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Treatment of urodelans based on temperature dependent infection dynamics of Batrachochytrium salamandrivorans

Treatment of urodelans based on temperature dependent infection dynamics of Batrachochytrium... OPEN Treatment of urodelans based on temperature dependent infection SUBJECT AREAS: ECOLOGICAL dynamics of Batrachochytrium EPIDEMIOLOGY FUNGAL BIOLOGY HERPETOLOGY salamandrivorans THERAPEUTICS 1,2 1 1 2 3 1 M. Blooi , A. Martel , F. Haesebrouck , F. Vercammen , D. Bonte & F. Pasmans Received Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 20 October 2014 9820 Merelbeke, Belgium, Centre for Research and Conservation, Royal Zoological Society of Antwerp, Koningin Astridplein 26, Accepted 2018 Antwerp, Belgium, Department of Biology, Terrestrial Ecology Unit, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, 24 December 2014 Belgium. Published 27 January 2015 The recently emerged chytrid fungus Batrachochytrium salamandrivorans currently causes amphibian population declines. We hypothesized that temperature dictates infection dynamics of B. salamandrivorans, and that therefore heat treatment may be applied to clear animals from infection. We examined the impact of environmental temperature on B. salamandrivorans infection and disease dynamics in fire salamanders Correspondence and (Salamandra salamandra). Colonization of salamanders by B. salamandrivorans occurred at 156C and 206C requests for materials but not at 256C, with a significantly faster buildup of infection load and associated earlier mortality at 156C. should be addressed to Exposing B. salamandrivorans infected salamanders to 256C for 10 days resulted in complete clearance of infection and clinically cured all experimentally infected animals. This treatment protocol was validated in M.B. (Mark.Blooi@ naturally infected wild fire salamanders. In conclusion, we show that B. salamandrivorans infection and UGent.be) disease dynamics are significantly dictated by environmental temperature, and that heat treatment is a viable option for clearing B. salamandrivorans infections. n the decades following the identification of Batrachochytrium dendrobatidis in 1999 it became apparent that this chytrid fungus was one of the biotic drivers of declines and extinctions of hundreds of amphibian species 2–5 I worldwide . However, the impact of B. dendrobatidis varies regionally from a dramatic decrease of amphi- bian diversity to a state of host-pathogen equilibrium. In one such region characterized by the co-existence of B. dendrobatidis with local amphibian communities , another recently described chytrid fungus, Batrachochytrium salamandrivorans , caused amphibian population declines. The reason for this obvious difference in disease dynamics between both chytrid fungi is not known. Disease dynamics are dictated by pathogen virulence, host factors and environmental determinants. Virulent strains of both chytrids, as well as susceptible host species are 8–11 12–14 present in the affected regions . For B. dendrobatidis, temperature is considered a key environmental factor . One major difference between both chytrids is their different thermal growth characteristics, which is probably due to differences in host spectrum, B. salamandrivorans being restricted to urodelan hosts . Knowledge of the infection dynamics of B. salamandrivorans at different temperatures may help to develop treatment proto- 16–18 19 cols . These are urgently needed as current therapies developed against B. dendrobatidis fail to eliminate B. salamandrivorans from infected amphibians (unpublished results). We hypothesized that temperature dictates infection and disease dynamics of B. salamandrivorans in salamanders, which may be applied to develop a heat treatment protocol to clear infection in animals. Results & Discussion Only after exposure at 15uCor20uC but not at 25uC, the salamanders were colonized by B. salamandrivorans.Ifa 20–22 10000 GE infection load per swab is considered indicative for a clinical threshold , this threshold was reached two times earlier at 15uC than at 20uC (on average 15 6 4 (SD) days and 31 6 12 days (SD) respectively, independent t-test p , 0.05) (Fig. 1). The faster buildup of B. salamandrivorans infection loads coincided with earlier mortality at 15uC than at 20uC (22 6 8 (SD) days and 35 6 14 (SD) days respectively, Cox regression analysis, x 5 3.941, df 5 1, p , 0.05) (Fig. 1 and 2). Besides preventing infection of salamanders with B. SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 1 www.nature.com/scientificreports Figure 1 | The course of Batrachochytrium salamandrivorans infection in fire salamanders at 15 and 206C. Each symbol represents the course of infection of an individual animal.Time of death of all animals is depicted beneath the graphs. The line represents the average increase in infection intensity in all tested individuals based on a repeated measure regression analysis. salamandrivorans, exposure of infected salamanders to a temper- 25uC on frogs was opposite to the pattern of temperature-dependent ature of 25uC during 10 days completely eliminated the infection growth at these temperatures in culture , and time until death in and resolved B. salamandrivorans lesions from all infected animals frogs infected with