International Journal of Food Microbiology 134 (2009) 163–175

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International Journal of Food Microbiology

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Review Biological risks associated with consumption of reptile products

Simone Magnino a,⁎, Pierre Colin b, Eduardo Dei-Cas c, Mogens Madsen d, Jim McLauchlin e, Karsten Nöckler f, Miguel Prieto Maradona g, Eirini Tsigarida h,1, Emmanuel Vanopdenbosch i, Carlos Van Peteghem j a Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna “Bruno Ubertini”, Sezione Diagnostica di Pavia, Strada Campeggi 61, 27100 Pavia, Italy b Université de Bretagne Occidentale, Ecole Supérieure de Microbiologie et de Sécurité Alimentaire, Technopôle Brest-Iroise, 29280 Plouzané, France c Parasitology-Mycology Service, Microbiology Department, EA3609 Faculty of Medicine, Lille 2 University, University Hospital Centre & IFR-142 Lille Pasteur Institute, France d Technical University of Denmark, DIANOVA, Incuba Science Park Skejby, 102 Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark e Health Protection Agency Food, Water & Environmental Microbiology Network, London WC1V 7PP, United Kingdom f Bundesinstitut für Risikobewertung, Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277 Berlin, Germany g Department of Food Hygiene and Technology, Veterinary Faculty, University of León, 24071 León, Spain h Scientific Panel on Biological Hazards, European Food Safety Authority, Largo N. Palli 5/A, 43100 Parma, Italy i Veterinary and Agrochemical Research Centre, Groeselenberg 99, B-1180 Brussels, Belgium j Faculty of Pharmaceutical Sciences, Laboratory of Food Analysis, University of Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium article info abstract

Article history: The consumption of a wide variety of species of reptiles caught from the wild has been an important source Received 27 March 2009 of protein for humans world-wide for millennia. Terrapins, snakes, lizards, crocodiles and iguanas are now Received in revised form 25 June 2009 farmed and the consumption and trade of their meat and other edible products have recently increased in Accepted 4 July 2009 some areas of the world. Biological risks associated with the consumption of products from both farmed and wild reptile meat and eggs include infections caused by bacteria (Salmonella spp., Vibrio spp.), parasites Keywords: (Spirometra, Trichinella, Gnathostoma, pentastomids), as well as intoxications by biotoxins. For crocodiles, Reptiles Salmonella spp. constitute a significant public health risk due to the high intestinal carrier rate which is Meat fl Salmonella spp. re ected in an equally high contamination rate in their fresh and frozen meat. There is a lack of information Spirometra about the presence of Salmonella spp. in meat from other edible reptilians, though captive reptiles used as Trichinella pets (lizards or turtles) are frequently carriers of these bacteria in Europe. Pentastomids Parasitic protozoa in reptiles represent a negligible risk for public health compared to parasitic metazoans, of which trichinellosis, pentastomiasis, gnathostomiasis and sparganosis can be acquired through consumption of contaminated crocodile, monitor lizard, turtle and snake meat, respectively. Other reptiles, although found to harbour the above parasites, have not been implicated with their transmission to humans. Freezing treatment inactivates Spirometra and Trichinella in crocodile meat, while the effectiveness of freezing of other reptilian meat is unknown. Biotoxins that accumulate in the flesh of sea turtles may cause chelonitoxism, a type of food poisoning with a high mortality rate in humans. Infections by fungi, including yeasts, and viruses widely occur in reptiles but have not been linked to a human health risk through the contamination of their meat. Currently there are no indications that natural transmissible spongiform encephalopathies (TSEs) occur in reptilians. The feeding of farmed reptiles with non-processed and recycled animal products is likely to increase the occurrence of biological hazards in reptile meat. Application of GHP, GMP and HACCP procedures, respectively at farm and slaughterhouse level, is crucial for controlling the hazards. © 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 164 2. Examples of farming systems for reptiles reared for meat ...... 164 2.1. Crocodilia ...... 164 2.2. Squamata ...... 165 2.2.1. Snakes ...... 165 2.2.2. Iguanas ...... 165

⁎ Corresponding author. Tel.: +39 0382 422006; fax: +39 0382 422066. E-mail address: [email protected] (S. Magnino). 1 Present address: Directorate of Research and Nutrition Policy, Hellenic Food Safety Authority, 124 Kifissias Av. & 2 Iatridou str., 11526 Athens, Greece.

0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.07.001 164 S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175

2.3. Testudines ...... 166 2.3.1. Soft-shelled terrapins ...... 166 2.3.2. Sea turtles ...... 166 3. Biological hazards ...... 166 3.1. Bacteria ...... 166 3.1.1. Salmonella spp...... 166 3.1.2. Vibrio spp...... 167 3.1.3. Other bacteria ...... 167 3.2. Fungi ...... 167 3.3. Parasites ...... 167 3.3.1. Protists and related eukaryotic microorganisms ...... 167 3.3.2. Metazoa ...... 167 3.4. Viruses ...... 171 3.5. Prions ...... 171 3.6. Biotoxins ...... 171 4. Exposure assessment ...... 171 5. Conclusions ...... 172 Acknowledgements ...... 172 References...... 173

1. Introduction as liver. Risks associated with other reptile products, such as skins, carapace, blood, eggs and medicinal products will only be briefly The class Reptilia includes numerous extinct taxons and four considered. Risks due to viruses, prions, bacteria, fungi, parasites, and extant orders: Crocodilia (crocodiles, caimans, alligators, gharials), biotoxins are considered, that are either present in the product at the Testudines (turtles, tortoises, terrapins), Squamata (lizards, geckos, time of slaughter or become a contaminant, regardless of the means iguanas, snakes) and Sphenodontia (tuatara), the latter being an by which they may be affected by storage, processing or retail of the endangered species which is not to be considered further. With product. The assessment of public health concerns is based on a ‘farm regard to the consumption of a wide variety of species of reptiles to fork’ approach and considers risks derived from farming practices caught from the wild, turtles (meat and eggs) are probably the most including the settings, housing, diet, veterinary interventions, meth- heavily exploited worldwide. In 1835 when in the Galapagos ods of slaughter, processing and retailing practices. Information Archipelago on the HMS Beagle, Charles Darwin reported meeting concerning the farming of reptiles for meat production, the biological a party of Spanish sailors who were salting giant tortoise meat to eat hazards associated with reptile meat, and data on consumption pat- on board ship, and when exploring inland “lived entirely upon terns is often fragmentary and not readily available. As a consequence, tortoise meat: the breast plate roasted… with flesh on it is very a risk profile approach will be presented in this paper. good; and young tortoises make excellent soup” (Darwin, 1854). Terrapins, crocodiles, snakes, iguanas and lizards are still today 2. Examples of farming systems for reptiles reared for meat locally important food sources (Klemens and Thorbjarnarson, 1995). The increasing demand of some reptile meat (e.g. from terrapins, Reptiles are farmed for human consumption in various parts of the crocodiles, caimans, alligators and iguanas) in some regions has world, including crocodiles in Zimbabwe, Papua New Guinea, resulted in the development of breeding programs in more than 30 Australia and England, alligators in North America, snakes in Asia countries in North, Central and South America, Africa, Asia and and North America, iguanas in Central and South America, and turtles Australia. Therefore, the consideration of food-borne disease from and terrapins in China, Japan and South-East Asia. Reptiles have many reptiles is becoming more important (Hutton and Webb, 2003). different feeding habits, and although some, such as the land tortoises, Reptiles can be carriers of a variety of disease causing agents and are completely vegetarian, most of them have a varied diet feeding on their meat can become contaminated depending on the housing, some form of animal life depending on the habitats they occur. The feeding, and hygienic practices under which they are reared or diet of reptiles therefore includes arthropods, insects, molluscs, slaughtered. For reptiles reared in aquatic environments, the quality amphibians, birds, mammals, fish, or other reptiles. For example, of water in which animals are raised is also important. However, alligators are almost totally carnivorous, and in the wild, small animals hygienic measures at farm level, slaughtering and processing will eat snails, frogs, insects, and small fish. As they become larger, procedures can significantly reduce the risk of a foodborne infection alligators will eat fish, turtles, snakes, waterfowl, small mammals and for consumer after consumption of meat originating from farmed even smaller alligators. reptiles. This review is based on the preparatory work of an ad hoc Working 2.1. Crocodilia Group set up for drafting an opinion subsequently issued by the Scientific Panel on Biological Hazards (BIOHAZ Panel) of the European The Nile crocodile (Crocodylus niloticus) is native to Africa, may Food Safety Authority (EFSA) on public health risks involved in the reach up to 