Characterization of European Yersinia enterocolitica 1A strains using restriction fragment length polymorphism and multilocus sequence analysis.

UNLABELLED: Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5. SIGNIFICANCE AND IMPACT OF THE STUDY: Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica.

Prevalence of Pathogenic Yersinia enterocolitica in Finnish Slaughter Pigs.

The prevalence of human pathogenic Yersinia enterocolitica was determined in tonsil and intestinal content samples from 388 healthy fattening pigs at the four biggest Finnish slaughterhouses. These slaughterhouses process 73% of pigs in Finland. Tonsil samples were tested by PCR targeted for yadA, and intestinal samples were cultured. All pathogenic Y. enterocolitica isolates represented bioserotype 4/O:3. The prevalence of Y. enterocolitica in tonsil samples was 60% (95% confidence limit, 55.4 to 65.1%), and its prevalence in intestinal samples was 26% (95% confidence limit, 22.1 to 31.2%). The prevalence of Y. enterocolitica in tonsil and intestinal samples varied between the four slaughterhouses. The tonsil prevalence of Y. enterocolitica was higher in slaughterhouse B, and the prevalence in intestinal content was higher in slaughterhouse C. There were more positive results in both tonsil and intestinal samples in pigs coming from fattening farms than in pigs coming from farrowing-and-fattening farms. A seasonal variation was observed in the prevalence of Y. enterocolitica in intestinal samples, with the highest prevalence during July and August, but no seasonal variation was detected in tonsil samples.

Bayesian Estimation of the True Prevalence and of the Diagnostic Test Sensitivity and Specificity of Enteropathogenic Yersinia in Finnish Pig Serum Samples.

Bayesian analysis was used to estimate the pig's and herd's true prevalence of enteropathogenic Yersinia in serum samples collected from Finnish pig farms. The sensitivity and specificity of the diagnostic test were also estimated for the commercially available ELISA which is used for antibody detection against enteropathogenic Yersinia. The Bayesian analysis was performed in two steps; the first step estimated the prior true prevalence of enteropathogenic Yersinia with data obtained from a systematic review of the literature. In the second step, data of the apparent prevalence (cross-sectional study data), prior true prevalence (first step), and estimated sensitivity and specificity of the diagnostic methods were used for building the Bayesian model. The true prevalence of Yersinia in slaughter-age pigs was 67.5% (95% PI 63.2-70.9). The true prevalence of Yersinia in sows was 74.0% (95% PI 57.3-82.4). The estimates of sensitivity and specificity values of the ELISA were 79.5% and 96.9%.

Bayesian modelling to identify the risk factors for Yersinia enterocolitica contamination of pork carcasses and pluck sets in slaughterhouses.

The probability of contamination by pathogenic Yersinia enterocolitica of carcasses and pluck sets at slaughterhouse was determined by means of a Bayesian analysis. Prior information of the prevalence of Y. enterocolitica in faeces and the seroprevalence of Yersinia in serum of pigs collected at farms were obtained from previous studies and introduced in the models as beta prior informative distributions. Samples of intestinal content, tonsils, and swabs of carcasses and pluck set surfaces were collected at slaughterhouses. The posterior probabilities, odds ratio (OR) and their probability interval (PI) were calculated by means of a generalized linear model constructed in WinBugs. Occurrence of Y. enterocolitica in intestinal content (OR: 35.6, 95%PI 2.8-8285), tonsils (OR: 38.4, 95%PI 5.0-854), and pluck set (OR: 16.6, 95%PI 1.9-1111) was a risk for the contamination of pork carcasses, and an increased risk of contaminated pluck set was observed when Y. enterocolitica was isolated in intestinal content (OR: 40.6, 95%PI 2.1-10510) and tonsils (OR: 17.6, 95%PI 3.4-230.6). This increased risk indicated a potential cross-contamination at the slaughterhouse.

Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

AIMS: Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. METHODS AND RESULTS: Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. CONCLUSIONS: There was a clear association between the presence of the host bacteria and specific phages in the stools. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples.

Introduction of infected animals to herds is an important route for the spread of Yersinia enterocolitica infection between pig farms.

