Prevalence and Dissemination of Salmonella Serotypes along the Slaughtering Process in Brazilian Small Poultry Slaughterhouses

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1 49 Journal of Food Protection, Vol. 3, No. 2, 2000, Pages Copyright, International Association for Food Protection Research Note Prevalence and Dissemination of Serotypes along the Slaughtering Process in Brazilian Small Poultry Slaughterhouses TERUMI O. FUZIHARA, SUELI A. FERNANDES, 2 AND BERNADETTE D. G. M. FRANCO 3 * Instituto Adolfo Lutz, Laboratório I, Santo André, São Paulo; 2 Instituto Adolfo Lutz, Laboratório Central, São Paulo, São Paulo; 3 Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Departamento de Alimentos e Nutrição Experimental, São Paulo, São Paulo, Brazil MS 00-09: Received 3 April 2000/Accepted 2 August 2000 ABSTRACT is the leading cause of human foodborne infections in Latin America, and poultry meat is one of the main vehicles. Small poultry slaughterhouses (fewer than 200 birds slaughtered per day) represent an important economic activity in certain regions. The slaughtering process in these abattoirs is manual and rudimentary, and frequently the hygienic conditions are poor. This study reports results of a detailed evaluation of the prevalence of serotypes in carcasses, utensils, and environmental samples collected in 0 small Brazilian slaughterhouses. In the second step of the study, one of these slaughterhouses was selected to monitor the dissemination of along the slaughtering process. For testing, conventional procedures were used: preenrichment in buffered peptone water (35 C for 24 h), selective enrichment in Selenite-cystine (35 C for 24 h), tetrathionate and Rappaport-Vassiliadis broths (42 C for 24 h), plating on bismuth-sulfite and brilliant green agars (35 C for 24 h), proper biochemical testing, and complete serotyping. Forty-one percent of samples harbored spp., including 42% of carcasses, 23.% of utensils,.4% of water, and.4% of freezers and refrigerators. Seventeen serotypes were detected. Enteritidis predominated (30%), followed by (2%), (2%), (0%), and I 4,2:z: (8%). All samples collected along the slaughtering process in the selected slaughterhouse were positive. Five serotypes were detected, including,,,, and. More than 30% of the samples contained more than one serotype, and 2.5% presented three serotypes. The widespread occurrence of in small slaughterhouses reinforces the need for implementation of effective control measures. spp. continue to be one of the leading causes of human foodborne infections in many countries ( 3, 5,, 8,, 2, 5). A recent review conducted in southern Latin America on foodborne outbreaks due to bacteria occurring between 995 and 998 indicated that spp. were responsible for most (3.8%) reported cases in the region (0). Eggs and mayonnaise were the most common food products associated with these outbreaks, but poultry meat was an equally important vehicle (0). In recent years, the importance of poultry as a vehicle of human salmonellosis has paralleled the marked increase in global consumption of poultry meat. Brazil is an important poultry-producing country and, allied to the low cost when compared with other types of meat, poultry meat is largely consumed in the country. In larger cities, poultry meat is usually supplied by large facilities, where thousands of birds are slaughtered per day. However, small poultry slaughterhouses, which slaughter fewer than 200 birds per day, represent an important economic activity in certain regions. The slaughtering process in these abattoirs is manual * Author for correspondence. Present address: Av. Prof. Lineu Prestes 580 Bloco 4, , São Paulo, SP, Brazil. Tel: ; Fax: ; bfranco@usp.br. and rudimentary, and frequently the hygienic conditions are poor. Whole carcasses and chicken parts are sold directly to the consumer without an effective control by local health authorities. Mauá is an important industrial city close to São Paulo, the capital of São Paulo State, Brazil. In 99, the number of inhabitants in Mauá was close to 400,000. This city is under the sanitary surveillance of the Santo André Regional Laboratory (Laboratório I) of Instituto Adolfo Lutz, São Paulo. This study was conducted to give a detailed evaluation of the prevalence of serotypes in carcasses, utensils, and environmental samples collected in 0 small poultry slaughterhouses located in Mauá. In the second step of the study, one of these slaughterhouses was selected to monitor the dissemination of along the slaughtering process. MATERIALS AND METHODS Carcass samples for the study of the prevalence of spp. Poultry carcasses, collected in 0 poultry slaughterhouses located in Mauá, São Paulo, Brazil, were tested. Carcasses were placed into sterile plastic bags, transported under refrigeration to the Food Microbiology Laboratory of Instituto Adolfo Lutz, Laboratório I, Santo André, São Paulo, Brazil, and

