Defining the caprine γδ T cell WC1 multigenic array and evaluation of its expressed sequences and gene structure conservation among goat breeds and relative to cattle.

Workshop cluster 1 (WC1) molecules are part of the scavenger receptor cysteine-rich (SRCR) superfamily and act as hybrid co-receptors for the γδ T cell receptor and as pattern recognition receptors for binding pathogens. These members of the CD163 gene family are expressed on γδ T cells in the blood of ruminants. While the presence of WC1+ γδ T cells in the blood of goats has been demonstrated using monoclonal antibodies, there was no information available about the goat WC1 gene family. The caprine WC1 multigenic array was characterized here for number, structure and expression of genes, and similarity to WC1 genes of cattle and among goat breeds. We found sequence for 17 complete WC1 genes and evidence for up to 30 SRCR a1 or d1 domains which represent distinct signature domains for individual genes. This suggests substantially more WC1 genes than in cattle. Moreover, goats had seven different WC1 gene structures of which 4 are unique to goats. Caprine WC1 genes also had multiple transcript splice variants of their intracytoplasmic domains that eliminated tyrosines shown previously to be important for signal transduction. The most distal WC1 SRCR a1 domains were highly conserved among goat breeds, but fewer were conserved between goats and cattle. Since goats have a greater number of WC1 genes and unique WC1 gene structures relative to cattle, goat WC1 molecules may have expanded functions. This finding may impact research on next-generation vaccines designed to stimulate γδ T cells.

Identification of Leptospiral Protein Antigens Recognized by WC1+ γδ T Cell Subsets as Target for Development of Recombinant Vaccines.

Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. γδ T cells play a role in the protective immune response in livestock species against Leptospira, while human γδ T cells also respond to Leptospira. Thus, activation of γδ T cells has emerged as a potential component in the optimization of vaccine strategies. Bovine γδ T cells proliferate and produce gamma interferon (IFN-γ) in response to vaccination with inactivated leptospires, and this response is mediated by a specific subpopulation of the WC1-bearing γδ T cells. WC1 molecules are members of the group B scavenger receptor cysteine-rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and γδ TCR coreceptors, the WC1 system has been proposed as a novel target to engage γδ T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ γδ T cells and identify two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific γδ T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+ γδ T cells respond. Identification of protein antigens will enhance our understanding of the role γδ T cells play in the leptospiral immune response and in recombinant vaccine development.

Goat γδ T cell subpopulations defined by WC1 expression, responses to pathogens and cytokine production.

The major functions of γδ T cells in mammals overlap with those of αß T cells but differ in that γδ T cells are rapid responders and see different types of antigens. While γδ T cells have been shown to be a major population of circulating lymphocytes in artiodactyl species such as cattle, sheep, and pigs, less is known about these cells in goats, an important agricultural species. We have recently shown that WC1, a γδ T cell-specific family of hybrid pattern recognition receptors/co-receptors, is a multigenic family in goats expanded beyond what occurs in cattle. This study was conducted to address some of the limitations of previous studies in determining the proportions of γδ T cells, WC1+ γδ T cells as well as the WC1.1+ and WC1.2+ subpopulations in blood and to evaluate their responses to various pathogens. Previously, the proportion of caprine γδ T cells was determined using a monoclonal antibody (mAb) 86D that we show here does not react with all γδ T cells thereby underestimating their contribution to the lymphocyte population. Using a mAb reactive with the TCRδ constant region we found the proportion of γδ T cells in blood was not significantly less than that of either CD4 or CD8 T cells and did not decrease with age after 6 months. γδ T cells that expressed WC1 ranged from ~20 to 85% of the total γδ T cells. Less than half of those were classified as WC1.1+ or WC1.2+ by mAb staining thus indicating a third major WC1+ population. We found that naïve γδ T cells proliferated in cultures of PBMC stimulated with antigens of Leptospira or Mycobacterium avium paratuberculosis (MAP) more than they did in control medium cultures or in those stimulated with M. bovis BCG antigens and that the responding γδ T cells included both WC1+ and WC1- cells. In ex vivo PMA/ionomycin-stimulated cultures of WC1- γδ T cells but not WC1+ cells produced both IL-17 and IFNγ. In longterm cultures with Leptospira or MAP both WC1- and WC1+ cells proliferated but only WC1- γδ T cells produced IL-17. In conclusion, goats have a substantial number of WC1- and WC1+ γδ T cells in PBMC that do not decrease with animal age after 6 months; both populations respond to bacterial antigens as naïve cells but in these cultures only the WC1- γδ cells produc IL-17 and IFNγ .

