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

Food Microbiology, 2000, 17, 93^101 Available online at http://www.idealibrary.com on

Article No. fmic.1999.0288

ORIGINAL ARTICLE

Prevalence and characterization of pathogenic Yersinia enterocolitica in pig tonsils from di¡erent slaughterhouses M. Fredriksson-Ahomaa*, J. BjÎrkroth, S. Hielm and H. Korkeala

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 61tonsils, 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, including13 yadA-negative isolates from di¡erent samples, were characterized with PFGE. Using NotI 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, de¢ned by combination of the various NotI and XbaI digestion pro¢les, were observed. Two to eight di¡erent 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 identi¢ed in eight out of nine slaughterhouses and the pulsotype 4g in # 2000 Academic Press seven slaughterhouses.

Introduction The epidemiology of Yersinia enterocolitica is obscure. It is considered to be a foodborne pathogen, even though pathogenic isolates have very seldom been recovered from suspect food sources (de Boer 1995). Healthy pigs have been found to carry human pathogenicY. enter*Corresponding author. Institute of Hygiene and Technology of Food of Animal Origin, VeterinÌrstr. 13, 80539 Munich, Germany. Fax: +49 -89 -2180 3872. E-mail: [email protected] 0740 -0020/00/010093 + 09 $35.00/0

ocolitica in the tonsils and oral cavity (Nesbakken 1985). In humans, Y. enterocolitica causes mainly gastroenteritis. Only a few bioserotypes are known to associate with human disease (Kapperud 1991). A prerequisite for expression of pathogenicity is a 70^75 kb virulence plasmid (pYV), which is present in all pathogenic Yersinia (Portnoy and Martinez 1985).The yadA gene, located on the virulence plasmid of all pathogenic serotypes, encodes for protein YadA, which is very important in the pathogenesis of Y. enterocolitica (Skurnik and WolfWatz 1989). # 2000 Academic Press

Received: 24 November1998 Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland

94 M. Fredriksson-Ahomaa et al.

Approximately 750 Y. enterocolitica cases (15 cases/100 000 inhabitants) in humans were reported in Finland in the year 1996 (Anonymous 1997).The most common serotype isolated from stool samples was O:3 (Kontiainen et al. 1994). The prevalence of Y. enterocolitica bioserotype 4/O:3 in pigs has been reported to be 17% in Finland overall (Asplund et al. 1988) and 36% in Southwest Finland (Asplund et al. 1990) utilizing a conventional culture method. The actual prevalence of Y. enterocolitica may well be higher because conventional culture methods may underestimate the occurrence of pathogenic strains (Nesbakken et al. 1991). Asplund et al. (1988) isolatedY. enterocolitica bioserotype 4/O:3 in Finland in only three out of seven pig slaughterhouses. Very little is yet known about geographical distribution and genetic diversity of pathogenicY. enterocolitica of animal origin. There is a need for subtyping bioserotypes of pathogenic isolates to get more information on epidemiology. Genotypic typing methods have greatly increased the ability to discriminate between micro-organisms and have become very common and powerful tools in the area of molecular epidemiology (Farber 1996). Several molecular typing methods have been applied to Y. enterocolitica (Andersen and Saunders 1990, Kapperud et al. 1990), of which pulsed¢eld gel electrophoresis (PFGE) is one of the most powerful tools available (Iteman et al. 1996). This technique seems to be very e¤cient for subtyping pathogenic isolates of Y. enterocolitica (Asplund et al. 1998; Buchrieser et al. 1994, Najdenski et al. 1994) and it might be a powerful epidemilogical tool for bioserotype 4/O:3.

The aim of this study was to examine the prevalence of pathogenicY. enterocolitica in pig tonsils obtained from slaughterhouses located in di¡erent parts of Finland, using both the PCR method targeting the yadA gene and conventional method. We also wanted to characterize the isolates with PFGE to study the genetic variation of the isolates obtained and to examine the value of PFGE in epidemiological studies, using NotI and XbaI enzymes.

