Biological Conservation 142 (2009) 1420–1426 Contents lists available at ScienceDirect Biological Conservation journal homepage: www.elsevier.com/locate/biocon Magnitude of the US trade in amphibians and presence of Batrachochytrium dendrobatidis and ranavirus infection in imported North American bullfrogs (Rana catesbeiana) Lisa M. Schloegel a,d,*, Angela M. Picco b, A. Marm Kilpatrick a,c, Angela J. Davies d, Alex D. Hyatt e, Peter Daszak a,d,* a Consortium for Conservation Medicine, Wildlife Trust, 460 West 34th Street, 17th Floor, New York, NY 10001, USA b United States Fish and Wildlife Service, Sacramento, CA 95825, USA c Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA 95064, USA d School of Life Sciences, Kingston University, Kingston-upon-Thames, Surrey KT1 2EE, UK e CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia article info abstract Article history: Amphibians are globally threatened by anthropogenic habitat loss, the wildlife trade and emerging dis- Received 14 October 2008 eases. Previous authors have hypothesized that the spread of the amphibian disease chytridiomycosis Received in revised form 31 January 2009 (Batrachochytrium dendrobatidis) and amphibian ranaviruses are associated with the international trade Accepted 8 February 2009 in live amphibians. The North American bullfrog (Rana catesbeiana) is thought to be a carrier of these Available online 5 April 2009 pathogens, is globally traded as a live commodity, and is sold live in US markets. We obtained importa- tion data for all live amphibians, and parts thereof, into three major US ports of entry (Los Angeles, San Keywords: Francisco and New York) from 2000 to 2005. Importation of live amphibians into these ports totaled Chytridiomycosis almost 28 million individuals over this 6-year period. We collected samples from freshly-imported mar- Batrachochytrium dendrobatidis Wildlife trade ket frogs and found infection with both pathogens in all three cities and all seasons, with an overall infec- Amphibian declines tion prevalence of 62% (306/493) and 8.5% (50/588) for B. dendrobatidis and ranaviruses, respectively, by Ranavirus PCR. This study definitively identifies these two important pathogens in recently imported live market frogs and suggests that the amphibian trade can contribute to introductions of these pathogens into new regions. It provides support for the recent listing of B. dendrobatidis and ranaviral diseases by the OIE, and provides evidence for measures to be taken to eradicate these pathogens from the trade. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction offs (Cunningham et al., 1996), but its capacity to cause sustained declines is unknown (Daszak et al., 2003). In recent years, two diseases (chytridiomycosis, caused by the Chytridiomycosis and amphibian ranaviral disease are classed zoosporic fungus Batrachochytrium dendrobatidis, and ranaviral dis- as emerging infectious diseases due to their recent spread into ease) have been identified as threats to amphibian populations new regions and, in the case of B. dendrobatidis, its significant im- (Daszak et al., 2003; Green et al., 2002). Outbreaks of chytridiomy- pact on amphibian populations in recent times (Daszak et al., cosis are often associated with high mortality rates in adult anu- 2003). There is increasing evidence that anthropogenic trade in, rans, and the disease has been implicated in a series of declines or the introduction of, amphibians is responsible for the recent and extinction events globally (Berger et al., 1998; Lips et al., spread of these diseases, with papers reporting the presence of B. 2005; Mendelson et al., 2006; Schloegel et al., 2006). Ranaviral dis- dendrobatidis in the pet trade, zoo animals, introduced species ease is known to cause amphibian mortality, often with large die- and the laboratory animal trade, among others (Daszak et al., 2003). Similarly, phylogenetic evidence strongly suggests that the trade in US salamanders as fish bait, and the introduction of species from the USA to the UK, resulted in the geographic expansion of * Corresponding authors. Address: Consortium for Conservation Medicine, Wild- ranaviruses (Hyatt et al., 2000; Storfer et al., 2007). life Trust, 460 West 34th Street, 17th Floor, NY 10001, USA. Tel.: +1 212 380 4460; The earliest identified infection by B. dendrobatidis was of an fax: +1 212 380 4475 (P. Daszak). E-mail addresses: [email protected] (L.M. Schloegel), Angela_Picco@ African clawed frog (Xenopus laevis) museum specimen dated fws.gov (A.M. Picco), [email protected] (A.M. Kilpatrick), ajdavies.russell@ 1938 (Weldon et al., 2004). This species appears to be resistant kingston.ac.uk (A.J. Davies), [email protected] (A.D. Hyatt), daszak@wildlifetrust. to the onset of disease when infected by B. dendrobatidis, leading org (P. Daszak). 