J. Sci. Dev. 2009, 7 (Eng.Iss.1): 54 - 61 HA NOI UNIVERSITY OF AGRICULTURE 54 Detection of pathogens causing food borne diseases in water used in small slaughterhouses in Hanoi, Vietnam Xác định một số loại vi sinh vật gây ngộ độc thực phẩm trong nước sử dụng tại các lò mổ tư nhân tại Hà Nội, Việt Nam Truong Ha Thai 1 , Yamaguchi Ryoji 2 , Chu Thi Thanh Huong 1 , Nguyen Thi Lan 1 1 Department of Microbiology – Infectious disease – Pathology, Faculty of Veterinary Medicine, HUA, Vietnam 2 Department of Pathology, Faculty of Agriculture, Miyazaki University, Japan TÓM TẮT Nước sử dụng trong hoạt động giết mổ được coi là một trong những nguồn gây nhiễm cho thân thịt trong quá trình giết mổ, đặc biệt ở các lò mổ tư nhân. Mục đích của nghiên cứu này là xác định một số loài vi sinh vật gây ngộ độc thực phẩm từ các mẫu nước sử dụng trong giết mổ như Coliforms, E. coli, Staphylococcus aureus, Salmonella, Clostridium perfringens. Kết quả đã chỉ ra mức độ ô nhiễm của nước tại các lò mổ này là đáng báo động. Cụ thể, tỷ lệ của mỗi loại mầm bệnh theo thứ tự nêu trên là 82,50%, 65,00%, 65,00%, 70,83% và 13,33%. Tỷ lệ phân lập được các loại mầm bệnh này trong các mẫu nước không có sự khác biệt giữa các loại lò mổ khác nhau (lò mổ trâu bò, lò mổ lợn và lò mổ gia cầm). Kết quả này cũng cho thấy mức độ ô nhiễm vi sinh vật trong nước sử dụng ở lò mổ cao hơn nhiều lần so với tiêu chuẩn cho phép của Bộ Nông nghiệp & Phát triển nông thôn và một số tổ chức khác. Nguyên nhân của tình trạng ô nhiễm này là do hoạt động thiếu chuyên nghiệp của công nhân giết mổ, sai sót trong quy trình giết mổ và điều kiện vệ sinh nguồn nước. Các số liệu được xử lý bằng phần mềm SAS, phiên bản 8.1 bằng cách chuyển các số liệu sang dạng log 10 , phép thử χ 2 với độ tin cậy 95%. Từ khóa: Hà Nội, lò mổ tư nhân, log 10 , mầm bệnh, nước, Việt Nam. SUMMARY Water used in small slaughterhouses is considered as one of the sources caused the contamination for carcass in food processing, especially, in un-hygienic conditions of small slaughterhouses in Vietnam. In this study, it was aimed at detecting some pathogens from water samples caused food borne disease such as Coliforms, E. coli, Staphylococcus aureus, Salmonella, Clostridium perfringens. The results indicated that the contamination level of water collected in studied slaughterhouses was alarming. In detail, the prevalence of each pathogens mentioned above was 82.50%, 65.00%, 65.00%, 70.83% and 13.33%, respectively. There was no difference in the presence of the pathogens among which kind of studied slaughterhouses (p>0.05). The results also reflected that the level contamination was much higher than the standard provided by the Ministry of Agriculture and Rural Development (MARD) as well as the permitted norms of other organizations. Reasons explaining for this situation may rely on the inappropriate activities of workers, the mistakes in procedures applied in the slaughterhouses and the water sanitary conditions. The data collected was transformed into log 10 and analyzed by using the SAS software, version 8.1. The prevalence was compared by χ2-test. All significant differences were determined at CI = 95%. Key words: Hanoi, log 10 , pathogens, small slaughterhouse, Vietnam, water sample. Detection of pathogens causing food borne diseases in water 55 1. INTRODUCTION Maize Nowadays, food safety has become a major concern in the public health throughout the world. In Vietnam, this has become a national problem and needs more research to identify factors effect on the quality of food in general and meat in particularly. Many researchers reported that raw meat (beef, pork, and chicken meat) sold in retail market in Vietnam was found to be contamination with pathogens like E. coli, Salmonella, Staphylococcus aureus, Campylobacter… with high prevalence and total bacteria in meat samples exceed the set standard of Ministry of Agriculture and Rural development (Van et al, 2007; Huong et al, 2006; Phan et al, 2005). A question was which source of contamination in meat? Cedric et al, 2006 reported that unhygienic conditions at slaughterhouses in Hanoi with 