Occurrence of Salmonella spp. in samples from pigs slaughtered for consumption: A comparison between ISO 6579:2002 and 23S rRNA Fluorescent In Situ Hybridization method

Contamination of pork products during slaughter represents an important vehicle for Salmonella spp. dissemination to humans. Salmonellosis poses an important risk for public health and presents an important economic issue to pork producers. This study aimed to evaluate the occurrence of this foodborne pathogen in pork carcasses and risk tissues (ileum, ileocolic and mandibular lymph nodes and tonsils) by two methods: the reference culture method (ISO 6579:2002) and a rapid Fluorescent In Situ Hybridization (FISH) method.The culture method identified the presence of Salmonella spp. in 13.7% of the samples, while the FISH technique revealed that 38.2% of the samples were positive. From these FISH positive samples, only 58 were concordant to the positive results obtained by the culture method.These results confirm the potential risk that pork represents in salmonellosis transmission, suggesting that additional measures should be taken during evisceration practices and extraction of tonsils and mandibular lymph nodes after slaughter, in order to achieve a better control of Salmonella contamination during slaughter. The FISH method showed to be a rapid screening tool for Salmonella spp. detection in pork samples.     

COMPARISON OF A REAL-TIME POLYMERASE CHAIN REACTION ASSAY WITH A CULTURE METHOD FOR THE DETECTION OF SALMONELLA IN RETAIL MEAT SAMPLES

A real‐time polymerase chain reaction (PCR) method developed in this study was compared with a conventional International Organization for Standardization culture method for the detection of Salmonella in Irish beef, chicken, pork and turkey. This also provided a snapshot of the incidence of Salmonella in such products. After selective enrichment, all meat samples (n = 100) were: (1) subjected to DNA extraction and real‐time PCR analysis using primers against Salmonella spp. specific gene (16S rRNA) or the invA virulence gene; and (2) plated on differential media and identified as Salmonella using immunological and sub‐typing methods. Retail samples (5/100) were shown to contain Salmonella using the 16S rRNA gene‐based real‐time PCR assay and the standard culture method. However, only two (of five) samples were shown to contain Salmonella using the invA gene‐based real‐time PCR assay. For the sample set examined, the developed 16S rRNA gene‐based real‐time PCR assay demonstrated comparable specificity and sensitivity to the currently used standard culture method but was considerably more rapid.     

Evaluation of a Polymerase Chain Reaction–Based System for Detecting Salmonella Species from Pork Carcass Sponge Samples

Pork carcass sponge samples (n = 230) collected from 10 Taiwanese slaughter plants were screened for Salmonella using 2 methods: BAX® for Screening/Salmonella, a polymerase chain reaction‐based detection system and a culture method using SM‐ID agar as the selective plating medium. The BAX method identified 14 samples as positive for Salmonella. The 8 samples identified as Salmonella‐positive by the SM‐ID method were also BAX‐positive. Inoculation studies showed BAX detected Salmonella in samples having initial 1.4 × 10¹ cfu/mL Salmonella inoculum prior to the enrichment process in the presence of 3.0 × 10⁶ cfu/mL of non‐Salmonella florae. BAX provides a rapid screening alternative to the culture method for Salmonella detection.     

Unveiling contamination sources and dissemination routes of Salmonella sp. in pigs at a Portuguese slaughterhouse through macrorestriction profiling by pulsed-field gel electrophoresis