B. dendrobatidis at 27uC, which is above B. den- (Fig. 3 and 4). However, 7 days exposure at 25uC did not result in drobatidis’ thermal preference , was shorter when compared to time fungal clearance since recrudescence of infection was observed in all until death of infected frogs kept at lower temperatures . The suit- these salamanders within 1–3 weeks after transferring them to an ability of raised ambient temperature as treatment option was vali- ambient temperature of 15uC (Fig. 4). This is remarkable since cul- dated by keeping 30 wild-caught B. salamandrivorans infected fire tures of the fungus are killed in vitro within 5 days of incubation at salamanders at 25uC during 10 days. Twenty-six animals were cured 25uC and shows that B. salamandrivorans is capable to persist in an of B. salamandrivorans infection after this treatment period, 2 died urodelan host experiencing temperatures that temporarily surpass early during treatment, and 2 needed an additional treatment period the fungal thermal maximum for up to one week. Exposure of the of 2 days in order to completely clear the infection (Fig. 5). This relapsing animals to 25uC for 10 days eliminated the infection. Our shows that heat treatment is a viable treatment option for B. sala- results reflect B. salamandrivorans growth curves obtained in vitro, mandrivorans infected amphibians when the clinical condition and with an optimal growth range around 15uC . In contrast, the pattern the thermal tolerance of the animal is taken into account. In order to of temperature-dependent growth of B. dendrobatidis at 15, 20 and completely eliminate B. dendrobatidis infections higher tempera- Figure 2 | The probability of survival of salamanders housed at 15 or 206C after infection with Batrachochytrium salamandrivorans. Survival probability was plotted based on a Cox regression analysis (x 5 3.981, df 5 1, p , 0.05). Time is displayed in days after initial infection. The dotted line represent survival probability of Batrachochytrium salamandrivorans infected salamanders housed at 15uC and the full line those housed at 20uC. SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 2 www.nature.com/scientificreports Figure 3 | Heat treatment of amphibians infected with Batrachochytrium salamandrivorans clears infection and resolves associated lesions. Batrachochytrium salamandrivorans associated skin lesions (A) are clearly reduced after the heat treatment composed of keeping the animals at 25uC during 10 days (B), and will eventually completely resolve. tures, composed of short exposure to 37uC or extended exposure to Methods All experiments were performed in accordance with the relevant guidelines and 30uC are required. These protocols are not suitable for treating regulations. All experiments with experimental animals were carried out with salamanders, as these temperatures surpass the upper thermal limit approval of the ethical committee of the Faculty of Veterinary Medicine, Ghent of most urodelans. The 2 animals that died were in poor clinical University. condition at the start of the treatment period, and probably died due to thermal shock as B. salamandrivorans loads were low at time Batrachochytrium salamandrivorans strain, culture conditions and experimental inoculation. The B. salamandrivorans type strain was grown in TGhL broth (16 g of death. This points out the narrow margin between the temperature tryptone, 4 g gelatin hydrolysate, 2 g lactose per liter of distilled water) in 25 cm cell able to eliminate B. salamandrivorans and the upper thermal limit culture flasks and incubated at 15uC. To obtain B. salamandrivorans zoospores, a most urodelans tolerate. Furthermore, these results show that the 2 ml aliquot of a 5-day-old culture was inoculated on TGhL agar plates (16 g course of infection should be carefully monitored since not all ani- tryptone, 4 g gelatin hydrolysate, 2 g lactose, 10 g bacteriological agar per liter of distilled water) and incubated at 15uC for 5–7 days. Zoospores were collected by mals tested negative for presence of Bs DNA after 10 days at 25uC. flooding the agar plates with 2 ml of distilled water and subsequent collection of the Although we do not think that this is a result of an active infection but fluid. A hemocytometer was used to count the number of zoospores present in the explained by presence of residual B. salamandrivorans DNA derived 3 suspension and the concentration of the zoospore suspension was adjusted to 5 3 10 from dead B. salamandrivorans cells, this remains uncertain. This zoospores per mL. Animals were inoculated with B. salamandrivorans by topically applying one mL of the inoculum on the intact skin. could have been further elucidated by transferring the animals back to 15uC after 10 days but we chose to keep them at 25uC until PCR Animals. Experimental animals. Fire salamanders (Salamandra salamandra) were results became negative. Thermal treatment of B. salamandrivorans experimentally infected with B. salamandrivorans to study temperature dependent infected amphibians would allow large groups of animals to be infection dynamics. The animal experiment was performed with the approval of the treated simultaneously at low costs and lacks the possible downsides ethical committee of the Faculty of Veterinary Medicine (Ghent University, EC2013/ 87). Twenty-five