Detection and confirmation of mycobacterium avium subspecies paratuberculosis using ELISA and PCR methodologies

Mycobacterium avium subsp. paratuberculosis (MAP) is the aetiological agent of Johne’s disease, which is a chronic enteritis of ruminants, resulting in reduced milk production, abundant diarrhoea, emaciation and eventually death. Infection usually occurs during their first year of life, however, the disease is usually not manifested clinically until the animal becomes a young adult. Subclinical infection is difficult to diagnose, presenting challenges to existing diagnostic techniques. This highly contagious disease has been implicated in the development of Crohn’s disease in humans via consumption of MAP contaminated milk and milk products. The purpose of this study was to evaluate existing testing methods for the diagnosis of Johne’s disease and to conduct a survey on the prevalence of Johne’s disease in Irish cattle. ELISA was used to determine if animals were antibody positive and PCR was used to confirm if MAP could be detected in serology positive animals. The results from the serology screen indicated a prevalence of 6.00% (95% CI: 4.51% - 7.48%) among individual animals tested (n=984). This is an increase on prevalence figures established from a serology screen conducted in 2005 by Good et al., (2009) where prevalence was determined to be 2.86% (95% CI: 2.76% – 2.97%) for individual animals. PCR detected MAP DNA in 33.33% of all faecal samples submitted from seropositive animals which indicates that not all seropositive animals were shedding when sampled. There was an observed correlation between the ELISA percentage positivity score value (S/P %) of a serum sample and the Ct value obtained from PCR analysis of a corresponding faecal sample proving that ELISA sensitivity increased as infection and shedding volume increased. A cross section of a known positive herd was also tested as a case study. The results indicated a prevalence of 27.27% (95% CI: 8.66% - 45.88%) by ELISA and a prevalence of 63.63% (95% CI: 43.53% - 83.74%) by PCR. PCR detected 2.33 times more positives than the ELISA method. Using these results to identify and cull infectious animals, the case study herd achieved an overall reduction in prevalence of 10.80% (95% CI: 7.89% - 13.72%) to 8.08% (CI: 4.76% - 11.39%) on seropositive animals from 2013 to 2014. Data from the ELISA screening supports higher incidence of Johne’s disease than previously recorded. PCR is a sensitive and specific method and provides a more practical and rapid approach to MAP confirmation than faecal culture. ELISA and PCR may be useful diagnostic tools in achieving reduction in prevalence of disease through accurate identification and confirmation of infection.