Low prevalence of yadA-positive Yersinia enterocolitica in sows

Yersinia enterocolitica 4/O:3 is commonly isolated from fattening pigs in Finland, but its prevalence in sows is relatively unknown. The current study determined the prevalence of yadA-positive Y. enterocolitica in sows and fattening pigs with polymerase chain reaction (PCR) and culture methods. The strains were characterized with pulsed-field gel electrophoresis (PFGE) using NotI DNA-restriction enzyme. Pathogenic Y. enterocolitica was detected in only 14% of sow tonsils compared with 56% of tonsils of fattening pigs. The prevalence varied between seven slaughterhouses from 0% to 30% in sows and from 30% to 87% in fattening pigs. A total of 37 NotI profiles were obtained from 148 Y. enterocolitica 4/O:3 strains isolated from 134 tonsil samples: eight profiles were obtained from 26 sow strains and 34 from 122 fattening pig strains. Two types predominated in both sows and fattening pigs. The prevalence of yadA-positive Y. enterocolitica in fattening pigs increased from 1995 versus 1999; the mean prevalence in five slaughterhouses for the 2 years was 33% and 64%, respectively. Seven NotI profiles, including the two common types, were found both years. In conclusion, pathogenic Y. enterocolitica was detected at a significantly lower rate in sows than in fattening pigs. Moreover, the prevalence of this pathogen in fattening pigs was significantly higher in 1999 than in 1995. Some Y. enterocolitica 4/O:3 strains persisting among slaughter swine were demonstrated to be very stable genetically. This study shows that fattening pigs are an important reservoir of different genotypes of Y. enterocolitica 4/O:3 strains.     

Correspondence of genotypes of sporadic Yersinia enterocolitica bioserotype 4/O:3 strains from human and porcine sources

The sources and transmission routes of sporadic Yersinia enterocolitica bioserotype 4/O:3 infections in Finland were studied. A total of 212 human strains were compared with 334 non-human strains, including 163 strains from pig slaughterhouses, 164 strains from retail outlets and 7 strains from pet animals. All strains were characterized using pulsed-field gel electrophoresis (PFGE) with NotI enzyme. When the 194 human and 287 non-human strains of 22 identical NotI profiles were further characterized with ApaI and XhoI enzymes, 126 genotypes (DI = 094) were distinguished. Of all 212 human strains, 80% were genetically indistinguishable from the strains found in samples of pig origin when characterized with the three enzymes. A major contamination source of sporadic Y. enterocolitica 4/O:3 infections was revealed to be edible pig offal: 71% of the human strains were indistinguishable from the strains isolated from tongues, livers, kidneys and hearts of pigs. These results reveal that in Finland contaminated pig offal is an important vehiclein the transmission of Y. enterocolitica bioserotype 4/O:3 from slaughterhouses to humans.     

Occurrence of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis in small wild rodents

Rodents are a potential source of pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. In order to study this, 190 rodents were captured and sampled on seven pig farms (n=110), five chicken farms (n=55) and six other locations (n=25) in Sweden. Pigs from three of the pig farms were also sampled (n=60). Pathogenic Y. enterocolitica was detected by TaqMan PCR in about 5% of rodent samples and 18% of pig samples. Only rodents caught on pig farms tested positive for the pathogen. Y. enterocolitica bioserotype 4/O:3 strains isolated from the rodent and pig samples were compared by pulsed-field gel electrophoresis and revealed a high degree of similarity, which was confirmed by random amplified polymorphic DNA. Y. pseudotuberculosis was only detected in one rodent sample. Thus, rodents may be vectors for the transmission of pathogenic Y. enterocolitica to pigs, acting as carriers rather than a reservoir, and should therefore remain an important issue in hygiene control measures on farms.     

