Regional heterogeneity and unexpectedly high abundance of Cooperia punctata in beef cattle at a northern latitude revealed by ITS-2 rDNA nemabiome metabarcoding.

BACKGROUND: The species composition of cattle gastrointestinal nematode (GIN) communities can vary greatly between regions. Despite this, there is remarkably little large-scale surveillance data for cattle GIN species which is due, at least in part, to a lack of scalable diagnostic tools. This lack of regional GIN species-level data represents a major knowledge gap for evidence-based parasite management and assessing the status and impact of factors such as climate change and anthelmintic drug resistance. METHODS: This paper presents a large-scale survey of GIN in beef herds across western Canada using ITS-2 rDNA nemabiome metabarcoding. Individual fecal samples were collected from 6 to 20 randomly selected heifers (n = 1665) from each of 85 herds between September 2016 and February 2017 and 10-25 first season calves (n = 824) from each of 42 herds between November 2016 and February 2017. RESULTS: Gastrointestinal nematode communities in heifers and calves were similar in Alberta and Saskatchewan, with Ostertagia ostertagi and Cooperia oncophora being the predominant GIN species in all herds consistent with previous studies. However, in Manitoba, Cooperia punctata was the predominant species overall and the most abundant GIN species in calves from 4/8 beef herds. CONCLUSIONS: This study revealed a marked regional heterogeneity of GIN species in grazing beef herds in western Canada. The predominance of C. punctata in Manitoba is unexpected, as although this parasite is often the predominant cattle GIN species in more southerly latitudes, it is generally only a minor component of cattle GIN communities in northern temperate regions. We hypothesize that the unexpected predominance of C. punctata at such a northerly latitude represents a range expansion, likely associated with changes in climate, anthelmintic use, management, and/or animal movement. Whatever the cause, these results are of practical concern since C. punctata is more pathogenic than C. oncophora, the Cooperia species that typically predominates in cooler temperate regions. Finally, this study illustrates the value of ITS-2 rDNA nemabiome metabarcoding as a surveillance tool for ruminant GIN parasites.

Characterization of the ß-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance.

BACKGROUND: The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding ß-tubulin proteins, the principal targets of BZ drugs. METHODOLOGY AND PRINCIPAL FINDINGS: First, the ß-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different ß-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. SIGNIFICANCE: This study demonstrated the presence of at least seven ß-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the ß-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of ß-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Multiple drug resistance in hookworms infecting greyhound dogs in the USA.

Ancylostoma caninum is the most prevalent nematode parasite of dogs. We confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the USA. Cases of MDR hookworms appear to be highly overrepresented in greyhounds. The aims of this study were to evaluate the drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from greyhounds of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite treatment with anthelmintics, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA), respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 54 animals. Mean and median IC50 and IC95 values for the EHA were 5.3 µM, 3.6 µM, and 24.5 µM, 23.4 µM, respectively. For the LDA, the median IC50 value was >1000 nM. These values ranged 62-81 times higher than our susceptible laboratory isolate. Only post-treatment samples were available. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools. Deep sequencing of the isotype 1 ß-tubulin gene only revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples. These clinical, in vitro, and genetic data provide strong evidence that greyhound dogs in the USA are infected with MDR A. caninum at very high levels in prevalence and infection intensity.

A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses.

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

The use of ITS-2 rDNA nemabiome metabarcoding to enhance anthelmintic resistance diagnosis and surveillance of ovine gastrointestinal nematodes.

A lack of quantitative information on the species composition of parasite communities present in fecal samples is a major limiting factor for the sensitivity, accuracy and interpretation of the diagnostic tests commonly used to assess anthelmintic efficacy and resistance. In this paper, we investigate the ability of ITS-2 rDNA nemabiome metabarcoding to enhance fecal egg count reduction testing by providing information on the effect of drug treatments on individual parasite species. Application of ITS-2 rDNA nemabiome metabarcoding to fecal samples from ewes from over 90 flocks across western Canada revealed high gastrointestinal nematode infection intensities in many flocks with Haemonchus contortus being the most abundant species followed by Teladorsagia circumcincta and then Trichostrongylus colubriformis. Integration of ITS-2 rDNA nemabiome metabarcoding with pre- and post-treatment fecal egg counting revealed consistently poor efficacy of producer-applied ivermectin and benzimidazole treatments against H. contortus, but much better efficacy against T. circumcincta and T. colubriformis, except for in a small number of flocks. Integration of nemabiome ITS-2 rDNA metabarcoding with Fecal Egg Count Reduction Tests (FECRT), undertaken on farm visits, confirmed that ivermectin and fenbendazole resistance is widespread in H. contortus but is currently less common in T. circumcincta and T. colubriformis in western Canada. FECRT/nemabiome testing did not detect moxidectin resistance in any GIN species but suggested the early emergence of levamisole resistance specifically in T. circumcincta. It also revealed that although poor efficacy to closantel was relatively common, based on total fecal egg counts, this was due to its narrow spectrum of activity rather than the emergence of anthelmintic resistance. This study illustrates the value of ITS-2 rDNA nemabiome metabarcoding to improve fecal egg count resistance testing, perform large-scale anthelmintic resistance surveillance and direct more targeted rational anthelmintic use.