88.77% tank water samples were positive for Salmonella may be one reason explaining for the contamination in carcass. Hanoi, the capital of Vietnam with more than 6 million habitants (Laodong, 2008), is facing a high demand for food supply. In detail, daily meat consumption in Hanoi is 280 - 300 tons, which includes 180 - 200 tons of pork (contributing to around 70% of all meat), 62 tons of poultry meat, and 40 tons of beef (Vnexpress, 2005). Almost the meat is processed in private and small slaughterhouses where the equipments and facilities are old, inadequate, or found in a desolate condition. During slaughtering and subsequent meat processing, the water used for processing becomes a contaminating source. Follow the standard of Ministry of Agriculture and Rural development, in the slaughtering process each cattle, pig and poultry need 300 to 500 liter of water, 100 liter of water and 30 liter of water, respectively. The quality of water used for slaughter processes plays an important role in the transmission of many pathogenic agents among animal food. Diseases can be transmitted to human through the animal origins were used water already contaminated by pathogenic. The aim of this research was to detect some of the food borne pathogens which can be originated from water used in slaughter processing. 2. METHODOLOGY 2.1. Study design Water samples were collected monthly from selected small slaughterhouses during a 12 month period from November 2007 to November 2008 in Hanoi. The samples were taken from each slaughterhouse in the early morning before slaughter processing. There were 3 kinds of slaughterhouses: slaughterhouses for killing cattle, pigs and poultry. 2.2. Sample collection and handling Water sample (200 ml for each sample) was selected in slaughterhouses aseptically in sterile containers before processing. Samples (n=120) were collected monthly and kept with ice and transported to the laboratory within 2 h of collection. Do not allow the sample to freeze. All samples were processed on the day of collection. Ten – fold serial dilutions were prepared using sterile 0.8% saline solution and 0.1ml of the dilution was plated onto duplicate selective agar plates. The inoculated plates were then incubated at the desired temperature for the microbial growth. The results were recorded by calculating colony forming units (cfu)/ml of the sample. 2.3. Enumeration of bacteria 2.3.1. Enumeration of total anaerobic bacteria (standard method) The samples were ten fold serial diluted with sterile 0.8% saline solution. An amount of 0.1 ml of each dilution was inoculated into each of 2 disks of Plate Count Agar (PO0158, Oxoid). The inoculated plates were incubated aerobically at 30 0 C for 24 h. C N (CFU/ml) = (D n + 0.1 x D (n+1) ) x 10 -n Note: N: Total aerobic bacteria per ml of sample C: The number of total colonies counted in 2 consecutive dilutions n: The first dilution counted D: Number of disks of each concentration counted. 2.3.2. Total Coliforms Using Brilliance E. coli/coliform Selective Agar (CM1046, OXOID) for the detection and enumeration of Escherichia coli and Coliforms water samples. The method for counting Coliforms was used like the method for counting total bacteria with purple and pink colonies. Truong Ha Thai, Yamaguchi Ryoji, Chu Thi Thanh Huong, Nguyen Thi Lan 56 2.3.3. Escherichia coli detection and enumeration Continue isolating E. coli form the purple colonies in Brilliance E. coli/coliform Selective Agar (CM1046, OXOID) by biochemical tests like lactose fermentation in 37 0 C/24h, Indol in 45 0 C/24h, VP – MR in 37 0 C/24h. Counting the number of bacteria by formula above. 2.3.4. Staphylococci detection and enumeration 0.1ml of each dilution of samples was plated onto Baird-Parker agar (BPA) (Oxoid Ltd., Basingstoke, and Hampshire, England) plates and incubated aerobically at 37 0 C for 48h. Representative colonies were Gram stained and those having the characteristic appearance of staphylococci counted and expressed as number of staphylococci/ml. 2.3.5. Clostridium perfringens detection and enumeration 1ml of each dilution of samples was put into Liver Broth (Merk). Incubation happened at 37 0 C for 48h. Use tube with gas inside for TSC agar (Oxoid) and Blood Agar (Merk). Growth bacteria at 37 0 C for 48h anaerobically. Representative colonies black coloured colonies with opaque halo in TSC and cause hemolysis in Blood agar were positive. 