Widespread Occurrence of an Emerging Fungal Pathogen in Heavily Traded Chinese Urodelan Species

Received: 31 October 2017 Revised: 14 December 2017 Accepted: 3 January 2018 DOI: 10.1111/conl.12436 POLICY PERSPECTIVE Widespread occurrence of an emerging fungal pathogen in heavily traded Chinese urodelan species Zhiyong Yuan1,2 An Martel3 Jun Wu4 Sarah Van Praet3 Stefano Canessa3 Frank Pasmans3 1 Key Laboratory for Forest Resources Conser- Abstract vation and Utilisation in the Southwest Moun- tains of China, Ministry of Education, South- Understanding introduction routes for wildlife pathogens is vital for the development west Forestry University, Yunnan 650224, of threat abatement plans. The chytrid fungus Batrachochytrium salamandrivorans China (Bsal) has recently emerged in Europe, where it is considered to be a serious threat 2Key Laboratory of Forest Disaster Warn- ing and Control of Yunnan Province, South- for urodelan conservation. If the highly diverse Chinese urodelans were to constitute west Forestry University, Kunming, Yunnan a Bsal reservoir, then the significant international trade in these species may vector 650224, China Bsal into naïve urodelan communities. Here, we analyzed a total of 1,143 samples, 3 Department of Pathology, Bacteriology representing 36 Chinese salamander species from 51 localities across southern China and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan for the presence of Bsal. We found Bsal was present across a wide taxonomic, geo- 133, Merelbeke, 9820, Belgium graphical, and environmental range. In particular, Bsal DNA was detected in 33 sam- 4Nanjing Institute of Environmental Sciences, ples from the genera Cynops, Pachytriton, Paramesotriton, Tylototriton, and Andrias, Ministry of Environmental Protection of China, Nanjing, Jiangsu 210042, China including the heavily traded species Paramesotriton hongkongensis and Cynops ori- Correspondence entalis. The true Bsal prevalence across our data set was estimated between 2% and Zhiyong Yuan, Key Laboratory for Forest 4%, with a maximum of 50% in a population of P. hongkongensis. Even at this overall Resources Conservation and Utilisation in the relatively low Bsal prevalence, the exportation of millions of animals renders Bsal Southwest Mountains of China, Ministry of Education. Southwest Forestry University, introduction in naïve, importing countries a near certainty, which calls for the urgent Kunming, Yunnan 650224, China. implementation of proper biosecurity in the international wildlife trade. Email: [email protected] Frank Pasmans, Department of Pathology, KEYWORDS Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Batrachochytrium salamandrivorans, biosafety, China, pet trade, salamander Salisburylaan 133, Merelbeke, 9820, Belgium. Email: [email protected] Funding information National Key R&D Program of China, Grant/Award Number: 2016YFC1200705; The First Class Discipline Construction Project for Forestry in Yunnan, Grant/Award Num- ber: 51600625; National Natural Science Foundation of China, Grant/Award Numbers: 31702008, 31401958; Fonds Wetenschap- pelijk Onderzoek, Grant/Award Number: FWO16/PDO/019 Editor Julie Lockwood This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright and Photocopying: © 2018 The Authors. Conservation Letters published by Wiley Periodicals, Inc. Conservation Letters. 2018;e12436. wileyonlinelibrary.com/journal/conl 1of8 https://doi.org/10.1111/conl.12436 2of8 YUAN ET AL. 1 INTRODUCTION 2 MATERIALS AND METHODS Emerging fungal diseases are major drivers of global Noninvasive skin samples were collected from Chinese sala- amphibian diversity loss (Fisher et al., 2012). Understanding mander species belonging to the Salamandridae across the pathways of pathogen introduction is key in shaping policy Chinese territory using cotton tipped swabs (Classical Swab, decisions aimed at the prevention of spillover into naïve Copan Diagnostics, Brescia, Italia) between August 2016 and regions. The chytrid fungus Batrachochytrium salamandrivo- August 2017. In total, 51 localities were surveyed, seeking to rans (Bsal) recently invaded northwestern Europe (Martel broadly encompass the distributions of Chinese Salamanders et al., 2013), causing extinction events in urodelan populations (Figure 1). Most animals sampled were wild, except for 16 (Stegen et al., 2017) and threatening the majority of urodelan Chinese giant salamanders (A. davidianus) that were farm- diversity in the western Palearctic (Martel et al., 2014). bred. To avoid cross-contamination, gloves were changed Current evidence suggests Bsal is probably absent from each time after handling and sampling an animal and before the highly urodelan diverse regions in Northern and Latin catching a new individual. Swabs were stored at -20◦C. Water America (Bales et al., 2015; Martel et al., 2014). In Asia, Bsal and air temperatures were measured at each site upon collec- appears to coexist with native urodelan communities in Viet- tion of the first salamander in the sample. nam (Laking et al. 2017), Thailand, and Japan (Martel et al., The presence of Bsal in the swab samples was determined 2014). This has led to the hypothesis that Bsal coevolved using simplex qPCR as described by Blooi et al. (2014). with Asiatic urodelans and has been vectored into Europe on Briefly, genomic DNA was extracted from the swabs using live amphibians through the pet trade (Martel et al., 2014). 