captive bred fire salamanders were housed individually in plastic linked to drug treatment like toxicity or development of acquired containers in a climatized room with an ambient temperature of 15uC. The animals antimicrobial resistance. were kept on a moist tissue, with access to a hiding place and water container. Crickets In conclusion, these results demonstrate that infection and disease powdered with mineral and vitamin supplement were provided ad libitum as food dynamics of B. salamandrivorans in urodelans are significantly dictated source. All animals were clinically healthy and free of B. dendrobatidis and B. salamandrivorans as determined by duplex real-time PCR of skin swabs .An by environmental temperature. The inability of B. salamandrivorans to acclimatization period of 1 week was admitted before the start of the experiment. survive for more than 10 days at 25uC inside its host, renders temper- ature treatment of infected urodelans a safe, effective and low-cost Field outbreak animals. Heat treatment to clear B. salamandrivorans infections in treatment option, when taking into account the host thermal tolerance. amphibians was validated on 30 wild fire salamanders found to be infected with B. Figure 4 | The effect of exposure to 256C for 7 and 10 days on the course of Batrachochytrium salamandrivorans infection in fire salamanders. After establishment of infection fire salamanders were subjected to an ambient temperature of 25uC for 7 days (A), or 10 days (B). Each symbol represents the course of infection of an individual animal. SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 3 www.nature.com/scientificreports 4. Wake, D. B. & Vredenburg, V. T. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. P. Natl. Acad. Sci. USA 105, 11466–11473 (2008). 5. Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484 (2012). 6. Spitzen-van der Sluijs, A. et al. Environmental determinants of recent endemism of Batrachochytrium dendrobatidis infections in amphibian assemblages in the absence of disease outbreaks. Conserv. Biol. B28, 1302–1311 (2014). 7. Martel, A. et al. Batrachochytrium salamandrivorans sp nov causes lethal chytridiomycosis in amphibians. P. Natl. Acad. Sci. USA 110, 15325–15329 (2013). 8. Pasmans, F. et al. Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans) in Belgium. Vlaams Diergen. Tijds. 79, 460–462 (2010). 9. Farrer, R. A. et al. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. P. Natl. Acad. Sci. USA 108, 18732–187368 (2011). 10. Garner, T. W. J. et al. Chytrid fungus in Europe. Emerg. Infect. Dis. 11, 1639–1641 (2005). 11. Martel, A. et al. The Absence of zoonotic agents in invasive bullfrogs (Lithobates Figure 5 | Heat treatment composed of exposure to 256C for 10 days of catesbeianus) in Belgium and The Netherlands. EcoHealth 10, 344–347 (2013). 12. Kriger, K. M. & Hero, J. M. Large-scale seasonal variation in the prevalence and fire salamanders naturally infected with Batrachochytrium severity of chytridiomycosis. J. Zool. 271, 352–359 (2007). salamandrivorans. After ascertaining presence of B. salamandrivorans in 13. Bosch, J., Carrascal, L. M., Duran, L., Walker, S. & Fisher, M. C. Climate change all animals they were subjected to an ambient temperature of 25uC for 10 and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; days. Each symbol represents the course of infection of an individual is there a link? P. Roy. Soc. B-Biol. Sci. 274 (2007). animal. Time of death for the 2 deceased animals is displayed beneath the 14. Berger, L. et al. Effect of season and temperature on mortality in amphibians due to chytridiomycosis. Aust. Vet. J. 82, 434–439 (2004). graph. 15. Martel, A. et al. Recent introduction of a chytrid fungus endangers Western Palearctic salamanders. Science 346, 630–631 (2014). salamandrivorans as determined by real-time PCR. These animals originated from a 16. Woodhams, D. C., Alford, R. A. & Marantelli, G. Emerging disease of amphibians population in Robertville Belgium (50u29’58.60N6u06’21.90E) undergoing a B. sal- cured by elevated body temperature. Dis. Aquat. Organ. 55, 65–67 (2003). amandrivorans outbreak event and were translocated to the research facility with 17. Chatfield, M. W. H. & Richards-Zawacki, C. L. Elevated temperature as a permission (2014/RS/nu23). Housing conditions of these animals were identical to treatment for Batrachochytrium dendrobatidis infection in captive frogs. Dis. the conditions described for the experimental animals. Aquat. Organ. 94, 235–238 (2011). 18. Geiger, C. C., Kupfer, E., Schar, S., Wolf, S. & Schmidt, B. R. Elevated temperature Temperature dependency of Batrachochytrium salamandrivorans infection clears chytrid fungus infections from tadpoles of the midwife toad, Alytes dynamics in salamanders. The experimental animals were randomly assigned to one obstetricans. Amphibia-Reptilia 32, 276–280 (2011). of the 5 groups (5 animals per group, kept individually). The purpose of the 5 groups 19. Martel, A. et al. Developing a safe antifungal treatment protocol to eliminate was to assess whether B. salamandrivorans was able to colonize the animals at Batrachochytrium dendrobatidis from amphibians. Med. Myc. 49, 143–149 different temperatures (groups 1 to 3), and whether temperature could be applied to (2011). clear B. salamandrivorans from colonized animals (groups 4 and 5). In group 1, 20. Blooi, M. et al. Duplex real-time PCR for rapid simultaneous detection of animals were inoculated and subsequently kept at 15uC, in group 2 kept at 20uCand Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in in group 3 kept at 25uC (the animals kept at 20 and 25uC were placed in incubators set amphibian samples. J. Clin. Microbiol. 