Nested PCR and its Comparison with other Diagnostic Test in the Diagnosis of Paratuberculosis (Johne’s Disease) in Goats

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. There are many methods for diagnosis of Johne’s disease in goats. Among these, bacterial isolation was still considered as reference standard for diagnosis of JD in spite of its long turn over time. Molecular targets like IS900 were routinely used for diagnosis, since it is present only in MAP. But lately presence of IS900 like sequence in closely related members of Mycobacterium avium complex (MAC) make IS900 based PCR a less sensitive method. Therefore, IS900 PCR positive sample should be confirmed by PCR assay targeting another gene within the genome of the organism. This led to the discovery of f57 gene, which is unique to this organism and not present in other members of MAC. The present study was carried out to evaluate the nested PCR method (targeting IS900 & f57 gene) to diagnose Johne’s disease in goats. The efficacy of this nested PCR was compared with other serological tests like agar gel immunodiffusion (AGID) and absorbed enzyme-linked immunosorbent assay (ELISA). Out of the 265 goat faecal and sera samples, positive results were; AGID 36 (13.59%); absorbed ELISA 51 (19.25%) and nested PCR 58 (21.88%). This nested PCR was also compared with intra-dermal Johnin test in 65 animals, of which, positive results recorded were; Johnin test 21 (32%) and nested PCR 28 (43%). The nested PCR showed higher sensitivity compared to other diagnostic tests. Hence, this method can be used for diagnosis of clinical and sub clinical JD in goats.