INCIDENCE OF SALMONELLA IN FISHERY PRODUCTS INTRODUCTION

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1 ' INCIDENCE OF SALMONELLA IN FISHERY PRODUCTS I SIFULAK SUWANRANGSI, KANOKPHAN SRIMATYOBHAS and SUWIMON KEERATIVIRIYAPORN Fish Inspection and Quality Control Division, Department of Fisheries, THAILAND ABSTRACT Contamination of Salmonella in various processed and non-processed fishery products was surveyed during May Samples of fishery products were collected from 72 prbcessors inthe central and eastern reiion of Thailand. Contamination by Salmonella spp. was found in 91 samples (1.74 percent). Twenty two serovars were detected. The top ten serovars found were Salmonella weltevreden, representing 45 percent, S. enteritidis 5 percent, S. senbenberg 5 percent, S. brunei 4 percent, S. anatum 3 percent, S. virchow 3 percent, S. stanley 3 percent, S. hadar 3 percent, S. paratyphi 3 percent and S. caen 3 percent. Products contaminated were frozen raw shrimp 1.6 percent, frozen processed shrimp 1.9 percent, fillets of marine fish 33 percent, frozen freshwater fish 5.8 percent, frozen cephalopods 1.4 and other products 1.2 percent. The sources of the serovars isolated from this survey were not only from fish handlers and process environment, but also from raw material and culture sites, particularly of aquacultured fish and shrimp. INTRODUCTION Frozen fishery products are important exports from Thailand worth at least two billion US dollars each year. Shrimp, cephalopods and fish fillets, in various types of ready to eat and processed products, are the main items. Quality problems, particularly pathogen contamination is still a factor resulting in product detention by some importing regulatory authorities, for instance, the United State Food and k g Administration (USFDA). Frozen fish and shrimp from some Thai exporters have been placed on import alert for suspected adulteration with Salmonella (USFDA 1996). As a result, products are automatically detained at the point of entry and released thereafter if the importers are able to prove that the products are free from Salmonella. This automatic detention procedure has resulted in extra-expenses for sample analysis, storage while awaiting for the results, and above of all loosing competitive opportunities in markets. During , frozen cephalopods from five processors were rejected by the Italian health authority due to Salmonella contamination and subsequently the said processor was banned from exporting to Italy. This incident also impacted the Thai fish industry to a certain extent. Salmonella are found naturally in the intestinal tracts of mammals, birds, amphibians and reptiles but not in fish, crustaceans or moliuscs. Salmonella is able to transfer to seafood through several means, for instance by sewage pollution of the coastal environment, contamination after harvest, or contamination during processing. Bangtrakulnon et al. (1997) reported that Salmonella serovars commonly found in humans were S. enteritidis, S. 1, 4, 5, 12; I:-, S. weltevreden, S. derby, S. typhimurium, S. anatuni, S. agona, S. stanley, S. virchow, S. paratyphi A, and S. typhi. The study also found that S. enteritidis has been increasing in humans and is the most common cause of Salmonella infection. This finding is more or less similar to the report of Gomez et al. (1997) who stated that serovars which commonly cause outbreak of Salmonellosis were S. enteritidis and S. fyphimurium.

