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The composition of cutaneous (skin-associated) bacterial communities of amphibians has been intensively studied in light of the potential of some of these commensal bacterial taxa to mitigate infection with the chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). However, surprisingly, the absolute densities in which these bacteria occur on the skin are only poorly known. We here combine quantification of bacterial 16SrDNA copies from skin swabs by quantitative PCR with counts from scanning electron microscopy (SEM) images to assess and compare bacterial abundances on the skin of various central European amphibians. We focus on the fire salamander (Salamandra salamandra) which is severely threatened by the spread of Bsal. Based on counts from SEM pictures of selected skin parts, local densities of ~43,000 bacteria per mm² were ascertained in frogs, although the data are insufficient to understand whether these values apply to the whole body surface. Bacterial densities are doubtless much lower in fire salamanders in which we observed almost no bacteria in SEM examination. From qPCR data, we find: (i) statistically relevant differences in bacterial abundances among species, with the lowest abundances in terrestrial salamanders and the highest abundances in toads; (ii) higher bacterial loads in captive compared to wild fire salamanders, and much higher loads in Bsal-infected captive specimens, in agreement with the hypothesis that Bsalinfection leads to blooming of opportunistic bacteria that may cause secondary infection; (iii) an only weak decrease of bacterial load after repeated swabbing, in agreement with the hypothesis that skin swabs capture only a part of the bacteria of the swabbed skin surface. We discuss the multiple sources of uncertainty in absolute estimates of abundances of cutaneous bacteria and suggest further research to clarify and reduce these uncertainties. [ABSTRACT FROM AUTHOR]

The chytrid fungus Batrachochytrium salamandrivorans (Bsal), recently introduced from Asia to Europe, causes mortality in numerous species of salamanders and newts and has led to catastrophic declines and local extinctions of the European fire salamander (Salamandra salamandra) in the Netherlands, Belgium, and Germany. Due to the continuous spread of the pathogen, Germany can be considered as the current 'hotspot' of Bsal-driven salamander declines. The pathogen was detected in 2015 in the Eifel Mountains where it probably has been present at least since 2004. Moreover, Bsal was found in 2017 in the Ruhr District where it also might occur since 2004. The Ruhr District is a heavily urbanized and industrialized region in western Germany, which offers an unprecedented opportunity to monitor range expansion and infection dynamics of Bsal in an area affected by intense human activities. We here review the current knowledge on Bsal in the Ruhr District where the pathogen by now has been recorded based on qPCR data from 18 sites distributed over eight cities. Transect counts (adult salamanders) and larval removal-sampling at two sites where Bsal was recorded in 2017 and 2018, confirm that fire salamander populations at the affected sites have declined dramatically. However, single negative-tested individuals were still observed and reproduction could be ascertained. Moreover, we successfully detected Bsal by analysing environmental DNA (eDNA) from samples obtained from a standing water body as well as a stream. Detailed monitoring of a site in Essen (Kruppwald) from January to May 2019 provided data on infection and disease dynamics during an acute Bsal-outbreak in a population of European fire salamanders. After initial observation of single dead infected salamanders in January and February 2019, the maximum Bsal loads in the population ranged from 7.90E+03 ITS copies in early March to 2.29E+09 ITS copies at the end of March. Prevalence of infection ranged from 4% to 50% and significantly increased over time; prevalence of externally visible disease symptoms peaked on May 2 and May 8. Single dead salamanders were encountered throughout the monitoring period. Recaptures of two infected salamanders indicated an increase of Bsal load by about one order of magnitude within one week. Infected salamanders showed small-sized regular round ulcerations usually of 0.25-1 mm but sometimes up to 2.5 mm in diameter, which gave the impression of outward growth from the centre of each ulceration. Among salamander individuals monitored in the Kruppwald, such ulcerations were only found in infected salamanders, but we found no significant correlation between the intensity of the ulcerations and Bsal load. Heat treatment proved effective to cure even deep ulcerations when salamanders were kept for 10 days at 25-27°C or 14 days at 25°C, but infection persisted and ulcerations reappeared six weeks after the end of the treatment; only heat treatment at 25°C for 21 days proved effective to reliably clear the infection in three tested salamanders. [ABSTRACT FROM AUTHOR]

