Peter Halvarsson

Background: Selective anthelmintic treatment, advocated due to evolving anthelmintic resistance, has been associated with an increase in Strongylus vulgaris prevalence. Reverting to routine interval anthelmintic treatments is not viable and therefore, identifying other management factors correlated with S. vulgaris infection is vital. Objectives: To investigate possible risk factors associated with the presence of S. vulgaris infection in resident horses on Swedish horse establishments. Study design: Internet‐based questionnaire survey. Methods: A questionnaire, created using the internet‐based survey platform Netigate, was distributed to owners of equine establishments throughout Sweden via established equine platforms and social media channels. The survey was available for response from 21 May until 1 September 2022. Questions were closed ended with branching logic paths. Results: Four factors were significantly associated with S. vulgaris infection, with an increased odds of infection seen in livery yards (odds ratio [OR] 1.67, 95% confidence interval [CI] 1.18–2.36, p = 0.004) and premises with more than 10 resident horses (OR 2.42, 95% CI 1.64–3.56, p < 0.001). A lower odds of infection were seen in establishments using quarantine routines (OR 0.69, 95% CI 0.50–0.96, p = 0.03) and anthelmintic treatment of new horses prior to arrival at the premise (OR 0.37, 95% CI 0.18–0.74, p = 0.005). Main limitation: Due to the presence of S. vulgaris infection in the present study being based on S. vulgaris diagnostics performed at the farm level, any association between faecal diagnostic use and risk of infection could not be investigated. Conclusions: Although the use of diagnostics for S. vulgaris can keep infection rates low, large farms or livery yards with many different horse owners, and those with low use of biosecurity measures as regards to new horses arriving at the premise, are associated with a higher risk of infection.

Genetic variation of immune genes is an important component of genetic diversity. Major histocompatibility complex (MHC) genes have been put forward as a model for studying how genetic diversity is maintained and geographically distributed in wild populations. Pathogen-mediated selection processes (i.e., heterozygosity advantage, rare-allele advantage or fluctuating selection) and demography are believed to generate and maintain the extreme diversity of MHC genes observed. However, establishing the relative importance of the different proposed mechanisms has proved extremely difficult, but heterozygote advantage is expected to be more detectable when multiple pathogens are considered simultaneously. Here, we test whether MHC diversity in three amphibian species ( Ichthyosaura alpestris, Pleurodeles waltl, and Pelophylax perezi ) is driven by pathogen-mediated selection. We examined the relationship between the individual MHC class II exon variability with individual infection status (infected or not), infection intensity, and co-infection of two main amphibian pathogens: Batrachochytrium dendrobatidis ( Bd ) and Ranavirus sp. ( Rv ). We found higher MHC class II exon 2 allelic diversity in I.alpestris and P. perezi than in P.waltl but no significant differences in allele frequencies between infection groups. We also observed significant differences in Bd infection intensity between Bd infected individuals and co-infected individuals depending on the number of MHC loci that an individual carries. For I. alpestris , we show stronger evidence for MHC associations with infection intensity and status when individuals carry specific alleles and supertypes. Our results suggest that studying the association between MHC genes and single and co-infected individuals might provide new insights into host-parasite evolution and a better understanding of evolutionary mechanisms driven by MHC diversity.

Gastrointestinal nematode parasites are of major concern for horses, where Strongylus vulgaris is considered the most pathogenic among the Strongylus species. Diagnosis of S. vulgaris infections can be determined with next generation sequencing techniques, which are inherently dependent on reference sequences. The best marker for parasitic nematodes is internal transcribed spacer 2 (ITS2) and we provide the first complete ITS2 sequences from five morphologically identified S. vulgaris and additional sequences from two S. edentatus. These sequences have high similarity to already published partial sequences and amplicon sequence variants (ASV) based on next generation sequencing (NGS). The ITS2 sequences from S. vulgaris matched available partial ITS2 sequences and the full ASVs, whereas the S. edentatus sequence matched another complete sequence. We also compare Sanger sequencing and NGS methods and conclude that the ITS2 variation is better represented with NGS methods. Based on this, we recommend that further sequencing of morphologically identified specimens of various species should be performed with NGS cover the intraspecific variation in the ITS2.

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

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

Background Wildlife hosts may serve as reservoirs for strongyles which can be transmitted to domestic livestock. Therefore, we aimed to assess the risk for transmission of important nematode pathogens to domestic sheep in Sweden. The main aim of the study was to assess the risk for transmission of important pathogens from four wildlife hosts to domestic sheep. 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 then 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 amongst which 26 (86%) to the species level These were found in different combinations, amongst which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer, and 10 (32%) in mouflon. Only five species 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. In addition, 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 between all three cervids (n = 8). Conclusion In contrast to our previous study with sheep, the relative abundance and prevalence of T. axei was generally much higher in wildlife than both H. contortus, and T. circumcincta which were negligible. Although the risk for cross-infections of nematodes between the studied wildlife hosts and sheep in Sweden cannot be ruled out, our findings indicate that the transmission potential appear to be relatively low for H. contortus and T. circumcincta, which are the two most pathogenic species in sheep.