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We assessed evidence of exposure to viruses and bacteria in an unmanaged and long-isolated population of Soay sheep (Ovis aries) inhabiting Hirta, in the St Kilda archipelago, 65 km west of Benbecula in the Outer Hebrides of Scotland. The sheep harbour many metazoan and protozoan parasites but their exposure to viral and bacterial pathogens is unknown. We tested for herpes viral DNA in leucocytes and found that 21 of 42 tested sheep were infected with ovine herpesvirus 2 (OHV-2). We also tested 750 plasma samples collected between 1997 and 2010 for evidence of exposure to seven other viral and bacterial agents common in domestic Scottish sheep. We found evidence of exposure to Leptospira spp., with overall seroprevalence of 6·5%. However, serological evidence indicated that the population had not been exposed to border disease, parainfluenza, maedi-visna, or orf viruses, nor to Chlamydia abortus. Some sheep tested positive for antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) but, in the absence of retrospective faecal samples, the presence of this infection could not be confirmed. The roles of importation, the pathogen-host interaction, nematode co-infection and local transmission warrant future investigation, to elucidate the transmission ecology and fitness effects of the few viral and bacterial pathogens on Hirta.

Atypical porcine pestivirus (APPV) is a novel member of the Pestivirus genus detected in association with congenital tremor (CT) type A-II outbreaks and from apparently healthy pigs, both as singular infection and as part of multi-pathogen infections. 'Classical' pestiviruses are known to cause immunosuppression of their host, which can increase susceptibility to secondary infections, severely impacting health, welfare, and production. To investigate APPV's effect on the host's immune system and characterise disease outcomes, 12 piglets from a natural APPV CT type A-II outbreak were experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV), a significant porcine pathogen. Rectal temperatures indicating febrile responses, viremia and viral-specific humoral and cellular responses were assessed throughout the study. Pathological assessment of the lungs and APPV-PRRSV co-localisation within the lungs was performed at necropsy. Viral co-localisation and pathological assessment of the lungs (Immunohistochemistry, BaseScope in situ hybridisation) were performed post-mortem. APPV status did not impact virological or immunological differences in PRRSV-infected groups. However, significantly higher rectal temperatures were observed in the APPV +ve /PRRSV +ve group over four days, indicating APPV increased the febrile response. Significant differences in the lung consolidation of the apical and intermediate lobes were also present, suggesting that APPV co-infection may augment lung pathology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Organisms have evolved diverse strategies to manage parasite infections. Broadly, hosts may avoid infection by altering behaviour, resist infection by targeting parasites or tolerate infection by repairing associated damage. The effectiveness of a strategy depends on interactions between, for example, resource availability, parasite traits (virulence, life-history) and the host itself (nutritional status, immunopathology). To understand how these factors shape host parasite-mitigation strategies, we developed a mathematical model of within-host, parasite-immune dynamics in the context of helminth infections. The model incorporated host nutrition and resource allocation to different mechanisms of immune response: larval parasite prevention; adult parasite clearance; damage repair (tolerance). We also considered a non-immune strategy: avoidance via anorexia, reducing intake of infective stages. Resources not allocated to immune processes promoted host condition, whereas harm due to parasites and immunopathology diminished it. Maximising condition (a proxy for fitness), we determined optimal host investment for each parasite-mitigation strategy, singly and combined, across different environmental resource levels and parasite trait values. Which strategy was optimal varied with scenario. Tolerance generally performed well, especially with high resources. Success of the different resistance strategies (larval prevention or adult clearance) tracked relative virulence of larval and adult parasites: slowly maturing, highly damaging larvae favoured prevention; rapidly maturing, less harmful larvae favoured clearance. Anorexia was viable only in the short term, due to reduced host nutrition. Combined strategies always outperformed any lone strategy: these were dominated by tolerance, with some investment in resistance. Choice of parasite mitigation strategy has profound consequences for hosts, impacting their condition, survival and reproductive success. We show that the efficacy of different strategies is highly dependent on timescale, parasite traits and resource availability. Models that integrate such factors can inform the collection and interpretation of empirical data, to understand how those drivers interact to shape host immune responses in natural systems., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant proteinase family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Telomere length (TL) is a biomarker hypothesized to capture evolutionarily and ecologically important physiological costs of reproduction, infection and immunity. Few studies have estimated the relationships among infection status, immunity, TL and fitness in natural systems. The hypothesis that short telomeres predict reduced survival because they reflect costly consequences of infection and immune investment remains largely untested. Using longitudinal data from a free-living Soay sheep population, we tested whether leucocyte TL was predicted by infection with nematode parasites and antibody levels against those parasites. Helminth parasite burdens were positively associated with leucocyte TL in both lambs and adults, which is not consistent with TL reflecting infection costs. We found no association between TL and helminth-specific IgG levels in either young or old individuals which suggests TL does not reflect costs of an activated immune response or immunosenescence. Furthermore, we found no support for TL acting as a mediator of trade-offs between infection, immunity and subsequent survival in the wild. Our results suggest that while variation in TL could reflect short-term variation in resource investment or environmental conditions, it does not capture costs of infection and immunity, nor does it behave like a marker of an individual's helminth-specific antibody immune response.

