Isolation of Campylobacter spp. from a pig slaughterhouse and analysis of cross-contamination

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1 International Journal of Food Microbiology 108 (2006) Isolation of Campylobacter spp. from a pig slaughterhouse and analysis of cross-contamination M. Malakauskas a,b,, K. Jorgensen b, E.M. Nielsen c,1, B. Ojeniyi b, J.E. Olsen b a Department of Food Safety and Animal Hygiene, Lithuanian Veterinary Academy, Tilžeṡ str. 18, LT-47181, Kaunas, Lithuania b Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark c Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen, Denmark Received 23 June 2004; received in revised form 23 October 2004; accepted 26 September 2005 Abstract The purpose of this study was to establish the prevalence and possible contamination routes of Campylobacter spp. in a pig slaughterhouse. Swab samples were taken from the last part of rectum, from the carcasses surface before meat inspection and from slaughter line surface from 4 different pig herds during slaughtering. Identification of Campylobacter isolates was determined by the use of phase-contrast microscopy, hippurate hydrolysis, indoxyl acetate hydrolysis tests and PCR based restriction fragment length polymorphism method (PCR-RFLP). Pulsed-field gel electrophoresis (PFGE) typing using two macro-restriction enzymes SmaI and SalI was applied to in-slaughterhouse contamination analysis of pig carcasses. The study showed that 28 (63.6%) of the 44 samples collected at slaughterhouse were contaminated by Campylobacter spp. Up to 5 different colonies were obtained from each swab sample and a total of 120 different isolates were collected. 23.4% (28 of 120) isolates were identified as C. jejuni (19 from carcasses and 9 from slaughter line surfaces) and 76.6% (92 of 120) isolates as C. coli (28 from faeces, 47 from carcasses and 17 from slaughter line surfaces). The typing results showed identity between isolates from successive flocks, different carcasses, and places in the slaughterhouse in contact with carcasses. The results suggest that cross-contamination originated in the gastro-intestinal tract of the slaughtered pigs and that cross-contamination happened during the slaughter process Elsevier B.V. All rights reserved. Keywords: Campylobacter jejuni; Campylobacter coli; Pigs; PCR-RFLP; PFGE; Cross-contamination 1. Introduction Campylobacter spp. has been recognized as the most common cause of the bacterial gastroenteritis in developed countries. In Denmark, the number of cases has more than quadrupled during the last 10-year period to a level of 82 registered cases of Campylobacter infections per inhabitants in 2002 (Anonymous, 2002). The same trend is seen in other industrialized countries (Anonymous, 2000). C. jejuni is responsible for 80 90% of human infections, while Corresponding author. Department of Food Safety and Animal Hygiene, Lithuanian Veterinary Academy, Tilžeṡ str. 18, LT-47181, Kaunas, Lithuania. Tel./fax: address: mindaugas@lva.lt (M. Malakauskas). 1 Present address: Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. C. coli is responsible for only about 7% and C. lari, C. hyointestinalis and C. upsaliensis for only about 1% of human cases (Nesbakken et al., 2002). Campylobacters colonize the gastro-intestinal tracts of a wide range of domestic and wild animals, especially animals raised for human consumption (Nielsen et al., 1997). Literature shows that Campylobacter spp. can be isolated from pig faeces at a wide range of frequencies up to 100% (Kwiatek et al., 1990; Meng and Doyle, 1998; Fermer and Engvall, 1999; Madden et al., 2000; Young et al., 2000; Nesbakken et al., 2002). Pigs carry higher proportion of C. coli than C. jejuni, whether they have enteritis or not (Harvey et al., 1999; Steinhauserova et al., 2001b). In comparison pig carcasses are not so frequently contaminated with campylobacters and reported range of contamination of pig carcasses varies from 2.9% to 10.3% (Oosterom et al., 1985; Pezzotti et al., 2003) /$ - see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.ijfoodmicro

