γδ TCRs Function as Innate-like Receptors in the Bovine γδ T Cell Response against Leptospira.

Leptospira serovar Hardjo are bacterial pathogens of cattle that also cause zoonotic disease in humans. Vaccine-mediated protection against Leptospira serovar Hardjo in cattle is associated with a workshop cluster 1 (WC1)+ γδ T cell response that can be recalled in vitro from PBMC by antigenic stimulation. This provides a model system in which to examine protective vaccine-induced γδ T cell responses in a γδ T cell high species. Only a small proportion (5-10%) of WC1+ γδ T cells from immunized cattle are Leptospira responders, implying that Ag specificity is determined by clonally distributed receptors. Both WC1 and TCR are known to be required for Leptospira-specific responses by bovine WC1+ γδ T cells. Through variegated expression patterns and V(D)J recombination, respectively, they have the capacity to confer Ag specificity. In this study, we develop and use a high-throughput TCR-sequencing approach to study the TCRγ and TCRδ repertoires of naive ex vivo PBMC, Leptospira-responding, and Leptospira nonresponding WC1+ γδ T cells to examine the potential role of γδ TCR in determining Ag specificity. Our results provide novel insights into the PBMC γδ TCR repertoires in cattle, demonstrating the TCRγ repertoire to be clonally stratified and essentially public, whereas the TCRδ repertoire shows much higher levels of clonal diversity and is essentially private. TCR repertoire analysis of Leptospira-responding WC1+ γδ T cells identifies no signature of TCR-mediated selection, suggesting that TCR functions largely as an innate-like receptor and does not act as a primary determinant of Ag specificity in the response to this pathogen.

Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions.

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Ocular mycobacterial lesions in cats.

Ocular mycobacterial infections are an under-recognized cause of morbidity in the domestic cat. This study aimed to explore the distribution, histopathological appearance, and severity of feline ocular mycobacterial lesions, and to characterize the immune cell population with immunohistochemistry. Routine histological staining with hematoxylin and eosin, and Masson's trichrome, was performed to identify ocular lesions and assign an inflammation score based on the number of cells present. Acid-fast bacilli were detected with Ziehl-Neelsen, and immunohistochemistry for ionized calcium-binding adaptor protein-1 (Iba1), calprotectin, cluster of differentiation 3 (CD3), and Pax5 was undertaken on formalin-fixed paraffin-embedded tissue samples from 24 cases of ocular mycobacteriosis. Posterior or panuveitis with concurrent retinitis was identified in 20/24 cases (83%), with retinal detachment in 16/20 (80%) of these cases. Choroidal lesions had the highest median inflammation score. Ziehl-Neelsen-positive organisms were detected in 20/24 cases (83%), with the highest prevalence of acid-fast bacilli detected in choroidal lesions (16/20, 80%). Lesions were typically granulomatous to pyogranulomatous, characterized by abundant numbers of Iba1-positive macrophages, followed by calprotectin-positive granulocytes and monocytes, fewer T cells, and rarer B cells. However, where iritis was identified, inflammation was typically lymphoplasmacytic (11/16 cases, 69%). Where diagnostic testing was performed, tuberculosis (ie, infection with Mycobacterium bovis, Mycobacterium microti, or a nonspeciated Mycobacterium tuberculosis-complex pathogen) was diagnosed in 20/22 cats (91%), with Mycobacterium lepraemurium infection identified in the other 2/22 cats (9%). These results suggest the choroid is the primary site of lesion development in most cases of feline ocular mycobacteriosis, and inflammatory changes are associated with the presence of mycobacteria localized to ocular tissues.

The UK Veterinary Immunological Toolbox Website: promoting vaccine research by facilitating communication and removing reagent barriers.

Using the best animal models to study immune responses against specific pathogens or vaccines can dramatically accelerate our understanding. Veterinary species are well studied, particularly livestock, to reduce their disease burden. They have also proven to be powerful models, especially for zoonotic pathogens and novel vaccination strategies. A prerequisite for any model selection is having the right quality and range of species-specific immunological reagents. To help promote the widest possible use of veterinary species, an open access website (https://www.immunologicaltoolbox.co.uk) has been created as a central community annotated hub for veterinary immunological reagents. The website is also the portal into services offered by the UK Immunological Toolbox project that includes antibody generation, sequencing and recombinant expression. The funding for this effort is linked into sustainable sources, but ultimate success relies on community engagement to continually increase the quality and quantity of information. It is hoped that as more users and reagent owners engage, it will become an essential resource for researchers, veterinarians and clinicians alike by removing barriers that prevent the use of the most informative animal models.

Retrospective application of transposon-directed insertion-site sequencing to investigate niche-specific virulence of Salmonella Typhimurium in cattle.

