Browsing by Author "Gilleard, John S"

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    A database for ITS2 sequences from nematodes
    (2020-07-10) Workentine, Matthew L; Chen, Rebecca; Zhu, Shawna; Gavriliuc, Stefan; Shaw, Nicolette; Rijke, Jill d; Redman, Elizabeth M; Avramenko, Russell W; Wit, Janneke; Poissant, Jocelyn; Gilleard, John S
    Abstract Background Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology. As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data. Results Here we present an ITS2 rDNA database for marker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is currently the most complete and up-to-date ITS2 database for nematodes and is able to reproduce previous analysis that used a smaller database. Conclusions This database is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy.
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    High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding
    (2020-09-10) Wang, Tong; Avramenko, Russell W; Redman, Elizabeth M; Wit, Janneke; Gilleard, John S; Colwell, Douglas D
    Abstract Background The ability of infective larvae of cattle gastrointestinal nematode (GIN) species to overwinter on pastures in northerly climatic zones with very cold dry winters is poorly understood. This is an important knowledge gap with critical implications for parasite risk assessment and control. Methods Infective third-stage larvae (L3) were quantified in samples of fecal pats, together with adjacent grass and soil, before and after winter on three farms in southern, central and northern Alberta. Nemabiome ITS2 metabarcoding was then performed on the harvested L3 populations to determine the species composition. Finally, parasite-free tracer calves were used to investigate if the L3 surviving the winter could infect calves and develop to adult worms in spring. Results Farm level monitoring, using solar powered weather stations, revealed that ground temperatures were consistently higher, and less variable, than the air temperatures; minimum winter air and ground temperatures were − 32.5 °C and − 24.7 °C respectively. In spite of the extremely low minimum temperatures reached, L3 were recovered from fecal pats and grass before and after winter with only a 38% and 61% overall reduction over the winter, respectively. Nemabiome ITS2 metabarcoding assay revealed that the proportion of L3 surviving the winter was high for both Cooperia oncophora and Ostertagia ostertagi although survival of the former species was statistically significantly higher than the latter. Nematodirus helvetinaus and Trichostrongylus axei could be detected after winter whereas Haemonchus placei L3 could not overwinter at all. Adult C. oncophora, O. ostertagi and N. helvetianus could be recovered from tracer calves grazing after the winter. Conclusions The largest proportion of L3 were recovered from fecal pats suggesting this is important refuge for L3 survival. Results also show that L3 of several GIN parasite species can survive relatively efficiently on pastures even in the extreme winter conditions in western Canada. Tracer calf experiments confirmed that overwintered L3 of both C. oncophora and O. ostertagi were capable of establishing a patent infection in the following spring. These results have important implications for the epidemiology, risk of production impact and the design of effective control strategies. The work also illustrates the value of applying ITS2 nemabiome metabarcoding to environmental samples.
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    High species diversity of trichostrongyle parasite communities within and between Western Canadian commercial and conservation bison herds revealed by nemabiome metabarcoding
    (2018-05-15) Avramenko, Russell W; Bras, Ana; Redman, Elizabeth M; Woodbury, Murray R; Wagner, Brent; Shury, Todd; Liccioli, Stefano; Windeyer, M. C; Gilleard, John S
    Abstract Background Many trichostrongylid nematode species are reported to infect bison, some of which are major causes of disase and production loss in North American bison herds. However, there is little information on the species distribution and relative abundance of these parasites in either commercial or conservation herds. This is largely because trichostrongylid nematode species cannot be distinguished by visual microscopic examination of eggs present in feces. Consequently, we have applied ITS2 rDNA nemabiome metabarcoding to describe the trichostrongyle parasite species diversity in 58 bison production groups derived from 38 commercial North American plains bison (Bison bison bison) herds from across western Canada, and two bison conservation herds located in Elk Island National Park (EINP) [plains bison and wood bison (Bison bison athabascae)] and one in Grasslands National Park (GNP) (plains bison). Results We report much higher infection intensities and parasite species diversity in commercial bison herds than previously reported in beef cattle herds grazing similar latitudes. Predominant trichostrongyle parasite species in western Canadian commercial bison herds are those commonly associated with Canadian cattle, with Ostertagia ostertagi being the most abundant followed by Cooperia oncophora. Combined with high fecal egg counts in many herds, this is consistent with significant clinical and production-limiting gastrointestinal parasitism in western Canadian bison herds. However, Haemonchus placei was the most abundant species in five of the production groups. This is both surprising and important, as this highly pathogenic blood-feeding parasite has not been reported at such abundance, in any livestock species, at such northerly latitudes. The presence of Trichostrongylus axei as the most abundant parasite in four herds is also unusual, relative to cattle. There were striking differences in parasite communities between the EINP and commercial bison herds. Most notably, Orloffia bisonis was the predominant species in the wood bison herd despite being found at only low levels in all other herds surveyed. Conclusions This study represents the most comprehensive description of parasite communities in North American bison to date and illustrates the power of deep amplicon sequencing as a tool to study species diversity in gastrointestinal nematode communities.
