The Incidence of Salmonella, Campylobacter, and Yersinia Enterocolitica in Swine Carcasses and the Slaughterhouse Environment

642

The Incidence of Salmonella, Campylobacter, and Yersinia enterocolitica in Swine Carcasses and the Slaughterhouse Environment

AKIER A. MAFU, R. HIGGINS*, M. NADEAU and G. COUSINEAU

Departement de Palhologie et Microbiologic. Facullede Medecine Velerinaire. Universite de Montreal. C. P. 5000. Saint-Hyacinthe. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/9/642/1658045/0362-028x-52_9_642.pdf by guest on 30 September 2021 Quebec. Canada. J2S 7C6.

(Received for publicalion October 25, 1988)

ABSTRACT the foods that are most frequently incriminated are those of animal origin, particularly poultry (14). Pigs are carriers of This study was done to evaluate the degree of contamination C. coli and C. jejuni (11,17), and those organisms are also of cooler-ready hog carcasses and the slaughterhouse environ isolated from pork (35). ment by Salmonella spp., Campylobacter spp. and Yersinia en Data concerning the importance of Yersinia spp. in food terocolitica. Samples from diaphragms and feces were collected are well documented in many countries (32). Y. entero from 200 market hogs in a Quebec slaughterhouse. Scalding-tank colitica is an important cause of human gastro-intestinal water and environmental swabs were also collected in the slaugh infections and has been isolated from a wide variety of terhouse. Specimens were tested for the presence of Salmonella foods (19). As a food animal, swine are a well recognized spp., Y. enterocolitica, and Campylobacter spp. Salmonella spp. were isolated from 45 (10%) of 448 samples. The distribution of reservoir of serotype 0:3, which infects humans in Europe the isolates were slaughtering floor (8.9%), feces (80.2%) , cold- (21), Japan (38), and Canada (34). Y. enterocolitica isolates room floor (4.4%), and diaphragms (6.7%). Campylobacter spp. have been retrieved from pigs in feces, mesenteric nodes, were isolated from 247 (61.7%) of 400 specimens, and C. colt, C. and meat samples (12,37), and to a greater extent from jejuni, and C. laridis accounted for 97%, 2%, and 1% of isolates, tonsils and tongues (8.22). respectively. Ninety-nine percent of fecal samples were positive This investigation was designed to determine the inci for the presence of C. coli. Y. enterocolitica was found in 42 dence of contamination of cooler-ready hog carcasses by (9.3%) of the 448 specimens. Of these, 85.7%, 11.9%, and 2.4% Salmonella spp., Campylobacter spp., and Y. enterocolitica of the isolates came from fecal, diaphragm, and cold-room floor organisms in Quebec. Fecal samples from the colon of pigs, samples, respectively. Neither Salmonella spp. nor Y. enterocolitica environmental swabs from floors in the plant, and scalding- were isolated from scalding-tank water. tank water were cultured to check for different sources of contamination of pork carcasses slaughtered on the same Meat and meat products have been implicated in the day. transmission of human pathogens such as Salmonella spp., Campylobacter spp., and Yersinia enterocolitica MATERIALS AND METHODS (5,7,15,30,35). Salmonella is presently the most important cause of foodborne infection. In 1981. Salmonella was Collection of samples recognized as the major cause of foodborne diseases in All samples were obtained from fattening pigs slaughtered Canada (16). Apparently healthy market swine may harbour between October 1985 and March 1986 in a Quebec abattoir. Salmonella, and, in addition to domestic animals and poul Two hundred animals were selected at random. A maximum of try, may serve as an important source for Salmonella con ten pigs were sampled on each visit. Thirty grams of feces and a tamination of food (13,23). Salmonellae present in slaugh piece of diaphragm were collected from each animal. Floor swabs terhouse environments may contaminate carcasses during and scalding tank water were also taken at each visit to the abattoir. processing (24). Immediately after evisceration and inspection, the terminal portion of the caecum was opened aseptically and approximately For more than 10 years, Campylobacter species have 30 g of caecal content was collected in a sterile container. Muscle been recognized as major enteropathogens in humans (6). sample was also taken from the diaphragm, with emphasis on These bacteria are responsible for approximately 10% of all surface area rather than depth of out. This tissue was collected cases of acute human enteritis (4). At the moment C. jejuni because of its lower commercial value, and because fluid drains and to a lesser extent C. coli, are among the most important over these areas. These were taken immediately prior to storage zoonotic agents of human gastro-intestinal infections (1,26). of the carcasses in the cold room. Swabs of both abattoir and cold Campylobacteriosis is considered a foodborne infection, and room floors were taken during slaughter. One hundred ml of