Sixty-nine isolates of Salmonella sp. isolated from the ileum, tonsils, carcass and mandibular and ileocolic lymph nodes of individual pigs slaughtered for consumption in one abattoir were analyzed using serotyping and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (RFLP-PFGE), in order to identify clonal relationships. XbaI macrorestriction was able to distinguish 18 genotypes among the eight identified serotypes: Salmonella Typhimurium (4 genotypes), Salmonella Rissen (3), Salmonella Tennessee (2), Salmonella Enteritidis (2), Salmonella 4,[5],12:i:- (4), Salmonella Give (1), Salmonella Anatum (1), and Salmonella Derby (1). Except for one sample, the serotype and the genotype identified in the samples from the same pork were always the same, allowing to unravel possible dissemination routes of Salmonella sp. through these pork tissues and equate presumptive sources of contamination or infection. Highly significant associations (p < 0.001) were observed for the presence of Salmonella sp. in the ileum and in the ileocolic lymph nodes, as well as between the carcass contamination and the presence of Salmonella sp. in others samples of the correspondent slaughtered pig, such as the ileum, the ileocolic and mandibular lymph nodes and the tonsils. Moreover, 80% of the pigs with ileum and ileocolic lymph nodes positive samples also presented the same salmonella genotype in the correspondent tonsils and, among pigs with positive tonsils, 70% also carried the same genotype in the corresponding mandibular lymph nodes. The occurrence of cross-contamination was also detected, since a genotype identified in other pigs slaughtered in the same day was found in 31% of positive carcasses. The global analysis of the genotypes suggested three different sources of pig infection: the farm of origin, the transportation and the lairage. A particular attention should be paid to the last one, since the majority of the isolates from pig samples were related to infection in the lairage. Since the presence of Salmonella sp. in the ileum of pigs and faeces ingestion promotes tonsils infection and internal dissemination of the agent through the mandibular lymph nodes, as well as drainage to the ileocolic lymph nodes, a potential risk exists at slaughter for Salmonella sp. contamination in the carcasses during pork processing. This risk may be increased by incorrect evisceration techniques and by hygienically inappropriate meat inspection procedures, especially those concerned to the mandibular lymph nodes incisions.     

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail−gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative.     

AN IMPROVED METHOD FOR RAPID ISOLATION OF SALMONELLA AGAINST PROTEUS IN CHICKEN CARCASSES

An improved method was developed for the rapid isolation and identification of Salmonella in chicken carcasses. Considering the heavy presence of Proteus mirabilis in the poultry‐production environment, we investigated combinations of selective bacterial growth media that promote the growth of Salmonella but inhibit that of P. mirabilis. The improved method was evaluated using standard methods described in the Bacteriological Analytical Manual (BAM) by the U.S. Food and Drug Administration (FDA), and the Microbiology Laboratory Guidebook published by the U.S. Department of Agriculture Food Safety Inspection Service (USDA/FSIS). A total of 44 chicken carcasses were analyzed for Salmonella using the three methods. The FDA/BAM method showed a poor sensitivity and specificity, especially when the tetrathionate (TT) broth was applied. The improved method offered an excellent specificity (>95%) and shortened the time of isolation and identification from 4 to 5 days to less than 3 days, consequently reducing the costs.     

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail−gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative. Received: January 30, 2007; Received in revised form: February 27, 2007; Accepted: February 28, 2007.     

COMPARISON OF REVERSE PASSIVE LATEX AGGLUTINATION TEST AND IMMUNOBLOTTING FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A AND B

Staphylococcal foodborne diseases resulting from consumption of food contaminated with staphylococcal enterotoxins (SEs) produced by certain strains of Staphylococcus aureus are the second most common foodborne illnesses in the world. Analytical methods are essential for routine monitoring purposes and safeguard public health. Different methods for SE detection have been proposed although their use in a complex matrix is often limited by the presence of substances that interfere with tests. In this article reverse passive latex agglutination (RPLA) and immunoblotting methods based on specific antibodies and currently available for SE detection have been compared. Culture filtrates from enterotoxin S. aureus strains isolated from cheese samples were identified by SET‐RPLA. Then the culture filtrates identified as staphylococcal enterotoxin A and staphylococcal enterotoxin B by RPLA test were analyzed with immunoblotting. The results obtained suggest that either SET‐RPLA or immunoblotting may be applied to culture filtrates for the detection of SEs with good correspondence of results. Although SET‐RPLA represents a simple method for routine monitoring purposes, a positive result by a rapid method (RPLA) is only regarded as presumptive and must be confirmed by standard methods ( Feng 1996 ), such as immunoblotting method.     