2 Seyeral investigations on isolation of Salmonella in animals have been conducted. Bangtrakulnon et al. (1989) reported that serovars naturally found in lizards were S. weltevreden, S. brunei, S. lexington, S. Newport, S. havana, S. derby, S. typhimurium, S. 18,2O:y:-, S. IY43,Z4:Zz3, S. stanley, S. anatum, S. saintpaul, S. agona, S. montevideo, and S. virchow. Sonsanit et al. (1989) also reported serovars commonly found in cockroaches were S. Lexington, S. weltevreden, S. brunei, S. agona, and S. IV43,Z4:&. Moreover, a survey on Salmonella naturally found in reptiles and other animals in Thailand conducted by Bangtrakulnon (1997) indicated that percent of lizards, 4.6 percent of cockroaches and 46.2 percent of rats had Salmonella. The study also stated that serovars isolated from lizards and cockroaches were similar to those found in humans and chicken. In addition, the epidemic study of Salmonella infection from chicken and its products revealed that contamination from this source was as high as 22 percent. The important serovars concerned were S. enteritidis, S. anatum, S. muenchen, S. blockley, S. agona, and S. typhimurium. Bangtrakulnon et al. (1991) isolated'salmonella from different internal organs of poultry such as spleen, intestine, liver, heart, and excrement. A total of different strains were found. The majority were S. Java, S hadar, S virchow, S typhimurium and S blockley. A review of the occurrence of Salmonella in cultured tropical shrimp by FA0 (Reilly et ~1.,1992)~ demonstrated that Salmonella in final products from shrimp aquaculture originate from the environment rather than as a result of poor standards of process hygiene and sanitation. This conclusion co-related with the study of Iyer and Varma (1990) on occurrence of Salmonella in shrimp processing operations. Results were shown as follows: Source Water from culture pond Mud from culture pond Coastal sea water Sea beach sand Process water Ice Floor, pre-processing unit Utensils Lizard droppings Rodent droppings Serotype isolated S. farmsen, S. weltevreden S. newport S. havana S. weltevreden, S. bareilly S. typhimurium S. bareilly S. bareilly, S. weltevreden S. weltevreden S. weltevreden S. weltevreden, S. typhimurium An incidence of Salmonella in frozen fishery products for export from Thailand was studied by Sajjapala et al. (1987). Contamination in shrimp was found to be 1.2 percent, marine fish 2.5 percent, cephalopods 0.8 percent, shellfish 2.7 percent, and freshwater fish 8.5 percent. However, there was no serovar isolation in this study. According to a report of USDA (1988), 40 percent of frozen poultry and its products were found to contain Salmonella. It is accepted that, despite research over the past twenty years, Salmonella incidence in chicken cannot be overcome. It is, therefore, the consumer's responsibility to destroy Salmonella by proper cooking before consumption. The objective of this study is to identify the contamination rate of Salmonella in exported fishery products of Thailand and to locate the sources of contamination which would be of use in establishing appropriate preventive measures for Salmonella.

3 MATERIAL AND METHODS A total of fishery product samples was collected from 72 processors in the central and eastern region from May 1996 to May Samples were transported to the laboratory in insulated containers to keep them frozen. Salmonella analysis was camed out by the procedure of the Compendium of Methods for the Microbiological Examination of Foods (Vanderzant and Splittstoesser,l992). Twenty-five gram of each sample was weighed, pre-enriched with lactose broth and incubated at 35OC for 24 hours. 1 ml of mixture was transferred to 10 ml tetrathionate broth (with brilliant green added) and selenite cystine broth, and incubated at 43 C and 35OC for 18 and 24 hours, respectively. The incubated samples were then streaked onto 3 selective media as follows: 1. Brilliant green agar (BG), incubated hours at 35OC. 2. Bismuth sulfite agar (BS), incubated hours at 35OC. 3. Xylose lysine desoxycholate agar (XLD), incubated hours at 35OC. After incubation, typical characteristics of Salmonella colonies were selected to inoculate into the slants of triple sugar iron agar (TSI) and lysine iron agar (LIA) together with McConkey agar plates. The inoculated media were incubated at 35OC for hours. The positive results of Salmonella were retained for biochemical tests and confirmed by using antiserum. The confirmed culture were further sent to the WHO Salmonella and Shigella Center, Department of Medical Science, Ministry of Public Health for specific serological identification. RESULTS AND DISCUSSION The results of this survey revealed that 91 of samples were found to be positive for Salmonella (1.74 percent) (Table 1). Products positive for Salmonella varied from month to month but included both raw and processed products such as black tiger shrimp, marine fwh, cuttlefish, squid, octopus, freshwater fish, cooked black tiger shrimp, dim sum, fish ball, imitation crab meat, and mixed seafood. August was the month with the highest rate of salmonella incidence (3.3 percent). However, export products were different in each -month. Twenty-two serovars were identified from 91 positive samples. The top ten serovars are ranked in Table 2. S. weltevreden was found to be the principal serovar (45 percent) contaminated in fishery products. h he rest, with a lesser extent, were S. enteritidis (5 percent), S. senftenberg (5 percent), S, brunei (4 percent), S. anatum (3 percent), S. virchow (3 percent), S. stanley (3 percent), S. hadar (3 percent), S. paratyphi (3 percent), and S. caen (3 percent).