Altogether, 369 pathogenic Yersinia enterocolitica isolates from 1,118 fecal samples collected from 22 pig farms of different production types were characterized by biotyping, serotyping, and genotyping using multiple-locus variable-number tandem repeats analysis. We investigated the distribution of the different genotypes at the farm level and their association with different farm conditions. Pigs were found to carry and transmit Y. enterocolitica between farms, because the same genotypes were found on farms that had previously transported the pigs between them. The purchase of new animals for the farms associated significantly with the number of different multiple-locus variable-number tandem repeats analysis types of Y. enterocolitica found within a farm. Some genotypes seemed to persist on farms for years. The results of this study show that pigs purchased from infected herds transmit Y. enterocolitica infection between farms. Certain pig farms may act as long-term sources of infection.

Infant botulism with prolonged faecal excretion of botulinum neurotoxin and Clostridium botulinum for 7 months.

In Finland in April 2010, a 3-month old baby was diagnosed with type A infant botulism. He excreted botulinum neurotoxin and/or Clostridium botulinum in his faeces until November 2010. Five months of excretion was after clinical recovery and discharge from hospital. C. botulinum isolates recovered from the household dust in the patient's home were genetically identical to those found in the infant's stool samples. Long-term faecal excretion of C. botulinum may pose a possible health risk for the parents and others in close contact with the infant.

Multiple-locus variable-number tandem-repeat analysis in genotyping Yersinia enterocolitica strains from human and porcine origins.

Sporadic and epidemiologically linked Yersinia enterocolitica strains (n = 379) isolated from fecal samples from human patients, tonsil or fecal samples from pigs collected at slaughterhouses, and pork samples collected at meat stores were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA) with six loci, i.e., V2A, V4, V5, V6, V7, and V9. In total, 312 different MLVA types were found. Similar types were detected (i) in fecal samples collected from human patients over 2 to 3 consecutive years, (ii) in samples from humans and pigs, and (iii) in samples from pigs that originated from the same farms. Among porcine strains, we found farm-specific MLVA profiles. Variations in the numbers of tandem repeats from one to four for variable-number tandem-repeat (VNTR) loci V2A, V5, V6, and V7 were observed within a farm. MLVA was applicable for serotypes O:3, O:5,27, and O:9 and appeared to be a highly discriminating tool for distinguishing sporadic and outbreak-related strains. With long-term use, interpretation of the results became more challenging due to variations in more-discriminating loci, as was observed for strains originating from pig farms. Additionally, we encountered unexpectedly short V2A VNTR fragments and sequenced them. According to the sequencing results, updated guidelines for interpreting V2A VNTR results were prepared.

Two cases of food-borne botulism in Finland caused by conserved olives, October 2011.

In October 2011 in Finland, two persons fell ill with symptoms compatible with botulism after having eaten conserved olives stuffed with almonds. One of these two died. Clostridium botulinum type B and its neurotoxin were detected in the implicated olives by PCR and mouse bioassay, respectively. The olives were traced back to an Italian manufacturer and withdrawn from the market. The public and other European countries were informed through media and Europe-wide notifications.

cspB encodes a major cold shock protein in Clostridium botulinum ATCC 3502.

The relative expression of three cold shock protein coding genes (cspA, cspB and cspC) of Clostridium botulinum ATCC 3502 was studied with quantitative RT-PCR analysis following a cold shock shift from 37 °C to 15 °C. A significant increase in the relative expression of all three genes was observed upon the temperature downshift. To validate these findings, single-gene insertional inactivation of cspA, cspB and cspC was undertaken with the ClosTron gene knock-out system. In growth experiments, mutations in cspB or cspC, but not cspA, resulted in a cold-sensitive phenotype. No growth of the cspB mutant was observed at 15°C over a ten day period, whereas at 20 °C the growth rate was 70% lower than that of wild type strain. The growth rate of cspC mutant was 70% and 80% lower than the growth rate of the wild type strain at 15 °C and 20 °C, respectively. At 37 °C the growth of cspB mutant did not differ from, but the growth rate of cspC mutant was 30% lower than, that of the wild type strain. The cspA mutant grew somewhat faster than the wild type strain at all studied temperatures. Since the inactivation of cspB resulted in the most prominent defect in growth at low temperatures, we suggest that cspB encodes the major cold shock protein of C. botulinum ATCC 3502. Understanding the mechanisms behind cold tolerance of C. botulinum helps to evaluate the safety risks this foodborne pathogen poses in the modern food industry.

Factors related to the prevalence of pathogenic Yersinia enterocolitica on pig farms.