JOURNAL OF FOOD PROTECTION. VOL. 52. SEPTEMBER 1989 SALMONELLA, CAMPYLOBACTER AND YERSINIA IN SWINE CARCASSES 643 scalding-tank water were taken before it was changed. All samples Samples that were negative after 21 d of cold enrichment were were carried in a refrigerated container to the laboratory and reincubated and checked at week six. All isolates were kept on cultured on the same day. BHI agar slants (Difco) and sent to Dr. S. Toma, Canadian National Reference Service for Yersinia, Toronto, for serotyping and bi- Isolation and identification of Salmonella spp otyping. Twenty-five grams of feces, 25 g specimens of diaphragm muscle, and 25 ml of scalding-tank water were each put into RESULTS Stomacher bags (Simport Plastique, Beloeil, Qc, Canada) with 225 ml of nutrient broth. They were then processed in a Colworth Salmonella spp. were isolated from 45 (10%) of 448 400 Stomacher (Lab-blender, London, Engl.) for one min, in order specimens tested, and from thirty-six (18%) of 200 feces, to obtain a semi-homogeneous suspension. Swabs were also placed three (1.5%) of 200 diaphragms, four (25%) of 16 floor into a tube containing 10 ml of nutrient broth. All samples were abattoir swabs, and two (12.5%) of 16 cold-room swabs incubated for 18-24 h at 37°C. (Table 1). These microorganisms were not retrieved from One ml of nutrient broth of each specimen submitted to the primary enrichment was inoculated into 9 ml of selenite broth scalding-tank water, when the temperature appeared to be (Difco, Detroit, MI) for secondary enrichment and incubated at relatively stable at 60°C. Although large numbers of Salmo Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/9/642/1658045/0362-028x-52_9_642.pdf by guest on 30 September 2021 42°C for 18-24 h. Then, one loopful of each selenite broth was nella spp. were isolated from environmental swabs and fecal inoculated onto a brilliant green sulfa agar (Difco, Detroit, MI), samples, the contamination level of diaphragms appeared to and incubated for 24 h at 37"C. Lactose negative colonies were be relatively low. submitted to biochemical testing as described by Ellis (10). Colo A total of nine Salmonella serotypes were indentified nies were then tested by agglutination against polyvalent O-antis- (Table 2). The most frequently isolated serotype was S. era, groups A and B (Difco, Detroit, Michigan, U.S.), and con brandenburg, which was found in 24 fecal samples and in firmed with the API 20E System (Analytab Products Inc., Saint- two abattoir-floor swabs. S. derby was isolated from four Laurent, Quebec). Salmonella isolates were serotyped by Dr. R. fecal samples, three muscle samples, and from one cold- C. Finlay. Animal Pathology Laboratory, Winnipeg, Manitoba. room swab. Other serotypes recovered were S. bredeny, S. Isolation and identification of Campylobacter spp hadar, S. heidelberg, S. infantis, S. london, S. saint-paul, Fecal swabs were cultured