51, 4173–4177 (2013). at the corresponding temperature). The animals in group 4 and 5 were inoculated at 21. Vredenburg, V. T., Knapp, R. A., Tunstall, T. S. & Briggs, C. J. Dynamics of an 15uC and put at 25uC for 7 or 10 days respectively, after B. salamandrivorans infection emerging disease drive large-scale amphibian population extinctions. P. Natl. was established (determined as an increase in infection load between two subsequent samplings). To determine whether the infection would recrudesce after the 25uC Acad. Sci. USA 107, 9689–9694 (2010). 22. Kinney, V. C., Heemeyer, J. L., Pessier, A. P. & Lannoo, M. J. Seasonal Pattern of exposure, salamanders of groups 4 and 5 were put back at 15uC afterwards and were followed up for another 3 weeks. In case of recrudescence of infection, the animals Batrachochytrium dendrobatidis infection and mortality in Lithobates areolatus: were put back at 25uC for 10 days. During the experiment, all animals were checked affirmation of Vredenburg’s ‘‘10,000 zoospore rule’’. Plos One 6, 1–10 (2011). daily for the presence of clinical signs. Skin swabs for B. salamandrivorans real-time 23. Raffel, T. R. et al. Disease and thermal acclimation in a more variable and PCR analysis were collected once every 7 days and/or at the time of death of the unpredictable climate. Nat. Clim. Change 3, 146–151 (2013). animals. An animal was considered negative for B. salamandrivorans infection after 4 24. Piotrowski, J. S., Annis, S. L. & Longcore, J. E. Physiology of Batrachochytrium consecutive negative real-time PCR results. Real-time PCR’s were performed on a dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96, 9–15 (2004). CFX96 Real Time System (Biorad, Hercules, California, USA) with amplification 25. Venables, W. N. & Ripley, B. D. Modern applied statistics with S. 4 edn (Springer, conditions, primer, and probe concentrations as described elsewhere . Infection New York, 2002). loads are presented as genomic equivalents (GE) of B. salamandrivorans zoospores. 26. Therneau, T. M. Package ’survival’ CRAN repository available at: http://r-forge.r- th Results were analyzed by means of independent t-test and Cox regression analysis project.org/ last accessed at the 10 of November 2014. 25 26 using respectively the mass and survival library in R . The censored response variable for the Cox regression analysis was time until death with temperature (15 or 20uC) as explanatory variable. Author contributions Thermal treatment of Batrachochytrium salamandrivorans infected salamanders. M.B., A.M. and F.P. designed the experiments. M.B. carried out the experiments. M.B., Based on the results of the thermal infection experiments, the B. salamandrivorans A.M., F.H., F.V., D.B. and F.P. contributed in writing and reviewing the manuscript. infected field outbreak animals were treated by putting them at 25uC for 10 days. Skin swabs for B. salamandrivorans real-time PCR analysis were collected after 10 days and subsequently every 7 days or at the time of death of the animals. Animals that Additional information remained positive after the heat treatment at 25uC during 10 days were kept at 25uC Competing financial interests: The authors declare no competing financial interests. and swabbed daily to follow up remaining infection intensities until the first negative How to cite this article: Blooi, M. et al. Treatment of urodelans based on temperature real-time PCR result and subsequently every 7 days. An animal was considered dependent infection dynamics of Batrachochytrium salamandrivorans. Sci. Rep. 5, 8037; negative for B. salamandrivorans infection after 4 consecutive negative real-time PCR DOI:10.1038/srep08037 (2015). results. Real-time PCR’s were performed as described above. This work is licensed under a Creative Commons Attribution-NonCommercial- NoDerivs 4.0 International License. The images or other third party material in 1. Longcore, J. E., Pessier, A. P. & Nichols, D. K. Batrachochytrium dendrobatidis gen this article are included in the article’s Creative Commons license, unless indicated et sp nov, a chytrid pathogenic to amphibians. Mycologia 91, 219–227 (1999). 2. Skerratt, L. F. et al. Spread of chytridiomycosis has caused the rapid global decline otherwise in the credit line; if the material is not included under the Creative and extinction of frogs. EcoHealth 4, 125–134 (2007). Commons license, users will need to obtain permission from the license holder 3. Daszak, P. et al. Emerging infectious diseases and amphibian population declines. in order to reproduce the material. To view a copy of this license, visit http:// Emerg. Infect. Dis. 5, 735–748 (1999). creativecommons.org/licenses/by-nc-nd/4.0/ SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 4 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Scientific Reports Springer Journals

Treatment of urodelans based on temperature dependent infection dynamics of Batrachochytrium salamandrivorans