2 296 M. Malakauskas et al. / International Journal of Food Microbiology 108 (2006) Since pigs are often carriers of Campylobacter, the slaughter process may be a source of cross-contamination, possibly leading to increased consumer risk. This study was set up to establish the prevalence and possible contamination routes of Campylobacter spp. in a pig slaughterhouse. Sampling of multiple colonies from each animal was applied to circumvent the problem of multi-clone infection, reported to be frequent (Weijtens et al., 1999) and PFGE typing was applied to in-slaughterhouse contamination analysis of pig carcasses. 2. Materials and methods 2.1. Design of the study and sample collection Samples were collected from one pig slaughterhouse in Denmark during one day. The sampling scheme was designed to trace groups of pigs from four herds identified by the herd number during slaughtering. Emphasis was put on identification and characterization of several isolates from each sample, rather than many samples per herd. Before slaughtering, six swab samples were taken to examine cleanness of the slaughter line after usual cleaning and disinfection from dehairer, slaughter line and tray-surface where intestines are placed after evisceration. During slaughtering, rectal swab samples were taken from gastrointestinal track of three randomly selected pigs from each herd immediately after evisceration and six samples from carcasses after splitting. Carcasses were swabbed over an area of approximately 100 cm 2, with particular attention to head, hind leg, tail and section line areas before meat inspection. At the same time, two approximately 100 cm 2 environmental samples were collected along the slaughter line, just before slaughtering of the pigs from next herd, from the chamber surface which collects drips from pigs' plugs and tray-surface where intestines are placed after evisceration. The same sampling sites were used for the sampling of all four pig herds. Sterile moistened cotton swabs were used to collect all samples. Direct streaking of swabs onto plates and enrichment of swab-content in broth were applied. The latter was included to increase recovery of Campylobacter spp. present in low numbers and especially C. jejuni which (if present) comprises a minor part of the initial Campylobacter population but has a selective advantage during enrichment (Madden et al., 2000) Detection of Campylobacter spp. Immediately after swabbing plates with modified Charcoal Cefoperazone Deoxycholate (mccda) agar (Oxoid, CM 739, Hampshire, England) with selective supplement (Oxoid, SR155E, Hampshire, England) and 0.2% Teepol 610 (Shell Chemicals, Amsterdam, the Netherlands), supplement was directly inoculated with swabs. Before use plates were dried at 37 C temperature for 10 h to get better isolated colonies. Inoculated plates were placed in a jar under microaerophilic conditions (85% nitrogen, 10% carbon dioxide, and 5% oxygen) generated by Oxoid CampyGen sachets (Oxoid, CN35, Hampshire, England) at 37 C for 48 h. Additionally the same swabs were placed in individual tubes with Mueller Hinton (MHB) broth (Oxoid, CM405, Hampshire, England) and the tubes were incubated at 37 C under microaerophilic conditions for 48 h. After incubation five presumptive Campylobacter colonies from each mccda plate were chosen after being tested by use of phase-contrast microscopy (characteristic morphology and motility) and transferred on Blood Agar Base No. 2 (Oxoid, CM0271, Hampshire, England) plates with 7% blood for purifications 2 times at 37 C under microaerophilic conditions for 48 h. After that bacterial cultures were stored at 70 C in special broth (Nutrient Broth No 2 (Oxoid,CM67, Hampshire, England), Agar No. 1 (Oxoid, L11, Hampshire, England) with 10% dimethyl sulphoxide supplement) until further investigation. If there was no growth of Campylobacter spp. on mccda plates after direct streaking after 48 h, additional plates were inoculated from the enrichment in MHB. After 48 h, enrichment samples were plated onto mccda with 10 μl loop and isolation was carried out as described above Differentiation of thermophilic Campylobacter spp. All isolates were examined by the use of phase-contrast microscopy (characteristic morphology and motility), hippurate hydrolysis test and indoxyl acetate hydrolysis. For hippurate hydrolysis test, C. jejuni NCTC was used as positive control, C. coli NCTC as negative control and C. jejuni NCTC as week positive control. Specification of isolates was also determined by PCR- RFLP. For bacterial DNA extraction, colonies identified as Campylobacter spp. were suspended in 500 μl 5% Chelex solution (BioRad, Hercules, USA) and mixed carefully, incubated at 56 C for 30 min, shaken and boiled for 10 min. After that tubes were shaken for 5 10 s and centrifuged 2 3 min at rpm. Supernatants were carefully transferred into new tubes and centrifuged again. After that they were carefully transferred to new tubes and stored at 20 C before use. A 491 bp of a highly polymorphic part of the 23S rrna gene was amplified using primer pair, THERM1 (5 -TATTCCAA- TACCAACATTAGT-3 ) and THERM4 (5 -CTTCGCTAA- TGCTAACCC-3 ) and species differentiation was achieved by digestion of the PCR amplicon with restriction enzymes, AluI (Amersham Pharmacia Biotech, E1004Z, China) and Tsp5091 (New England BioLabs Inc., R0576S, Beverly, MA), resulting in specific restriction fragments for thermophilic C. jejuni, C. coli, C. lari and C. upsaliensis, respectively (Fermer and Engvall, 1999) Subtyping of Campylobacter isolates by PFGE Subtyping of obtained isolates was done using two macrorestriction enzymes SmaI (Amersham Pharmacia Biotech,

