Analysis of National Animal Health Monitoring System Tonsil Samples for Presence of ail-bearing Yersinia enterocolitica by using a 5´ Nuclease Fluorogenic PCR (TaqMan) Assay

The fluorogenic 5′ nuclease polymerase chain reaction (TaqMan) assay, which was developed in this laboratory, was further optimized and used to screen hogs for Yersinia enterocolitica (4). Tonsil samples (n = 1,647) collected during the National Animal Health Monitoring System (NAHMS) Swine 2001 program were tested for the presence of the ail gene, which is present in virulent Y. enterocolitca. Tonsil swabs were collected on farms (n = 101) located in Arkansas, Colorado, Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Carolina, Ohio, Oklahoma, Pennsylvania, South Dakota, Texas and Wisconsin. Of the 1,647 tonsil samples, 11.4% (n = 188) were positive

A PCR ELISA Method For The Detection Of Yersinia Enterocolitica

The ELISA assay is a more rapid and sensitive method for PCR product detection than conventional gel electrophoresis. The PCR/ELISA allows for the processing of numerous samples, is relatively inexpensive and eliminates the need for electrophoretic and photographic equipment as well as the use of potential carcinogens such as ethidium bromide which is utilized in the gel electrophoretic detection of PCR products. The PCR ELISA is suitable for the rapid processing of samples required for field surveys of hogs and pork products

Evaluation of Reverse Transcriptase 5\u27 Nuclease Polymerase Chain Reaction assay for the Detection of Viable Heat-Injured and Resuscitated Listeria monocytogenes in Ground Pork

An anaerobic resuscitation-enrichment system was combined with a 5\u27 nuclease reverse transcriptase (RT) protocol for detecting Listeria monocytogenes Scott A from artificially inoculated ground pork. When irradiation-sterilized ground pork containing L. monocytogenes (~6 x 10 5 CFU/g) was heated (60 o C, 14 min), 100% of the cells were injured, as indicated by no growth on selective Modified Oxford (MOX) agar plates incubated aerobically. After resuscitation and enrichment (37°C) in anaerobic Penn State University (PSU) broth, L. monocytogenes was detected within 24 hours both by plating to MOX agar incubated in air and by a fluorogenic 5\u27 nuclease real-time RT-PCR assay. The RT-5\u27 nuclease polymerase chain reaction (PCR) assay targeting the hemolysin gene (hlyA) detected viable L. monocytogenes directly from the PSU within 24 hours, although a stronger signal was detected after 48 hours of resucitation. The RT-5\u27 nuclease PCR assay bypassed the need for subsequent plating of ground pork to selective agar and thus may shorten the interval to detect low numbers of viable L. monocytogenes following heating of naturally contaminated meat

Prevalence of Pathogenic \u3ci\u3eYersinia enterocolitica\u3c/i\u3e Strains in Pigs in the United States

Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United Sates using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5’ nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P \u3c 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans

The Cannulated Pig: A Model for Monitoring the Dynamics of Foodborne Pathogens In Vivo

We have developed a pig caecal cannulation model that allows us to evaluate the effects in vivo of feed withdrawal on (1) the caecal environment, including pH and volatile fatty acid (VFA) concentration, and (2) the growth of foodborne pathogens in the caecum. In vitro studies evaluated growth of Yersinia enterocolitica and Salmonella typhimurium at five concentrations of VFA at four pH levels. Minimal growth occurred in VFA and pH levels that simulated the caecum of a well-fed pig. Maximal occurs in the absence of VFA (0 mM/ml) at pH 7.0. When cultured in the caecal contents of a fasted pig, Yersinia and Salmonella replicate and survive. In contrast, caecal contents of a well-fed pig inhibit their growth in vitro. When instilled directly into the pig caecum, Y. enterocolitica and S. typhimurium were detected in fecal and cecal samples for up to 1 month. Infected pigs were subjected to four cycles of interrupted feeding. No predictable change occurs in the number of Yersinia or Salmonella in the caecum or in feces of pigs subjected to interrupted feedings compared with controls on a normal feeding regimen. In contrast, a fasting cycle predictably reduced VFA concentrations and increased the pH of the caecum. Thus, the pig caecal cannulation model is a practical way of monitoring the long-term dynamics of growth and survival of foodborne pathogens in the live animal

Rapid Detection of Listeria monocytogenes in Mechanically Separated Turkey Meat

The purpose of this study was to determine the level of Listeria spp., especially L. monocytogenes, in mechanically separated turkey (MST) meat samples. During four trials (n=150 samples), Listeria spp. were selected by using two enrichments (University of Vermont-modified I and II) and plating to selective Palcam agar base. A multiplex polymerase chain (PCR) reaction was used to confirm Listeria isolations. The specificity of the multiplex PCR assay was evaluated with reference strains of Listeria from the National Animal Disease Center (NADC) Culture Collection. The Listeria spp. yields a single 938-bp product, whereas L. monocytogenes yields the 938-bp product along with a 174-bp fragment. Results from trials I-IV indicated L. monocytogenes could not be detected by PCR in the UVM enrichment due perhaps to PCR inhibitors present in poultry fats and muscle myoglobin. The multiplex PCR performed from suspect colonies grown on Palcam, however, indicated 29 out of 150 (19%) of the MST meat harbored Listeria spp. Fifty-seven of 150 (38%) were confirmed positive for L. monocytogenes. Those confirmed as L. monocytogenes by PCR were serotyped and fingerprinted using enterobacterial repetitive intergeneic consensus (ERIC) motifs-PCR