Gastrointestinal (GI) parasites cause significant production losses in grazing ruminants which can be mitigated by breeding animals resistant to disease. Lymphocyte cytokine production and parasite-specific Immunoglobulin A (IgA) are adaptive immune traits associated with immunity to GI parasites. To explore the utility of these traits for selective breeding purposes, this study estimated the genetic parameters of the immune traits in sheep and assessed their relationship with disease and productivity traits. Whole blood stimulation assays were performed on 1 040 Scottish Blackface lambs at two months of age in 2016-2017. Blood was stimulated with either pokeweed mitogen (PWM), a non-specific activator of lymphocytes, and Teladorsagia circumcincta (T-ci) larval antigen to activate parasite-specific T lymphocytes. The type of adaptive immune response was determined by quantifying production of cytokines interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10, which relate to T-helper type (Th) 1, Th2 and regulatory T cell responses, respectively. Serum T-ci specific IgA was also quantified. Heritabilities were estimated for each immune trait by univariate analyses. Genetic and phenotypic correlations were estimated between different immune traits, and between immune traits vs. disease and productivity traits that were recorded at three months of age. Disease phenotypes were expressed as faecal egg counts (FEC) of nematode parasites (Strongyles and Nematodirus), faecal oocyst counts (FOC) of coccidian parasites, and faecal soiling score; production was measured as lamb live weight. Significant genetic variation was observed in all immune response traits. Heritabilities of cytokine production varied from low (0.14 ± 0.06) to very high (0.77 ± 0.09) and were always significantly greater than zero (P < 0.05). IgA heritability was found to be moderate (0.41 ± 0.09). Negative associations previously identified between IFN-γ production and FOC, and IL-4 production and strongyle FEC, were not evident in this study, potentially due to the time-lag between immune and parasitology measures. Instead, a positive genetic correlation was found between FOC and PWM-induced IFN-γ production, while a negative genetic correlation was found between FOC and T-ci induced IL-10. Live weight was negatively genetically correlated with IFN-γ responses. Overall, IFN-γ and IL-4 responses were positively correlated, providing little evidence of cross-regulation of Th1 and Th2 immunity within individual sheep. Furthermore, T-ci specific IgA was highly positively correlated with PWM-induced IL-10, indicating a possible role for this cytokine in IgA production. Our results suggest that while genetic selection for adaptive immune response traits is possible and may be beneficial for parasite control, selection of high IFN-γ responsiveness may negatively affect productivity., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Gastrointestinal parasitism represents a global problem for grazing ruminants, which can be addressed sustainably by breeding animals to be more resistant against infection by parasites. The aim of this study was to assess the genetic architecture underlying traits associated with gastrointestinal parasite resistance, immunological profile and production in meat sheep, and identify and characterise candidate genes affecting these traits. Data on gastrointestinal parasite infection (faecal egg counts for Strongyles (FEC S ) and Nematodirus (FEC N ) and faecal oocyst counts for Coccidia, along with faecal soiling scores (DAG), characterised by the accumulation of faeces around the perineum) and production (live weight (LWT)) were gathered from a flock Scottish Blackface lambs at three and four months of age. Data on the immune profile were also collected from a subset of these lambs at two and five months of age. Immune traits included the production of Interferon-γ (IFN-γ), Interleukin (IL)-4 and IL-10 following stimulation of whole blood with pokeweed mitogen (PWM) or antigen from the gastric parasite Teladorsagia circumcincta (T-ci), and serum levels of T. circumcincta-specific immunoglobulin A (IgA). Animals were genotyped with genome-wide DNA arrays, and a total of 1 766 animals and 45 827 Single Nucleotide Polymorphisms (SNPs) were retained following quality control and imputation. Genome-wide association studies were performed for 24 traits. The effects of individual markers with significant effects were estimated, and the genotypic effect solutions were used to estimate additive and dominance effects, and the proportion of additive genetic variance attributed to each SNP locus. A total of 15 SNPs were associated at least at a suggestive level with FEC S , FEC N , DAG, IgA, PWM-induced IFN-γ and IL-4, and T-ci-induced IL-10. This study uncovered 52 genes closely related to immune function in proximity to these SNPs. A number of genes encoding C-type lectins and killer cell lectin-like family members were close to a SNP associated with FEC N, while several genes encoding IL-1 cytokine family members were found to be associated with IgA. Potential candidate genes belonging to or in close proximity with the Major Histocompatibility Complex (MHC) were revealed, including Homeostatic Iron Regulator and butyrophilin coding genes associated with IFN-γ (PWM) , and IL-17 coding genes associated with IgA. Due to the importance of the MHC in the control of immune responses, these genes may play an important role in resistance to parasitic infections. Our results reveal a largely complex and polygenic genetic profile of the studied traits in this Scottish Blackface sheep population., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This work discusses and demonstrates the novel use of multivariate analysis and data dimensionality reduction techniques to handle the variety and complexity of data generated in efficacy trials for the development of a prototype vaccine to protect sheep against the Teladorsagia circumcincta nematode. A curated collection of data dimension reduction and visualisation techniques, in conjunction with sensible statistical modelling and testing which explicitly model key features of the data, offers a synthetic view of the relationships between the multiple biological parameters measured. New biological insight is gained into the patterns and associations involving antigen-specific antibody levels, antibody avidity and parasitological parameters of efficacy that is not achievable by standard statistical practice in the field. This approach can therefore be used to guide vaccine refinement and simplification through identifying the most immunologically relevant antigens, and it can be analogously implemented for similar studies in other areas. To facilitate this, the associated data and computer codes written for the R open system for statistical computing are made freely available., (© 2023. The Author(s).)

The rectal-anal junction (RAJ) is the major colonization site of Shiga toxin-producing Escherichia coli (STEC) O157 in beef cattle, leading to transmission of this foodborne pathogen from farms to food chains. To date, there is limited understanding regarding whether the mucosa-attached microbiome has a profound impact on host-STEC interactions. In this study, the active RAJ mucosa-attached microbiota and its potential role in host immunity-STEC commensal interactions were investigated using RAJ mucosal biopsies collected from calves orally challenged with two STEC O157 strains with or without functional stx2a (stx2a+ or stx2a-). The results revealed that shifts of microbial diversity, topology, and assembly patterns were subjected to stx2a production post-challenge and Paeniclostridium and Gallibacterium were the keystone taxa for both microbial interactions and assembly. Additional mucosal transcriptome profiling showed stx2a-dependent host immune responses (i.e. B- and T-cell signaling and antigen processing and presentation) post-challenge. Further integrated analysis revealed that mucosa-attached beneficial microbes (i.e. Provotella, Faecalibacterium, and Dorea) interacted with host immune genes pre-challenge to maintain host homeostasis; however, opportunistic pathogenic microbes (i.e. Paeniclostridium) could interact with host immune genes after the STEC O157 colonization and interactions were stx2a-dependent. Furthermore, predicted bacterial functions involved in pathogen (O157 and Paeniclostridium) colonization and metabolism were related to host immunity. These findings suggest that during pathogen colonization, host-microbe interactions could shift from beneficial to opportunistic pathogenic bacteria driven and be dependent on the production of particular virulence factors, highlighting the potential regulatory role of mucosa-attached microbiota in affecting pathogen-commensal host interactions in calves with STEC O157 infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Gastrointestinal nematodes (GIN) have enormous global impacts in humans, wildlife and grazing livestock. Within grazing livestock, sheep are of particular global importance and the economics and sustainability of sheep production are greatly constrained by GIN infections. Natural infections are composed of co-infections with multiple species, and while some past work suggests species can interact negatively with one another within the same host, there is wide variation in reported patterns. Here, we undertook a systematic literature search and meta-analysis of experimental GIN co-infections of sheep to determine whether these experimental studies support the hypothesis of antagonistic interactions between different co-infecting GIN, and test whether aspects of parasite biology or experimental design influence the observed effects. A systematic search of the literature yielded 4848 studies, within which, we identified 19 experimental sheep studies comparing post-mortem worm counts across two co-infecting GIN species. Meta-analysis of 67 effects obtained from these studies provides strong evidence for interactions between GIN species. There was wide variation in the strength and direction of these interactions, but the global effect was significantly antagonistic. On average, there was a decrease in the number of worms of one species when a co-infecting species was also present, relative to a mono-infection with that species alone. This effect was dependent on the infectious dose and was rapidly lost after anthelmintic treatment, suggesting that live worms are required for the effect to occur. Individual parasite species varied in the extent to which they both exerted, and were subject to, these interspecies interactions, and these differences are more complex than simply co-localisation within the gastrointestinal tract. Antagonistic interactions between co-infecting GIN may feedback into their epidemiology as well as potentially affecting the clinical impacts of infection. Furthermore, the consequences of these interactions may be heightened when clinical interventions affect only one species within the co-infecting network. Whilst it was not possible to identify the causes of variation between GIN species in the impact of co-infection, these findings point to new avenues for epidemiological, clinical and mechanistic research on GIN co-infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests., Competing Interests: Authors ES, RP, and JT were employed by the company Nimble Therapeutics, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bach, Fitzgerald, Williams-MacDonald, Mitchell, Golde, Longbottom, Nisbet, Dinkla, Sullivan, Pinapati, Tan, Joosten, Roest, Østerbye, Koets, Buus and McNeilly.)

