Prevalence of Salmonella spp. in Pet Reptiles in Japan

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1 NOTE Public Health Prevalence of Salmonella spp. in Pet Reptiles in Japan Aya NAKADAI 1,2), Toshiro KUROKI 3), Yukio KATO 2), Rieko SUZUKI 3), Shiro YAMAI 3), Chiharu YAGINUMA 2), Ryo SHIOTANI 4), Akira YAMANOUCHI 5) and Hideki HAYASHIDANI 1) * 1) Division of Animal Life Science, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo , 2) Department of Public Health II, School of Veterinary Medicine, Azabu University, Fuchinobe, Sagamihara, Kanagawa , 3) Kanagawa Prefectural Institute of Public Health, Shimomachiya, Chigasaki, Kanagawa , 4) Gentleman Loser, Fuchinobe-honcho, Sagamihara, Kanagawa and 5) Yamanouchi Iguana Laboratory, 103 Lions Plaza Sagami-ono, Sagamihara, Kanagawa , Japan (Received 28 May 2004/Accepted 27 August 2004) ABSTRACT. From November 2000 to July 2002, 112 fecal samples from pet reptiles, including 18 turtles, 71 lizards and 23 snakes, sold at a pet shop were examined for the prevalence of Salmonella spp. in Japan. Salmonella spp. were isolated from 83 (74.1%) of 112 samples, and a total of 112 Salmonella isolates were identified as subspecies I to IV. The majority of isolates (62.5%) belonged to subspecies I and 54 isolates could be identified as any of 28 serovars. The predominant serovars were found to be S. Bardo, S. Newport and S. Panama, which cause human salmonellosis. These results indicate that pet reptiles may be a potential infectious source of human salmonellosis in Japan. KEY WORDS: prevalence, reptiles, Salmonella. J. Vet. Med. Sci. 67(1): , 2005 Salmonellosis is known to be one of the most important cause of public health problems worldwide. In Japan, more than 460 cases of human salmonellosis occurred in 2002, and most of them resulted from consumption of foods of animal-origin contaminated with Salmonella [14]. Wild and pet reptiles are generally known to be asymptomatic carriers of several Salmonella serotypes, which are potentially pathogenic for humans. Recently, the number of exotic reptiles has been increasing in popularity as pets and the fact has led to an increase in the number of reptile-associated Salmonella infections in the United States [4 6, 18] and European countries [2, 7 9]. Majority of the patients infected with Salmonella spp. from reptiles are infants, and usually show gastroenteritis, less frequently, fatal meningitis or septicemia [5,7,8]. Recently, many reptiles have also been kept as pets in Japan as well as in other developed countries and it is possible that these animals can be a source of human salmonellosis. However, little is known about the prevalence of Salmonella spp. in reptiles except for aquarium turtle in Japan [13,19]. This study was designed to investigate the presence of Salmonella in pet reptiles, sold and kept in Japan. During the period from November 2000 through July 2002, a total of 112 fecal samples of 50 reptile species consisting of 12 families were collected (Table 1). All reptiles except green iguanas were sold as pets in a pet shop located in Kanagawa Prefecture, and green iguanas were kept at Yamanouchi Iguana Laboratory. Reptiles sold in the shop had been kept in individual cages immediately after they were introduced into the shop. Fecal samples were put into *CORRESPONDENCE TO: HAYASHIDANI, H., Division of Animal Science, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo , Japan. Carry-Blair transport medium (Eiken Chemical Co., Ltd., Tokyo, Japan), and immediately transported to the laboratory in an icebox. The fecal samples were suspended in 3 ml of sterile saline, and 1 ml of the suspension was inoculated into 10 ml of buffered peptone water (BPW, OXOID Ltd., Basingstoke, Hampshire, England). After incubation at 37 C for 24 hr, 1 ml of BPW culture was transferred to 10 ml of Hajna-tetrathionate broth (Eiken). The broth was incubated at 37 C for 24 hr, then one loopful of each tube was inoculated onto a plate of brilliant green agar (BGN, OXOID) supplemented with 20 µg