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    Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?
    (2019-12-09) Jimenez Castro, Pablo D; Howell, Sue B; Schaefer, John J; Avramenko, Russell W; Gilleard, John S; Kaplan, Ray M
    Abstract Background The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum. Methods Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene. Results Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases. Conclusions These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.
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    Partially redundant function of UDP glycosyltransferase (ugt) genes in the modulation of benzimidazole sensitivity in the nematode Caenorhabditis elegans
    (2021-12-10) Sharma, Nidhi; Gilleard, John S; Cobo, Eduardo; Mains, Paul E; McGhee, James D
    The benzimidazoles (BZ) are a family of anthelmintics used in livestock and human parasitic nematode control. Our previous work has shown that the free-living model nematode Caenorhabditis elegans and the parasite Haemonchus contortus detoxify BZ anthelmintics by conjugation with a glucose residue. This suggests the involvement of phase II drug metabolizing enzymes, specifically the UDP-glycosyltransferase (UGT) enzyme family, whose expression is induced by BZ drug exposure. First, the UGT gene family of C. elegans was characterized by phylogenetic analysis, RNA-seq expression data and an RNAi screen. Phylogenetic analysis revealed none of the C. elegans and H. contortus UGT enzymes or clusters shared a clear orthologous relationship to any of the human UGT families. Existing RNA-seq expression data of the C. elegans ugt gene family identified that most of the ugts were less expressed in all developmental stages and many of them displayed enriched expression in the gut. Next, an RNAi screening identified that knockdown of ugt-9, ugt-11 and ugt-22 make worms sensitive to albendazole (ABZ) treatment compared to wild type. However, the RNAi knockdown of ugt-9 and ugt-11 potentially have “off target” effects and, based on their high level of sequence identity with six other ugt genes that are tightly linked in a cluster on chromosome V. This “ugt-9 cluster” includes ugt-9 and ugt-11, but not ugt-22. Next, I undertook a detailed genetic analysis of the ugt-9 cluster genes and demonstrated the importance of both ugt-9 and ugt-11 in modulating the potency of BZ drugs in vivo. These two genes appear to have partially redundant functions with respect to modulating in vivo BZ sensitivity, both with each other and other genes of the ugt-9 cluster, as well as with ugt-22. Further, expression patterns of ugt-9, ugt-11 and ugt-22 reporter genes are consistent with a lack of complete functional redundancy. Furthermore, in vivo overexpression of ugt-9 was sufficient to confer ABZ resistance in wild-type worms, supporting the role of this gene in ABZ biotransformation. Overall, this work identified multiple UGT enzymes that can modulate BZ potency in C. elegans in a partially redundant manner due to their non-overlapping in vivo expression patterns. Equivalent enzymes in parasitic nematodes could potentially represent “druggable” targets to improve BZ potency and warrant further investigation for a potential role in anthelmintic resistance.