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.

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.

A database for ITS2 sequences from nematodes.

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.

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematodes, resistance in N. battus is likely to be conferred by single nucleotide polymorphisms (SNP) within the ß-tubulin isotype 1 locus at codons 167, 198 and 200. Pyrosequencing and deep amplicon sequencing assays were designed to identify the F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) SNPs. Nematodirus battus populations from 253 independent farms were analysed by pyrosequencing; 174 farm populations were included in deep amplicon sequencing and 170 were analysed using both technologies. F200Y was the most prevalent SNP identified throughout the UK, in 12-27% of the populations tested depending on assay, at a low overall individual frequency of 2.2 ±â€¯0.6% (mean ±â€¯SEM, based on pyrosequencing results). Four out of the five populations with high frequencies (>20%) of the F200Y mutation were located in NW England. The F167Y SNP was identified, for the first time in this species, in four of the populations tested at a low frequency (1.2% ±â€¯0.01), indicating the early emergence of the mutation. E198A or E198L were not identified in any of the isolates. Results obtained were comparable between both techniques for F200Y (Lins' CCC, rc = 0.96) with discrepancies being limited to populations with low frequencies. The recent emergence of resistance in this species will provide a unique opportunity to study the early stages of anthelmintic resistance within a natural setting and track its progress in the future.

Assessing anthelmintic resistance risk in the post-genomic era: a proof-of-concept study assessing the potential for widespread benzimidazole-resistant gastrointestinal nematodes in North American cattle and bison.

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 ß-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.

Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

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 ß-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 ß-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.

Survey of gastrointestinal nematodes in breeding-age heifers on 6 Saskatchewan dairy farms.

To determine the prevalence, mean fecal egg count intensities, and predominant gastrointestinal nematode species, fresh environmental fecal samples were collected from 30 grazing heifers at monthly intervals (June, July, and August) on 6 Saskatchewan dairy farms in 2016. The population average strongylid prevalence ranged from 67.2% [95% confidence interval (CI): 49.5% to 84.9%] in June to 88.3% (95% CI: 76.7% to 100.0%) in August (P = 0.05) while the mean fecal egg count ranged from 1.7 in June to 9.3 eggs/g of feces in August (P < 0.003). The predominant nematode species were identified using a deep-sequencing nemabiome assay targeting the internal transcribed spacer rDNA genomic locus of both L1 and L3-stage larvae. Cooperia oncophora and Ostertagia ostertagi were predominant on all farms, accounting for > 85% of the species. The results provide important previously unavailable data on the epidemiology of gastrointestinal nematodes (GIN) in fecal samples from these grazing heifers in Saskatchewan.

Examen des nématodes gastro-intestinaux chez les génisses en âge de se reproduire sur six fermes laitières de la Saskatchewan. Afin de déterminer la prévalence, l'intensité du dénombrement moyen d'oeufs dans les fèces et les espèces de nématodes gastro-intestinaux prédominants, des échantillons environnementaux de fèces fraîches furent prélevés de 30 génisses au pâturage à un intervalle mensuel (juin, juillet, et août) sur six fermes laitières de la Saskatchewan en 2016. La prévalence de la population moyenne de strongyles variait de 67,2 % [intervalle de confiance de 95 % (CI) : 49,5 % à 84,9 %] en juin à 88,3 % (CI 95 % : 76,7 % à 100,0 %) en août (P = 0,05) alors que le dénombrement moyen des oeufs dans les fèces variait de 1,7 en juin à 9,3 oeufs/g de fèces en août (P < 0,003). Les espèces de nématodes prédominantes furent identifiées à l'aide d'un test de séquençage profond du némabiome visant l'ADNr du locus génomique de l'espaceur interne transcrit des stades larvaires L1 et L3. Cooperia oncophora et Ostertagia ostertagi étaient prédominants sur toutes les fermes, représentant > 85 % des espèces. Ces résultats fournissent des données importantes non-disponibles auparavant sur l'épidémiologie de GIN dans des échantillons de fèces provenant de ces génisses au pâturage en Saskatchewan.(Traduit par Dr Serge Messier).