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Springer Journals
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Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
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Abstract

OPEN Treatment of urodelans based on temperature dependent infection SUBJECT AREAS: ECOLOGICAL dynamics of Batrachochytrium EPIDEMIOLOGY FUNGAL BIOLOGY HERPETOLOGY salamandrivorans THERAPEUTICS 1,2 1 1 2 3 1 M. Blooi , A. Martel , F. Haesebrouck , F. Vercammen , D. Bonte & F. Pasmans Received Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 20 October 2014 9820 Merelbeke, Belgium, Centre for Research and Conservation, Royal Zoological Society of Antwerp, Koningin Astridplein 26, Accepted 2018 Antwerp, Belgium, Department of Biology, Terrestrial Ecology Unit, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, 24 December 2014 Belgium. Published 27 January 2015 The recently emerged chytrid fungus Batrachochytrium salamandrivorans currently causes amphibian population declines. We hypothesized that temperature dictates infection dynamics of B. salamandrivorans, and that therefore heat treatment may be applied to clear animals from infection. We examined the impact of environmental temperature on B. salamandrivorans infection and disease dynamics in fire salamanders Correspondence and (Salamandra salamandra). Colonization of salamanders by B. salamandrivorans occurred at 156C and 206C requests for materials but not at 256C, with a significantly faster buildup of infection load and associated earlier mortality at 156C. should be addressed to Exposing B. salamandrivorans infected salamanders to 256C for 10 days resulted in complete clearance of infection and clinically cured all experimentally infected animals. This treatment protocol was validated in M.B. (Mark.Blooi@ naturally infected wild fire salamanders. In conclusion, we show that B. salamandrivorans infection and UGent.be) disease dynamics are significantly dictated by environmental temperature, and that heat treatment is a viable option for clearing B. salamandrivorans infections. n the decades following the identification of Batrachochytrium dendrobatidis in 1999 it became apparent that this chytrid fungus was one of the biotic drivers of declines and extinctions of hundreds of amphibian species 2–5 I worldwide . However, the impact of B. dendrobatidis varies regionally from a dramatic decrease of amphi- bian diversity to a state of host-pathogen equilibrium. In one such region characterized by the co-existence of B. dendrobatidis with local amphibian communities , another recently described chytrid fungus, Batrachochytrium salamandrivorans , caused amphibian population declines. The reason for this obvious difference in disease dynamics between both chytrid fungi is not known. Disease dynamics are dictated by pathogen virulence, host factors and environmental determinants. Virulent strains of both chytrids, as well as susceptible host species are 8–11 12–14 present in the affected regions . For B. dendrobatidis, temperature is considered a key environmental factor . One major difference between both chytrids is their different thermal growth characteristics, which is probably due to differences in host spectrum, B. salamandrivorans being restricted to urodelan hosts . Knowledge of the infection dynamics of B. salamandrivorans at different temperatures may help to develop treatment proto- 16–18 19 cols . These are urgently needed as current therapies developed against B. dendrobatidis fail to eliminate B. salamandrivorans from infected amphibians (unpublished results). We hypothesized that temperature dictates infection and disease dynamics of B. salamandrivorans in salamanders, which may be applied to develop a heat treatment protocol to clear infection in animals. Results & Discussion Only after exposure at 15uCor20uC but not at 25uC, the salamanders were colonized by B. salamandrivorans.Ifa 20–22 10000 GE infection load per swab is considered indicative for a clinical threshold , this threshold was reached two times earlier at 15uC than at 20uC (on average 15 6 4 (SD) days and 31 6 12 days (SD) respectively, independent t-test p , 0.05) (Fig. 1). The faster buildup of B. salamandrivorans infection loads coincided with earlier mortality at 15uC than at 20uC (22 6 8 (SD) days and 35 6 14 (SD) days respectively, Cox regression analysis, x 5 3.941, df 5 1, p , 0.05) (Fig. 1 and 2). Besides preventing infection of salamanders with B. SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 1 www.nature.com/scientificreports Figure 1 | The course of Batrachochytrium salamandrivorans infection in fire salamanders at 15 and 206C. Each symbol represents the course of infection of an individual animal.Time of death of all animals is depicted beneath the graphs. The line represents the average increase in infection intensity in all tested individuals based on a repeated measure regression analysis. salamandrivorans, exposure of infected salamanders to a temper- 25uC on frogs was opposite to the pattern of temperature-dependent ature of 25uC during 10 days completely eliminated the infection growth at these temperatures in culture , and time until death in and resolved B. salamandrivorans lesions from all infected animals frogs infected with