Reveal Salmonella enteritidis test for detection of Salmonella enteritidis in shell eggs and environmental samples

Reveal Salmonella Enteritidis (SE) is a lateral flow-based immunodiagnostic assay used for rapid detection of Salmonella enterica serovar Enteritidis from pooled shell eggs and environmental samples. This assay uses highly specific antibodies to accurately detect S. Enteritidis. Studies were conducted to compare the performance of this test against reference procedures for detection of S. Enteritidis from both pooled shell eggs and environmental samples. Pooled shell eggs were inoculated with low levels ofS. Enteritidis and were enriched according to the procedure prescribed by the U.S. Food and Drug Administration. Uninoculated samples were included in each trial. Reveal SE exhibited 100% sensitivity and 100% specificity in comparison to the reference method in all trials. An abbreviated 48 h/(no hold) enrichment procedure was also developed and validated for detection ofS. Enteritidis from pooled shell egg samples. This shortened enrichment procedure can be used in conjunction with the Reveal SE test and offers a significant enrichment time savings of 96 h. Chi-square analysis revealed that there was no significant difference between the abbreviated Reveal method and the reference procedure for detection ofS. Enteritidis from pooled shell egg samples. Out of 245 natural drag swabs screened internally, only three samples tested Reveal SE positive and were confirmed by the reference procedure, resulting in 100% sensitivity and 100% specificity. An external laboratory screened 147 poultry house environmental samples and obtained 35 Reveal SE confirmed positives for Reveal SE sensitivity of 100% and specificity of 90%. Inoculation trials with drag swabs resulted in 96% sensitivity and 100% specificity. Thus, these data demonstrate that Reveal SE is a highly sensitive and specific assay for the detection of S. Enteritidis from both pooled shell eggs and environmental samples.     

SDIX RapidChek Listeria F.A.S.T. environmental test system for the detection of Listeria species on environmental surfaces

The SDIX RapidChek Listeria F.A.S.T. test system was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) cultural reference method for the detection of Listeria species on stainless steel, plastic, rubber, and painted concrete. The SDIX method uses a proprietary RapidChek Listeria enrichment media for a one-step, 24-40 h enrichment at 30 degrees C, and detects Listeria on an immunochromatographic lateral flow device in 10 min. Different Listeria species were used to spike each of the environmental surfaces. Environmental surfaces were spiked at levels ranging from 50 to 400 CFU/surface (1 in.2 swabs for painted concrete, 4 in.(2) for sponge). A total of 120 spiked samples were tested by the SDIX method at 24 and 40 h and the cultural reference method. Total confirmed positives were 49, 54, and 48 for the SDIX 24 h method, the SDIX 40 h method, and the USDA-FSIS cultural reference method, respectively. Nonspiked samples from all environmental surfaces were reported as negative for Listeria spp. by all methods. The overall Chi square was 0.017 (P = 0.104) and 0.611 (P= 0.566) after a 24 and 40 h enrichment, respectively, indicating that the test method was equivalent in performance to the reference method at both enrichment times. The SDIX method was evaluated for the detection of 50 Listeria and 35 non-Listeria bacterial strains. All 50 Listeria strains were detected by the method (100% sensitivity). Five out of 35 non-Listeria species gave light test signals when grown in nonselective broth culture and tested undiluted. However, when grown in the RapidChek Listeria F.A.S.T. proprietary media, only one bacterial strain (Staphylococcus aureus) was detected, giving a very low test signal (97% specificity). The method was shown to be robust toward several alterations in testing and storage conditions.     

Development of an ELISA screening test for nitroimidazoles in egg and chicken muscle

An antibody was generated that can bind metronidazole (MNZ), a nitroimidazole drug used in veterinary medicine to treat poultry for coccidiosis and histomoniasis. A direct competitive enzyme-linked immunosorbent assay (cELISA) is described. It was used to characterise binding of this antibody to a number of nitroimidazole drugs. It displayed cross-reactivity with dimetridazole (DMZ), ronidazole (RNZ), hydroxydimetridazole (DMZOH), and ipronidazole (IPZ).Egg and chicken muscle samples were extracted with acetonitrile and de-fatted by washing with hexane. Detection capabilities (CCβ) were determined: dimetridazole, <1 ppb (egg) and <2 ppb (muscle); metronidazole, <10 ppb; ronidazole and hydroxydimetridazole, <20 ppb; ipronidazole, <40 ppb.     

Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.     

Detection of Salmonella in fresh cheese,poultry products,and dried egg products by the ISO 6579 Salmonella culture procedure and the AOAC official method: collaborative study

Three food types were analyzed for the presence of Salmonella by the AOAC culture method and by the International Organization for Standardization (ISO 6579:2002) culture method. Paired test portions of each food type were simultaneously analyzed by both methods. A total of 21 laboratories representing federal government agencies and private industry, in the United States and Europe, participated in this interlaboratory study. Foods were artificially contaminated with Salmonella and competing microflora if naturally contaminated sources were not available. No statistical differences (p < 0.05) were observed between the AOAC and ISO culture methods for fresh cheese and dried egg products. A statistically significant difference was observed for one of the 2 lots of poultry from the first trial. The poultry meat used in this run was radiation sterilized, artificially contaminated with Salmonella and competitive flora, and then lyophilized. A second trial was conducted with 2 separate lots of raw ground chicken that were naturally contaminated. The results from the second trial showed no statistical difference between the 2 culture methods. A third trial involving 4 laboratories was conducted on 2 separate lots of naturally contaminated raw poultry. Again, no statistically significant differences occurred. It is recommended that ISO 6579:2002 culture method for Salmonella be adopted Official First Action for the analysis of fresh cheese, fresh chilled and frozen poultry, and dried egg products.     