Materials and Methods Samples and sample preparation Tonsils of 185 pigs (Finnish Landrace^Yorkshire crosses) were sampled from nine slaughterhouses located in various parts of Finland during May and June. One sample was taken daily during 5 weeks in each slaughterhouse to avoid cross-contamination and to ensure sampling from di¡erent herds.The tonsils were cut out immediately after evisceration. A 10 gram tonsil sample was homogenised in 90 ml TSB (trypticase soya broth, Difco, Detroit, Michigan, USA) in a stomacher blender for 1 min. The homogenates were enriched at 228C for 16^18 h and, following overnight enrichment, studied with both the PCR and culture method (Fredriksson-Ahomaa et al. 1999).

Detection of yadA-positiveY. enterocolitica in pig tonsils using PCR The modi¢ed method of Kapperud et al. (1993) was used to detect pathogenic Y. enterocolitica

Table 1. The primers and the cycle pro¢les used in the nested PCR (Kapperud et al. 1993) Step 1

2

Primer

Sequence (5'^3')

The cycle pro¢le

1

TAA-GAT-CAG-TGT-CTC-TGC-GGC-A

2

TAG-TTA-TTT-GCG-ATC-CCT-AGC-AC

Initial denaturation at 958C for 3 min followed by 40 cycles of denaturation at 958C for 30 s, annealing at 588C for 60 s, extension at 728C for 90 s.

3

GCG-TTG-TTC-TCA-TCT-CCA-TAT-GC

4

GGC-TTT-CAT-GAC-CAA-TGG-ATA-CAC

Initial denaturation at 958C for 3 min followed by 20 cycles of denaturation at 958C for 30 s, annealing at 628C for 60 s, extension at 728C for 90 s.

PathogenicY. enterocolitica in pig tonsils 95

with nested PCR. A hundred microlitres from the enriched sample was centrifuged at 16 000 g for 10 min.The pellets were resuspended in 50 ml of 16PCR bu¡er (Finnzymes, Espoo, Finland) containing 0?2 mg of proteinase K (Finnzymes) ml71. After incubation at 378C for 1 h the suspension was boiled for 10 min and then centrifuged at 16 000 g for 5 min. Twenty ¢ve microlitres of the supernatant was used as a template in the ¢rst PCR step and 2 ml of the ¢rst PCR product was used as a template in the second PCR step. Two sets of Pharmacia Biotech (Vantaa, Finland) oligonucleotide primers (Kapperud et al. 1993), based on the nucleotide sequences of the yadA gene, were used for PCR ampli¢cation performed in a 16 well PTC-150 thermal cycler (MJ Research, Watertown, Massachusetts, USA) (Table 1). The sample volume was 50 ml and contained 1 U of Dynazyme DNA-polymerase (Finnzymes), 200 mM of each dNTP and 0?1 mM of each primer. The size of the ampli¢ed second PCR product (about 530 bp) was determined in 0?7% agarose gel by comparison to DNA molecular weight marker VI (Boehringer Mannheim, Mannheim, Germany).

Isolation and identi¢cation of yadA-positive Y. enterocolitica with the culture method Yersinia enterocolitica was isolated with the culture method, which included overnight enrichment, selective enrichment and cold enrichment. Every sample was initially enriched in TSB at 228C for 16^18 h and then further enriched at 48C for 4 days, after which 100 ml of this enrichment was inoculated into 10 ml of selective enrichment broth (MRB) incubated at 25 8C for 4 days. The TSB-homogenates were stored at 48C for 21 days for cold enrichment. Subculture on selective CIN agar plates (Yersinia Selective Agar Base, Oxoid, Basingstone, UK) was done after every enrichment step according to the method of the Nordic Committee on Food Analysis (1996).One to four suspect colonies of typical `bull's eye' appearance on the CIN agar plates were streaked onto blood agar plates to create a pure culture. One colony from a blood agar was inoculated onto a urea agar slant (Difco) and incubated for 1 day at 308C. Isolates showing urea hydrolysis were

further identi¢ed using the API 20E system (BioMe¨rieux, Marcy l'Etoile, France) and incubated at 258C for 18^20 h. Yersinia enterocolitica isolates were biotyped according to the revised scheme by Wauters et al. (1987) and serotyped by slide agglutination using O:3, O:5 and O:9 antisera (Denka Seiken, Tokyo, Japan). The yadA gene was detected in the pure culture using PCR (Kapperud et al. 1993). Four small (52 mm) colonies were boiled in 100 ml of water for 10 min and 2 ml of this boiled bacteria suspension was used as a template in the PCR.