Tuft cells have recently emerged as the focus of intense interest following the discovery of their chemosensory role in the intestinal tract, and their ability to activate Type 2 immune responses to helminth parasites. Moreover, they populate a wide range of mucosal tissues and are intimately connected to immune and neuronal cells, either directly or through the release of pharmacologically active mediators. They are now recognised to fulfil both homeostatic roles, in metabolism and tissue integrity, as well as acting as the first sensors of parasite infection, immunity to which is lost in their absence. In this review we focus primarily on the importance of tuft cells in the intestinal niche, but also link to their more generalised physiological role and discuss their potential as targets for the treatment of gastrointestinal disorders.

Shiga toxin-producing E. coli (STEC) infections associated with wildlife are increasing globally, highlighting many 'spillover' species as important reservoirs for these zoonotic pathogens. A human outbreak of STEC serogroup O157 in 2015 in Scotland, associated with the consumption of venison meat products, highlighted several knowledge gaps, including the prevalence of STEC O157 in Scottish wild deer and the potential risk to humans from wild deer isolates. In this study, we undertook a nationwide survey of wild deer in Scotland and determined that the prevalence of STEC O157 in wild deer is low 0.28% (95% confidence interval = 0.06-0.80). Despite the low prevalence of STEC O157 in Scottish wild deer, identified isolates were present in deer faeces at high levels (>10 4 colony forming units/g faeces) and had high human pathogenic potential based on whole genome sequencing and virulence gene profiling. A retrospective epidemiological investigation also identified one wild deer isolate from this study as a possible source of a Scottish human outbreak in 2017. These results emphasise the importance of food hygiene practices during the processing of wild deer carcasses for human consumption.

For the last two decades, the human infection frequency of Escherichia coli O157 (O157) in Scotland has been 2.5-fold higher than in England and Wales. Results from national cattle surveys conducted in Scotland and England and Wales in 2014/2015 were combined with data on reported human clinical cases from the same time frame to determine if strain differences in national populations of O157 in cattle could be associated with higher human infection rates in Scotland. Shiga toxin subtype (Stx) and phage type (PT) were examined within and between host (cattle vs human) and nation (Scotland vs England and Wales). For a subset of the strains, whole genome sequencing (WGS) provided further insights into geographical and host association. All three major O157 lineages (I, II, I/II) and most sub-lineages (Ia, Ib, Ic, IIa, IIb, IIc) were represented in cattle and humans in both nations. While the relative contribution of different reservoir hosts to human infection is unknown, WGS analysis indicated that the majority of O157 diversity in human cases was captured by isolates from cattle. Despite comparable cattle O157 prevalence between nations, strain types were localized. PT21/28 (sub-lineage Ic, Stx2a+) was significantly more prevalent in Scottish cattle [odds ratio (OR) 8.7 (2.3-33.7; P <0.001] and humans [OR 2.2 (1.5-3.2); P <0.001]. In England and Wales, cattle had a significantly higher association with sub-lineage IIa strains [PT54, Stx2c; OR 5.6 (1.27-33.3); P =0.011] while humans were significantly more closely associated with sub-lineage IIb [PT8, Stx1 and Stx2c; OR 29 (4.9-1161); P <0.001]. Therefore, cattle farms in Scotland were more likely to harbour Stx2a+O157 strains compared to farms in E and W ( P <0.001). There was evidence of limited cattle strain migration between nations and clinical isolates from one nation were more similar to cattle isolates from the same nation, with sub-lineage Ic (mainly PT21/28) exhibiting clear national association and evidence of local transmission in Scotland. While we propose the higher rate of O157 clinical cases in Scotland, compared to England and Wales, is a consequence of the nationally higher level of Stx2a+O157 strains in Scottish cattle, we discuss the multiple additional factors that may also contribute to the different infection rates between these nations.

Teladorsagia circumcincta is an abomasal parasitic nematode that can cause serious issues in small ruminant production, which are aggravated by drug resistance. Vaccines have been suggested as a feasible, long-lasting alternative for control since adaptation to the host's immune mechanisms by helminths develops at a much slower pace than anthelmintic resistance. Recently, a T. circumcincta recombinant subunit vaccine yielded over a 60% reduction in egg excretion and worm burden and induced strong humoral and cellular anti-helminth responses in vaccinated 3-month-old Canaria Hair Breed (CHB) lambs, but Canaria Sheep (CS) of a similar age were not protected by the vaccine. Here, we compared the transcriptomic profiles in the abomasal lymph nodes of such 3-month-old CHB and CS vaccinates 40 days after infection with T. circumcincta to understand differences in responsiveness at the molecular level. In the CS, differentially expressed genes (DEG) identified were related to general immunity processes such as antigen presentation or antimicrobial proteins and down-regulation of inflammation and immune response through regulatory T cell-associated genes. However, upregulated genes in CHB vaccinates were associated with type-2 oriented immune responses, i.e., immunoglobulin production, activation of eosinophils, as well as tissue structure and wound repair-related genes and protein metabolism pathways such as DNA and RNA processing. These results highlight potentially more optimal timing and orientation of immune responses in CHB sheep compared to CS associated with vaccine-induced protection. The data obtained in this study thus deepens our understanding of variations in responsiveness to vaccination in young lamb and provides insights for vaccine refinement strategies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jacqueline B. Matthews reports financial support was provided by European Commission. Tara Perez-Hernandez reports financial support was provided by Cabildo de Gran Canaria. Cynthia Machin reports financial support was provided by Agencia Canaria de Investigación, Innovación y Sociedad de la Información and European Social Fund. Julia N. Hernandez reports financial support was provided by Agencia Canaria de Investigación, Innovación y Sociedad de la Información. Tom N. McNeilly, Alasdair J. Nisbet, Stewart T. G. Burgess reports financial support was provided by Scottish Government Rural & Environment Science & Analytical Services Strategic Research Programme 2022–2027., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1β and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The bacterium Coxiella burnetii can cause the disease Q-fever in a wide range of animal hosts. Ruminants, including sheep, are thought to play a pivotal role in the transmission of C. burnetii to humans; however, the only existing livestock vaccine, namely, Coxevac ® (Ceva Animal Health Ltd., Libourne, France), a killed bacterin vaccine based on phase I C. burnetii strain Nine-Mile, is only approved for use in goats and cattle. In this study, a pregnant ewe challenge model was used to determine the protective effects of Coxevac ® and an experimental bacterin vaccine based on phase II C. burnetii against C. burnetii challenge. Prior to mating, ewes ( n = 20 per group) were vaccinated subcutaneously with either Coxevac ® , the phase II vaccine, or were unvaccinated. A subset of pregnant ewes ( n = 6) from each group was then challenged 151 days later (~100 days of gestation) with 10 6 infectious mouse doses of C. burnetii , Nine-Mile strain RSA493. Both vaccines provided protection against C. burnetii challenge as measured by reductions in bacterial shedding in faeces, milk and vaginal mucus, and reduced abnormal pregnancies, compared to unvaccinated controls. This work highlights that the phase I vaccine Coxevac ® can protect ewes against C. burnetii infection. Furthermore, the phase II vaccine provided comparable levels of protection and may offer a safer and cost-effective alternative to the currently licensed vaccine.

