B. N. Tripathi

Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne’s disease

A quantitative real-time PCR (qPCR) assay employing IS900 gene specific primers of Mycobacterium avium subsp. parartuberculosis (MAP) was compared with conventional PCR, bacterial culture and enzyme-linked immunosorbent assay in 38 sheep showing granulomatous enteritis and lymphadenitis with and without demonstration of acid-fast bacilli (AFB). The lesions were classified as multibacillary (MB) (n = 23), which had diffuse granulomatous lesions with abundant AFB, and paucibacillary (PB) (n = 15), which had focal or multifocal granulomatous lesions with few or no AFB. In the multibacillary group (MB), IS900 PCR detected 19 (82.6%), and qPCR detected all 23 (100%) sheep positive for MAP in the intestine and lymph node tissues. In the paucibacillary group (PB), IS900 PCR detected 2 (13.3%), and qPCR detected all 15 (100%) sheep positive for MAP in tissues. When results of both groups were taken together, IS900 PCR detected 21(55.2%), and qPCR detected all 38 (100%) animals positive for MAP genome either in the intestine or lymph node tissues. On Herrold egg yolk medium, tissues of 14 (60.9%) MB and 5 (33.3%) PB sheep were found to be positive for MAP. Out of 27 sheep (PB = 8, MB = 19) tested by an ELISA, 21 (77.7%) were found to be positive for MAP antibody, of which 25% (2/8) and 100% (19/19) sheep were from PB and MB sheep, respectively. Based on the results of the present study, it was concluded that qPCR was a highly sensitive test in comparison to conventional PCR, ELISA and bacterial culture for the diagnosis of paratuberculosis on infected tissues especially from paucibacillary sheep.

Epidemiological, bacteriological and molecular studies on caseous lymphadenitis in Sirohi goats of Rajasthan, India

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CL), a chronic debilitating disease of goats. In the present study, a total of 575 goats of Sirohi breed on an organized farm situated in the semi-arid tropical region of Rajasthan, India were clinically examined. Pus samples from superficial lymph nodes of 27 (4.7%) adult goats presenting clinical lesions suggestive of CL were collected for bacteriological and molecular analyses. Of these goats, 51.9% yielded C. pseudotuberculosis on the basis of morphological, cultural and biochemical characteristics. A polymerase chain reaction (PCR) assay targeting proline iminopeptidase gene specific to C. pseudotuberculosis was developed that confirmed all 14 bacterial isolates. The specificity of the PCR product was confirmed by sequencing of the 551-bp amplicon in both senses, showing 98–100% homology with published sequences. Thus, overall prevalence rate based on clinical, bacterial culture and PCR assay were found to be 4.7%, 2.4% and 2.4%, respectively. The PCR assay developed in this study was found to be specific and rapid, and could be used for confirmation of CL in goats as an alternative method to generally cumbersome, time-consuming and less reliable conventional methods.