Research in Veterinary Science 94 (2013) 209–213

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Research in Veterinary Science

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Prevalence and genetic diversity of enteropathogenic Yersinia spp. in pigsat farms and slaughter in Lithuania

Aleksandr Novoslavskij a,⇑, Loreta Šerniene a, Alvydas Malakauskas b, Riikka Laukkanen-Ninios c,Hannu Korkeala c, Mindaugas Malakauskas a

a Department of Food Safety and Quality, Faculty of Veterinary Medicine, Veterinary Academy, Lithuanian University of Health Sciences, Tilzes 18, LT-47181, Kaunas, Lithuaniab Department of Infectious Diseases, Faculty of Veterinary Medicine, Veterinary Academy, Lithuanian University of Health Sciences, Tilzes 18, LT-47181, Kaunas, Lithuaniac Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, P.O. Box 66 (Agnes Sjöbergin katu 2), University of Helsinki, FIN-00014, Helsinki, Finland

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 April 2012Accepted 29 September 2012

Keywords:Yersinia enterocoliticaYersinia pseudotuberculosisPrevalenceRisk factorsGenetic diversity

0034-5288/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.rvsc.2012.09.021

⇑ Corresponding author. Tel.: +370 37 362883; fax:E-mail address: aleksandr.novoslavskij@lva.lt (A. N

The prevalence of enteropathogenic Yersinia spp. in pigs at farms and slaughter in relation to potentialfarming risk factors in Lithuania was examined. Pig faeces and carcase swab samples from 11 farms werestudied at slaughterhouses. Nine of the 11 farms were visited again 3–5 months later, and pooled feacalsamples and environmental samples were collected. Pathogenic Yersinia enterocolitica was found in 64%and Yersinia pseudotuberculosis in 45% of the sampled pig farms. All obtained isolates belonged to bioser-otypes 4/O:3 and 2/O:3, respectively. Low biosecurity level was associated with a high prevalence of Y.enterocolitica on farms. Characterization with PFGE of 64 Y. enterocolitica and 27 Y. pseudotuberculosis iso-lates revealed seven and two different genotypes, respectively. Dominant enteropathogenic Yersinia spp.genotypes were obtained in both pig feacal and carcase samples. The high contamination of pig carcases(25%) with enteropathogenic Yersinia spp. may be an important factor contributing to the high incidenceof human yersiniosis in Lithuania.

� 2012 Elsevier Ltd. All rights reserved.

Yersinia enterocolitica and Yersinia pseudotuberculosis are impor-tant foodborne pathogens (Bottone, 1999; Nuorti et al., 2004). Yer-siniosis is one of the three leading foodborne zoonoses inLithuania, and an increase in the number of human cases overthe last decade has been reported (Anon., 2007, 2012). The inci-dence of 12.86 per 100,000 population in Lithuania was the highestamong European Union (EU) member states in 2010 (Anon., 2012).Several studies have linked outbreaks of human yersiniosis to theconsumption of contaminated foods, including pork meat and veg-etables, as well as water (Fredriksson-Ahomaa and Korkeala, 2003;Nuorti et al., 2004). Pigs are of particular importance in Yersiniaspp. epidemiology, as they are the main carriers and source ofhuman enteropathogenic Y. enterocolitica, especially bioserotype4/O:3, and Y. pseudotuberculosis bioserotype 2/O:3 (Bottone,1999; Niskanen et al., 2008). Pig carcases can be contaminatedwith enteropathogenic Yersinia spp. during slaughter, and contam-ination usually occurs from faeces and tonsils (Laukkanen et al.,2008, 2009).

Studies have shown that the prevalence of Y. enterocolitica andY. pseudotuberculosis in pigs varies considerably among farms(Andersen et al., 1991; Letellier et al., 1999; Gürtler et al., 2005;Niskanen et al., 2008). Some studies suggest that Y. enterocolitica

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+370 37 362417.ovoslavskij).

