NOTE Public Health

Prevalence of Salmonella spp. in Pet in Japan

Aya NAKADAI1,2), Toshiro KUROKI3), Yukio KATO2), Rieko SUZUKI3), Shiro YAMAI3), Chiharu YAGINUMA2), Ryo SHIOTANI4), Akira YAMANOUCHI5) and Hideki HAYASHIDANI1)*

1)Division of Life Science, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo 183–8509, 2)Department of Public Health II, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Sagamihara, Kanagawa 229–8501, 3)Kanagawa Prefectural Institute of Public Health, 1–3–1 Shimomachiya, Chigasaki, Kanagawa 253–0087, 4)Gentleman Loser, 5–29–7 Fuchinobe-honcho, Sagamihara, Kanagawa 229–0002 and 5)Yamanouchi Iguana Laboratory, 103 Lions Plaza 7–5–8 Sagami-ono, Sagamihara, Kanagawa 228–0803, 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 sub- species 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): 97–101, 2005

Salmonellosis is known to be one of the most important Carry-Blair transport medium (Eiken Chemical Co., Ltd., cause of public health problems worldwide. In Japan, more Tokyo, Japan), and immediately transported to the labora- than 460 cases of human salmonellosis occurred in 2002, tory in an icebox. The fecal samples were suspended in 3 ml and most of them resulted from consumption of foods of of sterile saline, and 1 ml of the suspension was inoculated animal-origin contaminated with Salmonella [14]. Wild and into 10 ml of buffered peptone water (BPW, OXOID Ltd., pet reptiles are generally known to be asymptomatic carriers Basingstoke, Hampshire, England). After incubation at of several Salmonella serotypes, which are potentially 37°C for 24 hr, 1 ml of BPW culture was transferred to 10 pathogenic for humans. Recently, the number of exotic rep- ml of Hajna-tetrathionate broth (Eiken). The broth was tiles has been increasing in popularity as pets and the fact incubated at 37°C for 24 hr, then one loopful of each tube has led to an increase in the number of -associated was inoculated onto a plate of brilliant green agar (BGN, Salmonella infections in the United States [4–6, 18] and OXOID) supplemented with 20 µg novobiocin/ml, desoxy- European countries [2, 7–9]. Majority of the patients cholate hydrogen sulfide lactose agar (DHL, Nissui Pharma- infected with Salmonella spp. from reptiles are infants, and ceutical Co., Ltd., Tokyo) and mannitol lysine crystal violet usually show gastroenteritis, less frequently, fatal meningi- brilliant green agar (MLCB, Nissui). These plates were tis or septicemia [5,7,8]. Recently, many reptiles have also incubated at 37°C for 24 hr and three suspicious colonies been kept as pets in Japan as well as in other developed morphologically similar to Salmonella spp. from each plate countries and it is possible that these can be a source were subcultured for biochemical examinations. Biochemi- of human salmonellosis. However, little is known about the cal characteristics were examined on triple sugar iron prevalence of Salmonella spp. in reptiles except for aquar- medium (Nissui), lysine indole motility medium (Nissui) ium turtle in Japan [13,19]. This study was designed to and Voges-Proskauer semisolid medium (Eiken). The sub- investigate the presence of Salmonella in pet reptiles, sold species of Salmonella isolates was confirmed by biochemi- and kept in Japan. cal examinations according to Holt et al. [12]. Serotyping of During the period from November 2000 through July Salmonella isolates was accomplished with commercial O 2002, a total of 112 fecal samples of 50 reptile species con- and H antisera (Denka Seiken Co., Ltd., Tokyo) according sisting of 12 families were collected (Table 1). All reptiles to the method of Popoff and Le Minor [16]. except green iguanas were sold as pets in a pet shop located Salmonella spp. were isolated from 83 (74.1%) of 112 in Kanagawa Prefecture, and green iguanas were kept at fecal samples of reptiles (Table 1). Salmonella spp. were Yamanouchi Iguana Laboratory. Reptiles sold in the shop isolated from 23 (100%) of 23 snakes, 13 (72.2%) of 18 tur- had been kept in individual cages immediately after they tles, and 47 (66.1%) of 71 lizards. The isolation rate from were introduced into the shop. Fecal samples were put into snakes was the highest of all. This high proportion of Sal- monella infection in captive reptiles, especially snakes, is *CORRESPONDENCE TO: HAYASHIDANI, H., Division of Animal Sci- ence, Institute of Symbiotic Science and Technology, Tokyo similar to the results of several previous studies [10, 15, 18]. University of Agriculture and Technology, 3–5–8 Saiwai-cho, Geue et al. [10] examined the prevalence of Salmonella in Fuchu, Tokyo 183–8509, Japan. pet reptiles in Germany and Austria, and reported that Sal- 98 A. NAKADAI ET AL.