The increasing resistance to anthelmintics has necessitated the exploration of alternative control strategies of gastrointestinal nematode (GIN) infections. A sustainable option is genetic selection based on differences in susceptibility to GIN infection between and within breeds of sheep. Here, three-month-old Canaria Hair breed (GIN-resistant) and Canaria Sheep breed (GIN-susceptible) showed no significant between-breed differences after trickle infection with Teladorsagia circumcincta , whereas considerable individual variability was found in both breeds. Next, data from lambs of both breeds were used to explore the relationships between parasitological variables and T. circumcincta -specific IgA levels, local immune cell populations, and abomasal lymph node gene expression to understand the possible mechanisms underlying resistance. Mucosal IgA levels as well as numbers of globular leukocytes and MHC-II + cells were associated with protection. Analysis of lymph node gene expression revealed the associations between lower parasite numbers and cumulative fecal egg counts and several immune pathways, such as leukocyte cell adhesion, activation and differentiation of T cells, in particular CD4 + and IL-4 production. The data obtained here may inform on the relationship between phenotypic resistance variability and protective responses at the humoral, cellular, and transcriptomic levels, thus contributing to identifying immune responses in young lambs that could be used as markers for selection.

Wild deer hunting is necessary in Scotland to control deer population density, with most carcasses being processed for human consumption. As limited information is available on the microbial condition of Scottish venison, we studied the variation of total coliforms and Escherichia coli (E. coli) on 214 wild deer carcasses collected from six approved establishments. Samples were collected from the hide, body cavity and external surface of each carcass and mean values were determined following bacterial plate counts. The mean log 10 /cm 2 coliforms were 5.78 (hide), 6.80 (body cavity) and 6.36 (external surface). The mean log 10 /cm 2 E. coli were 1.82 (hide), 2.27 (body cavity) and 2.17 (external carcass). Significantly higher coliforms counts were associated with storage-to-dressing times above 6 days and with longer transport distances. Risk factors that increased E. coli were red deer species, ambient temperature above 7 °C during hunting, dirty hides, faecal contamination and moisture or slimy film on the carcass. Although the bacterial counts obtained in this study indicated some hygienic processing, for around half of the carcasses, the E. coli counts were above 2 log 10 /cm 2 . Therefore, the above risk factors suggest a few handling hygiene practices that should be further improved to enhance quality and safety., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present study investigated the potential consequences, positive or negative, that selection for favorable production-related traits may have on concentrations of vitamin B 12 and key chemical elements in dairy cow milk and serum and the possible impact on milk healthiness, and associated benefits, for the dairy product consumer. Milk and serum samples (950 and 755, respectively) were collected from Holstein-Friesian dairy cows (n = 479) on 19 occasions over a 59-mo period, generating 34,258 individual records, and analyzed for concentrations of key trace and quantity elements, heavy metals, and milk vitamin B 12 . These data were then matched to economically important production data (milk, fat, and protein yield) and management data (dry matter intake, liveweight, and body condition score). Multivariate animal models, including full pedigree information, were used to analyze data and investigate relationships between traits of interest. Results highlighted negative genetic correlations between many quantity and trace elements in both milk and serum with production and management traits. Milk yield was strongly negatively correlated with the milk quantity elements Mg and Ca (genetic correlation between traits, r a = -0.58 and -0.63, respectively) as well as the trace elements Mn, Fe, Ni, Cu, Zn, and Mo (r a = -0.32, -0.58, -0.52, -0.40, -0.34, and -0.96, respectively); and in serum, Mg, Ca, Co, Fe, and Zn (r a = -0.50, -0.36, -0.68, -0.54, and -0.90, respectively). Strong genetic correlations were noted between dry matter intake with V (r a = 0.97), Fe (r a = -0.69), Ni (r a = -0.81), and Zn (r a = -0.75), and in serum, strong negative genetic correlations were observed between dry matter intake with Ca and Se (r a = -0.95 and -0.88, respectively). Body condition score was negatively correlated with serum P, Cu, Se, and Pb (r a = -0.45, -0.35, -0.51, and -0.64, respectively) and positively correlated with Mn, Fe, and Zn (r a = 0.40, 0.71, and 0.55, respectively). Our results suggest that breeding strategies aimed at improving economically important production-related traits would most likely result in a negative impact on levels of beneficial nutrients within milk for human consumption (such as Mg, Ca, Fe, Zn, and Se)., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Monitoring the prevalence and abundance of parasites over time is important for addressing their potential impact on host life histories, immunological profiles and their influence as a selective force. Only long-term ecological studies have the potential to shed light on both the temporal trends in infection prevalence and abundance and the drivers of such trends, because of their ability to dissect drivers that may be confounded over shorter time scales. Despite this, only a relatively small number of such studies exist. Here, we analysed changes in the prevalence and abundance of gastrointestinal parasites in the wild Soay sheep population of St. Kilda across 31 years. The host population density (PD) has increased across the study, and PD is known to increase parasite transmission, but we found that PD and year explained temporal variation in parasite prevalence and abundance independently. Prevalence of both strongyle nematodes and coccidian microparasites increased during the study, and this effect varied between lambs, yearlings and adults. Meanwhile, abundance of strongyles was more strongly linked to host PD than to temporal (yearly) dynamics, while abundance of coccidia showed a strong temporal trend without any influence of PD. Strikingly, coccidian abundance increased 3-fold across the course of the study in lambs, while increases in yearlings and adults were negligible. Our decades-long, intensive, individual-based study will enable the role of environmental change and selection pressures in driving these dynamics to be determined, potentially providing unparalleled insight into the drivers of temporal variation in parasite dynamics in the wild.

