γδ-Tlymphocytes characterization in tuberculin reactor cattle transiting early peripartum period

Abstract

Bovine tuberculosis is a zoonotic chronic granulomatous infectious disease that occurs in bovines and is caused by the intracellular pathogen Mycobacterium bovis. Cell mediated immunity with a T-helper 1 (Th1) bias is of great importance in the control of M. bovis and γδ-Tlymphocytes induce a Th1bias of αβ-Tlymphocytes in tubercoulous cattle. During early peripartum period (EPP) cattle also present an immunesupression. The objective of this job was to characterize γδ-Tlymphocytes in tuberculin reactor dairy cows during EPP. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation from noncoagulated blood from 9 ArgentineanHolstein cows older than 2 years, five were tuberculin-positive and transited EPP and four were tuberculin-negative and did not transit EPP. PBMC were indirectly immunolabeled with monoclonal antibodies in cocktail (IgG1/IgM) to detect LTCD4, LTCD8, γδ-Tlymphocytes and CD25 and after that with a cocktail 1:200 antiIgG1PE/antiIgMFITC, fixed and stored in dark until acquisition. 10000 events were acquired with a FACS-CANTO flow cytometer (Becton-Dickinson) and analyzed with FCSExpress software. In tuberculin reactor transiting EPP group it was observed a LTCD4:LTCD8 ratio of 0.95, 11.71% of γδTlymphocytes and a 5.45% of CD25 in control group values were 1.58, 13.37% and 13.10%, respectively. Even though we did not perform dual labelling of γδ-Tlymphocytes to detect CD25 in this subset and differences in CD25 percentages were not statistically significant it is important to notice that in tuberculin-positive cattle transiting EPP the Th1bias was lower and a half of the cells expressed CD25, a constitutive marker of bovine γδTlymphocytes.