Evaluation of a 5'-nuclease (TaqMan) assay with the thin agar layer oxyrase method for the detection of Yersinia enterocolitica in ground pork samples

A 5'-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5'-nuclease assay for detecting Y. enterocolitica 0:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5'-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of "Oxyrase for Agar" onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (-15 degrees C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5'-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.     

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.     

Application of multiplex PCR for monitoring colonization of pig tonsils by Yersinia enterocolitica, including biotype 1A, and Yersinia pseudotuberculosis

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n=6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n=31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid-bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.     

Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method

From October 1997 to April 1998, a survey was conducted to assess the occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products, using a traditional culturing method and a PCR assay. A total of 300 pork samples was examined. Five slaughterhouses in the Norwegian Meat Cooperative were represented with 249 samples and another 51 samples were obtained from retail outlets in the city of Oslo. Using the NMKL method, Y. enterocolitica 0:3 was isolated from six (2%) of the samples, while the PCR method indicated presence of pathogenic Y. enterocolitica in 50 (17%) of the samples. The results indicate that a reduction has occurred in the prevalence of pathogenic Y. enterocolitica in Norwegian pork products, as compared to a previous Norwegian study conducted in 1988-1989. The study also highlights the need for further development and improvement of methods applied for the detection of pathogenic Y. enterocolitica in foods.     

多重PCR快速检测三种食品病原菌

建立一种快速、经济、实用的可以同步检测食品中金黄色葡萄球菌、沙门氏菌、小肠结肠炎耶尔森氏菌的多重聚合酶链式反应(polymerase chain reaction,PCR)的方法。根据金黄色葡萄球菌的nuc基因,沙门氏菌的invA基因,小肠结肠炎耶尔森氏菌的ail基因,分别设计了三对引物,对单个基因PCR和单管多重PCR扩增进行特异性、灵敏性实验以及优化反应体系。三对引物能特异性扩增出236、475、127bp的目的条带。建立的多重PCR同时对3种食源性致病菌进行检测具有较高的灵敏度,灵敏度金黄色葡萄球菌为102CFU/mL、沙门氏菌为102CFU/mL、小肠结肠炎耶尔森氏菌为102CFU/mL。初步建立能同步、简便、快速、灵敏地检测食品中金黄色葡萄球菌、沙门氏菌和小肠结肠炎耶尔森氏菌的三重PCR方法。     

Isolation and characterization of Yersinia enterocolitica from swine feces recovered during the National Animal Health Monitoring System Swine 2000 study

A national study was conducted for the isolation of pathogenic Yersinia enterocolitica in pig feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from September 2000 to March 2001 from 77 production sites in 15 of the top 17 swine-producing states were tested for the presence of pathogenic Y. enterocolitica. After enrichment of swine fecal samples in irgasan-ticarcillin-potassium chlorate broth, the enriched cultures were plated on cefsulodin-irgasan-novobiocin agar for isolation of presumptive Y. enterocolitica. The isolates were confirmed as pathogenic Y. enterocolitica by the fluorogenic 5' nuclease PCR assay targeting the chromosomal attachment invasion ail gene. Of 2793 fecal samples tested, 106 (3.80%) ail-positive strains of Y. enterocolitica were isolated. These 106 ail-positive isolates originated from 7 of the 15 participating states. The predominant serotype O:3 (n = 79 of 106) was distributed in five states (n = 5 of 7). Serotype O:5 (n = 27 of 106) was also found in five states (n = 5 of 7). All isolates contained the virulence plasmid and expressed virulence-associated phenotypic characteristics. These results indicate that swine in the United Stares harbor Y. enterocolitica that can potentially cause human illness.     