7 m in length and is reared in many countries including human consumption of farmed or ranched reptile meat (EFSA, 2007a). Kenya, Zimbabwe, Tanzania, South Africa, Israel, Indonesia, France, The scope of this review is a wider assessment of biological risks that Japan and Spain, with a licence for its farming for meat awarded for the are associated with the consumption of reptile products, although first time in the UK in 2006. Other crocodilians are also commercially edible products derived from wild reptiles, either legally or illegally reared, in particular the saltwater crocodile (Crocodylus porosus) which caught and traded (the latter case, also known as “bush meat”) have may reach 9 m in length and is farmed in Australia, Papua New Guinea, not been considered in detail, unless relevant for risks that might Thailand and other countries, the freshwater crocodile (Crocodylus occur with farmed reptiles. The term of “reptile meat” is limited to johnstoni), also farmed in Australia, and a hybrid between the saltwater muscle tissue, blood confined to muscle vasculature, bone and bone crocodile and the Siamese crocodile (Crocodylus siamensis), which is marrow, and any other tissues (for example fat) that may be farmed extensively in Thailand and Cambodia. The American alligator considered inseparable from muscle, as well as internal organs such (Alligator mississippiensis) is typically about 4.5 m long and is farmed in S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 165

Southern US (Georgia, Florida, Texas, Louisiana) mostly for the indeed to ensure and preserve the quality of the skin. Contamination production of hides for leather products, but with an established of the meat is likely because the skin is valuable and must be removed market also for meat. The equivalent for crocodiles and alligators in carefully. Because the skin does not ‘peel’ off easily, crocodiles must be Central and South America are caimans. Among them, the species with skinned on a flat surface, which provides greater opportunity for the widest distribution is Caiman crocodilus, which is extremely contamination of the meat (Madsen et al., 1992). Meat harvested for adaptable to all lowland wetland and riverine habitats, and thus has human consumption almost exclusively comprises the tail and the become established from Mexico to Peru and Brazil. In comparison to dorsal fillets, while the rest of the carcass may be fed back to the other crocodilians, caimans are smaller, with males and females crocodiles. generally reaching 2.5 m and 1.5 m, respectively. Caimans supply the vast majority of the hide market in South America, and are also 2.2. Squamata exploited for their meat. Sustainable programs for these reptiles have been developed in Latin America, namely in Venezuela for C. crocodilus, Different species of iguanas and snakes are farmed and traded for and in Brazil and Argentina for the related species C. yacare (Larriera their meat, mostly in America and Asia. Farming of large lizards also and Imhof, 2006; Thorbjarnarson and Velasco, 1999). occurs, e.g. in Argentina where the omnivorous tegus (Tupinambis Rearing of crocodiles is performed in two ways, namely by spp.) have recently begun to be reared for their skins and meat, ranching or farming. Ranching depends on the presence of a whereas other large carnivorous lizards, such as the monitor lizards, sustainable wild population, as eggs or hatchlings which are collected are not farmed because they are considered difficult to feed and to from natural habitats and reared to harvest size in captivity. raise economically (NRC, 1991). Collections are carefully regulated by quotas and collectors' permits by wildlife authorities, and replenishment of the wild population 2.2.1. Snakes ensures restocking of a certain percentage of the animals reared in Farming of snakes is intended for different purposes such as venom captivity. Thus, permit holders in Zimbabwe must return 5% of the collection (for antivenom production), skin and meat production. There crocodiles reared in captivity to natural habitats (Child, 1984). Farm- are few reports available on commercial snake farms, e.g. in the central ing is based on captive breeding and although it provides better Terai district of Bara in central Nepal, where snakes are kept for the opportunities for controlled management, is more demanding on the production of venom, meat and skin in order to meet the demand in the provision of proper physical facilities for breeding animals, and international market. These goods are exported to many European and management of adults of considerable body sizes. Farming is less Asian countries and to Australia. New animals are provided to the farm favoured by wildlife conservationists, as it may exist as a closed from “snake collection centres” that operate in national parks and production system independent of (and with no particular interest in) wildlife reserves and are responsible for breeding. The snakes are sustainable natural crocodile populations. In practice, the crocodile supplied with water and fed with chicken and frogs at weekly or even industry is based on a mixture of ranching and farming. The main monthly intervals. Snakes (including rattlesnake and python) are also source of rearing stock is derived from collection of eggs or hatchlings farmed in the US for the production of meat for human consumption and from natural habitats, but many operators also supplement their stock are available in the EU market. Reticulated python (Python reticulatus) with offspring from captive breeding animals. Common for the two farms have become established over the last 20 years in many Asian systems is the incubation of fertilized eggs under controlled countries, in particular in Southeast Asia. Although the main interest of temperature and humidity conditions, the rearing of young hatchlings farming this species is for skin production, the meat is also harvested and in separate heated ponds, and the grow-out of rearing stock in earthen highly prized as a delicacy. Burmese pythons (Python molurus bivittatus) or concrete ponds to harvest size which is usually 2–3 years. The are farmed in Southeast Asia (Vietnam). Meat from this latter species is survival rate under captive rearing may approach 75% of viable eggs, currently imported into EU. while estimated survival rates in the wild are ~5% due to predation of eggs and hatchlings (Hutton and van Jaarsveldt, 1987). 2.2.2. Iguanas Crocodiles are carnivores, and their diet must be based on animal Traditionally, iguanas have been used as a source of food for more protein which may come from both aquatic and terrestrial animals. than 7000 years in part of Central and South America. However, due to Attempts to substitute animal protein partly or in full with vegetable excessive hunting of both animals and eggs and destruction of their sources have generally been unsuccessful, resulting in poor growth, tropical forest habitat, wild populations have become extinct in some runting and mortalities particularly in young hatchlings. However, countries and have hugely decreased in others. Breeding programs recent attempts of feeding pelleted diets to crocodiles have been originally intended to raise animals for their release back into the wild successful (Peucker and Jack, 2006). The natural diet of newly hatched have progressively developed in several countries, including Panama, crocodiles is insects, larvae etc. which animals must be taught to eat, Costa Rica, Guatemala, Nicaragua, Belize, Honduras, El Salvador, e.g. by farm attendants feeding insects, or hand-feeding with small Colombia, and Venezuela (Eilers et al., 2002). Further to the pieces of e.g. minced meat. After some months, crocodiles are fed conservation purpose, farming of iguanas has become an economical small fish which form the main part of the natural diet, that is only attractive alternative to cattle farming and a significant source of food occasionally supplemented with larger birds and mammals. The diet for local populations. of captive crocodiles at slaughter age usually reflects the available Two species are farmed for producing meat for human consump- animal protein sources that are locally available. In Zimbabwe, the tion: the green iguana (Iguana iguana), which is the most popular main diet is composed of kapenta fish supplemented with game meat species for farming, and the black iguana (Ctenosaura spp.). Iguana (particularly elephant) from hunting and culling (Hutton and van reproduction occurs in captivity as eggs are laid in artificial nests, Jaarsveldt, 1987), while the diet consists almost exclusively of fish in which increases the survival rate of hatched eggs to 90% and more Thailand and Australia (Suvanakorn and Youngprapakorn, 1987). In compared to 50% in the wild. Hatchlings are raised on farm until they Israel (Ben-Moshe, 1987) and also in England, culled poultry may are 6–10 months old, then they are released into forested areas close constitute 50% or more of the diet, and this practice carries a further to the farms until they are of marketable size. This takes about two risk of introducing pathogens such as Salmonella (especially S. years. Iguanas are herbivorous, and in the wild they feed primarily on Enteritidis) to the reptilian microflora. leaves, flowers and fruits. When farmed, they are fed with a mixed diet For public health purposes, it is important to note that crocodiles (“iguana chow”) consisting of broken rice, animal protein (meat, bone are primarily reared for their highly valued skins (leather), while meat and fish meal), fruit (e.g. papayas, mangos, bananas, avocados), leaves is usually a by-product. The main concern in carcass preparation is and flowers. 166 S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175

