SALAMANDRA 54(1) 87–91 15 February 2018 CoIrSrSeNsp 0o0n3d6e–n3c3e75 Correspondence First survey of the pathogenic fungus Batrachochytrium salamandrivorans in wild and captive amphibians in the Czech Republic Vojtech Baláž1,2, Milič Solský3, David Lastra González3, Barbora Havlíková3, Juan Gallego Zamorano3, Cristina González Sevilleja3, Laura Torrent3 & Jiří Vojar3 1) Department of Ecology and Diseases of Game, Fish and Bees, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1/3, 612 42 Brno, Czech Republic 2) Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1/3, 612 42 Brno, Czech Republic 3) Department of Ecology, Faculty of Environmental Sciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague 6, Czech Republic Corresponding author: Jiří Vojar, e-mail: [email protected] Manuscript received: 8 June 2017 Accepted: 27 July 2017 by Jörn Köhler The recently discovered fungal pathogen Batrachochytri­ exotic pet fairs take place on a regular basis (Havlíková um salamandrivorans (Martel et al. 2013) (hereinafter re- et al. 2015). Furthermore, Prague and its surroundings are ferred to as Bsal) has already received significant scientific known to harbour wild populations of at least four native and public attention (e.g., Martel et al. 2014, Van Rooij caudate species: fire salamander, smooth newt (Lissotriton et al. 2015, Yap et al. 2015, Stegen et al. 2017). The Bsal vulgaris), alpine newt (Ichthyosaura alpestris) and great epidemic has so far been limited to European newts and crested newt (Triturus cristatus) (Šťastný et al. 2015). All salamanders found in the wild (Belgium, Germany and the four are susceptible to Bsal-induced mortality (Martel Netherlands: Martel et al. 2013, Spitzen-van der Sluijs et al. 2013, 2014, Cunningham et al. 2015, Spitzen-van et al. 2016) and in captive populations (Germany: Sabino- der Sluijs et al. 2016, Stegen et al. 2017). The surround- Pinto et al. 2015; United Kingdom: Cunningham et al. ing areas of such large cities are likely to constitute areas of 2015). In the Netherlands, Bsal is responsible for the near high risk for wild populations of native amphibians. For extinction of wild fire salamander (Salamandra salaman­ this reason, we selected Prague and its surroundings as the dra) populations (Spitzen-van der Sluijs et al. 2013). first focal area for Bsal surveillance efforts in wild popula- The Bern Convention Standing Committee has there- tions of Czech caudate amphibians. Considering that Bsal fore announced Recommendation No. 176 on the preven- is spread through the pet trade in caudates originating in tion and control of the Bsal chytrid fungus. According to Asia (Martel et al. 2014), we also focused on captive col- this recommendation, European countries should adopt lections of caudate amphibians. measures that include establishment of monitoring pro- In total, 324 swab samples were tested for both Batracho­ grammes to control the possible further spread of the dis- chytrium dendrobatidis (Bd) and Bsal presence and preva- ease, especially in areas of high risk (e.g., areas near disease lence. In wild populations, 126 samples of three caudate outbreaks), and develop emergency action plans that will species (fire salamanders, smooth newts and alpine newts) allow prompt responses in case of Bsal occurrence (Coun- were collected at nine sites within Prague’s urban area and cil of Europe 2015). its surroundings during autumn 2015 and spring 2016 (Ta- The Czech Republic is a country with relatively high ble 1). Furtheremore, we analysed samples from five captive caudate species diversity (Sillero et al. 2014) and shares collections, including those of four private breeders and a western border with Germany, a country with previously Prague’s zoological garden during the period 2015–2016. proven Bsal occurrence (Sabino-Pinto et al. 2015, Spit- Within each collection, only subset of about two to four zen-van der Sluijs et al. 2016). The country, and espe- individuals were sampled from an aquarium. This Bsal-tar- cially the capital city of Prague, has an active and sizeable geted sampling in captivity was extended by re-analysing community of exotic pet keepers and pet shops, and large samples of caudates from previous surveillance projects © 2018 Deutsche Gesellschaft für Herpetologie und Terrarienkunde e.V. (DGHT), Mannheim, Germany Available at http://www.salamandra-journal.com 87 Correspondence Table 1. Numbers (No.) of individuals sampled within nine wild caudate populations in Prague and its surroundings. Date = date of sampling. Locality name Coordinates Species No. Date Podhořský potok, small stream, tributary of Vltava River 