directly on Columbia agar (base and S. thompson. CM331. Oxoid, Basingstoke, England) with 10% bovine blood A total of 247 (61.7%) samples out of 400 yielded and Campylobacter selective supplement SR98 (B laser-Wang, Campylobacter spp. (Table 3). The largest number of iso Oxoid) (2). Twenty five grams of muscle were emulsified in 100 lates came from fecal samples, and 198 (99%) of the iso ml of Rosef medium (27) for enrichment. All samples were incu lates were C. coli. Contamination of muscle samples was bated in anaerobic jars (Oxoid), under microaerophilic conditions quite high, and 41 (20.5%) of 200 diaphragms yielded C. for 24 and 48 h at 42°C. After 24 and 48 h, Rosef broths were streaked onto Columbia plates and incubated under the same conditions. Colony morphology was examined and motility was TABLE 1. Distribution of Salmonella spp. isolates among speci determined using darkfield microscopy. Other tests performed included catalase and oxidase reactions, growth at 42"C, growth mens. Number of at 25"C, sensitivity to nalidixic acid, and hydrolysis of hippurate. Number Specimens tested isolates Environmental swabs and water samples were not tested for % Feces 200 36 18 Campylobacter. Diaphragms 200 3 1.5 Slaughtering floor swabs 16 4 25 Isolation and identification of Yersinia enterocolitica Cold room swabs 16 2 12.5 Recovery of Y. enterocolitica was based on a cold enrich Scalding-tank water 16 - - ment technique (9,31). A half gram from each fecal sample was Total 448 45 placed in 10 ml of phosphate buffered saline (PBS, Ml5 pH 7.6) without peptone. Five grams of muscles were placed into a Stom acher bag containing 15 ml of the same buffer. Muscle specimens were emulsified as described earlier. The homogenate was placed TABLE 2. Distribution of Salmonella spp. serotypes according aseptically into plastic containers. Swabs were placed into 5 ml to their origin". PBS containing 10% peptone (Dubelco "A": Oxoid). Twenty ml Number of isolates Seotype Feces Diaphragms Slaughtering Cold room of scalding tank water were filtered through a 0.45 urn filter floor swabs swabs (Wathman Ltd, England), which was then placed into a tube S. brandenburg 24 containing 10 ml of PBS with peptone. All samples were refrig S. bredeny 1 erated at 4°C for six weeks. S. derby 4 At week three, samples were inoculated on "Yersinia agar S. hadar 1 base" (CIN, Oxoid) containing the selective supplement SRI09 S. heidelberg 1 (Oxoid) (29). CIN plates were incubated at 30°C for 24-48 h in S. infantis 2 an aerobic atmosphere. Typical colonies were inoculated on Triple S. london 2 sugar iron and Christensen's urea medium and were incubated at S. saint-paul 1 37°C for 24 h for presumptive identification. Additional tests such S. thompson - as growth on MacConkey agar, lactose, motility at 25°C, and API Total 36 3 20E system were used in order to complete the identification. aNo isolates from scalding-tank water.