Multicenter validation of PCR-based method for detection of Salmonella in chicken and pig samples

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.     

TECRA Unique test for rapid detection of Salmonella in food: collaborative study

The TECRA Unique Salmonella test uses the principle of immunoenrichment to allow rapid detection of Salmonellae in food. A collaborative study was conducted to compare the TECRA Salmonella Unique test with the reference culture method given in the U.S. Food and Drug Administration's Bacteriological Analytical Manual. Three food types (milk powder, pepper, and soy flour) were analyzed in Australia and 2 food types (milk chocolate and dried egg) were analyzed in the United States. Forty-one collaborators participated in the study. For each of the 5 foods at each of the 3 levels, a comparison showed no significant differences (p > or = 0.05) in the proportion of positive test samples for Unique and that for the reference method using the Chi-square test for independence with continuity correction.     

Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801

Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A-E in a variety of food and environmental samples. A Performance Tested Method validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A-E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.     

Clonal structure and variation in virulence of Salmonella enteritidis isolated from mice, chickens, and humans

The clonal analysis of a diverse collection of Salmonella Enteritidis indicates that most strains belong to a single multilocus genotype (i.e., ET-3) regardless of phage type, geographic origin, or time of isolation that spanned over 2 decades (1978 to 2004). Attachment and invasion assays, however, indicate that, among ET-3 isolates, there is a distinct invasive bacterial subpopulation that is more readily recovered from eggs and clinical cases in humans than from chicken cecal samples. These observations support the hypothesis that the specialized ability of S. Enteritidis to infect the avian reproductive tract and contaminate eggs has been critical in its emergence as a frequent cause of human illness.     

Evaluation of the BAX system for detection of Salmonella in selected foods: collaborative study

A multilaboratory study was conducted to compare the automated BAX System to the standard cultural methods for detection of Salmonella in selected foods. Five food types--frankfurters, raw ground beef, mozzarella cheese, raw frozen tilapia fish, and orange juice--at 3 inoculation levels, were analyzed by each method. A sixth food type, raw ground chicken, was tested using 3 naturally contaminated lots. A total of 16 laboratories representing government and industry participated. In this study, 1386 samples were analyzed, of which 1188 were paired samples and 198 were unpaired samples. Of the 1188 paired samples, 461 were positive by both methods and 404 were negative by both methods. Thirty-seven samples were positive by the BAX System but negative by the standard reference method, and 11 samples were positive by standard cultural method and negative by the BAX System. Of the 198 unpaired samples, 106 were positive by the BAX System and 60 were positive by the standard cultural method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, the BAX System demonstrated results comparable to those of the standard reference methods based on the Chi square results.     

Development of an optical surface plasmon resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry meat

The aim of this study was to develop an optical biosensor inhibition immunoassay, based on the surface plasmon resonance (SPR) principle, for use as a screening test for 13 (fluoro)quinolones, including flumequine, used as veterinary drugs in food-producing animals. For this, we immobilised various quinolone derivatives on the sensor chip and tested binding of a range of different antibodies (polyclonal and one engineered antibody) in the presence and absence of free (fluoro)quinolones. The main challenge was to detect flumequine in an assay giving good results for the other compounds. One antigen–antibody combination proved satisfactory: polyclonal antibodies raised against a dual immunogen and, on the sensor chip, a fluoroquinolone derivative. It was the first time that this concept of the bi-active antibody was described in the literature.The assay, optimised for detection in three matrices (poultry muscle, fish, and egg), was tested on incurred samples prepared by liquid extraction followed by two washing steps. This rapid, simple method proved adequate for detecting at least 13 (fluoro)quinolones at concentrations below established maximum residue levels (MRLs). The reference molecule norfloxacin could be detected in the range of 0.1–10 μg kg⁻¹ in extracts of egg and poultry meat and in the range of 0.1–100 μg kg⁻¹ in extracts of fish. The determined midpoints of these calibration curves were about 1, 1.5 and 3 μg kg⁻¹ in poultry meat, egg and fish, respectively.     