BACKGROUND: Salmonella enterica subspecies enterica is an animal and zoonotic pathogen of global importance. Cattle are a significant reservoir of human non-typhoidal salmonellosis and can suffer enteric and systemic disease owing to the ability of Salmonella to survive within the bovine lymphatic system and intestines. Contamination of food can occur due to the incorporation of contaminated peripheral lymph nodes or by direct contamination of carcasses with gut contents. It is essential to understand the mechanisms used by Salmonella to enter and persist within the bovine lymphatic system and how they differ from those required for intestinal colonization to minimize zoonotic infections. RESULTS: Transposon-directed insertion site sequencing (TraDIS) was applied to pools of mutants recovered from mesenteric lymph nodes (MLNs) draining the distal ileum of calves after oral inoculation with a library of 8550 random S. Typhimurium mini-Tn5Km2 mutants in pools of 475 mutants per calf. A total of 8315 mutants representing 2852 different genes were detected in MLNs and their in vivo fitness was calculated. Using the same improved algorithm for analysis of transposon-flanking sequences, the identity and phenotype of mutants recovered from the distal ileal mucosa of the same calves was also defined, enabling comparison with previously published data and of mutant phenotypes across the tissues. Phenotypes observed for the majority of mutants were highly significantly correlated in the two tissues. However, 32 genes were identified in which transposon insertions consistently resulted in differential fitness in the ileal wall and MLNs, suggesting niche-specific roles for these genes in pathogenesis. Defined null mutations affecting ptsN and spvC were confirmed to result in tissue-specific phenotypes in calves, thus validating the TraDIS dataset. CONCLUSIONS: This validation of the role of thousands of Salmonella genes and identification of genes with niche-specific roles in a key target species will inform the design of control strategies for bovine salmonellosis and zoonotic infections, for which efficacious and cross-protective vaccines are currently lacking.

Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle.

Salmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

Direct Manipulation of T Lymphocytes by Proteins of Gastrointestinal Bacterial Pathogens.

Gastrointestinal bacterial infection represents a significant threat to human health, as well as a burden on food animal production and welfare. Although there is advanced knowledge about the molecular mechanisms underlying pathogenesis, including the development of immune responses to these pathogens, gaps in knowledge persist. It is well established that gastrointestinal bacterial pathogens produce a myriad of proteins that affect the development and effectiveness of innate immune responses. However, relatively few proteins that directly affect lymphocytes responsible for humoral or cell-mediated immunity and memory have been identified. Here, we review factors produced by gastrointestinal bacterial pathogens that have direct T cell interactions and what is known about their functions and mechanisms of action. T cell-interacting bacterial proteins that have been identified to date mainly target three major T cell responses: activation and expansion, chemotaxis, or apoptosis. Further, the requirement for more focused studies to identify and understand additional mechanisms used by bacteria to directly affect the T cell immune response and how these may contribute to pathogenesis is highlighted. Increased knowledge in this area will help to drive development of better interventions in prevention and treatment of gastrointestinal bacterial infection.

Quantifying the Survival of Multiple Salmonella enterica Serovars In Vivo via Massively Parallel Whole-Genome Sequencing To Predict Zoonotic Risk.

Salmonella enterica is an animal and zoonotic pathogen of worldwide importance. Salmonella serovars that differ in their host and tissue tropisms exist. Cattle are an important reservoir of human nontyphoidal salmonellosis, and contaminated bovine peripheral lymph nodes enter the food chain via ground beef. The relative abilities of different serovars to survive within the bovine lymphatic system are poorly understood and constrain the development of control strategies. This problem was addressed by developing a massively parallel whole-genome sequencing method to study mixed-serovar infections in vivoSalmonella serovars differ genetically by naturally occurring single nucleotide polymorphisms (SNPs) in certain genes. It was hypothesized that these SNPs could be used as markers to simultaneously identify serovars in mixed populations and quantify the abundance of each member in a population. The performance of the method was validated in vitro using simulated pools containing up to 11 serovars in various proportions. It was then applied to study serovar survival in vivo in cattle challenged orally with the same 11 serovars. All the serovars successfully colonized the bovine lymphatic system, including the peripheral lymph nodes, and thus pose similar risks of zoonosis. This method enables the fates of multiple genetically unmodified strains to be evaluated simultaneously in a single animal. It could be useful in reducing the number of animals required to study mixed-strain infections and in testing the cross-protective efficacy of vaccines and treatments. It also has the potential to be applied to diverse bacterial species which possess shared but polymorphic alleles.IMPORTANCE While some Salmonella serovars are more frequently isolated from lymph nodes rather than the feces and environment of cattle, the relative abilities of serovars to survive within the lymphatic system of cattle remain ill defined. A sequencing-based method which used available information from sequenced Salmonella genomes to study the dynamics of mixed-serovar infections in vivo was developed. The main advantages of the method include the simultaneous identification and quantification of multiple strains without any genetic modification and minimal animal use. This approach could be used in vaccination trials or in epidemiological surveys where an understanding of the dynamics of closely related strains of a pathogen in mixed populations could inform the prediction of zoonotic risk and the development of intervention strategies.