B. dendrobatidis at 27uC, which is above B. den- (Fig. 3 and 4). However, 7 days exposure at 25uC did not result in drobatidis’ thermal preference , was shorter when compared to time fungal clearance since recrudescence of infection was observed in all until death of infected frogs kept at lower temperatures . The suit- these salamanders within 1–3 weeks after transferring them to an ability of raised ambient temperature as treatment option was vali- ambient temperature of 15uC (Fig. 4). This is remarkable since cul- dated by keeping 30 wild-caught B. salamandrivorans infected fire tures of the fungus are killed in vitro within 5 days of incubation at salamanders at 25uC during 10 days. Twenty-six animals were cured 25uC and shows that B. salamandrivorans is capable to persist in an of B. salamandrivorans infection after this treatment period, 2 died urodelan host experiencing temperatures that temporarily surpass early during treatment, and 2 needed an additional treatment period the fungal thermal maximum for up to one week. Exposure of the of 2 days in order to completely clear the infection (Fig. 5). This relapsing animals to 25uC for 10 days eliminated the infection. Our shows that heat treatment is a viable treatment option for B. sala- results reflect B. salamandrivorans growth curves obtained in vitro, mandrivorans infected amphibians when the clinical condition and with an optimal growth range around 15uC . In contrast, the pattern the thermal tolerance of the animal is taken into account. In order to of temperature-dependent growth of B. dendrobatidis at 15, 20 and completely eliminate B. dendrobatidis infections higher tempera- Figure 2 | The probability of survival of salamanders housed at 15 or 206C after infection with Batrachochytrium salamandrivorans. Survival probability was plotted based on a Cox regression analysis (x 5 3.981, df 5 1, p , 0.05). Time is displayed in days after initial infection. The dotted line represent survival probability of Batrachochytrium salamandrivorans infected salamanders housed at 15uC and the full line those housed at 20uC. SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 2 www.nature.com/scientificreports Figure 3 | Heat treatment of amphibians infected with Batrachochytrium salamandrivorans clears infection and resolves associated lesions. Batrachochytrium salamandrivorans associated skin lesions (A) are clearly reduced after the heat treatment composed of keeping the animals at 25uC during 10 days (B), and will eventually completely resolve. tures, composed of short exposure to 37uC or extended exposure to Methods All experiments were performed in accordance with the relevant guidelines and 30uC are required. These protocols are not suitable for treating regulations. All experiments with experimental animals were carried out with salamanders, as these temperatures surpass the upper thermal limit approval of the ethical committee of the Faculty of Veterinary Medicine, Ghent of most urodelans. The 2 animals that died were in poor clinical University. condition at the start of the treatment period, and probably died due to thermal shock as B. salamandrivorans loads were low at time Batrachochytrium salamandrivorans strain, culture conditions and experimental inoculation. The B. salamandrivorans type strain was grown in TGhL broth (16 g of death. This points out the narrow margin between the temperature tryptone, 4 g gelatin hydrolysate, 2 g lactose per liter of distilled water) in 25 cm cell able to eliminate B. salamandrivorans and the upper thermal limit culture flasks and incubated at 15uC. To obtain B. salamandrivorans zoospores, a most urodelans tolerate. Furthermore, these results show that the 2 ml aliquot of a 5-day-old culture was inoculated on TGhL agar plates (16 g course of infection should be carefully monitored since not all ani- tryptone, 4 g gelatin hydrolysate, 2 g lactose, 10 g bacteriological agar per liter of distilled water) and incubated at 15uC for 5–7 days. Zoospores were collected by mals tested negative for presence of Bs DNA after 10 days at 25uC. flooding the agar plates with 2 ml of distilled water and subsequent collection of the Although we do not think that this is a result of an active infection but fluid. A hemocytometer was used to count the number of zoospores present in the explained by presence of residual B. salamandrivorans DNA derived 3 suspension and the concentration of the zoospore suspension was adjusted to 5 3 10 from dead B. salamandrivorans cells, this remains uncertain. This zoospores per mL. Animals were inoculated with B. salamandrivorans by topically applying one mL of the inoculum on the intact skin. could have been further elucidated by transferring the animals back to 15uC after 10 days but we chose to keep them at 25uC until PCR Animals. Experimental animals. Fire salamanders (Salamandra salamandra) were results became negative. Thermal treatment of B. salamandrivorans experimentally infected with B. salamandrivorans to study temperature dependent infected amphibians would allow large groups of animals to be infection dynamics. The animal experiment was performed with the approval of the treated simultaneously at low costs and lacks the possible downsides ethical committee of the Faculty of Veterinary Medicine (Ghent University, EC2013/ 87). Twenty-five