Trade-offs between host resistance to parasites and host growth or reproduction can occur due to allocation of limited available resources between competing demands. To predict potential trade-offs arising from genetic selection for host resistance, a better understanding of the associated nutritional costs is required. Here, we studied resistance costs by using sheep from lines divergently selected on their resistance to a common blood-feeding gastro-intestinal parasite ( Haemonchus contortus ). First, we assessed the effects of selection for high or low host resistance on condition traits (body weight, back fat, and muscle thickness) and infection traits (parasite fecal egg excretion and loss in blood haematocrit) at various life stages, in particular during the periparturient period when resource allocation to immunity may limit host resistance. Second, we analysed the condition-infection relationship to detect a possible trade-off, in particular during the periparturient period. We experimentally infected young females in four stages over their first 2 years of life, including twice around parturition (at 1 year and at 2 years of age). Linear mixed-model analyses revealed a large and consistent between-line difference in infection traits during growth and outside of the periparturient period, whereas this difference was strongly attenuated during the periparturient period. Despite their different responses to infection, lines had similar body condition traits. Using covariance decomposition, we then found that the phenotypic relationship between infection and condition was dominated by direct infection costs arising from parasite development within the host. Accounting for these within-individual effects, a cost of resistance on body weight was detected among ewes during their first reproduction. Although this cost and the reproductive constraint on resistance are unlikely to represent a major concern for animal breeding in nutrient-rich environments, this study provides important new insights regarding the nutritional costs of parasite resistance at different lifestages and how these may affect response to selection., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Individuals vary broadly in their response to vaccination and subsequent challenge infection, with poor vaccine responders causing persistence of both infection and transmission in populations. Yet despite having substantial economic and societal impact, the immune mechanisms that underlie such variability, especially in infected tissues, remain poorly understood. Here, to characterise how antihelminthic immunity at the mucosal site of infection developed in vaccinated lambs, we inserted gastric cannulae into the abomasa of three-month- and six-month-old lambs and longitudinally analysed their local immune response during subsequent challenge infection. The vaccine induced broad changes in pre-challenge abomasal immune profiles and reduced parasite burden and egg output post-challenge, regardless of age. However, age affected how vaccinated lambs responded to infection across multiple immune pathways: adaptive immune pathways were typically age-dependent. Identification of age-dependent and age-independent protective immune pathways may help refine the formulation of vaccines, and indicate specificities of pathogen-specific immunity more generally., (© 2022. The Author(s).)

The development of three-dimensional cell culture systems representative of tissues from animals of veterinary interest is accelerating research that seeks to address specific questions tied to animal health. In terms of their relevance and complexity, these in vitro models can be seen as a midpoint between the more reductionist single-cell culture systems and complex live animals. Organoids in particular represent a significant development due to their organised multicellular structure that more closely represents in vivo tissues than any other cell culture technology previously developed. In this review, we provide an overview of the different three-dimensional cell culture systems available to veterinary researchers and give examples of their application in contexts relating to animal health., (© 2022 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Gastro-intestinal nematode (GIN) parasites are a major cause of production losses in grazing cattle, primarily through reduced growth rates in young animals. Control of these parasites relies heavily on anthelmintic drugs; however, with growing reports of resistance to currently available anthelmintics, alternative methods of control are required. A major hurdle in this work has been the lack of physiologically relevant in vitro infection models that has made studying precise interactions between the host and the GINs difficult. Such mechanistic insights into the infection process will be valuable for the development of novel targets for drugs, vaccines, or other interventions. Here we created bovine gastric epithelial organoids from abomasal gastric tissue and studied their application as in vitro models for understanding host invasion by GIN parasites. Transcriptomic analysis of gastric organoids across multiple passages and the corresponding abomasal tissue showed conserved expression of tissue-specific genes across samples, demonstrating that the organoids are representative of bovine gastric tissue from which they were derived. We also show that self-renewing and self-organising three-dimensional organoids can also be serially passaged, cryopreserved, and resuscitated. Using Ostertagia ostertagi , the most pathogenic gastric parasite in cattle in temperate regions, we show that cattle gastric organoids are biologically relevant models for studying GIN invasion in the bovine abomasum. Within 24 h of exposure, exsheathed larvae rapidly and repeatedly infiltrated the lumen of the organoids. Prior to invasion by the parasites, the abomasal organoids rapidly expanded, developing a 'ballooning' phenotype. Ballooning of the organoids could also be induced in response to exposure to parasite excretory/secretory products. In summary, we demonstrate the power of using abomasal organoids as a physiologically relevant in vitro model system to study interactions of O. ostertagi and other GIN with bovine gastrointestinal epithelium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This study received funding from Zoetis. The funder had the following involvement with the study: the decision to submit the manuscript for publication., (Copyright © 2022 Faber, Smith, Price, Steele, Hildersley, Morrison, Mabbott, Nisbet and McNeilly.)