is more common in conventional than organic production (Nowaket al., 2006; Virtanen et al., 2011) and on high-capacity farms thanlow-capacity farms (Laukkanen et al., 2009). However, more recentstudy suggests that no significant difference exists between pro-duction types or production capacities (Laukkanen et al., 2010).The prevalence of Y. pseudotuberculosis, by contrast, was higherin organic production than in conventional production and on con-ventional farms with high rather than low production capacity(Laukkanen et al., 2008). Thus, many factors may affect the preva-lence of Y. enterocolitica and Y. pseudotuberculosis on different pigfarms. Identification of these factors is crucial for establishing ade-quate control measures to minimize the prevalence of enteropath-ogenic Yersinia spp. at both the farm and slaughterhouse levels.Despite the impact of yersiniosis on human health in Lithuania,the prevalence and genetic diversity of enteropathogenic Yersiniaspp. have not been investigated. This study examined the preva-lence of Y. enterocolitica and Y. pseudotuberculosis in pigs on farmsand at slaughter in Lithuania, the genetic variability of enteropath-ogenic Yersinia spp., and potential risk factors contributing to thehigher prevalence of these pathogens in pigs.

Eleven pig farms located in seven of 10 counties in Lithuania,representing intensive pig production regions, were investigatedfor the prevalence of enteropathogenic Yersinia spp. in 2009–2010. Tested farms produce approx. 25% of the total pigs(800,000) grown in Lithuania each year. Fattening pigs aged

210 A. Novoslavskij et al. / Research in Veterinary Science 94 (2013) 209–213

6 months were sampled at slaughterhouses and farms. Pig feacaland carcase swabs were collected from two slaughterhouses atwhich the same slaughtering technique was used. Samplecollection was performed as described in our previous study(Novoslavskij et al., 2010). In total 110 feacal and 55 carcase swabsamples (10 feacal and five carcase swab samples per farm) werecollected from pigs originating from 11 farms. Three to five monthslater nine of these 11 farms were visited for sampling to examineon farm prevalence of Yersinia spp. Thus, 90 pooled feacal samples(10 pooled samples per farm) from fattening pigs and 45 environ-mental samples (five samples per farm) were collected. At eachfarm, fresh feacal samples collected from the floor of five fatteningpigs per pen were pooled together into a sterile plastic bag(5 � 1 g) and suspended in 50 ml of PMB (Phosphate-buffered sal-ine supplemented with 1% mannitol and 0.15% bile salts). Environ-mental samples were collected by swabbing the surface of the floorbetween pens (two samples per farm), the tread surface of boots offarm workers (one sample per farm), and the surfaces of rodenttraps (two samples per farm). Sample collection was performedusing a 7.5 cm sterile gauze square moistened with 10 ml of 0.1%peptone water, which was then transferred into bottles containing90 ml of PMB. All samples were transported at 4–6 �C to the labo-ratory on the day of collection.

Detection of enteropathogenic Yersinia spp. was performedusing the cold enrichment method (21 days at 4 �C) in PMB accord-ing to Korte et al. (2004) and Niskanen et al. (2002) with furtheridentification using the API 20E test (BioMèrieux, Marcy l‘Etoile,France).

Confirmation of Y. enterocolitica and Y. pseudotuberculosis iso-lates was performed with multiplex PCR targeting virF, ail, rfb,16SrRNA and wzz genes according to Thisted Lambertz andDanielsson-Tham (2005), with minor modifications as describedpreviously (Novoslavskij et al., 2010). Y. enterocolitica O:3 DSM13030 and Y. pseudotuberculosis III HH 146–36/84 strains wereused as positive controls. A negative control of sterile water insteadof the DNA template was also prepared.

Biotyping of Y. enterocolitica and Y. pseudotuberculosis isolateswas performed according to Wauters et al. (1987) and Tsubokuraand Aleksic (1995), respectively. Serotyping was done using a slideagglutination test with commercial antisera O:1–O:6 for Y. pseudo-tuberculosis and O:3 for Y. enterocolitica (Denka Seiken, Tokyo,Japan).

In total, 64 Y. enterocolitica and 27 Y. pseudotuberculosis isolateswere characterized by Pulsed-field gel electrophoresis method(PFGE) according to Fredriksson-Ahomaa et al. (1999). One to threeisolates for each Yersinia spp. positive sample were studied. Typingby PFGE was carried out as described by Fredriksson-Ahomaa et al.(1999) and Niskanen et al. (2002). Restriction patterns were ana-lyzed visually and with BioNumerics software version 5.1 (AppliedMaths, Sint Martens-Latem, Belgium).

