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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.

Abstract 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.

IntroductionWe previously demonstrated efficacy of an 8-antigen recombinant subunit vaccine against a single species homologous Teladorsagia circumcincta challenge in lambs and in lambing ewes in pen trials. We subsequently demonstrated efficacy of a simplified, 2-antigen, version of this vaccine in lambs in pen trials. Here, we test both vaccines in lambing ewes in a field setting.MethodsIn the work presented here, 12 adjacent plots were seeded with a mixed infection of several common species of parasitic nematodes of sheep in temperate regions, including T. circumcincta. Ewes (n = 144), in groups of 12, grazed for 2 years on these plots and, in the first year, six of these groups of ewes were vaccinated with a 2-antigen prototype vaccine against T. circumcincta prior to mating and then again prior to lambing. In the following year these ewes were immunised again, this time with the 8-antigen prototype vaccine against T. circumcincta prior to mating and then prior to lambing. Throughout both seasons antigen-specific serum antibody levels in ewes and faecal worm egg counts (FEC) in ewes and their lambs were monitored, along with nematode species diversity at lambing.ResultsImmunised ewes produced elevated serum antibody levels to each of the vaccine antigens following immunisation but their FEC levels were not statistically significantly impacted by vaccination with either vaccine. FEC levels were also not impacted in lambs co-grazing the pastures with these immunised ewes. Nematode species diversity was not significantly impacted by vaccination in either year.DiscussionThe immunosuppressive effects of co-infecting gastrointestinal nematodes, the absence of vaccine cross-protection against co-infecting species and the influence of the periparturient relaxation in immunity probably all contributed to the inability of either vaccine to protect against T. circumcincta infection in field trials in the work presented here.

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.

Abstract 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.

Abstract 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.

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.

Abstract 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.

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 (>104 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.

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.

Abstract 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.

Abstract 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, IgG2 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.

Abstract 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.

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.

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 106 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.

Abstract Background The multifaceted interactions between gastrointestinal (GI) helminth parasites, host gut microbiota and immune system are emerging as a key area of research within the field of host-parasite relationships. In spite of the plethora of data available on the impact that GI helminths exert on the composition of the gut microflora, whether alterations of microbial profiles are caused by direct parasite-bacteria interactions or, indirectly, by alterations of the GI environment (e.g. mucosal immunity) remains to be determined. Furthermore, no data is thus far available on the downstream roles that qualitative and quantitative changes in gut microbial composition play in the overall pathophysiology of parasite infection and disease. Results In this study, we investigated the fluctuations in microbiota composition and local immune microenvironment of sheep vaccinated against, and experimentally infected with, the ‘brown stomach worm’ Teladorsagia circumcincta, a parasite of worldwide socio-economic significance. We compared the faecal microbial profiles of vaccinated and subsequently infected sheep with those obtained from groups of unvaccinated/infected and unvaccinated/uninfected animals. We show that alterations of gut microbial composition are associated mainly with parasite infection, and that this involves the expansion of populations of bacteria with known pro-inflammatory properties that may contribute to the immunopathology of helminth disease. Using novel quantitative approaches for the analysis of confocal microscopy-derived images, we also show that gastric tissue infiltration of T cells is driven by parasitic infection rather than anti-helminth vaccination. Conclusions Teladorsagia circumcincta infection leads to an expansion of potentially pro-inflammatory gut microbial species and abomasal T cells. This data paves the way for future experiments aimed to determine the contribution of the gut flora to the pathophysiology of parasitic disease, with the ultimate aim to design and develop novel treatment/control strategies focused on preventing and/or restricting bacterial-mediated inflammation upon infection by GI helminths. Video Abstract

Abstract Genetic variation is associated with differences in disease resistance and susceptibility among individuals within a population. To date, molecular genetic analyses of host responses have relied on extraction of genomic DNA from whole blood or tissue samples. However, such samples are not routinely collected during large-scale field studies. We demonstrate that cell-free genomic DNA (cfDNA) may be extracted and amplified from archived plasma samples, allowing retrospective analysis of host genetic diversity. This technique was also applicable to archived serum samples up to 35 years old and to different ruminant species. As proof of concept, we used this cfDNA approach to genotype the major histocompatibility complex (MHC) class II DRB1 locus of 224 Merino sheep which had participated in field trials of a commercial Haemonchus contortus vaccine, Barbervax®, in Australia. This identified a total of 51 different DRB1 alleles and their relative frequencies. This is the first study to examine host MHC diversity using DNA extracted from archived plasma samples, an approach that may be applied to retrospective analyses of genetic diversity and responses to vaccination or infection across different species and populations.