DNA isolation, restriction enzyme digestions and PFGE A single colony grown on blood agar was inoculated into 5 ml TSB. After overnight enrichment at room temperature, DNA isolation was performed according to Maslow et al. (1993), with the following modi¢cations. The cell suspensions were mixed with an equal volume of 2% (wt/vol) low-melting-temperature agarose (InCert agarose; FMC Bioproducts, Rockland, Maine, USA). Instead of insert moulds, GelSyringe dispensers (New England Biolabs, Beverly, Massachusetts, USA) were used as speci¢ed by the manufacturer. Phenylmethylsulfonyl £uoride inactivation of proteinase K and restriction endonuclease digestion of the agarose embedded DNA were performed as described by New England Biolabs (1990). The DNA was digested with two rare-cutting enzymes, NotI and XbaI (New England Biolabs). The samples were electrophoresed at 128C through a 1% agarose gel in 0?56TBE (Amresco, Solon, Ohio, USA) at 200 V in a Gene Navigator system (Pharmacia, Uppsala, Sweden) with a hexagonal electrode. The pulse times were ramped from 1 to 18 s for 20 h, and from 1 to 15 s for 18 h, for NotI and XbaI, respectively. Lamda Ladder and Mid-Range I PFG markers were used as fragment size markers (New England Biolabs). The gels were stained with ethidium bromide and photographed using standard procedures (Sambrook et al. 1989).

Results Yersinia enterocolitica harbouring the yadA gene was detected in 68 (37%) out of 185 pig

96 M. Fredriksson-Ahomaa et al.

tonsils when both PCR and culture-positive results were included (Table 2). YadA-positive Y. enterocolitica could be detected in pig tonsils from all nine slaughterhouses, with prevalence varying from 13% to 45% between the slaughterhouses. Table 2 shows that more positive results were obtained using PCR. Of the 52 PCR-positive tonsil samples, 20 were culturenegative, while of the 48 culture-positive sample, 16 were PCR-negative. Yersinia enterocolitica belonging to the bioserotype 4/O:3 was the only pathogenic type found in this study. Bioserotype 4/O:3 could be isolated from 61 (33%) tonsils of which 48 (26%) were yadA-positive. Most of yadA-positive samples (41/48) were recovered directly following overnight enrichment in TSB, with some new positive samples obtained after selective and cold enrichments (Table 3). All the 61 isolates belonging to bioserotype 4/O:3, including 13 yadA-negative ones, were

characterised using PFGE. The 61 isolates yielded 20 and 17 PFGE patterns, with NotI (Fig. 1) and XbaI (Fig. 2), respectively. N1 (44%) and N4 (21%) were the most common PFGE patterns with NotI, and Xa (38%) and Xg (31%) with XbaI (Table 4). Up to nine band di¡erences were found with NotI, but only minor deviations in one to three fragments with XbaI were observed. A total of 26 di¡erent pulsotypes, de¢ned by combination of the various NotI and XbaI digestion pro¢les, were obtained (Table 4). The two most common pulsotypes were 1a (36%) and 4g (20%) (Table 4). Table 5 shows that 26 di¡erent pulsotypes were obtained in the slaughterhouses.The most common puslotypes, 1a and 4g, were found in most of the slaughterhouses. Pulsotype 1a was observed in eight out of nine slaughterhouses and type 4g, in seven slaughterhouses. Two to eight di¡erent pulsotypes were found in each slaughterhouse.

Table 2. Prevalence of yadA-positive Y. enterocolitica in 185 Finnish pig tonsils from nine di¡erent slaughterhouses Slaughterhouse

Number of samples

PCR positivea

Culture positiveb

PCR or culture positive

Prevalencec %

A B C D E F G H I Total

20 20 21 20 20 23 20 19 22 185

7 3 5 8 4 3 7 8 7 52

8 5 7 4 7 3 2 6 6 48

9 7 7 8 9 3 7 8 10 68

45 35 33 40 45 13 35 42 45 37

a

Number of yadA-positive tonsils detected with PCR. Number of yadA-positive isolates con¢rmed with PCR in tonsils with the culture method. c Number of yadA-positive tonsils when both PCR and culture positive results are included. b

Table 3. Number of pig tonsils contaminated with Yersinia enterocolitica bioserotype 4/O:3 using the culture method

Number of positive samples after: Bioserotype 4/O:3

Number of pig tonsils

Overnight enrichment

Selective enrichment

Cold enrichment

yadA-positive yadA-negative Total

48 13 61

41 7 48

‡3a ‡3 ‡6

‡4 ‡3 ‡7

a

New positive samples in addition to previous enrichment step.