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.     

RAPID METHOD FOR DETECTION OF SALMONELLAE ATTACHED TO CHICKEN SKIN1

A rapid, sensitive nitrocellulose membrane lifting technique was developed to identify Salmonella typhimurium attached to chicken skin. Basically, this method used a nitrocellulose membrane to remove salmonellae attached to chicken skin. The membrane with attached salmonellae was then incubated on xylose lysine deoxycholate (XLD) agar for 18 h at 37°C. Appearance of black colonies on the white membrane was considered a positive presumptive test for salmonellae. Immunostaining of colonies on the nitrocellulose membrane was then used to confirm presence or absence of salmonellae. This technique can be used to identify salmonellae contamination of chicken skin in less than 24 h and was shown to be more sensitive than presently used swabbing or washing techniques.     

Salmonella contamination in pigs at slaughter and on the farm: a field study using an antibody ELISA test and a PCR technique

An antibody ELISA test and a PCR method for identifying the risk of Salmonella contamination were compared in a field study on the same lots of animals in a slaughterhouse. The results were compared to investigations carried out on two farms with different prevalences of Salmonella antibody-positive animals. Salmonella antibody ELISA testing was carried out on all 383 meat juice samples derived from the diaphragm pillar muscle of each pig. Salmonella DNA analysis was performed by PCR technique on small intestine samples with lymph nodes from all 383 pigs, and on tonsils from the last 129 pigs. The 383 animals tested came from 32 different pig farms. Furthermore, the herd antibody blood serum status against Salmonella spp. of weaners was determined on two selected pig fattening farms, one with low and one with high seroprevalence in meat juice. A total of 7.0% (ELISA cut-off OD% > or =40) of the slaughtered pigs from 6 of 32 fattening farms were seropositive. Salmonella DNA was found in 16.4% of the jejunum/lymph nodes (383 animals) and in 15.5% of the tonsils (129 animals). Salmonella DNA was found in the jejunum/lymph nodes of 41% of the seropositive pigs. However, serotitres were also positive in only 17.5% of all pigs positive in the jejunum DNA test. Two farms were selected for further investigation: farm 13 (F13), with a high prevalence of seropositive pigs, 29.0%, Category II; and F11, with 9.4%, Category I. However, categorization according to the blood serum tests of the fattening pigs after on-farm testing was very different: F13 had 5% positive animals (Category I); and F11, 23.3% (Category II). The study led to the following results and recommendations: First, ELISA tests are useful for the detection of farms that are regularly contaminated with Salmonella, but such tests cannot give information on the infectious status of a single animal (or a group) at the point of slaughter. Second, it is crucial that management measures are taken to prevent the spread of infections by trade and transport: piglets should be supplied exclusively by a single, well-known producer, and finishers should be tested serologically on farm before going to slaughter. Third, ELISA tests and the PCR method are suitable for the detection of Salmonella and are recommended as analytical tools for all pork quality control programmes. Fourth, animals from suspicious farms should always be slaughtered at the end of the slaughter day, followed by thorough cleaning and disinfection.     

Multiplex PCR for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples

A multiplex PCR method was developed for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in meat samples. DNA detection sensitivity for this method was 10(3) CFU/ml for each pathogen. When this protocol was used for the detection of each of the above pathogenic bacteria in spiked pork samples, 1 cell per 25 g of inoculated sample could be detected within 30 h. In the samples of naturally contaminated meat, Salmonella spp., L. monocytogenes, and E. coli O157:H7 were detected over the same time period. Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method, which suggests that the multiplex PCR is a reliable and useful method for rapid screening of meat products for Salmonella spp., L. monocytogenes, and E. coli O157:H7 contamination.     

