Presence of Batrachochytrium Dendrobatidis in Amphibians from Central and Southern Hesse, Central Germany: Results from a Preliminary Regional Screening
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SALAMANDRA 48(3) 166–172 30 October 2012 CorrespondenceISSN 0036–3375 Correspondence Presence of Batrachochytrium dendrobatidis in amphibians from central and southern Hesse, central Germany: results from a preliminary regional screening Christiane Rasmussen 1, Tobias Eisenberg 2, Dirk Alfermann 3 & Jörn Köhler 1 1) Hessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany 2) Landesbetrieb Hessisches Landeslabor (LHL), Schubertstr. 60, 35392 Gießen, Germany 3) Arbeitsgemeinschaft Amphibien- und Reptilienschutz in Hessen e.V. (AGAR), Gartenstr. 37, 63517 Rodenbach, Germany Corresponding author: Christiane Rasmussen, e-mail: [email protected] Manuscript received: 21 March 2012 Chytridiomycosis is an emerging infectious amphibian cluding ten localities within Hesse. Of these ten localities, disease that has been associated with population declines four tested positive for the presence of Bd (T. Ohst pers. in different regions of the globe, but the geographic dis- comm.). tribution and actual effect of the fungus Batrachochytrium Given the fragmentary knowledge in Germany and the dendrobatidis (Bd) is still insufficiently known (Fisher et high potential threat of Bd, our small-scale study aimed at al. 2009, Ohst et al. 2011). Mass mortalities are apparently a first preliminary screening for Bd within a limited geo- restricted to certain regions, whereas in other cases, infect- graphical region. To assess the prevalence of Bd and esti- ed populations barely appear to suffer from their infection mate infection rates among amphibian species and popu- (e.g., Woodhams et al. 2007b, Kilpatrick et al. 2010). Re- lations, we selected certain localities and target taxa, hop- cently, Farrer et al. (2011) identified different strains of ing to provide useful new data for future in-depth research Bd and suggested that one highly virulent strain might be and conservation planning. responsible for the most dramatic consequences of the dis- The field survey included 14 collection sites in central ease. However, specific differences in susceptibility of host and southern Hesse, Germany (Fig. 1). All collection sites species, individual fitness, and/or environmental condi- range in altitude between 88 and 350 m above sea level. tions might also play important roles in the effects of Bd Geographical coordinates were geo-referenced by using infections (e.g., Blaustein et al. 2005, Woodhams et al. GoogleEarth (Tab. 1). We collected samples from 221 indi- 2007a, Hossak et al. 2010, Kilpatrick et al. 2010, Tobler viduals of adult amphibians representing eight species, as & Schmidt 2010, Ohst et al. 2011). Variable effects have well as three samples from juvenile brown frogs, between even been observed within individual populations of the February and June 2011. Additionally, we included 60 pre- same species (Tobler et al. 2010). served tissue samples from toe clippings collected during a Bd currently occurs on all continents (except Antarc- field survey at Heusenstamm in 2007. For easier sampling tica) and has just recently been detected in Asia (Mutsch- we mainly selected locations with fences established for mann et al. 2000, Garner et al. 2005, Bosch et al. 2007, amphibian protection. During an earlier study conducted Bielby et al. 2009, Kielgast et al. 2010, Ohst et al. 2011, by Ohst et al. (2011) between 2003 and 2010, four collec- Swei et al. 2011). Concerning Central Europe, the disease tion sites in Hesse had showed Bd infection (Fischborn, is well studied in Switzerland, where Bd could be found Wiesbaden-Auringen 1, Wiesbaden-Auringen 4, and Ep- at half of all water bodies north of the Alps (Garner et pertshausen; T. Ohst pers. comm.), two of which (Fisch- al. 2005, Tobler & Schmidt 2010). Given the data from born, Eppertshausen) were again included in this study. To Switzerland, it can be assumed that Bd is also widespread investigate the situation of the regionally endangered Rana and common in Germany, but the knowledge about Bd arvalis, we took samples from a spawning area in Rodgau distribution and its impact at national level is very frag- and a pond in Riedstadt-Erfelden near a spawning site, the mentary. Between 2003 and 2010, Ohst et al. (2011) test- latter being located within the conservation area Kühkopf- ed 2,100 samples from 156 German localities for Bd, in- Knoblochsaue. © 2012 Deutsche Gesellschaft für Herpetologie und Terrarienkunde e.V. (DGHT), Mannheim, Germany All166 articles available online at http://www.salamandra-journal.com