Evidence of Batrachochytrium dendrobatidis and other amphibian parasites in the Green toad (Bufotes viridis), syntopic amphibians and environment in the Cologne Bay, Germany

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 Thad, 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 cloaca' 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.)