Riikka Laukkanen

Contamination of Carcasses with Human Pathogenic Yersinia enterocolitica 4/O:3 Originates from Pigs Infected on Farms

Pigs are considered as a major reservoir of human pathogenic Yersinia enterocolitica and a source of human yersiniosis. However, the transmission route of Y. enterocolitica from farm to pork is still unclear. The transmission of pathogenic Y. enterocolitica from pigs to carcasses and pluck sets was investigated by collecting samples from 364 individual ear-tagged pigs on the farm and at the slaughterhouse. In addition, isolated strains were analyzed, using pulsed-field gel electrophoresis. Isolation of similar genotypes of pathogenic Y. enterocolitica 4/O:3 in animals on the farm and at the slaughterhouse and in carcasses shows that carcass contamination originates from the strains a pig carries during the fattening period. Direct contamination from the carrier pig to its subsequent pluck set is also the primary contamination route for pluck sets, but cross-contamination appears to have a larger impact on pluck set contamination than on carcasses. In this study, the within-farm prevalence of pathogenic Y. enterocolitica varied from 0% to 100%, indicating specific farm factors affect the prevalence of Y. enterocolitica in pigs. The association of farm factors with the high prevalence of pathogenic Y. enterocolitica on farms was studied for the first time, using correlation and two-level logistic regression analyses. Specific farm factors, i.e. drinking from a nipple, absence of coarse feed or bedding for slaughter pigs, and no access of pest animals to pig house, were associated with a high prevalence of pathogenic Y. enterocolitica 4/O:3.

Transmission of Yersinia pseudotuberculosis in the Pork Production Chain from Farm to Slaughterhouse

ABSTRACT The transmission of Yersinia pseudotuberculosis in the pork production chain was followed from farm to slaughterhouse by studying the same 364 pigs from different production systems at farm and slaughterhouse levels. In all, 1,785 samples were collected, and the isolated Y. pseudotuberculosis strains were analyzed by pulsed-field gel electrophoresis. The results of microbial sampling were combined with data from an on-farm observation and questionnaire study to elucidate the associations between farm factors and the prevalence of Y. pseudotuberculosis. Following the same pigs in the production chain from farm to slaughterhouse, we were able to show similar Y. pseudotuberculosis genotypes in live animals, pluck sets (containing tongue, tonsils, esophagus, trachea, heart, lungs, diaphragm, liver, and kidneys), and carcasses and to conclude that Y. pseudotuberculosis contamination originates from the farms, is transported to slaughterhouses with pigs, and transfers to pluck sets and carcasses in the slaughter process. The study also showed that the high prevalence of Y. pseudotuberculosis in live pigs predisposes carcasses and pluck sets to contamination. When production types and capacities were compared, the prevalence of Y. pseudotuberculosis was higher in organic production than in conventional production and on conventional farms with high rather than low production capacity. We were also able to associate specific farm factors with the prevalence of Y. pseudotuberculosis by using a questionnaire and on-farm observations. On farms, contact with pest animals and the outside environment and a rise in the number of pigs on the farm appear to increase the prevalence of Y. pseudotuberculosis.

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.

Listeria monocytogenes contamination in pork can originate from farms.

The presence of Listeria monocytogenes in the pork production chain was followed from farm to slaughterhouse by examining the farm and slaughterhouse levels in the same 364 pigs, and finally by analyzing the cut meats from the same pig lots. Both organic and conventional farms were included in the study. Altogether, 1,962 samples were collected, and the 424 L. monocytogenes isolates were analyzed by pulsed-field gel electrophoresis. The results from microbial analyses were combined with data from an on-farm observation and a questionnaire to clarify the associations between farm factors and prevalence of L. monocytogenes. The prevalence of L. monocytogenes was 11, 1, 1, 24, 5, 1, and 4% in feed and litter, rectal swabs, intestinal contents, tonsils, pluck sets (including lungs, heart, liver, and kidney), carcasses, and meat cuts, respectively. The prevalence was significantly higher in organic than in conventional pig production at the farm and slaughterhouse level, but not in meat cuts. Similar L. monocytogenes genotypes were recovered in different steps of the production chain in pigs originating from the same farm. Specific farm management factors, i.e., large group size, contact with pet and pest animals, manure treatment, use of coarse feed, access to outdoor area, hygiene practices, and drinking from the trough, influenced the presence of L. monocytogenes in pigs. L. monocytogenes was present in the production chain, and transmission of the pathogen was possible throughout the chain, from the farm to pork. Good farm-level practices can therefore be utilized to reduce the prevalence of this pathogen.

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.

Reduction of Enteropathogenic Yersinia in the Pig Slaughterhouse by Using Bagging of the Rectum

To evaluate the effectiveness of bagging of the rectum in mitigating the contamination of carcasses with enteropathogenic Yersinia at the slaughterhouse and to estimate the hidden prevalences of these pathogens in different farm types and capacities, samples from pigs, carcasses, and slaughterhouse environment were collected, and a Bayesian probability model was constructed. In addition, the contamination routes were studied with molecular typing of the isolated strains. According to the model, bagging of the rectum reduced carcass contamination significantly with pathogenic Yersinia enterocolitica, but not with Yersinia pseudotuberculosis, and alone it was insufficient to completely prevent the carcass contamination with enteropathogenic Yersinia. The hidden prevalence of pathogenic Y. enterocolitica was higher at high production capacity than it was in low production capacity, but the 95 % credible intervals overlapped. Slaughterhouse environments can contaminate carcasses with enteropathogenic Yersinia, but the plausible main contamination source is the pig carrying the pathogen.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

A multiplex real‐time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage λ; the latter was used as an internal amplification control. The Y. pestis‐specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10–100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

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.

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.

Causal hidden variable model of pathogenic contamination from pig to pork

Risk assessments relating to food safety over more than one step along a production chain are frequently hampered by lack of detailed quantitative data. This study set out to develop a Bayesian hidden variable model to integrate available limited data of the combined occurrence of three bacterial pathogens, Listeria monocytogenes, Yersinia enterocolitica and Yersinia pseudotuberculosis, with causal assumptions along three steps of pork production chain. The pathogen occurrence data were animal specific both on conventional and organic pig farms and at the abattoir, but merely farm specific at meat cutting plants. The model was able to incorporate all data concerning different types of testing at different steps of the chain, and missing data values were dealt with in a straightforward manner. It provides a tool for quantitative risk assessments and for estimating the causal risk mitigation effects by combining external data with the specific follow-up data. Intervention effects are provided with Bayesian credible intervals indicating the uncertainty due to all information sources included in the model. Combined prevalence in Finnish pork was estimated to be 1–11% and it could be reduced to 0–2% if head was removed intact and rectum sealed off.