A survey of 788 pigs from 120 farms was conducted to determine the within-farm prevalence of pathogenic Yersinia enterocolitica and a questionnaire of management conditions was mailed to the farms afterwards. A univariate statistical analysis with carriage and shedding as outcomes was conducted with random-effects logistic regression with farm as a clustering factor. Variables with a P value <0·15 were included into the respective multivariate random-effects logistic regression model. The use of municipal water was discovered to be a protective factor against carriage and faecal shedding of the pathogen. Organic production and buying feed from a certain feed manufacturer were also protective against total carriage. Tonsillar carriage, a different feed manufacturer, fasting pigs before transport to the slaughterhouse, higher-level farm health classification, and snout contacts between pigs were risk factors for faecal shedding. We concluded that differences in management can explain different prevalences of Y. enterocolitica between farms.

Fur animal botulism hazard due to feed.

To assess the botulism hazard in fur animal feed production, 236 fur animal feed components and feed samples were analysed for Clostridium botulinum by detecting BoNT-encoding genes (botA, botB, botC, botE or botF) by PCR and for sulphite-reducing clostridia (SRC) by iron sulphite agar. The quality of the hazard analysis of critical control points (HACCP) -based in-house control system (IHCS) was evaluated with respect to botulism risk in feed plants (n=32). The overall prevalence of C. botulinum was 13% in different feed components and 5% in feed. The estimated MPN count of C. botulinum in feed components was 6.4 × 10(3)/kg at the highest and was shown to poorly correlate with SRC count. The critical control points in IHCSs were variable, and control limits were improperly set in most feed-producing plants. C. botulinum possesses a persistent safety hazard for fur animals by feed production, and control practices should be reassessed.

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content.

AIMS: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. METHODS AND RESULTS: The four methods comprised of 15 isolation steps using selective enrichments (irgasan-ticarcillin-potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25 degrees C. Salmonella-Shigella-desoxycholate-calcium chloride agar, cefsulodin-irgasan-novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre-enrichment step with further selective enrichment showed the highest sensitivities (55-66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. CONCLUSIONS: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.

Botulism and hot-smoked whitefish: a family cluster of type E botulism in France, September 2009.

A family cluster of three cases of type E botulism were identified in south-east France in September 2009. The suspected food source of infection was a vacuum packed hot-smoked whitefish of Canadian origin purchased by the family during a visit to Finland and consumed several weeks later in France on the day prior to symptom onset. No leftover fish was available to confirm this hypothesis. Vacuum packed hot-smoked whitefish has previously been associated with cases of type E botulism in multiple countries, including Finland, Germany, the United States and Israel.

Characterisation of non-pathogenic Yersinia pseudotuberculosis-like strains isolated from food and environmental samples.

Non-pathogenic Yersinia pseudotuberculosis-like strains were recovered from Finnish food and environmental samples. These strains could not be differentiated from Y. pseudotuberculosis strains using API 20E or other phenotypical tests. However, all of the strains were inv-, and virF-negative with polymerase chain reaction (PCR), while all Y. pseudotuberculosis strains used as controls were inv-positive and fresh Y. pseudotuberculosis strains were also virF-positive, indicating that the Y. pseudotuberculosis-like strains were non-pathogenic. Using pulsed-field gel electrophoresis (PFGE) with NotI enzyme and ribotyping with EcoRI and HindIII enzymes, the Y. pseudotuberculosis-like strains, which grouped genetically together, could be differentiated from true Y. pseudotuberculosis strains and from strains belonging to other sucrose-negative Yersinia species. In addition, the O-antigen gene cluster of one Y. pseudotuberculosis-like strain was characterized, and it differed from those of known Y. pseudotuberculosis serotypes. This study demonstrates that identification of Y. pseudotuberculosis from food and environmental sources using solely biochemical reactions can be incorrect, and when a strain cannot be serotyped to known Y. pseudotuberculosis serotypes, the pathogenic potential of isolates should be determined.

Type C bovine botulism outbreak due to carcass contaminated non-acidified silage.

The first reported bovine botulism outbreak in Finland is described. Nine out of 90 cattle on a dairy farm died after being fed non-acidified silage contaminated by animal carcasses. Type C botulinum neurotoxin gene was detected in one heifer by polymerase chain reaction (PCR) and the neurotoxin was detected by the mouse bioassay. Clostridium botulinum type C was isolated from liver samples. The isolated strain was identified with amplified fragment length polymorphism (AFLP) analysis as group III C. botulinum. To our knowledge, this is the first time that a type C bovine botulism outbreak has been diagnosed by PCR and confirmed by subsequent isolation and AFLP identification of the disease strain. The importance of the acidification process in silage production to inhibit C. botulinum toxin production in silage and thus to prevent further botulism outbreaks is emphasized. Nevertheless, preformed toxin in the carcass is not destroyed by acid.