Chytridiomycosis, a disease induced by the chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), has strongly contributed to the ongoing worldwide amphibian conservation crisis. While Bd infection has caused amphibian declines for decades on several continents, Bsal is a novel threat to Central European salamanders and newts, being responsible for the collapse of Fire Salamander populations in the Netherlands, Belgium, and Germany. However, numerous other parasites causing harm to amphibians exist, yet have received much less attention than the chytrid fungi. The goal of the present study was to contribute to the understanding of declines of the Green Toad, Bufotes viridis, at its northwestern distribution border, in the area of Cologne, Germany. We combined the data from four years of Bd monitoring with a metabarcoding approach to detect other, mainly unicellular parasites, from amphibian feces and environmental samples, and also report results from Bsal testing in 2019. Skin swabs of approximately 280 amphibians were tested for Bd and 66 for Bsal, and 150 cloacal swabs and environmental samples from five sites were tested for other pathogens and parasites.We found Bd in all sampled locations with high prevalences and partly high individual infection loads but without clinical signs of chytridiomycosis. None of the samples tested for Bsal was positive for this pathogen. We further detected eight additional potential amphibian pathogens from fecal samples: three metamonads (Tritrichomonas augusta, Trichomitus batrachorum and Hexamita inflata), three ciliates (Balantidium duodeni, Nyctotherus cordiformis and N. hubeiensis), one stramenopile (Blastocystis sp.) and one metazoan (the nematode Rhabdias ranae). In the environmental samples, we detected OTUs of nine organisms potentially harmful for amphibians: Blastocystis sp., Hexamita inflata, Tritrichomonas augusta, Trichomitus batrachorum, two oomycetes (Leptolegnia sp., Saprolegnia sp.), two ichthyosporeans (Amphibiocystidium ranae, Anurofeca sp.) and the myxozoan Myxobolus sp. [ABSTRACT FROM AUTHOR]

The infamous chytrid fungus Batrachochytrium salamandrivorans (Bsal) recently led to the collapse of European fire salamander populations (Salamandra salamandra) in The Netherlands. Currently, the pathogen has been rapidly expanding its range and threatens salamander populations throughout Europe, including Germany. Here, Bsal is known from wild and captive amphibians and has mostly been reported from the federal state of Northrhine-Westphalia. Due to the geographical proximity of infected areas, its dispersal into neighbouring states is possible. A Bsal taskforce was therefore recently formed in the state of Hesse that aims to implement preparative measures for Bsal mitigation at different levels. Based on the known Bsal susceptibility of salamanders in captivity and their inherent threat potential towards natural populations, an epidemiological screening for Bsal prevalence in private amphibian collections throughout the state of Hesse was conducted. We analysed a total of 174 samples from nine private collections of different urodelan species via qPCR and did not detect Bsal. We discuss our results and their implications for salamander conservation relative to other surveys of this kind and underscore the importance of tight cooperation between private keepers and conservation scientists in order to protect wild amphibians from the lethal Bsal fungus. [ABSTRACT FROM AUTHOR]

Emerging infectious diseases are one of the main suggested reasons for global amphibian decline. Fungal agents play a key role. Since its introduction, the Asian pathogen Batrachochytrium salamandrivorans has driven the European fire salamander, Salamandra salamandra, to the edge of extinction in the Netherlands and caused severe population declines in Belgium and Germany. We screened 1,526 amphibians (1,431 urodelans and 95 anurans) from 50 sites in a 1,500 km² large area in Germany, south of the next known infected populations. Furthermore, we conducted a presence-absence mapping of larval salamanders in 88 randomly selected creeks and creeks where salamanders had been reported in the past using a standardized removal sampling approach. Our results revealed an expanded distribution of the pathogen in Western Germany and we could detect seven infected urodelan populations including the southernmost locality of the fungus in its exotic range. Larval salamanders were found in 54 out of 63 creeks south, but only in seven out of 25 creeks north of a highway that divides the study area. Bsal infection could mainly be detected in newts (Ichthyosaura alpestris and Lissotriton helveticus) because many previously known European fire salamander populations most likely disappeared in the affected forest regions, however, silently and without observed mass mortalities. The only detectable salamander population north of the highway was found to be Bsal-infected at high prevalence in 2019, but not from 2016 to 2018, suggesting a recent infection event. Overall, prevalence at the individual (2.6%) and population level (14%) was very low. Moreover, modelling habitat suitability in seemingly unaffected areas suggests that most reproduction creeks and surrounding land habitats in the seemingly affected area are still suitable for S. salamandra, supporting our suspicion that the absence of the species is disease-related rather than habitat related. [ABSTRACT FROM AUTHOR]

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