novobiocin/ml, desoxycholate hydrogen sulfide lactose agar (DHL, Nissui Pharmaceutical Co., Ltd., Tokyo) and mannitol lysine crystal violet brilliant green agar (MLCB, Nissui). These plates were incubated at 37 C for 24 hr and three suspicious colonies morphologically similar to Salmonella spp. from each plate were subcultured for biochemical examinations. Biochemical characteristics were examined on triple sugar iron medium (Nissui), lysine indole motility medium (Nissui) and Voges-Proskauer semisolid medium (Eiken). The subspecies of Salmonella isolates was confirmed by biochemical examinations according to Holt et al. [12]. Serotyping of Salmonella isolates was accomplished with commercial O and H antisera (Denka Seiken Co., Ltd., Tokyo) according to the method of Popoff and Le Minor [16]. Salmonella spp. were isolated from 83 (74.1%) of 112 fecal samples of reptiles (Table 1). Salmonella spp. were isolated from 23 (100%) of 23 snakes, 13 (72.2%) of 18 turtles, and 47 (66.1%) of 71 lizards. The isolation rate from snakes was the highest of all. This high proportion of Salmonella infection in captive reptiles, especially snakes, is similar to the results of several previous studies [10, 15, 18]. Geue et al. [10] examined the prevalence of Salmonella in pet reptiles in Germany and Austria, and reported that Sal-

2 98 A. NAKADAI ET AL. Table 1. Distribution of samples examined, number of isolates and serovars of Salmonella associated with reptile species Reptile taxonomy No. of samples (%) a) No. of Subspecies and Serovars Common name Nomenclature isolates (No. of isolates) Order Testdines (Turtles) 13/ 18 (72.2) 20 Family Testdidae Red-footed Tortoise Geochelone carbonaria 0/ 1 (0.0) 0 Indian Starred Tortoise Geochelone elegans 1/ 1 (100.0) 1 Salmonella subsp.i (UT b) ) (1) Leopard Tortoise Geochelone pardaris 7/ 8 (87.5) 11 S. Minnesota (3) S. Montevideo (1) Salmonella subsp.i (UT) (5) Salmonella subsp.iiib (UT) (2) Greace Tortoise Testudo graeca ibera 1/ 1 (100.0) 1 S. Bareilly (1) Central Asian Tortoise Testudo horsfieldi 1/ 1 (100.0) 3 S. Horsham (1) S. 13:z 29 :1,5 (subsp.ii) (1) Salmonella subsp.ii (UT) (1) Southern Spider Tortoise Pyxis arachnoides oblonga 0/ 1 (0.0) 0 Madagascar Flat-shelled Tortoise Pyxis planicauda 0/ 1 (0.0) 0 Family Emydidae Three-toad Box Turtle Terrapene carolina 2/ 2 (100.0) 2 Salmonella subsp.i (UT) (2) Family Chelydridae Snapping Turtle Chelydra serpentina 0/ 1 (0.0) Alligator Snapping Turtle Macroclemys temminckii 1/ 1 (100.0) 2 S. Haifa (1) S. Potsdam (1) Order Squamata Suborder Sauria (Lizards) 47/ 71 (66.1) 57 Family Agamidae Inland Bearded Dragon Pagona vitticeps 15/ 16 (93.8) 16 S. Amsterdam (1) S. Beaudesert (1) S. Kisarawe (1) S. Minnesota (1) S. Rissen (1) S. 16:z 4 : (subsp.iv) (1) Salmonella subsp.i (UT) (3) Salmonella subsp.ii (UT) (5) Salmonella subsp.iv(ut) (2) Egyptian Spiny-tailed Agama Uromastyx aegypticus 1/ 1 (100.0) 1 Salmonella subsp.ii (UT) (1) Indian Spiny-tailed Agama Uromastyx hardwickii 3/ 4 (75.0) 3 Salmonella subsp.i (UT) (3) Family Iguanidae Green Iguana Iguana iguana 3/ 12 (25.0) 3 Salmonella subsp.iv (UT) (3) Family Scincidae Monky-tailed Skink Corucia zebrata 0/ 1 (0.0) 0 Pygmy Spiny-tailed Skink Egernia depressa 1/ 2 (50.0) 1 Family Cordylidae Rough-scaled Plated Lizard Gerrhosaurus major 2/ 2 (100.0) 2 S. Amsterdam (1) Family Helodermatidae Banded Gila Monsters Heloderma S. cinctum 1/ 1 (100.0) 1 S. Poona or Farmsen c) (1) Reticulated Gila Monsters Heloderma S. Suspectum 1/ 1 (100.0) 2 S. Alachua (1) Family Varanidae Gray s Monitor Varanus olivaceus 1/ 1 (100.0) 3 S. Haifa (1) Roughneck Monitor Varanus rudicllis 1/ 1 (100.0) 2 S. Kentucky (1) S. Schwarzengrund (1) Family Gekkonidae Texas Banded Gecko Coleonyx brevis 1/ 2 (50.0) 1 S. Beaudesert (1) Central American Banded Gecko Coleonyx mitratus 0/ 2 (0.0) Malayan Bow-fingered Gecko Cyrtodactylus pulchellus 0/ 1 (0.0) Leopard Gecko Eublepharis macularius 1/ 5 (20.0) 1 S. Amsterdam (1) Japanese Gecko Gekko japonicus 1/ 1 (100.0) 2 S. Bardo (1) Continued on following page.

