Local cytokine transcription in naïve and previously infected sheep and lambs following challenge with Teladorsagia circumcincta

<b>Background</b><p></p> The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites affecting sheep in temperate regions. Infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all classes of anthelmintic drugs, teladorsagiosis is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Protective immunity develops slowly, wanes rapidly and does not appear to be as effective in young lambs. To investigate the development of immunity to T. circumcincta in sheep and lambs, we used cytokine transcript profiling to examine differences in the abomasal mucosa and gastric lymph node of naïve and previously infected sheep and lambs following challenge.<p></p> <b>Results</b><p></p> The results of these experiments demonstrated that the abomasal mucosa is a major source of cytokines during abomasal helminth infection. A local Th2-type cytokine response was observed in the abomasal mucosa and gastric lymph node of the previously infected sheep and lambs when compared with those of the naïve during the early stages of infection. In contrast, a pro-inflammatory component more was evident in the abomasal mucosa and gastric lymph node of the naïve sheep when compared with those of the previously infected, which was not observed in the lambs.<p></p> <b>Conclusions</b><p></p> The greater levels of Th2-type cytokine transcripts in both the abomasum and gastric lymph node of the previously infected compared with naïve sheep and lambs emphasises the importance of these mechanisms in the immune response to T. circumcincta infection. Younger lambs appear to be able to generate similar Th2-type responses in the abomasum suggesting that the increased morbidity and apparent lack of resistance in younger lambs following continuous or repeated exposure to T. circumcincta is unlikely to be due to a lack of appropriate Th2-type cytokine production

Theileria annulata-transformed cell lines are efficient antigen-presenting cells for in vitro analysis of CD8 T cell responses to bovine herpesvirus-1

Continuously growing cell lines infected with the protozoan parasite Theileria annulata can readily be established by in vitro infection of leukocytes with the sporozoite stage of the parasite. The aim of the current study was to determine whether such transformed cell lines could be used as antigen presenting cells to analyse the antigenic specificity of bovine CD8 T cell responses to viral infections. Bovine herpes virus 1 (BHV-1), which is known to induce CD8 T cell responses, was used as a model. T. annulata- transformed cells were shown to express high levels of CD40 and CD80 and were susceptible to infection with BHV-1, vaccinia and canarypox viruses. The capacity of the cells to generate antigen-specific CD8 T cell lines was initially validated using a recombinant canarypox virus expressing a defined immunodominant T. parva antigen (Tp1). Autologous T. annulata-transformed cells infected with BHV-1 were then used successfully to generate specific CD8 T cell lines and clones from memory T cell populations of BHV-1-immune animals. These lines were BHV-1-specific and class I MHC-restricted. In contrast to previous studies, which reported recognition of the glycoproteins gB and gD, the CD8 T cell lines generated in this study did not recognise these glycoproteins. Given the ease with which T. annulata-transformed cell lines can be established and maintained in vitro and their susceptibility to infection with poxvirus vectors, these cell lines offer a convenient and efficient in vitro system to analyse the fine specificity of virus-specific CD8 T cell responses in cattle

Genomic analysis reveals extensive gene duplication within the bovine TRB locus

<p>Abstract</p> <p>Background</p> <p>Diverse TR and IG repertoires are generated by V(D)J somatic recombination. Genomic studies have been pivotal in cataloguing the V, D, J and C genes present in the various TR/IG loci and describing how duplication events have expanded the number of these genes. Such studies have also provided insights into the evolution of these loci and the complex mechanisms that regulate TR/IG expression. In this study we analyze the sequence of the third bovine genome assembly to characterize the germline repertoire of bovine TRB genes and compare the organization, evolution and regulatory structure of the bovine TRB locus with that of humans and mice.</p> <p>Results</p> <p>The TRB locus in the third bovine genome assembly is distributed over 5 scaffolds, extending to ~730 Kb. The available sequence contains 134 TRBV genes, assigned to 24 subgroups, and 3 clusters of DJC genes, each comprising a single TRBD gene, 5–7 TRBJ genes and a single TRBC gene. Seventy-nine of the TRBV genes are predicted to be functional. Comparison with the human and murine TRB loci shows that the gene order, as well as the sequences of non-coding elements that regulate TRB expression, are highly conserved in the bovine. Dot-plot analyses demonstrate that expansion of the genomic TRBV repertoire has occurred via a complex and extensive series of duplications, predominantly involving DNA blocks containing multiple genes. These duplication events have resulted in massive expansion of several TRBV subgroups, most notably TRBV6, 9 and 21 which contain 40, 35 and 16 members respectively. Similarly, duplication has lead to the generation of a third DJC cluster. Analyses of cDNA data confirms the diversity of the TRBV genes and, in addition, identifies a substantial number of TRBV genes, predominantly from the larger subgroups, which are still absent from the genome assembly. The observed gene duplication within the bovine TRB locus has created a repertoire of phylogenetically diverse functional TRBV genes, which is substantially larger than that described for humans and mice.</p> <p>Conclusion</p> <p>The analyses completed in this study reveal that, although the gene content and organization of the bovine TRB locus are broadly similar to that of humans and mice, multiple duplication events have led to a marked expansion in the number of TRB genes. Similar expansions in other ruminant TR loci suggest strong evolutionary pressures in this lineage have selected for the development of enlarged sets of TR genes that can contribute to diverse TR repertoires.</p