captive bred fire salamanders were housed individually in plastic linked to drug treatment like toxicity or development of acquired containers in a climatized room with an ambient temperature of 15uC. The animals antimicrobial resistance. were kept on a moist tissue, with access to a hiding place and water container. Crickets In conclusion, these results demonstrate that infection and disease powdered with mineral and vitamin supplement were provided ad libitum as food dynamics of B. salamandrivorans in urodelans are significantly dictated source. All animals were clinically healthy and free of B. dendrobatidis and B. salamandrivorans as determined by duplex real-time PCR of skin swabs .An by environmental temperature. The inability of B. salamandrivorans to acclimatization period of 1 week was admitted before the start of the experiment. survive for more than 10 days at 25uC inside its host, renders temper- ature treatment of infected urodelans a safe, effective and low-cost Field outbreak animals. Heat treatment to clear B. salamandrivorans infections in treatment option, when taking into account the host thermal tolerance. amphibians was validated on 30 wild fire salamanders found to be infected with B. Figure 4 | The effect of exposure to 256C for 7 and 10 days on the course of Batrachochytrium salamandrivorans infection in fire salamanders. After establishment of infection fire salamanders were subjected to an ambient temperature of 25uC for 7 days (A), or 10 days (B). Each symbol represents the course of infection of an individual animal. SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 3 www.nature.com/scientificreports 4. Wake, D. B. & Vredenburg, V. T. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. P. Natl. Acad. Sci. USA 105, 11466–11473 (2008). 5. Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484 (2012). 6. Spitzen-van der Sluijs, A. et al. Environmental determinants of recent endemism of Batrachochytrium dendrobatidis infections in amphibian assemblages in the absence of disease outbreaks. Conserv. Biol. B28, 1302–1311 (2014). 7. Martel, A. et al. Batrachochytrium salamandrivorans sp nov causes lethal chytridiomycosis in amphibians. P. Natl. Acad. Sci. USA 110, 15325–15329 (2013). 8. Pasmans, F. et al. Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans) in Belgium. Vlaams Diergen. Tijds. 79, 460–462 (2010). 9. Farrer, R. A. et al. Multiple emergences of genetically diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. P. Natl. Acad. Sci. USA 108, 18732–187368 (2011). 10. Garner, T. W. J. et al. Chytrid fungus in Europe. Emerg. Infect. Dis. 11, 1639–1641 (2005). 11. Martel, A. et al. The Absence of zoonotic agents in invasive bullfrogs (Lithobates Figure 5 | Heat treatment composed of exposure to 256C for 10 days of catesbeianus) in Belgium and The Netherlands. EcoHealth 10, 344–347 (2013). 12. Kriger, K. M. & Hero, J. M. Large-scale seasonal variation in the prevalence and fire salamanders naturally infected with Batrachochytrium severity of chytridiomycosis. J. Zool. 271, 352–359 (2007). salamandrivorans. After ascertaining presence of B. salamandrivorans in 13. Bosch, J., Carrascal, L. M., Duran, L., Walker, S. & Fisher, M. C. Climate change all animals they were subjected to an ambient temperature of 25uC for 10 and outbreaks of amphibian chytridiomycosis in a montane area of Central Spain; days. Each symbol represents the course of infection of an individual is there a link? P. Roy. Soc. B-Biol. Sci. 274 (2007). animal. Time of death for the 2 deceased animals is displayed beneath the 14. Berger, L. et al. Effect of season and temperature on mortality in amphibians due to chytridiomycosis. Aust. Vet. J. 82, 434–439 (2004). graph. 15. Martel, A. et al. Recent introduction of a chytrid fungus endangers Western Palearctic salamanders. Science 346, 630–631 (2014). salamandrivorans as determined by real-time PCR. These animals originated from a 16. Woodhams, D. C., Alford, R. A. & Marantelli, G. Emerging disease of amphibians population in Robertville Belgium (50u29’58.60N6u06’21.90E) undergoing a B. sal- cured by elevated body temperature. Dis. Aquat. Organ. 55, 65–67 (2003). amandrivorans outbreak event and were translocated to the research facility with 17. Chatfield, M. W. H. & Richards-Zawacki, C. L. Elevated temperature as a permission (2014/RS/nu23). Housing conditions of these animals were identical to treatment for Batrachochytrium dendrobatidis infection in captive frogs. Dis. the conditions described for the experimental animals. Aquat. Organ. 94, 235–238 (2011). 18. Geiger, C. C., Kupfer, E., Schar, S., Wolf, S. & Schmidt, B. R. Elevated temperature Temperature dependency of Batrachochytrium salamandrivorans infection clears chytrid fungus infections from tadpoles of the midwife toad, Alytes dynamics in salamanders. The experimental animals were randomly assigned to one obstetricans. Amphibia-Reptilia 32, 276–280 (2011). of the 5 groups (5 animals per group, kept individually). The purpose of the 5 groups 19. Martel, A. et al. Developing a safe antifungal treatment protocol to eliminate was to assess whether B. salamandrivorans was able to colonize the animals at Batrachochytrium dendrobatidis from amphibians. Med. Myc. 49, 143–149 different temperatures (groups 1 to 3), and whether temperature could be applied to (2011). clear B. salamandrivorans from colonized animals (groups 4 and 5). In group 1, 20. Blooi, M. et al. Duplex real-time PCR for rapid simultaneous detection of animals were inoculated and subsequently kept at 15uC, in group 2 kept at 20uCand Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in in group 3 kept at 25uC (the animals kept at 20 and 25uC were placed in incubators set amphibian samples. J. Clin. Microbiol. 