Optimisation of a new two‐plate screening method for the detection of antibiotic residues in meat

A two‐plate microbiological method to screen residues of the most commonly used antibiotics in animal production, named new two‐plate test (NTPT), has been optimised and validated, according to criteria derived from the European Commission Decision 2002/657/CE. This screening method used two media at different pH seeded with a single bacteria strain (Bacillus subtilis). The method consists of a simple extraction, followed by the application of the extract on Petri plates. The method detected, in pork and chicken muscles, most of the antibiotics from six groups (tetracyclines, (fluoro)quinolones, penicillins, macrolides, aminoglycosides and sulfonamides) and florfenicol at concentration very close to maximum residue limits used in the EU. The screening capacity of the NTPT was compared with another screening technique, the Premi‐Test^®, by analysing spiked samples as well as real samples of meat from pork and chicken sold for local consumption, in the Red River Delta region (Vietnam). The NTPT described here appeared to detect more samples than the Premi‐Test^®, showing its interest for Vietnamese control laboratories, as a screening method to monitor antibiotic residues in chicken and pork meat, before sending the suspected samples to the confirmatory step.     

EXTERNAL VALIDATION AND TRANSFERABILITY OF NIRS MODELS DEVELOPED FOR DETECTING AND QUANTIFYING MBM IN INTACT COMPOUND FEEDING STUFFS

ABSTRACT

The ban on the use of animal‐origin by‐products such as meat and bone meal (MBM) in compound feeds was one of the measures implemented in the European Union to stop the spread of bovine spongiform encephalopathy (BSE) and to prevent its reoccurrence. It is now clear that the blanket ban will only be lifted if reliable analytical methods are available that ensure the detection of animal by‐products. Near‐infrared spectroscopy (NIRS) is likely to be the most rapid method for testing feedingstuffs, enabling a substantial increase in the number of samples tested and providing instant detection of adulteration, especially when samples are analyzed in intact form. This study aimed to demonstrate the feasibility of NIRS predictive models for detecting and quantifying MBM in intact compound feedingstuffs, and to demonstrate the transferability of calibration models between two NIRS instruments.

PRACTICAL APPLICATIONS

This study shows the potential of near infrared spectroscopy (NIRS) as a fast screening method for detecting the adulteration of compound feedingstuffs with animal‐origin meals, useful for the feed industry and the inspection bodies. NIRS could provide the first line of defense of the food chain, allowing a large‐scale analysis and making more costly methods to be used more productively on suspect specimens.     

DEVELOPMENT OF A CHEMILUMINESCENT ELISA FOR DETERMINING OKADAIC ACID IN SHELLFISH

ABSTRACT

An indirect competitive enzyme‐linked immunosorbent assay with chemiluminescent (CL‐ELISA) detection for okadaic acid (OA) in mussel muscle was developed. A hybridoma cell line secreting monoclonal antibody against OA was established after immunization of BALB/c mice with artificially synthesized OA‐bovine serum albumin as antigen. Luminol solution was used as the substrate for horseradish peroxidase. The detection limit was 0.175 ng/g. The range of average fortified recovery was 91.8–102.6% when OA was spiked in mussel muscle at levels of 50, 160 and 800 µg/kg. The standard curve for OA showed good linearity over the concentration range of 0.08125–20 ng/mL. The cross‐reactivity with dinophysistoxin1 and saxitoxin was 51.82 and 0%, respectively. In a residue study, the results obtained by CL‐ELISA correlated well within those obtained using the commercial ABRAXIS kit (ABRAXIS, Warminster, PA). The developed method is therefore suitable for detecting the residues of OA in shellfish.

PRACTICAL APPLICATIONS

Diarrhetic shellfish poisoning is a gastrointestinal syndrome that occurs in humans following the consumption of bivalve mollusks contaminated with OA with the main symptoms of diarrhea, nausea, vomiting and abdominal pain. OA widely exists at marine products and it is very important to develop a technique to monitor this toxin. In this study, a sensitive competitive indirect enzyme‐linked immunosorbent assay with chemiluminescence for determination of OA in mussel soft tissues was investigated. Chemiluminescent ELISA (CL‐ELISA) is a good alternative method for screening samples. This technique has the potential to improve the sensitivity of the immunoassays by at least two to three orders of magnitude compared with conventional colorimetric detection.     