Abstract Understanding the immunological basis of resistance to gastrointestinal nematode infections in livestock is important in order to develop novel methods of parasite control such as vaccination or genetic selection for parasite resistance. The present study aimed to investigate differences in immune response between parasite resistant Santa Ines and susceptible Ile de France sheep breeds to natural Haemonchus contortus infection. Parasitological parameters, humoral immunity, local and circulating cellular immune responses were evaluated in 19 Santa Ines and 19 Ile de France lambs undergoing different anthelmintic treatments regimens: suppressive treatments (SUP) or targeted selective treatments (TST) over a 5-month grazing period. Santa Ines lambs had significantly lower Haemonchus faecal egg count and worm burden compared to Ile de France regardless of treatment regime. In addition, circulating blood eosinophils count and parasite-specific IgG levels were significantly higher and more rapidly induced in Santa Ines lambs. Abomasal immune responses were generally greater in the resistant breed, which had significantly higher levels of parasite-specific IgA in mucus, and elevated number of globule leukocytes and CD3+ T cells within the abomasal mucosal. Furthermore, numbers of POU2F3+ epithelial cells, a tuft-cell specific transcription factor, were also elevated in the Santa Ines breed, suggesting that this breed is better able to initiate T-helper type 2 immune responses within the abomasum. In conclusion, the differential immunological responses detailed here are relevant to understanding resistance to gastrointestinal nematodes in other host breeds, as well as to resistance breeding as a sustainable control approach for parasitic infections.

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.

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.

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.

Abstract Background The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory–secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory–secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae). Results In this study excretory–secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory–secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells. Conclusions In this study, we identified differences in the excretory–secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory–secretory proteins of potential importance for parasite survival and/or host immune modulation.

Abstract Vaccines and genetic resistance offer potential future alternatives to the exclusive use of anthelmintics to control gastrointestinal nematodes (GIN). Here, a Teladorsagia circumcincta prototype vaccine was administered to two sheep breeds which differ in their relative levels of resistance to infection with GIN. Vaccination of the more susceptible Canaria Sheep (CS) breed induced significant reductions in worm length and numbers of worm eggs in utero (EIU) when compared to control CS sheep. In the more resistant Canaria Hair Breed (CHB), although vaccination induced a reduction in all parasitological parameters analysed, differences between vaccinated and control sheep were not statistically significant. Such interactions between sheep breed and vaccination may allow better integrated control of GIN in future.

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.

Maedi-visna (MV) is a complex lentiviral disease syndrome characterised by long immunological and clinical latencies and chronic progressive inflammatory pathology. Incurable at the individual level, it is widespread in most sheep-keeping countries, and is a cause of lost production and poor animal welfare. Culling seropositive animals is the main means of control, but it might be possible to manage virus transmission effectively if its epidemiology was better quantified. We derive a mathematical epidemiological model of the temporal distributions of seroconversion probabilities and estimate susceptibility, transmission rate and latencies in three serological datasets. We demonstrate the existence of epidemiological latency, which has not explicitly been recognised in the SRLV literaure. This time delay between infection and infectiousness apparently exceeds the delay between infection and seroconversion. Poor body condition was associated with more rapid seroconversion, but not with a higher probability of infection. We estimate transmission rates amongst housed sheep to be at about 1,000 times faster than when sheep were at grass, when transmission was negligible. Maternal transmission has only a small role in transmission, because lambs from infected ewes have a low probability of being infected directly by them, and only a small proportion of lambs need be retained to maintain flock size. Our results show that MV is overwhelmingly a disease of housing, where sheep are kept in close proximity. Prevalence of MV is likely to double each year from an initial low incidence in housed flocks penned in typically-sized groups of sheep (c. 50) for even a few days per year. Ewes kept entirely at grass are unlikely to experience transmission frequently enough for MV to persist, and pre-existing infection should die out as older ewes are replaced, thereby essentially curing the flock.

