Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection may be detected by measuring antigen specific cell-mediated immune responses by the interferon-gamma (IFN-¿) assay.
Available IFN-¿ assay use purified protein derivate of Johnin (PPDj) leading to low specificity. The objectives of the study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-¿ assay and to assess the consistency of IFN-¿ responses with repeated samplings.
The antigens used were 4 ESAT-6 family members, 4 latency proteins, 4 secreted proteins including Ag85B, 3 other antigens and PPDj. The study included blood samples from 26 heifers of a MAP infected herd, collected three times with 4 and 5 week interval and blood samples from 60 heifers of a MAP noninfected herd collected once. The IFN-¿ responses of the non-infected heifers were used to establish cutoff values for each antigen. A case was defined as an animal with =2 positive tests for =4 antigens, resulting in 13 cases and 13 non-cases. Based on the case-definition, immunogenicity and specificity of each antigen were calculated. IFN-¿ levels against each of the antigens of the infected and non-infected herds were significantly (P<0.05) different and IFN-¿ levels against each of the antigens of cases were significantly higher than non-cases (P<0.05 for all antigens). The results of the IFN-¿ assay using PPDj did not correlate well with the results using the novel antigens since 5 of the 17 animals that were positive to PPDj were non-cases and one case was negative to PPDj but positive to all other tested antigens. Furthermore, PPDj produced elevated IFN-¿ responses in both the infected and non-infected herds and showed low consistency. Immunogenicity was highest for the group of latency proteins (0.65-
0.85) which also had high specificity (0.92-1.00). Three latency proteins showed positive IFN-¿ tests that correlated highly with the case definition and one of these antigens (LATP-2) had no homologue sequence in the M. avium subsp. avium or M. bovis genome and could be a promising diagnostic antigen. The combination of antigens for use as a cocktail should be further investigated. However, to detect all the animals defined as cases, 8 of the novel antigens and Ag85B would have to be included.