Identification of contaminating bacteria when attempting to isolate Mycobacterium avium subsp. paratuberculosis (MAP) from bovine faecal and tissue samples using the BACTEC MGIT 960 system

Steuer, Pamela; Waard, Jacobus De; Collins, Maxwell D.; Troncoso, E.; Martínez, OA; Tejeda, Carlos; Salgado, Miguel

doi:10.4067/s0301-732x2015000100016

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“…Therefore, the identification of infected animals by direct detection of MAP in ruminant faeces is an indispensible tool for eradication. The diagnostic "gold standard" is faecal culture, which allows MAP-detection with high specificity and sensitivity but consumes up to 3 months until visible bacterial growth (Whitlock et al, 2000;Zare et al, 2014;Steuer et al, 2015). The diagnostic sensitivity of faecal culture can additionally be reduced by chemical decontamination steps which are necessarily implemented before cultivation to remove typical enteric bacteria.…”

Section: Introductionmentioning

confidence: 99%

Comparative Evaluation of PCR Assay for Direct Detection of Mycobacterium avium subsp. paratuberculosis in Ruminant

Selim¹,

Gaede²

2015

Asian J. of Animal and Veterinary Advances

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Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne´s disease) in ruminants. The infected and carrier animals shed the microorganism intermittently in faeces. In order to ensure the sensitive identification of MAP-shedders by the examination of faecal samples. Real-time PCR assays were compared which amplify the insertion sequences IS900 and ISMAV2 and furthermore the genomic element F57. The assays were designed as duplex-PCR including the amplification of PUC19-plasmid as internal control. The analytical sensitivity of the assays was determined using DNA of 6 different isolates of MAP in broad linear range (50 ng-5 fg μLG 1). The specificity was validated using 23 known species and subspecies of the Mycobacteriacea and 18 other non-Mycobacteriacea pathogens. The sensitivity for detection of MAP-DNA was 5 fg/ reaction targeting IS900. Reproducible detection limit for real-time PCR targeting ISMAV2 and F57 was 50 fg reaction. All Mycobacteriacea different from MAP and non-Mycobacteriacea gave negative results for ISMAV2 and F57 sequence. For IS900 weak positive signals were observed with highly concentrated DNA (5 ng μLG 1) of 3 Mycobacterium avium subsp. avium strains from cattle and poultry but not with low concentrated DNA (5 fg μLG 1). Thus false-positive results should not be found if analyzing ruminant faeces directly with IS900-PCR. ISMAV2-PCR and F57-PCR has to be preferred to IS900-PCR if real-time PCR is intended for the specification of cultured Mycobacteria.

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Section: Introductionmentioning

confidence: 99%