Prevalence of pathogenic Yersinia enterocolitica in pigs slaughtered at a Swiss abattoir

Human yersiniosis is the third most common enteric disease after campylobacteriosis and salmonellosis in many European countries. However, epidemiological data on the prevalence of pathogenic Yersinia enterocolitica in animals and humans is insufficient. Pigs are assumed to be the main reservoir of pathogenic Y. enterocolitica because pig is so far the only animal species from which pathogenic strains have frequently been isolated. This work was conducted to study the frequency of ail-positive Y. enterocolitica in pigs slaughtered at a Swiss abattoir. In total, 212 pig tonsils were screened by real-time PCR and culture methods. The prevalence rate of ail-positive Y. enterocolitica in pigs at slaughter was 88% and 34% with PCR and culture methods, respectively. The 148 ail-positive isolates from the 72 culture-positive tonsils were bio-and serotyped. The most common bioserotype was 4/O:3 found in 96% (69/72) of the culture-positive samples. However, pig was also shown to be a reservoir for ail-positive Y. enterocolitica belonging to bioserotypes 2/O:5,27 and 2/O:9, which were detected in 8% (6/72) and 1% (1/72) of the culture-positive samples, respectively. Using PFGE with NotI, only a limited number of different patterns was found. In all, 6 genotypes were obtained when 86 isolates of bioserotype 4/O:3 from 69 samples were characterised and two genotypes (N1 and N4) dominated. The biotype 4 differs clearly from biotype 2 with PFGE. Antimicrobial resistance testing of 77 ail-positive Y. enterocolitica isolates from 72 samples studied with disc-diffusion revealed that all strains were sensitive to cefotaxime, chloramphenicol, ciprofloxacin and tetracycline, which are antimicrobial agents used for treatment of human disease. The isolates of bioserotype 2/O:5,27 differed from the isolates of bioserotypes 2/O:9 and 4/O:3 in resistance to ampicillin and amoxicillin/clavulanic acid.     

Antimicrobial resistance of Yersinia enterocolitica strains from human patients, pigs and retail pork in Switzerland

To obtain basic data for future resistance monitoring programs, 386 Yersinia enterocolitica strains from human patients, raw retail pork and pig feces were tested for their susceptibilities to 16 antimicrobial agents and two antimicrobial growth promoters (carbadox and olaquindox). No strains were resistant to ceftriaxone, cefuroxime, ciprofloxacine, gentamicin, kanamycin, neomycin or polymyxin. Although in Switzerland carbadox and olaquindox were used as growth promoters for pigs for over 25 years, all strains were susceptible to them. In contrast, there were high levels of resistance to ampicillin, cefalothin and amoxicillin/clavulanic acid. Less than 10% of clinical isolates and strains from pig feces were resistant to streptomycin, sulfonamide, trimethoprim/sulfamethoxazole, tetracyclin, trimethoprim and chloramphenicol, but strains from retail pork were all susceptible to these antimicrobial agents. This finding suggested that pork is probably not a major source of Y. enterocolitica that cause human infections in Switzerland. A difference between clinical isolates and strains from pork was also shown by serotyping. Clinical isolates frequently belonged to the O3 and O9 groups whereas these two serotypes were not found in strains from pork. Resistance to multiple antimicrobial agents was rare. When examined by pulsed field gel electrophoresis (PFGE), two strains of fecal origin with an identical pattern of resistance to six antimicrobial agents were shown to be unrelated. Of four clinical isolates with resistances to five antimicrobial agents, two were of the same pulsotype. Retrospectively, it was found that these strains came from two members of the same household and thus represented a mini-outbreak.     

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.     

Contamination of freshly slaughtered pig carcasses with human pathogenic Yersinia enterocolitica

Evidence is presented for the extent of contamination of freshly slaughtered pig carscasses with human pathogenic Yersinia enterocolitica and shows the significance of faecal contamination as a source of infection. Swab samples collected from the rectum and the surface of a total of 1458 pig carcasses were examined for the presence of human pathogenic Y. enterocolitica Y. enterocolitica, biovar IV, serogroup 0:3, were isolated from the rectum of 360 pigs (24.7%). The organism was isolated from carcass surfaces with varying frequencies depending on the evisceration technique. Manual evisceration was found to correspond with high frequencies of contamination: 26.3% on the medial hind limb and 12.9% on the split sternum. The use of a mechanised bung cutter was found to reduce the rate of contamination, especially when the bung cutter was used in connexion with enclosing the anus and rectum in a plastic bag to minimise faecal contamination. When carcasses were eviscerated in this way, it was possible to reduce carcass contamination to 1.9% on the medial hind limb, 1.0% in the pelvic duct, and 2.2% on the split sternum.     

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.     

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.     

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).     

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.     

