Detection of Mycobacterium avium subspecies paratuberculosis (MAP) in alcohol-fixed tissues of sheep by ISMav2 gene PCR and its comparison with histopathology, bacterial culture and IS900 PCR

The objective of the present investigation was to study the efficiency of an ISMav2 polymerase chain reaction (PCR) in detecting Mycobacterium avium subspecies paratuberculosis (MAP) DNA in archived alcohol-fixed sheep tissues and compare with Ziehl Neelesen (ZN) staining, bacterial culture on Herrold's egg yolk medium and IS900 PCR on fresh tissues. Tissue samples preserved in 70% alcohol for 6-8 months from 23 naturally infected paratuberculosis sheep and 7 healthy sheep were used for DNA extraction. In PCR amplification targeting ISMav2 gene of MAP, 19 (82.6%) were found to be positive. Bacterial culture, ZN and fresh tissue IS900 PCR detected 65%, 100%, and 95% cases, respectively. It was concluded that alcohol could be an alternative fixative for transportation of tissues for molecular detection of MAP genome in tissues by ISMav2 PCR, which compared well with fresh tissue IS900 PCR for the diagnosis of paratuberculosis in sheep. This may be useful in tropical countries, where shipment of fresh tissues for molecular diagnosis may be expensive proposition and most of the times facilities for maintaining cold chain are not available.