Evaluation of a 5'-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples.

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5'-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with "gold standard" methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5'-nuclease assay was able to identify the organism in 100% of the repetitions when 10(2) CFU/ml or more organisms were present in pure cultures and 10(3) CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10(5) CFU/ml or more organisms were present in pure culture and 10(6) CFU/g or more organisms were present in inoculated ground pork. The 5'-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5'-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5'-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica.

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5' nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay.

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when >/=10(4) CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >/=10(2) CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >/=10(4) CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

Effect of endotoxin on interleukin-6 secretion and messenger ribonucleic acid in porcine anterior pituitary cells.

The pleiotropic cytokine interleukin-6 (IL-6) is produced in and secreted from anterior pituitary (AP) cells of a number of species. Bacterial endotoxin (END) may enhance the transcription of IL-6 and its secretion from the AP. In the studies presented here, we evaluated pig AP cells for the presence of IL-6 mRNA. In addition, because we had observed previously that END stimulated the secretion of prostaglandin E2 from cultured porcine AP cells, the effects of the inhibition of END-stimulated cyclooxygenase products on IL-6 mRNA abundance and the secretion of IL-6 were evaluated. In the first experiment, RNA was extracted from cultured pig AP cells that had been treated with END for 0.5 or 1 hr and subjected to reverse transcription followed by polymerase chain reaction and hybridization after Southern transfer. Bands of expected amplified product size, corresponding to IL-6, were observed only from cells treated with END, although specific hybridization was observed from both control and END-treated wells. In the next experiment, RNA was extracted from cultured AP cells treated with END or END in the presence of the cyclooxygenase inhibitor indomethacin (IND). Amplification of the expected product could be observed from all cultured cells except those treated with IND. However, hybridization data indicated that IND did not eliminate IL-6 mRNA entirely. Next, we measured IL-6 secretion from cultured AP cells exposed to END or END and IND. Treatment with END stimulated IL-6 secretion (P < 0.001) above controls, whereas IND blocked END stimulation of IL-6 secretion (P < 0.001). Finally, using immunostaining, we confirmed the presence of CD14, an END receptor, in cultured pig AP cells. These studies clearly establish the presence of IL-6 mRNA and secretion of the cytokine from cultured porcine AP cells. In addition, END stimulates the secretion of IL-6, perhaps through cells expressing CD14, and END-stimulated IL-6 secretion appears to be mediated by products of the cyclooxygenase pathway.

Combined PCR-oligonucleotide ligation assay for rapid detection of Salmonella serovars.

We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens. This method utilizes a short cultivation period followed by PCR. For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used. In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria. In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria. With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect > or = 20 CFU per assay or > or = 80 fg of chromosomal DNA (corresponding to 160 molecules of DNA). Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100%. In all experiments, the PCR-OLA was as sensitive as membrane hybridization. These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation.

Enhanced karyotype resolution of Cryptosporidium parvum by contour-clamped homogeneous electric fields.

Previous karyotype analysis with contour-clamped homogeneous electric fields (CHEF) indicated that the Cryptosporidium parvum karyotype comprises a minimum of five chromosomes all less than 1.6 Mb in size. In this study we were able to improve the resolution of the karyotype using a modified CHEF gel electrophoretic procedure that allows for the routine identification of seven C. parvum chromosomes ranging in size from about 0.945 to 2.2 Mb. Our data also suggest that an eighth chromosome is present and comigrates with another chromosome at about 1.3 Mb.

Detection of Salmonella typhimurium from rectal swabs of experimentally infected beagles by short cultivation and PCR-hybridization.

A rapid and sensitive cultivation and PCR-hybridization procedure for the detection and identification of Salmonella typhimurium was evaluated over a 42-day period with eight experimentally infected beagles. Rectal swabs were taken at several times postinfection, inoculated into selenite-cystine broth, and plated onto Hektoen-Enteric Enteric agar immediately after incubation for 4 and 24 h. PCRs and hybridizations were also conducted with each sample, and the results were compared with those of standard culture techniques to evaluate the efficiency of the PCR-hybridization procedure. The PCR-hybridization procedure was more sensitive than standard culture techniques at each enrichment incubation (P < 0.05). In addition, the PCR-hybridization procedure was significantly better than culture up through 3 days postinfection (P < 0.05). A nonspecific amplified product, relatively close in size to the 457-bp specifically amplified product, did not hybridize to an internal oligonucleotide probe or to a random-primed labeled probe. Subsequent sequence information revealed that the product had very little similarity to the 457-bp product but had significant similarity to an Escherichia coli aldehyde dehydrogenase gene. This study indicated that a cultivation and PCR-hybridization procedure is significantly better than culture for the identification of S. typhimurium. Additionally, the results confirm the importance of determining specificities of PCR products beyond the gel electrophoresis level by hybridization with a specific probe.

Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure.

