Interferon gamma ;production in lymphocyte proliferative assay from tuberculin reactor dairy cattle transiting early peripartum period.

TRAVERSA M.J.; PAOLICCHI F.A.; MORSELLA C.; ESTEIN S.M.; RODRÍGUEZ E.; JORGE M.C.

La Habana

Congreso; Modern biotechnologies for animal health. Biotecnología Habana 2011; 2011

Center for Genetic Engineering and Biotechnology

Mycobacterium bovis causes bovine and zoonotic tuberculosis (TB) and triggers a cellular immune response. In dairy cattle during early peripartum period (EPP) this response is suppressed but few studies link this to TB. In our studies we found that lymphocytes were increased in tuberculin (Tbc) reactors during EPP. When we characterized lymphocytes subpopulations by flow cytometry CD4 cells were increased, CD14 cells were decreased and gd cells did not show significant differences. The objective of this project was to characterize interferon-g (IFNg) production during lymphocytes proliferative assay. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation from non-coagulated blood from 10 Argentinean Holstein cows transiting EPP; 5 were Tbc positive and 5 Tbc negative. The first group belonged to a dairy farm with endemic TB and the second to a TB free dairy farm. 500 μL (1x106) of PBMC suspended in RPMI 1640 complete media were added per duplicated microfuge tube. As antigen 500 μL of bovine Tbc in a 5 μg/mL concentration was used. PBMC were incubated for six days at 37°C in 5% CO . Supernatants were separated and stored at -20 °C until IFN g 2 determination with a commercially Ag capture ELISA kit. Results were expressed as the mean optical density (OD) of duplicates plus the standard deviation. OD from each experimental group were compared using an unpaired t test 95% of confidence verifying variance homogeneity (SAS version 9.2). No significant differences between Tbc positive and Tbc negative cows transiting EPP were found (p > 0.05). Our findings suggest that although infection with M. bovis alters the frequency of CD4 and CD14 cells in Tbc reactors transiting EPP no difference in the production of IFN g a major proinflammatory cytokine is detected. That is why studies are needed to determine the interaction of lymphocytes subpopulations and cytokines patterns during the immune process of Tbc reactors transiting EPP.