Distribution of virF/lcrF-positive Yersinia pseudotuberculosis serotype O:3 at farm level.

The distribution and persistence of pathogenic, virF/lcrF-positive Yersinia pseudotuberculosis were investigated in pigs and in the pig house environment during rearing to determine possible contamination routes of early infections. Based on Y. pseudotuberculosis-positive tonsils of slaughter pigs in our previous study, Y. pseudotuberculosis-positive animals were traced back to the farms. Eight farms were visited from 6-10 months later, and a total of 155 pooled and six individual faecal samples from pigs and 116 pooled environmental samples were collected for analysis by different culture methods. Four of the eight farms were found to be Y. pseudotuberculosis-positive. All positive faecal samples were obtained from fattening pigs, with prevalence varying from 5% to 71% on positive farms. Sows, boars and suckling piglets were Y. pseudotuberculosis-negative on all farms. Most Y. pseudotuberculosis-positive farms (three of four) were on a one-site production system, which had a higher prevalence of Y. pseudotuberculosis (5-26%) among fattening pigs than the all-in, all-out system (1-5%). All Y. pseudotuberculosis isolates belonged to serotype O:3 and carried the virF/lcrF gene on the virulence plasmid. Biotypes 2 and 3 were involved, the latter in one isolate and not being previously reported in pigs. Altogether 53 isolates from 16 positive samples were characterized with pulsed-field gel electrophoresis (PFGE). Using SpeI, NotI and XbaI enzymes, four, three and two different PFGE patterns were obtained respectively. A total of nine different genotypes were identified when the profiles of the enzymes were combined. The most common genotypes were gIV, found on three, and gXII, found on two of the four Y. pseudotuberculosis-positive farms. The same genotypes previously detected in pig tonsils were present in pig faeces from the same farm, indicating that some Y. pseudotuberculosis strains can persist in the pig house environment.

Acid and heat tolerance of persistent and nonpersistent Listeria monocytogenes food plant strains.

AIMS: Acid and heat tolerance of 17 persistent and 23 nonpersistent Listeria monocytogenes strains, recovered from three meat-processing plants, were investigated. METHODS AND RESULTS: The isolates were genotyped by pulsed-field gel electrophoresis and categorized into persistent strains according to the frequency of the strain and duration of the contamination. The persistent and nonpersistent strains were challenged to acidic conditions (pH 2.4 for 2 h, 1 mol l(-1) HCl were used to acidify the suspension) and to heat (55 degrees C for 40 min) to receive a reduction in cell count. Listeria monocytogenes strains showed large variation in acid tolerance (over 6 log units) and in heat tolerance (3 log units). The persistent strains showed higher tolerance to acidic conditions than the nonpersistent strains (Student's t-test, P = 0.02), but significant differences in heat tolerance between persistent and nonpersistent strains were not observed. CONCLUSIONS: The results indicate that acid tolerance may have an effect on the persistence of L. monocytogenes contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the fact that there are great differences in acid and heat tolerances between L. monocytogenes strains, and the preventive measures should be designed to be effective against the most tolerant strains.

Listeria monocytogenes is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain.

AIMS: To evaluate the prevalence and genetic diversity of Listeria monocytogenes in wild birds and to compare the genotypes with isolates previously collected from foods and food processing environments. METHODS AND RESULTS: Samples of wild birds' faeces (n = 212) were collected from a municipal landfill site and from urban areas in the Helsinki region and analysed by two-step enrichment and plating onto L. monocytogenes-selective agar. The overall prevalence of L. monocytogenes in bird faeces was 36% (95% CI 30-43%), and prevalence on the landfill site was significantly higher. All isolates were analysed with pulsed-field gel electrophoresis and compared with the L. monocytogenes profiles in an existing collection. Similar pulsotypes were found in birds and in isolates collected along the food chain. CONCLUSIONS: Birds commonly carry L. monocytogenes, and strains are frequently similar with those detected in foods and food processing environments. Thus, birds may disseminate L. monocytogenes in nature and may also contaminate foods when entering the food processing environments and outdoor market places. SIGNIFICANCE AND IMPACT OF THE STUDY: Populations of L. monocytogenes in wild birds and along the food processing chain overlap. Our findings add to the epidemiological data on this significant foodborne pathogen.