To overcome problems associated with application of PCR to clinical samples, we have combined a short cultivation procedure with a Salmonella-specific PCR-hybridization assay to specifically identify Salmonella serovars from clinical samples of various animal species. The technique was investigated by using fecal samples seeded with known numbers of Salmonella organisms and cultivated for different lengths of time in assorted selective and nonselective enrichment media. The ability of PCR to amplify a Salmonella-specific DNA product (457-bp sequence covering the Salmonella invE and invA genes) was examined in Southern hybridizations with an internal oligonucleotide probe. Forty-seven Salmonella isolates representing 32 serovars were evaluated, and all Salmonella isolates resulted in a 457-bp product that hybridized with the oligonucleotide probe, whereas no hybridizations were evident with 53 non-Salmonella organisms. The assay detected as few as 9 CFU of Salmonella organisms in pure culture and as little as 300 fg of purified chromosomal DNA. Rappaport-Vassiliadis and tetrathionate broths were inhibitory to PCR, whereas brain heart infusion and selenite-cystine broths were not. The PCR-hybridization assay coupled with a brain heart infusion enrichment culture incubated for 2 h detected as few as 80 CFU of Salmonella organisms in seeded feces. We have successfully identified Salmonella serovars in clinical samples from swine, horses, and cattle more rapidly than with conventional culture techniques. The sensitivity and specificity of this assay were both 100% compared with culture results. These results indicate that a combined cultivation-PCR-hybridization assay could be applicable and advantageous in the rapid identification of Salmonella serovars in routine diagnostic situations.

Experimental infections and natural outbreaks of eperythrozoonosis in pigs identified by PCR-DNA hybridizations.

Eperythrozoon-specific DNA amplification reactions and subsequent hybridizations with an eperythrozoon DNA probe (KSU-2) were used in experimental infection studies to identify Eperythrozoon suis DNA in the blood of splenectomized and nonsplenectomized pigs. The results indicate that E. suis DNA is present in nonsplenectomized pigs at levels that can be amplified by polymerase chain reaction (PCR) and identified in DNA hybridizations within 24 hours after infection. The ability of the E. suis PCR/hybridization assay to detect eperythrozoonosis was further demonstrated in blood samples collected from pigs in 2 separate natural outbreaks in Oklahoma. Results from these initial samplings indicate that pigs infected with E. suis from geographically distinct locations can be identified using the eperythrozoon-specific PCR/hybridization assay, which offers many advantages over conventional laboratory procedures for diagnosing eperythrozoonosis in pigs.

Characteristic differences in reverse transcription-polymerase chain reaction products of ovine, bovine, and human respiratory syncytial viruses.

In reverse transcription-polymerase chain reactions (RT-PCR) and DNA hybridizations using primers and an oligonucleotide probe to the fusion (F) protein mRNA of bovine respiratory syncytial virus (BRSV), all the BRSV isolates and a goat isolate could be distinguished from prototype isolates of human respiratory syncytial viruses (HRSV) and ovine (sheep and bighorn sheep) respiratory syncytial viruses (RSV). However, RT-PCR amplifications with primers to sequences of the HRSV F protein mRNA resulted in amplified products of approximately 243 bp if mRNA templates of subgroup A HRSV strains were present and slightly larger amplified products with subgroup B HRSV strains. No amplified products were observed in HRSV-primed RT-PCR with BRSV or goat or ovine RSV mRNA templates. Although the ovine RSV isolates were antigenically cross-reactive with the goat RSV, HRSV and BRSV isolates, they were no amplified with either HRSV- or BRSV-specific primers in RT-PCR. These results confirm previous immunological comparisons suggesting that some ovine RSV isolates should be considered as distinct respiratory syncytial viruses.

Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations.

An assay to identify tissue culture cells infected with bovine respiratory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide hybridization probe has been developed. The RT-PCR assay uses a BRSV-specific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucleotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isolates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specific to BRSV, as demonstrated in hybridizations with a positive-sense oligonucleotide probe complementary to internal sequences and in sequence comparisons with the F protein of BRSV 391-2. In analysis of the BRSV RT-PCR assay with prototype strains of human RSV subgroups A and B, amplification of a similar 381-bp RT-PCR product was not evident, and no RT-PCR product hybridized with the internal probe. We conclude that the specific ability to amplify DNA sequences of BRSV F protein mRNA by RT-PCR and then to demonstrate the presence of the amplified product with a BRSV-specific oligonucleotide probe will greatly add to the speed, sensitivity, and specificity of BRSV diagnostics.

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multiocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella cholerasuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.

Detection of Eperythrozoon suis using the polymerase chain reaction.

Eperythrozoon suis is an extracellular red blood cell parasite that causes icteroanemia in acutely ill pigs and a variety of syndromes in chronically infected pigs. Current techniques to detect E. suis infection are limited by variability of parasitemias and antibody responses in infected animals. The polymerase chain reaction (PCR) was investigated to determine its potential as a means of detecting E. suis infection in pigs. With DNA samples extracted from either purified E. suis organisms or E. suis-infected pig blood, PCR produced an amplification product 492 base pairs in length. This amplification product hybridized successfully with the fragment of the DNA probe from which the primer sequences had been selected. Sensitivity studies indicated that the PCR protocol was capable of amplifying total genomic E. suis DNA in quantities as low as 450 pg. When PCR was used with DNA from blood samples from a splenectomized pig that had been infected with E. suis, amplification products were detectable as early as 24 hours postinfection. This preliminary analysis indicates that PCR shows promise as a means of efficiently detecting E. suis infection in pigs.