Herpetology Notes, volume 14: 777-784 (2021) (published online on 16 May 2021) Incidence of emerging pathogens in the legal and illegal amphibian trade in Spain Barbora Thumsová1,2, Jaime Bosch3,2,*, and Albert Martinez-Silvestre4 Abstract. Amphibians are threatened globally and emerging diseases are some of the most important drivers of their catastrophic situation. There is increasing evidence that the international trade in live amphibians is one of the most important mechanisms driving pathogen pollution. Here, we report the presence of Batrachochytrium dendrobatidis (Bd) and Ranavirus in 11% of tested individuals in legal amphibian trade fairs in Spain. Although none of the Bd infected animals in trade fairs presented disease symptoms, symptoms of ranavirosis were observed in some specimens, which were nonetheless still offered for sale. None of the traders who were selling infected animals showed interest in engaging in collaboration to control infections when offered for free. In addition, a large private urodele collection confiscated by the police in Barcelona comprised a number of illegally wild-caught species. Many confiscated individuals presented signs of poor welfare and several were positive for Bd. Our results indicate the urgent need for implementation of real sanitary regulations or effective legislation governing the practice of trade in living amphibians to prevent pathogen spread in Europe. Keywords. Batrachochytridium dendrobatidis, chytridiomycosis, Ranavirus, ranavirosis, pet keeping Introduction al., 2005; Collins and Crump, 2009; Rowley et al., 2010; Pasmans et al., 2017). In fact, of the 5,579 vertebrate Wildlife continues to be threatened by a number of species affected by the global wildlife trade, 9.4% are anthropogenic activities that have compromised the amphibians (Scheffers et al., 2019). Although some integrity and persistence of natural ecosystems over traded amphibian species are monitored by international time (Ceballos et al., 2015; Wilson et al., 2019). In conventions such as the Convention on International recent decades, amphibians have become an iconic Trade in Endangered Species of Wild Fauna and Flora example of species extinction and population extirpation (CITES), regulations for 98% of all amphibian species due to increased human pressure (Skerratt et al., 2007; are not still established (Auliya et al., 2016a). As a Blaustein et al., 2011). As a result, forty-one percent of result, the great majority of traded amphibians are wild- known amphibian species are now listed as threatened, caught, which are mostly harvested in an unsustainable and many are experiencing global population declines manner despite posing risks for population persistence (IUCN, 2020). (Schlaepfer et al., 2005; Auliya et al., 2016b). Among Commercial trade in amphibians for food, pets or other things, the increasing demand from keepers traditional medicine is one of the most prominent to include rare or newly described species in their drivers of species extinction risk globally (Schlaepfer et collections stimulates illegal trade in amphibians (Courchamp et al., 2006; Natusch and Lyons, 2012; Cooney et al., 2015; Auliya et al., 2016b). In fact, the 1 Asociación Herpetológica Española, José Gutiérrez Abascal 2, black market in animal trade is one of the most lucrative 28006 Madrid, Spain. illicit activities in the world (TRAFFIC, 2008; Esmail 2 Museo Nacional de Ciencias Naturales-CSIC, José Gutiérrez et al., 2020), generating a turnover of around 20 billion Abascal 2, 28006 Madrid, Spain. dollars annually (Nellemann et al., 2016). 3 Research Unit of Biodiversity, Gonzalo Gutiérrez Quirós s/n, Both legal and illegal exotic pet trades are widely Oviedo University - Campus Mieres, Edificio de Investigación, considered as important mechanisms of intercontinental 33600 Mieres, Spain. and international pathogen pollution (e.g., Fisher 4 Catalonian Reptiles and Amphibians Rescue Centre, 08783 Barcelona, Spain. and Garner, 2007; O’Hanlon et al., 2018), including * Corresponding author. E-mail address: [email protected] the chytrids Batrachochytrium dendrobatidis (Bd; O’Hanlon et al., 2018), B. salamandrivorans (Bsal; © 2021 by Herpetology Notes. Open Access by CC BY-NC-ND 4.0. Martel et al., 2014) and ranaviruses (RV; Schloegel et 778 Barbora Thumsová et al. al., 2009). Both chytrid fungi and ranaviruses are listed thoroughly inspected by the police and the CITES by the World Organisation for Animal Health (Schloegel national department. Illegally acquired individuals et al., 2010; OIE, 2019) and therefore, in theory, control were confiscated and transported to the Catalonian measures by all countries importing amphibians must Reptiles and Amphibians Rescue Centre (CRARC). All be implemented to ensure