4 Table 1. Salmonella contamination in fishery products. Month No. of No. YO Product types Nr May 96 Jun samples detected (& Salmonella) BIT(*,*,*), M/R fish (W) MR fish (*,W,K,W), B/T (W,W), cooked BIT (W), Dimsum (w) Jul fish ball (W), MIR fish (S,W,W,W), B/T(W,W,Z), squid (W,W), imitation crab meat (W) Aw cuttlefish(a,b), mixed seafood (W,W,*), M/R fish(*,h), cooked B/T(W,Br), crocodile meat(v,h), squid(*), F/W fish (E), B/T(W,T,W,W,W), scallops (St), tuna loins(k) S~P mixed seafood (*,W), B/T(w,G,W), processed BlT(W), M/R fish (S), octopus (W), F/W fish (P,W) Oct processed BIT@, crocodile meat (M,Hv,M,A), F/W fish (Ty,V), M/R fish (W), B/T(St,*), F/W shrimp(w,*), imitation crab meat(w) Nov squid@, B/T (W,W), cuttlefish (B,B,S), M/R fish 6) Dec Jan 97 Feb Mar AP~ May Total F/W fish (Pa,*), dimsurn (W,W) processed shrimp (Mb), cuttlefish (*), BIT (*) S/W shrimp (P), F/W fish (E,E) fish ball (Mt) S/W shrimp (W), B/T(S), F/W fish (E) F/W fish (Ty) BIT (W,Ha), crocodile meat (H) *= not identified W = Welteweden K = Kentucky S = Senftenberg A = Anatum B = Braenderup V = Virchow P = Paratyphy B Bio Java BIT = black tiger shrimp F/W fish = freshwater fish E = Enteritidis St = Stanley T = Thomson H = Hadar Br = Brunei G = Give Z= Subspecies IV 43 :Z4 223 Ha= Haardt M/R fish = marine fish S/W shrimp = sea water shrimp M = Muenchen Ty= Typhimwium Hv= Hvittingfoss L = London Pa= Panama Mb= Mbandaka Mt= Montevideo C = Caen

5 Table 2. Top ten serovars isolated from fishery products (expressed as percentage of total Salmonella). Serovars YO S. weltevreden 45 S. enteritidis 5 S. senpenberg 5 S. brunei 4 S. anaturn 3 S. virchow 3 S. stanley 3 S. hadar 3 S. paratyphi 3 S. caen 3 From Table 3, it appears that frozen freshwater fish has the highest contamination (5.8 percent) whereas marine fish, raw black tiger shrimp, and cooked shrimp were found to be at the rate of 2.9, 1.7 and 1.6 percent, respectively. Table 3. Percentage of Salmonella contamination based on product types. Product types Yo Frozen raw black tiger shrimp 1.7 Frozen cooked shrimp Frozen marine fish Frozen freshwater fish Frozen cuttlefish others S. weltevreden was also the main serovar found in contaminated shrimp and marine fish products, wbile S. enteritidis was dominant in freshwater fish and S. Braenderup in cephalopods (Table 4). This finding is similar to a review of Salmonella incidence in aquaculture shrimp by Reilly et al. (1992). The review concluded that S. weltevreden was identified as the principal species found in aquacultured shrimp due to its culture site environment such as mud, pond, water being inherently contaminated with Salmonella.