2 50 FUZIHARA ET AL. J. Food Prot., Vol. 3, No. 2 FIGURE. Flowchart of the slaughtering process (Ca-, Ca-2, Ca-3, and Ca-4 carcasses; T-, T-2, and T-3 immersion tanks; P- and P-2 containers with water). Gray rectangles are points tested for. tested in less than 2 h. In the laboratory, test samples (25 g) of each carcass, comprising randomly obtained pieces of the chest, thighs, wings, and skin, were homogenized with 225 ml of sterile buffered peptone water, using a Stomacher 400 Lab Blender (Seward Medical Ltd., London, England). Utensils and environmental samples for the study of the prevalence of spp. This group of samples included 5 knives, chopping boards, water from drinking water containers, and internal walls and shelves of freezer and refrigerators. For sampling of knives, sterile swabs, moisturized with sterile buffered peptone water, were applied to both sides of blades. For sampling of boards, walls, and shelves, sterile swabs, moisturized with sterile buffered peptone water, were applied to a 0-cm 2 area, using a sterile aluminum template. Swabs were introduced in tubes containing 5 ml of sterile buffered peptone water. For sampling of water from tanks and containers, 00 ml was transferred to a sterile flask. Tubes with swabs and water samples were transported under refrigeration to the Food Microbiology Laboratory of Instituto Adolfo Lutz, Laboratório I, Santo André, São Paulo, Brazil, and tested in less than 2 h. Samples for the monitoring of spp. during slaughtering. One poultry slaughterhouse was randomly selected among those tested in the first part of this study (see above). Figure shows the flowchart of the slaughtering process in this place and the points (gray rectangles) sampled for. Six trials were accomplished in distinct days. Carcass samples for the monitoring of spp. during slaughtering. Poultry carcasses were numbered using a label firmly tied to the skin. After defeathering, the carcasses (Ca- ) were put into bags containing 225 ml of sterile buffered peptone water. After hand massaging, they were removed from the bags and returned to the slaughtering process. The same carcasses were tested again for after evisceration (Ca-2), after immersion in tank T- (Ca-3), and after immersion in tank T-2 (Ca-4). The peptone water from the bags was transferred to sterile flasks and tested for as described below. Parts for the monitoring of spp. during slaughtering. Gizzards, backs, necks, and feet samples were withdrawn from the plastic boxes used to collect them. Each box contained approximately 5 kg of parts. Four gizzards, randomly selected, comprised one sample. One back and the correspondent neck comprised one sample. Each feet sample contained eight randomly selected feet. Wing, thigh, chest, liver, and heart samples were withdrawn from the water of plastic storage containers. One sample of wing contained four randomly selected wings. Livers and hearts were sampled together, and each sample contained two of each. For testing, samples were transferred to sterile plastic bags and hand massaged with 225 ml of sterile buffered peptone water. This rinse solution was transferred to a sterile flask and tested for as described below. Water from tanks T-, T-2, and T-3 and from plastic containers P- and P-2. Water samples were collected in sterile flasks. For testing, 00 ml of each sample was added to 00 ml of double-strength sterile buffered peptone water and tested for as described below. Feces of handlers. Approximately 2goffeces of five employees was homogenized with 0 ml of sterile saline and tested for as described below. Microbiological analyses of carcasses, parts, swabs, and water samples. Conventional procedures, recommended by