2.3. Testudines Hawksbill turtles (Erethmochelys imbricata) are also farmed, although globally in smaller number. Although a completely closed aquaculture Although the generic terms “turtle” and “chelonian” may be used system has been attempted, an ‘Ocean Pasture Plan’ was described by for all members of the order Testudines, different names are usually Ikenoue and Kafuku (1992b). This involved either collection of eggs from used for turtles that live in different habitats: terrapins are freshwater the wild where the eggs are laid in unfavourable conditions (and where turtles that live in fresh and brackish waters (rivers, pond, lakes), a high level of mortality occurs) or the catching of mature adult females while tortoises are land-dwelling species, and sea turtles (also known and placing them in breeding ponds adjacent to artificial nesting as marine turtles) inhabit marine waters. Most information concern- grounds. Hatchlings are collected and the animals are raised in large, ing farmed species for meat production refers to terrapins. well aerated seawater tanks and fed on pelleted sea turtle food (“turtle Several terrapin and turtle species are commercially reared for chow” formulated with ideal rations of fiber, fat, protein and nutrients) meat, including (most importantly) the Chinese soft-shelled terrapin as well as on fish, fish offal, squid, seaweed, and vegetables. Turtles are (Pelodiscus sinensis) which is farmed in China, Japan, Thailand, grown to 40 kg after about 2 years (fully grown adults are about 200 kg) Malaysia, the diamondback terrapin (Malaclemmys terrapin) in the and slaughtered on farm. The meat and internal organs are frozen, while US, and the green sea turtle (Chelonia mydas) in the Cayman Islands, the plastron is processed into the ingredients for canned turtle soup. The Suriname, Japan and Réunion. The green sea turtle is also farmed for carapace, skin and scutes are processed for ornaments (Ikenoue and leather and decorative products. The soft-shelled terrapin and the Kafuku, 1992b). Export of meat from sea turtles is prohibited under the diamondback terrapin compete on the market with the wild-caught CITES Treaty. The meat can be consumed only by the local population animals (Wood, 1991). In general, hard-shelled turtles are less and is sold in the domestic market. attractive for farming, in comparison with soft-shelled terrapins, due to their slow growth. 3. Biological hazards

2.3.1. Soft-shelled terrapins This section deals with biological hazards for which there is Soft-shelled terrapins inhabit warm ponds, marshes, lakes and evidence for a demonstrable public health risk from consumption of rivers and feed on crustaceans (e.g. crabs, shrimps) as well as small reptile meat. fish and snails. They grow to a maximum of 5 kg and can live for more than 50 years. Animals over 8 years are used as parents and are only 3.1. Bacteria obtained from cultivated sources. Mating takes place in May to June and females spawn 3–4 times each year laying clutches of eggs in 3.1.1. Salmonella spp. specially designed sandy pens. Eggs hatch after 50 to 60 days and Salmonella spp. are considered the major bacterial hazard that may newly hatched terrapins are 3–4 g and take about three years to grow occur in reptile meat since this genus of bacteria occurs as commensals to 500 g when they are ready for market. The animals are cultured in of their intestinal flora (Minette, 1984; Zwart et al., 1970). Intestinal freshwater ponds 3–33 m2 with sandy beds with water to a depth of carriage rates of salmonellae exceeding 50% in asymptomatic reptiles 20–40 cm and are segregated on size since cannibalism is common. have been reported (Chiodini and Sundberg, 1981; Geue and Loschner, Ponds are oxygenated by inclusion of algae (Microcystis spp.) and 2002; Woodward et al.,1997). Although the occurrence of salmonellae unless heated, induce the animals to hibernate in the winter. The in reptiles kept as pets (mainly iguanas, turtles, and snakes) or farmed younger animals are fed mainly with water fleas and polychaetes for the supply of the pet market has been repeatedly documented in which propagate naturally in the pond. Larger animals are fed with the US (CDC, 1992a,b, 2003, 2007) and in Europe (Geue and Loschner, solid food (shrimp, boiled egg, minced fish, meat and specially 2002; Schroter et al., 2004), limited information is available about the formulated feeds) placed on a ramp outside the water. Sulphona- presence of salmonellae in reptiles, other than crocodilians, farmed for mides, antibiotics and vaccines can be added to the feed (Ikenoue and the production of meat for human consumption. Studies on salmo- Kafuku, 1992a). nellae in crocodiles are few, but consistently report a very common The Chinese soft-shelled terrapin (P. sinensis) is cultivated in Japan, occurrence of a great number of serotypes in clinically healthy farmed Korea, China, Taiwan, Thailand, Malaysia, Vietnam and Indonesia. Lake as well as in wild crocodiles (Madsen et al., 1998; Manolis et al., 1991; and river turtles are also cultivated in Japan, Taiwan, and Korea for Obwolo and Zwart, 1993; van der Walt et al., 1997). The rates of meat for human consumption (Silpachai, 2001). The largest produc- intestinal carriage of Salmonella spp. have been reported as 16–27%, tion is of soft-shelled terrapins, 94% of which exports come from and the identified serotypes cover a broad range within S. enterica Southern China. However this is a growing market: for example, the subspecies enterica, salamae, arizonae and diarizonae. Salmonellae production of soft-shelled terrapins in Taiwan grew from 22 to were recovered from cloacal swabs collected from 4 out of 29 farmed 3782 tons between 1992 and 1998, in China from 4400 tons to alligators and from 2 out of 71 wild alligators in Texas and Louisiana 92,000 tons between 1993 and 2000 and in Japan from 25 to 700 tons (Scott and Foster, 1997). Salmonella was also detected in 8 out of 20 between 1970 and 1990 (Ikenoue and Kafuku, 1992a; Li, 2001; hatchlings, 3 out of 20 adults and 7 out of 16 egg surfaces of farmed Silpachai, 2001). The popularity of the Chinese soft-shelled terrapins green iguanas in El Salvador (Mitchell and Shane, 2000). for commercial farming depends on many characteristics, such as its Studies on crocodile meat (Madsen,1993, 1996; Manolis et al., 1991) rapid growth rate and high annual reproductive output, the consumer have documented the common presence of salmonellae in both fresh interest and the wide experience on the condition of its farming chilled and frozen meat for human consumption. In Australia, (CITES, 2003). Harvest of soft-shelled terrapins occurs by a number of salmonellae were recovered from 16% of fresh carcasses of farmed C. different routes including transport of live animals, or on farm killing. johnstoni and C. porosus, while similar studies on fresh and frozen meat The carcasses are processed and the meat can be frozen or canned. of farmed C. niloticus in Zimbabwe revealed recovery percentages of 20– Recycling of carcass residues is likely. 33%. Serotype distribution showed a great variety within S. enterica subspecies enterica, salamae and diarizonae. Although many of these 2.3.2. Sea turtles serotypes may be considered as types rarely or never associated with Artificial hatching and rearing of several species of marine turtle is human disease, 40% or more of isolates belong to subsp. enterica, which practiced in various tropical regions, almost exclusively for conservation. comprises potential human pathogens, e.g. S. Typhimurium and S. However, there is certainly a tradition of meat production from the Enteritidis. Limited data are available on the presence of Salmonella spp. green sea turtle (C. mydas) in different parts of the world such as in the in the meat of other reptilians. Salmonella Chester has been isolated in Grand Cayman Island, Réunion and in the Ogasawara Islands of Japan. Australia from marine turtle meat (O'Grady and Krause,1999) and more S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 167 recently, S. Typhimurium was isolated in Japan from snapping turtle reptiles are rarely recognized. Skin mycoses are not relevant in a meat (Fukushima et al., 2008). Salmonellae isolated from alligator meat perspective of reptile consumption, therefore only deep-seated in Florida have been recently typed as S. Anatum and S. Baildon (Xia mycoses are considered here. Deep-seated mycoses are usually not et al., 2009). transmissible among animals, except for pneumocystosis, which is a Salmonella contamination of crocodile meat occurs on the surface strictly host-species specific mammalian infection (Dei-Cas et al., although no quantitative data are available. This contamination is due 2006). Reptiles, like humans, usually acquire deep-seated fungal to the common intestinal carriage of Salmonella spp. (Chiodini and infections from the soil, which is the major reservoir. Although several Sundberg, 1981; Foggin,1987; Madsen et al.,1998; Manolis et al.,1991; fungal species involved in reptilian deep-seated mycoses can be also Obwolo and Zwart, 1993; Zwart et al., 1970), and the resulting high involved in human disease, no risk from reptile products consumption crocodile pond water concentrations of Salmonella spp. (Madsen, could be identified. 