Sequencing ofhsp65Distinguishes among Subsets of theMycobacterium aviumComplex

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Abstract
TheMycobacterium aviumcomplex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of thehsp65gene has proven to efficiently classify isolates within theMycobacteriumgenus but provides low resolution for distinguishing among members of theM. aviumcomplex. Therefore, in this study, we have targeted the more variable 3′ region of thehsp65gene to determine whether it can effectively discriminateM. aviumcomplex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified asM. aviumcomplex. Sequences obtained indicate thatM. intracellulareis genetically divergent fromM. aviumorganisms, and distinct sequevars were obtained forM. aviumsubsets, includingM. aviumsubsp.avium(bird type),M. aviumsubsp.hominissuis, andM. aviumsubsp.paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains ofM. aviumsubsp.paratuberculosis. A unique profile forM. aviumsubsp.silvaticumwas not obtained. These results indicate that sequencing the 3′ region of thehsp65gene can simply and unambiguously distinguish species and subspecies of theM. aviumcomplex.

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