2.3.6. Salmonella detection Fresh samples (1 ml) of the tanks water were pre-enriched using lactose broth and the solution incubated aerobically at 42 0 C for 24 h. The samples (1 ml) were then enriched by placing in selenite cystine and tetrathionate broths (9 ml each) and incubating at 42 0 C and 37 0 C respectively for 24 h. Samples were inoculated onto Xylose-lysine-desoxycholate agar (XLD, Oxoid Ltd., Basingstoke, Hampshire, England) and Brilliant green agar (BGA, Oxoid Ltd., Basingstoke, Hampshire, England), incubated aerobically at 37 0 C and examined after 24 h of incubation. Suspected colonies were inoculated onto nutrient agar for biochemical tests (MacFaddin, 2000). 2.4. Data analyse Counts expressed as colony forming units (cfu)/ml were transformed into log10 prior to statistical analysis using the SAS, version 8.1. Data was analyzed statistically using one way analysis of variance for each type of microorganism and differences in counts determined by Fischer’s Least Square difference test. Prevalence was compared by χ 2 - test. All significant differences were determined at P < 0.05. 3. RESULTS AND DISCUSSION 3.1. Enumeration of total aerobic bacteria From table 1, it was showed that 100% samples tested were found aerobic bacteria. The log 10 mean  log 10 SD counted per ml water sample ranged from 3.92  0.59 in poultry slaughterhouse, 3.97  0.45 in pig slaughterhouse to 4.03  0.55 in cattle slaughterhouse. However there were not significant difference about the number of total bacteria in water in each kind of slaughterhouses (P = 0.6422). Total aerobic bacteria criteria have been used to assess the hygiene of processing plants (Lillard et al., 1984). The contamination caused by microbial built up as well as the water source. During slaughtering and subsequent meat processing, the water becomes polluted by livestock manure Moreover, in our study, these slaughterhouses used water from unhygienic water supply (pond and drill well). Beside, the pipe – borne water supply used in slaughterhouses could also be a contamination source of aerobic bacteria to water because it was always put on the floor and be used for next times without any clean or washing methods. One reason was water kept in tank for a long time without tip. 3.2. Prevalence of Coliforms in water samples collected from slaughterhouses Most Coliforms present in intestinal flora of humans as well as other warm-blooded animals, and are found in fecal wastes. As a consequence, Coliforms, which was detected in higher level than other pathogens, are used as an index of the potential presence of entero-pathogens in water environments. The measurement of Coliforms group has been used extensively as an indicator of water quality. Historically, the ability to measure Coliforms in water has translated into the concept of public health protection. As can be seen from table 2 that, there was not a remarkable difference in the prevalence of Coliforms in water in these slaughterhouses (P = 0.3916 > 0.05). Fecal Coliforms were detected in almost samples. The prevalence was 82.5%, 85.0% and 92.5% in Detection of pathogens causing food borne diseases in water 57 poultry, pig and cattle slaughterhouse, respectively. The number of Coliforms were counted at a range of log 10 mean  log 10 SD per 100 ml from 2.23  0.26 at pig slaughterhouse and 2.27  0.40 at cattle slaughterhouse to 2.35  0.40 at poultry slaughterhouse. The level of Coliforms contamination in water collected in slaughterhouses was similar (P = 0.3007 >0.05) with a quite severity. The number of Coliforms isolated from water in studied slaughterhouses was higher nearly 4 times than the Standard of MARD. It reflected that slaughterhouses were not usually cleaned and washed carefully in process of slaughter. These factors created opportunity for feces of animals contaminated to water through tools and practice of worker at slaughterhouse. 