6 Table 4. Serovars isolated from fishery products. No. of No. Products samples positive Serovars samples I Frozen shrimp 1, black hger shrimp 24 Welteweden(l3), #(5), give, Stanley, Senftenberg, Haardt, Subspecies IV 43:24:223, Thomson. freshwater shrimp Weltevreden, # pink shrimp Processed and cooed Weltevreden, Paratyphi Wdtwd), Caeri, Mhandaka, Bmei shrimp Frozen marine fish fillets Frozen freshwater fish Erozen cqhalopods squid cuttlefish octopus crumbed squid ring Others crocodile meat mixed seafood imitation crab stick fish ball scallop roe off tuna loins (skipjack) - Total 5, # : not confirmed serovars stated without brackets equal to one sample WelteM.edmf'f), S*bpr&), #(2), Hadar, Zbndon,&nmky Enteritidis(d), xyptrimuriurn(2), Weltevrnden, Parmyphi B BEo Java, Vimhow, Panama, # Weltevreden(2), # BraenderupO), Anatum, Senftenberg, P: Weltevreden Caen Muenchen(2), Hadar(2), Virchow, Hvittingfoss, Anatum Weltevreden(3), #(2) Wekeweden(2) Welteweden, Montevideo Stanley Kentucky The incidence of Salmonella in raw material potentially exists in Thailand, since the climate and culture environment are suitable to promote the growth of this organism. Types of raw material used and products including processing environment and transportation conditions also influence the contamination rate of final products. Freshwater fish tends to be more contaminated than others due to its culture condition and the feed used. Contamination of freshwater fish was found to be at the rate of 5.8 percent. S. enteritidis was the majority (4 in 11 positive samples, or 36 percent). S. paratyphi and S. typhimurium were also detected. USFDA reported the incidence of Salmonella in freshwater fish from Thailand in 1988 to be 40 percent of samples taken for analysis. As a result, USFDA have placed this product on an automatic detention. As the matter of fact, freshwater fish are mostly from farms where raw feed such as chicken intestines, other internal organs, and chicken excreta may be used for feeding. There is no doubt about the existence of Salmonella in the culture site, since S. enteritidis is transmitted to fish through chicken manure and intestinal organs as fish feed (USDA, 1988). Additionally, the limitation of water circulation and accumulation of feed in ponds also increases the potential for Salmonella contamination. The only means to eliminate the incidence

7 of Salmonella in the final product is to avoid buying raw material from farms that use the practices mentioned above. For fiozen marine fish, 1.7 percent were positive for Salmonella; S. weltevreden was the most frequent, 7 m 14 positive samples (50 percent), then S. senftenberg, 4 in 14 (29 percent). These serovars could be transmitted to fish through the contaminated coastal sea water, sea birds at the fishing port, or unhygienic practices in fish handling at processing plants. The hygienic practices can be improved by application of HACCP, but contamination from the culture environment is more difficult to control. Salmonella contamination was found in 1.6 percent of raw black tiger shrimp. Again S. weltevreden was the main serovar detected in 13 out of 24 positive samples (54 percent). As noted, sources of S. weltevreden are commonly reptiles and birds, and transfer to shrimp could be during the farming period. Other serovars found were also correlated to those found in various fishery products in the study of Reilly et al. (1992). In comparison with the study carried out by Sajjapala et al. (1987), it was found that the contamination rate of Salmonella in shrimp had increased to 1.2 percent. This is probably because the export of shrimp from aquaculture has been increasing compared to wild caught shrimp. The chance for farmed shrimp to become contammated is higher than for the wild caught product, especially by S. weltevreden, the dominant serovar inherently found in shrimp ponds (Reilly et al., 1992). The shrimp culture environment is one of the main factors affecting the control of Salmonella, as animals such as lizards and birds are commonly seen. A group of experts from FAO/NACA/WHO (1997) concluded that Salmonella contamination in cultured shrimp is mainly from birds and reptiles and is unavoidable. It is not because of unhygienic practices in processing plants. In processed shrimp and cephalopods, 1.6 and 1.4 percent, respectively, were positive with S. weltevreden as the main serovar for processed shrimp (6 in 9 samples, or 67 percent). For cephalopods, the serovars varied, possibly indicating contaminated raw material for shrimp and contamination during processing, due to poor sanitation and personal hygiene, for squid. These consequences are clearly reflected particularly for cooked products. To minimize the incidence of Salmonella in products, many practical preventive measures have been broadly recommended. Examples are listed below. ' 1. Pest control to eliminate S. weltevreden, S, brunei, S. lexington, S. newport, S. havana, S. derby, etc.; 2. Sanitation control to avoid S. weltevreden, S. brunei, S. panama, S. anatum, S. montevideo, S. virchow (Iyer and Verma, 1990); 3. Personal hygiene control to eliminate S. enteritidis, S, paratyphi, S hadar (Bangtrakulnon, 1997); 4. Appropriate temperature control during transportation and on line processing. USFDA (1996) has suggested product temperature should be maintained below 5 C throughout processing, since exposure of raw material above 21 "C for 2 hours could allow Salmonella to grow. CONCLUSION FAO/?JACA/WHO (1997) and Gomez et al. (1997) quote reports of studies on the epidemiology of Salmonella conducted by the health authorities in Europe and America. These reveal that the epidemiological spread of Salmonella from fishery products is low compared to chicken and dairy products. Also that the rate of Salmonella contamination in fishery products was low and hence the risk to public health less. Results of the present study indicate that the contamination rate in finished products is still low at 1.74 percent. S. weltevreden was the dominant serovar found in fishery products, specifically black tiger shrimp and marine f sh, while S. enteritidis was the main serovar in freshwater fish. Other serovars isolated were shown to vary depending on the type of products. However, they were similar to those found in humans, lizards, cockroaches and other animals. These indicate that Salmonella in final products could come both from the

