Evaluation of motility enrichment on modified semi-solid Rappaport-Vassiladis medium (MSRV) for the detection of Salmonella in foods

The detection and identification of Salmonella spp. is still troublesome and time consuming to the food industry. Employing the modified semi-solid Rappaport-Vassiliadis medium (MSRV), presumptive results for Salmonella can be obtained in 48 h, representing an interesting alternative to the standard methods. The specificity and sensitivity of the MSRV method were evaluated in this research. The efficiency of this method was also compared with the methodology recommended by the US Food and Drug Administration (FDA) using bismuth sulfite agar, XLT4 agar and Rambach agar. A total of 146 food samples comprised of 41 chicken thighs, 35 Brazilian fresh pork sausages, 35 samples of cocoa powder and/or granulated cocoa and 35 samples of grated fresh coconut, were examined. Overall, the rapid method (direct + indirect) and the standard culture detected 96.1% and 84.6% of the positive samples, respectively. No Salmonella was detected in the coconut or cocoa samples by any of the methods. Eighteen (43.9%) chicken thigh samples were contaminated with the microorganism. The rapid method (direct + indirect) and the standard culture detected 94.4% and 88.9% of these, respectively. Salmonella was detected in eight (22.8%) fresh pork sausage samples. The MSRV method detected Salmonella in all eight samples, while the standard gave positive results in six (75%). When compared with the standard method, the indirect method showed 86.4% sensitivity and 96.8% specificity, while the direct MSRV showed a sensitivity of 71.4% and specificity of 99.2%. Combined, both MSRV methods showed 95.5% sensitivity and 96.8% specificity. The MSRV medium also reduces the time necessary for the isolation of Salmonella from foods.     

A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed using monoclonal antibodies (MAbs) as a rapid, economical alternative to culture isolation procedures for detection of Salmonella. Four MAbs previously shown to react with Salmonella strains representing 18 different serogroups were evaluated as capture antibodies and, after biotinylation, as detection antibodies. One MAb (M183) was selected for use in the ELISA to capture and detect Salmonella antigens. The detection limit of the ELISA was evaluated using Salmonella enterica subspecies enterica serovar Typhimurium and various selective and nonselective Salmonella enrichment media. The highest detection limit (ca. 10(4) CFU/ml) was achieved using an enrichment broth containing brain heart infusion, yeast extract, sodium hydrogen selenite, and sodium cholate (BYSC) after preenrichment in buffered peptone water. The ELISA detected all Salmonella serovars tested, which included representative serovars of serogroups B, C, D, and E and gave negative results for all non-Salmonella species tested. Samples (106) from various sources, including fecal samples from humans and pigeons, chicken carcass rinses, chicken parts, feed, and the environment, were used to evaluate the performance of the ELISA. The ELISA had a specificity and sensitivity of 100 and 91%, respectively, and a kappa value of 0.93 relative to the culture methods. Such an ELISA has the potential to be used in the implementation of the pathogen reduction and hazard analysis critical control point systems as well as in clinical laboratories.     

Detection of Salmonella from chicken rinses and chicken hot dogs with the automated BAX PCR system

The BAX system with automated PCR detection was compared with standard cultural procedures for the detection of naturally occurring and spiked Salmonella in 183 chicken carcass rinses and 90 chicken hot dogs. The automated assay procedure consists of overnight growth (16 to 18 h) of the sample in buffered peptone broth at 35 degrees C, transfer of the sample to lysis tubes, incubation and lysis of the cells, transfer of the sample to PCR tubes, and placement of tubes into the cycler-detector, which runs automatically. The automated PCR detection assay takes about 4 h after 16 to 24 h of overnight preenrichment. The culture procedure consists of preerichment, enrichment, plating, and serological confirmation and takes about 72 h. Three trials involving 10 to 31 samples were carried out for each product. Some samples were spiked with Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Montevideo, and Salmonella Enteritidis at 1 to 250 cells per ml of rinse or 1 to 250 cells per g of meat. For unspiked chicken rinses, Salmonella was detected in 2 of 61 samples with the automated system and in 1 of 61 samples with the culture method. Salmonella was recovered from 111 of 122 spiked samples with the automated PCR system and from 113 of 122 spiked samples with the culture method. For chicken hot dogs, Salmonella was detected in all 60 of the spiked samples with both the automated PCR and the culture procedures. For the 30 unspiked samples, Salmonella was recovered from 19 samples with the automated PCR system and from 10 samples with the culture method. The automated PCR system provided reliable Salmonella screening of chicken product samples within 24 h.     

