Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.Mycobacterium avium subspecies paratuberculosis is the etiologic agent of a chronic granulomatous enteritis of ruminants known as Johne's disease (12). M. avium subsp. paratuberculosis has also been suspected to be involved in the chronic inflammatory bowel disorder in humans known as Crohn's disease (5, 10, 27). Although prevalent mostly in the bovine host species, this organism has been found to be an infectious agent of numerous mammals and birds alike (6, 9). M. avium subsp. paratuberculosis infections are transmitted through the fecaloral route by contaminated feed sources, and human cases are suspected to be due to contaminated milk (28).Many reports have examined the genetic differences and strain diversity among M. avium subsp. paratuberculosis isolates (1, 8, 29-31, 33, 35). These studies report that genetic differences exist between isolates from differing host species, locations, and strain types and that these genetic differences can range from single-nucleotide polymorphisms (SNP) to large genomic rearrangements. Previous data have confirmed that amplified fragment length polymorphism (AFLP) analysis is capable of detecting large polymorphic differences between isolates (29) and can also detect genetic differences due to SNPs at the AFLP restriction sites. Furthermore, the highly sensitive AFLP technique can distinguish between isolates with epigenetic differences at the AFLP restriction sites if the restriction site differences are due to methylation differences (36). Although this information does not allow for the identification of the methylated DNA base responsible for the polymorphism, it serves as a starting point for future epigenetic studies involving M. avium subsp. paratuberculosis isolates. The importance of this information has been demonstrated in multiple previous studies in which DNA methylation has been found to play a key role in the differential regulation of the corresponding gene products and to b...

No abstract