Gastrointestinal nematode (GIN) infections are a serious drawback on small ruminant production. Since anthelmintic resistance has extended, optimisation of alternative non-chemical control strategies has attracted interest. Recently, a prototype recombinant vaccine protected immunologically mature sheep from Texel-cross and Canaria Sheep breeds against Teladorsagia circumcincta. The level of protective immunity stimulated by the vaccine varied between individuals and with age. Previous studies suggest that Canaria Hair Breed (CHB) sheep is naturally resistant to GIN infection, with some evidence suggesting that this protection is present in young lambs. Here, we sought to enhance this resistance by immunising three-month-old CHB lambs with a T. circumcincta prototype recombinant vaccine. Following vaccination and a larval challenge period, levels of protection against T. circumcincta infection were compared in CHB lambs with Canaria Sheep (CS) lambs (a breed considered less resistant to GIN). Lambs from the resistant CHB breed appeared to respond more favourably to vaccination, shedding 63% fewer eggs over the sampling period than unvaccinated CHB lambs. No protection was evident in CS vaccinated lambs. At post-mortem, CHB vaccine recipients had a 68% reduction in mean total worm burden, and female worms were significantly shorter and contained fewer eggs in utero compared to unvaccinated CHB lambs. A higher anti-parasite IgG 2 level was detected in immunised CHB lambs compared to unvaccinated control CHB animals, with data suggesting that IgA, globular leucocytes, CD45RA + , CD4 + and CD8 + T cells are implicated in this protective response. The development of effective immunity in vaccinated CHB lambs did not reduce lamb growth rate as immunised CHB lambs had a significantly higher average daily weight gain after challenge than their unvaccinated counterparts. Therefore, the protection of CHB lambs was enhanced by immunisation at weaning, suggesting a synergistic effect when combining vaccination with presumed genetic resistance., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The adaptive immune system is critical to an effective response to infection in vertebrates, with T-helper (Th) cells pivotal in orchestrating these responses. In natural populations where co-infections are the norm, different Th responses are likely to play an important role in maintaining host health and fitness, a relationship which remains poorly understood in wild animals. In this study, we characterised variation in functionally distinct Th responses in a wild population of Soay sheep by enumerating cells expressing Th-subset specific transcription factors and quantifying Th-associated cytokines. We tested the prediction that raised Th1 and Th2 responses should predict reduced apicomplexan and helminth parasite burdens, respectively. All measures of Th-associated cytokine production increased with age, while Th17- and regulatory Th-associated cytokine production increased more rapidly with age in males than females. Independent of age, sex, and each other, IL-4 and Gata3 negatively predicted gastro-intestinal nematode faecal egg count, while IFN-γ negatively predicted coccidian faecal oocyst count. Our results provide important support from outside the laboratory that Th1 and Th2 responses predict resistance to different kinds of parasites, and illustrate how harnessing specific reagents and tools from laboratory immunology will illuminate our understanding of host-parasite interactions in the wild., (© 2022. The Author(s).)

The liver fluke Fasciola hepatica is an economically important global pathogen of humans and their livestock. To facilitate host invasion and migration, F. hepatica secretes an abundance of cathepsin peptidases but prevents excessive damage to both parasite and host tissues by co-secreting regulatory peptidase inhibitors, cystatins/stefins and Kunitz-type inhibitors. Here, we report a vaccine strategy aimed at disrupting the parasite's protease/anti-protease balance by targeting these key inhibitors. Our vaccine cocktail containing three recombinant stefins (rFhStf-1, rFhStf-2, rFhStf-3) and a Kunitz-type inhibitor (rFhKT1) formulated in adjuvant Montanide 61VG was assessed in two independent sheep trials. While fluke burden was not reduced in either trial, in Trial 1 the vaccinated animals showed significantly greater weight gain ( p < 0.05) relative to the non-vaccinated control group. In both trials we observed a significant reduction in egg viability (36-42%). Multivariate regression analyses showed vaccination and increased levels of IgG2 antibodies specific for the F. hepatica peptidase inhibitors were positive indicators for increased weight gain and levels of haemoglobin within the normal range at 16 weeks post-infection (wpi; p < 0.05). These studies point to the potential of targeting peptidase inhibitors as vaccine cocktails for fasciolosis control in sheep.

This review article provides an historical perspective on some of the major research advances of relevance to ruminant livestock gastrointestinal nematode control over the last 50 years. Over this period, gastrointestinal nematode control has been dominated by the use of broad-spectrum anthelmintic drugs. Whilst this has provided unprecedented levels of successful control for many years, this approach has been gradually breaking down for more than two decades and is increasingly unsustainable which is due, at least in part, to the emergence of anthelmintic drug resistance and a number of other factors discussed in this article. We first cover the remarkable success story of the discovery and development of broad-spectrum anthelmintic drugs, the changing face of anthelmintic drug discovery research and the emergence of anthelmintic resistance. This is followed by a review of some of the major advances in the increasingly important area of non-pharmaceutical gastrointestinal nematode control including immunology and vaccine development, epidemiological modelling and some of the alternative control strategies such as breeding for host resistance, refugia-based methods and biological control. The last 50 years have witnessed remarkable innovation and success in research aiming to improve ruminant livestock gastrointestinal nematode control, particularly given the relatively small size of the research community and limited funding. In spite of this, the growing global demand for livestock products, together with the need to maximise production efficiencies, reduce environmental impacts and safeguard animal welfare - as well as specific challenges such as anthelmintic drug resistance and climate change- mean that gastrointestinal nematode researchers will need to be as innovative in the next 50 years as in the last., (Copyright © 2021 Australian Society for Parasitology. All rights reserved.)

Helminth parasite infections of humans and livestock are a global health and economic problem. Resistance of helminths to current drug treatment is an increasing problem and alternative control approaches, including vaccines, are needed. Effective vaccine design requires knowledge of host immune mechanisms and how these are stimulated. Mouse models of helminth infection indicate that tuft cells, an unusual type of epithelial cell, may 'sense' infection in the small intestine and trigger a type 2 immune response. Currently nothing is known of tuft cells in immunity in other host species and in other compartments of the gastrointestinal (GI) tract. Here we address this gap and use immunohistochemistry and single cell RNA-sequencing to detail the presence and gene expression profile of tuft cells in sheep following nematode infections. We identify and characterize tuft cells in the ovine abomasum (true stomach of ruminants) and show that they increase significantly in number following infection with the globally important nematodes Teladorsagia circumcincta and Haemonchus contortus . Ovine abomasal tuft cells show enriched expression of tuft cell markers POU2F3, GFI1B, TRPM5 and genes involved in signaling and inflammatory pathways. However succinate receptor SUCNR1 and free fatty acid receptor FFAR3 , proposed as 'sensing' receptors in murine tuft cells, are not expressed, and instead ovine tuft cells are enriched for taste receptor TAS2R16 and mechanosensory receptor ADGRG6. We also identify tuft cell sub-clusters at potentially different stages of maturation, suggesting a dynamic process not apparent from mouse models of infection. Our findings reveal a tuft cell response to economically important parasite infections and show that while tuft cell effector functions have been retained during mammalian evolution, receptor specificity has diverged. Our data advance knowledge of host-parasite interactions in the GI mucosa and identify receptors that may potentiate type 2 immunity for optimized control of parasitic nematodes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hildersley, McNeilly, Gillan, Otto, Löser, Gerbe, Jay, Maizels, Devaney and Britton.)

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.