50.129947°N 14.404111°E Salamandra salamandra 31 07.10.2015 Únětice, unnamed tributary of Únětický potok 50.144853°N 14.384502°E Salamandra salamandra 25 15.10.2015 Levý Hradec, unnamed tributary of Vltava River 50.169883°N 14.377429°E Salamandra salamandra 12 20.10.2015 Úholičky, unnamed tributary of Podmoráňský potok 50.170698°N 14.344784°E Salamandra salamandra 8 09.11.2015 Lhotecký potok, tributary of Vltava River 49.956059°N 14.411423°E Salamandra salamandra 7 15.10.2015 Chalupecká strouha, near confluence with Zvolský potok 49.930541°N 14.390361°E Salamandra salamandra 1 17.11.2015 Baně, unnamed tributary of Vltava River 49.961229°N 14.392828°E Salamandra salamandra 2 17.11.2015 Ohrobecké údolí, unnamed tributary of Vltava River 49.943775°N 14.413338°E Salamandra salamandra 10 21.10.2015 Botanická zahrada, artificial pond in botanic garden 50.070429°N 14.421077°E Lissotriton vulgaris 28 01.07.2016 Botanická zahrada, artificial pond in botanic garden Ichthyosaura alpestris 2 01.07.2016 searching for Bd presence in captive amphibians (Hav- ples from captivity the 99% confidence limits are 0.0–2.6% líková et al. 2015), including 18 individuals of the largest (Rósza et al. 2000). This does not directly mean that Bsal is amphibian species, the Chinese giant salamander (Andrias not present in the Czech Republic. Because the disease out- davidianus), reared in Prague’s zoological garden. In total, breaks can occur at very low host densities in wild popula- 198 samples of 60 caudate (sub)species were analysed in tions (Schmidt et al. 2017), all host populations of suscep- captive collections (Table 2). tible European caudate species (Martel et al. 2014) are at Sampling and DNA extraction were performed accord- risk from Bsal (Schmidt et al. 2017). In the case of asymp- ing to procedures used in amphibian chytridiomycosis re- tomatic Asian caudates in captive collections, infection may search (Boyle et al. 2004). The first sample subset, consist- be present in such small prevalence (Martel et al. 2014, ing of 98 wild and 56 captive samples, was checked for Bsal Laking et al. 2017) that our sampling was not sufficient. presence by SYBRGreen quantitative polymerase chain re- On the other hand, because the intensive sampling of wild action (qPCR) following the method described in Blooi fire salamanders covered nearly all localities within Prague et al. (2013) as one possible detection option. Bovine se- where the species presently is known to occur (Šťastný et rum albumin (BSA) was added to reduce PCR inhibition al. 2015) and no sampled individual exhibited visible disease (Garland et al. 2010).The identity of amplified DNA was symptoms, we conclude that Bsal probably has not invaded checked by melt curve analysis and compared to results for Prague’s fire salamander population, at least for now. Simi- genomic standards of Bsal provided by An Martel (Ghent lar results of pathogen absence have been found in stud- University). We later adopted the duplex Bd+Bsal qPCR ies focused on fire salamanders in Austria (Gimeno et al. (Blooi et al. 2013) and used it for additional samples. In 2015), eastern hellbenders (Cryptobranchus alleganiensis) this assay, we used genomic standards of Bd equivalent to in the U.S. (Bales et al. 2015), Japanese giant salamanders 0.1, 1, 10 and 100 zoospores per 5 µl (strain IA042, Ibon (Andrias japonicus) in Japan (Bletz et al. 2017a), Chinese Acherito, Pyrenees, 2004) obtained from the Institute of amphibians (Zhu et al. 2014), five species of newts and fire Zoology, Zoological Society of London. A single quan- salamanders in most of tested localities in Belgium, Ger- tity sample of Bsal genomic DNA was used as a positive many and the Netherlands (Spitzen-van der Sluijs et al. control. If any sample showed fluorescence growth in the 2016), alpine newts, smooth newts and great crested newts wavelength of the Bsal probe, it would be re-analysed with in Germany (Bletz et al. 2017b), and in a study by Parrot the full set of Bsal standards. In this way, we slightly re- et al. (2016) on 17 caudate species across three continents. duced the cost of analysis. In both detection assays, we We used two available detection assays in our study, used duplicates of all analysed samples, standards, as well both based on DNA amplification with the same pair of as positive and negative controls. Bsal primers (Blooi et al. 2013) and differing only in the All tested samples yielded negative results for the pres- detection format of the amplicon. The SYBR Green qPCR ence of Bsal. Bd was detected in three individuals of wild assay often produced detectable fluorescence growth of