that animals are free of these individuals were housed separately in tanks, and were pathogens (OIE, 2019). immediately checked visually for disease symptoms, The European Union (EU) has been documented as and sampled as described above. For the specimens a top importer of legally and illegally collected wild found dead, a toe-clip and a small piece of liver were animals (Engler and Parry-Jones, 2007). Within the also taken and stored in 70% ethanol. EU, Spain houses a sizable community of keepers, with DNA was extracted from swab samples using PrepMan extensive amphibian collections containing specimens Ultra reagent, and was extracted from tissue samples imported from all around the world (e.g., Fitzpatrick with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, et al., 2018; Lastra-González et al., 2020). The largest Germany) following the manufacturer’s instructions. amphibian and reptile trade fairs of Southern Europe Real-time Taqman PCR assays were conducted take place in some of Spain’s main cities. Local traders, following the protocols suggested by Boyle et al. (2004) as well as traders from all over Europe, gather four for Bd, Blooi et al. (2013) for Bsal, and Leung et al. times a year at these events to sell a great variety of (2017) for RV. Samples were amplified on a MyGo exotic species. Despite the fact that imported exotic mini PCR machine in duplicate and against negative individuals may serve as vectors of both fungal and and positive controls with known concentrations of viral infections, sanitary regulations and legislation genomic equivalents of zoospores/virions. A sample was governing the practice to prevent pathogen spread in assigned as positive when the infection load was equal Spain are still not officially established. to or higher than 0.1 zoospores for Bd/Bsal or 3 virions Here we report the results of Bd, Bsal and RV testing for RV, and when the amplification curve presented a in four trade fairs that took place in Spain between 2017 robust sigmoidal shape. Where just one replicate of a and 2019, and also within a large illegal private collection sample was amplified, the sample was analysed a third recently confiscated in the centre of Barcelona city. time and considered positive only if the curve of the third amplification yielded a positive result. Materials and Methods Results Sampling at Spanish trade fairs took place between 2017–2019, three times in Madrid, and once in Over the four sampling events in trade fairs, a total of Barcelona. All pet traders and all amphibian species 82 individuals comprising 18 families and 34 species were sampled as described below. At least one sample from 20 traders were sampled (Table 1). Seventy-seven was collected per container when multiple animals were samples were tested for Bd, fifty-five for Bsal and housed together. Additionally, five imported terrapins forty-six for RV presence. While around 10% of the (two individuals of Graptemys pseudogeographica specimens tested positive for Bd and 2.2% of specimens (Gray, 1831), one individual of Cuora amboinensis tested positive for RV, no individuals tested positive for (Daudin, 1802), and two individuals of Kinosternon Bsal. Infected individuals were detected at all sampling baurii (Garman, 1891)) were sampled and tested for events. Bd infection was detected in individuals RV presence. The whole body of each individual was belonging to the families of Ambystomatidae (1/6 swabbed with a sterile cotton-tipped dry swab (MWE individuals), Arthroleptidae (1/2), Ceratophryidae medical wire, UK), and a new pair of disposable nitrile (2/9), Hylidae (1/1), Pipidae (2/3), and Pyxicephalidae gloves was used for each animal. Samples were stored (1/4). No Bd infected individuals presented disease dry and refrigerated until laboratory analyses within 1-2 symptoms. Ranavirosis symptoms were observed in weeks. three specimens of Litoria caerulea (White, 1790) In the spring of 2020, a police intervention was carried (Pelodryadidae), of which one individual also tested out in a private collection of urodele amphibians in positive for RV (Fig. 1A). All sampled terrapins were the centre of Barcelona city. The collection comprised negative for RV. Bd or RV infection was detected in a great number of exotic and Iberian-native urodele a total of five traders. One trader sold Bd-infected species. For the administrative and court proceedings, animals repeatedly across different trade fairs (3 out 4), the origin and legal status of all individuals were all of which were imported. Despite traders being very Incidence of emerging pathogens in the legal and illegal amphibian trade in Spain 779 Table 1. For each pathogen the number of positive animals and sample size are shown. The proportion of infected animals appears in