Survey for Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Latvian Water Frogs

The amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) is widely distributed in Europe (Allain and Duffus 2019). This fungus has practically a global distribution and has measurable effects on populations of amphibians (Berger et al. 2016).

Latvia is home to 13 amphibian species (11 anurans and two caudates; Pupins and Pupina 2011). None of the species is classified as globally threatened, but seven species show a global trend of population decline (IUCN 2020). Decline has been observed in some previously known populations in Latvia (Epidalea calamita, Pelobates fuscus, Bufotes viridis, Pelophylax esculentus complex; Čeirāns and Pupins 2019; M.P. pers. comm.). Nothing is known about the effect of pathogens in Latvian amphibians. Batrachochytrium dendrobatidis had not been detected in wild Latvian amphibian populations in a previous screening (Ouellet et al. 2005), and no screening for the salamander pathogen Batrachochytrium salamandrivorans (Bsal) had been performed. Amphibians positive for Bd have been detected in Riga Zoo (in three of 58 samples from various species; Saare et al. 2021). Our study aimed to investigate the presence of Bd in Latvia as a potential threat to local amphibian populations. We used P. esculentus complex water frogs for the study because they are widespread, inhabit various types of waterbodies, and may have high Bd prevalence with no apparent deleterious effect, which combined with their high dispersal ability, predispose them potentially to spread chytridiomycosis (Baláž et al. 2014a). The nearest known point of Bsal in wild amphibian populations is in Germany (Lötters et al. 2020); it has not been detected in any wild or captive amphibians in Estonia and Latvia (Saare et al. 2021). The presence of Bsal in P. esculentus complex was considered very unlikely but was tested for because of the chosen method of duplex quantitative PCR (Blooi et al. 2013).

In August 2017, we sampled 135 adult P. esculentus complex water frogs from 41 sites in Latvia. All frogs were sampled by taking skin swabs from the pelvic patch and the ventral surface of the abdomen, legs, and feet, following the standard protocol recommended by Hyatt et al. (2007). Swabs were preserved in 75% ethanol then stored at –20 C. We followed all applicable institutional and national guidelines for the care and use of animals. Sample processing took place at the University of Veterinary Sciences Brno following the guidelines of Blooi et al. (2013). The presence of Bd and Bsal DNA was detected by quantitative PCR; all samples were run in duplicate. Bovine serum albumin was added to reduce PCR inhibition (Garland et al. 2010). We considered a sample positive if the result fulfilled these criteria: mean of measured genomic equivalents of zoospore (GE) representing infection intensity was over 0.1, and the sample produced a clear sigmoidal growth curve in both wells. Samples with any equivocal result were reanalyzed. Four quantification standards of 10-fold dilutions (0.1 to 100 GE) were used for quantification of Bd. These were made from stock Bd DNA of the globally distributed pandemic lineage, strain IA042 and were received from the Institute of Zoology, Zoological Society of London, UK. A sample of Bsal DNA used as a positive control was provided by An Martel, Ghent University, Belgium.

We detected Bd in 53 (39%) samples (Table 1); water frogs in 18 sites (44%) were infected. The highest prevalence (100%) was noted in the Daugavpils region near place Ilgas; GE values reached from 1.69 to 119.00±34.73 (mean±SD; Table 1 and Fig. 1). The highest recorded infection intensity was 143.00 GE in a sample from a locality near Jurmala. None of the infected frogs showed any apparent signs of disease. We did not detect Bsal in any samples.

The infection prevalence and intensity of Bd in P. esculentus complex in Latvia and the distribution of infected habitats closely reflects the situation observed across the distributional range of P. esculentus complex in Europe (e.g., Ouellet et al. 2005; Ohst et al. 2013; Baláž et al. 2014b). Our data clearly indicate that Bd is widely distributed in Latvia, with prevalence and GE similar to the rest of Europe. Although chytridiomycosis outbreaks have never been observed in Latvia, it cannot be excluded that Bd infections may affect the dynamics of local amphibian populations if other stressors are present (Kolenda et al. 2017).

Latvian populations of P. esculentus complex infected with Bd share habitats with European rare and protected amphibian species, for example in Nature Reserve Ilgas (southeastern Latvia) with Bombina bombina, Triturus cristatus (Bern Convention Appendix 2 – European Treaty Series 1979; Habitats Directive Appendices II, IV – Publications Office of the European Union 1992), P. fuscus, Rana arvalis (Bern Convention Appendix 2 – European Treaty Series 1979; Habitat Directive IV – Publications Office of the European Union 1992; Pupina and Pupins 2007; Čeirāns et al. 2020; M.P. pers. comm.) and in Nature Park Dvietes paliene with E. calamita (Bern Convention Appendix 2 – European Treaty Series 1979; Habitat Directive IV – Publications Office of the European Union 1992; Čeirāns and Pupins 2019).

We detected Bd in protected natural areas such as Silene Nature Park (including the Ilgas Nature Reserve; sites 13, 18), Dridzis Lake (site 26), Dviete water-meadows (sites 34, 39), and Augsdaugava protected landscape area (site 19); these are all designated as Natura 2000 sites. In light of our data, it is necessary to evaluate the risk Bd presence poses to the cohabiting species, to assist in developing and implementing conservation and management measures in nature conservation plans of the territories.

We thank Siarhei Uhlianets for help with the map. The research was partly supported by the Administration of Latvian Environmental Protection Fund, projects 1-08/153/ 2017 and 1-08/189/2018 and by grant 224/ 2016/FVHE of the Internal Grant Agency of the University of Veterinary Sciences Brno, Czech Republic. We are thankful for the cooperation of the project Pond aquaculture production and ecosystem service innovative research with modeling of the climate impact to tackle horizontal challenges and improve aquaculture sustainability governance in Latvia (lzp-2020/2-0070) financed by Fundamental and Applied Research Projects (FLPP) and the Joint Latvian-Ukrainian project The ecological and biological triggers of expansion of the invasive fish, Chinese sleeper (Perccottus glenii), in Eastern Europe co-financed by the State Education Development Agency of Latvia (LV-UA/2018/6) and the Ministry of Science and Education of Ukraine (0119U101806).