Validation of an in house iELISA for serodiagnosis of caprine brucellosis and evaluation of the performance of a B. neotomae lysate for the detection of anti-smooth Brucella specific antibodies in ruminants

Abstract

Enzyme-linked immunosorbent assay (ELISA) is a widely used and effective tool for detection of anti-Brucella antibodies in serum, easy to perform with high sensitivity and specificity. In this study, we validated an in-house indirect ELISA using B. melitensis whole cell lysate as antigen (Bm-WCL iELISA) for the serodiagnosis of caprine brucellosis and evaluated the use of BSL-2 B. neotomae in replacement of BSL-3 Brucella species as an antigen for the detection of Brucella-specific antibodies in ruminant sera. Using 724 serum samples from female crossbred goats classified as brucellosis-positive or -negative by both the buffered plate antigen (BPA) and the complement fixation (CF) tests, the Bm-WCL iELISA was successfully validated with a sensitivity (Se) of 91.83% (88.51% to 94.25%) and a specificity (Sp) of 97.41% (95.41% to 98.70%). In addition, the Bm-WCL iELISA showed a great concordance with a commercial iELISA kit (k = 0.94) in a subset of 217 serum samples. To avoid working with a BSL-3 Brucella for antigen preparation, we replaced it with a less virulent Brucella species such as B. neotomae. A total of 214 goat and 220 cow serum samples were evaluated for the diagnosis of brucellosis using the B. neotomae whole cell homogenate (Bn-WCL) iELISA. The analysis of the ROC curves suggested cut-off values of 63.83 PP for goats and 24.04 PP for cattle, with associated Se and Sp of 98.18% (93.61% to 99.68%) and 90.38% (83.20% to 94.69%) respectively in goat sera, and 95.45% (89.80% to 98.04%) and 96.36% (91.02% to 98.58%) of Se and Sp, respectively in cattle. These results confirm the utility of the in house Bm-WCL iELISA and encourage validation of the Bn-WCL iELISA for the serodiagnosis of ruminant brucellosis in resource-limited areas where the disease is endemic.