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Abstract Background There is a higher risk for nematode infections associated with outdoor-reared pigs. Next to Ascaris suum, Oesophagostomum dentatum and Trichuris suis, there is the potential of infections with other nodular worm species, Hyostrongylus rubidus, Stongyloides ransomi and Metastrongylus spp. lungworms. Next-generation sequencing methods describing the nemabiome have not yet been established for porcine nematodes. Methods FLOTAC was used for faecal egg counts of porcine gastrointestinal nematodes and lungworms in piglets, fatteners and adults individually. A nemabiome analyses based on ITS-2 gene region metabarcoding was used to differentiate strongyle species. Additionally, questionnaire data was analysed using mixed-effect regression to identify potential risk factors associated with parasite occurrences and egg shedding intensity. Results On 15 of 17 farms nematode eggs were detected. Ascaris suum, strongyles and T. suis were detected on 82%, 70% and 35% of the 17 farms, respectively. Lungworms were detected on one out of four farms with access to pasture. Strongyloides ransomi was not detected. 32% (CI 28–36%), 27% (24–31%), 5% (4–7%) and 3% (0.9–8%) of the samples where tested positive for strongyles, A. suum, T. suis and lungworms, respectively. The nemabiome analysis revealed three different strongyle species, with O. dentatum being the most common (mean 93.9%), followed by O. quadrispinulatum (5.9%) and the hookworm Globocephalus urosubulatus (0.1%). The bivariate and multivariate risk factor analyses showed among others that cleaning once a week compared to twice a week increased the odds significantly for being infected with A. suum (OR 78.60) and strongyles (2077.59). Access to pasture was associated with higher odds for A. suum (43.83) and strongyles (14.21). Compared to shallow litter systems, deep litter and free range systems resulted in significant higher odds for strongyles (85.74, 215.59, respectively) and T. suis (200.33, 623.08). Conclusions Infections with A. suum, O. dentatum, O. quadrispinulatum, T. suis, Metastrongylus spp. and G. urosubulatus are present in German outdoor-reared pigs. This is the first report of G. urosubulatus in domestic pigs in Europe. Metabarcoding based on the ITS-2 region is a suitable tool to analyse the porcine nemabiome. Furthermore, management practices have the potential of reducing the risk of parasite infections.

Cyathostomins are the most abundant equid endoparasites globally. There are approximately fifty cyathostomin species and, whilst they occupy distinct niches within the large intestine, they are generally considered to share similar characteristics in terms of pathogenicity and response to drug treatment. There are three classes of anthelmintic licensed in the UK to treat cyathostomins (benzimidazoles, tetrahydropyrimidines and macrocyclic lactones) and cases of resistance have been documented for all classes. Previously, faecal egg count reduction tests (FECRT) on four UK Thoroughbred studs revealed multidrug resistant cyathostomins on one stud (A), with evidence of resistance to the macrocyclic lactones (MLs) ivermectin (IVM) and moxidectin (MOX), and to pyrantel (PYR). The remaining three studs (B-D) lacked resistance to IVM and MOX but had a shortened egg reappearance period post treatment.To determine whether specific species could be associated with the observed resistance and shortened egg reappearance period, strongyle eggs collected from between six and 15 individual horses per stud were copro-cultured to third larval stage (L3), before and after anthelmintic treatment, over a three-year timeframe (2021–2023). Quantitative DNA metabarcoding of the ITS-2 region was carried out on all samples.On stud A, single but differing species were found to be responsible for ML and pyrantel resistance in yearlings, Cyathostomum catinatum and Cylicocyclus nassatus, respectively. On studs B-D, with shortened egg reappearance periods, species composition remained largely unchanged post treatment.This study is the first to quantitatively profile cyathostomin species composition pre- and post-treatment in a multidrug resistant population in the UK, revealing that resistance in cyathostomins was species specific. This raises the question of whether these species may be responsible for ML and PYR resistance more widely and indicates that anthelmintic resistance in cyathostomins may not be a multi-species phenomenon.

Anthelmintic resistance occurs worldwide in strongyles of ruminants but data from low-income countries are sparse and rarely apply most up to date methods, while effects of management practices in these countries are poorly documented. In Mozambique, benzimidazole resistance has been previously reported; the present study followed this up in detail, applying in vivo faecal egg count (FEC) reduction test (FECRT), in vitro egg hatch test (EHT) and molecular deep amplicon sequencing approaches targeting the internal transcribed spacer 2 (ITS-2, nemabiome) and the isotype 1 β-tubulin gene to determine the resistance status on farms and the strongyle species involved. Adult Landim goats (433) from six semi-intensive and ten extensive farms (22–30 animals/farm) from Maputo Province were visited April 2021 to February 2022. Fenbendazole (5 mg/kg bw, Panacur®) was administered orally and FEC determined using Mini-FLOTAC. The eggCounts package was used to calculate FECRs with 90% confidence intervals from paired day 0 and 14 data. In vivo and in vitro tests detected AR on 5/16 (31%) farms. This included 1/10 extensive and 4/6 semi-intensive farms. The odds of finding resistant strongyles on a semi-intensive commercial farm was 40-fold higher than on an extensive farm (p = 0.016, logistic regression). A strong, negative correlation was observed between FECRT and EHT EC50 values (Pearson's R = −0.83, P = 0.001; Cohen's κ coefficient 1.0). Nemabiome data showed that Haemonchus contortus, Trichostrongylus colubriformis and unclassified Oesophagostomum closely related to Oesophagostomum columbianum were most abundant before treatment and in particular H. contortus frequencies increased after treatment. Benzimidazole resistance associated polymorphisms were detected in H. contortus and T. colubriformis. Moreover, there were hints that resistance alleles were present in Trichostrongylus axei and Teladorsagia circumcincta. Farmers should regularly test the efficacy of anthelmintics used and consider more sustainable worm control approaches to reduce selection for resistance.