Epidemiological data on pathogenic Yersinia enterocolitica in Southern Germany during 2000-2006

Yersinia enterocolitica is the most common species causing enteric yersiniosis, which is still the third most frequently reported foodborne gastroenteritis in Europe. Y. enterocolitica generally causes sporadic human infections, and outbreaks are rare. The most important infection source of yersiniosis is believed to be contaminated pork and pork products. Data on the prevalence of pathogenic Y. enterocolitica in animals and foodstuffs are very limited and old; thus, more information on the extent and range of the prevalence of this enteropathogen in nonhuman sources is needed. In this work, prevalence of pathogenic Y. enterocolitica in different sources in Bavaria is presented. Further, the antimicrobial resistance of human and nonhuman strains is reported. The highest isolation rate of pathogenic Y. enterocolitica (67%) was found in tonsils of slaughter pigs. No pathogenic strains were isolated from cattle, sheep, turkey, and horses. ail-Positive Y. enterocolitica was detected in dogs (5%), cats (3%), and rodents (3%) by real-time PCR. Pathogenic Y. enterocolitica was isolated only from raw pork, especially from edible offal (51%). Surprisingly, 38% of game was contaminated with this pathogen when the samples were studied with PCR. Additionally, some raw pork sausages and one poultry sample were PCR positive. All pathogenic Y. enterocolitica isolates from nonhuman sources were belonging to bioserotype 4/O:3. Antimicrobial resistance of 60 human and 140 porcine strains of bioserotype 4/O:3 was tested by the agar disc diffusion method to 15 different antimicrobial agents. All Y. enterocolitica 4/O:3 strains were susceptible to most of the tested antibacterial agents.     

Effect of sampling and short isolation methodologies on the recovery of human pathogenic Yersinia enterocolitica from pig tonsils

The objective of this study was to determine the effect of sampling (swab samples compared to destructive samples) on isolation rates of human pathogenic Yersinia enterocolitica from pig tonsils. Moreover, the relative efficiency of different rapid, routinely applicable isolation methods was evaluated. Therefore, swab and destructive samples from tonsils of 120 pigs at slaughter were analyzed in parallel using direct plating and different enrichment methods. Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) agar, cefsulodin-irgasan-novobiocin (CIN) agar, and Yersinia enterocolitica chromogenic medium (YeCM) were used as selective agar media. For enrichment, irgasan-ticarcillin-potassium chlorate (ITC) broth and peptone-sorbitol-bile (PSB) broth were incubated at 25°C for 48 h. Overall, 55 tonsils (45.8%) were positive for Y. enterocolitica bioserotype 4/O:3. Recovery was significantly higher using the destructive method compared to the swabbing method. Direct plating resulted in 47 and 28 Y. enterocolitica-positive destructive and swab samples, respectively. Alkali treatment of PSB and ITC enrichment broths significantly increased recovery of pathogenic Y. enterocolitica from destructive tonsil samples. The performance of YeCM for qualitative and quantitative isolation of pathogenic Y. enterocolitica from pig tonsils was equal to SSDC and CIN. In conclusion, direct plating and ISO 10273: 2003 with minor modifications are suitable and rapid methods for isolation of pathogenic Y. enterocolitica from destructive tonsil samples.     

The ail gene is present in some Yersinia enterocolitica biotype 1A strains

One chromosomal virulence marker of Yersinia is the gene ail, which encodes Ail, an outer membrane protein that promotes attachment and invasion. A high correlation has been found between the ail gene and the virulence of Yersinia. Here, we report two Yersinia enterocolitica biotype 1A strains that are usually nonpathogenic and carry the ail gene. The ail gene sequences of biotype 1A strains displayed similarity to the bioserotype 1B/O:8 strain 8081. The finding suggests that ail-based detection methods for Y. enterocolitica alone are insufficient to detect real pathogenic strains.     

Antibodies against Yersinia among farmers and slaughterhouse workers.