Primers specific for the fimbrial major subunit gene stdA can be used to detect Salmonella enterica serovars

The feasibility of using two primers internal to the stdA gene (which encodes the fimbrial major subunit of the std fimbrial gene cluster in Salmonella enterica serovar Typhi) to detect Salmonella by PCR was explored. The 518-bp stdA specific sequence was conserved among 268 strains from 45 serovars of S. enterica. One Salmonella bongori CCUG 30042 strain and 34 non-Salmonella strains did not possess this sequence. A sensitivity test revealed that the stdA-specific primer set detected 3.4 x 10(-1) pg of genomic DNA and 3.0 x 10(5) CFU/ml with serial dilutions of Salmonella Typhimurium cells. In vitro testing for specificity using pig carcass sponge samples contaminated with Salmonella Typhimurium also was performed. An initial Salmonella Typhimurium inoculum of 4.4 x 10(1) CFU/ml in pig carcass exudates reached the stdA primer detection level after preenrichment in buffered peptone water at 37 degrees C for 18 h in the presence of indigenous non-Salmonella flora at 4.0 X 10(7) CFU/ml, but the detection level decreased to 4.4 x 10(0) CFU/ml after selective enrichment in Rappaport-Vassiliadis R10 broth for 18 h at 42 degrees C. The PCR method with primers specific for stdA is a quick and sensitive tool for detecting S. enterica, which is an important cause of foodborne disease.     

Rapid detection of Salmonella in chicken washes by immunomagnetic separation and flow cytometry.

Use of flow cytometry to rapidly detect Salmonella in chicken carcass washes was investigated. A direct immunomagnetic separation method was used to prepare samples and was found to be an effective method for separating target cells from debris in chicken carcass washes. When flow cytometry was combined with immunomagnetic separation, the average lowest detectable level of Salmonella detected was 2.3 x 10(4) CFU/ml. Fifty of 100 wash samples from six groups were inoculated with 2 x 10(-1) CFU of Salmonella Typhimurium per milliliter. After 18 h of enrichment at 37 degrees C, all samples were tested for Salmonella using flow cytometry and conventional culture methods. An identification correlation of 96% was found between flow cytometry and xylose-lysine-tergitol agar plating. Quantitative analysis established a significant linear relationship between the enumeration results of flow cytometry and xylose-lysine-tergitol agar plate counts (R2 = 0.796). Time required for flow cytometry, including sample processing and flow cytometric analysis, was less than 1 h.     

Comparison between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork carcass sponge samples

VIDAS Salmonella (VIDAS-SLM) is an automated system that uses the enzyme-linked fluorescent assay method to detect Salmonella species. This study evaluated the efficacy of the VIDAS-SLM method in detecting Salmonella species in pork carcass sponge samples gathered from 10 slaughter plants in Taiwan. Two hundred fifty-seven pork carcass sponge samples were screened by the VIDAS-SLM method and by the culture method in parallel. While 18 sponge samples were found to test positive by both methods, the VIDAS-SLM method detected four additional positive samples for which the culture method failed to recover Salmonella. The specificity of the VIDAS-SLM method was found to be 0.98, and its sensitivity was 1.0, since no false-negative results occurred. Artificially inoculated Salmonella at concentrations as low as 5.0 x 10(0) CFU/ml was detected in the heat-inactivated sponge sample in the presence or absence of 5.0 x 10(4) CFU of Citrobacter freundii per ml. Thus, the VIDAS-SLM method is a rapid screening method and a potential alternative to the time- and labor-intensive culture method.     