3 J. Food Prot., Vol. 3, No. 2 SALMONELLA IN BRAZILIAN POULTRY SLAUGHTERHOUSES 5 TABLE. Incidence of spp. in poultry slaughterhouses in Mauá, São Paulo, Brazil Type of sample Carcasses Water Utensils Knives Boards Subtotal Equipment Freezers Refrigerators Subtotal No. of samples Tested Positive (%) (42.0) 5 (.4) 3 (20.0) 3 (2.3) (23.) (00) 4 (.) 5 (.4) Total 00 4 (4.0) American Public Health Association (9), were used. Preenrichment was performed by incubating buffered peptone water at 35 C for 24 h. Selective enrichment was performed in Selenite-cystine broth, incubated at 35 C for 24 h, and in tetrathionate and Rappaport-Vassiliadis broths, incubated at 42 C for 24 h. Each enrichment broth was streaked onto bismuth-sulfite agar and brilliant green agar plates and incubated at 35 C for 24 h for isolation of colonies. Positive colonies were transferred to triple sugar iron agar and lysine iron agar slants, and those presenting typical results were confirmed in API 20E system. After confirmation of results for through agglutination tests with polyvalent somatic and flagellar sera produced by the Brazilian Serotyping Reference Laboratory at Instituto Adolfo Lutz, Seção de Bacteriologia, the positive strains were sent to this reference laboratory for complete serotyping. Microbiological analyses of feces. Each homogenate was preenriched in Selenite-cystine broth, incubated at 35 C for 24 h, and in tetrathionate and Rappaport-Vassiliadis broths, incubated at 42 C for 24 h. Bismuth-sulfite agar and brilliant green agar, incubated at 35 C for 24 h, were used for isolation of colonies. Each homogenate was also directly streaked onto Bismuth-sulfite agar and brilliant green agar and incubated at 35 C for 24 h. Complete identification of colonies and serotyping were done as described above. RESULTS AND DISCUSSION The frequency of spp in the slaughterhouses is presented in Table. As shown, 4% of samples harbored. The pathogen was detected in 42% of the carcasses,.4% of the water samples from drinking water containers, 23.% of the utensil samples, and.4% of freezer and refrigerator samples. These results indicate that disseminates very easily within the slaughterhouse and is able to survive in hostile environments, such as refrigerators and freezers. Seventeen serotypes were detected, besides one nontypable rough isolate (Table 2). Enteritidis was the predominant serotype. and were the second most common serotypes, followed by, I 4,2:z:, and Infantis. Enteritidis was detected in seven carcasses TABLE 2. serotypes isolated in poultry slaughterhouses in Mauá, São Paulo, Brazil Serotypes No. (%) Enteritidis I 4,2:z: Infantis Bredeney Braenderup Typhimurium Mbandaka Saint Paul I,3,9: I,,:r: I,:z0: I,:eh: I 4,2:r: I 4,2: :,: Rough strain (30) (0) (8) (4) Total 50 (00) and eight environmental samples. The prevalence of this serotype in many parts of the world is well known (4, 3, 4, 8), but the reasons for its increased incidence in certain foods after 90 are not well understood. According to Cox (), it is possible that new virulence factors, combined with previous pathogenicity factors, contributed to establishment of systemic infections and consequently a prevalence of this serotype in birds. The monitoring of dissemination of serotypes during slaughtering indicated that the prevalence in carcasses after defeathering was 83.3% (Table 3). This index did not change after immersion of the carcasses in tank T- but increased to 00% after immersion in tank T-2. The prevalence of in water samples collected from tanks T-, T-2, and T-3 and from plastic containers P- and P-2 was high (., 00, 00, 00, and.%, respectively). The high frequency of in water samples and the increased percentage of in carcasses and parts after immersion were not surprising. These recipients were sanitized only at the end of the day, the water was not substituted during the slaughtering process, and chlorination was not controlled. The temperature of the water varied between 25 and 2 C, contributing to survival and growth of the pathogen. According to D Aoust (), the larger the period of contact between the carcass and the water, the greater will be the number of the pathogens adhering to the skin. The Food Safety and Inspection Service () demonstrated that only 3 to 4% of the birds entering a slaughterhouse are positive for, whereas 35% of the carcasses leaving the same slaughterhouse become positive. As shown in Table 3, almost all samples of wings, thighs, chests, backs, necks, feet, hearts, livers, and gizzards selected for monitoring the critical points of contamination during slaughtering were positive. The frequen-

4 52 FUZIHARA ET AL. J. Food Prot., Vol. 3, No. 2 TABLE 3. Distribution of serotypes in a poultry slaughterhouse in Mauá, São Paulo, Brazil a Sampling date Immersion water T- T-2 T-3 P- P-2 Carcasses Ca- Ca-2 Ca-3 Ca-4 Wing Thigh and chest Parts Back and neck Feet Liver and heart Gizzard I II III IV V nd nt nt VI nd nt nt nt nt nt nt nt nt nd nd nd nd nd nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt Positives 4 (.) 5 (00) 3 (00) 3 (00) 2 (.) 5 (83.3) 4 (.) 5 (83.3) 5 (.) 3 (00) 3 (00) 3 (00) 2 (.) 3 (00) 3 (00) b a T-, T-2, T-3, immersion tanks; P-, P2, plastic containers; Ca-, postdefeathering; Ca-2, postevisceration; Ca-3, post-t-; Ca-4, post-t-2; nt, nontested; nd, nondetected. b No. (%).