Correspondence Individuals were captured with dip-nets, fish traps or 1.4 mm ceramic spheres (Lysing Matrix D; mp biomedi- along the fences with pitfall traps. To avoid the further cals, Eschwege, Germany) for 45 s with intensity 6.5 using spread of Bd, sampling was subjected to the recommenda- a cell disrupter (Ribolyser Fastprep 120 Bio101, Thermo tions of Schmidt et al. (2009). We used Virkon S (2 g/l) Electron, Osterode, Germany). One millilitre of the sam- for disinfecting dip-nets, fish traps and waders, and 70% ple solution was then centrifuged (7,500 rpm for 10 min.) ethanol for smaller items like torch lights. For collect- and the cell pellet further processed for nucleic acid extrac- ing zoospores, we used disposable gloves and sterile cot- tion. DNA was extracted from all samples using a commer- ton swabs (Sarstedt, Nümbrecht, Germany), which were cial kit (Qiagen DNeasy Blood & Tissue Kit, Hilden, Ger- firmly swept over the skin of ventral and dorsal surfaces, many) according to the manufacturer’s recommendations thighs and webbing of the foot as described by Kriger et (methods not specifically adapted for Bd). Briefly, the cell al. (2006). Dry swabs were stored in tubes and frozen at pellet was suspended in 200 µl reaction buffer with pro- -20ºC following sampling. Tissue samples obtained by toe teinase K, incubated at 56°C for 60 min and centrifuged clippings in 2007 were preserved in small tubes (1.5 ml) (8,000 rpm for 1 min.) following the addition of buffer and containing 90% ethanol. ethanol. Eluted DNA was extracted from DNeasy columns Swabs were eluted in 1.0 ml PBS buffer by an ultrasound after several centrifugation steps and 5 µl template DNA treatment for 5 min. After removal of the cotton tip, a cen- were utilized for PCR. A realtime PCR protocol for the trifugation step (7,500 rpm for 10 min.) was performed and specific detection of Bd was employed according to Boyle the supernatant carefully removed. Toe clippings were di- et al. (2004) with the minor modifications mentioned be- luted 1:5 in PBS buffer and homogenised in vials containing low. The final reaction volume of 25 µl contained templates Figure 1. Schematic map of Germany (lower right) and southern Hesse (left) showing the sampled areas. Localities with positive re- sults for Batrachochytrium dendrobatidis (Bd) are indicated by black dots, those with doubtful results by grey dots, and those without positive results by circles, respectively. 167 Correspondence Table 1. Number of samples taken per locality and species. Abbreviations used refer to B.b. (Bufo bufo), L.v. (Lissotriton vulgaris), M.a. (Mesotriton alpestris), P.spp. (Pelophylax spp.), R.a. (Rana arvalis), R.d. (Rana dalmatina), R.t. (Rana temporaria), P.f. (Pelobates fuscus) and juv. (juvenile Rana temporaria). *) Tissue samples collected by toe clipping in 2007. Locality/coordinates B.b. L.v. M.a. P. spp. P.f. R.a. R.d. R.t. juv. total Gedern 6 6 4 0 0 0 0 0 0 16 50°26’40’’ N, 9°11’10’’ E Fischborn 6 0 6 6 0 0 0 6 0 24 50°23’15’’ N, 9°17’43’’ E Fulda 6 6 6 0 0 0 0 5 0 23 50°35’52’’ N, 9°42’26’’ E Bad Camberg 10 0 0 0 0 0 0 0 0 10 50°18’03’’ N, 8°19’18’’ E Rosbach 13 0 1 0 0 0 0 0 0 14 50°16’58’’ N, 8°42’31’’ E Freigericht 10 0 0 0 0 0 0 0 0 10 50°08’11’’ N, 9°10’07’’ E Wiesbaden 12 5 1 0 0 0 0 6 0 24 50°05’49’’ N, 8°11’09’’ E Mühlheim am Main 0 0 0 0 0 0 0 12 3 15 50°06’22’’ N, 8°50’46’’ E Rodgau 0 3 0 7 0 1 2 9 0 22 49°59’10’’ N, 8°51’56’’ E Eppertshausen 6 0 8 6 0 0 6 4 0 30 49°56’58’’ N, 8°49’23’’ E Riedstadt 0 0 0 12 0 0 0 0 0 12 49°50’27’’ N, 8°26’42’’ E Reinheim 10 0 0 0 0 0 0 0 0 10 49°49’07’’ N, 8°50’26’’ E Bensheim 4 3 2 1 2 0 0 2 0 14 49°43’21’’ N, 8°33’12’’ E Heusenstamm* 0 0 40 0 0 0 13 7 0 60 50°03’45’’ N, 8°47’46’’ E 83 23 68 32 2 1 21 51 3 284 or controls (5 µl), primers (each 1.1 µl with 20 pmol/µl), We detected Bd at three out of the fourteen sampling and taqman probe (0.6 µl with 10 pmol/µl) as well as an sites (Figs. 1, 2). The samples from Eppertshausen, near internal control reaction (Hoffmann et al. 2006), and 2x the conservation area Rallenteich, were notable for their Quantitect Probe PCRmaster mix (Qiagen). After centrifu- high detection rates with a prevalence of approximate- gation (7,000 rpm for 20 s), the PCR was processed on a ly 50% (CI 32–68). In addition, Bd was recorded in one realtime thermocycler (iCycler, Bio-Rad Laboratories Inc., water frog (Pelophylax sp.) at the Rana arvalis spawning Munich, Germany; initial denaturation 95°C for 15 min., site in Rodgau, but samples from a pond near the large 45 cycles with 94°C for 60 s and 60°C for 60 s, and finally conservation area Kühkopf-Knoblochsaue tested nega- 40°C for 30 s) without quantification standard.