Table 1. Distribution of samples examined, number of isolates and serovars of Salmonella associated with reptile species Reptile 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 (UTb)) (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:z29:1,5 (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 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:z4: – (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 Salmonella subsp.I (UT) (1) Family Cordylidae Rough-scaled Plated Lizard Gerrhosaurus major 2/ 2 (100.0) 2 S. Amsterdam (1) Salmonella subsp.I (UT) (1) Family Helodermatidae Banded Gila Monsters S. cinctum 1/ 1 (100.0) 1 S. Poona or Farmsenc) (1) Reticulated Gila Monsters Heloderma S. Suspectum 1/ 1 (100.0) 2 S. Alachua (1) Salmonella subsp.I (UT) (1) Family Gray’s Monitor Varanus olivaceus 1/ 1 (100.0) 3 S. Haifa (1) Salmonella subsp.I (UT) (1) Salmonella subsp.IIIb (UT) (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) S. Panama (1) Continued on following page. 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 Salmonella subsp.IIIb (UT) (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. Panama (1) S. Potengi (1) S. Soananina or Sundsvallc) (2) Salmonella subsp.I (UT) (1) 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) Salmonella subsp.IIIb (UT) (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 Salmonella subsp.IIIb (UT) (1) Green Python Morelia viridis 1/ 1 (100.0) 1 S. Amsterdam (1) Family Colubridae Japanese Ratsnake Elaphe climacophora 1/ 1 (100.0) 1 Salmonella subsp.IIIb (UT) (1) Japanese Four-lined Ratsnake Elaphe quadrivirgata 1/ 1 (100.0) 1 Salmonella subsp.IIIb (UT) (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 Floridac) (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 Floridac) (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) Salmonella subsp.IIIb (UT) (1) Blotched Kingsnake Lampropeltis getula goini 1/ 1 (100.0) 1 Salmonella subsp.IIIb (UT) (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 Salmonella subsp.IIIb (UT) (1) Pueblan Milksnake Lampropeltis triangulum camphelli 1/1 (100.0) 1 Salmonella subsp.IIIb (UT) (1) Sinaloan Milksnake Lampropeltis triangulum sinaloae 1/1 (100.0) 2 S. Bardo (1) S. Panama (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 it may suggest that reptiles that are sold at pet shops in Japan and 71.6%(48/67) of snakes. Although there are few reports can be a potential source of human salmonellosis. It is about pet reptiles in Japan, Iida et al. [13] showed that Sal- unclear whether these reptiles acquired Salmonella in monella spp. were isolated from five (22.7%) of 22 pet rep- nature, or during their captivity by ingestion of contami- tiles kept at shops and household. The rate of isolation of nated prey, or by contact with contaminated feces of other Salmonella in reptiles in the present study is very high, and reptiles. In the present study, since we focused our attention 100 A. NAKADAI ET AL.