Inhibition of Antigen-Specific and Nonspecific Stimulation of Bovine T and B Cells by Lymphostatin from Attaching and Effacing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally encoded 365-kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonization of cattle by EHEC serogroup O5, O111, and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδ T cell receptor [γδ-TCR]) and cytokines examined (interleukin 2 [IL-2], IL-4, IL-10, IL-17A, and gamma interferon [IFN-γ]) and rendered T cells refractory to mitogen for a least 18 h after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavalin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB; L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood.

Natural killer (NK) cells are widely distributed in lymphoid and non-lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady-state and also for determining the roles for NK cells in vaccine-induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady-state conditions was investigated in cattle using the pseudo-afferent lymphatic cannulation model. CD2+ CD25lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2- CD25hi NK cells were the main subset present within the skin-draining afferent lymphatic vessels and lymph nodes, indicating that CD2- NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2- subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady-state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady-state conditions and therefore may be important during immune responses to vaccination or infection.

Bovine cryptosporidiosis: impact, host-parasite interaction and control strategies.

Gastrointestinal disease caused by the apicomplexan parasite Cryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. At present, effective therapeutic and preventative measures are not available and a better understanding of the host-pathogen interactions is required. Cryptosporidium parvum is also an important zoonotic pathogen causing severe disease in people, with young children being particularly vulnerable. Our knowledge of the immune responses induced by Cryptosporidium parasites in clinically relevant hosts is very limited. This review discusses the impact of bovine cryptosporidiosis and describes how a thorough understanding of the host-pathogen interactions may help to identify novel prevention and control strategies.

Enhancing the toolbox to study IL-17A in cattle and sheep.

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.

Interactions between natural killer cells and dendritic cells favour T helper1-type responses to BCG in calves.

Vaccination of neonatal calves with BCG induces a significant level of protection from infection with Mycobacterium bovis, the causative agent of bovine tuberculosis. Since neonatal vaccination of humans with BCG induces activation of NK cells, and young calves have high circulating numbers of these cells, we hypothesised that NK cells are important in the protective response to BCG. Furthermore, since NK cells play a role in shaping adaptive immune responses through interactions with DCs, we investigated the interactions between NK cells and DCs in the context of BCG. DCs infected with BCG expressed significantly higher levels of MHC class II and the co-stimulatory molecules CD40 and CD80, alongside augmented production of the Th1 polarising cytokine IL-12, when compared with uninfected DCs. Following in vitro co-culture with BCG-infected DCs, NK cells increased their expression of the activatory molecule CD25, with preferential activation of the CD2- NK cell subset. NK cell effector function, as measured by production of IFN-γ, was also significantly enhanced following co-culture with BCG-infected DCs. This study provides novel evidence to demonstrate that NK cells phenotypically and functionally mature after interactions with DCs in the context of BCG. Furthermore, through the production of IFN-γ and IL-12 by NK cells and DCs respectively, this interaction may drive protective Th1-type immune responses to Mycobacteria.

Immunity, safety and protection of an Adenovirus 5 prime--Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves.

Vaccination is the most cost effective control measure for Johne's disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4+, CD8+ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1ß and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Canine tuberculosis: A review of 18 new and 565 previously reported confirmed cases.

Cases of canine tuberculosis, a zoonotic infection of significant public health significance, are typically only sporadically reported in the literature. For this observational study, case details were collated both retrospectively and prospectively for dogs infected with Mycobacterium tuberculosis-complex (MTBC) organisms. A total of 18 previously unreported cases as well as 565 historically reported confirmed cases were reviewed. A variety of diagnostic techniques were used to make a confirmed diagnosis of tuberculosis (culture, interferon-gamma release assay [IGRA], and PCR). The reference standard for diagnosis is culture; however, this was negative or not attempted in some dogs. Where fully speciated, all cases were caused by infection with one of three MTBC organisms: M. tuberculosis, Mycobacterium bovis, or Mycobacterium microti. This study includes the first documented canine infections with M. microti in the UK. All cases were assigned to one of four clinical groups based on the presenting signs: 44.1% were primarily pulmonary, 14.5% were primarily abdominal, and the remainder were disseminated or miscellaneous. The development of adjunctive tests remains necessary to support early treatment decisions pending reporting of culture for MTBC organisms, which can take weeks to months. Definitive treatment, where attempted, was successful in most cases. Of the 13 dogs treated by the authors with triple combination antimicrobial therapy, a good clinical outcome was seen in 12 (92%) of them.