3 SALMONELLA FROM PET REPTILES 99 Table 1. Continued. Reptile taxonomy No. of samples (%) a) No. of Subspecies and Serovars Common name Nomenclature isolates (No. of isolates) Indian House Gecko Hemidactylus flaviridas 1/ 1 (100.0) 1 Masobe Madagascar Ground Gecko Paroedura masobe 1/ 1 (100.0) 1 S. Enteritidis (1) Madagascar Ground Gecko Paroedura pictus 6/ 9 (66.7) 8 S. Hvittingfos (1) S. Potengi (1) S. Soananina or Sundsvall c) (2) Salmonella subsp.ii (UT) (2) Carter s Rock Gecko Pristurus rupestoris 1/ 1 (100.0) 1 S. Stanley (1) Crested Gecko Rhacodactylus ciliatus 3/ 3 (100.0) 4 S. Braenderup (2) Salmonella subsp.iv (UT) (2) Carrot-tailed Viper Gecko Teratolepis fasciata 2/ 2 (100.0) 2 S. Panama (2) Fantastic Flat-tailed Gecko Uroplatus phantasticus 1/ 1 (100.0) 2 S. Muenchen (1) Order Squamata Suborder Ophidia (Snakes) 23/ 23 (100.0) 35 Family Boidae Children s Python Antaresia childreni 1/ 1 (100.0) 1 Salmonella subsp.ii (UT) (1) Garden Tree Boa Corallus hortulaus 2/ 2 (100.0) 3 S. Kentucky (1) Salmonella subsp.iiib (UT) (2) Kenyan Sand Boa Gongylophis colubrinus 1/ 1 (100.0) 1 Green Python Morelia viridis 1/ 1 (100.0) 1 S. Amsterdam (1) Family Colubridae Japanese Ratsnake Elaphe climacophora 1/ 1 (100.0) 1 Japanese Four-lined Ratsnake Elaphe quadrivirgata 1/ 1 (100.0) 1 Great Plains Ratsnake Elaphe guttata emori 1/ 1 (100.0) 1 S. Bardo (1) Corn Snake Elaphe guttata guttata 2/ 2 (100.0) 5 S. Midway or Florida c) (1) S. Newport (2) S. Othmarschen (1) Salmonella subsp.ii (UT) (1) Greenish Ratsnake Elaphe obsoleta obsoletax 1/ 1 (100.0) 2 S. Midway or Florida c) (1) Salmonella subsp.iiia (UT) (1) Amur Ratsnake Elaphe schrenchi 1/ 1 (100.0) 2 S. Newport (1) Salmonella susbp.iiib (UT) (1) Beauty Snake Elaphe taeniura 1/ 1 (100.0) 2 S. Bardo (1) Blotched Kingsnake Lampropeltis getula goini 1/ 1 (100.0) 1 California Kingsnake Lampropeltis getula californiae 5/ 5 (100.0) 8 S. Newport (1) S. Othmarschen (2) Salmonella subsp.iiia (UT) (1) Salmonella subsp.iiib (UT) (4) Desert Kingsnake Lampropeltis getula splendida 1 /1 (100.0) 1 Pueblan Milksnake Lampropeltis triangulum camphelli 1/1 (100.0) 1 Sinaloan Milksnake Lampropeltis triangulum sinaloae 1/1 (100.0) 2 S. Bardo (1) San Diego Mountain Kingsnake Lampropeltis zonata pulchra 1/ 1 (100.0) 2 S. Bardo (1) S. Newport (1) Total 83/ 112 (74.1) 112 a) No. of positive samples/no. of samples examined (%). b) UT = untypable. c) Serovar was not typed completely. monella was isolated from 45.5%(86/189) of pet reptiles and 71.6%(48/67) of snakes. Although there are few reports about pet reptiles in Japan, Iida et al. [13] showed that Salmonella spp. were isolated from five (22.7%) of 22 pet reptiles kept at shops and household. The rate of isolation of Salmonella in reptiles in the present study is very high, and it may suggest that reptiles that are sold at pet shops in Japan can be a potential source of human salmonellosis. It is unclear whether these reptiles acquired Salmonella in nature, or during their captivity by ingestion of contaminated prey, or by contact with contaminated feces of other reptiles. In the present study, since we focused our attention