Table 2. Distribution of Salmonella subspecies in pet reptiles Subspecies Order of Total reptile I II IIIa IIIb IV 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 Japan, such as S. Enteritidis, S. Newport and S. Bareilly, and serovars in reptiles, different infection rates among were also identified [14]. The Centers for Disease Control groups divided by origin of reptiles (ex. wild or breeding, and Prevention (CDC) [1] defined some serovars such as S. domestic or foreign) or diet patterns (ex. meat or vegetable, Hvittingfoss and S. Kisarawe, as “reptile-associated” sero- mouse or insect) were not identified clearly. vars. Of 83 positive samples, 59 samples yielded only one A total of 112 Salmonella isolates were obtained from 83 serovar or subspecies, 16 yielded two, seven yielded three, Salmonella positive-samples. These isolates were identified and one sample yielded four different serovars or subspe- as subspecies I to IV (Table 2). Of 112 Salmonella isolates, cies. Some researchers also reported that different plural 70(62.5%) belonged to subspecies I, 19(17.0%) to IIIb, Salmonella serovars or subspecies were sometimes isolated 12(10.7%) to II, nine(8.0%) to IV, and two(1.8%) to IIIa. from reptile [10, 15]. These facts suggest that the distribu- Subspecies I are usually isolated from humans and warm- tion of Salmonella in reptiles may reflect their living envi- blooded animals, and serovars belonging to this group are ronments. designated with the name related to the geographical place Iguana has been reported as an important source of infec- where the serovar was first isolated. Subspecies II, IIIa, tion in many cases of human salmonellosis and most of Sal- IIIb, IV are usually isolated from cold-blooded animals and monella isolates from iguanas belong to subspecies IV [1, the environment. The high prevalence of subspecies I in pet 3–6, 21]. In the present study, 25.0% (3/12) of iguanas har- reptiles in the present study is in accordance with the results bored Salmonella (Table 1). One year after the first investi- of other studies concerning captive reptiles [10, 13, 15, 19]. gation, the prevalence of Salmonella was examined again in Geue et al. [10] reported that about 45 % of Salmonella iso- same iguanas in order to investigate the persistence of the lates originated from pet reptiles belonged to subspecies I, pathogen. Three of 10 iguanas (except for two dead ones) and 30% to IIIb, 6% to IIIa, 3% to II and 2% to IV. It has still harbored Salmonella spp. in their intestines. Salmo- been recognized that human salmonellosis is caused mainly nella isolates obtained during the two investigations by subspecies I and our result may indicate that Salmonella belonged to subspecies IV, and were indistinguishable by carried by pet reptiles can potentially affect humans. It has pulsed-field gel electrophoresis (data not shown). Our been reported that Salmonella subspecies II, IIIa, IIIb, and results indicate that iguana may carry Salmonella for a long IV are usually isolated from reptiles and rarely from humans period of time and serve as a potential source of human sal- or domestic animals [17, 20] and that both pet and wild monellosis. snakes are the main reservoir of subspecies IIIb [10, 11, 17, In the present study, all reptiles examined were kept in 18, 20]. Sakazaki [17] reported that about 99% of Salmo- individual cages, and each cage was washed and disinfected nella isolates from humans or domestic animals belong to well after use. Therefore, a wide variety of serovars in rep- subspecies I, while 49% of isolates from turtles and 64% tiles may not be attributable to cross contamination of Sal- from snakes belong to IIIb. In the present study, the major- monella in the shop investigated in the present study. It is ity of Salmonella isolates from turtles (80%) and lizards possible that reptiles were infected by Salmonella before (65%) belonged to subspecies I, but 40% of isolates from they were carried into the shop. A high isolation rate of Sal- snakes belonged to subspecies IIIb and 48% to I (Table 2). monella in reptiles occurred at quarantine in a zoo [19] and The reason of this different distribution of Salmonella sub- the fact may support our results. The high isolation rate of species in reptiles is still unclear. Salmonella in pet reptiles and identification of major sero- Of the 112 Salmonella isolates, 54 could be identified as vars of the isolates in the present study indicate a potential 28 serovars. The remaining 58 isolates were untypable with human risk of Salmonella infection from pet reptiles in commercial antisera. The most frequently isolated serovars Japan as well. However, there is no restriction of import or were S. Bardo (n=5), S. Newport (n=5), S. Panama (n=5), S. sale of reptiles in Japan and many reptiles are traded freely Amsterdam (n=4), and S. Minnesota (n=4). These serovars now. In the future, it may be necessary to regulate the trade were isolated from more than two reptile species. The of pet reptiles in Japan. Since only one pet shop specializing majority of the serovars found in the present study have also in reptiles could be surveyed in the present study, further been reported in other studies of wild or captive reptiles [10, extensive investigations should be done to clarify the preva- 21]. The serovars associated with human gastroenteritis in lence and serovars of Salmonella in pet reptiles in Japan. SALMONELLA FROM PET REPTILES 101

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