Associations between enteropathogenic Yersinia spp. and farmfactors were evaluated according to data collected using a ques-tionnaire submitted to veterinarians and the first author’s personalon-farm observations. The questionnaire focused on productiontype, management practices, pest and pet monitoring and control,and farm hygiene (Table 1). Farm biosecurity level was assessedaccording to the presence or lack of disinfection barriers for farmincoming transport, disinfection barriers for worker’s footwearnear the entrance to piggeries and a fence around the farm. Biose-curity was rated high only if all these measures were present.

SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for sta-tistical analyses. A two-stage procedure (univariable and multino-mial logistic regression) was applied to assess the relationshipbetween explanatory variables on the questionnaire and the num-ber of Y. enterocolitica and Y. pseudotuberculosis-positive samplesfound on farms. Correlations between farm factors and the number

of positive Y. enterocolitica and Y. pseudotuberculosis feacal samplescollected on farms were also calculated.

Pathogenic Y. enterocolitica and Y. pseudotuberculosis were foundin 64% and 45% of sampled pig farms, respectively, in Lithuania. Afarm was considered positive when at least one feacal sample waspositive for Y. enterocolitica or Y. pseudotuberculosis. The number ofcontaminated pig pens among different positive farms varied from10% to 70% for Y. enterocolitica and from 10% to 40% for Y. pseudo-tuberculosis (Table 2). Environmental samples from the floor, boots,and rodent traps collected at eight of nine tested farms were neg-ative for enteropathogenic Yersinia spp. One floor swab samplefrom farm B and one from worker’s boots were positive for Y.enterocolitica and Y. pseudotuberculosis, respectively. No seasonalityin shedding enteropathogenic Yersinia spp. in our study was tested.Other studies have shown the farm prevalence of enteropathogenicYersinia spp. in pigs varies and could reach 100% depending on thecountry, and the selected farms and detection methods used (OrtizMartínez et al., 2009, 2011). In this study, the farm prevalence ofenteropathogenic Yersinia spp. in pigs (64%) and the detection rateof Y. enterocolitica-positive (18% and 22%) and Y. pseudotuberculo-sis-positive (10% and 9%) feacal samples collected at slaughter-houses and farms in Lithuania are similar to those reported inneighboring countries such as Latvia and the Leningrad region ofRussia (Ortiz Martínez et al., 2009; Terentjeva and Berzins, 2010).We were unable to detect enteropathogenic Yersinia spp. on 36%of farms (4/11, farms: C, E, G and J). This finding is consistent withother studies that propose that enteropathogenic Yersinia-negativeherds can exist despite high prevalence rates (Pilon et al., 2000;Ortiz Martínez et al., 2009; Terentjeva and Berzins, 2010). Whileenteropathogenic Yersinia spp. are known to be present in pig ton-sils, lymphatic nodes and digestive tract, levels in these sites varywith age. In infected herds, young pigs start to shed in their faecesat 12–14 weeks, with maximal sheding at 19–20 weeks (Gürtleret al., 2005; Nesbakken et al., 2006). At slaughter age (26 weeks)pigs can carry enteropathogenic Yersinia spp. in their tonsils with-out shedding the agent in the faeces (Niskanen et al., 2008;Laukkanen et al., 2009). On this basis and given the age of pigs atthe time of sampling in this study, false-negative detection mayhave occurred.

Samples from pig carcases were collected after the carcaseinspection when tonsils and tongues are already removed. Swabsampling of the area of the removed tonsils was done in additionto sampling of tonsils to enable evaluation of carcase cross-con-tamination. Twenty-five percent of carcases tested in this studywere contaminated with enteropathogenic Yersinia spp. This rateis higher than reported in some other studies (Fukushima et al.,1989; Frederiksson-Ahomaa et al., 2000; Nesbakken et al., 2003;Gürtler et al., 2005). Although, one recent study reported the rateof enteropathogenic Y. enterocolitica-contaminated pig carcaseswas as high as 26% (Laukkanen et al., 2010). The high prevalenceof enteropathogenic Yersinia spp. on pig farms and poor slaughterhygiene resulting in carcase contamination may represent a signif-icant public health risk, particularly in light of the high rates of hu-man yersiniosis in Lithuania.

In total, 63 (virF, ail, rfb, and 16SrRNA-positive) and one (virF-negative; ail, rfb, and 16SrRNA-positive) Y. enterocolitica and 27(virF and wzz-positive) Y. pseudotuberculosis isolates were recov-ered from samples collected at slaughterhouses and pig farms.All Y. enterocolitica isolates were confirmed as bioserotype 4/O:3and all Y. pseudotuberculosis isolates as bioserotype 2/O:3. Theseresults support the finding of other studies indicating that Y.enterocolitica 4/O:3 is the most common bioserotype circulatingin the Baltic region and in continental Europe (Ortiz Martínezet al., 2009, 2011).

From 64 Y. enterocolitica isolates (collected from 54 positivesamples), seven and three PFGE patterns were obtained using NotI

Table 1Explanatory variables included in the analysis of Yersinia spp. prevalence on Lithuanian pig farms.

Definition of variables Variable Sampling at farm level

Yersinia enterocolitica Yersinia pseudotuberculosis

Positive/(%) Negative/(%) Positive/(%) Negative/(%)

Breed Mix 20/(25) 60/(75) 8/(10) 72/(90)Purebredb 0/(0) 10/(100) 0/(0) 10/(100)

Production system All in–all out 16/(20) 64/(80) 4/(5) 76/(95)One-sitec 4/(40) 6/(60) 4/(40) 6/(60)

Farm capacity (pigs/year) Low (<5000) 4/(13) 26/(87) 4/(13) 26/(87)Average (5000–25,000) 5/(17) 25/(83) 3/(10) 27/(90)High (>25,000) 11/(37) 19/(63) 1/(3) 29/(97)

Herd size (No. of fattening pigs) <450 9/(23) 31/(77) 4/(10) 36/(90)>450 11/(22) 39/(78) 4/(8) 46/(92)

Vaccination 2–3 diseases 1/(5) 19/(95) 2/(10) 18/(90)4–6 diseases 19/(27) 51/(73) 6/(9) 64/(91)

Stocking density <0.7 m2/pig 3/(30) 7/(70) 0/(0) 10/(100)>0.7 m2/pig 13/(16) 67/(84) 8/(10) 72/(90)

Pen floor type Concrete 4/(40) 6/(60) 1/(10) 9/(90)Combined (concrete + grating) 16/(20) 64/(80) 7/(9) 73/(91)

Pig cleanliness Low 5/(13) 35/(87) 6/(15) 34/(85)Average 15/(38) 25/(62) 2/(5) 38/(95)High 0/(0) 10/(100) 0/(0) 10/(100)

Slurry accumulation in pens Yes 9/(23) 31/(77) 7/(18) 33/(82)No 11/(22) 39/(88) 1/(2) 49/(98)

Bedding Yes 4/(40) 6/(60) 1/(10) 9/(90)No 16/(20) 64/(80) 7/(9) 73/(91)

Ventilation system Natural + automatic 16/(23) 54/(77) 7/(10) 63/(90)Automatic 4/(20) 16/(80) 1/(5) 19/(95)

Possibility of feces spread between pens Yes 12/(17) 58/(73) 3/(4) 67/(96)No 8/(40) 12/(60) 5/(25) 15/(75)

Wet area around drinkers Yes 13/(65) 57/(35) 8/(11) 62/(89)No 7/(35) 13/(65) 0/(0) 20/(100)

Biosecurity level Low 11/(55)a 9/(45) 4/(20) 16/(80)High 9/(13)a 61/0(87) 4/(6) 66/(94)

Insect level inside farm Low 11/(28) 29/(72) 5/(13) 35/(87)High 9/(18) 41/(82) 3/(6) 47/(94)

Dogs have access to piggery Yes 15/(30) 35/(70) 5/(10) 45/(90)No 5/(13) 35/(87) 3/(8) 37/(92)

Birds have access to piggery Yes 20/(25) 60/(75) 8/(10) 72/(90)No 0/(0) 10/(100) 0/(0) 10/(100)

Rodents inside piggery Yes 15/(38) 25/(62) 5/(10) 45/(90)No 5/(10) 45/(90) 3/(8) 37/(92)

a P = 0.004.b Landrase, Duroc, Yorkshire.c All stage of rearing are under continuous production.

