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

Ganesh G Sonawane,Bhupendra N Tripathi

doi:10.1186/2193-1801-2-45

Abstract

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.

Highlights

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne’s disease mainly of cattle, goats, sheep and other domestic and wild ruminants
We report the efficacy of an IS900 quantitative real-time PCR (qPCR) in detection and quantification of MAP in sheep tissues with two distinct types of pathology i.e. PB and MB and its comparison with commonly used methods such as bacterial culture, conventional IS900 PCR and ELISA
In early stages of the infection, where excretion of the bacilli is too low to be detected by culture or PCR, regular screening of morbid tissue materials in histopathology and subsequent confirmation by culture or genetic test appear to be a logical option for detecting incursion of MAP infection in the farm

Summary

Introduction

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne’s disease mainly of cattle, goats, sheep and other domestic and wild ruminants. Animals become infected early in the life but clinical disease develops only in the adult stage Lilenbaum et al (2007). Animal remains in paucibacillary and subclinical state for a quite long time, excreting none or low numbers of bacilli in the faeces and milk. In these animals, inflammatory responses are quite characteristic to MAP infection, but organisms are difficult to demonstrate (Lilenbaum et al 2007; Perez et al 1997). Over a period in the infected host, MAP organisms proliferate extensively in tissues which could be demonstrable in tissues and faeces by bacterial culture and genetic tests

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Conclusion