5 J. Food Prot., Vol. 3, No. 2 SALMONELLA IN BRAZILIAN POULTRY SLAUGHTERHOUSES 53 TABLE 4. serotypes isolated along the slaughtering process in one poultry slaughterhouse in Mauá, São Paulo, Brazil Serotypes No. (%) (35.2) (3) (4.2) (9.8) (9.8) Total (00) cy of the pathogen in these samples was 00%, except for feet, where a lower prevalence (.%) was detected. Among isolates obtained in the slaughterhouse, only five serotypes were detected (Table 4). was the predominant serotype. In the first trial, this serotype was detected in seven of nine sampling points and also in gizzards, livers, and hearts. In the second trial, predominated, but continued to be detected in high frequency, along with. In the third trial, besides, two new serotypes ( and ) were detected, but was not detected anymore. Curiously, in the fourth trial, predominated and was detected in almost all samples. In the fifth trial, the incidence of decreased significantly, and reappeared. The same occurred in the last trial, when was detected again and could not be detected anymore. This fluctuation suggests that a variety of serotypes of may be present in a poultry slaughterhouse. As shown in Table 3, a significant number of samples contained more than one serotype. This was observed in 30.2% of positive samples, whereas 2.5% of them contained three serotypes simultaneously. All isolates were lysine decarboxylase negative, whereas all Typhimurium and Enteritidis isolates were unable to ferment inositol. All fecal samples were negative for. Despite the reduced number of tests, human feces do not seem to play a significant role as source of contamination. The widespread occurrence of several serotypes of in small poultry slaughterhouses reinforces the need for the implementation of effective control measures. REFERENCES. Bean, N. H., and P. M. Griffin Foodborne disease outbreaks in the United States, 93 98: pathogens, vehicles, and trends. J. Food Prot. 53: Bean, N. H., J. S. Goulding, M. T. Daniels, and F. J. Angulo. 99. Surveillance for foodborne disease outbreaks: United States, J. Food Prot. 0: Bryan, F. L. 98. Current trends in foodborne salmonellosis in the United States and Canada. J. Food Prot. 44: Carramiñana, J. J., J. Yangüela, D. Blanco, C. Rota, A. I. Agustin, A. Ariño, and A. Herrera. 99. incidence and distribution of serotypes throughout processing in a Spanish poultry slaughterhouse. J. Food Prot. 0: Chalker, R. B., and M. J. Blaser A review of human salmonellosis, III: magnitude of infection in the United States. Rev. Infect. Dis. 0: 24.. Cox, J. M enteritidis: virulence factors and invasive infection in poultry. Trends Food Sci. Technol. : D Aoust, J. Y. 989., p In M. P. Doyle (ed.), Bacterial foodborne pathogens. Marcel Dekker, New York. 8. D Aoust, J. Y. 99. species, p In M. P. Doyle, L. K. Beuchat, and T. J. Montville (ed.), Food microbiology fundamentals and frontiers. American Society for Microbiology, Washington, D.C. 9. Flowers, R. S., J. Y. D Aoust, W. H. Andrews, and J. S. Bailey. 992., p In C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, D.C. 0. Franco, B. D. G. M., M. Landgraf, M. T. Destro, and D. Gelli. Foodborne diseases in Southern South America. In M. Miliotis and J. Bier (ed.), International handbook of foodborne pathogens. Marcel Dekker, New York, in press.. Green, S. S. 98. Results of a national survey: in broilers and overflow chill tank water, , United States Department of Agriculture, Food Safety and Inspection Service, Science, Washington, D.C , Apud. J. Food Prot. 52: Jackson, G. J., C. F. Langford, and D. L. Archer. 99. Control of salmonellosis and similar foodborne infections. Food Control : Machado, J., and F. Bernardo Prevalence of in chicken carcasses in Portugal. J. Appl. Bacteriol. 9: Plummer, R. A. S., S. J. Blissett, and C. E. R. Dodd contamination of retail chicken products sold in the UK. J. Food Prot. 58: Roberts, D Sources of infection: food. Lancet 33: Rusul, G., J. Khair, S. Radu, C. T. Cheah, and R. M. Yassin. 99. Prevalence of in broilers at retail outlets, processing plants and farms in Malaysia. Int. J. Food Microbiol. 33: Sakai, T., and T. Chalermchaikit. 99. The major sources of enteritidis in Thailand. Int. J. Food Microbiol. 3: Uyttendaele, M. R., J. M. Debevere, R. M. Lips, and K. D. Neyts Prevalence of in poultry carcasses and their products in Belgium. Int. J. Food Microbiol. 40: 8.