Comparison of assurance gold salmonella EIA, BAX for screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays for detection of Salmonella in alfalfa sprouts and sprout irrigation water

The Assurance Gold Salmonella EIA, BAX for Screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays were compared with official cultural methods described in the Bacteriological Analytical Manual (BAM) for analysis of alfalfa sprouts and sprout irrigation water for the presence of Salmonella. The lower limits of detection of 4 serovars of Salmonella cells (S. tennessee, S. muenchen, S. mbandanka, and S. cubana) in pure culture were determined as approximately log10 2, 5, and 6 for the BAX, GENE-TRAK, and Gold EIA, respectively. Despite its low detection limit, the BAX did not perform as well as the other assays in analyzing contaminated sprouts and sprout irrigation water. For 4 different lots of sprouts and sprout irrigation water samples inoculated with the 4 serovars at low [1-2 colony forming units (CFU/g)] and high (68-180 CFU/g) levels, the BAX detected Salmonella in 58/64 (90.6%) of the samples, compared with 64/64 (100%) by the GENE-TRAK, Gold EIA, and BAM methods. Assay performance was also compared for analysis of naturally contaminated sprouts and sprout irrigation water with 3 lots of alfalfa sprouted seeds associated with different salmonellosis outbreaks. Positive assay results for the naturally contaminated samples were Gold EIA 41, GENE-TRAK 36, BAM 33, and BAX 13.     

Evaluation of applied biosystems MicroSEQ real-time PCR system for detection of Salmonella spp. in food

Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested Method validation program. In addition, the performance of the MicroSEQ system was evaluated for the detection of Salmonella in peanut butter as a part of the Emergency Response Validation Program sponsored by the AOAC Research Institute. The system was compared to the ISO 6579 reference method using a paired-study design for detecting Salmonella spp. in raw ground beef, raw chicken, raw shrimp, Brie cheese, shell eggs, cantaloupe, chocolate, black pepper, dry infant formula, and dry pet food. For the peanut butter study, the system was compared to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures using an unpaired-study design. No significant difference in performance was observed between the MicroSEQ Salmonella spp. detection system and the corresponding reference methods for all 11 food matrixes. The MicroSEQ system detected all Salmonella strains tested, while showing good discrimination against detection of an exclusivity panel of 30 strains, with high accuracy.     

An enzyme linked immunoassay for the determination of deoxynivalenol in wheat based on chicken egg yolk antibodies

An indirect competitive enzyme linked immunoassay (ELISA) for the detection of the Fusarium mycotoxin deoxynivalenol (DON) in wheat was developed. Instead of the much more common antibody isolation from mammal serum, DON specific antibodies were, for the first time, isolated from the eggs of previously immunized hens. The limit of detection was 2 μg/L for standard curves and spiked wheat extracts. Recoveries for naturally contaminated samples (200–525 μg/kg) were between 80 and 125% compared with GC-ECD data. Concentrations for naturally contaminated samples were chosen with regard to current Austrian guidelines concerning DON levels in produce intended for human consumption, recommending a maximum of 500 μg DON/kg.     

An enzyme linked immunoassay for the determination of deoxynivalenol in wheat based on chicken egg yolk antibodies

An indirect competitive enzyme linked immunoassay (ELISA) for the detection of the Fusarium mycotoxin deoxynivalenol (DON) in wheat was developed. Instead of the much more common antibody isolation from mammal serum, DON specific antibodies were, for the first time, isolated from the eggs of previously immunized hens. The limit of detection was 2 microg/L for standard curves and spiked wheat extracts. Recoveries for naturally contaminated samples (200-525 microg/kg) were between 80 and 125% compared with GC-ECD data. Concentrations for naturally contaminated samples were chosen with regard to current Austrian guidelines concerning DON levels in produce intended for human consumption, recommending a maximum of 500 microg DON/kg.     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat, and peanut in foods

The labeling of 5 major allergenic ingredients (egg, milk, wheat, buckwheat, and peanut) is mandatory in Japan, and 2 series of enzyme-linked immunosorbent assay (ELISA) kits have been established as official screening methods. However, these official methods have not provided the necessary sensitivity, due in part to poor extraction efficiency. To address this need, 2 novel ELISA kits have been developed: the FASTKIT ELISA Ver. II Series and the FASPEK Allergenic Substances Detection Kit. The new kit systems use an improved extraction buffer that can extract insoluble proteins produced by processing and feature new antibodies that bind to the denatured proteins extracted with the new extraction buffer. The analytical performances of the 2 new ELISA kit series were evaluated in an interlaboratory study. Ten laboratories participated in the study and determined the major allergenic ingredients contained in 5 types of model processed food. The 2 ELISAs displayed fairly good reproducibility and sufficient recovery.     

A simple,one‐tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria

Coccidiosis is a costly worldwide enteric disease of chickens caused by parasites of the genus Eimeria. At present, there are seven described species that occur globally and a further three undescribed, operational taxonomic units (OTUs X, Y, and Z) that are known to infect chickens from Australia. Species of Eimeria have both overlapping morphology and pathology and frequently occur as mixed‐species infections. This makes definitive diagnosis with currently available tests difficult and, to date, there is no test for the detection of the three OTUs. This paper describes the development of a PCR‐based assay that is capable of detecting all ten species of Eimeria, including OTUs X, Y, and Z in field samples. The assay is based on a single set of generic primers that amplifies a single diagnostic fragment from the mitochondrial genome of each species. This one‐tube assay is simple, low‐cost, and has the capacity to be high throughput. It will therefore be of great benefit to the poultry industry for Eimeria detection and control, and the confirmation of identity and purity of vaccine strains.