Antibodies to immunoglobulins (Ig) M, G, and A against Yersinia enterocolitica serotypes O:3, O:5, O:8, and O:9 and Yersinia pseudotuberculosis serotypes I and III were analyzed by enzyme immunoassay of the serum samples of 161 slaughterhouse workers, 147 pig farmers, and 114 grain or berry farmers. The crude risk ratios for elevated serum antibody concentrations were calculated with the use of the grain and berry farmers as the reference population. The risk for an elevated Y enterocolitica O:3 Ig G concentration was 3.0 (95% confidence interval (95% CI) 1.3-7.1) for the pig farmers and 1.8 (95% CI 0.7-4.4) for the slaughterhouse workers and the respective risks for elevated Y enterocolitica O:9 Ig G were 2.4 (95% CI 1.1-5.5) and 1.7 (95% CI 0.7-4.0). Since these two serotypes are commonly associated with swine, the increased number of subjects with elevated antibody levels could be causally related to occupational contact with this animal.     

1997-2010年宁夏回族自治区小肠结肠炎耶尔森菌致病性的基因特征

目的 了解宁夏回族自治区小肠结肠炎耶尔森菌主要毒力基因分布情况和致病性小肠结肠炎耶尔森菌分子分型特征。方法 于1997-2010年在宁夏回族自治区共分离得到283株小肠结肠炎耶尔森菌,用PCR法分析其黏附侵袭位点基因(ail)、耐热肠毒素A基因（ystA）、ystB、黏附素基因（yadA）、毒力活化因子基因（virF）;应用限制性内切酶Not Ⅰ酶切致病性小肠结肠炎耶尔森菌染色体DNA进行脉冲场凝胶电泳(PFGE),利用BioNumerics软件进行聚类分析。结果 209株O∶3、O∶9血清型小肠结肠炎耶尔森菌中ail、ystA、yadA、virF毒力基因阳性,ystB为阴性的占97.6％( 204/209);O∶8血清型和未开展血清分型的菌株5种毒力基因全部阴性;11株O∶5血清型有9株5种毒力基因全部阴性。将致病性菌株进行PFGE分型,根据染色体DNA的Not Ⅰ酶切图谱,将29株O∶3血清型分成12个PFGE带型,包含5株以上的优势PFGE带型有2种。180株O∶9血清型菌株分成13个PFGE带型,包含10株以上的优势PFGE带型有4种,各自是从同一地区猪与家鼠、猪与犬、猪与野兔分离。结论 宁夏地区O∶3、O∶9血清型小肠结肠炎耶尔森菌具有致病性,O∶3逐步成为如今的优势血清型;O∶5、O∶8与血清未分型的菌株无致病性。     

Detection of natural inter- and intra-genotype recombination events revealed by cap gene analysis and decreasing prevalence of PCV2 in wild boars

Porcine circovirus type 2 (PCV2), the causative agent of a number of PCVAD (porcine circovirus associated diseases), is ubiquitous in domestic pig and wild boar populations. In the present study, using recombination detection program, phylogenetic analysis and base-by-base comparison of 28 PCV2 ORF2s (capsid protein coding gene) from wild boars and 8 from domestic pigs of Transylvania, recent natural intra- (PCV2b-1B/PCV2b-1C) and inter-genotype (PCV2a-2D/PCV2b-1C) recombination events were detected. Notably, one potential recombinant (F1-21) was detected in domestic pig with possible parental strains of wild boar origin. The estimated recombinant breakpoints comprised epitopes A, B and C of ORF2, without major changes in amino acid sequences. The prevalence of PCV2 in the wild boar population during the 5-year period following the first outbreaks of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs in Romania showed a decrease from 13.4% to 8.3%. To our knowledge, this is the first study to show the existence of ORF2-based intra- and inter-genotype recombination in wild boar populations and the possible recombination between PCV2 strains of wild boars and domestic pigs. Our results suggest a certain independence of PCV2 infection in wild boar populations and demonstrate the possibility of infection with multiple PCV2 genotypes under natural circumstances. On the other hand, PCV2 genotypes specific for wild boars could be detected in domestic pig at lower frequency suggesting the possible spread of wild boar PCV2 to domestic swine. The recombination events described here may contribute to the genetic diversity of PCV2 and may also be the source of emergence of new PCV2 strains.     