Multiplication and motility of Salmonella enterica serovars enteritidis, infantis, and montevideo in in vitro contamination models of eggs

The invasive ability of Salmonella enterica serovars Enteritidis, Infantis, and Montevideo in eggs was examined. Strains of these serovars originating from egg contents, laying chicken houses, and human patients were experimentally inoculated (0.1-ml dose containing 78 to 178 cells) onto the vitelline membrane of eggs collected from specific-pathogen-free chickens and incubated at 25 degrees C. The test strains were detected in 25 of 138 yolk contents by day 6, indicating the penetration of Salmonella organisms through the vitelline membrane. There were no significant differences in overall rates of penetration between serovars. The organisms were also detected in the albumen from 125 of 138 eggs tested by day 6. Growth to more than 10(6) CFU/ml was observed in 48 of the 125 albumen samples. An inoculum of 1000 Salmonella cells was added to 15 ml of albumen at the edge of a petri plate. A 10-mm-diameter cylindrical well, the bottom of which was sealed with a polycarbonate membrane with 3.0-microm pores, was filled with egg yolk and placed into the albumen at the center of the dish, which was maintained at 25 degrees C. Experiments were performed in triplicate with each strain. Salmonella organisms in all the albumen samples were detected by day 11. However, motility of the organisms toward the yolk was observed in only two dishes inoculated with the Salmonella Enteritidis strain from a human patient and in one dish inoculated with the Salmonella Infantis strain from liquid egg. The albumen samples obtained from the dishes inoculated with the Salmonella Enteritidis strain had high numbers of bacteria (>10(8) CFU/ml). The present study suggests that Salmonella organisms in egg albumen are unlikely to actively move toward the yolk, although depositionon or near the vitelline membrane can be advantageous for proliferation.     

实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌

根据沙门氏菌invA基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立了沙门氏菌实时荧光定量环介导等温扩增检测方法。结果表明,检测过程仅需45 min,灵敏度达6 CFU/管,而且细菌数量对数值（lg（CFU/管））与扩增荧光指数增加时间（Tp值）具有良好正相关线性关系,R2为0.985 1。通过模拟沙门氏菌人工污染25 g鸡肉样品,经37 ℃保温4 h,提取样品制备DNA模板用于实时荧光定量环介导等温扩增检测,结果表明灵敏度达450 CFU/g,共需时约7 h。随机从市场购买冷冻鸡肉样品21 份,采用国标沙门氏菌检测方法和鸡肉样品实时荧光定量环介导等温扩增检测进行对比检测,结果两种方法均检测出同一份样品为沙门氏菌阳性,其余20 份样品均为沙门氏菌阴性,表明本研究建立的鸡肉沙门氏菌实时荧光定量环介导等温扩增检测,其灵敏性和准确性与国标检测方法相当,但检测时间可缩短至7 h。     

Preliminary evaluation of flow-through immunocapture followed by real-time PCR for the detection of Salmonella serovars on tomato surfaces within 8 hours

This study reports a preliminary evaluation of flow-through immunocapture (FTI) followed by real-time PCR (FTI-PCR) for the detection of Salmonella serovars on tomato surfaces within 8 h. The FTI-PCR method was compared with real-time PCR, direct plating of FTI beads on xylose lysine desoxycholate (XLD), and the conventional culture method for Salmonella found in the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). Unwaxed green tomatoes were spot inoculated with a five-serovar Salmonella cocktail on smooth surfaces at levels of 10(0) to 10(4) CFU per tomato and washed in lactose broth (LB) using a shake-rub method. The resulting LB rinse was incubated at 37 degrees C for 4 h prior to analysis by FTI-XLD, real-time PCR, or FTI-PCR and for 24 h as the first step in the BAM Salmonella culture method. For FTI-XLD, the observed lowest detection level (LDL) was 4.6 x 10(1) CFU per tomato. There was no significant difference in performance between the FTI-XLD method and the BAM Salmonella culture method (P > 0.05); however, the FTI-XLD method reduced the overall assay time by 48 h. For real-time PCR and FTI-PCR, the observed LDLs were 4.6 x 10(1) and 9.2 x 10(0) CFU per tomato, respectively. The FTI-PCR method was superior to the BAM Salmonella culture method (P < 0.05) for the detection of Salmonella serovars on tomato surfaces and was completed within 8 h.     