1994), both of which act as important sources of meat contamination Microsporidia, which were recently assigned to the group of Fungi, during slaughter and dressing. There is a high degree of handling are recognized pathogens of reptiles, especially for weakened animals during skinning, where great care is taken to avoid damage to the (Graczyk and Cranfield, 2000). Microsporidian species pathogenic to valuable skin and consequently less attention is paid to possible humans have not been identified in reptiles hitherto, and no data contamination of the meat (Madsen et al., 1992). Since high rates of suggesting microsporidiosis transmission from reptiles to humans intestinal carriage of Salmonella spp. often occur among reptiles, in the have been reported to the authors' knowledge. absence of qualitative and quantitative data, it is likely that meat from species other than crocodiles presents a similar hazard. 3.3. Parasites Cases of human salmonellosis (due to S. Montevideo, S. Thompson, S. Paratyphi B and S. Typhimurium) have been reported in Japan 3.3.1. Protists and related eukaryotic microorganisms following consumption of raw blood, viscera and raw meat as well as Reptiles host a variety of enteric unicellular pathogens, including cooked meat of the soft-shelled terrapin Trionyx sinensis japonicus those in the genera Blastocystis, Cryptosporidium, Eimeria, Entamoeba, (Fukushima et al., 2008; K. Kubota, National Institute of Health Sciences, Giardia and Isospora, as well as blood Apicomplexa (Hepatocytozoon Tokyo, personal communication). An outbreak of salmonellosis with 36 spp., Haemogregarina spp.) (Bhattacharya et al., 1988; Ladds and Sims, human cases occurred in 1998 in an Aboriginal community in Northern 1990; Lainson et al., 2003; Noel et al., 2005; Upton and Zien, 1997; Australia after consumption of the meat of a green sea turtle, which is a Xiao et al., 2004). However, the majority of these protozoa are specific popular food source in coastal communities in the Pacific(O'Grady and for reptiles and are of no public health importance. Krause,1999). The potential for contamination of other reptile species is Xiao et al. (2004) detected Cryptosporidium parvum and Cryptos- illustrated by cases of systemic infection (some of which were fatal) by poridium muris (both human pathogens, the latter very rarely Salmonella enterica subsp. arizonae in the US following consumption of reported) in the faeces of lizards and snakes, but they concluded dried rattlesnake meat used as a medicinal product among Hispanic that the parasites had probably infected the rodents ingested by those communities (Bhatt et al., 1989; Kelly et al., 1995; Riley et al., 1988; carnivorous reptiles. Cysts or oocysts from other protozoan parasites Waterman et al., 1990). No information has been provided as to the (including the above mentioned, plus additional species such as Tox- origin of the meat, either from a wild or farmed rattlesnake. oplasma gondii and Cyclospora cayetanensis) may contaminate reptile meat if harvested from water contaminated by other animals' faeces. 3.1.2. Vibrio spp. As to the tissue cysts of T. gondii, they are not found in reptile meat, Turtles and terrapins and water from their breeding pools have since the life cycle of the parasite is restricted to warm-blooded been identified as the samples most contaminated by Vibrio cholerae animals (mammals and birds). In conclusion, reptile meat has not in an investigation on 12,104 seafood and aquatic products from 12 been implicated with transmission of protozoa to humans. provinces of China (Zhang et al., 2007). Cholera toxin producing V. cholerae (non-O1) and Vibrio mimicus were also detected in soft- 3.3.2. Metazoa shelled terrapins in Japan (K. Kubota, National Institute of Health Cestodes, acanthocephalans, nematodes, leeches, pentastomids and Sciences, Tokyo, personal communication). arthropods are frequently found parasitizing reptiles, some of which Consumption of raw eggs from Olive Ridley sea turtles (Lepidochelys (e.g. cestodes, nematodes, and pentastomids) are pathogenic to their olivacea) was associated with an outbreak among 33 hospitalised reptile hosts (especially under captive conditions) and can be patients with severe diarrhoea associated with cholera toxin producing transmitted to humans. This is the case for sparganosis and pentasto- V. mimicus in Costa Rica (Campos et al., 1996). V. mimicus was also miasis that can be contracted by humans through snake meat isolated from the sea turtle eggs collected both from a market and consumption (Dei-Cas, 1996), and for trichinellosis which can develop directly from the wild (Campos et al., 1996). after ingestion of meat from monitor lizards, and possibly from other reptilians (crocodiles). 3.1.3. Other bacteria Most published information about bacterial genera other than 3.3.2.1. Cestodes: Spirometra. Spirometra is a genus of pseudophylli- Salmonella and Vibrio refers to their detection in reptiles kept as pets dean cestode tapeworms that reproduces in canines and felines or in zoos. Although some of those genera (e.g., Aeromonas, Myco- (definitive hosts) and requires two intermediate hosts (Beaver et al., bacterium, Chlamydia) have been also isolated from wild, captive and/ 1984). The first intermediate host is a copepod (planktonic crusta- or farmed reptiles (Ariel et al., 1997; Gorden et al., 1979; Huchzer- cean) of the genus Cyclops which ingests the coracidium (free-living, meyer et al., 2008), there are limited or no data concerning their ciliated embryos) that develops from Spirometra eggs in water presence in reptile meat and other products (Madsen, 1993, 1996). contaminated with the faeces from infected definitive hosts. In the Consequently, the relevance of such bacterial genera for public health tissues of the copepod, the coracidium turns into the first-stage larva, following consumption of reptile products is considered very low or or procercoid. When a second intermediate host ingests an infected even negligible. copepod, the procercoid develops into a second larval form, the plerocercoid or sparganum. The plerocercoid larva can be harboured 3.2. Fungi by many vertebrates, including amphibians, reptiles, birds, small mammals (rodents and insectivores), humans, other primates, and Superficial or systemic fungal diseases have been reported in swine which may function as paratenic or transport hosts by feeding captive reptiles (Cheatwood, 2000), however mycoses in free-living on animals infected with encysted plerocercoids that pass through the 168 S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 intestinal wall and migrate to other tissues (Denegri and Reisin, 1993; but they are more common in Asia, particularly in Korea, China, Japan, Khahil et al., 1994). In the intestine of the definitive hosts, the Taiwan, Vietnam, and Thailand (Beaver et al., 1984). In Korea, 3 human plerocercoid or sparganum attaches to the mucosa where it matures cases were reported for the first time in 1924 (Kobayshi, 1925), but into an adult cestode of about 25 cm length (in the case of S. more than 100 cases were subsequently documented before the end of mansonoides) within 10 to 30 days. The adult cestode generally does the '80s (Min, 1990). not affect the health of the host, but in cats it may produce weight loss, Foodborne sparganosis cases have been traced to the consumption irritability, and emaciation, together with an abnormal or exaggerated of snake meat but not to crocodiles, lizards or turtles. Plerocercoid appetite (Hendrix, 1995). The infection of the second intermediate larvae have the ability to migrate to any part of the human body (Cho host by plerocercoid or sparganum can be clinically apparent when et al., 1975; Min, 1990), including the brain (Anders et al., 1984) and the number of ingested larvae in the intestinal tract is large and the oral cavity (Iamaroon et al., 2002). However, based on the analysis especially when they invade other organs (Acha and Szyfres, 1994). of 135 cases reported in the Republic of Korea up to 1987, preferred Spirometra is found throughout the world. Infections in the Far East sites include the abdomen (38 cases; 28.1%), urogenital organs (30; are due to plerocercoid larvae of S. mansoni, in the United States to S. 22.2%), extremities (24; 17.8%), central nervous system (16; 11.9%), mansonoides, in Europe to S. europaei, in Africa to S. theileri, and in chest (14; 10.4%), the orbital region (11; 8.1%) and breast (2; 1.5%) Australia to S. erinacei. However, the identification of Spirometra (Min, 1990). species can be difficult and the taxonomy is uncertain. Among reptiles, The incubation period of sparganosis is not well defined, and the snakes are important intermediate hosts. For example, 91% of 1240 parasite is thought to live up to 20 years in the human host. Clinical snakes captured between 1980 and 1988 in Ehime Prefecture, Japan, signs of sparganosis vary according to the tissues and organs into were infected with S. erinacei (Sato et al., 1992). In another study, S. which the parasite migrates, and the deriving inflammation and pain mansoni was recovered in 61% of 100 snakes from 11 of 13 localities of may persist after the death of the sparganum. When the sparganum southern Korea examined during 1981 and 1982 (Cho et al., 1982). invades the subcutaneous tissue, a nodule will form under the skin, Snakes also play the most important role, in regard to reptile meat, as and the lesion will be usually referred to as “creeping tumor” due to infection source for acquiring sparganosis. the parasite migrating to other sites through the tissues. A severely The majority of human foodborne sparganosis cases have been painful lesion may develop in case of inflammation and edema of the recognized following the consumption of undercooked meat of either tissues around the