小肠结肠炎耶尔森菌在猪肉中失活/存活模型的初步研究

采用预测微生物学的基本方法和程序,研究小肠结肠炎耶尔森菌在特定条件下的生长规律,并用CurveExpertl.38软件作为辅助工具,从中选取了Linear模型对实验数据进行拟合.初步建立了肉汤中小肠结肠炎耶尔森菌55、60℃的热失活模型,以及猪肉中小肠结肠炎耶尔森菌的速冻失活模型和冷冻存活模型.     

Comparison of methods for the isolation of Yersinia enterocolitica from porcine tonsils and pork

Three enrichment procedures and three plating media were evaluated for their efficiency in isolating Yersinia enterocolitica from porcine tonsils and pork. Cold enrichment in phosphate-sorbitol-bile medium (PBSSB) with alkali treatment before plating resulted in higher isolation rates than enrichment in modified Rappaport broth (MRB) and bile-oxalate-sorbose broth (BOS). Post-enrichment alkali treatment gave a considerable increase in isolation rate with all the enrichment media tested. A sample inoculum of 10 g showed a better recovery than 0.2 g. Cefsulodin-irgasan-novobiocin (CIN) agar was slightly better than MacConkey agar and MacConkey agar + Tween 80 as isolation medium for Yersinia spp. from porcine tonsils. Most of the serotype 0:3 and 0:9 strains were isolated after enrichment in MRB and PBSSB. Enrichment in PBSSB resulted in the isolation of some other potentially virulent Yersinia strains. For the isolation of Yersinia spp. from pork and porcine tonsils an inoculum of 10 g, parallel use of MRB and PBSSB, post-enrichment alkali treatment and isolation on CIN agar is recommended.     

Prevalence and characterization of pathogenic Yersinia enterocolitica in pig tonsils from different slaughterhouses

The prevalence of yadA-positive Yersinia enterocolitica was determined in 185 pig tonsils from nine slaughterhouses using both the PCR and culture method. The mean prevalence was 37%, varying from 13% to 45% when both PCR and culture-positive results were included. Of the 52 PCR-positive tonsil samples, 20 were culture-negative, while of the 48 culture-positive, 16 were PCR-negative. Using the culture method, Y. enterocolitica belonging to the bioserotype 4/O:3 was found in 61 tonsils, of which 48 were yadA-positive. Type 4/O:3 was the only pathogenic bioserotype found in this study. Most of the yadA-positive samples (85%) were recovered already after overnight enrichment. A total of 61 isolates, including 13 yadA-negative isolates from different samples, were characterized with PFGE. UsingNotI and XbaI, 20 and 17 PFGE patterns were obtained, respectively. Although the patterns were not identical, most of them played only minor deviations. A total of 26 pulsotypes, defined by combination of the various NotI andXbaI digestion profiles, were observed. Two to eight different pulsotypes were observed in each slaughterhouse, The most common pulsotypes, 1a and 4g, were found in 36% and 20% of the tonsils, respectively and these pulsotypes were widely distributed to most of the slaughterhouses. The pulsotype 1a was identified in eight out of nine slaughterhouses and the pulsotype 4g in seven slaughterhouses.     

自腹泻病例和家用冰箱分离的小肠结肠炎耶尔森菌病原特征分析

目的研究自腹泻病例和家用冰箱中分离的小肠结肠炎耶尔森菌(Yersinia enterocolitica,Ye)的病原学特征,为科学防控Ye的污染提供依据。方法采集2017年北京市顺义区2家哨点医院肠道门诊腹泻患者病例粪便标本和顺义区83户家用冰箱中的涂抹拭子样品,对分离的Ye进行毒力基因、脉冲场凝胶电泳(PFGE)分子分型检测和耐药试验。结果腹泻病例粪便标本中Ye阳性率为0.27%(1/372),家用冰箱中Ye阳性率为6.02%(5/83);冰箱分离株仅携带ystB基因,腹泻病例分离株携带ail、ystA、virF、yadA基因;腹泻病例和冰箱分离株PFGE带型亲缘关系较远。全部Ye对氨苄西林、阿莫西林/克拉维酸、头孢唑林均耐药,腹泻病例分离株对萘啶酸耐药。结论 Ye在北京市顺义区腹泻病例中流行强度不高,家用冰箱受Ye污染较为严重,腹泻病例和冰箱中分离的Ye病原学特征具有一定差异。     

Enrichment, isolation, and virulence of freeze-stressed plasmid-bearing virulent strains of Yersinia enterocolitica on pork

The influence of freeze stress at -20 degrees C on the enrichment, isolation, detection, presence of virulence plasmid, and expression of virulence of plasmid-bearing Yersinia enterocolitica (YEP+) inoculated on pork chop medallions was assessed. Pork chop medallions (10 cm2) artificially contaminated with 10, 1, and 0.5 CFU/cm2 of YEP+ strains (serotype O:3) were placed in sterile petri dishes at -20 degrees C for 24 h. The medallions were swabbed when frozen, after thawing at room temperature for 1.5 h and after thawing at 4 degrees C for 18 h. Swabs were enriched and YEP+ were detected and isolated using the Congo red-binding and low-calcium-response assays. The YEP+ were isolated under all conditions on pork chop medallions inoculated with 10 CFU/cm2 and at a level of 1 CFU/cm2 when thawed at room temperature and at 4 degrees C but not from frozen pork chop medallions. The YEP+ were not isolated from pork chop medallions inoculated with 0.5 CFU/cm2 and then frozen, whereas YEP+ were recovered when inoculated at this level from pork chop medallions not subjected to freezing. Virulence of the strains isolated from frozen pork chop medallions was confirmed by PCR and the expression of plasmid-associated phenotypes. These results indicate that YEP+ subjected to freezing on pork are potentially capable of causing foodborne illness and that freezing is not a substitute for safe handling and proper cooking of pork.     