PathogenicY. enterocolitica in pig tonsils 97

Figure 1. NotI pro¢les of 20 isolates of Yersinia enterocolitica bioserotype 4/O:3 from pig tonsils.

Discussion

Figure 2. XbaI pro¢les of 17 isolates of Yersinia enterocolitica bioserotype 4/O:3 from pig tonsils.

A fragment of about 48 kb with NotI and 50 kb with XbaI was observed in PFGE patterns of all yadA-positive isolates, but not in PEGE patterns of yadA-negative isolates.

Yersinia enterocolitica carrying the yadA gene was found to be common in Finnish pig tonsils. Pathogenic Y. enterocolitica was detected from all nine slaughterhouses, but the prevalences varied signi¢cantly between the slaughterhouses, which may be due to the di¡erent prevalences of infected herds in di¡erent areas. It is not known how many herds are infected with pathogenic Y. enterocolitica in Finland. In Norway, Nesbakken and Skjerve (1996) revealed with serological investigations that 63?5% of herds were infected with Y. enterocolitica O:3, and that there were signi¢cantly fewer positive combined herds of farrowing and fattening units than herds of only fattening units. Risk of transmittingY. enterocolitica into a fattening herd is high when pigs are collected from many farrowing herds (Nesbakken and Skjerve 1996). All tonsil samples were taken on di¡erent days to avoid cross-contamination during removal of tonsils and to get samples from di¡erent herds because the prevalence is high among infected herds and pigs are slaughtered penwise, not randomly (Christensen 1980, Andersen et al. 1991, Nielsen and Wegener 1997). The tonsil samples were homogenised because

98 M. Fredriksson-Ahomaa et al.

Table 4. Di¡erent pulsotypes obtained with

combination of NotI and XbaI digestion pro¢les of 61 Yersinia enterocolitica bioserotype 4/O:3 isolates from di¡erent pig tonsils PFGE pattern Pulsotype

No, of isolates

NotI

XbaI

1a 1b 1c 1d 1e 2f 3a 4g 4n 5h 6g 7i 8k 9g 10l 11m 12j 13g 14n 14o 15n 16g 17g 18p 19q 20n

22 1 1 2 1 1 1 12 1 1 2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1

N1 N1 N1 N1 N1 N2 N3 N4 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N14 N15 N16 N17 N18 N19 N20

Xa Xb Xc Xd Xe Xf Xa Xg Xn Xh Xg Xi Xk Xg Xl Xm Xj Xg Xn Xo Xn Xg Xg Xp Xq Xn

Nesbakken (1985) found that more positive samples were obtained when the tonsils were homogenised compared to swabbing. The primary advantage of the swabbing method is its convenience in studying a large number of samples, but it is less sensitive (Nesbakken et al. 1985, Andersen et al. 1991, Funk et al. 1998). PathogenicY. enterocolitica carrying the yadA gene was detected with both the PCR and culture method in this study.The PCR method presented by Kapperud et al. (1993) was chosen because it was developed for natural samples and there is no need for DNA isolation before PCR.The method is targeting the yadA gene located on the virulence plasmid, which is essential for the full pathogenicity of Y. enterocolitica. Due to the instability of the virulence plasmid at 378C, the samples were not exposed to

Table 5. Di¡erent pulsotypes of 61 Yersinia enterocolitica bioserotype 4/O:3 isolates from pig tonsils in nine slaughterhouses Slaughterhouse

