Improving detection of Mycobacterium avium subsp paratuberculosis infection in cattle by anamnestic ELISA

A practical approach to the indirect diagnosis of Mycobacterium avium subsp. paratuberculosis (Map) infection in cattle is performed by an ELISA test. However, ELISA has been criticized for not being sufficiently sensitive because the Immoral response detected by the ELISA appears in the advanced stages of infection and usually after bacterial excretion in feces has began. A commercially available indirect ELISA test (IDEXX) allows to discriminate serum samples on the basis of three possible results: negative (net sample OD less than or equal to 15% of net positive control OD), positive (net sample OD greater than or equal to 30% of net positive control OD) and doubtful (net sample OD values ranging from 15 to 30% of net positive control OD). To improve test sensitivity and to differentiate doubtful animals into positive or negative, an anamnestic ELISA was applied. Johnin was intradermally inoculated into 300 animals aged 2 - 4 years, of which 100 were medium/strong-negative (net sample OD less than or equal to 5% of net positive control OD), 100 weak-negative (net sample OD values ranging from 5 to 15% of net positive control OD) and 100 doubtful, as detected by the ELISA. In addition, 300 animals aged 2 - 4 years, of which 100 animals were medium/strong-negative, 100 weak-negative and 100 doubtful, were kept as non-inoculated controls. Twenty days post-inoculation, sera were collected and subjected to the ELISA (anamnestic ELISA). Fifty four doubtful and 20 weak-negative animals tested positive after johnin inoculation, no medium/strong-negative animals seroconverted. Only 22 doubtful and 4 weak-negative controls became spontaneously positive at second sampling. Statistical analysis (chi-square) pointed out a significant difference of seroconversion rate between johnin-treated and controls for either doubtful or weak-negative animals. Therefore, our anamnestic ELISA may be considered a promising tool to improve sensitivity of serological diagnosis of paratuberculosis infection in cattle. However, at present we cannot exclude that a repeated inoculation of johnin, in paratuberculosis-free animal, could elicit an antibody production. In this case the anamnestic ELISA should be used only one time on the same animal.