Polymerase Chain Reaction Identification of Mycobacterium Avium in Formalin-Fixed, Paraffin-Embedded Animal Tissues

Abstract

Abstract.A PCR procedure previously developed for identification of Mycobacterium bovis in formalinfixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections. Eradication of bovine tuberculosis in the UnitedStates has been a long-term goal that is now nearing attainment. Early in the campaign, the major effort was directed toward identification of infected herds by systematic skin testing. As incidence of the disease declined, the focus of attention shifted to a surveillance program that is based on detection of suspect lesions in slaughter animals, with subsequent histopathologic examination and culture of the tissues. If culture results indicate the presence of Mycobacterium bovis, epidemiologic investigations are begun to locate the potential source of infection and to identify other animals that might have been exposed. Unfortunately, several weeks may elapse before this effort can be initiated because M. bovis is such a slow-growing organism. To provide a more rapid diagnostic method, a polymerase chain reaction (PCR) procedure was developed that allows specific identification of M. tuberculosis complex bacteria (M. tuberculosis, M. bovis, M. africanum, and M. microti) in formalin-fixed, paraffin-embedded tissues. 14 When positive results are obtained with this procedure, it is possible to make a

Histopathological Classification of Lesions Observed in Natural Cases of Paratuberculosis in Free-ranging Fallow Deer (Dama dama)

“…The specificity of the insertion sequence IS900 has been demonstrated by different investigators (Kunze et al, 1992;Challans et al, 1994;Pe´rez et al, 1994;Miller et al, 1999;Coetsier et al, 2000;Garrido et al, 2000;Collins et al, 2002) and may provide the basis for an alternative to culture. The technique failed, however, to detect a significant proportion of animals with focal lesions; this may have been due to inability to detect small numbers of mycobacteria or to the presence of unusual forms of mycobacteria (Pe´rez et al, 1994).…”

Determination of Sheep Prion Gene Polymorphisms from Paraffin-Embedded Tissue

“…This PET DNA extraction method, principally consisting of repeated cycles of boiling and freezing, was originally applied to the PCR-based diagnosis of mycobacterioses, [10][11][12] diseases in which serologic diagnosis is confounded by cross-reactive dominant antigens, and isolation is delayed by the indolent growth characteristics of the bacteria. Application of the method to PrP genotyping for retrospective studies should be a simple undertaking for any institution currently using this particular technique for diagnosis of tuberculosis.…”

Examination of Mycobacterium avium subsp. avium distribution in naturally infected hens by culture and triplex quantitative real time PCR

“…identification and species identification with insertion sequence-specific PCR reactions (Miller et al, 1999;Ikonomopoulos et al, 2009). Our detection method was based on a specific and sensitive triplex reaction which enables identification of MAA .…”