Genetic analysis of mycobacterium avium subspecies paratuberculosis reveals sequence and epigenetic variation among field isolates

Previous research performed in 1999 by Harris et al. has shown that many varieties of ruminants serve as the host species for Mycobacterium avium subspecies paratuberculosis (MparaTb) infections. Gene sequencing has supported the contention that organisms isolated from different hosts harbor different gene sequences; this has been exemplified by Amonsin et al. in 2004 with the sequencing of the mfd (transcription-repair coupling factor) and by Motiwala et al. in 2005 through sequence analysis of phosphatidylethanolaminebinding proteins which reveal a host-specific correlation of isolates. Some contradicting reports from Bannantine et al. from 2003 have further claimed that MparaTb is a monogenic organism based upon sequence data from regions flanking the origin of replication and the 16s rRNA. One of the drawbacks to the techniques implemented in these reports is the extremely restricted region of the bacterial genome that was analyzed; furthermore, only a select number of isolates were analyzed. In the present studies, amplified fragment length polymorphism (AFLP) was used as a tool for a genome scale comparison of MparaTb isolates from differing isolation types as well as a comparison of MparaTb isolates to the genetically similar yet avirulent Mycobacterium avium subspecies avium isolates. AFLP data reveals the MparaTb genome to be much more plastic and polymorphic than previously thought. These polymorphic regions were identified and characterized and are shown to be unique to the organism when compared to an array of Mycobacterial isolates of differing species. These polymorphic regions were also utilized in polymerase chain reaction (PCR) based diagnostic as well as epidemiologic tests. Furthermore, AFLP comparative analysis of intracellular and fecal MparaTb isolates reveals polymorphic regions unique to each isolate type. While these genomic differences are not based upon differences in the genetic code, they are based upon epigenetic modifications such as DNA methylation. These DNA methylation patterns are unique to intracellular MparaTb isolates as opposed to isolates from fecal material. Furthermore, AFLP comparisons of fecal MparaTb isolates that were passaged through the bovine ileum revealed banding pattern differences as compared to the original inoculum.