A STUDY ON SUITABILITY OF FOUR ENRICHMENT BROTHS FOR PCR-BASED DETECTION OF LISTERIA MONOCYTOGENES FROM RAW MEAT

Four enrichment broths were evaluated for their compatibility with the polymerase chain reaction (PCR) for detection of Listeria monocytogenes from raw meat after single‐step enrichment. Standardized PCR protocols for listeriolysin O (hlyA) gene were used for the species‐specific identification of L. monocytogenes. Four broths, namely, modified University of Vermont broth (MUVM), Listeria enrichment broth (LEB), Fraser broth (FB) and polymyxin, acriflavin, lithium chloride, ceftazidime, aesculin, mannitol, egg yolk broth (PALCAM) , were inoculated with L. monocytogenes. The enriched cultures were subjected for PCR. Similarly, meat samples were artificially spiked with various concentrations of L. monocytogenes, these spiked samples were enriched in the above‐mentioned four broths and subjected to PCR to determine the medium that was most compatible for PCR‐based detection of L. monocytogenes. The aliquots taken during different incubation periods were subjected to three different procedures for the concentration of the target organism for use in PCR. Results revealed that MUVM was better than other broths for the detection of L. monocytogenes by both PCR and cultural method; moreover, it was able to support the growth of as low as 10 cfu/g of meat. Concentration of the target organisms by centrifugation and washing with PCR buffer was the most suitable method for improving PCR performance for detection of L. monocytogenes. Goat (n = 67) and buffalo (n = 45) meat samples from local markets were also screened by both PCR and cultural method to validate the results obtained from the spiking studies. Both results were in agreement in spiking studies as well as screening of market meat samples.     

Comparison of TaqMan Salmonella amplification/detection kit with standard culture procedure for detection of Salmonella in meat samples

We evaluated the TaqMan PCR Salmonella amplification/detection kit (PE Applied Biosystems) for rapid detection of Salmonella from a variety of meat 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 detection sensitivity of the kit, using 2 kinds of DNA extraction protocols, was compared with that obtained with 4 protocols of official culture methods. A total of 98 meat samples (16 raw beef, 31 pork and 51 chicken) were tested. The results of the TaqMan PCR method and the combined results of the 4 cultural protocols showed excellent agreement. However, no single culture protocol showed optimal recovery of Salmonella comparable to the PCR method. These results suggest that the TaqMan PCR method is a reliable and rapid method useful for detecting Salmonella in meat products.     

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.     

FREQUENCY OF SALMONELLA, CAMPYLOBACTER, LISTERIA AND ENTEROBACTERIACEAE DETECTION IN COMMERCIALLY COOL WATER-WASHED SHELL EGGS

The effect of cool water washing on shell egg temperature and pathogen detection was examined. Three temperature schemes were utilized in commercial dual washer systems: (1) HH = 48.9C, 48.9C; (2) HC = 48.9C, 23.9C; and (3) CC = 23.9C, 23.9C. HH eggsmaintainedthe highest surface temperature (26.25C in‐line, 20.25C off‐line and 23.25C combined, P < 0.05). The lowest temperatures were found in the CC eggs (21.25C in‐line, 17.25C off‐line and 19.25C combined). The frequency of Enterobacteriaceae detection in shell and membrane emulsions was greatest for the CC eggs (P < 0.05 for in‐line and combined). There was no difference in Enterobacteriaceae detection for the off‐line facility. Salmonella was detected in three of 384 samples from the in‐line facility. They were found in HC (2) and CC (1) shell emulsions. Two of 384 samples were positive for Campylobacter from the in‐line facility (CC). Three wash water samples were positive for Listeria in the off‐line facility (1 HC, 2 CC). No pathogens were detected in the egg contents during this study. The results of this study indicate that warm followed by cool water washing has the potential of decreasing egg temperature while maintaining surface microbiology at an acceptable level.