Monochloramine versus sodium hypochlorite as antimicrobial agents for reducing populations of bacteria on broiler chicken carcasses

Studies were conducted to compare the effect of sodium hypochlorite (SH) versus monochloramine (MON) on bacterial populations associated with broiler chicken carcasses. In study 1, nominal populations (6.5 to 7.5 log CFU) of Escherichia coli, Listeria monocytogenes, Pseudomonas fluorescens, Salmonella serovars, Shewanella putrefaciens, and Staphylococcus aureus were exposed to sterilized chiller water (controls) or sterilized chiller water containing 50 ppm SH or MON. SH at 50 ppm eliminated all (6.5 to 7.5 log CFU) viable E. coli, L. monocytogenes, and Salmonella serovars; 1.2 log CFU of P. fluorescens; and 5.5 log CFU of S. putrefaciens. MON eliminated all (6.5 to 7.5 log CFU) viable E. coli, L. monocytogenes, S. putrefaciens, and Salmonella serovars and 4.2 log CFU of P. fluorescens. In study 2, chicken carcasses were inoculated with P. fluorescens or nalidixic acid-resistant Salmonella serovars or were temperature abused at 25 degrees C for 2 h to increase the populations of naturally occurring E. coli. The groups of Salmonella serovar-inoculated or temperature-abused E. coli carcasses were immersed separately in pilot-scale poultry chillers and exposed to tap water (controls) or tap water containing 20 ppm SH or 20 ppm MON for 1 h. The P. fluorescens-inoculated group was immersed in pilot-scale poultry chillers and exposed to tap water (controls) or tap water containing 50 ppm SH or 50 ppm MON for 1 h. Carcasses exposed to the SH treatment had nominal increases (0.22 log CFU) in E. coli counts compared with controls, whereas exposure to MON resulted in a 0.89-log reduction. Similarly, average nalidixic acid-resistant Salmonella serovar counts increased nominally by 34% (41 to 55 CFU/ml) compared with controls on carcasses exposed to SH, whereas exposure to MON resulted in an average nominal decrease of 80% (41 to 8 CFU/ml). P. fluorescens decreased by 0.64 log CFU on carcasses exposed to SH and decreased by 0.87 log CFU on carcasses exposed to MON. In study 3, SH or MON was applied to the chiller in a commercial poultry processing facility. E. coli counts (for carcass halves emerging from both saddle and front-half chillers) and Salmonella prevalence were evaluated. Data from carcasses exposed to SH during an 84-day historical (Hist) and a 9-day prepilot (Pre) period were evaluated. Other carcasses were exposed to MON and tested during a 27-day period (Test). E. coli counts for samples collected from the saddle chiller were 25.7, 25.2, and 8.6 CFU/ml for Hist, Pre, and Test, respectively. E. coli counts for samples collected from the front-half chiller were 6.7, 6.9, and 2.5 CFU/ml for Hist, Pre, and Test, respectively. Salmonella prevalence was reduced from 8.7% (Hist + Pre) to 4% (Test). These studies indicate that MON is superior to SH in reducing microbial populations in poultry chiller water.     

Growth of Salmonella enterica serovar Enteritidis in albumen and yolk contents of eggs inoculated with this organism onto the vitelline membrane

By using an in vitro model simulating the potential opportunities for Salmonella enterica serovar Enteritidis (SE) to proliferate within eggs contaminated with this organism following oviposition, we investigated growth of SE in eggs. Seventy to 140 CFU of one of three SE strains originating either from egg contents, chicken meat, or a human infection were experimentally inoculated onto the vitelline membrane of eggs collected from specific-pathogen-free flocks of chickens and incubated at 25 degrees C. SE organisms were detected in 6 of 71 yolk contents of the eggs inoculated with any of the test strains attaining levels ranging from 2.0 x 10(2) to 4.2 x 10(8) CFU/ml by day 6. The organisms were also detected in the albumen from 38 of 55 eggs tested, growing to levels ranging from 1.0 x 10(2) to 4.3 x 10(8) CFU/ml by day 6 after inoculation. An additional three yolk contents and 15 albumen samples were culture positive for SE following enrichment. There was no correlation between the number of the organisms in the yolk contents and that in the albumen from each of the eggs. When 73 to 91 CFU of the egg strain were inoculated into samples of separated albumen obtained from eggs that were stored at 4 degrees C for 1 to 4 weeks or at 25 degrees C for 1 week, slight growth (3.0 x 10(2) to 7.4 x 10(3) CFU/ml) was found in only 3 of the 60 albumen samples by day 6 after inoculation, but the organisms were recovered from 52 samples following enrichment. The results suggest that the environment on or near the vitelline membrane can be conducive to SE proliferation over time.     