No. of isolates

Pulsotype

No. of isolates

A

10

1a 1d 4g 5h 13g 15n 16g

2 2 1 1 1 1 2

B

6

1a 1e 17g 18p

3 1 1 1

C

9

1a 2f 3a 4g 6g 7i 8k 9g

2 1 1 1 1 1 1 1

D

6

4g 10l 11m 12j 14n

2 1 1 1 1

E

8

1a 1c 4g 6g 19q

3 1 2 1 1

F

4

G

4

1a 1b 4g 1a 14o

1 1 2 3 1

H

7

1a 4g 4n 20n

2 3 1 1

I

7

1a 4g

6 1

temperatures above 308C to avoid the possibility of losing the plasmid.To increase the sensitivity and to restrict the detection to culturable organisms, only enriched samples were used in this study. The prevalence was only slightly higher with PCR (28%) compared to the culture method (26%), which was unexpected

PathogenicY. enterocolitica in pig tonsils 99

since when we studied the minced meat samples with the same PCR and culture methods, we found signi¢cantly more PCR-positive samples compared to culture-positive ones (Fredriksson-Ahomaa et al. 1999). In this study, 16 samples were PCR-negative but culture-positive. One reason for these false negative results may be the high contamination level of pathogenic Y. enterocolitica in the pig tonsils, which inhibits PCR reaction, and another reason is the presence of inhibitory factors, which are always a problem when natural samples are studied (Rossen et al. 1992). No easy method has yet been devised for overcoming this problem (Olsen et al. 1997). The isolation rate of bioserotype 4/O:3 was comparable to many other studies from di¡erent countries (Andersen et al. 1991, de Boer and Nouws 1991, De Giusti et al. 1995, Hariharan et al. 1995, Borie et al. 1997). The culture method used in this study included three enrichment steps: overnight, selective and cold enrichment. It was surprising that most (41/48) of the yadA-positive samples were already recovered after overnight enrichment because when we studied pig tongues and minced meat samples with the same culture method, most of pathogenic isolates were found after selective enrichment (Fredriksson-Ahomaa et al. 1999). A possible explanation for the high recovery rate after overnight enrichment might be the high number of pathogenic Y. enterocolitica strains colonised in pig tonsils (Shiozawa et al. 1991) and/or low background £ora on the CIN agar plates. Bioserotype 4/O:3 was the only pathogenic type found in this study. It is the most common pathogenic bioserotype isolated in man in most countries, including Finland (Kapperud 1991, Anonymous 1997). However, all isolates belonging to type 4/O:3 did not carry the yadA gene and could only be considered as potential pathogens. PCR was used to con¢rm the isolates carrying the yadA gene as, at present, no isolation procedure exist, which can clearly di¡erentiate pathogenic from nonpathogenic variants. Using PFGE, we were able to con¢rm that all yadA-positive isolates did carry the virulence plasmid, which was expected since yadA is located on the pYV. PFGE was e¤cient in subtyping isolates belonging to bioserotype 4/O:3 originating from

pig tonsils. In this study, NotI and XbaI, were used because they have given good resolution for all Y. enterocolitica serotypes tested (Iteman et al. 1991, Buchreiser et al. 1994, Najdenski et al 1994, Saken et al. 1994). However, a large number of restriction fragments and too many fragments of the same size range made it di¤cult to interpret these pro¢les. Despite this, 20 and 17 PFGE pro¢les were observed with NotI (Fig. 1) and XbaI (Fig. 2), respectively. Although the pro¢les were not identical, most of them displayed only minor deviations. Genetic variation of Y. enterocolitica bioserotype 4/O:3 of pig tonsils appears to be quite limited in Finland. In each slaughterhouse, di¡erent pulsotypes, obtained by combining di¡erent NotI and XbaI pro¢les, were recognized and no predominant pulsotype was found in most of the slaughterhouses (Table 5). However, two common pulsotypes, 1a and 4g, were obtained and these pulsotypes were widely distributed to most of the slaughterhouses. Isolates of the same pulsotype recovered from di¡erent slaughterhouses may have derived from a common source. On the other hand, unrelated isolates may have indistinguishable genotypes if the genetic diversity is restricted within the bioserotype (Tenover et al. 1995). Using additional enzymes, these unrelated isolates may also be distinguished. If the most common pulsotypes cannot be further subdivided with other enzymes, the value of PFGE would be limited in epidemiological studies.

Acknowledgements We wish to thank Sirkku EkstrÎm for technical assistance. This work was supported by the Technology Development Centre (TEKES), Finland.

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