Development of an indirect ELISA system to diagnosis paratuberculosis using surface antigens of Mycobacterium avium subsp. paratuberculosis in cattle

Abstract

Background/objectiveMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), also called as Johne’s disease, and is considered as the cause of irreparable economic losses in livestock industry. This bacterium is excreted in large amounts through the milk and feces of infected animal. For the detection of the PTB, indirect ELISA has been highly considered as a simple method with high sensitivity and specificity. Accordingly, this study aims at designing a system of indirect ELISA for the detection of PTB. MethodsIn this study, 100 serum samples from 10 herds of dairy farms in Tehran and Alborz provinces, where PTB has been proven by culture, were selected and surveyed using the IDXX commercial kit, and the in-house ELISA system was designed. To design ELISA system, surface antigens and real positive and negative serum samples were used, and checkerboard titration was performed. To determine the cutoff point, the results of the commercial kit were used as gold standard and mean+2SD formula was also used in this regard. ResultsAccording to the commercial ELISA kit results (15 positive samples and 85 negative samples), the best concentration of antigen and antibody dilution were evaluated as 1.2μg and 1/100, respectively. Furthermore, the cutoff point was determined as 0.44. The sensitivity and specificity were evaluated as 67% and 100%, respectively. ConclusionSurface antigens in MAP are sensitive to detect the infected animals, and the indirect ELISA system can be used to detect antibody in the early stages of the disease.