Evaluation of cytokines expression by Real time PCR from organs and blood of persistent infected animals by BVDV

The aim of this study was to apply the Real time PCR technique to tissues and blood samples of persistently infected animals and healthy animals in order to evaluate the expression of cytokines as INF-γ, IL-8, IL-1β and IL-10. Eighty-one samples of organs from 15 animals both BVDV persistently infected (PI) and healthy were collected and analyzed for cytokines expression. Following cytokines were quantify with a real time PCR technique: INF-γ, IL-8, IL-1β and IL-10. The quantification of cytokines expression from organs by Real time PCR showed differences between PI and healthy animals. Both INF-γ and IL-8 expressions were down-regulated in the organs from PI animals compared to healthy animals, and the INF-γ decrease was statistically significant (P< 0,001) in each group of organs analyzed. The expression of each cytokine evaluated on buffy coat was different depending on the time point considered. Only IL-8 was quantified in each time point reaching a peak of expression at the ninth blood collection and its level was higher than that one of other cytokines. INF-γ expression was detectable among the seventh and the ninth blood collection; IL-1β among the second and the sixth blood collection and IL-10 between the sixth and the seventh blood collection. Cytokine levels of PI and healthy animals were almost comparable and no statistically significant differences were observed. This study put in evidence that Real time PCR is a suitable technique for the quantification of cytokines. We found a reduction of cytokines expression (INF-γ and IL-8) from organs of PI animals compared to control animals confirming the activity of BVDV on the immune system. The INF-γ down-regulation could be due to the immunotolerance phenomenon and to the ability of the virus to infect persistently without triggering an antiviral state. In blood samples of PI and healthy animals cytokine levels were almost comparable, but the expression curves were different. The detection of INF-γ only at 3 time points out of 10 confirms the hypothesis that in PI animals there is not an effective antiviral state and that CD4+ and CD8+ depletion decreases the expression of this cytokine. As demonstrated on organs analysis there is no difference between the expression of IL-8 in PI and healthy animals probably because of its function as neutrophil chemotactic factor at the inflammation site. IL-10 is a modulatory cytokine mainly decreasing other soluble factor as IL-1 β. The reduction of this cytokine since the sixth blood collection could be correlated to the higher level of IL-10. In conclusion our results demonstrate that Real time PCR is a useful technique in the study of cytokine expression and further studies are justified to increase the panel of cytokines to be evaluated in order to better understand the role of these soluble factors in the immune response against BVDV