Detection, isolation and enumeration of Yersinia enterocolitica from raw pork

The methods available for the isolation of Yersinia enterocolitica from foods are generally considered to be less than optimal, and methods for estimation of numbers are lacking. Such methods are needed to understand better the significance of foodborne yersiniosis and to provide data for exposure assessment. We describe a method for the detection and enumeration of Y. enterocolitica containing the pYV virulence plasmid (YeP+) in samples from pork surfaces. The method uses a multiplex PCR targeting the ail and virF genes to detect Y. enterocolitica after incubation of surface swabs in Yersinia enrichment broth according to Ossmer. Enumeration was achieved by adapting the enrichment to a most probable number (MPN) method format. A presumptive result was available within 24 h of sample receipt, and YeP+ isolates were confirmed within four days. The presence/absence and MPN methods were evaluated in a pilot survey of 34 packs of raw pork meat purchased from retail outlets in Christchurch, New Zealand. YeP+ was detected by PCR on meat from 32% of the packs, and YeP+ isolates were obtained from 18% of the samples. YeP+ were present at numbers ranging from 0.30 to 5.42 MPN/cm(2). This improved method for the detection and enumeration of YeP+ from meat samples can be used for microbiological surveys to obtain data for assessments of consumer exposure to virulent Y. enterocolitica, and in outbreak investigations.     

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.     

Effect of different concentrations of carbon dioxide and oxygen on the growth of pathogenic Yersinia enterocolitica 4/O:3 in ground pork packaged under modified atmospheres

The influence on Yersinia enterocolitica counts of a gradual increase of carbon dioxide concentrations (percentage by volume in air) during packaging and storage of ground pork meat artificially contaminated with this pathogen was evaluated. Ground meat was packaged under customary conditions using modified atmospheres with various carbon dioxide percentages (0, 30, 50, 70, and 100% CO2 by volume; for atmospheres of less than 100% CO2, the rest of the gas was O2). The packs were stored at 2 degrees C for 12 days. During the entire storage time, counts of Y. enterocolitica were determined by the spread plate method for direct plate counts (DPCs). Microbiological shelf life of the stored ground pork also was assessed by total mesophilic aerobic bacterial plate counts (APCs). Y. enterocolitica counts were not significantly different (P > or = 0.05) in the ground pork packaged under the various CO2-enriched atmospheres. The growth of Y. enterocolitica was nearly entirely inhibited in all tested modified atmospheres containing the protective CO2. However, in ground pork packaged with 100% oxygen, there was a significant decrease (P < or = 0.05) in the DPC for Y. enterocolitica from 4.30 log CFU/g (day 0) to 3.09 log CFU/g at the end of the storage time (day 12). The decrease was presumably due to the marked increase in APC seen only in those packages stored under 100% O2. Packaging with high CO2 concentrations had significant inhibitory effect (P < or = 0.05) on the growth of mesophilic aerobic bacteria.     

Prevalence, antibiotic susceptibility, and virulence factors of Yersinia enterocolitica and related species from ready-to-eat vegetables available in Korea

A total of 673 ready-to-eat vegetable samples were collected in Korea from 2001 to 2002 and analyzed for the presence of Yersinia spp. We analyzed biotypes, serotypes, and susceptibility to 12 antibiotics and tested for virulence genes of pathogenic Yersinia enterocolitica isolates by PCR assay. Among the samples, 27 (4.0%) were found to be contaminated with Yersinia spp. Among the 27 strains of Yersinia spp. isolates, 18 strains (66.7%) of Y. enterocolitica, 5 strains (18.5%) of Y. frederiksenii, 3 strains (11.1%) of Y. intermedia, and 1 strain (3.7%) of Y. kristensenii were identified. According to the serotypes of Y. enterocolitica isolates, O:3 (11.1%) and O:5 (11.1%) were the most predominant, followed by O:8 (5.6%) and others (72.2%). For biotypes of Y. enterocolitica isolates, 1A (77.8%) was the most predominant, followed by 3B (11.1%), 3 (5.6%), and 5A (5.6%). Also, an antibiotic susceptibility test showed that Y. enterocolitica isolates were very susceptible to the antibiotics tested but highly resistant to ampicillin (94%), cephalothin (100%), and carbenicillin (83%). PCR assays with specific primers derived from yst and ail genes of Y. enterocolitica were applied to confirm the presence of pathogenic Y. enterocolitica. Among the 18 strains of Y. enterocolitica isolates, only 3 strains (O:3/1A, UT/3B, and UT/1A isolated from Chinese cabbage, onion, and spinach, respectively) were shown to have a virulence gene.     