Liver flukes (Fasciola spp.) are important parasites of ruminant livestock worldwide, causing profound damage to animal health and productivity. Many reviews have discussed the results of decades of research on the impact of fluke on livestock traits such as weight gain and milk production, but there have been no known attempts to collate previous research in a quantitative manner or to determine the factors that vary between studies that find substantial effects of fluke and others concluding that effects of fluke are negligible. Here, we use meta-analysis to provide quantitative "global" estimates of the impact of liver fluke on animal performance, and to identify elements of study design ("moderators") that influence variation between studies in their outcome. A literature search provided 233 comparisons of performance in fluke-infected and uninfected animals. We standardised these data as log response ratios and calculated effect size variances to weight studies by the accuracy of their estimates. We performed multi-level meta-analysis to estimate effects of fluke infection on daily weight gain, live weight, carcass weight, total weight gain and milk production. There were statistically-supported negative effects of fluke infection on daily weight gain, live weight and carcass weight (9%, 6% and 0.6% reductions, respectively), but not on total weight gain or milk production. A mixed-effects meta-analysis revealed that studies of younger animals found more severe effects of fluke on weight gain and that effects on live weight increased with time since infection. Limitations to the data that could be analysed, including a lack of statistical reporting in older papers and variation in the outcome variables measured, may have explained the relatively limited influence of modifiers that we detected. Our results provide, to our knowledge, the first quantitative estimate of the impact of liver fluke on performance across studies and highlight some elements of study design that can influence conclusions., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta , demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smith, Price, Burrells, Faber, Hildersley, Chintoan-Uta, Chapuis, Stevens, Stevenson, Burgess, Innes, Nisbet and McNeilly.)

Objectives: Studies that measure the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ('seroprevalence') are essential to understand population exposure to SARS-CoV-2 among symptomatic and asymptomatic individuals. We aimed to measure seroprevalence in the Scottish population over the course of the COVID-19 pandemic - from before the first recorded case in Scotland through to the second pandemic wave., Study Design: The study design of this study is serial cross sectional., Methods: We tested 41,477 residual samples retrieved from primary and antenatal care settings across Scotland for SARS-CoV-2 antibodies over a 12-month period from December 2019-December 2020 (before rollout of COVID-19 vaccination). Five-weekly rolling seroprevalence estimates were adjusted for the sensitivity and specificity of the assays and weighted to reference populations. Temporal trends in seroprevalence estimates and weekly SARS-CoV-2 notifications were compared., Results: Five-weekly rolling seroprevalence rates were 0% until the end of March, when they increased contemporaneously with the first pandemic wave. Seroprevalence rates remained stable through the summer (range: 3%-5%) during a period of social restrictions, after which they increased concurrently with the second wave, reaching 9.6% (95% confidence interval [CI]: 8.4%-10.8%) in the week beginning 28th December in 2020. Seroprevalence rates were lower in rural vs. urban areas (adjusted odds ratio [AOR]: 0.70, 95% CI: 0.61-0.79) and among individuals aged 20-39 years and 60 years and older (AOR: 0.74, 95% CI: 0.64-0.86; AOR: 0.80, 95% CI: 0.69-0.91, respectively) relative to those aged 0-19 years., Conclusions: After two waves of the COVID-19 pandemic, less than one in ten individuals in the Scottish population had antibodies to SARS-CoV-2. Seroprevalence may underestimate the true population exposure as a result of waning antibodies among individuals who were infected early in the first wave., (Copyright © 2021 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.)

The immunomodulatory capacity of F. hepatica antigens is probably one of the main reasons for the development of a driven non-protective Th2 immune response. In this study, we analysed the cellular response of hepatic lymph node cells and CD4 + T cells in terms of proliferative response, efficiency of antigen presentation and cytokine production, to F. hepatica-derived molecules, at early and late stages of the infection. Thirty-one sheep were allocated into five groups and were slaughtered at 16 dpi and 23 wpi. In order to analyse antigen-specific response, the following F. hepatica recombinant molecules were used: rFhCL1, rFhCL2, rFhCL3, rFhCB1, rFhCB2, rFhCB3, rFhStf-1, rFhStf-2, rFhStf-3 and rFhKT1. A cell proliferation assay using hepatic lymph node cells and an antigen presentation cell assay using CD4 + T cells were performed. At 16 dpi, all molecules but rFhStf-2 and rFhKT1 elicited a significant cell proliferative response on hepatic lymph node cells of infected animals. At both early and late stage of the infection, antigen presentation of rFhCB3 and rFhCL2 resulted in higher stimulation index of CD4 + T cells which was IL-2 mediated, although no statistically significant when compared to uninfected animals. Significant cytokine production (IL-4, IL-10 and IFN-γ) was conditioned by the antigen-specific cell stimulation. No CD4 + T cell exhaustion was detected in infected sheep at the chronic stage of the infection. This study addressed antigen-specific response to F. hepatica-derived molecules that are involved in key aspects of the parasite survival within the host.

Due to increased anthelmintic resistance, complementary methods to drugs are necessary to control gastrointestinal nematodes (GIN). Vaccines are an environmentally-friendly and promising option. In a previous study, a Teladorsagia circumcincta recombinant sub-unit vaccine was administered to two sheep breeds with different levels of resistance against GIN. In the susceptible Canaria Sheep (CS) breed, vaccinates harboured smaller worms with fewer eggs in utero than the control group. Here, we extend this work, by investigating the cellular and humoral immune responses of these two sheep breeds following vaccination and experimental infection with T. circumcincta. In the vaccinated CS group, negative associations between antigen-specific IgA, IgG 2 and Globule Leukocytes (GLs) with several parasitological parameters were established as well as a higher CD4 + /CD8 + ratio than in control CS animals, suggesting a key role in the protection induced by the vaccine. In the more resistant Canaria Hair Breed (CHB) sheep the vaccine did not significantly impact on the parasitological parameters studied and none of these humoral associations were observed in vaccinated CHB lambs, although CHB had higher proportions of CD4 + and CD8 + T cells within the abomasal lymph nodes, suggesting higher mucosal T cell activation. Each of the component proteins in the vaccine induced an increase in immunoglobulin levels in vaccinated groups of each breed. However, levels of immunoglobulins to only three of the antigens (Tci-MEP-1, Tci-SAA-1, Tci-ASP-1) were negatively correlated with parasitological parameters in the CS breed and they may be, at least partially, responsible for the protective effect of the vaccine in this breed. These data could be useful for improving the current vaccine prototype.

