Vol. 109: 165–175, 2014 DISEASES OF AQUATIC ORGANISMS Published May 13 doi: 10.3354/dao02732 Dis Aquat Org Batrachochytrium dendrobatidis prevalence and haplotypes in domestic and imported pet amphibians in Japan Kenichi Tamukai1, Yumi Une2,*, Atsushi Tominaga3, Kazutaka Suzuki4, Koichi Goka4 1Den-en-chofu Animal Hospital, 2-1-3 Denenchofu, Ota-ku, Tokyo 145-0071, Japan 2Laboratory of Veterinary Pathology, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan 3Department of Natural Sciences, Faculty of Education, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 901-0213, Japan 4Invasive Alien Species Research Team, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan ABSTRACT: The international trade in amphibians is believed to have increased the spread of Batrachochytrium dendrobatidis (Bd), the fungal pathogen responsible for chytridiomycosis, which has caused a rapid decline in amphibian populations worldwide. We surveyed amphibians imported into Japan and those held in captivity for a long period or bred in Japan to clarify the Bd infection status. Samples were taken from 820 individuals of 109 amphibian species between 2008 and 2011 and were analyzed by a nested-PCR assay. Bd prevalence in imported amphibians was 10.3% (58/561), while it was 6.9% (18/259) in those in private collections and commercially bred amphibians in Japan. We identified the genotypes of this fungus using partial DNA sequences of the internal transcribed spacer (ITS) region. Sequencing of PCR products of all 76 Bd-positive samples revealed 11 haplotypes of the Bd ITS region. Haplotype A (DNA Data Bank of Japan accession number AB435211) was found in 90% (52/58) of imported amphibians. The results show that Bd is currently entering Japan via the international trade in exotic amphibians as pets, sug- gesting that the trade has indeed played a major role in the spread of Bd. KEY WORDS: Chytrid · Amphibian disease · Batrachochytrium dendrobatidis · Chytridiomycosis · Pet trade · ITS · Japan Resale or republication not permitted without written consent of the publisher INTRODUCTION time, emerging pathogens that threaten wild am phi - bian populations have been identified. The World As a consequence of economic globalization and Organisation for Animal Health (OIE) has listed the changes in transportation in recent years, goods Batrachochytrium dendrobatidis (Bd) and ranavirus are now being distributed rapidly on a massive scale as notifiable diseases under the Aquatic Animal over vast areas. This has been accompanied by an Health Code and is working to control the spread of increase in the number of animals being traded inter- these pathogens (Schloegel et al. 2010, OIE 2012). nationally. Many wild animals, in particular reptiles Bd, the fungal pathogen that causes chytridio - and amphibians, are sold commercially as pets, for mycosis, is responsible for a worldwide decline in am- food, or for traditional medicines (Mazzoni et al. phibian populations. This pathogen has decimated 2003, Schlaepfer et al. 2005, Fisher & Garner 2007, amphibian populations in the tropical rain forests of Garner et al. 2009, Smith et al. 2009). At the same Panama and Australia, and has been detected in wild *Corresponding author: [email protected] © Inter-Research 2014 · www.int-res.com 166 Dis Aquat Org 109: 165–175, 2014 and captive populations of amphibians in Africa, as endangered and near-threatened on the Red List South America, Central America, North America, Eu- compiled by the Ministry of the Environment Gov- rope, and Asia (Berger et al. 1998, 1999, Lips 1999, ernment of Japan (2012). There is therefore a need to Mutschmann et al. 2000, Bosch et al. 2001, Weldon assess the effect on Japan’s native fauna of patho- 2002, Bradley et al. 2002, Une et al. 2008). gens that are unintentionally introduced by imported There have been a number of reports on the origin amphibians. and spread of Bd, and one hypothesis is that it origi- Bd has a broad host range, is highly infectious, and nated in the African clawed frog Xenopus laevis. has a high case fatality rate. Bd zoospores in infected This frog was used in human pregnancy testing in water can cause the infection to spread rapidly over a the late 1930s and as a consequence it was exported wide area (Johnson & Speare 2003). Animals that from its native South Africa to countries around the have been artificially raised are sometimes aban- world (Weldon et al. 2004). Xenopus spp. were fur- doned as unwanted pets (Karesh et al. 2005, Hols- ther distributed for use in various fields of research beek et al. 2008). Consequently, any international (Weldon et al. 2007). The wide distribution of this amphibian trade is potentially linked to the spread of