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    Population genomic and evolutionary modelling analyses reveal a single major QTL for ivermectin drug resistance in the pathogenic nematode, Haemonchus contortus
    (2019-03-15) Doyle, Stephen R; Illingworth, Christopher J R; Laing, Roz; Bartley, David J; Redman, Elizabeth; Martinelli, Axel; Holroyd, Nancy; Morrison, Alison A; Rezansoff, Andrew; Tracey, Alan; Devaney, Eileen; Berriman, Matthew; Sargison, Neil; Cotton, James A; Gilleard, John S
    Abstract Background Infections with helminths cause an enormous disease burden in billions of animals and plants worldwide. Large scale use of anthelmintics has driven the evolution of resistance in a number of species that infect livestock and companion animals, and there are growing concerns regarding the reduced efficacy in some human-infective helminths. Understanding the mechanisms by which resistance evolves is the focus of increasing interest; robust genetic analysis of helminths is challenging, and although many candidate genes have been proposed, the genetic basis of resistance remains poorly resolved. Results Here, we present a genome-wide analysis of two genetic crosses between ivermectin resistant and sensitive isolates of the parasitic nematode Haemonchus contortus, an economically important gastrointestinal parasite of small ruminants and a model for anthelmintic research. Whole genome sequencing of parental populations, and key stages throughout the crosses, identified extensive genomic diversity that differentiates populations, but after backcrossing and selection, a single genomic quantitative trait locus (QTL) localised on chromosome V was revealed to be associated with ivermectin resistance. This QTL was common between the two geographically and genetically divergent resistant populations and did not include any leading candidate genes, suggestive of a previously uncharacterised mechanism and/or driver of resistance. Despite limited resolution due to low recombination in this region, population genetic analyses and novel evolutionary models supported strong selection at this QTL, driven by at least partial dominance of the resistant allele, and that large resistance-associated haplotype blocks were enriched in response to selection. Conclusions We have described the genetic architecture and mode of ivermectin selection, revealing a major genomic locus associated with ivermectin resistance, the most conclusive evidence to date in any parasitic nematode. This study highlights a novel genome-wide approach to the analysis of a genetic cross in non-model organisms with extreme genetic diversity, and the importance of a high-quality reference genome in interpreting the signals of selection so identified.
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    Treatment efficacy and re-infection rates of soil-transmitted helminths following mebendazole treatment in schoolchildren, Northwest Ethiopia
    (2020-11-12) Zeleke, Ayalew J; Bayih, Abebe G; Afework, Solomon; Gilleard, John S
    Abstract Background Transmission of soil-transmitted helminth (STH) infection remains high in Ethiopia. This study aimed at assessing the therapeutic efficacy of mebendazole against soil-transmitted helminths and determining the re-infection rates of the parasites among schoolchildren in Northwest Ethiopia. Methods A school-based cross-sectional study was conducted. Data was collected using a structured questionnaire. Stool specimens were examined using direct wet mount microscopy and Kato-Katz methods. Schoolchildren who tested positive for soil-transmitted helminths were treated with 500 mg single-dose of mebendazole. Cure and egg reduction rates were evaluated 2 to 3 weeks post treatment. Moreover, the re-infection rate of these parasites among those who were cured was determined 1 year after treatment. Data were analyzed using SPSS version 20. P value < 0.05 was considered as statistically significant. Result A drug efficacy study was conducted on 62, 52, and 14 children infected by Ascaris lumbricoides (A. lumbricoides), hookworm, and Trichuris trichiura (T. trichiura), respectively. The cure rates (CR) of mebendazole against A. lumbricoides, hookworm, and T. trichiura were found to be 96.9%, 23.1%, and, 28.6%, respectively. The egg reduction rate (ERR) of A. lumbricoides was found to be 99.6% whereas 49.6% and 56.3% were reported for hookworm and T. trichiura, respectively. Eighty schoolchildren who were treated and cured from any STH infections were included for the determination of re-infection rate. Out of 80 children, 36.3% (29/80) were found to be re-infected after 1 year: 22 (75.9%), 6 (20.7%), and 1 (1.3%) of study participants were re-infected with A. lumbricoides, hookworm, and both infections, respectively. All re-infections were grouped under the “light infection” category. Conclusion Mebendazole was found to be highly effective against A. lumbricoides, but had relatively low efficacy against hookworms and T. trichiura. These results bring into question the use of mebendazole in STH mass drug administration (MDA) programs in this region if albendazole, a drug with higher efficacy against hookworms, is available. Moreover, a significant number of treated children were re-infected with either or both of A. lumbricoides or hookworms 1 year after treatment emphasizing the need for better integrated intestinal helminthiasis control measures.