Validation of ITS-2 rDNA nemabiome sequencing for ovine gastrointestinal nematodes and its application to a large scale survey of UK sheep farms.

We have validated ITS-2 rDNA nemabiome next-generation amplicon sequencing to determine relative species abundance of gastrointestinal nematode species in ovine fecal samples. In order to determine species representation biases, ITS-2 rDNA amplicon sequencing was applied to mock communities or field populations with known proportions of L3 for eight of the major ovine gastrointestinal nematode species: Teladorsagia circumcincta, Trichostrongylus vitrinus, Haemonchus contortus, Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, Chabertia ovina and Oesophagostumum venulosum. Correction factors, calculated from this data, were shown to reduce species representation biases when applied to an independent set of field samples of known composition. We compared ITS-2 rDNA amplicon sequencing data that was generated from harvested eggs, freshly hatched L1 or L3 larvae following fecal culture and no statistically significant differences were found for the more abundant parasite species. We then applied the validated ITS-2 rDNA nemabiome amplicon sequencing assay to a set of archived L1 gastrointestinal nematode populations, collected in 2008 from fecal samples from 93 groups of 20 ewes and 61 groups of 20 lambs derived from 99 UK sheep farms. The presence of the major gastrointestinal nematode species had previously been determined on this large sample set by species-specific PCR. We show how the ITS-2rDNA amplicon sequencing data provided much more detailed information on species abundance than the previous species-specific PCR. This new data represents the most comprehensive overview of the relative abundance of the major gastrointestinal nematode species across UK sheep farms to date. Substantial variance in the relative abundance of both T. circumcincta and T. vitrinus between farms was revealed with the former species being of statistically significantly higher abundance in all three regions sampled (England, Scotland and Wales). The data also revealed that the relative abundance of T. circumcinta was statistically significantly higher in ewes than in lambs with the opposite pattern being the case for T. vitrinus. The nemabiome sequencing data also clearly illustrated the sporadic nature and skewed distribution of H. controtus across UK sheep farms as well as a higher relative abundance on farms from England compared to Wales and Scotland. The nemabiome survey also provides the first widescale data on the relative abundance of the two major large intestinal nematodes C. ovina and O. venulosum. This work validates ITS-2 rDNA nemabiome sequencing for use in sheep and illustrates the power of the approach for large scale surveillance of ovine gastrointestinal nematodes.

Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations.

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the ß-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.

High species diversity of trichostrongyle parasite communities within and between Western Canadian commercial and conservation bison herds revealed by nemabiome metabarcoding.

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.

The use of nemabiome metabarcoding to explore gastro-intestinal nematode species diversity and anthelmintic treatment effectiveness in beef calves.

Next-generation deep amplicon sequencing, or metabarcoding, has revolutionized the study of microbial communities in humans, animals and the environment. However, such approaches have yet to be applied to parasitic helminth communities. We recently described the first example of such a method - nemabiome sequencing - based on deep-amplicon sequencing of internal transcribed spacer 2 (ITS-2) rDNA, and validated its ability to quantitatively assess the species composition of cattle gastro-intestinal nematode (GIN) communities. Here, we present the first application of this approach to explore GIN species diversity and the impact of anthelmintic drug treatments. First, we investigated GIN species diversity in cow-calf beef cattle herds in several different regions, using coproculture derived L3s. A screen of 50 Canadian beef herds revealed parasite species diversity to be low overall. The majority of parasite communities were comprised of just two species; Ostertagia ostertagi and Cooperia oncophora. Cooperia punctata was present at much lower levels overall, but nevertheless comprised a substantive part of the parasite community of several herds in eastern Canada. In contrast, nemabiome sequencing revealed higher GIN species diversity in beef calves sampled from central/south-eastern USA and Sao Paulo State, Brazil. In these regions C. punctata predominated in most herds with Haemonchus placei predominating in a few cases. Ostertagia ostertagi and C. oncophora were relatively minor species in these regions in contrast to the Canadian herds. We also examined the impact of routine macrocyclic lactone pour-on treatments on GIN communities in the Canadian beef herds. Low treatment effectiveness was observed in many cases, and nemabiome sequencing revealed an overall increase in the proportion of Cooperia spp. relative to O. ostertagi post-treatment. This work demonstrates the power of nemabiome metabarcoding to provide a detailed picture of GIN parasite community structure in large sample sets and illustrates its potential use in research, diagnostics and surveillance.

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.