Validation of a real-time polymerase chain reaction assay for the detection of Salmonella in crops of broiler chickens.

Salmonella is one of the frequent causes of bacterial foodborne diseases with major public health impact in industrialized countries. Food-producing animals, in particular poultry, are major sources of human salmonellosis. Salmonella is normally found in the gastrointestinal tract of animals and can contaminate the carcass during the slaughtering process. In poultry, crops are also colonized by this pathogen. Crops are more likely to get ruptured during evisceration and contaminate the carcass and therefore present a health risk to consumers. Reducing Salmonella colonization in crops could decrease carcass contamination and is considered a potential preharvest critical control point in poultry production. Furthermore, rapid and reliable diagnostic methods to detect Salmonella are needed to monitor crop colonization to help ensure food safety. However, detection of Salmonella by bacteriological methods is time consuming and labor intensive and is not suitable for routine screening of a large number of samples. Therefore, this study was undertaken to validate a real-time PCR (RPCR) assay for the detection of Salmonella spp. in crop samples of broiler chickens. In total, 997 crop samples (35 spiked, 962 field) were processed by both RPCR and culture. The RPCR correctly identified all spiked crop samples. Out of 962 field crop samples, 100 tested positive by RPCR and 88 tested positive by culture for Salmonella, giving a sample level prevalence of 10.4 % (95% CI: 8.54 to 12.50%) and 9.1% (95% CI: 7.40 to 11.15%), respectively. The agreement beyond chance between RPCR and culture was 92% (P < 0.001) and 100% (P < 0.001) for field and spiked samples, respectively. Compared with culture, the sensitivity and specificity of RPCR were 98.86 and 98.51% for field samples and 100 and 100% for spiked samples, respectively. Where bacterial speciation is required, only the positive samples would be cultured. Therefore, RPCR can be used as a good screening tool for Salmonella spp. in crops by eliminating the time-consuming and labor-intensive culture of negative samples.

A microbiological survey of selected Alberta-grown fresh produce from farmers' markets in Alberta, Canada.

Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh produce grown in Alberta, Canada. Baseline information on the occurrence and levels of Escherichia coli and the prevalence of foodborne pathogens in selected produce items available to consumers from farmers' and public markets in two large urban centers and surrounding areas in Alberta was obtained. A total of 10 large markets with between 1 and 12 produce vendors and 26 small markets with between 1 and 6 produce vendors were sampled from 21 June to 7 October 2007. Lettuce (128 samples), spinach (59 samples), tomatoes (120 samples), carrots (206 samples), green onions (129 samples), and strawberries (31 samples) were analyzed for E. coli, Salmonella, E. coli O157:H7, and Campylobacter spp. Lettuce, spinach, green onion, and strawberry samples were also tested for the presence of Cryptosporidium spp. Information on whether produce was grown using organic or conventional practices was obtained from the produce vendors. E. coli was isolated from 8.2% of the samples that included lettuce, spinach, carrots, and green onions. The bacterial counts ranged from <0.48 to >3.04 Log most probable number per g. E. coli was not isolated from tomatoes or strawberries. The percentage of positive samples ranged from 4.4% for carrots to 27.1% for spinach. Salmonella, E. coli O157:H7, and Campylobacter spp. were not isolated from any of the samples. Cryptosporidium was identified by PCR in one sample of spinach (0.6% of the samples).

A real-time PCR assay for the detection of Salmonella in a wide variety of food and food-animal matricest.

Conventional culture methods have traditionally been considered the "gold standards" for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada.

A total of 800 meat and poultry products were purchased from the retail marketplace in Edmonton, Alberta, Canada. The products consisted of raw ground beef, chicken legs, pork chops, and ready-to-eat fermented sausage, roast beef, processed turkey breast, chicken wieners, and beef wieners. The samples were analyzed to determine the prevalence of Shiga toxin-producing Escherichia coli, Salmonella, Campylobacter spp., and Listeria monocytogenes. Shiga toxin-producing E. coli 022: H8 was found in one raw ground beef sample. Salmonella and Campylobacter were found in 30 and 62% of raw chicken legs, respectively. L. monocytogenes was found in 52% of raw ground beef, 34% of raw chicken legs, 24% of raw pork chops, 4% of fermented sausages, 3% of processed turkey breast, 5% of beef wieners, and 3% of chicken wieners. The occurrence of pathogens in this study is similar to that in retail products in many other international locales.

