Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle

Abstract

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

FIG. 1

Multiple nucleotidic sequence alignment of M. bovis (MB), M. tuberculosis (MT), M. avium subsp. paratuberculosis (MPT), and M. avium subsp. (MA) us-p34 genes. Gaps between sequences are indicated by dots. Vertical bars indicate identity across sequences. The start codon (ATG) for the p34 open reading frame is in bold. Primer sequences are indicated by shaded boxes. The vertical arrows indicate point mutations between M. tuberculosis and M. bovis and between M. avium subsp. paratuberculosis and M. avium subsp. The horizontal arrows indicate directions of transcription.

FIG. 2

Duplex PCR strategy. Amplified DNA fragments from M. bovis (lane 1), M. avium subsp. (lane 2), and M. avium subsp. paratuberculosis (lane 3) were separated by electrophoresis in a 2% agarose gel. The lengths (in base pairs) of the amplified fragments, as well as of the BglI- and HinfI-cleaved pBR328 DNA fragments (molecular weight marker) (lane 4), are indicated on the left- and righthand sides of the gel, respectively.

FIG. 3

PCR duplex analysis of DNA purified from formalin-fixed and paraffin-embedded paratuberculosis tissues. DNA samples (100 ng) isolated from mesenteric lymph node biopsy specimens and were used in a duplex PCR procedure. (A) Aliquots of PCR-amplified DNA (50 μl of each amplified sample) were analyzed by 2% agarose gel electrophoresis. (B) Hybridization analysis of the same amplified products with the DIG-labeled f57 and us-p34 probes. Lanes 1, BglI- and HinfI-cleaved pBR328 weight marker; lanes 2 and 3, specimens from pluribacillary cows; lanes 4 and 5, specimens from paratuberculous cows (lanes of panel B are as labeled in panel A).

FIG. 4

Ziehl-Neelsen stained, formalin-fixed, paraffin-embedded tissues (intestinal mucosa). (A) Negatively stained tissue; (B) paucibacillary tissue disclosing rare acid-fast bacilli; (C) pluribacillary tissue containing clumps of acid-fast organisms.