species supports the hypothesis that it has caused the Bd and should be considered a significant risk. spread of Bd. Likewise, the cane toad Bufo marinus We therefore surveyed imported and captive exotic can asymptomatically harbor Bd infection (Berger et amphibians in Japan in order to understand the al. 1998, 1999), and the intentional introduction of this prevalence of Bd and its haplotypes with the aim of species as a biological pest control into different determining the extent to which the international regions around the world is another likely cause of trade has exacerbated the spread of Bd. the spread of Bd (Daszak et al. 1999). Moreover, the Ame rican bullfrog Lithobates catesbeianus, commonly used for human consumption, is raised MATERIALS AND METHODS throughout the world and traded internationally. This species is not susceptible to Bd but is a carrier, Sample collection and is now equal to the African clawed frog in terms of the spread of Bd (Mazzoni et al. 2003, Garner et al. Samples were obtained from 820 adult individuals 2006, Schloegel et al. 2009, 2012). of 109 amphibian species that had been imported or As well as these amphibians which have high- raised and sold as pets during the 4 yr period from value uses in the fields of medicine and agriculture, 2008 to 2011. Of these, 561 individuals of 93 species the tiger salamander Ambystoma tigrinum, which is were imported and 259 individuals of 47 species used as live bait for fishing, may also have con- were captive amphibians. tributed to the spread of Bd and ranavirus (Picco & Amphibians were imported by 4 animal import Collins 2008, Picco et al. 2010). In addition, many companies (2 in Tokyo, 1 in Osaka, and 1 in Saitama) other amphibians are traded around the world as from 21 countries. Swab samples for Bd detection pets and for zoological and aquarium collections were either taken immediately or within 7 d of arrival (Schlaepfer et al. 2005, Andreone et al. 2006, Forzán in Japan. The species, country of export, date of et al. 2008, Nijman & Shepherd 2011, Spitzen-van import, and date of swab sample were all recorded. der Sluijs et al. 2011). Captive amphibians which had been kept for a This international trade in amphibians has facili- long period of time were either from private house- tated the spread of Bd, providing new habitats and hold collections and were examined at veterinary hosts for Bd, and is the major cause of the pandemic clinics or had been bred in Japan as pets. (Daszak et al. 2000, Skerratt et al. 2007, Kriger & The swab samples for Bd detection were collected Hero 2009). The first outbreak of Bd in Asia occurred with great care to avoid contamination. Amphibians in 2006 in Japan, where it caused a significant num- were sampled individually, using fresh rubber gloves ber of exotic pet amphibian deaths (Une et al. 2008). for each animal, by swabbing the animal’s ven - At that time the distribution pathways in Japan, from tral surface, inner thigh, and toe pads with cotton breeding facilities to the homes of hobbyists, allowed swabs (Men-tip 1P1501, Nihon-Menbo). A nested- for disease spread and many epidemics were seen (Y. PCR assay was used to detect Bd infection. All sam- Une et al. unpubl. data). ples were treated according to the same protocol, Japan is home to 66 native species of amphibians and were kept in microtubes at −28°C until DNA (39 anurans and 27 urodeles), 60 of which are en - analysis. PCR assays were performed within 15 d of demic to Japan. Of these, 42 species (67%) are listed sampling. Tamukai et al.: Bd prevalence in Japanese pet amphibians 167 DNA extraction and nested-PCR assay Cloning Kit (Novagen, EMD Bioscience) and trans- formed into Escherichia coli in accordance with the Each swab was placed in a microtube containing manufacturer’s protocol. Three positive cloned frag- 200 µl of lysis buffer (1 mg ml−1 Proteinase K, 0.01 M ments for each nested PCR product were sequenced NaCl, 0.1 M EDTA, 0.01 M Tris-HCl pH 8.0, 0.5% using an ABI3730 Sequencer (Applied Biosystems) Nonidet P-40). The microtube was then vortexed at and T7 promoter and U19 reverse primers. 15°C for 1 min. After removing the swab, the tube containing the extract was incubated at 50°C for 120 min and then at 95°C for 20 min. After incuba- RESULTS tion, the extract was diluted to 10% of its original concentration in TE buffer (0.001 M EDTA, 0.01 M In total, 76 samples (9.3%) from 820 individual am- Tris-HCl, pH 8.0) and used as the DNA template in phibians were identified as Bd-positive (Table 1). The the PCR assay. Bd-positive samples were as follows: Anura, 8.9% We followed the method of Goka et al. (2009) for (64/722 individuals); Caudata, 12.5% (12/96); Gym- the nested-PCR assay.