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Beverly Byrum

Publications and source records attributed to Beverly Byrum.

6 recordsLinked to original sources
PubMed2006

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis in bovine feces utilizing the ESP para-JEM liquid culture system.

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in broth cultures of bovine fecal samples carried out in ESP para-JEM System was evaluated. The scheme included acid-fast staining (on signal-positive and signal-negative samples), and confirmation by PCR for 2 MAP-specific targets and subculture of all acid-fast positive PCR-negative samples. Two hundred and fifty bovine fecal samples were evaluated for the presence of MAP using this scheme. Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed testing scheme was used, compared to 14 (6%) positive results when using the standard ESP para-JEM protocol (requiring samples to have a positive signal from the system, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on Herrold egg yolk (HEY) media. A preliminary comparison of real-time and conventional PCR on DNA extracted from 15 MAP-positive broth cultures by 3 different protocols suggested that conventional PCR may be a better choice for the confirmation of the presence of MAP in the liquid cultures than real-time PCR.

Animals↗
PubMed2005

Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures.

A liquid culture followed by molecular confirmation was evaluated for potential to improve sensitivity and reduce time to diagnosis of Mycobacterium avium subsp. paratuberculosis infection. Fecal samples from 240 animals from Ohio farms were assessed for presence of M. avium subsp. paratuberculosis using four different protocols: (i) sedimentation processing followed by inoculation on Herrold's Egg Yolk media (HEYM) slants (monitored biweekly up to 16 weeks), (ii) double centrifugation processing followed by inoculation on HEYM slants (monitored biweekly up to 16 weeks), (iii) liquid-solid double culture method using modified 7H9 broth (8 weeks) followed by subculture on HEYM slants (monitored up to 8 weeks), and (iv) liquid culture using modified 7H9 broth (8 weeks) followed by molecular assays for the presence of two M. avium subsp. paratuberculosis-specific targets. The number of positive samples detected by each protocol was 37, 53, 65, and 76, respectively. Twenty-seven samples were positive by all four methods. Based on samples positive by at least one method (n = 81), the sensitivities for sedimentation processing, double centrifugation processing, liquid-solid double culture, and liquid culture followed by molecular confirmation were 46%, 65%, 80%, and 94%, respectively. Fingerprinting of the positive samples using two polymorphic (G and GGT) short sequence repeat regions identified varying levels of within-farm and between-farm diversity. Our data indicate that liquid culture followed by molecular confirmation can significantly improve sensitivity and reduce time-to-diagnosis (from 16 to 8 weeks) of M. avium subsp. paratuberculosis infection and can also be efficiently employed for the systematic differentiation of M. avium subsp. paratuberculosis strains to understand the epidemiology of Johne's disease.

Bacterial Typing Techniques↗
PubMed2004

Evaluation of multiple genomic targets for identification and confirmation of Mycobacterium avium subsp. paratuberculosis isolates using real-time PCR.

Specificity of six previously published Mycobacterium avium subsp. paratuberculosis (MAP) genomic loci, including 10, 38, 56, 93, 251, and 252 were evaluated in this study. Target 251 which was identified as MAP-specific was further evaluated in 210 MAP isolates, 14 non-MAP mycobacterial species, 7 atypical mycobacterial isolates, and 9 other bacterial species using real-time PCR. A previously published IS900 primer and probe combination was used as a positive control along with a universal ribosomal DNA gene sequence (UVA) as an internal control to evaluate PCR inhibition. All MAP isolates were positive with IS900, 251, and UVA by real-time PCR. All non-MAP mycobacterial species except one atypical mycobacterial isolate and other bacterial species used in this study were negative for IS900. All of these species were negative for 251. The atypical mycobacterial isolate, positive for IS900 and UVA, was negative for 251. A combination of IS900 and 251 PCR is ideal for sensitive and specific confirmation of MAP isolates from conventional fecal cultures. This study also evaluated the specificity of 251 real-time PCR, on broth cultures from 50 known bovine fecal samples. Acid fast staining followed by IS900 and 251 real-time PCR can be used for accurate identification and confirmation of MAP from broth cultures.

Animals↗
PubMed2004

Evaluation of an automated system for non-radiometric detection of Mycobacterium avium paratuberculosis in bovine feces.

Cultivation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from feces remains the most reliable method to detect infected animals. The purpose of this study was to evaluate a broth-based automated system used for cultivation of mycobacteria such as M. tuberculosis from human hosts, for the detection of M. paratuberculosis in bovine feces. Bovine feces was spiked with tenfold serial dilutions of M. paratuberculosis (5x10(5) to 5x10(-1) organisms), then processed with a double-centrifugation technique that included disinfection prior to inoculation into broth tubes. The same pathogen dilution series was also inoculated directly into broth and broth with uninfected processed feces. All of the system signal-positive bottles were identified within 30 days, with the highest concentration of M. paratuberculosis detected by the system in as few as 8 days. The presence of the pathogen was confirmed with acid-fast staining and an IS900-based PCR assay when growth of M. paratuberculosis was indicated by the system. However, some of the signal-negative cultures inoculated with the equivalent of 0.5 organisms tested PCR-positive 56 days post-inoculation, indicating that longer culture periods may lead to detection of small quantities of the organisms. Additionally, it was indicated that the processing step had a detrimental effect on detection of the organism. Comparison of the broth- and Herrold's egg yolk medium (HEYM) solid media-based culture methods with defined check test specimens corroborated the experimental evaluation of this system, indicating that broth-based detection could provide a more rapid assay for M. paratuberculosis. These results suggest that this automated system could be used to detect this organism in bovine feces, but that new approaches to processing the feces for culture should be explored.

Animals↗
PubMed2003

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis.

The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.

Animals↗