eye. snakes or frogs. Sato et al. (1992) reported that the number of Seroepidemiological observations in the normal adult population plerocercoids collected from snakes ranged from 0 to 427 per snake and in epileptic patients revealed prevalences of 1.9% and 2.5%, with a median of 12 (for Elaphe quadrivirgata), and from 0 to 130 with respectively, in Korea (Kong et al., 1994). Ultrasonographic findings a median of 9 (for Rhabdophis tigrinus tigrinus). The parasite burden in may also be useful for the pre-operative diagnosis of breast or other snakes increased with age, and it was suggested that E. quadrivirgata organs sparganosis (Cho et al., 2000; Kim et al., 2005). had more parasites than R. tigrinus tigrinus mainly because the former had a longer life span. Although freezing of snake meat would 3.3.2.2. Nematodes: Trichinella. Nematode worms belonging to the probably destroy plerocercoids, there is no data on the appropriate genus Trichinella have a very broad range of host species (mammals, treatment to prevent foodborne sparganosis from consumption of birds and reptiles) including humans. They have been detected in all snake meat. continents except Antarctica (Pozio, 2007b). Natural Trichinella infec- In crocodile meat, S. erinacei was detected in Australia in 1987 in tions have been reported in more than 100 species of mammals, seven the meat of two freshwater crocodiles (C. johnstoni)(Bodger and avian species, and three reptile species (Pozio, 2005). At present,12 taxa Goulding, 2003; Melville, 1988), and spargana were also found in the with eight species and four genotypes are recognized in the genus meat of dwarf crocodiles (Osteolaemus tetraspis) at markets in the Trichinella, namely Trichinella spiralis (T1), T. nativa (T2) and its related Democratic Republic of the Congo (Huchzermeyer, 1997). However, genotype Trichinella T6, T. britovi (T3) and its related genotype Trichi- Spirometra larvae have not been found in more recently farmed nella T8, T. pseudospiralis (T4), T. murrelli (T5) and its related genotype crocodiles, which may be due to changes in the management systems Trichinella T9, T. nelsoni (T7), T. papuae (T10), and T. zimbabwensis since the parasite was first identified. Although gourmet restaurants (T11) (Pozio, 2007a). Trichinella T12 is a new genotype recently usually prefer fresh (chilled) rather than frozen meat, crocodile meat detected in a mountain lion from Argentina (Krivokapich et al., 2007). must be frozen to eliminate the risk of tapeworm (S. erinacei) Since all taxa are morphologically indistinguishable at all develop- infection (Bodger and Goulding, 2003). As an example, according to mental stages, only biochemical or molecular methods can reliably the Australian regulation for the export control of fish and fish identify the genotype of the parasite (Pozio and La Rosa, 2003). products (Federal Register of Legislative Instruments F2006C00346), The life cycle of this viviparous nematode parasite includes two the meat of a crocodile exposed to or suspected of being infested by S. generations in the same host. After ingesting the meat of an infected erinacei must (a) immediately after processing be placed in a host, first-stage larvae are released following gastric digestion in the refrigeration chamber and (b) be held at a temperature of minus new host, subsequently reach the duodenum and, while embedded in 12 °C or cooler at the thermal centre for a minimum of 5 days; or the intestinal mucosa, undergo four moults to develop into the adult subjected to such other temperature controls that achieve the stage within 2 days. Following mating of male and female larvae, the destruction of all viable S. erinacei in the crocodile meat. In the latter females start to deliver newborn larvae 5 to 7 days post infection. case, the applicable approved arrangement must validate that: (a) the Newborn larvae migrate directly into lymphatic and blood vessels of alternative temperature controls will be achieved; and (b) the way in the host, subsequently penetrate into striated muscle cells by the use which the controls are to be applied will be efficient to destroy all of a stiletto apparatus and lytic enzymes, and develop to infective viable stages of S. erinacei in the crocodile meat. In this way, the risk of muscle larvae as early as 17 days post infection (Capo et al., 1998). sparganosis after consumption of crocodile meat is negligible. Larval metabolism is basically anaerobic which favors the survival of Human sparganosis is caused by the ingestion of procercoid larvae the parasite in decaying tissues (Despommier, 1990), where hypobio- from unfiltered water contaminated with copepods harbouring the tic first stage larvae are maintained until they are ingested by a new parasite (Cho et al., 1975), or by the ingestion of plerocercoid larvae in host. Within several weeks, an immune mediated host response raw or insufficiently cooked meat of reptiles (or amphibians), or by affects the viability of the adult parasites, which results in their traditional medicine remedies, e.g. by topically applying snake or frog expulsion from the intestine (Pozio, 2007a). skin as poultices to eyes which can result in ocular sparganosis (Cho Genetically and biologically, Trichinella species and genotypes are et al., 1975; Min, 1990). Sparganosis cases are reported world-wide, classified into two distinct clades, with the main criterion being the in S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 169 vivo presence or absence of an intramuscular collagen capsule are highly susceptible to infection with several Trichinella species and surrounding the parasite (Zarlenga et al., 2006). One clade comprises genotypes. Thus, although trichinellosis cases have not been reported for all species that encapsulate in host muscle tissue of mammals only (T1 all species and genotypes so far, all trichinellae are considered to be to T9, and T12), while the other one includes those worms that do not pathogenic for humans. Whereas infections with few Trichinella larvae encapsulate after muscle cell invasion and that can also occur in birds can remain asymptomatic, higher larval burdens can cause gastro- (T4) and reptiles (T10 and T11) (Pozio and Murrell, 2006). intestinal as well as generalized clinical signs and symptoms such as Data from several studies suggest that body temperature in the fever and myalgia which are directly related to the development of the host plays a key role for the infectivity of Trichinella. According to parasitic cycle in the human host. A minimal infectious dose of 100 to results from experimental infections in mammals, birds and reptiles, 300 larvae can cause clinical disease in humans (Dupouy-Camet and the non-encapsulated species T. papuae and T. zimbabwensis can Bruschi, 2007). Differences as to signs, symptoms, and clinical course develop at temperatures ranging from 26 °C to 40 °C, in both cold- and have been observed, but it is not known whether they are due to the warm-blooded animals (i.e. reptiles and mammals). In comparison, different Trichinella species and genotypes involved (Bruschi and the non-encapsulated species T. pseudospiralis can develop at 37 °C to Murrell, 2002; Dupouy-Camet and Bruschi, 2007). 42.5 °C (i.e. mammals and birds), and all of the encapsulated species Trichinellosis in humans is associated with the consumption of raw are adapted to a body temperature of 37 °C to 40 °C (i.e. mammals) or undercooked contaminated meat. Consumer habits such as the (Pozio et al., 2004a). Therefore, experimental infections of reptiles consumption of traditionally prepared food based on raw or under- such as Texas horned lizards (Phrynosoma cornutum) and common cooked meat or derived products play an important role in the caimans (Caiman sclerops) with T. spiralis, which is well adapted to the epidemiology of the disease. Conversely, when a population uniquely body temperature of warm blooded animals except birds, were not consumes well cooked meat, trichinellosis cases are lacking or very successful at their usual activity temperature of 20 °C to 28 °C (Jordan, rare, despite a persisting transmission among wildlife (Pozio, 2007b). 1964; Kapel et al., 1998), but conversely 1 month post infection, viable Till now, only two human trichinellosis outbreaks due to the con- larvae were recovered from lizards when kept at 37 °C (Jordan, 1964). sumption of reptile meat from a monitor lizard (Varanus nebulosus) In 1995, Trichinella was detected for the first time in farmed Nile and a turtle (unidentified species) have been documented in Thailand crocodiles (C. niloticus) in Zimbabwe (Foggin et al., 1997). At this time, (Khamboonruang, 1991), possibly due to infection with T. papuae, out of 29 farms examined, 18 (62.1%) were positive for Trichinella although the involved nematode species could not be confirmed muscle larvae with 256 (39.1%) out of 648 animals being infected. The (Pozio et al., 2007). feeding of meat from other crocodiles slaughtered at the farms was Since meat of reptiles may be infectious for humans, specific identified as a potential infection source. Although a program for the control measures have been implemented for consumer protection. control of Trichinella was established, 11 (40.7%) of 27 farms in Several countries including Australia prescribe the systematic inspec- Zimbabwe had infected crocodiles 7 years later (Pozio et al., 2007). tion of crocodile meat for Trichinella larvae (Anonymous, 2000a). Studies on the etiological agent revealed a new non-encapsulated According to the legislation of the European Union, namely Regulation species, namely T. zimbabwensis, which infects both