Phylogeny and evolutionary genetics of porcine parvovirus in wild boars

Porcine parvovirus (PPV) is widespread among swine and is responsible for reproductive failure of susceptible sows, characterized by embryonic and fetal death. Studies showed that PPV in domestic pig is genetically diverse and some strains differ from the ones used for vaccination. Organ samples from wild boars and domestic pigs were collected in Transylvania (Romania) and tested for the presence of PPV by polymerase chain reaction. Positive samples were grouped and 14 from the wild boar and 1 from the domestic pig PPVs were selected for VP1/VP2 sequence analysis and comparison with available GenBank data. The molecular clock analysis revealed that PPV has a relatively recent evolutionary history, originated approximately 120 years ago and the main divergence occurred in the last 20-60 years. Phylogenetic and residue substitution analysis showed that the viruses could be divided into 6 distinct clusters and that wild boar PPVs were partially different and independent from domestic pig PPVs. PPVs of wild boars proved to be more diverse than viruses of domestic pigs. The presence of the highly virulent 27a-like PPV strains in wild boars was also detected.     

Induction of CD8+ T cell responses by Yersinia vaccine carrier strains

Yersinia enterocolitica employs a type III secretion system (TTSS) to target virulence factors (e.g. YopE) into the cytosol of the host cells. We utilized the TTSS to introduce a recombinant antigen directly into the cytosol of host cells and to investigate the potential of Y. enterocolitica and Y. pseudotuberculosis as live carrier for vaccines. The model antigen ovalbumin (Ova) was fused to defined secretion or translocation domains of the Yersinia effector protein YopE and introduced into attenuated mutant strains of Y. enterocolitica and Y. pseudotuberculosis. In vitro experiments showed secretion and translocation of YopE-Ova hybrid proteins into host cells. To investigate the resulting immune responses, mice expressing transgenic Ova-specific T cell receptors were used. Both Y. enterocolitica and Y. pseudotuberculosis mutants induced efficaciously Ova-specific CD8+ T cell responses. The translocation domain of YopE was required for induction of CD8+ T cell responses in vivo, but not for T cell responses induced in vitro. The in vivo frequency of Ova-specific splenic T cells was up to six-fold higher in mice immunized with YopE-Ova-translocating Y. enterocolitica/Y. pseudotuberculosis mutants than in control mice. The Ova-specific T cells were shown to produce high amounts of IFN-gamma. We did not observe significant Ova-specific CD4+ T cell or antibody responses upon vaccination with either of the strains. In conclusion, Yersinia live carrier vaccine strains are suitable to target antigens into the MHC class I pathway and stimulate CD8+ T cell responses and thus, might be useful in vaccine approaches against intracellular pathogens.     

Isolation of Yersinia, campylobacter, Plesiomonas and Aeromonas from environmental water and fresh water fishes]

Yersinia, Campylobacter, Plesiomonas and Aeromonas are known causative agents in waterborne diseases. For about 10 years, outbreak of diarrhea has been observed, especially among children, in the mountain areas of Okayama. Y. pseudotuberculosis recently isolated from non-chlorinated drinking water sources such as mountain streams and wells has been suspected to be the causative bacteria of the disease. Attempts were made to isolate Yersinia and Campylobacter from water samples from rivers in rural areas and Plesiomonas and Aeromonas from samples of well water and fresh water fishes in Okayama prefecture from 1987 to 1990. The isolation rate of Yersinia from river water samples was 7.5% with a higher rate in the mountain areas than in the nonmountain areas. While Y. enterocolitica was isolated throughout the year, Y. pseudotuberculosis was only seen during the winter. Various serogroups including human types of Y. enterocolitica and serogroup 2B, 4A and UT of Y. pseudotuberculosis were detected. Campylobacter was isolated from 0.5% of river water samples. Plesiomonas from 1.5% of fresh water fishes, and Aeromonas was isolated from 6.9% of well water samples and 47.1% of fresh water fishes.     

