AUTHOR=Ossiboff Robert J. , Towe Anastasia E. , Brown Melissa A. , Longo Ana V. , Lips Karen R. , Miller Debra L. , Carter E. Davis , Gray Matthew J. , Frasca Salvatore 

TITLE=Differentiating Batrachochytrium dendrobatidis and B. salamandrivorans in Amphibian Chytridiomycosis Using RNAScope®in situ Hybridization

JOURNAL=Frontiers in Veterinary Science

VOLUME=6

YEAR=2019

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2019.00304

DOI=10.3389/fvets.2019.00304

ISSN=2297-1769

ABSTRACT=<p><italic>Batrachochytrium dendrobatidis</italic> and <italic>B. salamandrivorans</italic> are important amphibian pathogens responsible for morbidity and mortality in free-ranging and captive frogs, salamanders, and caecilians. While <italic>B. dendrobatidis</italic> has a widespread global distribution, <italic>B. salamandrivorans</italic> has only been detected in amphibians in Asia and Europe. Although molecular detection methods for these fungi are well-characterized, differentiation of the morphologically similar organisms in the tissues of affected amphibians is incredibly difficult. Moreover, an accurate tool to identify and differentiate <italic>Batrachochytrium</italic> in affected amphibian tissues is essential for a specific diagnosis of the causative agent in chytridiomycosis cases. To address this need, an automated dual-plex chromogenic RNAScope<sup>®</sup><italic>in situ</italic> hybridization (ISH) assay was developed and characterized for simultaneous detection and differentiation of <italic>B. dendrobatidis</italic> and <italic>B. salamandrivorans</italic>. The assay, utilizing double Z target probe pairs designed to hybridize to 28S rRNA sequences, was specific for the identification of both organisms in culture and in formalin-fixed paraffin-embedded amphibian tissues. The assay successfully identified organisms in tissue samples from five salamander and one frog species preserved in formalin for up to 364 days and was sensitive for the detection of <italic>Batrachochytrium</italic> in animals with qPCR loads as low as 1.1 × 10<sup>2</sup> zoospores/microliter. ISH staining of <italic>B. salamandrivorans</italic> also highlighted the infection of dermal cutaneous glands, a feature not observed in amphibian <italic>B. dendrobatidis</italic> cases and which may play an important role in <italic>B. salamandrivorans</italic> pathogenesis in salamanders. The developed ISH assay will benefit both amphibian chytridiomycosis surveillance projects and pathogenesis studies by providing a reliable tool for <italic>Batrachochytrium</italic> differentiation in tissues.</p>