4 100 A. NAKADAI ET AL. Table 2. Distribution of Salmonella subspecies in pet reptiles Order of Subspecies reptile I II IIIa IIIb IV Total Turtles 16 (80.0) a) 2 (10.0) 0 2 (10.0) 0 20 (100.0) Lizards 37 (64.9) 8 (14.0) 0 3 ( 5.3) 9 (15.8) 57 (100.0) Snakes 17 (48.6) 2 ( 5.7) 2 (5.7) 14 (40.0) 0 35 (100.0) Total 70 (62.5) 12 (10.7) 2 (1.8) 19 (17.0) 9 ( 8.0) 112 (100.0) a) No. (%) of isolates. on the prevalence and distribution of Salmonella subspecies and serovars in reptiles, different infection rates among groups divided by origin of reptiles (ex. wild or breeding, domestic or foreign) or diet patterns (ex. meat or vegetable, mouse or insect) were not identified clearly. A total of 112 Salmonella isolates were obtained from 83 Salmonella positive-samples. These isolates were identified as subspecies I to IV (Table 2). Of 112 Salmonella isolates, 70(62.5%) belonged to subspecies I, 19(17.0%) to IIIb, 12(10.7%) to II, nine(8.0%) to IV, and two(1.8%) to IIIa. Subspecies I are usually isolated from humans and warmblooded animals, and serovars belonging to this group are designated with the name related to the geographical place where the serovar was first isolated. Subspecies II, IIIa, IIIb, IV are usually isolated from cold-blooded animals and the environment. The high prevalence of subspecies I in pet reptiles in the present study is in accordance with the results of other studies concerning captive reptiles [10, 13, 15, 19]. Geue et al. [10] reported that about 45 % of Salmonella isolates originated from pet reptiles belonged to subspecies I, and 30% to IIIb, 6% to IIIa, 3% to II and 2% to IV. It has been recognized that human salmonellosis is caused mainly by subspecies I and our result may indicate that Salmonella carried by pet reptiles can potentially affect humans. It has been reported that Salmonella subspecies II, IIIa, IIIb, and IV are usually isolated from reptiles and rarely from humans or domestic animals [17, 20] and that both pet and wild snakes are the main reservoir of subspecies IIIb [10, 11, 17, 18, 20]. Sakazaki [17] reported that about 99% of Salmonella isolates from humans or domestic animals belong to subspecies I, while 49% of isolates from turtles and 64% from snakes belong to IIIb. In the present study, the majority of Salmonella isolates from turtles (80%) and lizards (65%) belonged to subspecies I, but 40% of isolates from snakes belonged to subspecies IIIb and 48% to I (Table 2). The reason of this different distribution of Salmonella subspecies in reptiles is still unclear. Of the 112 Salmonella isolates, 54 could be identified as 28 serovars. The remaining 58 isolates were untypable with commercial antisera. The most frequently isolated serovars were S. Bardo (n=5), S. Newport (n=5), S. Panama (n=5), S. Amsterdam (n=4), and S. Minnesota (n=4). These serovars were isolated from more than two reptile species. The majority of the serovars found in the present study have also been reported in other studies of wild or captive reptiles [10, 21]. The serovars associated with human gastroenteritis in Japan, such as S. Enteritidis, S. Newport and S. Bareilly, were also identified [14]. The Centers for Disease Control and Prevention (CDC) [1] defined some serovars such as S. Hvittingfoss and S. Kisarawe, as reptile-associated serovars. Of 83 positive samples, 59 samples yielded only one serovar or subspecies, 16 yielded two, seven yielded three, and one sample yielded four different serovars or subspecies. Some researchers also reported that different plural Salmonella serovars or subspecies were sometimes isolated from reptile [10, 15]. These facts suggest that the distribution of Salmonella in reptiles may reflect their living environments. Iguana has been reported as an important source of infection in many cases of human salmonellosis and most of Salmonella isolates from iguanas belong to subspecies IV [1, 3 6, 21]. In the present study, 25.0% (3/12) of iguanas harbored Salmonella (Table 1). One year after the first investigation, the prevalence of Salmonella was examined again in same iguanas in order to investigate the persistence of the