3 M. Malakauskas et al. / International Journal of Food Microbiology 108 (2006) E1085W, China) and SalI (New England BioLabs Inc., B0138S, Beverly, MA) as described by Ribot et al. (2001) with minor changes as follows. Adjustment of the concentration of cell suspension was made using standard McFarland tube No. 2. Lambda Ladder PFG Marker (New England BioLabs Inc., N0340S, Beverly, MA) was used as the molecular size marker. For the macro-restriction with SalI electrophoresis was carried out for 21.5 h at 220 V and 14 C constant temperature in a CHEF-DRIII system (BioRad, Hercules, USA) with pulse time ramped from 4 to 50 s Computer analysis of PFGE patterns After electrophoresis, the PFGE patterns were analysed using GelCompare v4.1 software (Applied Maths, Belgium). The saved gel images were converted to.tif files and normalized by aligning the peaks of the size molecular size marker (Lambda Ladder PFG Marker N0340S, New England BioLabs Inc., US), which was loaded on four lines in each gel. Matching and dendogram UPGMA (unweighted pair group method with averages) analysis of the PFGE patterns was performed using the Dice coefficient with a 2% tolerance window Serotyping C. jejuni isolates representing all groups clustered according to PFGE profiles were serotyped according to the Penner scheme for heat-stable antigens (Penner and Hennessey, 1980) by the use of passive haemagglutination using three dilutions of antisera (1 in 80, 1 in 640, 1 in 5120) and the full set of antisera as described previously (Nielsen et al., 1997). 3. Results 3.1. Isolation of Campylobacter spp. from collected samples The study showed that 7 out of 12 (58%) of faecal samples, 15 out of 20 (62%) of carcass samples and 6 out of 8 (75%) of samples from slaughter line surfaces were culture positive for Campylobacter spp. Up to 5 different colonies were obtained from each swab sample and a total of 120 different isolates were collected and used for analysis of cross-contamination (Table 1). All samples taken before slaughtering were negative Species distribution of isolates According to the PCR-RFLP results, 28 of 120 isolates (23.4%) were identified as C. jejuni (19 from carcasses and 9 from slaughter line surfaces) and 92 isolates (76.6%) as C. coli (28 from faeces, 47 from carcasses and 17 from slaughter line surfaces). Only samples taken in connection with the slaughter of herds B and C showed positive samples for C. jenuni, and with herd C, only environmental samples were positive. No C. jejuni isolates were obtained from pig faeces samples. All 28 C. jejuni isolates showed positive hippurate hydrolysis test, whereas the 92 C. coli isolates showed negative reaction. Table 1 Isolation of Campylobacter spp. from faeces and carcasses from four different pigs' herds slaughtered on the same day and from the slaughterhouse environment Samples from Faeces (N=3 samples) Carcass (N=6 samples) A Positive swab samples Number of isolates a B Positive swab samples Number of isolates C Positive swab samples Number of isolates D Positive swab samples Number of isolates Total number of isolates a Up to five different isolates were obtained per sample Cross-contamination demonstrated by PFGE Slaughter line (N=2 samples) The 120 isolates generated 31 different PFGE patterns after restriction with SmaI and 27 different patterns when restricted with SalI, respectively. Some of the patterns were displayed by only one isolate while others were presented by up to 12 isolates when restriction was done with SmaI and up to 17 isolates when restriction was performed with SalI. From one faecal sample five isolates displayed four different SmaI and four SalI patterns. Isolates from two samples taken from faeces and slaughterhouse environment generated three different SmaI and three and two SalI patterns, respectively. Isolates obtained using direct plating generated more different PFGE profiles in comparison with isolates obtained after enrichment in MHB (data not shown). Analysis of the obtained PFGE profiles showed a high degree of genetic diversity among isolates. Fig. 1 shows a cluster analysis of the 31 SmaI distinct profiles generated by PFGE analysis. Results generated by SalI digestion supported the results obtained with SmaI and are not shown. Isolates clustered into two major groups, as C. jejuni clustered separately from C. coli, and within both of these we found evidence of crosscontamination or conditions that could lead to crosscontamination during slaugher. The SmaI type four of C. jejuni was demonstrated from two different carcasses (No. I and II) of herd B, from the swab No. 27 taken from the surface which had contact with pig intestines during slaughtering of the pigs of herd B and from the swab sample No. 39 taken from the chamber surface when pigs from herd C were slaughtered. In addition, SmaI type five of C. jejuni was obtained from the samples taken from the mentioned No. II pig carcass and from the swab No. 27 mentioned before. The SmaI type three of C. jejuni was isolated from two other different pig carcasses (III and IV) of the heard B pigs. SmaI type two and SmaI type one were isolated from pig carcasses No. IV and No. V, respectively. The SmaI type twenty three of C. coli was obtained from three different carcasses (No. I, II and VI) of herd D. Two different SmaI types of C. coli (8 and 25, respectively) were found from the swab No. 28 taken

4 298 M. Malakauskas et al. / International Journal of Food Microbiology 108 (2006) Discussion C. coli C. jejuni from the chamber surface which collects drips from pigs' plugs of heard B Serotype distribution Carcass (31) Faeces (30) Carcass (29) Faeces (28) Faeces (27) Chamber (26) Faeces (25) Carcass (24) Carcass (23) Pad (22) Carcass (21) Faeces (19,20) Faeces (18) Faeces (16,17) Faeces (14,15) Pad (13) Faeces (12) Faces (11) Faeces (9) Carcass (10) Chamber (8) Carcass (7) Carcass (6) Carcass (5) Pad (5) Chamber (4) Pad (4) Carcass (4) Carcass, Chamber (3) Carcass (2) Carcass (1) Chamber (1) Fig. 1. Dendrogram generated from the similarity matrix determined from the different SmaI banding patterns of 120 Campylobacter isolates obtained from pig carcasses, pig faeces and slaughterhouse environment. Matching and dendogram UPGMA (unweighted pair group method with averages) analysis of the PFGE patterns was performed using the Dice coefficient with a 2% tolerance window in GelCompare (numbers in parentheses indicate different PFGE types). Seventeen C. jejuni isolates representing all PFGE profiles (SmaI and SalI) were serotyped and only two different serotypes were identified: 35 and 23, 36. The present study showed that 63.6% of swab samples collected at a pig slaughterhouse were contaminated with Campylobacter spp. These results are in agreement with other studies which suggest that Campylobacter spp. can be isolated from 0% to 100% of the pig intestinal tract and up to 10.3% of pig carcasses depending on the farm and the sampling technique (Kwiatek et al., 1990; Meng and Doyle, 1998; Harvey et al., 1999; Madden et al., 2000; Nesbakken et al., 2002; Pezzotti et al., 2003). In recent years species identification of Campylobacter has been facilitated by several PCR-RFLP and multiplex PCR techniques (Fermer and Engvall, 1999; Steinhauserova et al., 2001a; Cloak and Fratamico, 2002; Moore et al., 2002; Moreno et al., 2002; Jauk et al., 2003), and in the present investigation, the PCR-RFLP test of Fermer and Engvall (1999) identified 23% of the selected isolates as C. jejuni and 76.6% isolates as C. coli in full agreement with the biochemical identification, i.e. the hippurate test. Either C. jejuni or C. coli was identified in each individual sample irrespective of the fact that five colonies per sample were tested. However, other investigations have shown that pigs can be infected by both C. coli and C. jejuni simultaneously (Madden et al., 2000). The majority of pig carcasses were contaminated with C. coli. This has been viewed as of minor importance to human health, however, recent publication has suggested that its health burden may be considerable and greater than previously thought (Tam et al., 2003). Unexpectedly, C. jejuni was isolated from five of 16 swab samples collected from pig carcasses and from two environment samples. No C. jejuni bacteria were isolated from faecal samples by the chosen technique. However, the most likely explanation for the finding on carcasses is faecal contamination. We speculate that pigs may be carriers of C. jejuni in low numbers that go undetected using direct plating. As C. jejuni is significantly more resistant to the selective stresses than C. coli (Madden et al., 2000), they will however be detected using selective enrichment. Since we only analysed enrichment broth in the