Abstract Background Mixed strongylid infections significantly impact equine health and performance. Traditional microscopy-based methods exhibit limitations in accurately identifying strongylid species. Nemabiome deep amplicon sequencing approach previously succeeded in describing the strongylid communities in livestock including equids. However, there are no available studies that describe the structural communities of strongylid parasites in horses in Thailand. Therefore, this study was undertaken encompassing the ITS-2 rDNA metabarcoding assay to characterize strongylid species within horse fecal samples collected from a cohort of yearlings at the largest domesticated stud farm in Thailand. In addition, to investigate the capability of ITS-2 rDNA in assessing the phylogenetic relationships among the identified strongylid species. Results The study identified 14 strongylid species in the examined equine populations, each with varying prevalence. Notably, Cylicocyclus nassatus and Cylicostephanus longibursatus were identified as the predominant species, with Strongylus spp. conspicuously absent. The phylogenetic analysis of 207 amplicon sequence variants (ASVs) displayed a complex relationship among the investigated cyathostomin species, with some species are positioned across multiple clades, demonstrating close associations with various species and genera. Conclusion The ITS-2 nemabiome sequencing technique provided a detailed picture of horse strongylid parasite species in the studied population. This establishes a foundation for future investigations into the resistance status of these parasites and enables efforts to mitigate their impact.

Abstract Background Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. Methods Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and ‘nemabiome’ internal transcribed spacer 2 (ITS-2) amplicon sequencing. Results Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. Conclusions These data represent the first ‘nemabiome’ sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas. Graphical Abstract

Abstract Background Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. Methods Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. Results Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. Conclusions DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume. Graphical Abstract

Abstract Background Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden. Methods First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R. Results Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8). Conclusion In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored. Graphical Abstract

Abstract Background Next-generation sequencing (NGS) has provided an alternative strategy to study the composition of nematode communities with increased resolution and sensitivity. However, the handling and processing of gigabytes worth of amplicon sequence data produced by an NGS platform is still a major hurdle, limiting the use and adoption of faster and more convenient analysis software. Methods In total 32 paired, fecal samples from Swedish sheep flocks were cultured and the larvae subsequently harvested subjected to internal transcribed spacer 2 (ITS2) amplicon sequencing using the PacBio platform. Samples were analyzed with three different bioinformatic pipelines, i.e. the DADA2, Mothur and SCATA pipelines, to determine species composition and richness. Results For the the major species tested in this study (Haemonchus contortus, Teladorsagia circumcinta and Trichostrongylus colubriformis) neither relative abundances nor species diversity differed significantly between the three pipelines, effectively showing that all three analysis pipelines, although different in their approaches, yield nearly identical outcomes. In addition, the samples analyzed here had especially high frequencies of H. contortus (90–95% across the three pipelines) both before and after sample treatment, followed by T. circumcinta (3.5–4%). This shows that H. contortus is the parasite of primary importance in contemporary Swedish sheep farms struggling with anthelmintic resistance. Finally, although on average a significant reduction in egg counts was achieved post-treatment, no significant shifts in major species relative frequencies occurred, indicating highly rigid community structures at sheep farms where anthelmintic resistance has been reported. Conclusions The findings presented here further contribute to the development and application of NGS technology to study nemabiome compositions in sheep, in addition to expanding our understanding about the most recent changes in parasite species abundances from Swedish sheep farms struggling with anthelmintic resistance. Graphical Abstract

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.

Identifying factors that drive among-individual variation in mixed parasitic infections is fundamental to understanding the ecology and evolution of host–parasite interactions. However, a lack of non-invasive diagnostic tools to quantify mixed infections has restricted their investigation for host populations in the wild. This study applied DNA metabarcoding on parasite larvae cultured from faecal samples to characterize mixed strongyle infections of 320 feral horses on Sable Island, Nova Scotia, Canada, in 2014 to test for the influence of host (age, sex and reproductive/social status) and environmental (location, local density and social group membership) factors on variation. Twenty-five strongyle species were identified, with individual infections ranging from 3 to 18 species with a mean richness (±1 s.d.) of 10.8 ± 3.1. Strongyle eggs shed in faeces were dominated by small strongyle (cyathostomins) species in young individuals, transitioning to large strongyles (Strongylus spp.) in adults. Egg counts were highest in young individuals and in the west or centre of the island for most species. Individuals in the same social group had similar parasite communities, supporting the hypothesis that shared environment may drive parasite assemblages. Other factors such as local horse density, sex, date and reproductive/social status had minimal impacts on infection patterns. This study demonstrates that mixed infections can be dynamic across host ontogeny and space and emphasizes the need to consider species-specific infection patterns when investigating mixed infections.