Development of a qPCR assay for diagnosis of Mycobacterium avium subsp. Paratuberculosis (MAP) infection in Deer.

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of Johne’s disease, a chronic infectious enteritis, in ruminants. Johne’s disease causes significant losses due to clinical disease in deer <15 months of age. The most common route of infection is ingestion via contaminated pasture, food and water contaminated by MAP infected faeces. For management of JD, early identification of heavy shedders from a faecal sample would be a useful tool in preventing further pasture contamination and spread of infection. Several diagnostic tests are available, including faecal culture and an serological immunoassay, which are limited by lack of sensitivity, specificity and long incubation times. Polymerase Chain Reaction (PCR) assays have been applied for detection of MAP, these PCR assays have shown to be sensitive and specific for the detection of MAP and have reduced time for detection to 2-3 days. The central objective of this study was to develop a fast, robust and reproducible qPCR assay for MAP in faecal material and tissue samples of deer. Two different MAP sequence targets were compared (F57 and IS900) and two different detection chemistries were compared (SYBR Green vs. TaqMan). A comparison with an established immunological bioassay, an ELISA (Paralisa) has been made. A significant correlation between the Cervine matched blood, (Paralisa Johnin results), and faecal samples(qPCR CT values) is verified, p < 0.001. Finally the effect of lab contamination was evaluated. This study revealed that it is possible to do a qPCR assay to detect MAP.