登封市屠宰场生猪小肠结肠炎耶尔森菌病原学研究

目的 了解登封市屠宰场生猪小肠结肠炎耶尔森菌带菌率、血清分型、毒力基因分布等情况,为制定预防措施提供参考依据。方法 从屠宰场生猪扁桃体及回盲部内容物中分离小肠结肠炎耶尔森菌,并进行生化鉴定、血清分型、毒力基因PCR检测。结果 从196份生猪咽拭子中分离出107株小肠结肠炎耶尔森菌,检出率为54.59%;从196份生猪肛拭子中分离出36株小肠结肠炎耶尔森菌,检出率为18.37%。检出菌血清型为O∶3,生物型为3型,Ⅰ型（ail＋、ystA＋、ystB-、yadA＋、virF＋）占93.71%（134/143）,Ⅱ型（ail＋、ystA＋、ystB-、yadA-、virF-）占6.29%（9/143）。结论 登封市生猪小肠结肠炎耶尔森菌以致病性菌株为主,今后应加强该菌的进一步监测工作。     

Pig herds free from human pathogenic Yersinia enterocolitica

Pork products are a substantial source of human yersiniosis, a foodborne disease caused by Yersinia enterocolitica. Thus, the ability to eliminate this agent from pig herds would be an important step in producing human pathogen-free pork. Pig herds free from Y. enterocolitica O:3/biovar 4 have been established and maintained. According to serologic and cultural testing results, 15 of 16 specific pathogen-free herds were free from Y. enterocolitica O:3/biovar 4; this closed breeding pyramid has remained free from this organism since 1996. Pig herds free from human pathogenic Y. enterocolitica suggest that human pathogen-free herds could be attained to provide pork free from zoonotic agents.     

Yersinia in effluents from the food-processing industry.

Yersinia enterocolitica and Yersinia pseudotuberculosis are current sources of pathogenic strains in humans and animals. Yersiniae infections occur throughout the world, but are most prevalent in regions with moderate and subtropical climates. In Australia, Central Europe and North America, cases of human infections with Yersinia enterocolitica now rank in third place. The food-processing industry may influence the epidemiological situation in different ways. Effluents which contaminate the environment may originate from slaughterhouses; e.g. from sewage contaminated with faeces from the lairage or contaminated effluents from the actual slaughter areas. The carcasses may serve as carriers of the organisms to the food-processing plants where they eventually contaminate the processed foods. Rodents and pests may also be carriers. Pathogenic Y. enterocolitica and Y. pseudotuberculosis strains mainly occur in swine and pork. The ability to multiply under refrigeration and in vacuum-packaged products means that pathogenic Y. enterocolitica can cause foodborne diseases. If a plant harbours any pathogenic Yersiniae, transfer of the contaminant to the sewage is possible. Although pathogenic Yersiniae from infected animals can survive in sewage and in surface waters, the role of properly treated sewage in the transmission of yersiniosis seems to be of minor importance. If the recommendations for modern slaughter techniques are properly followed, the spread of pathogens in the slaughterhouses and, subsequently, into other food-processing plants can be minimised.     

Hepatitis E as zoonosis

The hepatitis E virus (HEV) the causative agent of hepatitis E, is a non-enveloped RNA virus. HEV is transmitted through oral consumption of contaminated food and water According to the currently knowledge now be considered as zoonosis. The main reservoir of HEV are pigs, boars and deer. For the first time HEV was isolated from animals (pigs) in 1997 in the U.S. Genetic analysis of strains isolated from pigs showed high similarity to strains HEV isolated from humans. This was the first evidence showing that HEV is a zoonosis. Further studies have shown that occupational groups e.g. veterinarians, swine breeders with close contact to pigs have an increased risk for HEV infections. The additional evidence supported the zoonotic potential of HEV were reports of acute hepatitis E after the consumption of undercooked meat from deer and wild boar. Infection of HEV in the domestic pig and wild boar population in Europe is widespread.     