Prevalence of Yersinia enterocolitica in fattening pigs

The prevalence of pathogenic Yersinia enterocolitica in pig herds was monitored during six trials (at four different farrow-to-finisher farms). Samples were taken throughout the whole rearing period from birth of the piglets to the final fattening stage, and different samples were taken from these pigs during the slaughter process. Environmental samples also were evaluated to identify potential sources of on-farm infection. Y. enterocolitica was isolated using irgasan-ticarcillin-potassium chlorate broth enrichment and cefsulodin-irgasan-novobiocin agar culture. Colonies were identified using bio- and serotyping methods and by PCR assay. Pathogenic Y. enterocolitica were not isolated from fecal samples from piglets and weaners. The only fecal samples positive for Y. enterocolitica were obtained during the fattening stage. The prevalence of Y. enterocolitica in fattening pig herds ranged between 0 and 65.4%. Y. enterocolitica isolates were detected at the abattoir in 38.4% of the tonsils, in 3.8% of the ileocecal lymph nodes, on 0.3% of the carcass surfaces before chilling, and on 0% of the carcass surfaces after chilling. Almost all isolates belonged to bioserotype 4/O:3. Only one strain was identified as O:9. All isolates contained the ail gene. The yopT gene was found in 99.1% of the farm isolates but in only 76.6% of the isolates found at the abattoir from the corresponding carcasses. Although a direct link between porcine isolates and human infection has not been demonstrated, the similarity of the bioserotypes in infected pigs and humans and the presence of virulence factors in porcine isolates should encourage further studies to determine the risk of transmission of Y. enterocolitica to humans from pigs and pork products.     

Evaluation of a combined culture and PCR method (NMKL-163A) for detection of presumptive pathogenic Yersinia enterocolitica in pork products

A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food (NMKL-163A) was evaluated by testing samples of artificially and naturally contaminated pork. The performance of the pre-PCR sample treatment, buoyant density centrifugation, was first compared with two commercially available methods (DNeasy tissue kit and PrepMan). We found that similar sensitivity was reached (i.e., 25 CFU/g of food was detected by single PCR) with the buoyant density centrifugation and the DNeasy Tissue kit when tested on overnight enrichments. However, the DNeasy tissue kit was superior when tested on nonenriched homogenates; the detection limit was 25 CFU/g in minced beef by single PCR and 25 CFU/g in sausage by nested PCR. We then analyzed 100 raw minced pork samples. Thirty-five tested positive for presumptive pathogenic Y. enterocolitica when analyzed by the NMKL-163A method, whereas none tested positive when analyzed in parallel by a standard culture method (ISO 10273). We also analyzed 97 samples of cold-smoked pork sausage, of which approximately 11% tested positive by the NMKL-163A method. This study showed that sensitivities such as those obtained by nested PCR were required for detection of the pathogen in naturally contaminated samples, and therefore the nested PCR primers, which are included in the NMKL-163A method only as an option, need to be validated and applied routinely.     

Slaughter pigs and pork as a source of human pathogenic Yersinia enterocolitica.

Pathogenic Yersinia enterocolitica strains (serogroups 0:3;0:9 and 0:5.27) were isolated from 36 (42%) of 86 porcine tonsils, 8 (20%) of 40 tongues, 17 (17%) of 100 rectal swabs and from 4 (1%) of 400 pork samples. Pathogenic Yersinia strains were not isolated from samples of 210 pig carcasses and from 20 samples of porcine head meat. These results confirm that pigs are an important reservoir of pathogenic Y. enterocolitica. However, contamination of carcasses during the slaughtering process with Yersinia from either faecal material or from the tonsillary region does not seem to occur frequently and this may also explain the low contamination rate of pathogenic Y. enterocolitica found for pork. For the isolation of pathogenic Y. enterocolitica strains from foods, enrichment in irgasan-ticarcillin-chlorate broth (ITC) and isolation on SS-deoxycholate-calcium agar (SSDC) is recommended.     

Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation

A Yersinia PCR-Compatible Enrichment (YPCE) medium was developed, which removes the necessity for sample pretreatment before PCR-based detection of Yersinia enterocolitica. The medium was designed through a sequence of independent screening and factorial design experiments to study the PCR inhibition and growth characteristics of medium components. The compatibility of the YPCE medium was evaluated using real-time PCR. The real-time PCR assay, based on the fluorescent double-stranded DNA binding dye SYBR green, generated approximately a 4-log linear range of amplification and in the range of 10(5)-10(8) (CFU/ml), the coefficient of variation <5%. When a background flora was present at concentrations > or = 10(6) (CFU/ml), the DNA amplification was influenced and a change in the log-linear slope leading to a lower amplification efficiency was observed. To study the dynamic detection range and relative amplification precision during enrichment. Y. enterocolitica and background flora were inoculated at various concentrations. It was possible to detect inoculation concentrations of 10(1) (CFU/ml) Y. enterocolitica in the presence of at least an inoculation concentration of 10(3) (CFU/ml) of an undefined background flora and the optimal conditions for sample withdrawal was in the range of 9 to 18 h enrichment. The YPCE medium can, especially for swab samples, form part of a simple analysis procedure allowing high throughput PCR.     

Detection, semiquantitative enumeration, and antimicrobial susceptibility of Yersinia enterocolitica in pork and chicken meats in Italy

Yersinia enterocolitica is recognized as an etiological agent of gastroenteritis, lymphadenitis, and chronic sequelae. During 2006 and 2007, 205 samples (125 pork and 80 chicken meats) were collected in Italy and tested for detection and most-probable-number (MPN) enumeration of Y. enterocolitica organisms. The microorganism was isolated from 45 samples (21.9%): 19 (15.2%) pork samples and 26 (32.5%) chicken samples. Y. enterocolitica MPN contamination levels were low, ranging from 0.30 to 1.50/g. Most (94.4%) Y. enterocolitica strains were biotype 1A (serotypes O:3; O:5; O:6,30; O:6,30-6,31; O:7,8-8-8,19; O:8; O:9; O:25,35; O:36; and O nontypeable), and 5.6% of the isolates were bioserotype 2/O:9. All isolates were tested for yadA, ail, inv, ystA, and ystB virulence sequences. The yadA gene was detected in two strains (3.7%) isolated from chicken samples: one Y. enterocolitica 2/O:9 yadA+ ail+ ystA+, and one Y. enterocolitica 1A/O:7,8-8-8,19 yadA+ inv+ ystB+. Two (3.7%) 2/O:9 strains, isolated from pork products, were ail+ ystA+. Most biotype 1A strains were ystB+ (84.3%) and inv+ (39.2%). All strains were sensitive to cefotaxime, ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, sulfonamide, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole. Resistance to gentamicin and aztreonam was observed in 1.9% of the isolates. High levels of resistance were detected toward amoxicillin-clavulanic acid (27.8%), ampicillin (75.9%), and erythromycin (100%). The authors hypothesize that Y. enterocolitica pathogenic biotypes are rather uncommon in foods when compared with their isolation rates from animal sources and that chicken meat could be contaminated as well as pig meat and its derived products.     

Extent of microbial contamination in United States pork retail products

To determine the extent of microbiological contamination of U.S. pork, 384 samples of retail pork were collected from 24 stores in six cities, including (i) whole-muscle, store-packaged pork; (ii) fresh, store-packaged ground pork and/or pork sausage; (iii) prepackaged ground pork and/or pork sausage; and (iv) whole-muscle, enhanced (injected or marinated; 60% store-packaged, 40% prepackaged) pork. Additional samples (n = 120) of freshly ground pork and/or pork sausage were collected from two hot-boning sow/boar sausage plants, two slaughter and fabrication plants, and two further-processing plants. Samples were analyzed for aerobic plate counts (APC), total coliform counts (TCC), Escherichia coli counts (ECC), and incidences of Salmonella spp., Listeria monocytogenes, Campylobacter jejuni, Campylobacter coli, and Yersinia enterocolitica. Mean log APC and TCC were highest (P < 0.05) for store-ground pork, while whole-muscle, enhanced products and prepackaged ground products had the lowest (P < 0.05) APC. Mean log APC and TCC were higher (P < 0.05) in samples from the slaughter and fabrication plants than in samples from hot-boning and further processing plants. Mean log ECC were lower (P < 0.05) in samples from further-processing plants compared to slaughter and fabrication plants and hot-boning, sow and boar sausage plants. L. monocytogenes was detected in 26.7% of plant samples and 19.8% of retail samples and was present more frequently in ground products. Y. enterocolitica was detected most often in whole-muscle, store-packaged cuts (19.8%) and in store-ground product (11.5%). Salmonella spp. were found in 9.6% of retail samples and 5.8% of plant samples, while C. jejuni and C. coli were found in 1.3% of retail samples and 6.7% of plant samples. Pork products exposed to the most handling and processing appeared to be of the poorest microbiological quality. These results should be useful in risk assessments that are directed at the identification of actions that could enhance food safety.     