smooth newts and in one reared ribbed newt (Pleurodeles nonspecific products, thus complicating interpretation of waltl) in a captive collection, albeit with no visible signs the results. In several cases, we ran standard PCR followed of the chytridiomycosis. Low Bd prevalence in caudates by gel electrophoresis with samples of equivocal results to corresponds well with our previous findings in Czech cap- confirm the identity of PCR products. Our results indicat- tive collections (Havlíková et al. 2015), and wild popula- ed a mean melting temperature (Tm) for Bsal standards of tions of caudates in Central and east Europe (Baláž et al. 77.21°C (SD = 0.29), which differs slightly from the pub- 2014a,b, Vojar et al. 2017). lished value of 75.5°C (Blooi et al. 2013). For monitoring The 0% Bsal prevalence in wild caudates has Sterne-Wald Bsal presence in wild and captive amphibians, we later ad- 99% confidence limits of 0.0–4.2%, while in the case of sam- opted and recommend the use of duplex Bd+Bsal qPCR, 88 Correspondence Table 2. List of surveyed species and numbers (No.) of individuals sampled in captivity. Species No. Species No. Ambystoma mexicanum 3 Neurergus deryugina deryugina 2 Ambystoma tigrinum 2 Neurergus strauchii barani 3 Andrias davidianus 18 Neurergus strauchii strauchii 3 Calotriton asper 3 Ommatotriton ophryticus nesterovi 3 Cynops ensicauda ensicauda 3 Pachyhynobius shangchengensis 1 Cynops ensicauda popei 4 Pachytriton sp. 2 Cynops orientalis 1 Paramesotriton caudopunctatus 7 Cynops pyrrhogaster 3 Paramesotriton deloustali 6 Cynops pyrrhogaster “Kanagawa” 6 Paramesotriton guangxiensis 4 Cynops pyrrhogaster “Yubana” 2 Paramesotriton hongkongensis 3 Euproctus platycephalus 2 Paramesotriton chinensis 12 Hynobius dunni 1 Paramesotriton sp. “helm” 1 Hynobius leechii 2 Paramesotriton sp. “red” 6 Hynobius lichenatus 1 Paramesotriton yunwensis 2 Hynobius quelpartensis 2 Pleurodeles nebulosus 2 Hynobius retardatus 2 Pleurodeles waltl 4 Hypselotriton cyanurus 2 Salamandra algira tingitana 2 Hypselotriton cyanurus cyanurus 2 Siren intermedia 1 Hypselotriton chenggongensis 3 Triturus anatolicus 2 Hypselotriton orientalis 1 Triturus blasii 3 Ichthyosaura alpestris 3 Triturus carnifex 9 Laotriton laoensis 3 Triturus cristatus 2 Lissotriton boscai 3 Triturus dobrogicus dobrogicus 2 Lissotriton graecus 3 Triturus dobrogicus macrosoma 3 Lissotriton helveticus 3 Triturus ivanbureschi 1 Lissotriton italicus 3 Triturus karelinii 2 Lissotriton malcani 3 Triturus macedonicus 10 Lissotriton meridionalis 3 Triturus marmoratus 2 Lissotriton montandoni 3 Triturus pygmaeus 3 Neurergus crocatus complex 3 Tylototriton shanjing 2 which is designed specifically to detect either of the patho- detection in captive collections of amphibians, forming a gen species (Blooi et al. 2013). The higher cost of analy- network of continuously monitored localities in proxim- ses versus using fluorescent probes is counterbalanced by ity to larger cities, and preparing an action plan in case of clearer and more specific results. Bsal occurrence in collaboration with the Nature Conser- As not only newts, but also infected anurans and even vation Agency of the Czech Republic, the State Veterinary waterfowl via scales on their feet, may promote fungal Authority, and the Czech Ministry of Environment. spread over large spatial distances (Stegen et al. 2017), the spread of this emerging pathogen is difficult to predict, and we can expect the distribution of Bsal to change consider- Acknowledgements ably over time. The risk that new points of entry for Bsal into Europe will occur via the pet trade is a constant threat We thank A. Martel from Ghent University for providing the that can be alleviated by collaboration among pet owners, genomic standards for Bsal, T. Garner from the Institute of Zoo- the pet trade, veterinary authorities, and conservationists logy, Zoological Society of London, for providing the genomic (Sabino-Pinto et al. 2015). It is essential to prevent this standards for Bd, and G. A. Kirking for useful comments on the manuscript. We also thank all those zookeepers, institutions, pathogen entering the wild amphibian populations (Cun- private amphibian breeders, and pet shop sellers who cooperat- ningham et al. 2015), because there is no effective meth- ed voluntarily and provided amphibians for sampling. The study od to reduce the impact of chytridiomycosis in the field was supported by the Czech Ministry of Environment and by the (Garner et al. 2016). Therefore, our next planned steps in University of Life Sciences Prague (grant no. 20164245). 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