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Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples

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Abstract

Comparative efficacy of faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was investigated in 40 clinically suspected cases of Johne’s disease in dairy cattle. The sensitivity of faecal culture and PCR assay in this study was 52.5% (21/40) and 90% (36/40) respectively. All isolates appeared only on the mycobactin J supplemented Herrold’s egg yolk medium (HEYM) at 8–16 weeks post-inoculation, were acid-fast and were positive for IS900 PCR yielding a single amplicon of 217 bp. A total of 28 faecal samples out of 40 were positive by IS900 primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product of size 217 bp. Twelve faecal samples, which gave negative results in the primary PCR, were subjected to secondary PCR assay. Of the 12 samples, 8 gave positive results in the IS900 nested PCR (nPCR), which yielded a PCR product of 167 bp, proving better sensitivity of nPCR assay than single amplification PCR. PCR could detect additionally 15 samples as positive which were negative by faecal culture. The chi-square analysis showed a highly significant difference between the tests (P< 0.01). This study suggests that IS900-PCR-based detection of Map could be used as a potential diagnostic tool for rapid and effective Johne’s disease surveillance.

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Abbreviations

PCR:

Polymerase chain reaction

HEYM:

Herrold’s egg yolk medium

Map:

Mycobacterium avium subsp. paratuberculosis

OIE:

Office International des Epizooties

nPCR:

Nested PCR

HPC:

Hexadecylpyridinium chloride

VAN:

Vancomycin, Amphotericin B and Nalidixic acid

SDS:

Sodium dodecyl sulphate

CTAB-NaCl:

Cetyl trimethyl ammonium bromide-Sodium chloride

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Acknowledgements

The authors thank The Dean, Rajiv Gandhi College of Veterinary and Animal Sciences, Puducherry, for providing facilities to carry out the work.

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Authors and Affiliations

  1. Department of Veterinary Microbiology, Rajiv Gandhi College of Veterinary and Animal Sciences, Kurumbapet, Puducherry, 605009, India

    M. P. Soumya, R. M. Pillai, P. X. Antony & H. K. Mukhopadhyay

  2. Animal Husbandry Expert, Kudumbashree District Mission, Local Self Government Department, Govt. of Kerala, Pattom Palace P.O., Thiruvananthapuram, 695004, Kerala, India

    M. P. Soumya

  3. Department of Veterinary Medicine, Ethics and Jurisprudence, Rajiv Gandhi College of Veterinary and Animal Sciences, Kurumbapet, Puducherry, 605009, India

    V. N. Rao

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  1. M. P. Soumya
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Correspondence to M. P. Soumya.

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Soumya, M.P., Pillai, R.M., Antony, P.X. et al. Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples. Vet Res Commun 33, 781–791 (2009). https://doi.org/10.1007/s11259-009-9226-3

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