poikilothermic (EC) No. 2075/2005, all reptile meat intended for human consump- vertebrate species and mammals including primates (Pozio et al., tion has to be examined for Trichinella larvae with one of the approved 2002). Later studies were conducted in wild monitor lizards (Varanus methods (European Community, 2005). niloticus) and wild Nile crocodiles in Zimbabwe and Mozambique, Meat of reptiles not subjected to Trichinella meat inspection should respectively. T. zimbabwensis was found in 5 (17.6%) of 28 monitor be processed using methods recommended by the International lizards from an area close to a crocodile farm which was known to rear Commission on Trichinellosis (ICT) in order to inactivate the parasite infected crocodiles. In one monitor lizard from Victoria Falls, the larval (Gamble et al., 2000). Freezing is an efficient method to kill the larvae, burden ranged from 4 to 8.3 larvae per g of examined muscles. In 8 if the meat is frozen for 20 days at −15 °C (5°F), 10 days at −23 °C (20%) of 40 crocodiles from Zimbabwe and in one farmed crocodile (−10°F), or 6 days at −29 °C (−20 °F), provided that the meat is less from Ethiopia, non-encapsulated larvae were identified. Larval density than about 15 cm (6 in.) thick. Cooking is another post-harvest in muscle samples of four examined wild crocodiles from Zimbabwe processing method for inactivating muscle larvae. The ICT recom- ranged between 2 and 42 larvae per g (Pozio et al., 2007). mends that fresh meat should be cooked to an internal temperature of After discovery of the non-encapsulated species T. papuae in wild at least 71 °C (160°F). Inactivation of Trichinella larvae can depend on and domestic pigs in 1999, 46 farm- and 72 wild-borne saltwater several factors such as salt concentration, moisture, and temperature, crocodiles (C. porosus) from Papua New Guinea were tested for Tri- hence consumer protection by salting, drying, smoking, or preserving chinella. Whereas all farm-borne crocodiles were negative, 16 (22.2%) crocodile meat is not reliable (Gamble et al., 2000). wild-borne crocodiles were positive for T. papuae with an average larval burden of 7 larvae per g of muscle (Pozio et al., 2004a). 3.3.2.3. Nematodes: Anisakidae. Ascaridoidea nematodes of the Besides field studies in reptiles, experimental infections have family Anisakidae, genera Contracaecum, Anisakis and Pseudoterranova demonstrated that other reptile species, especially caimans (C. are intestinal parasites from fish-eating birds, Cetaceans (dolphins, sclerops) and varans (Varanus exanthemicus), and to a lower degree porpoises, whales) or Pinnipedians (seals, sea lions, walruses), turtles (Pelomedusa subrufa) and pythons (P. molurus bivittatus) are respectively. Females shed undeveloped eggs in host faeces, then eggs susceptible to T. zimbabwensis and T. papuae (Pozio et al., 2004b). embryonate and hatch in water releasing free-swimming larvae that are After inoculation of 3000 larvae per animal, the average larval burden ingested by crustaceans such as euphausiaceans, decapods, copepods for T. zimbabwensis was 1, 7, 15 and 589 larvae per g in the muscles of and amphipods, where they localize within the haemocoel (Chai et al., pythons, turtles, caimans and varans, respectively, while for T. papuae, 2005; Dei-Cas, 1996). Definitive avian or mammalian hosts may acquire the larval density calculated for the aforementioned reptile species the infection directly by ingesting infected crustaceans. However, was 0.5, 8, 30 and 1074 larvae per g, respectively. infected crustaceans are more frequently ingested by teleostean fish Since both T. zimbabwensis and T. papuae can be easily transmitted or cephalopod molluscs. Predators of infected fish or cephalopods can in to mammals, the discarded parts of crocodile carcasses should be turn get infected and play the role of paratenic or transport hosts. properly destroyed to avoid infection of synanthropic animals, and the Anisakidae larvae (10–30 mm×1 mm) penetrate the intestines and waste products should not be fed to domestic animals unless the meat invade internal tissues to become the source of infection for definitive is frozen or cooked before use (Pozio et al., 2004b). bird or mammalian hosts or for humans, who constitute dead-end Contrary to animals, where susceptibility to natural infection can hosts. In fish, anisakid larvae can be found inside the gastro-intestinal remarkably vary depending on the Trichinella species involved, humans tract, in the pleuro-peritoneal cavity, liver, gonads or embedded in 170 S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 muscles (Mudry et al., 1986). These larvae are infectious either to were recently assigned to the Crustacean group of Maxillopoda, which definitive hosts (piscivorous birds or mammals) or to other fish-eating includes also copepods, cirripeds, ostracods and other organisms vertebrates such as larger fish, sea turtles (Burke and Rodgers, 1982)or (Lecointre and Le Guyader, 2001). Pentastomids are worm-like even crocodiles, which are potential paratenic hosts. Ichthyophagous arthropoda, also known as ‘tongue worms’, measuring 1–10 cm length reptiles, like most crocodiles, are probably the most exposed reptiles to (with females larger than males) that dwell in the rhino-pharynx and anisakid larval infections. Contracaecum larvae have been in fact sinus cavities of snakes, crocodiles and numerous wild or domestic reported in crocodilians (Acha and Szyfres, 1994; Goldberg et al., carnivorous mammals (Dei-Cas, 1996). 1991; Moravec, 2001). These findings suggest that consumption of raw Armillifer life cycle involves transmission between snakes (defini- or lightly cooked anisakid-infected crocodile meat could cause tive hosts) and their prey, i.e. rodents (intermediate hosts). Eggs anisakiasis in humans. The risk to humans could be relatively low containing the infective larva or nymph are shed by adult pentasto- when crocodiles are born in farms and fed with artificial food or frozen mids in the upper respiratory tract of the snake, and are disseminated fish (−20 °C for more than 24 h). Apart from crocodiles, anisakid in the environment by sneezing, expectoration or, after swallowing, parasitism does not seem to be usual in reptiles though anisakid larvae through the faeces. After ingestion by the intermediate hosts, the egg have been also found in the green sea turtle (C. mydas)(Burke and hatches in the gut and releases the nymph that has 4 typical hook- Rodgers, 1982). shaped appendices. The nymph can then pass through the intestinal In humans, anisakid larvae can penetrate the gastric or intestinal wall and invades abdominal or thoracic organs such as lymphatic wall where they may induce eosinophilic granulomas. In some cases, nodes, liver, spleen or lungs. Nymphs reach the infectious stage 250– the larvae migrate to the peritoneal cavity and invade adjacent organs 350 days after the infection of the intermediate host (Acha and (liver, pancreas) or, more rarely, distant organs (Acha and Szyfres, Szyfres, 1994). They measure 5 mm length and are similar to the adult 1994; Dei-Cas, 1996; Mudry et al., 1986). The infection can be life cycle stage. When the definitive host ingests the nymphs with the asymptomatic or, alternatively, cause disease of varying severity tissues of its prey, nymphs leave the stomach and migrate to the upper depending on both the number of larvae and the intensity of the respiratory tract, where they develop into sexually mature female and inflammatory response. Recent works showed that human disease males. Mating takes place and females begin egg production. associated with allergic reactions against anisakid larvae also occurs In crocodiles, two pentastomid families occur: Sebekidae, with the (Daschner et al., 2005), even when larvae are dead. Thus, anisakid genera Sebekia, Leiperia, Alofia, Selfia and Agema, and Subtriquetridae larvae might represent a neglected cause of allergic diseases in with only one genus, Subtriquetra (Anonymous, 2000a). The life cycle Europe. However, despite the occurrence of this parasite in reptiles, of these pentastomids is not well known. Fish may play the role of human disease has not yet been linked to reptile meat consumption. intermediate hosts as suggested by the finding of Subtriquetra larvae in two Cichlidae species (Junker et al., 1998) from South Africa, which 3.3.2.4. Other nematodes. Eustrongylides spp. (Nematoda: Diocto- are often predated by the Nile crocodile (C. niloticus). In Northern phymatoidea) are intestinal parasites of aquatic birds. Their larvae can areas of Australia, pentastomid larvae were identified in the rainbow be found in tissues of fish, amphibians, reptiles and humans. In these fish (Melanotaenia maccullochi) and in perchlets (Ambassis sp.) hosts, Eustrongylides larvae (25–150 mm×2 mm) are able to cause (Anonymous, 2000a). In the New World, the fish Gambusia affinis,a intestinal perforation (Eberhard et al., 1989), or visceral lesions well known predator of mosquito larvae, is the intermediate host of at (Beaver et al., 1984). The zoonotic potential of these larvae, which are least two pentastomid species of alligators (Anonymous, 2000a). found in crocodiles (Acha and Szyfres, 1994; Fang et al., 1991; In crocodiles, pentastomids can cause either sub-clinical infection Goldberg et al., 1991; Junker et al., 2006) is significant. Fish-eating or severe disease (Buenviaje et al., 1994). Tissue migration of nymphs crocodiles and other fish-eating vertebrates such as water snakes and encysting of larvae in different organs and muscles can induce (Bursey, 1986) or humans (Eberhard et al., 1989; Narr et al., 1996) may