Detection by multiplex real-time polymerase chain reaction assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef.

Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 42°C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx(1), stx(2), and genes in the O-antigen gene clusters of the six serogroups, [corrected] and then immunomagnetic separation (IMS) followed by plating onto Rainbow® Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25 g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24 h (6 h at 37°C and 18 h at 42°C). The detection limit of the real-time multiplex PCR assays was ∼50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.

Performance comparison of a fliC(h7) real-time PCR assay with an H7 latex agglutination test for confirmation of the H type of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. Positive identification of E. coli O157:H7 is made using biochemical tests and specific antisera or latex agglutination reagents for the O157 and H7 antigens. However, under certain conditions, some E. coli O157:H7 isolates can appear to be nonreactive with H7 antisera and may require multiple passages on motility medium to restore H7 antigenicity. In this study, we compared the performance of a real-time PCR test with that of a method using latex agglutination reagents to detect the presence of the fliC(h7) gene or the H7 antigen, respectively, in E. coli O157:H7 isolates. One hundred twenty-six E. coli strains were tested including reference strains and strains isolated from meat. Lyophilized E. coli O157:H7 isolates were rehydrated and were plated on sheep blood agar without passage on motility medium. All strains were analyzed in parallel by a real-time PCR test targeting the fliC(h7) gene and by a latex agglutination test that detects the H7 antigen. The real-time PCR assay showed 100% agreement with the H7 status reported for reference strains and E. coli O157:H7 meat isolates. The latex agglutination test results agreed with the H7 status reported for the E. coli O157:H7 reference strains and non-O157:H7 strains, except for one, E. coli O117:H7; however, 42% (42 of 100) of the E. coli O157:H7 meat isolates tested negative for the H7 antigen by latex agglutination. The real-time fliC(h7) PCR test can be used to confirm E. coli O157:H7 strains that are not expressing the immunoreactive H7 antigen.

Thermal injury and recovery of Salmonella enterica serovar enteritidis in ground chicken with temperature, pH, and sodium chloride as controlling factors.

Cells of Salmonella enterica serovar Enteritidis were grown at 25 and 35 degrees C, heat injured (55, 60, and 62.5 degrees C), and recovered in tryptic soy broth (TSB) at various NaCl concentrations (2.0 and 3.5%) and pH levels (5.5 and 6.5). To assess the interactions of growth temperature, heating temperature, NaCl concentration and pH on the thermal injury and recovery of Salmonella Enteritidis in ground chicken, a randomized design with each experimental combination was used. When a logistic equation for nonlinear survival curves was used, D-values of cells of Salmonella Enteritidis grown at 25 degrees C were 7.60, 5.73, and 4.81 min at 55, 60, and 62.5 degrees C, respectively. For cells grown at 35 degrees C, the D-values were 12.38, 7.45, and 5.70 min at 55, 60, and 62.5 degrees C. The influence of tryptic soy agar and double modified lysine agar (DMLIA) on the recovery of heat-injured cells was determined. Recovery was significantly reduced on DMLIA at increased pH levels and NaCl concentrations. Higher numbers of cells were recovered in TSB with 2.0% NaCl than in TSB with 3.5% NaCl. It was observed that the rate of recovery of heat-injured cells was similar at each pH. Therefore, a pH range of 5.5 to 6.5 does not have a major inhibitory effect on the recovery of Salmonella Enteritidis.

Effect of refrigerating delayed shipments of raw ground beef on the detection of Salmonella Typhimurium.

In eight separate trials, four groups of raw ground beef samples were inoculated with 0.04 to 0.3 CFU/g of Salmonella Typhimurium (DT 104). Each group consisted of four 25-g samples (three inoculated and one uninoculated). After inoculation, these samples were shipped by overnight courier in Shipping containers with ice packs from the U.S. Department of Agriculture (USDA), Eastern Regional Research Center, in Wyndmoor, Pa., to the U.S. Food Safety and Inspection Service (FSIS), Eastern Laboratory, in Athens, Ga. A total of 128 samples (32 in each of four groups) were shipped. A temperature data logger was placed inside each shipping container to record the temperature during shipping and storage. The first group of ground beef samples was analyzed within approximately 1 h of arrival. The second group of samples was left in the original containers, with a gel ice pack, for 24 h before processing. The third and fourth groups of samples were removed from the original shipping containers and stored at room temperature (21 +/- 2 degrees C) for 6 h and then in a refrigerator at 4 +/- 2 degrees C for 24 and 48 h, respectively, before analysis. The samples were analyzed for the presence of Salmonella according to the USDA/FSIS Microbiological Laboratory Guidebook, chapter 4.02. There was no significant difference in the presence and levels of Salmonella in ground beef among the four test groups. These data show that it is acceptable to process the late-arriving ground beef samples for the detection of Salmonella if they are kept in a refrigerator (4 +/- 2 degrees C) for 24 to 48 h or when the shipments arrive late (24 h in the container with ice pack).

Molecular characterization of reticuloendotheliosis virus insertions in the genome of field and vaccine strains of fowl poxvirus.

Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent "variant" FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5' long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3' LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3' LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5' LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3' LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5' LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5' LTR sequences, positive PCR results with primers that amplify the 5' LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3' LTR PCR.

Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

Latex agglutination assays for detection of non-O157 Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145.

Latex agglutination assays utilizing polyclonal antibodies were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups. Rabbit antisera were affinity purified through protein A/G columns, and the isolated immunoglobulins (IgGs) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 0.20 to 0.28 mg/ml in a 1% latex suspension. Optimum conditions for the agglutination assay consisted of utilizing 20 µm l of latex-IgG reagent containing 2.0 to 2.8 µm g IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly after mixing the colonies with the latex-IgG, indicating STEC strains. More than 100 target and nontarget strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) cross-reacted with some other STECs. The anti-O103 and anti-O145 latex reagents cross-reacted with O26 strains, and the anti-O26 cross-reacted with O103 strains. The latex-IgG reagents are stable for at least 1 year and are easy to prepare. These agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. The techniques used to prepare the latex reagents also can be utilized for testing other STEC serogroups, other E. coli serotypes, or other pathogens to ensure safe foods to consumers.

Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment.

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.