JOURNAL OF FOOD PROTECTION, VOL. 52, SEPTEMBER 1989 644 MAFU. HIGGINS. NADEAU AND COUSINEAU

TABLE 3. Distribution of Campylobacter spp. isolates in speci only 1.5% of the muscle samples tested yielded Salmonella. mens. This is in accordance with a similar study by Fin lay et al Specimens Number of Number of isolates (percentage) (11), in which 1.7% of muscles were contaminated with Specimens C. jejuni C. coli C. laridis this organism. Finally, it is noteworthy that the scalding- Feces 200 1(0.5) 198(99) 1(0.5) tank water was negative for the presence of Salmonella, Diaphragms 200 4(2) 41(20.5) 2(1) Total 400 5(1.2) 239(59.7) 3(0.7) indicating that the temperature of water remained very stable (60°C) and that this is not a factor in the contamination of carcasses in this slaughterhouse. Rosset and Lameloise reported the killing of Enterobacteriaceae by water at such coli. C. jejuni and C. laridis represented only 3% of Campy temperatures (28). lobacter isolates. C. coli was isolated from intestinal contents of almost Y. enterocolitica was isolated from 42 (9.3%) of 448 all pigs (99%) in this study. Other authors have reported a specimens (Table 4). Isolates came from 36 (18%) of 200 prevalence between 46.5 and 100% in similar studies (25). fecal samples, five (2.5%) of 200 diaphragms, and one Luechtenfeld and Wang (20) suggested that C. coli could be Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/9/642/1658045/0362-028x-52_9_642.pdf by guest on 30 September 2021 (6.2%) of 16 cold-room swabs. Y. enterocolitica was not found in healthy pigs as often as was C. jejuni in chicken. recovered from the abattoir floor or from scalding-tank Our results regarding muscle contamination with C. coli are water. Serotypes and biotypes of Y. enterocolitica are pre in accordance with those of Finlay et al (11) with preva sented in Table 5. Of the 42 Y. enterocolitica isolates, 29 lences of 20.5% in diaphragm muscles and 19.6% in neck belonged to serotype 0:3 biotype 4. Serotypes 0:5 and 0:16 muscles, respectively. The procedures used in our study did were isolated three times each while serotypes 0:4, 0:7,8, not allow for the isolation of non-thermophilic 0:3,13, 0:34, 0:41,43, and 0:46 were isolated only once each Campylobacter, like C. hyointestinalis or C. sputorum ssp. from fecal samples. mucosalis, which are also likely to be found in the swine intestinal tract (3). The low number of C. jejuni and C. DISCUSSION laridis isolates from fecal and muscle samples confirms the results of Turnbull and Rose (35). This study indicated that a large proportion (18%) or The presence of Y. enterocolitica in 18% of pig fecal healthy pigs were carriers of Salmonella. The absence of S. samples is in accordance with other reports that identify typhimurium has to be noted as well as the host-specific 5. swine as the main reservoir for this microorganism (32,33). choleraesuis which is not likely to be isolated by the tech Most isolates of Y. enterocolitica in this study belonged to nique used. The high prevalence of Salmonella on the cold serotype 0:3, biotype 4. This serotype is regularly isolated room floor (12.5%) could be due to the coming and going from pig feces and is the main serotype isolated from humans of workers between the slaughterhouse floor (25% preva in Canada (18,33). The presence of this bacterium on the lence) and this room. In spite of this high level of contami cold-room floor, and its ability to grow at low temperatures nation in the intestinal tract of pigs and in the environment, could become a health hazard in plants where disinfection is neglected.

TABLE 4. Distribution of Yersinia enterocolitica isolates in speci ACKNOWLEDGMENT mens". Specimens Number of Number of % specimens isolates The authors acknowledge the financial support of the Canadian Agency for International Aid. Feces 200 36 18 Diaphragms 200 5 2.5 Cold room swabs 16 1 6.2 Total 448 42 9.3 REFERENCES "Slaughtering floor swabs (16) and scalding-tank water (16) speci 1. Blaser, M. J., D. N. Taylor, and R. A. Feldman. 1983. Epidemiology mens were negative for Y. enterocolitica. of Campylobacter jejuni infection. Epidemiol. Rev. 5:157-176.

TABLE 5. Distribution of Yersinia enterocolitica serotypes and biotypes in the different specimens. Specimens Number of Serotypes Biotypes' isolates 0:3 0:4 0:5 0:7,8 0:7,13 0:16 0:34 0:41,43 0:46 NT" Feces 36 25 1 2 1 3 1 1 1 26 Diaphragms 5 4 1 4 Cold-room swabs 1 - - 1 Total 42 29 1 3 1 I 3 1 30 'According to Wauters' scheme (36). bNon Typable. No isolates were recovered from slaughtering floor swabs or scalding-tank water.