8 processing plants, as a result of unhygienic practices, and from beyond the processors' responsibility such the cultures environment. It must be accepted that no preventative measures can be taken by the processor to achieve absolute control of Salmonella if the organism is present in the raw material as a result of environmental contamination. This is true even if a well managed HACCP system, and QMP, is in place. Howeyer, for raw products that will be cooked before consumption, Salmonella is not a significant hazard and should be accepted as a part of the natural flora as it is in chicken. REFERENCES Bangtrakulnon, A., Bangtrakulnon, S., Manrim, N. and Cheawsilp, D Salmonella contamination in frozen chicken for export. Conference on Diarrhea the 10th. Faculty of Tropical Medicine, Mahidol University, 2 May. (Thai). Bangtrakulnon, A., Bangtrakulnon, S., Pornruhngwong, S., Kaewkangwan, A. and Sooksringam, B Epidemiological of Salmonella in lizards. Journal of Medical Science Department 31(1) Jan-May. (Thai). Bangtrakulnon, A., Manrim, N. and Pornruangwong, S Salmonella analysis in food products : Salmonella incidence in Thailand. Microbiology seminar on Recent Advance in Detection of Pathogenic Bacteria, Merck Ltd. 14 October. (Thai). FAOINACAIWHO Report of A Joint FAOINACAIWHO Study group on food safety issues associated with products from aquaculture by Network of Aquaculture Centers in Asia(NACA), Bangkok, Thailand July. Gomez, T.M., Motarjemi, Y., Miyagawa, S., Kaferstein, F.K. and Stohr, K Foodborne Salmonellosis in: World Health Statistics Quarterly 50(1/2), WHO, Geneva. Iyer, P.G.A. and Varma, P.R Source of contamination with Salmonella during processing of frozen shrimp. Fishery Technology 27,60-3. Reilly, P.J.A. and Twiddy, D.R Salmonella and Vihrio chlolerae in brackish water cultured tropical prawns. International Journal of Food Microbiology, 16(1992) Reilly, P.J.A., Twiddy, D.R. and Fuchs, R.S Review of the occurrence of Salmonella in cultured tropical shrimp. Fisheries Circular No Food and Agriculture Organization of the United Nations. Rome. Sajjapala, T., Solprom, A., Srisomwong, P., Juengmanukul, P. and Wilaipan, P Study on Salmonella incidence in frozen fishery products. Report of the National Seminar on Epidemiology the 5", Ministry of Public Health, Bangkok, Thailand. (Thai). Sonsanit, T., Sooksringam, B. and Bangtrakulnon, A Epidemiology of salmonella in cockroaches. Journal of Srinakarinvirote and Development 1(3), p 26. (Thai). USDA Salmonella and food safety. US Department of Agriculture/Food Safety and Inspection Services. Background Document. Washington, DC. USFDA Foods, Except Dairy Products - Adulteration with Salmonella (CPG ). Sec (Rev. 3/95). Compliance Policy Guides August Department of Health and Human Services, Food and Drug Administration, Washington, DC.

9 USFDA Appendix 4: Bacterial pathogen growth. In Fish and Fishery Products Hazards and Controls Guide, 1" ed Department of Health and Human Services, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Seafood, Washington, DC. Vanderzant, C. and Splittstoesser, D.F. (ed.) Compendium of methods for the microbiological examination of foods, 3d edition. American public Health Association, Washington, DC. World Health Organization World Health Statistics Quarterly. Food Safety and Foodborne Diseases. Vol. 50 No. 112, World health Organization, Geneva.