Abstract
Controlled exposure experiments can be very informative, however, they are based on the assumption that pathogens maintained on artificial media under long-term storage retain the infective and pathogenic properties of the reproducing pathogen as it occurs in a host. We observed that JEL284, an in vitro cultured and maintained isolate of Batrachochytrium dendrobatidis (Bd), was becoming less infectious with successive uses. We hypothesized that passing an isolate propagated on artificial media through an amphibian host would make the isolate more infectious and pathogenic in subsequent exposures. To test our hypothesis, we used two discreet steps, a reisolation step (step 1) and a comparative exposure step (step 2). In step 1, we exposed eastern spadefoot toads, Scaphiopus holbrooki, to JEL284 and JEL197, another isolate that had been maintained in vitro for over six years. We then re-isolated JEL284 only from a successful infection and named this new isolate JEL284FMBa. JEL197 did not infect any amphibians and, thus, did not proceed to step 2. In step 2, we compared infectivity and pathogenicity (mortality and survival time) of JEL284 and JEL284FMBa by exposing 54 naïve S. holbrooki to three treatments (JEL284, JEL284FMBa, and negative control) with 18 individuals per group. We found that JEL284FMBa caused higher mortality and decreased survival time in infected individuals when compared to JEL284 and negative controls. Thus, our data show that pathogenicity of Bd can decrease when cultured successively in media only and can be partially restored by passage through an amphibian host. Therefore, we have demonstrated that pathogenicity shifts can occur rapidly in this pathogen. Given the potential for shifts in pathogenicity demonstrated here, we suspect Bd to have similar potential in natural populations. We suggest that, when possible, the use of freshly isolated or cryopreserved Bd would improve the quality of controlled exposure experiments using this pathogen.
Highlights
Experimental disease ecology has relied heavily on controlled laboratory experiments when investigating the biology of hostpathogen systems
Step 1 – Exposure and Reisolation of Cultured Batrachochytrium dendrobatidis (Bd) Isolates Using light microscopy of skin scrapings we found that JEL284 infected 4 of 20 (0.20, 95% CL 0.06–0.44) and both JEL197 and the negative control infected 0 of 20 (0.00, 95% CL 0.00–0.17)
Our work shows that passing long-term cultured Bd through an amphibian host can increase the pathogenicity of the isolate in a subsequent exposure (Figures 1 and 2)
Summary
Experimental disease ecology has relied heavily on controlled laboratory experiments when investigating the biology of hostpathogen systems. Controlled exposure experiments have been extensively used to examine the relationship between Batrachochytrium dendrobatidis (Bd), the etiological agent causing the disease amphibian chytridiomycosis, and its amphibian hosts Such experiments have been used to investigate determinants of infectivity (ability to infect) and pathogenicity (ability to cause disease given infection) of Bd [3,4,5], and were effectively used to demonstrate how Bd kills amphibians [6]. Controlled exposure experiments have proven useful for investigations of potential interactions between amphibian species and Bd isolates [7,8,9] All such studies are inherently based on the assumption that laboratory host-pathogen models accurately reflect natural host-pathogen interactions, and that pathogens raised strictly in culture retain the same infectivity and pathogenicity as those that reproduce in a host. These assumptions need to be tested, because loss of infectivity and/or pathogenicity has been documented in some fungal pathogens maintained in vitro outside their natural host(s) [10]
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