Analysis of interferon-gamma producing cells during infections by Yersinia enterocolitica O:9 and Brucella abortus in cattle.

One of the major constraints in the diagnosis of animal brucellosis is the cross-reactivity that occurs between Brucella and Yersinia surface antigens. With the aim to find a method to distinguish Brucella from Yersinia infection, the expansion of interferon gamma producing (IFN-γ+) T cell subsets obtained from peripheral blood mononuclear cells (PBMC) isolated from cattle either infected by Brucella abortus or experimentally immunized with Yersinia enterocolitica O:9 were compared. The lymphocytes were analyzed by flow cytometry after PBMC were in vitro re-exposed to Yersinia or Brucella antigens. The results highlighted a statistically significant difference in the expansion of the CD4+ and CD8+ IFN-γ+ T cells occurring when PBMC of animals immunized with Yersinia are in vitro exposed to Y. enterocolitica O:9 antigen but not to Brucella antigen. This method could thus be suggested in those cases where results obtained by serodiagnosis need to be further clarified.

Timing of activation of CD4+ memory cells as a possible marker to establish the efficacy of vaccines against contagious agalactia in sheep.

Mycoplasma agalactiae is a major pathogen of sheep and goats in many areas of the world and particularly in Mediterranean countries. It causes contagious agalactia, an infectious disease primarily affecting mammary glands. Many vaccines against the pathogen are currently under development. The aim of the study was to investigate the involvement of T cell-mediated immunity during vaccination and challenge experiments against Mycoplasma agalactiae. A comparison of the antigen-specific expansion of interferon gamma positive T cell memory and naïve subsets was performed between vaccinated and non-vaccinated sheep to identify cellular subsets whose activation was different between protected and non-protected sheep. Data reported in this manuscript demonstrated that two out of the three vaccines used in this study protected sheep from the disease. In the protected groups CD4(+) memory interferon-γ(+) T cells underwent an early expansion (p<0.05 when compared to unprotected groups), whilst memory CD8(+) Interferon-γ(+) T cells increased in non-protected animals 7 days after infection (p<0.05). γδ(+) Interferon-γ(+) T cells reached peaks of expansion in infected and in two vaccinated groups thus indicating that these cells are not preferentially involved in protection or pathogenesis (p<0.05). Hereby we propose that the early activation of CD4(+) memory Interferon-γ(+) T cells could be considered as a marker of protection from the disease as well as a tool to establish vaccine efficacy.

Development and validation of two PCR tests for the detection of and differentiation between Anaplasma ovis and Anaplasma marginale.

Anaplasma ovis and Anaplasma marginale are tick-transmitted bacteria that cause anaplasmosis in domestic and wild animals. Recent results show that some domestic and wild animals and ticks are susceptible to both A. ovis and A. marginale, thus supporting the need to differentiate between these species in hosts and ticks diagnosed with Anaplasma infection. However, although anaplasmosis is one of the most common diseases of grazing animals worldwide, rapid and effective tests are not available for the detection of and discrimination between these 2 Anaplasma species. The objective of this research was to develop an easy and reliable method to identify and discriminate between the closely related pathogens A. ovis and A. marginale. A. ovis and A. marginale major surface protein 4 (msp4) gene sequences were retrieved from different geographic strains and aligned to design 2 sets of primers in a region with significant differences between the 2 species, but completely conserved among strains. PCR reactions using these primers were 100% species-specific and detected all strains from each pathogen previously identified with other methods. The 2 sets of primers designed for the specific PCR amplification of A. ovis and A. marginale allow easy-to-detect and discriminate between the 2 pathogens, thus avoiding the time-consuming sequencing or multi-gene amplification procedures. This PCR provides a tool for the detection of A. ovis and A. marginale in ticks and in wildlife and domestic hosts.

Antigen-specific T cells and cytokines detection as useful tool for understanding immunity against zoonotic infections.

Zoonoses include a broad range of diseases, that are becoming of great interest, due to the climate changing, that cause the adaptation of vectors to new niches and environments. Host immune responses play a crucial role in determining the outcome of infections, as documented by expansion of antigen-specific T cells during several zoonotic infections. Thus, understanding of the contribution of antigen-specific T-cell subsets in the host immune response is a powerful tool to evaluate the different immunological mechanisms involved in zoonotic infections and for the development of effective vaccines. In this paper we discuss the role of T cells in some eukaryotic and prokaryotic infectious models.