0006-3207/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocon.2009.02.007 L.M. Schloegel et al. / Biological Conservation 142 (2009) 1420–1426 1421 to speculation that the widespread distribution of X. laevis in the mal parts and derivatives), source population, and purpose. Wild- 1940s and 1950s for human pregnancy testing disseminated the life source codes indicate whether the animal was taken from the pathogen to differing regions of the world (Weldon et al., 2004). wild, originated from a ranching operation, bred in captivity, and There is a discrepancy in the overlap of X. laevis distributions and more. For our analyses, all shipments whose source population the nature of B. dendrobatidis outbreaks, however, suggesting addi- did not come from the wild were grouped as originating from cap- tional hosts must play a role in the spread of this disease (Rach- tive populations, excluding those whose origins were unknown. owicz et al., 2005). Purpose codes identified whether the shipment was commercial, The North American bullfrog (Rana catesbeiana) is another po- intended for a zoological institution, for scientific purposes, per- tential carrier of B. dendrobatidis, exhibiting no clinical signs when sonal, for biomedical research or educational, among others. experimentally or naturally-infected with the fungus (Daszak et al., Shipment size was recorded in one of four unit measures: num- 2004). This species is farmed for the international food and pet ber of individuals, kilograms, pounds, or gallons. More than 99% of trades, and large numbers are imported into the USA annually for all shipments recorded in units of kilograms, pounds, or gallons human consumption (Schlaepfer et al., 2005). Previous studies were identified as Ranid shipments. Therefore, all live shipments have established the presence of B. dendrobatidis in farmed and es- recorded in a weighted measure were converted to number of indi- caped bullfrogs in South America (Hanselmann et al., 2004), and in viduals based on the average weight of adult R. catesbeiana sam- other regions (Garner et al., 2006). The international trade in live pled at each of the markets (0.4 kg, n = 200). All shipments amphibians is also implicated in the spread of ranaviruses. Ranav- denoted as SKI (skins) and SPE (scientific specimens including iruses isolated from pig frogs (Rana grylio) farmed in China (RGV- blood, tissue and histological preparations) were excluded from 9506, RGV-9807 and RGV-9808) are genetically similar to Frog our analyses. virus 3 (FV3), originally isolated from R. pipiens in North America (Zhang et al., 2001). The pig frog is native to North America and 2.2. Sample collection was exported to China specifically for the frog farming industry (Chinchar, 2002; Zhang et al., 2001). Evidence also suggests that We obtained samples from shops selling live amphibians for ranaviruses linked to seasonal mortalities in amphibians in the human consumption in three major US cities: 1 in Los Angeles, Cal- western US may have spread throughout the region via the trade ifornia (LA); 13 in New York, New York (NY); and 4 in San Fran- in salamanders as bait for freshwater fishing (Jancovich et al., cisco, California (SF). The market frogs were on display in large 2005; Picco and Collins, 2008). This and other evidence prompted plastic bins or trash cans, typically containing water (Fig. 1). The the World Organization for Animal Health (OIE – Office Interna- number of frogs per container was variable and could be as few tional des Epizooties) to list B. dendrobatidis and ranaviral diseases as 10 or >50. of amphibians as ‘notifiable’ (www.oie.int/eng/maladies/en_classi- The animals were purchased from the shops, killed by the shop fication2009.htm?e1d7, 2009, accessed 29 January). owners (we were informed by shop owners in California that the Despite interest in the origins of these pathogens, there are no sale of live frogs is illegal in that state, but calls to contacts at published reports on the presence of B. dendrobatidis or ranaviruses the appropriate authorities have been unable to verify this state- in Ranid frogs imported into the USA, nor accurate data on the vol- ment) and transported to the laboratory post mortem on ice. We ume and geographical pattern of these trades. To assess overall requested each animal be placed in a separate bag for transporta- trends in the amphibian trade in the USA, and their potential to tion to prevent any contamination that could result from the mix- spread these diseases, we analyzed data collected by the US Fish ing of blood from one individual to the next. Due to language and Wildlife Service from three major US ports of entry over a restrictions, however, there were several occasions where the ani- six-year period (2000–2005) and tested live amphibians sold for mals were bagged collectively. To prevent additional cross-con- human consumption in each of these three cities for B. dendrobat- tamination of residual DNA, a fresh pair of gloves was used for idis and ranaviruses. each frog and equipment was sterilized with ethanol and subse- quently flamed between specimens. Each individual was swabbed along the ventral surface and between the digits (i.e.