JOURNAL OF FOOD PROTECTION. VOL. 52. SEPTEMBER 1989 SALMONELLA, CAMPYLOBACTER AND YERSINIA IN SWINE CARCASSES 645

2. Blaser, M. J., H. L. Hardesty, B. Powers, and W-L. L. Wang. 1980. 20. Leuchtenfeld, N. W., and W. L. L. Wang. 1981. Campylobacter fetus Survival of Campylobacter fetus subsp jejuni in biologicial milieus. J. subsp. jejuni in a turkey processing plant. J. Clin. Microbiol. 13:266- Clin. Microbiol. 11:309-313. 268. 3. Boosinger, T. R„ H. L. Thacker, and C. H. Armstrong. 1985. Campy 21. Mollaret, H. N., J. M. Alonso, et H. Bercovier. 1976. Sur 1'inegalite lobacter sputorum subsp., mucosalis, and Campylobacter hyointesti- de frequence dans I'isolement de Yersinia enterocolitica de part et nalis infections in the intestine of gnotobiotic pigs. Am. J. Vet. Res. d'autre de la frontiere franco-beige. Med. Mai. infect. 6:102-107. 46:2152-2156. 22. Narucka, U., and J. F. Westendoorp. 1977. Studies for the presence of 4. Bruce, D. W. Zochowski, and I. R. Ferguson. 1977. Campylobacter Yersinia enterocolitica and Yersinia pseudotuberculosis in clinically enteritis. Br. Med. J. 5:1219. normal pigs. Tijdschr. Diergeneeskd. 102:299-303. 5. Bryan, F. L. 1980. Foodbome disease in the United States associated 23. Nelson, J. W., R. A. Robinson, M. M. Pullen, and L. Rotanahakorm. with meat and poultry. ]. Food Prot. 43:140-150. 1982. The prevalence of Salmonella in various age groups of market 6. Butzler, J. P., and M. B. Skirrow. 1979. Campylobacter enteritis. Acta swine. Proc. 86,h Ann. Meet. U.S. Anim. Hlth. Ass. 424-444. Ppaediatr. Bclg. 32:89. 24. Oosterom, J., and S. Notermans. 1983. Further research into a possi 7. Doyle, M. P. 1981. Campylobacter fetus subsp. jejuni. An old patho bility of Salmonella-free fattening and slaughter of pigs. J. Hyg. Camb. gen of new concern. J. Food Prot. 44:480-488. 91:59-69. 8. Doyle, M. P., M. B. Hugdahl, and S. L. Taylor. 1981. Isolation of 25. Oosterom, J., G. J. A. De Wilde, E. De Boer, L. H. De Blaauw, and virulent Yersinia enterocolitica from porcine tongues. Appl. Environ. H. Karman. 1983. Survival of Campylobacter jejuni during process

