Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds

The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens.

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Growth and Destruction of Salmonella typhimurium in Egg White Foam Products Cooked by Microwaves

Currently, in both home and institutional food preparation, attempts are being made to produce high quality foods with a minimum of time and effort. Research is being carried out to develop equipment capable of cooking foods in a fraction of the time required by conventional methods; as a result, the problem arises as to the bacteriological safety of these products. We investigated the microbiological aspects of lemon and chocolate foam pies before and after cooking by microwaves for less than 2 min. Pies prepared with sterile equipment under sanitary conditions were inoculated with washed cells from a 24-hr broth culture of Salmonella typhimurium and were incubated for 24, 48, and 72 hr at 33 C. The same procedures were followed in model systems to determine the effects of various sugar and pH levels on the survival of S. typhimurium. No S. typhimurium was detected in inoculated cooked or uncooked lemon pies by the plating method; with the Lactose Broth pre-enrichment method, survivors were detected in lemon pies immediately after preparation. After electronic cooking, no survivors were detected in lemon pies by plate counts, whereas cells were recovered from chocolate pies by the Lactose Broth method. Both chocolate and lemon pies had lower counts throughout the 72-hr incubation period than the model systems compared to them. With the model systems, at pH 7.3, media containing sugar inhibited the growth of S. typhimurium but did not cause a significant reduction in counts during the incubation times studied. At pH 3.7, media without sugar yielded no cells with the Lactose Broth pre-enrichment method after 48 hr of incubation, whereas media with sugar were not sterile until after 72 hr of incubation. Apparently, the presence of sugar in the medium had a protective influence which made the lethal effect of the low pH less severe.     

A NEW RAPID TECHNIQUE FOR SALMONELLA ANTIGEN DETECTION USING A PARTICLE CONCENTRATION FLUORESCENCE IMMUNOASSAY (PCFIA)

A fluorescent assay employing polystyrene beads coated with antibody to a common structural antigen (CSA) of salmonellae has been developed to detect the presence of Salmonella. This sensitive technique is very fast, relatively simple and with further testing may be suitable for screening food products as well as clinical diagnostics. Comparison was made with a commercially available Salmonella antigen capture enzyme immunoassay (EIA). As little as one single colony-forming unit (CFU) could be detected by fluorescence after only 16 h of enrichment. the procedure requires only 1 h to run and uses proportionately smaller volumes of sample and reagents than EIA.     

Automated detection of Salmonella spp. in foods.

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.     

Salmonellae Associated with Further-processed Turkey Products

"Further-processed" turkey products, prepared from chilled, eviscerated, and thawed carcasses at two commercial turkey-processing plants, were evaluated, for the presence of salmonellae. These organisms were isolated from swab samples from 12% of chilled, eviscerated turkey carcasses, 27% of finished products, and 24% of processing equipment. The same serotypes as those found throughout a plant on any one visit were recovered from 31% of rinse-samples taken from hands and gloves of processing personnel. Salmonellae were found in samples taken on 37 of 48 visits; a greater number of recoveries were made on days when freshly killed turkeys were processed (87%) than when frozen-defrosted carcasses were processed (59%). The predominant serotype isolated from meat and environment usually changed from visit to visit. Salmonella sandiego and Salmonella anatum were the most frequent among 23 serotypes recovered. Most of the isolated serotypes are commonly associated with turkeys and have been incriminated as causative agents of human salmonellosis. The implication is that further-processed turkey products, if inadequately cooked by the consumer and if improperly refrigerated between the time of manufacture and consumption, could directly transmit salmonellae. These same products might also contaminate other foods by introducing salmonellae into food-preparation areas.     

Effect of Brilliant Green on the Motility of Salmonellae and Other Selected Microorganisms

The effect of Brilliant Green on motility was studied with Salmonella anatum, S. derby, S. tennessee, Escherichia coli, Proteus vulgaris, and Pseudomonas aeruginosa. Semisolid tryptic soy-agars containing 0, 20, or 40 mg of Brilliant Green per liter were used as the motility media. Both concentrations of Brilliant Green inhibited the growth of the non-Salmonella species through the semisolid agar. For 24 hr, the Brilliant Green appeared to limit the growth of the salmonellae; however, by 48 hr the salmonellae were able to grow through the semisolid agars. The presence of Brilliant Green in the motility media aided in the detection of Salmonella when mixed cultures were used.     

APPLICATION OF POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS

We have tested a rapid and sensitive DNA-based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella-specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA-based microtiter plate format. The combined cultivation and PCR-OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty-one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR-OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR-OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR-OLA (96.1% specificity). All positive PCR-OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR-OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR-OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.     

Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples.     

Application of BAX system, Tecra Unique Salmonella test, and a conventional culture method for the detection of Salmonella in ready-to-eat and raw foods

AIMS: To compare the BAX system, the Tecra Unique Salmonella test, and a conventional culture method for the detection of Salmonella in various foods. METHODS AND RESULTS: Ready-to-eat and raw foods were inoculated with Salmonella serotype Typhimurium, Salmonella serotype Enteritidis, Salmonella serotype Typhi, or Salmonella serotype Derby. Incubated pre-enrichment cultures were examined using the BAX system, the Tecra Unique Salmonella test, and a conventional culture method. Salmonella could be detected in all ready-to-eat food samples inoculated with S. Typhimurium, S. Enteritidis, or S. Derby, with any of the three test methods. However, false negatives were obtained with the Tecra test and the culture method when samples with higher background flora were inoculated with S. Typhi. Sensitivity test results suggested the two rapid tests performed as well as the culture method in the detection of 10(1) CFU of S. Typhimurium in 25-g cooked or raw food. CONCLUSIONS: The BAX system and the Tecra Unique Salmonella test demonstrated results comparable with those of the culture method in the detection of Salmonella serotypes used except S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evaluation of the BAX system, the Tecra Unique Salmonella test, and a culture method in the detection of Salmonella in a variety of western and oriental foods.     