NKp46 defines ovine cells that have characteristics corresponding to NK cells

Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16 - characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species

A comparative study of single Theileria lestoquardi and mixed infections with Theileria ovis

Abstract Background Epidemiological surveys in Oman have revealed a high prevalence of the co-occurrence of the pathogenic Theileria lestoquardi and the non-pathogenic Theileria ovis among sheep in the Barka region, Oman. Our most recent data illustrated an interaction and reduced mortality risk in animals co-infected with T. lestoquardi and T. ovis, suggesting that the latter confers protection against pathogenicity of T. lestoquardi. The present study extends the above findings and examines disease outcomes; clinical markers, hematological parameters, and parasite density in mixed and single T. lestoquardi infections. Methods A total of 390 blood samples were collected from 16 sheep pens located in Barka, Oman between July and November 2019. Theileria spp. were detected and quantified using qPCR assay targeting 18S rRNA, and the extent of genetic diversity was estimated by a panel of T. lestoquardi specific micro- and mini-satellites. The association of some disease markers with the presence of Theileria spp. and genetic diversity was tested. Results Theileria spp. were detected in 75 (19.2%) sheep; of these 65 (86.7%) had mixed infections (T. lestoquardi plus T. ovis), 8 (10.6%) were infected with T. lestoquardi alone, and 2 (2.7%) with only T. ovis. Exotic breeds had a higher risk for Theileria spp. infection. The density (18S rRNA gene copies) of both parasites was higher in single infection against mixed infection, and there was a relatively lower density of T. lestoquardi in mixed infections. However, there was no difference in hematological indices between single T. lestoquardi and mixed infections. High genetic diversity was observed among T. lestoquardi in Barka, with no differences of T. lestoquardi in single and mixed infections. The extent of diversity seen in Barka was higher (He = 0.772) than that reported in Oman in 2019 (He = 0.582), with distinct T. lestoquardi genotypes. Conclusion The lower density of T. lestoquardi as mixed infection with T. ovis compared to single infection supports the hypothesis that T. ovis confers protection against lethal T. lestoquardi infection. However, there were no differences in disease correlations (clinical markers, hematological parameters, and density of parasites) or the extent of diversity of T. lestoquardi between the two types of infection. The presence of distinct T. lestoquardi genotypes in Barka, compared to that reported earlier in Oman, likely reflects movement of carrier animals and highlights the need for further analysis of the parasite populations to inform novel approaches for controlling malignant ovine theileriosis. Graphical Abstrac

Protection associated with a TB vaccine is linked to increased frequency of Ag85A-specific CD4⁺ T cells but no increase in avidity for Ag85A

AbstractThere is a need to improve the efficacy of Bacille Calmette-Guérin (BCG) vaccination against tuberculosis in humans and cattle. Previously, we found boosting BCG-primed cows with recombinant human type 5 adenovirus expressing antigen 85A (Ad5-85A) increased protection against Mycobacterium bovis infection compared to BCG vaccination alone. The aim of this study was to decipher aspects of the immune response associated with this enhanced protection. We compared BCG-primed Ad5-85A-boosted cattle with BCG-vaccinated cattle. Polyclonal CD4+ T cell libraries were generated from pre-boost and post-boost peripheral blood mononuclear cells – using a method adapted from Geiger et al. (2009) – and screened for antigen 85A (Ag85A) specificity. Ag85A-specific CD4+ T cell lines were analysed for their avidity for Ag85A and their Ag85A epitope specificity was defined. Boosting BCG with Ad5-85A increased the frequencies of post-boost Ag85A-specific CD4+ T cells which correlated with protection (reduced pathology). Boosting Ag85A-specific CD4+ T cell responses did not increase their avidity. The epitope specificity was variable between animals and we found no clear evidence for a post-boost epitope spreading. In conclusion, the protection associated with boosting BCG with Ad5-85A is linked with increased frequencies of Ag85A-specific CD4+ T cells without increasing avidity or widening of the Ag85A-specific CD4+ T cell repertoire

Simple model for tuberculosis in cattle and badgers

As an aid to the study of bovine tuberculosis (TB), a simple model has been developed of an epidemic involving two species, cattle and badgers. Each species may infect the other. The proportion of animals affected is assumed relatively small so that the usual nonlinear aspects of epidemic theory are avoided. The model is used to study the long-run and transient effect on cattle of culling badgers and the effect of a period without routine testing for TB, such as occurred during the 2001 epidemic of foot-and-mouth disease in Great Britain. Finally, by examining the changes in cattle TB over the last 15 years, and with some other working assumptions, it is estimated that the net reproduction number of the epidemic is 1.1. The implications for controlling the disease are discussed