Isolation of Yersinia enterocolitica from ready-to-eat foods and pork by a simple two step procedure

Out of 119 ready-to-eat food samples and pork processed for isolation of Yersinia enterocolitica, 15 samples (12·6%) were positive for the presence of Y. enterocolitica by a two step procedure in a modified trypticase soy broth containing 0·25% yeast extract, 0·2% bile salts and 4 μg ml^-1 Irgasan at pH 7·6 and upon incubating at 10°C and 22°C for 6 days. Only five samples (4·2%) were positive by cold enrichment in trypticase soy broth containing 0·2% yeast extract, incubated at 4°C for 14 days. Four strains of Y. enterocolitica and one strain of Y. intermedia were isolated from pork samples processed by cold enrichment. Out of 15 strains of Y. enterocolitica isolated from pork (four strains) and ready-to-eat food samples (10 strains) by two step procedure only three strains belonged to pathogenic serotype O:3 and which were isolated only from pork samples. Overall recovery of Y. enterocolitica was much better by the two step procedure as compared to cold enrichment (P < 0·05).     

Prevalence of virulent Yersinia enterocolitica in bulk raw milk and retail cheese in northern-west of Iran

To determine the prevalence of virulent Yersinia enterocolitica, 554 samples consisting of 354 bulk raw milks and 200 traditional cheeses were collected from different parts of Eastern-Azerbaijan province, during a 23-month period from 2008 to 2010. The occurrence of virulent strains of Y. enterocolitica in samples enriched in peptone sorbitol bile broth (PSBB) was evaluated via the detection of attachment invasion locus (ail) gene by PCR. The viability of virulent Y. enterocolitica in the PCR-positive samples was tested using conventional culture method and the isolates were confirmed by the second-phase ail-PCR. According to the results, 8.66% of total samples including 7.62% of bulk raw milks and 10.5% of raw milk cheeses were found ail-positive by PCR method; subsequently Y. enterocolitica was isolated by the culture method and confirmed by the second phase ail-PCR in 2.88% of total samples including 2.26% of raw milks and 4% of cheese samples. It was concluded that, a sample enrichment followed by ail-PCR was more sensitive and robust to detect and distinguish the virulent strains of Y. enterocolitica compared to the conventional culture method.     

Occurrence of Yersinia enterocolitica in milk and dairy products in Morocco.

A total of 227 samples of milk and dairy products were examined for the presence of Yersinia enterocolitica. Yersinia spp. were recovered from 11 of 30 raw milks (36.6%), one of 20 pasteurized milks (5%), 15 of 63 traditional fermented milks (23.8%), seven of 94 cheeses and one of 20 cream samples (5%). The overall incidence of Y. enterocolitica in milk and dairy products was 6.6%. The other Yersinia species were Y. intermedia, Y. kristensenii, Y. frederiksenii and Y. pseudotuberculosis. Y. enterocolitica was detected only in raw milk (30% of the samples), in traditional fermented milks (6.3%) and in raw milk-made cheese (4%). The majority of the Y. enterocolitica isolates were of biotype 1 (environmental strains). The Celfulodin-Irgasan-Novobiocin (CIN) Agar was found to be more efficient than the Mac Conkey Agar in the isolation of Yersinia organisms.