Ambient-temperature primary nonselective enrichment for isolation of Salmonella spp. from an estuarine environment.

A primary, nonselective, ambient-temperature enrichment procedure for isolation of Salmonella spp. is described. The procedure was superior to elevated-temperature selective enrichment for Salmonella when estuarine water samples were examined. Five Chesapeake Bay stations were monitored, over an 8-month period, for the presence of salmonellae. Of 72 water and sediment samples collected, 17 (23.6%) yielded Salmonella spp. Seven serotypes were identified among the isolates. A seasonal pattern was noted for the incidence of the salmonellae. A most probable number procedure, performed by membrane filtration and nonselective enrichment, yielded Salmonella most probable number indices as high as 110 per 100 g of sediment. The results suggest that new methods, such as the one described in this report, are required for isolation of human intestinal pathogens from estuaries and coastal waters.     

Ambient-temperature primary nonselective enrichment for isolation of Salmonella spp. from an estuarine environment.

A primary, nonselective, ambient-temperature enrichment procedure for isolation of Salmonella spp. is described. The procedure was superior to elevated-temperature selective enrichment for Salmonella when estuarine water samples were examined. Five Chesapeake Bay stations were monitored, over an 8-month period, for the presence of salmonellae. Of 72 water and sediment samples collected, 17 (23.6%) yielded Salmonella spp. Seven serotypes were identified among the isolates. A seasonal pattern was noted for the incidence of the salmonellae. A most probable number procedure, performed by membrane filtration and nonselective enrichment, yielded Salmonella most probable number indices as high as 110 per 100 g of sediment. The results suggest that new methods, such as the one described in this report, are required for isolation of human intestinal pathogens from estuaries and coastal waters.     

Slow rehydration for detection of Salmonella spp. in feeds and feed ingredients

Rehydration and equilibration (4 h) of feeds and feed ingredients at water/sample ratios (vol/wt) of 1.4 to 3.2 did not markedly increase recovery of Salmonella spp. in the slurry when analyzed by standard cultural and direct enrichment methods. Of 143 naturally contaminated samples examined, equilibration increased levels of detection from 106 to 109 positive samples by the standard cultural method and from 103 to 112 by direct enrichment. Results suggest that nonhomogeneous distribution of low incident numbers of salmonellae in test samples rather than an equilibration-dependent response provided for the observed heterogeneity in recovery patterns. The novel equilibration approach is of limited application and requires validation on an individual food basis.     

Slow rehydration for detection of Salmonella spp. in feeds and feed ingredients.

Rehydration and equilibration (4 h) of feeds and feed ingredients at water/sample ratios (vol/wt) of 1.4 to 3.2 did not markedly increase recovery of Salmonella spp. in the slurry when analyzed by standard cultural and direct enrichment methods. Of 143 naturally contaminated samples examined, equilibration increased levels of detection from 106 to 109 positive samples by the standard cultural method and from 103 to 112 by direct enrichment. Results suggest that nonhomogeneous distribution of low incident numbers of salmonellae in test samples rather than an equilibration-dependent response provided for the observed heterogeneity in recovery patterns. The novel equilibration approach is of limited application and requires validation on an individual food basis.     

Comparison of commercially available kits with standard methods for detection of Salmonella strains in foods.

Six commercial kits were compared with the U.S. Food and Drug Administration (USFDA) method and the Japanese standard method for Salmonella isolation in foods. When only Salmonella serovars were tested, many of the methods performed well; however, when foods were artificially inoculated, only the USFDA method and immunomagnetic separation coupled with the xylose-lysine-brilliant green agar method (MS-XLBG) could positively detect Salmonella serovars. All seven wild-type Salmonella serovars were detected by the USFDA method, and the MS-XLBG method detected salmonellae from six samples.     

Salmonellae in health foods

Various health food products of different brands were purchased from stores in the metropolitan Atlanta area. These foods were examined for the presence of salmonellae by fluorescent-antibody and cultural methods. Included in the study were tablets of alfalfa, parsley, kelp, wheat bran, enzyme, bone meal, and vitamins. Beef liver powder and tablets and granola cereal were also studied. Salmonella minnesota, Salmonella anatum, and Salmonella derby were isolated from two of three lots of beef liver powder from one manufacturer. All other products were negative.     

Salmonellae in health foods.

Various health food products of different brands were purchased from stores in the metropolitan Atlanta area. These foods were examined for the presence of salmonellae by fluorescent-antibody and cultural methods. Included in the study were tablets of alfalfa, parsley, kelp, wheat bran, enzyme, bone meal, and vitamins. Beef liver powder and tablets and granola cereal were also studied. Salmonella minnesota, Salmonella anatum, and Salmonella derby were isolated from two of three lots of beef liver powder from one manufacturer. All other products were negative.     

The Use of Enrichment Serology for Salmonella Detection in Human Foods and Animal Feeds

S ummary . Samples (2208) of food raw materials and products were examined for the presence of salmonellae by use of conventional salmonella detection procedures and the enrichment serology (ES) techniques described by Sperber & Deibel (1969); 348 samples were positive for salmonellae by the conventional procedures. Using the ES technique with a 24 h elective enrichment step, 93–98% of samples positive by the conventional procedures were also positive by the ES technique. Selective enrichment of food samples using tetrathionate broth containing novobiocin, incubated at 41°, led to the best recovery of salmonellae by both the conventional and ES techniques.     

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.