pathogen. Three of 10 iguanas (except for two dead ones) still harbored Salmonella spp. in their intestines. Salmonella isolates obtained during the two investigations belonged to subspecies IV, and were indistinguishable by pulsed-field gel electrophoresis (data not shown). Our results indicate that iguana may carry Salmonella for a long period of time and serve as a potential source of human salmonellosis. In the present study, all reptiles examined were kept in individual cages, and each cage was washed and disinfected well after use. Therefore, a wide variety of serovars in reptiles may not be attributable to cross contamination of Salmonella in the shop investigated in the present study. It is possible that reptiles were infected by Salmonella before they were carried into the shop. A high isolation rate of Salmonella in reptiles occurred at quarantine in a zoo [19] and the fact may support our results. The high isolation rate of Salmonella in pet reptiles and identification of major serovars of the isolates in the present study indicate a potential human risk of Salmonella infection from pet reptiles in Japan as well. However, there is no restriction of import or sale of reptiles in Japan and many reptiles are traded freely now. In the future, it may be necessary to regulate the trade of pet reptiles in Japan. Since only one pet shop specializing in reptiles could be surveyed in the present study, further extensive investigations should be done to clarify the prevalence and serovars of Salmonella in pet reptiles in Japan.

5 SALMONELLA FROM PET REPTILES 101 ACKNOWLEDGEMENT. The authors wish to thank Dr. Rita M. Sakala (Azabu University) for critical reading of this manuscript. REFERENCES 1. Ackman, D.M., Drabkin, P., Birkhead, G. and Cieslak, P Pediatr. Infect. Dis. J. 14: Andreaccio, A. and Miller, F Orthopedics 23: Burnham, B.R., Atchley, D.H., DeFusco, R.P., Ferris, K.E., Zicarelli, J.C., Lee, J.H. and Angulo, F.J J. Am. Vet. Med. Assoc. 213: Centers for Disease Control Morb. Mortal. Wkly. Rep. 44: Centers for Disease Control Morb. Mortal. Wkly. Rep. 48: Centers for Disease Control Morb. Mortal. Wkly. Rep. 52: Communicable Disease Surveillance Centre. 2000a. Commun. Dis. Rep. Wkly. 10: 49, Communicable Disease Surveillance Centre. 2000b. Commun. Dis. Rep. Wkly. 10: Cyriac, J. and Wozniak, E.R Commun. Dis. Public Health. 3: Geue, L. and Loschner, U Vet. Microbiol. 84: Greenberg, Z. and Sechter, I Isr. J. Med. Sci. 47: Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Williams, S.T pp In: Bergey s Manual of Determinatve Bacteriology 9th ed., Williams&Wilkins, Baltimore, U.S.A. 13. Iida, T., Kanzaki, M., Watabe, H., Miyazaki, T. and Maruyama, T J. Jpn. Vet. Med. Assoc. 52: (in Japanese with English summary). 14. Infectious Disease Surveillance Center, National Institute of Infectious Diseases Infect. Agents Surveillance Rep. 24: 179' 180'. 15. Onderka, D.K. and Finlayson, M.C Can. J. Comp. Med. 49: Popoff, M.Y. and Le Minor, L Antigenic Formulas of the Salmonella Serovars 8th ed. WHO Collaborating Centre for Reference and Research on Salmonella, Institute Pasteur, Paris, France. 17. Sakazaki, R pp In: Proceeding of the International Symposium on Salmonella (Snoyenbos, G. H. ed.), Am. Assoc. Avian Pathol., Pennsylvania. 18. Schroter, M., Roggentin, P., Hofmann, J., Speicher, A., Laufs, R. and Mack, D Appl. Environ. Microbiol. 70: Tatara, S., Kataoka, M. and Kinuta, T J. Jpn. Assoc. Zool. Aqua. 40: (in Japanese with English summary). 20. Thomas, A.D., Forbes-Faulkner, J.C., Speare, R. and Murray, C J. Wildl. Dis. 37: Woodward, D.L., Khakhria, R. and Johnson, W.M J. Clin. Microbiol. 35: