebout, Peter Geldhof and Jozef Vercruysse.Peer reviewe

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

A case of canine Angiostrongylus vasorumin 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

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 population

<i>Teladorsagia circumcincta</i>: The transcriptomic response of a multi-drug-resistant isolate to ivermectin exposure <i>in vitro</i>

The emergence and spread of anthelmintic resistance in parasitic nematodes is a serious threat to the sustainability of the livestock industry. Resistance has a genetic component but the underlying mechanisms and the means by which resistant parasites survive anthelmintic treatment are still poorly understood. Differential gene expression may be implicated, especially in multi-drug resistant parasites. In this study, we investigated the transcriptomic response of a triple drug-resistant isolate of Teladorsagia circumcincta to ivermectin exposure in vitro, using Roche 454 sequencing. The study generated similar to 100,000 new EST sequences, similar to 50,000 each from the ivermectin-exposed and -unexposed pools of parasites. Bioinformatic analysis of the expression profiles revealed statistically significant differences in the mean expression levels of four KEGG orthologous groups, namely 'translation', 'amino acid metabolism', 'carbohydrate metabolism' and 'xenobiotic degradation and metabolism'. Notably, candidate resistance genes such as p-glycoproteins and cytochrome P450s were poorly represented in both datasets. Clusters of sequences, containing both exposed and unexposed ESTs, also revealed statistically significant differences. Four clusters were identified as cytochrome c oxidase subunits, two of these clusters had a statistically significant increase in the number of exposed ESTs compared to unexposed ESTs. Four clusters were identified as vitellogenin; three of these clusters had a statistically significant decrease in number of exposed ESTs compared to unexposed ESTs

A survey of the trichostrongylid nematode species present on UK sheep farms and associated anthelmintic control practices

A survey of sheep farms from across the UK was conducted to establish information on farming practices, the trichostrongylid nematode species present and anthelmintic usage. Questionnaires and faecal samples were returned from 118 farms. First stage larvae (L1) were cultured from faecal samples and used for PCR analysis to determine the presence/absence of selected trichostrongylid species. Teladorsagia circumcincta was the only species present on 100% of farms. Haemonchus contortus was found on ∼50% of farms and was widespread throughout the UK. The most common Trichostrongylus spp. was T. vitrinus, found on 95% of farms. Determining the anthelmintic dose rate based on the weight of the heaviest animal in the flock to avoid under dosing was carried out on 58% of farms and was associated with a significantly lower mean epg (p &#60; 0.001) in lambs. However, the weight of animals was only estimated (as opposed to animals weighed) on 32% of farms. Macrocyclic lactones (ML) were the most commonly used anthelmintic class for ewes, whilst benzimidazoles (BZ) were the most widely used in lambs. Twenty-two of the surveyed farms had confirmed anthelmintic resistance, of these, 18 had BZ resistance, one had levamisole (LEV) resistance and 3 had resistance to both BZ and LEV. No farms in this survey reported resistance to ML. Location had a significant effect on the incidence of anthelmintic resistance on the farms in this survey (p = 0.002). There was evidence of a lower risk of anthelmintic resistance occurring on farms from Scotland compared to those in England (pf = 0.047) and Wales (pf = 0.012). Farm type, flock type and open or closed status did not have any significant effect on the incidence of anthelmintic resistance when all other factors were taken into consideration