cases where no colonies were obtained by direct plating, the prevalence estimated in the present study may consequently be too low. The finding of C. jejuni on the pig carcasses is important as the majority of human cases of campylobacteriosis are caused by C. jejuni (Skirrow, 1990; Nielsen et al., 1997; Burnett et al., 2002; Kapperud et al., 2003). All C. jejuni isolates of this study were serotype 35 or serotype 23, 36. These serotypes have consistently been the most common serotypes among C. jejuni isolates from pigs in Denmark (E.M. Nielsen, unpublished data). These serotypes are also found in Danish patients, however, in relatively low frequency. Previous investigations, like the present, have detected high diversity of campylobacter types not only within group of pigs as a whole, but even within individual faecal samples (Weijtens et al., 1999). Therefore, when investigating the epidemiology of campylobacter in pigs it is important to type more than one colony per sample. However, even when typing more colonies, the diversity may be underestimated (Weijtens et al., 1999; Kramer et al., 2000; Dickins et al., 2002). The high diversity

5 M. Malakauskas et al. / International Journal of Food Microbiology 108 (2006) may be due to infection of the pigs with various campylobacter types from different sources and routes of infection (Weijtens et al., 1999), and such mixed infection may complicate source tracing (Newell et al., 2001; Schouls et al., 2003). However, genomic instability has also been suggested to underlie diversity (On, 1998; Wassenaar et al., 1998). We believe that the diversity observed in the current investigation represents true diversity and not instability during the culturing and preparation for PFGE. Nielsen et al. (2001) did not observe changes in ribotypes, PFGE or random amplified polymorphic DNA analysis when three strains of C. jejuni were subcultured 50 times in triplicate, nor after colonizing mice for up to 26 days. In addition, eleven other C. jejuni strains of four different serotypes were subcultured ten times to screen for instability and none of these showed instability using PFGE and serotyping (Nielsen et al., 2001). The fact that up to five isolates from the same initial sample (after direct plating or enrichment in MHB) showed the same PFGE pattern also indicate minimal effect of plating and subculturing on the genomic instability of Campylobacter spp. In the present study we used direct streaking on the plates which enabled us to assess more accurately the diversity of isolates (PFGE patterns) by elimination of the enrichment step. However, we could not obtain Campylobacter spp. isolates after direct plating from 15 samples but isolated Campylobacter from these after enrichment in MHB. These results indicate that although direct plating is desirable when typing investigation is performed in order to understand the epidemiology of Campylobacter spp., the enrichment step is needed to ensure detection of stressed bacteria or low numbers of viable campylobacters. Our results suggested that contamination of carcasses and slaughterhouse environment was likely to originate from the gastro-intestinal tracts of the slaughtered pigs, as swab samples taken from surfaces which had direct contact only with pig intestines and which collect drops from pigs' plugs were positive. Several other places could be contaminated from the pig gastro-intestinal tract: pens where pigs are kept before slaughtering, the scalding tank, dehairing and polishing machines, knifes and workers hands, and the slaughter process thus offers a multitude of cross-contamination possibilities, especially if requirements of GHP and HACCP are not kept in the slaughterhouse. Gill and Bryant (1993) previously reported that detritus from dehairing machines may be a source of enteric bacteria such as Escherichia coli, Campylobacter and Salmonella, and Borch et al. (1996) also stressed the difficulty of cleaning polishing machines, where bacteria may become established, which often constitute a source of cross-contamination for carcasses. Pearce et al. (2003) indicate that Campylobacter spp. are highly prevalent in the intestinal tracts of swine arriving at the slaughter facility. However, they also found that Campylobacter does not propagate and spread easily during the slaughtering operation. Therefore sporadic contamination of the slaughter line need not lead to massive crosscontamination. This could explain why we found C. jejuni only on the pig carcasses of heard B and not from the samples of other herds slaughtered after. 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