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

A one health-focused literature review on bovine and zoonotic tuberculosis in Pakistan from the past two decades: challenges and way forward for control.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a globally prevalent zoonotic infectious disease. World Organization for Animal Health (WOAH) estimates indicate that up to 10% of the total human TB cases in developing countries are attributed to M. bovis. Pakistan ranks 4th in global milk production with a livestock population of over 212 million animals. Over 8 million families are involved in raising these animals as a means of livelihood. To date, there is an absence of national-level data on the prevalence of bTB and an effective control program is still lacking. The multifaceted impacts and substantial economic losses render addressing bTB a daunting, but highly important challenge. In this review, we summarise all the freely available literature on M. bovis infection from Pakistan using Google scholar and PubMed databases. A total of 40 animal studies were identified using search terms: "bovine tuberculosis in Pakistan, bTB, Pakistan, Mycobacterium bovis in Pakistan, M. bovis in Pakistan"; while seven human studies were identified using the terms: zoonotic tuberculosis in Pakistan', 'M. bovis in humans Pakistan', 'zTB in TB patients in Pakistan". We have summarized all these studies to identify critical risk factors involved in transmission of bTB among animals and humans. Despite lack of comprehensive and geographically representative studies, the literature suggests a varying prevalence of bTB in animals, ranging from as low as 2% to as high as 19%. Regarding zTB prevalence in humans, estimates range from 1.5% to 13% in high-risk group of farm and abattoir workers, with notably higher percentages in extra-pulmonary TB cases. The review also addresses the challenges that Pakistan faces in formulating an effective policy for the control and eradication of bTB. We conclude with one-health based recommendations as a way forward for controlling TB caused by M. bovis in cattle and humans.

Expression and functional characterization of bovine receptor activator of NF-κB ligand (RANKL).

Receptor activator of nuclear factor Kappa-B Ligand (RANKL) is a member of the tumor necrosis factor ligand (TNF) family involved in immune responses and immunomodulation. Expressed in various cells types around the body, RANKL plays a crucial role in bone remodeling and development of the thymus, lymph nodes and mammary glands. Research in other species demonstrates that RANKL is required for the development of microfold cells (M cells) in the gut, however limited information specific to cattle is available. Cloning and expression of bovine RANKL (BoRANKL) was carried out and bioactivity of the protein was demonstrated in the induction of osteoclast differentiation from both bovine and ovine bone marrow cells. The effects of BoRANKL on particle uptake in bovine enteroids was also assessed. The production of cross-reactive bovine RANKL protein will enable further investigations into cell differentiation using the available ruminant organoid systems, and their role in investigating host-pathogen interactions in cattle and sheep.

A novel monoclonal antibody against porcine macrophage colony-stimulating factor (CSF1) detects expression on the cell surface of macrophages.

Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system through binding to the receptor CSF1R. The expression and function of CSF1 has been well-studied in rodents and humans, but knowledge is lacking in other veterinary species. The development of a novel mouse anti-porcine CSF1 monoclonal antibody (mAb) facilitates the characterisation of this growth factor in pigs. Cell surface expression of CSF1 was confirmed on differentiated macrophage populations derived from blood and bone marrow monocytes, and on lung resident macrophages, the first species for this to be confirmed. However, monocytes isolated from blood and bone marrow lacked CSF1 expression. This species-specific mAb delivers the opportunity to further understanding of porcine myeloid cell biology. This is not only vital for the role of pigs as a model for human health, but also as a veterinary species of significant economic and agricultural importance.

Bovine NK subsets in the afferent lymph and lymph nodes have distinct expression of naïve and activation-associated cell surface expressed molecules, and are differentially stimulated by BCG vaccination.

Bovine natural killer (bNK) cells are heterogeneous cell populations defined by constitutive expression of the natural cytotoxicity receptor, NKp46 (CD335). Two major subsets of bNK cells, classified by differential expression of CD2, display divergent functions in innate immunity, and are hypothesised to contribute to adaptive immunity following vaccination. Here we characterised phenotypic variation of bNK cells within afferent lymph and lymph node (LN) tissues and between CD2+ and CD2- bNK subsets, and report phenotypic changes induced by BCG vaccination. CD2- bNK cells, which dominate in the afferent lymph and LN, displayed lower expression of the activation marker CD25 within the LN, with CD25+ cells being less than half as frequent as in afferent lymph. Furthermore, we found bNK cells had a lower expression of CD45RB, associated in cattle with naïve cell status, within LN compared to afferent lymph. Following BCG vaccination, bNK cells in afferent lymph draining the vaccination site showed increased CD2-CD25+ frequencies and increased expression of CD25 on CD2+ bNK cells, although the frequency of these cells remained unchanged. In summary, we provide an overview of the phenotype of bNK cells within bovine lymphatic tissues, and provide an indication of how subsets may diverge following BCG vaccination.