Use of Conventional and Real-Time Polymerase Chain Reaction for Confirmation of Mycobacterium Avium Subsp. Paratuberculosis in a Broth-Based Culture System ESP II

Abstract

Abstract. The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture-positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II-negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2%) PCR confirmation assays were performed. Of those, 2,259 (80%) were both ESP and PCR positive; 104 (4%) were ESP positive and PCR negative; 423 (15%) were ESP negative and PCR positive; 28 (1%) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15%) more PCRpositive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5%) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non-M. paratuberculosis isolates.

Distribution of Mycobacterium avium subsp.paratuberculosis in tissues of naturally infected cattle as affected by age

“…Kim et al (2004) assumed that faecal culture was an ineffective method for the detection of low bacterial shedders and according to Whitlock et al (2000) the sensitivity of the method for such animals may be as low as 33%. Giese and Ahrens (2000) established 100 CFU/g faeces as a detection limit for culture examination.…”

Comparison of Three Methods for Extraction of Mycobacterium Avium Subspecies Paratuberculosis DNA for Polymerase Chain Reaction from Broth-Based Culture Systems

“…?show "fnote_aff1"$^! "content-markup(./author-grp [1]/aff|./author-grp [1]/dept-list)> Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle and several other domestic and wild animals causes chronic granulomatous enteritis. 1,6 Clinical signs include weight loss, fatigue, diarrhea, decreased milk production, and mortality, resulting in substantial economic loss.…”

“…1,6 Clinical signs include weight loss, fatigue, diarrhea, decreased milk production, and mortality, resulting in substantial economic loss. 1 Recently, MAP was associated with Crohn disease in humans, thus making it a pathogen of public health concern. 2 For decades, identification of MAP-infected animals was dependent on fecal culture on solid media, which takes up to 16 weeks to conclude because of the slow growth rate of the bacteria.…”

A Testing Scheme for the Detection of Mycobacterium Avium subsp. Paratuberculosis in Bovine Feces Utilizing the ESP Para-JEM Liquid Culture System

“…9 However, this assumption should be interpreted with caution since the number of organisms present in a clinical sample is not the only factor influencing a PCR result.…”