3.3. Prevalence of E. coli in water samples collected from slaughterhouses Escherichia coli is commonly found in the lower intestine of warm - blooded animals. Most E.coli strains are harmless, especially, serotype O157:H7 can cause serious food poisoning in humans. Common routes of transmission may include unhygienic food preparation or direct consumption of sewage-contaminated water. Among Coliforms group, the specific determination of Escherichia coli contamination can be performed as one of the best mean of estimation the degree of recent fecal pollution (Edberg et al., 2000). The rate of positive samples with E. coli among 3 kinds of slaughterhouse was not different (P=0.7716, >0.05). The rates were 62.5%, 65.0% and 70.0% in cattle, poultry and pig slaughterhouse, respectively. However, the level of contamination E. coli was different (P<0.0001) among slaughterhouses. In detail, the highest level was in pig slaughters, the log 10 mean ± log 10 SD per 100 ml was 2.07 ± 0.49. Following were poultry slaughterhouses (1.75 ± 0.45) and cattle slaughterhouses (1.43 ± 0.30). There were many opportunities existing at specific points in the slaughter process for E. coli transferred from animals as well as intestine’s content to water such as the level of hygiene, the dressing procedures and the general condition of the plant. That may effect on the overall level of E. coli contamination. Comparison with the Standard of MARD, the level of E. coli contamination in water using in slaughterhouses were higher from 10 to 100 times. Table 1. The result of total bacteria enumeration in water using in slaughterhouse Variable No. of observation Minimum Maximum Mean ± Std Dev Cattle slaughterhouse 40 2.30 5.24 4.03 ± 0.55 a Pig slaughterhouse 40 3.20 4.50 3.97 ± 0.45 a Poultry slaughterhouse 40 2.48 4.64 3.92 ± 0.59 a (P= 0.6422) Table 2. The result of Coliforms isolation in water using in slaughterhouses Positive sample Variable No. of observation No. of sample Rate (%) Min Max Mean ± Std Dev Cattle Slaughterhouse 40 37 92.50 A 1.11 2.96 2.27 ± 0.40 a Pig Slaughterhouse 40 34 85.00 A 1.32 2.79 2.23 ± 0.26 a Poultry Slaughterhouse 40 33 82.50 A (P=0.3916) 1.56 4.36 2.35 ± 0.40 a (P= 0.3007) Truong Ha Thai, Yamaguchi Ryoji, Chu Thi Thanh Huong, Nguyen Thi Lan 58 3.4. Prevalence and enumeration of Staphylococcus aureus in water samples collected from slaughterhouses Staphylococcus aureus is found on human, environment, dust, air, and sewage normally. The bacteria can spread primarily by food handlers using poor sanitary practices. No statistically significant difference was noted for the prevalence and numbers of Staphylococcus aureus in water samples in what kinds of slaughterhouses (P = 0.46 and P = 0.66). This prevalence was ranged from 65.00% at poultry slaughterhouse, 70.0% at cattle slaughterhouse to 77.5% at pig slaughterhouse. In this study, Staphylococcus aureus were isolated from water samples at levels ranging from log 10 mean  log 10 SD 1.43  0.27 (poultry slaughterhouse), 1.48  0.41 (cattle slaughterhouse) to 1.50  0.42 (pig slaughterhouse) per ml. This result was hardly a surprise since workers handle carcasses with their bare hands. Cross- contamination of carcasses with S. aureus constitutes part of the normal flora of the human hand (Ayc- ic-ek et al., 2004). Enterotoxigenic strains of S. aureus exist and there is potential risk of food-borne staphylococcal intoxication. S. aureus intoxications are often associated with institutions such as unhygienic slaughterhouses where animals are often killed in the dirty floor with unhygienic water supplies. While heat processing and normal cooking temperatures are sufficient to kill the bacterial cells, the enterotoxins are stable and not inactivated. The appearance of Staphylococcus aureus in water may be transferred to the carcass, therefore, can not ensure that food from animals which were killed in these slaughterhouses is safe. Table 3. The result of E. coli isolation in water using in slaughterhouse