Abstract Background The active form of the vitamin D3, 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to have major effects not only on physiological processes but also on the regulation of the immune system of vertebrates. Dendritic cells are specialised antigen presenting cells which are in charge of the initiation of T-cell dependant immune responses and as such are key regulators of responses towards pathogens. In this study we set out to evaluate the effects of 1,25-(OH)2D3 on the phenotype of cattle monocyte-derived dendritic cells (MoDCs) and how the conditioning with this vitamin affects the function of these myeloid cells. Results MoDCs were generated from CD14+ monocytes with bovine IL-4 and GM-CSF with or without 1,25-(OH)2D3 supplementation for 10 days. Vitamin D conditioned MoDCs showed a reduced expression of co-stimulatory and antigen presenting molecules, as well as a reduced capability of endocytose ovalbumin. Furthermore, the capacity of MoDCs to induce proliferation in an allogeneic mixed leukocyte reaction was abolished when MoDCs were generated in presence of 1,25-(OH)2D3. LPS induced maturation of 1,25-(OH)2D3conditioned MoDCs resulted in lower secretion of IL-12 and higher IL-10 than that observed in MoDCs. Conclusions The typical immunotolerant phenotype observed in cattle DCs after exposure to 1,25-(OH)2D3 has a significant effect on the functionality of these immune cells, inhibiting the T-cell stimulatory capacity of MoDCs. This could have profound implications on how the bovine immune system deals with pathogens, particularly in diseases such as tuberculosis or paratuberculosis.

Abstract Metazoan parasites have to survive in many different niches in order to complete their life-cycles. In the absence of reliable methods to manipulate parasite genomes and/or proteomes, identification of the molecules critical for parasite survival within these niches has largely depended on comparative transcriptomic and proteomic analyses of different developmental stages of the parasite; however, changes may reflect differences associated with transition between developmental stages rather than specific adaptations to a particular niche. In this study, we compared the transcriptome of two fourth-stage larval populations of the nematode parasite, Teladorsagia circumcincta, which were of the same developmental stage but differed in their location within the abomasum, being either mucosal-dwelling (MD) or lumen-dwelling (LD). Using RNAseq, we identified 57 transcripts which were significantly differentially expressed between MD and LD larvae. Of these transcripts, the majority (54/57) were up-regulated in MD larvae, one of which encoded for an ShKT-domain containing protein, Tck6, capable of modulating ovine T cell cytokine responses. Other differentially expressed transcripts included homologues of ASP-like proteins, proteases, or excretory-secretory proteins of unknown function. Our study demonstrates the utility of niche- rather than stage-specific analysis of parasite transcriptomes to identify parasite molecules of potential importance for survival within the host.

Abstract The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

Abstract Background Polymorphonuclear neutrophil (PMN) and eosinophil extracellular trap (ETs) formation has recently been described as an important host effector mechanism against invading pathogens. So far, scarce evidence on metazoan-triggered ET formation has been published. We here describe for the first time Haemonchus contortus-triggered ETs being released by bovine PMN and ovine eosinophils in response to ensheathed and exsheathed third stage larvae (L3). Methods The visualization of ETs was achieved by SEM analysis. The identification of classical ETs components was performed via fluorescence microscopy analysis. The effect of larval exsheathment and parasite integrity on ET formation was evaluated via Pico Green®- fluorescence intensities. ETs formation under acidic conditions was assessed by using media of different pH ranges. Parasite entrapment was evaluated microscopically after co-culture of PMN and L3. ET inhibition experiments were performed using inhibitors against NADPH oxidase, NE and MPO. Eosinophil-derived ETs were estimated via fluorescence microscopy analysis. Results L3 significantly induced PMN-mediated ETs and significant parasite entrapment through ETs structures was rapidly observed after 60 min of PMN and L3 co-culture. Co-localization studies of PMN-derived extracellular DNA with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite-entrapping structures confirmed the classical characteristics of ETs. Haemonchus contortus-triggered ETs were significantly diminished by NADPH oxidase-, NE- and MPO-inhibition. Interestingly, different forms of ETs, i.e. aggregated (aggETs), spread (sprETs) and diffused (diffETs) ETs, were induced by L3. AggETs and sprETs firmly ensnared larvae in a time dependent manner. Significantly stronger aggETs reactions were detected upon exposure of PMN to ensheathed larvae than to exsheathed ones. Low pH conditions as are present in the abomasum did not block ETosis and led to a moderate decrease of ETs. Eosinophil-ETs were identified extruding DNA via fluorescence staining. Conclusion We postulate that ETs may limit the establishment of H. contortus within the definitive host by immobilizing the larvae and hampering them from migrating into the site of infection. Consequently, H. contortus-mediated ET formation might have an impact on the outcome of the disease. Finally, besides PMN-triggered ETs, we here present first indications of ETs being released by eosinophils upon H. contortus L3 exposure.