Experimental methodologies can affect pathogenicity ofBatrachochytrium salamandrivoransinfections

Abstract

25Controlled experiments are one approach to understanding the pathogenicity of etiologic agents 26 to susceptible hosts. The recently discovered fungal pathogen, Batrachochytrium 27 salamandrivorans (Bsal), has resulted in a surge of experimental investigations because of its 28 potential to impact global salamander biodiversity. However, variation in experimental 29 methodologies could thwart knowledge advancement by introducing confounding factors that 30 make comparisons difficult among studies. Thus, our objective was to evaluate if variation in 31 experimental methods changed inferences made on the pathogenicity of Bsal. We tested whether 32 passage duration of Bsal culture, exposure method of the host to Bsal (water bath vs. skin 33 inoculation), Bsal culturing method (liquid vs. plated), host husbandry conditions (aquatic vs. 34 terrestrial), and skin swabbing frequency influenced diseased-induced mortality in a susceptible 35 host species, the eastern newt (Notophthalmus viridescens). We found that disease-induced 36 mortality was faster for eastern newts when exposed to a low passage isolate, when newts were 37 housed in terrestrial environments, and if exposure to zoospores occurred via water bath. We did 38 not detect differences in disease-induced mortality between culturing methods or swabbing 39 frequencies. Our results illustrate the need to standardize methods among Bsal experiments; we 40 provide suggestions for future experiments in the context of hypothesis testing. 41 42 43 44 4 45 46Batrachochytrium salamandrivorans (Bsal) is an emerging fungal pathogen of global 47 conservation concern(1-3). Bsal has been identified as the causal agent in recent near extirpations 48 of wild fire salamanders (Salamandra salamandra) in Belgium and the Netherlands(4, 5), and 49 has been detected in live amphibians in captivity and moving through international trade(6-8). 50Controlled experiments where hosts are exposed to Bsal under standardized conditions suggest 51 that the pathogen has a broad host range, including several salamander and anuran families (4, 9). 52A central tenet to understanding Bsal invasion threat is estimating host susceptibility to infection, 53 and whether host infection progresses to clinical disease hence its pathogenicity (10). 54One approach to estimating pathogenicity of etiologic agents is using exposure 55 experiments(11, 12). However, variation in pathogen culturing methods, host husbandry 56 conditions, and pathogen exposure methods could result in different inferences made on 57 pathogenicity (13). For example, Martel et al. (9) inferred that the eastern newt (Notophthalmus 58 viridescens) was hyper-sensitive to Bsal infection, because 100% of individuals exposed to 59 5x10 3 zoospores died within 34 days. Longo et al. (14) reported ca. 50% mortality of eastern 60 newts exposed to the same dose of Bsal over 18 weeks, with some individuals clearing the 61 pathogen and surviving to the end of the experiment. Several explanations were offered for the 62 difference in findi...