Genetic analysis of Mycobacterium avium complex strains used for producing purified protein derivatives

Abstract

For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established due in part to limited biologic and immunologic standardization. Because there is little information about the strains used to produce these reagents (avian PPD, intracellulare PPD, scrofulaceum PPD, and Johnin), we have performed genetic characterizations of strains used to produce these products. Sequence analysis of 16S rRNA and the hsp65 gene provided results concordant with species designations provided for M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum organisms. For M. avium strains, comparative genomic hybridization was performed on a whole-genome DNA microarray, revealing one novel 7.9-kilobase genomic deletion in certain Johnin-producing strains, in addition to genomic variability inherent to the particular M. avium subspecies. Our findings indicate that considerable genomic differences exist between organisms used for reagents and the infecting organism being studied. These results serve as a baseline for potency studies of different preparations and should aid in comparative studies of newly discovered antigens for the diagnosis of infection and disease by M. avium complex organisms.

FIG. 1.

Schematic representation of large sequence polymorphism Johnin. Coordinates on the genome are given as base pairs starting from the first nucleotide of the start codon of dnaA in M. avium 104 and M. avium subsp. paratuberculosis K10, respectively. White boxes represent homologous sequences across M. avium 104, M. avium subsp. paratuberculosis K10, and M. avium subsp. paratuberculosis strains used to produce Johnin. The striped box represents a large sequence (LSP^p4) that is present in M. avium subsp. paratuberculosis but missing in M. avium 104. LSP^jn is depicted by the gray box and is missing from M. avium subsp. paratuberculosis strains III, IV, C286, C300, III.V, 3+5, and C. Thick arrows represent primers flanking LSP^jn (bridging primers); a PCR product is obtained if the region is missing. Thin arrows represent primers targeting a sequence within LSP^jn; a PCR product is obtained if the sequence is present.