Optimization and validation of a simple method using P22::luxAB bacteriophage for rapid detection of Salmonella enterica serotypes A, B, and D in poultry samples

A simple method was developed for the fast and inexpensive detection of Salmonella Typhimurium using a recombinant P22::luxAB phage. All the steps from phage production to detection were considered. A strain of Salmonella Typhimurium harboring the prophage P22::luxAB was grown in batch culture to produce spontaneously the recombinant bacteriophage. Batch production to stationary phase was better for propagation of the phage and led to a total population of 4.3 x 10(9) (+/-4.3 x 10(9)) PFU/ml of P22, including only 1.4 x 10(6) (+/-1 x 10(6)) PFU/ml harboring the luxAB genes. After preenrichment, a simple four-step bioassay was tested and optimized for several parameters. The detection limit of the luminometer was only 5 x 10(2) (+/-1.75 x 10(2)) CFU Salmonella Typhimurium per ml, but increased to 1.5 x 10(4) (+/-1.17 x 10(4)) CFU Salmonella Typhimurium per ml when the cells were in a complex matrix. The detection limit after the preenrichment was 6.5 x 10(3) (+/-1.5 x 10(3)) CFU Salmonella Typhimurium per ml, but the detection limit after the preenrichment also increased markedly to 1.65 x 10(5) (+/-0.15 x 10(5)) CFU Salmonella Typhimurium per ml when Salmonella Typhimurium was in a complex matrix. Finally, the bioassay was applied to the detection of Salmonella Typhimurium LT2 in 14 different feed and environmental samples (including duck feed, litters, and feces) spiked either before or after the preenrichment process. It was possible to detect Salmonella Typhimurium LT2 in all samples within 16 h.     

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples

The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non-salmonella strains yielded no amplification products. The melting temperature (Tm=79 degrees C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle (C(T)) versus copy numbers of Salmonella Enteritidis showed good linearity in broth (R2=0.994; slope=3.256) and sterilized milk (R2=0.988; slope=3.247), and the minimum levels of detection were >10(2) and >10(3) colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10(0) to 10(5) CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10(3) to 10(4) CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.     

Development of a rapid response biosensor for detection of Salmonella typhimurium.

An integrated optic interferometer for detecting foodborne pathogens was developed. The interferometer is a planar waveguide with two thin antibody-coated channels of immunochemically selective agents that interact with antigen molecules. One channel is coated with antibody to Salmonella as a sample, and the other is coated with human immunoglobulin G as a reference channel by using reductive amination. Salmonella was introduced onto the sensing channels through the flow cell on the channels. Phase shift (pi) generated by refractive index variation, as determined by interfering the perturbed sample channel with an unperturbed reference channel and observing the fringe shift, was used for detection. Salmonella Typhimurium (heat-treated or boiled) was detected by binding to antibody against Salmonella common structural antigen immobilized on a silane-derived sensor surface at concentrations in the range of 1x10(5) to 1x10(7) CFU/ml. Salmonella (1x10(7) CFU/ml) mixed with Escherichia coli (1x10(7) CFU/ml) were readily detected without any decrease in sensitivity by the direct assay. Application of a sandwich assay with a second antibody or a gold-conjugated antibody increased the detection limit to 1x10(5) CFU/ml within a 10-min reaction time. Various methods for the immobilization of the capture antibody to the biosensor channels were compared. The greatest binding response was observed in a direct reductive amination method with a long reaction period and increased the detection limit of direct binding of Salmonella antigen to 1x10(4) CFU/ml. The biosensor was able to detect Salmonella Typhimurium in chicken carcass wash fluid originally inoculated at a level of 20 CFU/ml after 12 h of nonselective enrichment. The planar optic biosensor shows promise as a fast, sensitive, reliable, and economical means of detecting food pathogens in the future.     

Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).     