mild or severe lesions according to the number of parasites, host acquire the infection from infected fish. Humans may also become susceptibility and occurrence of other underlying diseases. The adult infected by consuming raw or lightly cooked infected reptile meat, pentastomids are able to cause severe lesions, and even to go across though no case has been reported to date. lung tissue and to reach the skin surface through the thoracic wall Gnathostomiasis is a zoonotic parasitic disease caused by larvae or (Anonymous, 2000b). These parasites can cause meningitis, pneu- immature adults of spirurid nematodes of the genus Gnathostoma. monia and significant mortality, especially in young crocodiles in Gnathostomiasis is frequent in the Far East, especially in Japan and captivity. If no intermediate fish hosts are introduced into crocodile Thailand, but it was also reported in Mexico and Central America breeding farms, the circulation of pentastomids in the breeding (Ligon, 2005). Two species are most frequently involved: G. facilities should be self limiting. Conversely, the introduction of spinigerum, from domestic or wild felids, and G. hispidum from wild infected fish to feed crocodiles in Australian breeding farms was boar and pigs (Dei-Cas, 1996). However, two other species have an associated with the emergence of pentastomiasis (Buenviaje et al., increasing impact in humans in Japan (Nawa, 1991): G. nipponicum 1994). Crocodiles might however be infected also with pentastomid and G. doloresi. G. hispidum larvae were recently reported from the eggs from the environment. pit-viper Agkistrodon brevicaudus in Korea (Sohn and Lee, 1998). Humans may get infected with pentastomids by ingesting raw or Gnathostomiasis is usually contracted by ingestion of raw or lightly insufficiently cooked snake or crocodilian meat contaminated with cooked food prepared from freshwater fish, frogs, snakes, birds or the larval stage of the parasite, or by ingesting infectious eggs that mammals. The disease in humans includes a great variety of clinical contaminate the carcasses of infected reptiles, water or food manifestations caused by cutaneous and/or visceral larva migrans (Anonymous, 2000a; Dei-Cas, 1996). Human pentastomiasis is usually syndrome. The development of a cutaneous superficial or creeping caused by Armillifer species (A. armillatus, A. grandis in sub-Saharan eruption, and a migrating erythema, is common. Among visceral Africa; A. moniliformis, A. agkistrodontis in Asia) which are parasites of involvements, neurological forms can be associated with significant pythons or other snakes, or by Linguatula serrata that parasitizes nasal morbidity and mortality (Ligon, 2005). A human case of G. doloresi cavity, frontal sinuses and eardrum cavities of dogs, other canids and infection with the clinical presentation of colonic ileus has been felids. Infection by Armillifer spp. is surely frequent in Africa, referred to the consumption of meat of the snake Agkistrodon halys particularly in the Republic of Congo, but it is usually asymptomatic. (Seguchi et al., 1995). The Armillifer nymphs are preferentially found in the intestinal mucosa, peritoneal cavity and liver (du Plessis et al., 2007), although 3.3.2.5. Crustaceans: Pentastomids. Previously included among the spleen, lungs and conjunctiva can be also affected. The infection, “Pararthropoda”, pentastomids (70 species, mostly parasites of reptiles) usually asymptomatic, can be discovered incidentally at the time of a S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 171 surgical intervention (Tappe and Büttner, 2009). Clinical disease 3.6. Biotoxins occurs when eyes are invaded or if nymphs cause intestinal occlusion. Fatal forms are unusual (Yapo Ette et al., 2003). The histological The term chelonitoxism refers to an intoxication following examination of visceral lesions can reveal the nymphs surrounded by ingestion of the flesh of sea turtles (Champetier de Ribes et al., a granulomatous tissue with eosinophilic abscesses (Dei-Cas, 1996). 1997; Fussy et al., 2007; Gatti et al., 2008; Yasumoto, 1998). Fussy et al. Lesions often evolve towards calcification. (2007) listed 34 incidents of chelonitoxism poisoning in the Caribbean, Indian and Pacific Oceans involving 1880 cases with 267 3.4. Viruses deaths. The hawksbill turtle (Eretmochelys imbricata) and, less frequently, the green sea turtle (C. mydas) are the two species mainly There are several specific viral diseases of reptiles which are not implicated in this type of food poisoning, but the leatherback turtle transmissible to humans. Huchzermeyer (1997) reported that no viral (Dermochelys coriacea) and the loggerhead turtle (Caretta caretta) agent has been recognized as transmissible from crocodilians to may also be involved. The toxic compounds identified to date as the humans. Experimental data suggest that turtles and lizards can be causative agents are lyngbyatoxins, which are accumulated through overwintering reservoirs of some arboviruses that are infectious to the food chain into the flesh of marine turtles following ingestion of humans, such as the Eastern Equine Encephalitis (EEE) virus, the the cyanobacterium Lyngbya majuscula, that grows on seagrass, sand Western Equine Encephalitis (WEE) virus and the Japanese Encepha- and rocky outcrops (Osborne et al., 2001). In addition, other litis (JE) virus (Bowen, 1977; Doi et al., 1983; Oya et al., 1983; Smith unidentified toxins might also be involved, particularly in carnivorous and Anderson, 1980). In addition, two epizootics due to West Nile sea turtles such as E. imbricata. No effect on the health of the sea turtle Virus (WNV) infection occurred in 2001 and 2002 among captive itself has been reported, but all organs of the animal are potentially American alligators (A. mississippiensis) in the US (Miller et al., 2003), toxic regardless of the heat treatment, which suggests that the toxin is and WNV infection was reported in Nile crocodiles reared in a thermoresistant. In humans, clinical signs due to sea turtle poisoning commercial farm in Israel (Steinman et al., 2003). However, involve the upper digestive tract with nausea, vomiting, epigastric arboviruses have not been shown to be transmissible to humans pain, and sometimes diarrhea. General signs such as dizziness, malaise through consumption of meat of infected reptiles, thus the deriving and sweating may also be observed. Recovery may occur over a week public health risk is considered to be negligible. without further signs. Severe cases develop glossitis, dysphagia, drowsiness, multiorgan failure (tubular nephropathy, liver cytolysis, 3.5. Prions respiratory distress) leading to a coma and to high mortality or neurological sequelae in survivors (Aguirre et al., 2006). Breast-fed The prion protein is a normal cellular protein (PrPc) exhibiting children may also get poisoned as the toxins pass into milk (Fussy unique properties, due to its ability of adopting an abnormal et al., 2007). conformation (usually abbreviated as PrPsc, sc from scrapie, the prototype animal TSE) that is associated with transmissible spongi- 4. Exposure assessment form encephalopathies (TSEs) or prion diseases in humans and animals. PrPsc is the main if not only constituent of the infectious TSE Biological hazards of reptile meat either originate from infection or agents or prions. The appearance of a prion disease in cattle (bovine contamination of live reptilians or are a secondary result from spongiform encephalopathy, BSE) and its transmission to humans, contamination at slaughter or through processing. The application of causing variant Creutzfeldt-Jakob disease (vCJD), discloses potential Good Hygienic and Good Manufacturing Practices (GHP, GMP) and impact for food safety if similar events would occur in reptiles. Hazard Analysis Critical Control Points (HACCP) systems, respectively at Currently there are no indications that natural TSEs occur in farm and slaughterhouse level, is crucial for controlling the hazards. The reptiles. However, this is based on no or very limited data. Data on PrP decrease or elimination of hazards in reptile meat may be also sequences are only available in one reptile species, the turtle accomplished with different results through further processing (e.g. (Trachemys scripta), indicating an identity degree to mammalian freezing, cooking) or by applying preservative methods (e.g. drying, prion proteins of about 40% (Simonic et al., 2000), suggesting a high smoking, curing). Adequate cooking is effective for inactivating bacteria species barrier for transmission of mammalian prions to reptiles. and parasites. Freezing will not inactivate bacteria, but is likely to kill Mammalian products are frequently fed to carnivorous reptiles, Spirometra spp., Trichinella spp. and pentastomids, however there is hence intra-species and intra-order recycling via feed could enable incomplete information on the effectiveness and optimal treatments for mammalian TSE agents to establish themselves in animals currently different parasites or reptile species. Spirometra spp. and Trichinella spp. believed to be not susceptible, including reptiles, and for species are killed in crocodile meat if specified temperature–time combinations adaptation of such agents to occur. To this regard and in analogy with (e.g. −12 °C for at least 5 days) are applied. There is no information as to fish, risks may exist, linked to feeding possibly TSE-contaminated freezing conditions for inactivating pentastomids in reptile meat. The feeds to animals currently believed to be not susceptible, including effect of drying, smoking and curing for