Isolation of Yersinia enterocolitica from foods.

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24 degrees C; plating on SSDC agar (2 days at 30 degrees C). (2) Pre-enrichment in TSB for 1 day at 24 degrees C; enrichment in BOS for 5 days at 24 degrees C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24 degrees C).     

Differences in heat resistance among pathogenic Yersinia enterocolitica depended on growth temperature and serotype

To gain a better understanding about the effect of growth temperature on heat resistance of Yersinia enterocolitica, we determined decimal reduction times at 60 degrees C (D60-values) for O:3; O:5,27; O:8; and O:9 strains harboring virulence plasmid coding for Yersinia outer membrane protein and experimentally virulence plasmid-deleted strains after they were grown to stationary phase at 7, 25, or 37 degrees C. Bacteria were inoculated into Trypticase soy broth and were incubated at several temperatures. D60-values of O:3; O:5,27; and O:8 strains were larger when they were grown at 37 degrees C than at 7 or 25 degrees C, despite the presence or absence of virulence plasmids. However, similar D60-values were observed in O:9 strains, despite growth at 7, 25, or 37 degrees C. The results indicate two types of Y. enterocolitica strains, growth temperature-dependent and -independent, and a Yersinia outer membrane protein that is not directly involved in growth temperature-dependent heat resistance.     

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.     

Testing of pathogenic Yersinia enterocolitica in pig herds based on the natural dynamic of infection

This study was performed to evaluate testing methods of pathogenic Yersinia enterocolitica in pigs at different ages. Relevant tools and procedures are crucial if pig herds should be declared free from pathogenic Y. enterocolitica. Historical data based on serology showed that the two farms investigated in this study (herds A and B) were contaminated with Y. enterocolitica O:3 since at least 1995. Laboratory investigations of 60 pigs were sampled one to four times (herd A) and 20 pigs were sampled one to three times (herd B) at different ages were the basis for this report. The following testing procedures could be used to conclude that a herd is free from pathogenic Y. enterocolitica:--serological testing of pigs could be performed as a basis for categorisation for all ages from about 100 days including at slaughter when the pigs are 150-180 days old, --bacteriological examination of faeces could be used as a basis for categorisation at all ages from 85 days until about 135 days, --bacteriological examination of tonsils could be used as a basis for categorisation at all ages from 85 days including at slaughter when the pigs are 150-180 days old. However, due to animal welfare aspects, one should avoid sampling of tonsils. Accordingly, the serological method or bacteriological examination of faeces at relevant ages should be preferred. One aspect related to slaughter hygiene is that in pigs slaughtered at the age of 135 days or more, the tonsils may be a more significant source of human pathogenic Y. enterocolitica than faeces.     

The effect of blast chilling on occurrence of human pathogenic Yersinia enterocolitica compared to Campylobacter spp. and numbers of hygienic indicators on pig carcasses

In this study, the occurrence of Yersinia enterocolitica on pig carcasses was compared to the occurrence of Campylobacter spp., and to the numbers of aerobic micro-organisms, coliform bacteria, thermotolerant coliform bacteria, and Escherichia coli before and after blast chilling. Y. enterocolitica O:3/biovar 4 was isolated from five (8.3%) of 60 carcasses before blast chilling, and also from five of them 1 h after blast chilling. Therefore this procedure does not seem to have a significant effect on the occurrence of pathogenic Y. enterocolitica on pig carcasses. Y. enterocolitica O:9/biovar 2 was isolated from a pig source in Norway for the first time when this sero/biovariant was isolated from one of the carcasses before blast chilling. Campylobacter spp. was isolated from 34 (56.7%) of 60 carcass samples before blast chilling. After blast chilling Campylobacter spp. was isolated from only one (1.7%) of the 60 carcasses. There was a significant decrease of the numbers of coliform bacteria, thermotolerant coliform bacteria and E. coli after blast chilling. The number of aerobic micro-organisms did not decrease after this step. In contrast to the drastic decrease in the occurrence of campylobacter-positive carcasses and the significant decrease of the numbers of coliform bacteria, thermotolerant coliform bacteria and E. coli, blast chilling does not seem to have a significant effect on the occurrence of human pathogenic Y. enterocolitica on pig carcasses.