Proceedings of the 10th International Colloquium on Paratuberculosis

#17 Molecular characterization <strong>of</strong> Mycobacterium avium subsp. paratuberculosis isolates
from different host and geographical origins
Ganesh G Sonawane, Bhupendra N. Tripathi, Karen Stevenson
Central Sheep and Wool Research Institute, Awikanagar, India; Indian Veterinary Research Institute, Izatnagar
India; Moredun Research Institute, United Kingdom
The IS900 and IS1311 PCR and IS1311 PCR-REA assay was used in <strong>the</strong> present study to analyse 60 MAP
isolates from various species and geographic regions to assess <strong>the</strong>ir strain types. Five non-MAP reference
strains were also included in <strong>the</strong> study. All <strong>the</strong> 35 MAP strains tested from India and <strong>the</strong> UK (27) were positive
for both PCRs. The reference strain M. avium was negative for IS900 and positive for IS1311 PCR. A non-MAP
reference strain M. bovis was found to be negative by both PCRs. All <strong>the</strong> sheep isolates (17) from CSWRI,
Avikanagar farms produced 3 bands on REA (67 bp, 218 bp and 323 bp) and were identified as “bison” B type
strains. MAP isolates <strong>of</strong> cattle (7), goat (8) and sheep origin (2) obtained from nor<strong>the</strong>rn and sou<strong>the</strong>rn parts <strong>of</strong>
India and border region, were identified as ‘B’ type. One MAP isolate <strong>of</strong> goat origin from Bareilly region was
identified as ‘C’ type. The MAP isolates <strong>of</strong> cattle (8), sheep (6), goat (4), crow (1), badger (1), deer (1), moufflon
(1), rat (1), rook (1) and stoat (1) obtained from Scotland were found to be cattle (C) type by REA as <strong>the</strong>y
yielded 4 bands (67 bp, 218 bp, 285 bp and 323 bp). Three MAP reference strains (K10, ATCC 19698, 316F
MTCC) were ‘C’ type. M. avium (M120/04 UK) gave M. a. avium (Maa) type REA pattern as expected. The
results suggested that in India bison type strains were more prevalent than o<strong>the</strong>r types. In <strong>the</strong> UK cattle type
strains were prevalent. Also, since most MAP strains irrespective <strong>of</strong> <strong>the</strong>ir host and geographic origins are <strong>of</strong>
similar type on PCR-REA, <strong>the</strong> diagnostics and vaccine reagents prepared from one MAP strain (Bison type) <strong>of</strong>
Indian origin may act effectively in all species <strong>of</strong> animals.
#18 Efficacy <strong>of</strong> a PCR targeting <strong>the</strong> 251 gene <strong>of</strong> Mycobacterium avium subsp.
paratuberculosis for <strong>the</strong> diagnosis <strong>of</strong> Johne’s disease in goats
T Rajendra Kumar, Bhupendra N. Tripathi, Shirish Narnaware, Karen Stevenson
Indian Veterinary Research Institute, Izatnagar India; Moredun Research Institute, United Kingdom
The diagnostic potential <strong>of</strong> a recently identified 251 gene locus specific to Mycobacterium avium subsp. paratuberculosis
(MAP) has been found to be 100% sensitive and specific in a study on MAP isolates (Rajeev et
al., 2005). In <strong>the</strong> present study, polymerase chain reaction (PCR) using specific primers to <strong>the</strong> 251 gene <strong>of</strong>
MAP was first carried out on 62 MAP strains and 5 non-MAP strains <strong>of</strong> different geographical (Indian and UK)
and host origins, and <strong>the</strong>n applied to clinical samples. PCR amplification <strong>of</strong> <strong>the</strong> gene was obtained for all MAP
strains, but not for o<strong>the</strong>r non-MAP strains.
The PCR was performed on formalin-fixed paraffin embedded tissue sections from 38 naturally and
10 experimentally infected goats with different grades <strong>of</strong> pathology. All natural (100%) and 9 experimental
goats (90%) were positive in <strong>the</strong> PCR. The results showed that while <strong>the</strong> PCR was specific it was also a
sensitive method <strong>of</strong> diagnosis. The sensitivity <strong>of</strong> this PCR (97.9%) was found to be higher than ZN (84.2%)
that detects acid fast bacilli. The assay was fur<strong>the</strong>r carried out on 70 fresh tissue samples from 70 suspected
paratuberculous goats. Positive amplifications were obtained in 26 cases (37.1%). On histopathology, all
<strong>of</strong> <strong>the</strong>se goats showed chronic enteritis and lymphadenitis with and without distinct granulomas formation.
Acid-fast bacilli were rarely observed in <strong>the</strong> tissue sections <strong>of</strong> <strong>the</strong>se goats (paucibacillary). Nucleotide
sequencing <strong>of</strong> amplified 251-gene locus products from a bovine strain (accession no.EU348778) and from a
goat tissue sample (accession no.EU366498) showed 100% similarity with respect to <strong>the</strong> 251-gene locus <strong>of</strong>
Mycobacterium avium subsp. paratuberculosis K-10 strain.
The results suggested that 251 gene locus PCR was a highly specific and sensitive test for <strong>the</strong> diagnosis
<strong>of</strong> paratuberculosis. A larger number <strong>of</strong> samples should be tested to assess it’s suitability for <strong>the</strong> routine identification
<strong>of</strong> MAP isolates and diagnosis <strong>of</strong> Johne’s disease.
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