Proceedings of the 10th International Colloquium on Paratuberculosis

#51 A new marker IS1311 PCR-REA assay for rapid differentiation <strong>of</strong> ‘Indian Bison type’
Mycobacterium avium subspecies paratuberculosis isolates
Jagdeep Singh Sohal, Shoor Vir Singh, Pravin Kumar Singh, Ajay Vir Singh, M C Sharma
Indian Council <strong>of</strong> Agricultural Research, India; Central Institute for Research on Goats, India
Objective: Studies in last two years identified a unique molecular signature in some loci <strong>of</strong> IS1311 element
capable <strong>of</strong> distinguishing native ‘Indian Bison type’ Mycobacterium avium subspecies paratuberculosis (MAP)
from o<strong>the</strong>r non-Indian MAP isolates. Objective <strong>of</strong> <strong>the</strong> study was to design a specific test based on unique
molecular signature for <strong>the</strong> specific detection and differentiation <strong>of</strong> native ‘Indian Bison type’ MAP.
Materials and Methods: Bioinformatics tools were applied on <strong>the</strong> IS1311 sequences <strong>of</strong> native MAP and
IS1311 sequences retrieved from GenBank database including MAP K10 sequences to identify a restriction
enzyme capable <strong>of</strong> differentiating two sets <strong>of</strong> IS1311 sequences from ‘Indian Bison type’ MAP and ‘non-Indian’
MAP strinas based on its restriction pr<strong>of</strong>ile. Criteria <strong>of</strong> selection <strong>of</strong> restriction enzyme was ei<strong>the</strong>r loss or gain <strong>of</strong>
new restriction site due to presence <strong>of</strong> unique molecular signature in IS1311 sequences <strong>of</strong> native ‘Indian Bison
type’ MAP compared to ‘non-Indian’ MAP.
IS1311 sequences from MAP ‘Indian Bison type’ isolates recovered from different geographical regions
and different host species <strong>of</strong> <strong>the</strong> country were amplified and processed for PCR-REA using <strong>the</strong> restriction enzyme
selected in <strong>the</strong> above step. Sequencing <strong>of</strong> PCR products was also done in order to confirm <strong>the</strong> presence/
absence <strong>of</strong> molecular signature.
Results and Conclusions: Based on <strong>the</strong> criteria described above BsaJI enzyme was selected for
PCR-REA studies. PCR-REA was successfully optimized based on this enzyme capable <strong>of</strong> distinguishing
native ‘Indian Bison type’ MAP from ‘non-Indian’ MAP. Results revealed that all ‘Indian Bison type’ MAP
isolates carried molecular signature in <strong>the</strong>ir IS1311 sequences despite <strong>the</strong>ir origin from different species.
Sequencing also confirmed results <strong>of</strong> PCR-REA. Thus PCR-REA optimized in this study may be used for
rapid differentiation <strong>of</strong> ‘Indian Bison type’ isolates. These findings may give new directions to <strong>the</strong>
paratuberculosis research in <strong>the</strong> country.
#58 Environmental fecal samples for <strong>the</strong> detection <strong>of</strong> MAP in small cattle herds
Johannes Lorenz Khol, Matthias Vill, Martina Mattes, Walter Baumgartner
Clinic for Ruminants, Department for Farm Animals and Veterinary Public Health, University <strong>of</strong> Veterinary Medicine
Vienna, Austria
Detection <strong>of</strong> MAP in environmental fecal samples to determine <strong>the</strong> paratuberculosis herd status <strong>of</strong> large dairy
herds has recently been described. Two studies were performed at <strong>the</strong> Clinic for Ruminants at <strong>the</strong> University
<strong>of</strong> Veterinary Medicine in Vienna to evaluate this new approach for <strong>the</strong> assessing <strong>of</strong> <strong>the</strong> paratuberculsois herd
level in small farms.
Austrian cattle herds with and without known paratuberculosis status were sampled for MAP periodically
by collecting <strong>of</strong> environmental fecal samples. Environmental samples were taken from different sites within <strong>the</strong>
stable and manure storage and tested for MAP by bacterial culture and PCR (Polymerase Chain Reaction).
The correct MAP infection status was assigned in almost 70 % <strong>of</strong> <strong>the</strong> herds by fecal culture <strong>of</strong> <strong>the</strong> environmental
samples. When 3 consecutive environmental samples were tested for MAP, 80 % <strong>of</strong> <strong>the</strong> farms could be
assigned <strong>the</strong> correct herd infection status. All farms with a high within herd prevalence <strong>of</strong> MAP or an increasing
frequency <strong>of</strong> clinical cases <strong>of</strong> Paratuberculosis could be detected by environmental fecal sampling.
These results indicate that environmental fecal samples could be used to detect <strong>the</strong> paratuberculosis herd
status <strong>of</strong> small farms as a cheap alternative to o<strong>the</strong>r testing schemes. Repeated testing for MAP by bacterial
culture seems to be <strong>the</strong> most effective way to determine paratuberculosis herd status according to <strong>the</strong>se
results and might be more accurate and economic than o<strong>the</strong>r screening methods.
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