Positive sample Variable No. of observation No. of sample Rate (%) Min Max Mean ± Std Dev Cattle Slaughterhouse 40 25 62.50 A 1.01 2.51 1.43 ± 0.30 c Pig Slaughterhouse 40 28 70.00 A 1.19 2.86 2.07 ± 0.49 a Poultry Slaughterhouse 40 26 65.00 A (P=0.7716) 1.10 2.58 1.75 ± 0.45 b (P <0.0001) Table 4. The result of Staphylococcus aureus isolation in water using in slaughterhouses Positive sample Variable No. of observation No. of sample Rate (%) Min Max Mean ± Std Dev Cattle Slaughterhouse 40 28 70.00 A 0.63 2.51 1.48 ± 0.41 a Pig Slaughterhouse 40 31 77.50 A 0.88 3.20 1.50 ± 0.42 a Poultry Slaughterhouse 40 26 65.00 A (P=0.4647) 0.64 1.94 1.43 ± 0.27 a (P= 0.6643) Detection of pathogens causing food borne diseases in water 59 3.5. Prevalence of contamination Salmonella spp in water samples collected from slaughterhouses Salmonella Salmonella is one of major causes food-borne illness throughout the world. The bacteria is generally transmitted to human through consumption of contaminated food of animal origin, mainly meat and poultry. The symptoms of Salmonella infection usually appear 12 - 72 hours after infection, including fever, abdominal pain, diarrhea, nausea and sometimes vomiting. The results of Salmonella isolation in water using in slaughterhouse were represented in table 5. Salmonella was isolated from 85 out of the 120 samples tested (70.83%). No significant difference was found among in what kinds of slaughterhouses (P = 0.5919 >0.05). The prevalence of contamination Salmonella spp detected from water in poultry slaughterhouse, pig slaughterhouse and cattle slaughterhouse were 65.0%, 72.0% and 75.0%, respectively. The higher prevalence of Salmonella infection of water in pig slaughterhouse (88.77%) was reported by Cedric et al. (2006). Large commercial processing plant may be explained for higher prevalence of contamination Salmonella in this research. In fact, there has been no Standards for Salmonella criteria in water using in slaughterhouse in Vietnam. However, following the Standards of MARD, establishes guidelines for microbiological contamination in fresh meat: Salmonella should be absent in 25 g of sample. Consequently, nobody can ensure that no carcass or fresh meat contaminated by Salmonella before it’s comes to consumer with prevalence of Salmonella infection in water at slaughterhouses above. 3.6. Prevalence of Clostridium perfringens in water samples collected from slaughterhouses Clostridium perfringens Clostridium perfringens are Gram-positive, anaerobic sporeforming rods. They are widely distributed in the environment and frequently occur in the intestines of humans and many domestic and feral animals. The spores are capable of surviving in soil, sediments and areas subject to fecal contamination. The spores are also extremely heat resistant and have been reported to survive boiling for several hours. Contaminated meat is usually responsible for outbreaks of Clostridium perfringens food poisoning. In this study, Clostridium perfringens were detected 16/120 samples tested (13.33%). There were not remarkable difference of the prevalence of Clostridium perfringens in water samples collected (P = 0.6036 > 0.05). This prevalence were from 10.0% in poultry slaughterhouse to 17.50% at cattle slaughterhouse. And as regards to pig slaughterhouse, the rate was 12.50%. These results reflected that there was contamination from feces of animals to water at slaughterhouses. The results obtained here indicated that microbiological contamination of water in the slaughterhouses in Hanoi was high. The water was contaminated with both anerobic bacteria like Clostridium perfringens and aerobic bacteria such as Salmonella, E. coli, Staphylococcus aureus. These bacteria are considered a risk to human health. Practice in slaughterhouse may explain for the high prevalence of bacteria isolated in Vietnam. At slaughterhouse, animals were slaughter in the floors which was very dirty. In addition, viscera were also done in this place because almost slaughterhouses in Vietnam were not divided to separated areas for each production chain. Cross-contamination