Goat γδ T cells.

Goats are important food animals and are disseminated globally because of their high adaptability to varying environmental conditions and feeding regimes that provide them with a comparative advantage. Productivity is impacted by infectious diseases; this then contributes to societal poverty, food insecurity, and international trade restrictions. Since γδ T cells have been shown to have vital roles in immune responses in other mammals we reviewed the literature regarding what is known about their functions, distribution in tissues and organs and their responses to a variety of infections in goats. It has been shown that caprine γδ T cells produce interferon-γ and IL-17, are found in a variety of lymphoid and nonlymphoid tissues and constitute a significant population of blood mononuclear cells. Their representation in tissues and their functional responses may be altered concomitant with infection. This review summarizes caprine γδ T cell responses to Brucella melitensis, Fasciola hepatica, Mycobacterium avium paratuberculosis, caprine arthritis encephalitis virus (CAEV), and Schistosoma bovis in infected or vaccinated goats. Caprine γδ T cells have also been evaluated in goats infected with M. caprae, Ehrilichia ruminantium, Haemonchus contortus and peste des petits ruminants (PPR) virus but found to have an unknown or limited response or role in either protective immunity or immunopathogenesis in those cases.

γδ T cells in livestock: Responses to pathogens and vaccine potential.

The immediate objective of our research is to understand the molecular mechanisms underlying activation and potentiation of the protective functional response of WC1+ γδ T cells to pathogens afflicting livestock species. The long-term goal is to incorporate stimulation of these cells into the next generation of vaccine constructs. γδ T cells have roles in the immune response to many infectious diseases including viral, bacterial, protozoan and worm infections, and their functional responses overlap with those of canonical αß T cells, for example they produce cytokines including interferon-γ and IL-17. Stimulation of non-conventional lymphocytes including γδ T cells and αß natural killer T (NKT) cells has been shown to contribute to protective immunity in mammals, bridging the gap between the innate and adaptive immune responses. Because of their innate-like early response, understanding how to engage γδ T-cell responses has the potential to optimize strategies of those that aim to induce pro-inflammatory responses as discussed here.

Prevalence and antimicrobial resistance of Salmonella isolated from lactating cows and in contact humans in dairy farms of Addis Ababa: a cross sectional study.

BACKGROUND: Salmonella are the major pathogenic bacteria in humans as well as in animals. Salmonella species are leading causes of acute gastroenteritis in several countries and salmonellosis remains an important public health problem worldwide, particularly in the developing countries. The situation is more aggravated by the ever increasing rate of antimicrobial resistance strains. Cattle have been implicated as a source of human infection with antimicrobial resistant Salmonella through direct contact with livestock and through the isolation of antimicrobial resistant Salmonella from raw milk, cheddar cheese, and hamburger meat traced to dairy farms. Despiite the presence of many studies on the prevalence and antimicrobial susceptibility pattern of Salmonella in Ethiopia, nothing has been said on the degree of the situation among apparently healthy lactating cows and in contact humans. Hence this study was conducted to determine the prevalence and antimicrobial resistance pattern of Salmonella isolates from lactating cows and in contact humans in dairy farms of Addis Ababa. METHODS: a cross sectional study was conducted in Addis Ababa by collecting milk and faecal samples from lactating cows and stool samples from humans working in dairy farms. Samples were pre-enriched in buffered peptone water followed by selective enrichment using selenite cysteine and Rapaport-Vassilidis broths. Isolation and identification was made by inoculating the selectively enriched sample on to Xylose Lysine Deoxycholate agar followed by confirmation of presumptive colonies using different biochemical tests. The Kibry Bauer disk diffusion method was used for antimicrobial sensitivity testing. RESULTS: 10.7% (21/195) of cows and 13.6% (3/22) of the human subjects sheded Salmonella. 83% resistance to two or more antimicrobials and 100% resistance to ampicillin were observed. Most of the isolates were relatively sensitive to ciprofloxacin, cotrimoxazole, and chloramphenicol. CONCLUSION: High proportion of Salmonella isolates developed resistance to the commonly prescribed antimicrobials and this may be a considerable risk in the treatment of clinical cases. So, wise use of antimicrobials must be practiced to combat the ever increasing situation of antimicrobial resistance.