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1.  A case of bovine raw milk contamination with Listeria monocytogenes 
Irish Veterinary Journal  2012;65(1):13.
During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.
doi:10.1186/2046-0481-65-13
PMCID: PMC3526472  PMID: 22769601
Listeria monocytogenes; Raw milk; Subclinical mastitis; Milk quality
2.  Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Ireland using both traditional culture and molecular based methods 
Gut Pathogens  2010;2:11.
Background
Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic gastroenteritis affecting many species. Johne's disease is one of the most widespread and economically important disease of ruminants. Since 1992 and the opening of the European market, the exposure and the transmission of MAP in cattle herds considerably increased. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. In total, 290 cattle from seven Irish herds with either a history or a strong likelihood of paratuberculosis infection were selected by a veterinary team over 2 years. Faecal samples (290) were collected and screened for MAP by a conventional culture method and two PCR assays. In order to further evaluate the usefulness of molecular testing, a nested PCR was also assessed.
Results
M. paratuberculosis was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. A complete correlation (100%) was observed between the results of both molecular targets (IS900 and ISMAP02). Sensitivity was increased by ~10% with the inclusion of a nested PCR for ISMAP02 (29 further samples were positive). When culturing and PCR were retrospectively compared, every culture positive faecal sample also yielded a PCR positive result for both targets. Alternatively, however not every PCR positive sample (n = 105, 36%) produced a corresponding culture isolate. Interestingly though when analysed collectively at the herd level, the correlation between culture and PCR results was 100% (ie every herd which recorded at least 1 early PCR +ve result later yielded culture positive samples within that herd).
Conclusion
PCR on bovine faecal samples is a fast reliable test and should be applied routinely when screening for MAP within herds suspected of paratuberculosis. Nested PCR increases the threshold limit of detection for MAP DNA by approximately 10% but proved to be problematic in this study. Although slow and impractical, culturing is still regarded as one of the most reliable methods for detecting MAP among infected cattle.
doi:10.1186/1757-4749-2-11
PMCID: PMC2954866  PMID: 20875096
3.  Optimization of a Rapid Viability Assay for Mycobacterium avium subsp. paratuberculosis by Using alamarBlue▿  
Applied and Environmental Microbiology  2009;75(24):7870-7872.
A microtiter alamarBlue assay was adapted and optimized for Mycobacterium avium subsp. paratuberculosis. Using cell concentrations ranging from 104 to 108 CFU/ml, a minimum incubation time to indicate viability was obtained after 24 h. Rifampin (rifampicin) was used to demonstrate that this method has applications for high-throughput screening against M. avium subsp. paratuberculosis.
doi:10.1128/AEM.01203-09
PMCID: PMC2794114  PMID: 19837835
4.  Rapid Multiplex PCR and Real-Time TaqMan PCR Assays for Detection of Salmonella enterica and the Highly Virulent Serovars Choleraesuis and Paratyphi C▿ †  
Journal of Clinical Microbiology  2008;46(12):4018-4022.
Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.
doi:10.1128/JCM.01229-08
PMCID: PMC2593286  PMID: 18923008

Results 1-4 (4)