Protein turnover in compensated chronic aortic regurgitation.

We recently demonstrated dynamic alterations in protein turnover 3 days and 1 month after surgical induction of aortic regurgitation (AR). To characterize protein synthesis and degradation during the long-term plateau phase, we performed [3H]-leucine infusion 2.5 years after induction of AR in 10 New Zealand White rabbits and 12 sham-operated controls. Protein fractional synthesis rates were obtained by analyses of plasma and protein hydrolysates, growth rates from protein concentration and heart weight measurements, and degradation rates by subtraction of growth from synthesis rates. AR (regurgitant fraction 25 +/- 11%) caused a 57% increase in left ventricular (LV) weight in comparison with controls (7.4 +/- 1.7 vs. 4.7 +/- 0.6 g, p < 0.001) and no evidence of heart failure. Although concentrations of total cardiac protein, myosin heavy chain and actin were similar, the enlarged AR hearts had increased amounts of total cardiac protein (1,009 +/- 312 vs. 682 +/- 120 mg/LV, p < 0.05), myosin heavy chain (148 +/- 91 vs. 81 +/- 29 mg/LV, p < 0.05), and actin (73 +/- 42 vs. 44 +/- 16 mg/LV, p < 0.06). Individual protein fractional synthesis and degradation rates were closely balanced. However, myosin fractional synthesis rates were 152% (p < 0.01) greater than those of total cardiac protein in AR animals, while only 52% (p < 0.05) greater in controls (AR vs. controls, p = 0.05). Variations in actin turnover between AR and control animals did not attain statistical significance. Myosin and actin fractional synthesis rates correlated closely in AR rabbits (R = 0.81, p < 0.02), but not among controls (R = 0.41, NS). Thus, selective alterations in myofibrillar protein turnover contribute to the maintenance of increased myofibrillar protein content in the 'compensatory' LV hypertrophy of chronic AR.

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains.

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.

Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction.

In this study, the polymerase chain reaction (PCR) was used in the detection of the attaching and effacing (eae) gene of Shiga-like toxin-producing Escherichia coli (SLT-EC). Oligonucleotide primers, complementary to the 5' portion of the eae gene of the enteropathogenic E. coli E2348/69 (O127:H6) and of SLT-EC CL8 and EDL933 (O157:H7), generated PCR products of the predicted sizes with DNA from the majority of human clinical SLT-EC strains tested from O serogroups 5, 26, 103, 111, 121, 128, 145, and 157; all SLT-EC strains of O serogroups 5, 26, and 111 from cattle; and a minority of porcine SLT-EC strains (one strain each from O serogroups 107 and 130 and one rough strain). Five HaeIII digestion profiles were obtained for PCR products generated by amplification of a 2.3-kb DNA fragment from the 5' end of eae. The HaeIII profiles for SLT-EC O serogroups, such as 26, 103, and 157, differed from each other but were consistent among strains within these O serogroups. Oligonucleotide primer pairs complementary to the 3' end of either the O127:H6 E. coli or the O157:H7 eae nucleotide sequence only amplified DNA from E. coli strains from a few of the SLT-EC O serogroups examined. One primer pair with homology to the 3' nucleotide sequence of eae from E. coli O157:H7 appeared to be relatively specific for this O serogroup by PCR. No PCR products were obtained in amplification experiments with the eae primers using DNA from human SLT-EC of O serogroups 38 (1 0f 1) and 91 (3 or 3), 15 of 15 SLT-EC strains from edema disease, or 29 of 29 non-SLT-EC strains from pigs and calves with diarrhea.

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.