A. Novoslavskij et al. / Research in Veterinary Science 94 (2013) 209–213 211

and XhoI enzymes, respectively. In total, seven different genotypes(YEgI to YEgVII) were identified by combining the various NotI andXhoI digestion patterns. No different Y. enterocolitica genotypeswere found in isolates obtained from the same sample. For Y. pseu-dotuberculosis, two genotypes (YPgI and YPgII) were observed; inthis case NotI and SpeI PFGE digestion patterns were used to differ-entiate isolates. Among the Lithuanian pig isolates, two dominantgenotypes (YEgII and YPgI) were detected in 42% and 89% of theY. enterocolitica and Y. pseudotuberculosis isolates, respectively.Among the carcase swab isolates, there are four examples of iso-lates with genotypes that were not detected in any other isolatefrom that particular farm (YEgV from farm D, YEgI from farm B, YE-gII and YEgIII from farm J). While these isolates may have arisenfrom the respective farms, the possibility exists that these isolatesarise from cross-contamination at the slaughterhouse, furtheremphasizing the importance of slaughterhouse hygiene to themanagement of this zoonotic disease (Fredriksson-Ahomaa et al.,2000; Laukkanen et al., 2010). Interestingly, the prevalence and ge-

netic diversity of Y. enterocolitica 4/O:3 at farm level and in theslaughtering pigs of farm D were different when compared to theother farms. Higher genetic diversity (6 genotypes) was observedamong Y. enterocolitica 4/O:3 isolates (Table 2), and farm D wasthe only farm with Y. pseudotuberculosis on pig carcases indicatinga high prevalence of both enteropathogenic Yersinia spp. on thisfarm. Furthermore, Y. enterocolitica 4/O:3 genotypes YEgVI andYEgVII found in feacal samples collected on this farm were not de-tected in any other tested samples. Such findings indicate a possi-ble limited distribution of some genotypes on pig farms inLithuania, as described previously by the study in Germany andFinland (Fredriksson-Ahomaa et al., 2006b). Correlation betweenPFGE genotypes and sample sources was tested. However, no sta-tistically significant differences were observed.

According to correlation (r = �0.392, P = 0.0001) and logisticregression analysis results (B = �19.86 ± 0.58, P = 0.0001), a lowbiosecurity level (noted for farms H and D) was associated with ahigher prevalence of Y. enterocolitica on farms. However, the low

Table 2Positive Yersinia enterocolitica and Yersinia pseudotuberculosis samples and different genotypes found in pig farms.

Farm Yersinia enterocolitica Yersinia pseudotuberculosis

Slaughterhousea Farmb Slaughterhouse Farm

Fecal Carcass swabs Fecalc Ed Fecal Carcassswabs

Fecal E

No. of positive samples (%)/No. of samples; genotype (No.) No. of positive samples (%)/No. of samples; genotype (No.)

A 7 (70)/10; YEgI (6), YEgII(1)

3 (60)/5; YEgI (3) 4 (40)/10; YEgI (4) 0 (0)/5 0 (0)/10 0 (0)/5 1 (10)/10; YPgI(1)

0 (0)/5

B 0 (0)/10 1 (20)/5; YEgI (2) 1 (10)/10H; YEgII (1) 1 (20)/5e;YEgII (1)

4 (40)/10;YPgI (4)

0 (0)/5 2 (20)/10; YPgI(2)

1 (20)/5f;YPgI (1)

C 0 (0)/10 0 (0)/5 0 (0)/10H 0 (0)/5 0 (0)/10 0 (0)/5 0 (0)/10D 0 (0)/5D 3 (30)/10; YEgII (1),

YEgIII (3), YEgIV (1)5 (100)/5; YEgIII (6),YEgIV (3), YEgV (1)

4 (40)/10; YEgIV (2),YEgVI (1), YEgVII (1)

0 (0)/5 4 (40)/10;YPgI (7)

2 (40)/5;YPgI (3)

4 (40)/10C,E,G,H,I; YPgI(5)

0 (0)/5

E 0 (0)/10 0 (0)/5 0 (0)/10H 0 (0)/5 0 (0)/10 0 (0)/5 0 (0)/10D 0 (0)/5F 1 (10)/10; YEgII (1) 0 (0)/5 4 (40)/10; YEgII (4) 0 (0)/5 2 (20)/10;