Microbiol. 42:661-666. ing and pig slaughtering. J. Food Prot. 46:702-706. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/52/9/642/1658045/0362-028x-52_9_642.pdf by guest on 30 September 2021 9. Eiss, J. 1975. Selective culturing of Yersinia enterocolitica at a low 26. Prescott, J. F., and D. L. Munroe. 1982. Campylobacter jejuni enteri temperature. Scand. J. Infect. Dis. 7:249-251. tis in man and domestic animals. J. Am. Vet. Med. Assoc. 18:1524- 10. Ellis, E. M. (ed.), J. E. Williams, E. T. Mallinson, G. H. Snoeyenbos, 1530. and W. J. Martin. 1976. Culture methods for the detection of animal 27. Rosef, O. 1981. Isolation of Campylobacter fetus subsp. jejuni from salmonellosis and arizonosis; pp. 48-54. In E. M. Ellis (Ed). A manual the gallbladder of normal slaughter pigs, using an enrichment proce of the American Association of Veterinary Laboratory Diagnosticians. dure. Acta Vet. Scand. 22:149-151. Iowa State University Press, Ames, Iowa. 28. Rosset, R., et P. Lameloise. 1984. Microbiologie de la viande. pp. 11. Finlay, R. C, E. D. Mann, and J. L. Homing. 1986. Prevalence of 131-133. Dans Les viandes. Informations Techniques Services Veteri- Salmonella and Campylobacter contamination in Manitoba swine car naires. Paris, Cedex 13. casses. Can. Vet. J. 27:185-187. 29. Schiemann, D. A. 1979. Synthesis of a selective agar medium for 12. Fukushima, H. 1985. Direct isolation of Yersinia enterocolitica and Yersinia enterocolitica. Can. J. Microbiol. 25:1298-1304. Yersinia pseudotuberculosis from meat. Appl. Environ. Microbiol. 30. Schiemann, D. A. 1980. Isolation of toxinogenic Yersinia enterocolitica 50:710-712. from retail pork products. J. Food Prot. 43:360-365. 13. Gloves, B. I., N. A. Fish, and W. R. Mitchell. 1971. The occurrence 31. Schiemann, D. A. 1982. Development of a two-step enrichment pro of Salmonella infection in market swine in South Western Ontario. cedure for recovery of Yersinia enterocolitica from food. Appl. Environ. Can. Vet. J. 12:11-14. Microbiol. 43:14-27. 14. Grant, 1. H., N. J. Richardson, and V. D. Bokkenhcuser. 1980. Broiler 32. Swaminathan, B., M. C. Harmon, and I. J. Mehlman. 1982. Yersina chickens as potential source of Campylobacter infection in humans. J. enterocolitica. J. Appl. Bacteriol. 52:151-183. Clin. Microbiol. 11:508-510. 33. Toma, S., and V. R. Deidrick. 1975. Isolation of Yersinia entero 15. Harris, N. V., N. S. Weiss, and C. M. Nolan. 1986. The role of poultry colitica from swine. J. Clin. Microbiol. 2:478-481. and meats in the etiology of Campylobacter jejuni!coli enteritis. Am. 34. Toma, S., and L. Lafleur. 1981. Yersinia enterocolitica in Canada J. Public. Health 76:407-411. 1966 to August 1978. pp. 183-191. In E. J. Bottone (Ed.) Yersinia en 16. Health Protection Branch. 1981. Food-borne and water-borne diseases terocolitica. CRC Press Inc., Boca Raton, Florida. in Canada. Annual Summary 1977: Health Protection Branch, Health 35. Turnbull, P. C. B., and P. Rose. 1982. Campylobacter jejuni and Sal- and Welfare Canada, Ottawa. monella in raw red meats. J. Hyg. Camp. 88:29-37. 17. Jorgensen, K. 1979. The occurrence of Campylobacter in swine. Nord. 36. Wauters, G. 1970. Contribution a l'etude de Yersina enterocolitica. Vet. Med. 31:534. Louvain, Belgium: These Vander. 18. Lafleur, L., O. Hammerberg, G. Delage, and C. H. Pai. 1979. Yersinia 37. Zen-Yoji, H., S. Sakai, T. Maruyama, and Y. Yanagawa. 1974. Iso enterocolitica infection in children: 4 years experience in the Mon lation of Yersinia enterocolitica and Yersinia pseudotuberculosis from treal Urbain Community. Contrib. Microbiol. Immunol. 5: 298-303. swine, cattle, and rats at an abattoir. Jpn. J. Microbiol. 18:103-105. 19. Lee, W. H., C. Vanderzant, and N. Stem. 1981. The occurrence of 38. Zen-Yoji, H. 1981. Epidemiologic aspects of yersiniosis in Japan, pp. Yersinia enterocolitica in foods, pp. 161-171. In Yersinia enterocolitica. 205-216. In E. J. Bottone (Ed.) Yersinia enterocolitica CRC Press E. J. Bottone ed. CRC Press Inc., Boca Raton, Florida. Inc., Boea Raton, Florida.

JOURNAL OF FOOD PROTECTION. VOL. 52. SEPTEMBER 1989