Proceedings of the 10th International Colloquium on Paratuberculosis

#162 Comparison <strong>of</strong> faecal culture, ELISA and IS900 PCR assay for <strong>the</strong> detection <strong>of</strong>
Mycobacterium avium subsp. paratuberculosis infection in cattle
P Muralidharan Soumya, R Madhusoodanan Pillai, Prabhakar Xavier Antony, Hirak Kumar Mukhopadhyay, V N Rao
Department <strong>of</strong> Veterinary Microbiology, RAGACOVAS, Puducherry, India; Department <strong>of</strong> Veterinary Medicine, Ethics
and Jurisprudence, RAGACOVAS, Puducherry, India
Comparative efficacy <strong>of</strong> faecal culture, Enzyme-linked immunosorbent assay (ELISA) and IS900 Polymerase
chain reaction (PCR) assay <strong>of</strong> faecal samples was investigated in 40 clinically suspected cases <strong>of</strong> Johne’s
disease in dairy cattle. The sensitivity <strong>of</strong> faecal culture, ELISA and PCR assay in this study was 52.5% (21/40),
87.5% (35/40) and 90% (36/40) respectively. All isolates appeared only on <strong>the</strong> 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 <strong>of</strong> 217 bp. Of <strong>the</strong> 40 serum samples tested by <strong>the</strong> indigenous ELISA kit using
soluble protoplasmic antigen from ‘Bison type’ genotype <strong>of</strong> Map <strong>of</strong> goat origin developed by Central Institute for
Research on Goats (CIRG), Makhdoom, 17 (42.5%) were strong positive, 17 (42.5%) were positive, 1 (2.5%)
was low positive and 5 (12.5%) were negative. A total <strong>of</strong> 28 faecal samples out <strong>of</strong> 40 were positive by IS900
primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product
<strong>of</strong> size 217 bp. Twelve faecal samples, which gave negative results in <strong>the</strong> primary PCR, were subjected to
secondary PCR assay. Of <strong>the</strong> 12 samples, 8 gave positive results in <strong>the</strong> IS900 nested PCR (nPCR), which
yielded a PCR product <strong>of</strong> 167 bp, proving better sensitivity <strong>of</strong> nPCR assay than single amplification PCR. The
chi-square test showed a highly significant difference between faecal culture and ELISA and faecal culture and
PCR (P< 0.01) whereas <strong>the</strong> analysis indicated no significant difference between ELISA and PCR assay (P>
0.05). Among <strong>the</strong> tests employed in <strong>the</strong> present study, IS900 PCR assay and ELISA showed highest sensitivity.
This study suggests that IS900 PCR or ELISA based detection <strong>of</strong> Map could be used as a potential diagnostic
tool for rapid and effective Johne’s disease surveillance.
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