Background: Growing evidence points towards a role of gastrointestinal (GI) helminth parasites of ruminants in modifying the composition of the host gut flora, with likely repercussions on the pathophysiology of worm infection and disease, and on animal growth and productivity. However, a thorough understanding of the mechanisms governing helminth-microbiota interactions and of their impact on host health and welfare relies on reproducibility and replicability of findings. To this aim, in this study, we analysed quantitative and qualitative fluctuations in the faecal microbiota composition of lambs vaccinated against, and experimentally infected with, the parasitic GI nematode Teladorsagia circumcincta over the course of two separate trials performed over two consecutive years., Methods: Two trials were conducted under similar experimental conditions in 2017 and 2018, respectively. In each trial, lambs were randomly assigned to one of the following experimental groups: (i) vaccinated/infected, (ii) unvaccinated/infected and (iii) unvaccinated/uninfected. Faecal samples collected from individual animals were subjected to DNA extraction followed by high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene and bioinformatics and biostatistical analyses of sequence data., Results: Substantial differences in the populations of bacteria affected by immunisation against and infection by T. circumcincta were detected when comparing data from the two trials. Nevertheless, the abundance of Prevotella spp. was significantly linked to helminth infection in both trials., Conclusions: Despite the largely conflicting findings between the two trials, our data revealed that selected gut microbial populations are consistently affected by T. circumcincta infection and/or vaccination. Nevertheless, our study calls for caution when interpreting data generated from in vivo helminth-microbiome interaction studies that may be influenced by several intrinsic and extrinsic host-, parasite- and environment-related factors.

Gastrointestinal parasitism is a global problem for grazing ruminants which can be addressed in a sustainable way through breeding animals to be more resistant to disease. This study estimates the genetic parameters of common and new disease phenotypes associated with natural nematode and coccidian infection in Scottish Blackface sheep to underpin future genetic improvement strategies for parasite control. Data on faecal egg counts (FEC) from different species of strongyle parasites and faecal oocyst counts (FOC) from coccidian parasites were collected on 3-month-old lambs together with a faecal soiling score in the breech area dagginess (DAG) and live weight (LWT). Faecal count data were obtained for Strongyles (FEC S ), Nematodirus (FEC N ) and Coccidia (FOC). Data from 3 731 lambs sampled between 2011 and 2017 were included. Faecal egg counts and DAG records were log-transformed prior to analysis. Data were analysed using linear mixed models. Average age at sampling was 92 days with a mean LWT of 24.5 kg. Faecal soiling was not evident in 69% of lambs. Coccidia were the most prevalent parasite (99.5%), while Strongyles and Nematodirus had a prevalence of 95.4% and 72.7%, respectively. Heritability estimates (±SE) were 0.16 ± 0.03, 0.17 ± 0.03, 0.09 ± 0.03, 0.09 ± 0.03 and 0.33 ± 0.04 for FEC S , FEC N , FOC, DAG and LWT, respectively. Strongyles faecal egg count had a strong and positive genetic correlation with FEC N (0.74 ± 0.09) and a moderate positive correlation with FOC (0.39 ± 0.15) while DAG was negatively genetically correlated with LWT (-0.33 ± 0.15). The significant positive genetic correlations between FEC S , FEC N and FOC at 3 months of age show that co-selection of sheep for resistance to these different parasites is feasible. Selection for increased resistance to parasite infection is not expected to adversely affect live BW, as no significant antagonistic genetic correlations were found between LWT and FEC. There were significant antagonistic phenotypic and genetic relationships between DAG and LWT being -0.19 ± 0.02 and -0.33 ± 0.15, respectively, indicating that the expression of the manifestation of disease in lambs may be a more meaningful indicator of the impact of parasite burden on productivity., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

A wealth of research is dedicated to understanding how resistance against parasites is conferred and how parasite-driven pathology is regulated. This research is in part driven by the hope to better treatments for parasitic diseases of humans and livestock, and in part by immunologists who use parasitic infections as biomedical tools to evoke physiological immune responses. Much of the current mechanistic knowledge has been discovered in laboratory studies using model organisms, especially the laboratory mouse. However, wildlife are also hosts to a range of parasites. Through the study of host-parasite interactions in these non-laboratory systems we can gain a deeper understanding of parasite immunology in a more natural, complex environment. With a focus on helminth parasites, we here explore the insights gained into parasite-induced immune responses through (for immunologists) non-conventional experimental systems, and how current core findings from laboratory studies are reflected in these more natural conditions. The quality of the immune response is undoubtedly a central player in susceptibility versus resistance, as many laboratory studies have shown. Yet, in the wild, parasite infections tend to be chronic diseases. Whilst reading our review, we encourage the reader to consider the following questions which may (only) be answered by studying naturally occurring parasites in the wild: a) what type of immune responses are mounted against parasites in different hosts in the wild, and how do they vary within an individual over time, between individuals of the same species and between species? b) can we use wild or semi-wild study systems to understand the evolutionary drivers for tolerance versus resistance towards a parasite? c) what determines the ability of the host to cope with an infection and is there a link with the type of immune response mounted? d) can we modulate environmental factors to manipulate a wild animal's immune response to parasitic infections, with translation potential for humans, wildlife, and livestock? and e) in context of this special issue, what lessons for Type 2 immunity can we glean from studying animals in their natural environments? Further, we aim to integrate some of the knowledge gained in semi-wild and wild settings with knowledge gained from traditional laboratory-based research, and to raise awareness for the opportunities (and challenges) that come with integrating a multitude of naturally-occurring variables into immunoparasitological research., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Liver flukes (Fasciola spp.) are important helminth parasites of livestock globally and cause substantial reductions in health and productivity of beef cattle. Attempts to control fluke have been thwarted by the difficulty of vaccine design, the evolution of flukicide resistance and the need to control the intermediate snail host. Mechanisms to reduce the impact of parasites on animal performance have typically focused on promoting host resistance - defined as the ability of the host to kill and remove the parasite from its system - and such strategies include improving protein nutrition or selective breeding for resistance. Organisms, however, have another broad mechanism for mitigating the impact of parasites: they can show tolerance, defined as the ability to maintain health or performance under increasing parasite burden. Tolerance has been studied in the plant literature for over a century, but there are very few empirical studies of parasite tolerance in livestock. In this study, we used data collected from >90 000 beef cattle to estimate the impact of the severity of liver fluke infection on performance and variation in tolerance of fluke. Severity of liver fluke infection was estimated using liver "fibrosis score" on a scale of 0-3 and performance estimated as (1) age at slaughter and (2) daily dead weight gain. Animals with higher fibrosis scores were slaughtered around 2 weeks later than animals with no fluke and gained around 10 g less weight per day. There was also considerable variation in these effects of fibrosis score, such that animals from different producers and breeds varied in their tolerance of fluke infection. While breeds did not vary in the association between fibrosis and age at slaughter, there was considerable variation among producers: high fibrosis score delayed slaughter by up to 50 days in some producers, but not at all in others. Meanwhile, there was support for variation in the slope of daily dead weight gain on fibrosis score among both breeds and producers, with some unaffected by high fluke scores and some breeds and producers experiencing a 20 g/day lower weight gain under high fluke scores. Our results point to the potential for both environmental and genetic variation in tolerance of liver fluke in cattle, paving the way for quantitative genetic and nutritional research into the feasibility of promoting tolerance as a disease mitigation strategy., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)