Specificity of a conductance assay for enumeration of Escherichia coli from broiler carcass rinse samples containing genetically similar species

Experiments were conducted to evaluate the specificity of a rapid method for enumeration of Escherichia coli from fresh broiler chicken carcasses. In three separate trials, E. coli, Citrobacter freundii, Salmonella Enteritidis, and Shigella sonnei were serially diluted and then inoculated into identical broiler chicken carcass rinses. Inoculated rinses were mixed with double-strength Coliform Medium supplemented with 2% dextrose. This mixture was placed in a Bactometer module in duplicate, and conductance was measured at 44 degrees C. Results indicated that C. freundii did not grow to an appreciable degree in the selective medium at 44 degrees C. Salmonella Enteritidis grew similarly to E. coli; however, an initial level of 10(6) Salmonella in the food product would be required for Salmonella to interfere with enumeration of E. coli using this method. S. sonnei grew at a more rapid rate than E. coli; however, there was an interaction between the regression lines formed when serial dilutions (log10 CFU/ml) were compared to E. coli detection times for these two species of bacteria. Therefore, high levels of S. sonnei in a food sample may interfere with the enumeration of E. coli. In general, Salmonella and Shigella are not found at high enough levels on poultry products to interfere with enumeration of E. coli using this method and, if found at high levels, would be detected and rejected using this procedure. Hence, the presence of organisms that are genetically and phylogenetically similar to E. coli would not preclude enumeration of E. coli using conductance under these conditions.     

A comparison of sample weight and culture methods for the detection of Salmonella in pig feces

Five protocols were compared to determine the combined effects of different sample weights and culture methods for the recovery of Salmonella from 310 pig cecal samples taken in abattoirs as part of the Canadian Integrated Program for Anti-microbial Resistance Surveillance. Sample weights evaluated were 1 and 10 g. Culture methods used with each sample weight were modified semisolid Rappaport-Vassiliadis agar (MSRV) and brilliant green agar with sulfa and novobiocin (BGSN) and xylose-lysine-tergitol-4 agar (XLT4). A preliminary sample preparation step in saline was also evaluated using a 10-g sample and MSRV. The Salmonella recovery rate varied from 20% for the saline MSRV 10-g protocol to 32% for the MSRV 10-g and the BGSN-XLT4 10-g protocols. A good agreement (K > 0.8) was observed between pairs of protocols except whenever the saline MSRV 10-g and the MSRV 1-g protocols were compared. Larger samples (10 g) yielded higher detection of Salmonella than 1-g samples for the MSRV protocol (32 versus 25%), whereas the differences were not statistically significant for the BGSN-XLT4 protocols. Protocols using the BGSN-XLT4 agar yielded higher detection rates of Salmonella compared with MSRV with 1-g samples (30 versus 25%), whereas it was equivalent with 10-g samples. Considering a greater recovery rate, the ease of use, and a better time and resource efficiency, the MSRV 10-g protocol was therefore adopted by the Canadian Integrated Program for Antimicrobial Resistance Surveillance.     

Development and use of polymerase chain reaction for the specific detection of Salmonella Typhimurium in stool and food samples.

Salmonella Typhimurium is one of the most important Salmonella serovars that may cause foodborne disease and human salmonellosis infection. Detection of this organism in the clinical samples of persons with gastroenteritis and the food samples associated with such persons may allow us to trace the cause of disease. Because malic acid dehydrogenase, an enzyme of the citric acid cycle, is common to organisms, the gene (mdh) coding for this enzyme was selected for the design of Salmonella Typhimurium-specific polymerase chain reaction (PCR) primers. By comparison of the mdh gene sequences of Salmonella Typhimurium and other Salmonella serotypes and of some isolates of other genera, two oligonucleotides were designed and used as PCR primers for the specific detection of Salmonella Typhimurium. The molecular weight of the PCR product was 261 bp as expected. Salmonella serovars other than Salmonella Typhimurium and isolates of other genera in the Enterobacteriaceae that is closely related to Salmonella did not generate any false-positive results. When this primer pair was used for the detection of Salmonella Typhimurium cells artificially inoculated into human stool specimens and food samples, such as milk and raw chicken meat, levels as low as 10(0) CFU per 0.1 g of stool specimen or per ml of milk or food homogenate could be detected if an 8- to 12-h preculture step using combined lactose-tetrathionate broth was performed prior to the PCR.     

Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp.

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.     

Rapid, specific detection of Salmonella Enteritidis in pooled eggs by real-time PCR

An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5' nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye-labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non-group D Salmonella and other non-Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 10(2) to 10(9) CFU/ml in phosphate-buffered saline and 10(3) to 10(8) CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.