The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes

Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.     

Identification and typing of Yersinia enterocolitica biotypes and serotypes isolated

OBJECTIVE: To assess the presence of Yersinia enterocolitica in otherwise healthy pigs slaughtered for human consumption. METHODS: One hundred pharyngeal tonsils were sampled in a slaughterhouse in the state of Mexico. The minimum sample size (n=100) was calculated based on a preliminary sample of 20 cases, which had 20% positive cases. The collected tonsil samples were inoculated in Rappaport broth, and Salmonella-Shigella and McConkey media. The biotyping identification process was based on biochemical and serological tests using O:3, O:8 and O:9 antisera. RESULTS: Twenty-two isolates were obtained. Most were biotype 1 (8 cases of O:3 and 8 cases of O:9), but 6 cases could not be serotyped. None of the isolates were of O:8 group. CONCLUSIONS: This was the first time that Y. enterocolitica serotypes were isolated from pig tonsils in Mexico. Its importance rely on the fact that the isolated serotypes are the most commonly found in public health problems.     

中国六省致病小肠结肠炎耶尔森菌的脉冲场凝胶电泳分析

目的 对中国致病小肠结肠炎耶尔森菌分离株进行脉冲场凝胶电泳分析。方法 参照美国疾病预防控制中心PulseNet实验方法对中国6省165株致病小肠结肠炎耶尔森菌进行脉冲场凝胶电泳分析，使用BioNumerics软件进行聚类分析，并按照PulseNet命名原则对带型进行命名。结果 将114株O：3血清型小肠结肠炎耶尔森菌分成25个带型；K6GNllC30012（50株）、K6GNllC30015（19株）及K6GNllC30016（10株）是其主要带型，三个主要带型之间聚类相似性很高。将51株O：9血清型小肠结肠炎耶尔森菌分为14个带型；其中K6GNllC90004（22株）与K6GNllC90010（13株）是其主要带型，K6GNllC90004与K6GNllC90010带型之间有一定的差异。O：3与O：9血清型的主要带型之间有明显的差异。结论 O：3血清型的小肠结肠炎耶尔森菌可能起源于一个克隆系，且在不同地区和年代变异很小；O：9血清型的小肠结肠炎耶尔森菌可能起源于两个不同的克隆系，且各自有一定的变异。O：3与O：9血清型的小肠结肠炎耶尔森菌亲缘关系较远。     

An evaluation of methods for the isolation of Yersinia enterocolitica from surface waters in the Grand River watershed

Yersinia enterocolitica is a foodborne pathogen, but the importance of water as a route of exposure for human infection is not well known. Y. enterocolitica isolation methods were developed primarily for food and clinical samples, and may not be effective for use with environmental samples. The objective of this study was to assess the recovery of Y. enterocolitica from surface water used for drinking water treatment. Four enrichment broths and an alkaline treatment protocol were compared for the isolation of Y. enterocolitica bioserogroup 4/O:3 spiked into surface water samples. Results showed that the methods tested were not effective for the recovery of Y. enterocolitica, primarily due to inadequate inhibition of interfering background microorganisms. Using one method that showed the most potential for recovery, Yersinia spp. were isolated from rivers in southwestern Ontario, Canada, over a 17-month period. Of 200 samples analysed, Yersinia spp. were isolated from 52 samples. All river isolates belonged to non-pathogenic sub-groups, including Y. enterocolitica biotype 1A, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. mollaretii. Results of this study show that method improvements are required to more fully understand the role of water as a source of clinically important Yersinia strains.