O6-6 Preliminary study of interferon gamma test used for diagnosis of brucellosis in the water buffalo

Abstract

Brucellosis (BRC) is a worldwide zoonosis with a strong economic and public health impact. In water buffalo, BRC is sustained by Brucella abortus and B. melitensis. Currently, diagnosis in ruminants is based on indirect serological assays required by actual legislation, the Rapid Rose Bengal Serum Agglutination (RBT) and the Complement Fixation (CFT) tests, that mainly detected antibodies against lipopolysaccharide (S-LPS) and thus, reveal the humoral immune response. In contrast, little is known about the cell-mediated (CM) responseduring brucellosis infection in buffalo. The aim of this study is to develop a diagnostic test based on the assessment of CM immunity. To this aim we are evaluating the use of the interferon-gamma test (IFN-γ), recently introduced for the diagnosis of bovine tuberculosis also in the buffalo species. Preliminary results of a field study are herein described. Serum and heparinised blood samples, collected from buffaloes, in 11 BRC-free herds (261 samples) and in 15 brucellosis-infected herds (158 samples), that were confirmed by bacteriological test, were analyzed. RBT and CFT assays were carried out according to the WOAH official procedures. Blood samples (1 mL) for the IFN-γ test were stimulated by two different concentrations of Brucellergene OCB (Zoetis, France) (40 U/well and 100 U/well) introducing phosphate-buffered saline (PBS), as negative control for the basal value of IFN-γ. The IFN-γ production was detected by the BOVIGAM TB kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To evaluate the relationship between nonparametric values of CFT and IFN-γ assays, Chi-square statistic test was performed. Statistical analysis highlighted a higher association rate between CFT and IFN-γ tests positivity using the antigenic stimulus at 100 U/well (phi=0.32, P<0.0001) in respect to 40 U/well (phi=0.26, P<0.001). Although previous studies in bovine have been carried out at 40 U/well, our results suggest that in buffalo the antigen stimulation should be done at 100 U/well. These preliminary results suggest that the IFN-γ test could be used in buffalo as an ancillary test as its performance are similar to CFT test. Further studies are needed to evaluate the accuracy of the test.