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Genetic Diversity Applied to the Diagnosis and Epidemiology of Mycobacterium Avium Paratuberculosis


<OL> <LI> Characterize the extent of genetic diversity among and between M. avium avium (M. avium) and M. avium paratuberculosis (MapTB) isolates using amplified fragment length polymorphism (AFLP). <LI>Characterize specific genetic differences by isolation and sequence analysis of AFLP fragments. <LI>Utilize diversity data to develop PCR primers for diagnostics and epidemiology.

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NON-TECHNICAL SUMMARY: Johne's disease ('paratuberculosis') is a chronic, infectious, wasting disease with significant impact on herd productivity resulting in a corresponding economic loss to U.S. dairy operations. According to the USDA National Animal Health Monitoring System's (NAHMS) 1996 national dairy study, Johne's-positive herds experience an economic loss of almost $100-200 per cow resulting from reduced milk production and increased cow-replacement costs. When averaged across all herds, Johne's disease costs the US dairy industry $200-250 million annually in reduced productivity. The experiments described are designed to enhance early detection of Johne's disease and to correctly identify and track virulent isolates. We have developed a sensitive detection method for Johne's disease organisms which employs magnetic bead recovery of organisms from cattle milk or fecal samples. We are also utilizing a newly developed genetic approach, Amplified Restriction Fragment Polymorphism (AFLP) in combination with bioinformatics to develop new tools for tracking individual and herd isolates of Johne's organisms. Research here will provide new tools for diagnosis and control of subclinical infection and permit the elimination of infected animals before the organism has a chance to spread within herds.

APPROACH: AFLP, a relatively new experimental approach, will be used to reveal polymorphisms useful in cataloging the genetic diversity of of MapTB clinical isolates from Central and North America. This approach will also be used in subdividing and tracking clinical isolates of MapTB. AFLP will first be used to establish relationships among MapTB isolates and between MapTB and nonpathogenic M. avium. Secondly, AFLP fragments unique to MapTB will be isolated and sequenced and the sequences compared to that of M. avium. PCR primers capable of specifically detecting MapTB isolates will be derived from this data and used in diagnostic and epidemiological analysis.
PROGRESS: 2002/09 TO 2003/08<BR>
One hundred ninety two AFLP primer sets have been employed to characterize 20 Mycobacterium avium subspecies paratuberculosis (MparaTb) field isolates, one ATCC MparaTb isolate (ATCC 19698), and two M. avium avium isolates (ATCC 35716 and Mac 104) totaling 4,416 independent PCR reactions. Specifically, 16 MseI primer sets bearing selective dinucleotides at the 3'end were used in combination with 12 PstI primers bearing 3' selective dinucleotides. Diagnostic patterns have been established for the identification of MparaTb isolates and five AFLP fragments unique to the MparaTb genome have been cloned and sequenced. BLAST search results for these five AFLP fragments reveals sequence homology to genes such as the primosomal protein N' (PPN') of Brucella melitensis, P44k protein of Rhodococcus erythropolis, putative polyketide type 1 synthase (PPKS) of Streptomyces, replication protein of Rhodococcus erythropolis, and one region showed no significant similarity to any known protein. Primers were generated that were internal to these regions and when applied to the 20 MparaTb field isolates in this study an appropriate PCR product was obtained in 99 of 100 reactions. The M. avium avium isolates failed to act as templates for PCR amplification in 9 of 10 reactions. This work revealed the presence of polymorphisms in the genomes of MparaTb and M. avium avium. In addition to these PCR reactions we also carried out PCR with 4 out of the 5 MparaTb specific primer sets on an NVSL Johnes test set. This test set consisted of 25 samples, 17 determined to be MparaTb and 8 were determined to be negative for MparaTb genomic DNA through IS900 PCR and verification from NVSL. PCR amplification with diagnostic primer sets for regions 1-4 designed in this study were performed on these 25 NVSL isolates. Region 1 primer set amplified its corresponding region in 15 of 17 MparaTb isolates. Region 2 primer set amplified its corresponding region in 11 of the 17 MparaTb isolates. Region 3 primer set amplified its corresponding region in 12 of the 17 MparaTb isolates. Finally, Region 4 primer set amplified its corresponding region in only 7 of the 17 MparaTb isolates. One of the 17 MparaTb isolates (#15) showed no amplification with any primer set and none of the MparaTb-specific primer sets described here produced amplification products from the negative samples. This data suggests that there is a high degree of heterogeneity among these 17 MparaTb isolates and that these polymorphic regions are not conserved in all MparaTb isolates. Examining one of the MparaTb specific sequences more closely revealed a 5,145bp region with no homolog in the M. avium avium genome. Within this region are genes responsible for integrase/recombinase function. An additional MparaTb specific sequence revealed a 15kb region with no homologue in the M. avium avium genome. Both of these missing regions were verified to be absent from the M. avium avium genome through PCR primer walks. Three additional MparaTb-specific regions have been cloned, revealing a number of house keeping genes; all were evaluated for their diagnostic and epidemiological value.
IMPACT: 2002/09 TO 2003/08<BR>
The work we have completed will enable a rapid diagnostic test to be put in place utilizing isolation of organisms from milk or feces followed by PCR. This test will enable a diagnosis within a matter of days rather than the 6 or more weeks normally required for culture growth and identification. As we continue to investigate a spectrum of field isolates and analyze them via AFLP, we will identify more DNA targets and additional PCR diagnostic primer sets that will facilitate not only identification of M. paratuberculosis isolates, but epidemiological tracking of organisms.

Adams, L. Garry; Ficht, Thomas; Ficht, Allison
Texas A&M University
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