51, 4173–4177 (2013). at the corresponding temperature). The animals in group 4 and 5 were inoculated at 21. Vredenburg, V. T., Knapp, R. A., Tunstall, T. S. & Briggs, C. J. Dynamics of an 15uC and put at 25uC for 7 or 10 days respectively, after B. salamandrivorans infection emerging disease drive large-scale amphibian population extinctions. P. Natl. was established (determined as an increase in infection load between two subsequent samplings). To determine whether the infection would recrudesce after the 25uC Acad. Sci. USA 107, 9689–9694 (2010). 22. Kinney, V. C., Heemeyer, J. L., Pessier, A. P. & Lannoo, M. J. Seasonal Pattern of exposure, salamanders of groups 4 and 5 were put back at 15uC afterwards and were followed up for another 3 weeks. In case of recrudescence of infection, the animals Batrachochytrium dendrobatidis infection and mortality in Lithobates areolatus: were put back at 25uC for 10 days. During the experiment, all animals were checked affirmation of Vredenburg’s ‘‘10,000 zoospore rule’’. Plos One 6, 1–10 (2011). daily for the presence of clinical signs. Skin swabs for B. salamandrivorans real-time 23. Raffel, T. R. et al. Disease and thermal acclimation in a more variable and PCR analysis were collected once every 7 days and/or at the time of death of the unpredictable climate. Nat. Clim. Change 3, 146–151 (2013). animals. An animal was considered negative for B. salamandrivorans infection after 4 24. Piotrowski, J. S., Annis, S. L. & Longcore, J. E. Physiology of Batrachochytrium consecutive negative real-time PCR results. Real-time PCR’s were performed on a dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96, 9–15 (2004). CFX96 Real Time System (Biorad, Hercules, California, USA) with amplification 25. Venables, W. N. & Ripley, B. D. Modern applied statistics with S. 4 edn (Springer, conditions, primer, and probe concentrations as described elsewhere . Infection New York, 2002). loads are presented as genomic equivalents (GE) of B. salamandrivorans zoospores. 26. Therneau, T. M. Package ’survival’ CRAN repository available at: http://r-forge.r- th Results were analyzed by means of independent t-test and Cox regression analysis project.org/ last accessed at the 10 of November 2014. 25 26 using respectively the mass and survival library in R . The censored response variable for the Cox regression analysis was time until death with temperature (15 or 20uC) as explanatory variable. Author contributions Thermal treatment of Batrachochytrium salamandrivorans infected salamanders. M.B., A.M. and F.P. designed the experiments. M.B. carried out the experiments. M.B., Based on the results of the thermal infection experiments, the B. salamandrivorans A.M., F.H., F.V., D.B. and F.P. contributed in writing and reviewing the manuscript. infected field outbreak animals were treated by putting them at 25uC for 10 days. Skin swabs for B. salamandrivorans real-time PCR analysis were collected after 10 days and subsequently every 7 days or at the time of death of the animals. Animals that Additional information remained positive after the heat treatment at 25uC during 10 days were kept at 25uC Competing financial interests: The authors declare no competing financial interests. and swabbed daily to follow up remaining infection intensities until the first negative How to cite this article: Blooi, M. et al. Treatment of urodelans based on temperature real-time PCR result and subsequently every 7 days. An animal was considered dependent infection dynamics of Batrachochytrium salamandrivorans. Sci. Rep. 5, 8037; negative for B. salamandrivorans infection after 4 consecutive negative real-time PCR DOI:10.1038/srep08037 (2015). results. Real-time PCR’s were performed as described above. This work is licensed under a Creative Commons Attribution-NonCommercial- NoDerivs 4.0 International License. The images or other third party material in 1. Longcore, J. E., Pessier, A. P. & Nichols, D. K. Batrachochytrium dendrobatidis gen this article are included in the article’s Creative Commons license, unless indicated et sp nov, a chytrid pathogenic to amphibians. Mycologia 91, 219–227 (1999). 2. Skerratt, L. F. et al. Spread of chytridiomycosis has caused the rapid global decline otherwise in the credit line; if the material is not included under the Creative and extinction of frogs. EcoHealth 4, 125–134 (2007). Commons license, users will need to obtain permission from the license holder 3. Daszak, P. et al. Emerging infectious diseases and amphibian population declines. in order to reproduce the material. To view a copy of this license, visit http:// Emerg. Infect. Dis. 5, 735–748 (1999). creativecommons.org/licenses/by-nc-nd/4.0/ SCIENTIFIC REPORTS | 5 : 8037 | DOI: 10.1038/srep08037 4

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Published: Jan 27, 2015

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