YPgII (2)0 (0)/5 1 (10)/10; YPgII

(1)0 (0)/5

G 0 (0)/10 0 (0)/5 0 (0)/10H 0 (0)/5 0 (0)/10 0 (0)/5 0 (0)/10D 0 (0)/5H 5 (50)/10; YEgII (5) 1 (20)/5; YEgII (1) 7 (70)/10B,C,E,G,I; YEgII (7) 0 (0)/5 0 (0)/10 0 (0)/5 0 (0)/10D 0 (0)/5I 3 (30)/10; YEgII (1),

YEgIV (3)2 (40)/5; YEgII (2) 0 (0)/10H 0 (0)/5 1 (10)/10;

YPgI (1)0 (0)/5 0 (0)/10D 0 (0)/5

J 0 (0)/10 2 (40)/5; YEgII (1), YEgIII(1)

NSg NS 0 (0)/10 0 (0)/5 NS NS

K 1 (10)/10; YEgII (1) 0 (0)/5 NS NS 0 (0)/10 0 (0)/5 NS NSTotal 20 (18)/110 14 (25)/55 20 (22)/90 1 (2)/45 11 (10)/110 2 (4)/55 8 (9)/90 1 (2)/45

Y. enterocolitica: differences between farms B, C, E, G, and I and farm H are significant (P = 0.011).Y. pseudotuberculosis: differences between farms C, E, G, H, and I and farm D are significant (P = 0.045).

a Samples collected at slaughterhouses.b Samples collected on farms.c One pooled feacal sample per pig pen; 10 pooled samples per farm.d E – environmental; samples were collected by swabbing the surface of the floor between pigs pens (two samples per farm), the boot surface of farm workers (one sample

per farm), and the surface of rodent traps (two samples per farm).e Floor swab sample positive for Y. enterocolitica.f Swab sample from workers boots positive for Y. pseudotuberculosis.g NS, not studied.

212 A. Novoslavskij et al. / Research in Veterinary Science 94 (2013) 209–213

explanatory value r2 of this factor (�0.392) suggests that otherfarm factors can also affect the higher prevalence of Y. enterocoliticaon farms. The absence of fence around the pig farms increased thepossibility of wild fauna and domestic pet access to the piggery.This factor, together with observed inadequate rodent control,might explain the high prevalence of Y. enterocolitica on farms Hand D, as pathogenic Y. enterocolitica has been isolated fromrodents and pets in previous studies (Aldová et al., 1977;Zheng and Xie, 1996). In addition, a higher prevalence ofY. pseudotuberculosis was also associated with a large number ofpests and pets, which was implicated in the spread of infectionon organic farms in Finland (Laukkanen et al., 2008). Althoughrecent reports suggest that transmission from pigs to rodentsrather than from rodents to pigs is more likely (Wang et al.,2009; Backhans et al., 2010). The higher genetic diversity of Y.enterocolitica isolates found on farm D in comparison to the othersampled farms may also be associated with the low biosecurity le-vel found on this farm. The low number of positive environmentalsamples in our study may indicate pig-to-pig transmission beingmore likely than environmental transmission (Pilon et al., 2000).The detection of the enteropathogenic Yersinia spp. in environmen-tal samples indicates formite-mediated transmission is possibleand suggests a value to the use of simple biosecurity measuressuch as the disinfection of boots.

In conclusion, the high prevalence of pathogenic Yersiniaenterocolitica (64%) and Yersinia pseudotuberculosis (45%) was foundamong sampled pig farms. Y. enterocolitica bioserotype 4/O:3 and Y.pseudotuberculosis bioserotype 2/O:3 were predominant in pigsfrom these farms. The high prevalence of these pathogens on farmsmay have an impact on the highest incidence of human yersiniosisin Lithuania among EU countries because pork is commonly

associated with human cases of yersiniosis (Tauxe et al., 1987;Fredriksson-Ahomaa et al., 2006a).

Acknowledgments

This study was supported by the Lithuanian State Science andStudies Foundation (project No. T-26/09) and by the ResearchCouncil of Lithuania. PFGE typing was done at the Department ofFood Hygiene and Environmental Health, University of Helsinki,Finland.

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