bacteria from feces, sewage in the floor to water may also have occurred as a result of using the tools or basin to take water in the tanks and then put these tools in the dirty floor. These tools will be used for the next time without clean or disinfection. Beside, animals were carried from different sources, a part of them were not kept in quarantine by veterinarian. Moreover, at slaughterhouses, animals were usually killed when they were dirty, hungry and thirsty. Therefore, when Ministry of Agriculture and Rural Development (MARD) mission visited one of these slaughterhouses in Hanoi, the leader had said that “This area only is gathered for slaughter and not slaughterhouse” because both of inside and outside of this area were very dirty and unhygienic. These processing plants cannot guarantee that whether the products will be free of bacterial pathogens, ever effort must be made to decrease the incidence of pathogens and therefore reduce the potential risks for the consumers. Truong Ha Thai, Yamaguchi Ryoji, Chu Thi Thanh Huong, Nguyen Thi Lan 60 Table 5. The prevalence of Salmonella in water using in slaughterhouse Positive sample Negative sample Variable No. of observation No. of samples Rate (%) No. of samples Rate (%) Cattle Slaughterhouse 40 30 75.00 a 10 25.00 Pig Slaughterhouse 40 29 72.50 a 11 27.50 Poultry Slaughterhouse 40 26 65.00 a 14 35.00 Total 120 85 70.83 (P=0.5919) 35 29.17 Table 6. Test results of Clostridium perfringens isolation in water using in slaughterhouse Positive sample Negative sample Variable No. of observation No. of samples Rate (%) No. of samples Rate (%) Cattle Slaughterhouse 40 7 17.50 a 33 82.50 Pig Slaughterhouse 40 5 12.50 a 35 87.50 Poultry Slaughterhouse 40 4 10.00 a 36 90.00 Total 120 16 13.33 (P=0.6036>0.05) 104 86.67 4. CONCLUSION The quality of water using in slaughterhouses in Hanoi, Vietnam was alarmed. Detection some pathogens caused food borne disease from these water samples indicated that the prevalence of these bacteria was much higher than the set of Standard. Especially, the rate of Salmonella and Clostridium perfringens were high compare to the Negative Requirement of Standard. The main reasons of the situation were activities and practice of workers in slaughterhouses as well as the sanitary condition in slaughterhouses. This problem may lead the contamination for carcass and from that it will be the source of food borne diseases. Acknowledgement The present studies were supported by the special fund of Hanoi University of Agriculture for young researchers. The author would like to thank the students and technicians who help with sample collection and processing and colleagues at Miyazaki University for their helps. We also thank to the hosts of slaughterhouses and individuals participated in this study who offered us great cooperation in our field collection. We would like to express our deep gratitude to them for their kindness and helps. REFERENCES Aycicek, H., Aydogan, H., Kucukkaraaslan, A., Baysallar, M., Basustaoglu, A.C., (2004). Assessment of the bacterial contamination on hands of hospital food handlers. Food Cont. 15, 253–259. CDC (Centers for Diseases control and Prevention)., January, 10, (2005). http://www.cdc.gov/ncidod/dbmd/diseaseinfo/fo odborneinfections_g.htm#howmanycases. 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Detection of Salmonella spp. in Retail Fresh Food Samples from Vietnam and Characterization of Their Antibiotic Resistance. American Society for Microbiology, Vol. 73, No. 21, p. 6885-6890. . Water used in small slaughterhouses is considered as one of the sources caused the contamination for carcass in food processing, especially, in un-hygienic conditions of small slaughterhouses. hours after infection, including fever, abdominal pain, diarrhea, nausea and sometimes vomiting. The results of Salmonella isolation in water using in slaughterhouse were represented in table. MARD, the level of E. coli contamination in water using in slaughterhouses were higher from 10 to 100 times. Table 1. The result of total bacteria enumeration in water using in slaughterhouse