100 l of Prepman Ultra reagent (Applied Biosystems, Foster The large-scale harvest of Asian urodelans for trade has City, CA), following the DNA extraction method described been shown to drive declines in at least some species (Rowley by Hyatt et al. (2007). We used a specific real-time PCR et al., 2016). Chinese species dominate this trade, with high procedure described by Blooi et al. (2015) on CFX96 real- numbers of specimens from the genera Cynops, Pachytriton, time system (Bio-Rad Laboratories, Hercules, CA). Genomic Tylototriton, and Paramesotriton having been traded over the equivalents (GE) of Bsal zoospores were calculated per past decades. For the United States alone, pet trade imports swab sample. Samples were run in duplicate and were from China have included over 2.3 million specimens of considered positive based on a combination of: (1) shape of Cynops orientalis during the period 2001-2009 (Herrel & Der amplification curves (2) positive results in both duplications, Meijden, 2014) and an estimated over 300,000 specimens of and (3) returning GE values above the detection threshold. Paramesotriton hongkongensis during the period 2005-2010 All positive samples were confirmed using sequencing of the (Lee, Lau, & Chan, 2004; Xie et al., 2007). To date, firm evi- amplicons. dence for the potential of Chinese species as vectors for Bsal is In addition to the sample prevalence (the number of posi- lacking and the only study performed on Chinese amphibians tive individuals in each sample), we sought to obtain estimates did not demonstrate the presence of the pathogen in Chinese of the true prevalence in the sampled population, accounting amphibian communities (Zhu et al., 2014). However, only a for the uncertainty determined by the variable sample sizes. limited number of salamanders (16 samples of Andrias david- We assumed the number n of infected individuals out of a sam- ianus and 19 samples of C. orientalis, which were from farms ple of size N to be a binomially distributed random variable or food markets) were tested by Zhu et al. (2014). Given the n∼Bin (p, N), where p is the true prevalence of infection in the volume of trade originating from China, conclusive evidence sampled population. This estimate provides a more realistic of Bsal presence on widely traded Chinese urodelans would representation of our current knowledge of Bsal prevalence, further reinforce the need to speed up decision processes in and can, for example, be used as a prior belief for the design the European Union and to strengthen existing trade bans in of surveillance monitoring and biosecurity controls. We esti- the United States and Canada. Moreover, quantifying the prior mated the average true prevalence across the entire data set, belief about the probability of presence and prevalence of the as well as true prevalence at the species, genus, and province pathogen is key to devising effective biosecurity control and level. We implemented the estimation in a Bayesian frame- surveillance monitoring (Runge et al., 2017). work, using the JAGS software (Plummer, 2005). We used Here, we present this evidence, obtained from a large-scale uninformative beta-distributed priors for p at all levels (over- field sampling for Bsal in Chinese urodelans, including the all, species, genus, and province), and drew 100,000 samples most widely traded salamandrid genera, which demonstrates from three separate Markov chains after a burn-in of 50,000 presence of Bsal in several trade species, and remarkably high iterations. We assessed convergence by visual inspection of prevalence in some of them. These findings support urgent the chain histories and using the R-hat statistic, and summa- calls for immediate actions to restrict trade in live amphibians rized estimates using the mean and 95% credible intervals in order to prevent pathogen pollution and potential massive (CRI) of the posterior distribution of p. Finally, we used logis- biodiversity loss. tic regression to model p across the entire data set as

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