inactivating bacteria and reptiles. In other words, after eating TSE agents, reptiles might be a parasites in reptile meat will differ according to the specified parameters possible reservoir of the infectious agent, even if not undergoing any of the treatments. Cooking or freezing is unlikely to inactivate marine overt disease. biotoxins occurring in sea turtles. Feeding experiments in rainbow trout indicate that PrPsc remained Reptiles are an important source of protein for human populations no longer than 15 days in the fish intestine and did not cross the in many parts of the world other than Europe and North America, intestinal barrier. This experimental work shows that there is a where consumption is comparatively much lower. In Europe, there is potential risk of residual TSE infectivity in fishmeal produced from fish limited tradition of reptile meat consumption, although there have recently fed with TSE contaminated feed. After oral inoculation of been examples in the past of consumption of some species, such as the scrapie prions, mouse bioassay became positive only in one single Ocellated lizard (Timon lepidus) in some parts of Spain up to few intestinal sample one day after infection, while all other organs decades ago, and turtle soup, which was a delicacy in the UK and in remained negative up to 90 days after inoculation (EFSA, 2007a,b). By other European countries during the 19th and early 20th centuries. analogy with fish, there is a potential risk of residual TSE infectivity in In tropical marine environments, turtles are taken from the wild reptiles acting as temporary carriers when recently fed with TSE and their eggs and meat are consumed in stocks and stews. This is now contaminated feed. Experimental work would be needed to clarify this less common but still occurs in some parts. Turtle soup is also made issue, however the risk of transmission of TSEs is considered unlikely. from the plastron (ventral shell). Soft-shelled terrapin is cultivated 172 S. Magnino et al. / International Journal of Food Microbiology 134 (2009) 163–175 and widely consumed in China, Taiwan, Japan and Korea, either raw or Table 1 prepared as a soup or as broiled meat. In Japan, terrapin meat, eggs Incidents of infections and intoxications associated with consumption of reptile meat and other products. and blood are eaten raw as well as cooked (Campos et al., 1996; Ikenoue and Kafuku, 1992a) and the plastron is boiled and allowed to Hazard Reptile (food type) Country References solidify in Japan and China. Turtle meat is also popular in Southern US Salmonella Soft-shelled terrapin Japan Fukushima et al. (2008); K. (e.g. Louisiana and Florida). spp. (cooked meat and raw Kubota, National Institute Snake is widely consumed in the Far East. In the Republic of Korea, blood, viscera or meat) of Health Sciences, Tokyo, fi personal communication consumption of snake meat is viewed as an aphrodisiac and as eld Green sea turtle Australia O’Grady and Krause (1999) food during military survival training (Cho et al., 1974, 1975; Min, (partially cooked 1990). Rattlesnake is eaten in Southwestern US (Noel et al., 2005) and meat) dried snake meat and snake meat powders are consumed as a Rattlesnake (dried USA, Mexico Bhatt et al. (1989); Kelly meat) et al. (1995); Riley et al. Mexican–American folk remedy (Bhatt et al., 1989). (1988); Waterman et al. Consumption of crocodile, caiman and alligator meat occurs (1990) mostly in Australia, Thailand, South Africa and the US, often where Vibrio mimicus Olive Ridley sea turtle Costa Rica Campos et al. (1996) the meat is a by-product of leather farming of crocodile and alligator (raw eggs) (Millan et al., 1997). Iguanas are widely consumed in Central America, Spirometra Snake (raw or Korea, China, Beaver et al. (1984); Cho spp. insufficiently cooked Japan, Taiwan, et al. (1975); Min (1990), where they are often even preferred to other species' meat. Lizards of meat) Vietnam, Wiwanitkit (2005) various species are traditionally consumed by Australian Aboriginal Thailand communities. Trichinella sp. Monitor lizard, turtle Thailand Khamboonruang (1991); Consumers of reptile meat in Europe and North America mainly (papuae?) (cooked meat ?) Pozio et al. (2007) Gnathostoma Snake (raw meat) Japan Seguchi et al. (1995) belong to two categories: communities of immigrants with traditional doloresi habits of eating reptile meat, and customers of gourmet restaurants or Pentastomids Snake (raw or West Africa, du Plessis et al. (2007); specialty shops that prepare or sell reptile meat as a delicacy. The insufficiently cooked Central Africa, Prathap et al. (1969); Yapo pattern of consumption is likely to differ between these two meat) Malaysia Ette et al. (2003) categories. Imported frozen crocodile, alligator, caiman, rattlesnake Chelonitoxism Sea turtle (cooked Caribbean, Fussy et al. (2007), Gatti meat) Indian and et al. (2008); Yasumoto and python meat are now readily available from specialist meat Pacific Oceans (1998) suppliers in the EU Member States, however since local production may occur in the near future for some of these species, fresh meat may become available. Reptile meat is consumed in a variety of ways, Salmonella spp. in meat from farmed reptilians other than crocodilians. generally following cooking, although dried and raw meat is some- Salmonella spp. constitute a significant public health risk due to the times consumed. Recipes for crocodile carpaccio can be found in the documented high intestinal carrier rate in live crocodilians that is Internet. The meat is generally consumed as whole pieces although reflected in an equally high contamination rate in their fresh and frozen processed meat may be used for preparing sausages and burgers as meat. Parasites causing sparganosis, pentastomiasis, gnathostomiasis well (Bodger and Goulding, 2003). and trichinellosis have been transmitted to humans through consump- tion of contaminated snake meat, and monitor lizard and turtle meat, 5. Conclusions respectively. Other reptiles, e.g. crocodilians, although found to be parasitized by the above parasites, have not been implicated with their The consumption of reptile meat and other reptile products occurs transmission to humans through meat consumption. throughout the world and probably represents similar biological Conversely, consumption of reptile products may be related to hazards to the consumption of meat of any other animal. These hazards biological hazards which pose a negligible risk for consumers. For can occur from pathogenic agents within the meat or other products instance, infections by fungi, including yeasts, widely occur in reptiles (including the reptilian enteric tract), or as a result from cross but have not been linked to the contamination of their meat. Parasitic contamination of food poisoning agents from food contact surfaces or protozoa represent a negligible risk for public health after reptile meat the meat of other species during slaughter or butchery and processing. consumption compared to parasitic metazoa. At present, there is no Although relatively small in number, reports of food poisoning from evidence that viruses infecting reptilians can be transmitted to reptile products illustrate their potential to cause disease which is likely humans through consumption of reptile meat. Currently there are to increase if consumption of these products becomes more common. no indications that natural TSEs occur in farmed reptilians. However, Reported cases of human disease associated with the consumption of by analogy with fish, there is a potential risk of residual TSE infectivity food from reptiles are listed in Table 1. To this regard, it should be noted in reptiles acting as temporary carriers when recently fed with TSE that most probably several cases of disease fail to be linked to the contaminated feed. consumption of reptile products and are consequently underreported. The feeding of reptiles with non-processed and recycled animal The BIOHAZ Panel of EFSA concluded that risks due to the products is likely to increase the occurrence of biological hazards in consumption of reptile meat are related to hazards caused by certain reptile meat. The application of GHP, GMP and HACCP procedures, bacteria (Salmonella spp.) and parasites (Spirometra spp., Trichinella respectively at farm and at slaughterhouse level is crucial for spp., pentastomids) (EFSA, 2007a). Other hazards related to the controlling the hazards. Freezing treatment inactivates Spirometra consumption of reptile products are caused by V. mimicus and by spp. and Trichinella spp. in crocodile meat. However, the effectiveness marine biotoxins which may occur, respectively, in eggs and meat of of freezing for other reptilian meat is unknown. sea turtles, and by Gnathostoma doloresi that may be found in snake meat. Although these hazards are well documented in the literature, Acknowledgements there is limited knowledge concerning other risks which may arise for humans after consumption of reptile meat. Apart from farmed Research for this review was performed by an ad hoc Working crocodilians, there is limited information related to biological hazards Group set up by the BIOHAZ Panel of EFSA. The scientific opinion from meat of other farmed reptilians. “Public health risks involved in the human consumption of reptile Reptiles are well-known reservoirs for Salmonella species, and the meat” was published (http://www.efsa.europa.eu/cs/BlobServer/ risks of getting infected from reptilians kept as pets are well docu- Scientific_Opinion/biohaz_op_ej578_reptile_en.pdf) and provides mented. 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