Partial Validation And Application Of Henneguya Species Specific In Situ Hybridization Illustrates Mixed Species Infections In Proliferative Gill Disease Of Catfish Aquaculture

 1Justin M. Stilwell, 1Alvin C. Camus, 2Ethan T. Woodyard, 2,3Cyndi Ware, 2T. Graham Rosser, 2,4Mackenzie Gunn, 2,4Adrian Lopez-Porras, 1John H. Leary, 2,3Lester H. Khoo, 2,4David J. Wise, and 2,3Matt J. Griffin

 1Department of Pathology, University of Georgia, Athens, GA 30602; 2College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762; 3Thad Cochran National Warmwater Aquaculture Center, Stoneville, MS 38776; 4Mississippi Agriculture and Forestry Experiment Station, Mississippi State, MS  39762

ABSTRACT

The myxozoan Henneguya ictaluri is cited as the causative agent of proliferative gill disease (PGD) in channel (Ictalurus punctatus) and channel × blue (I. furcatus) catfish. Despite evidence of decreased H. ictaluri infection rates and impaired parasite development in hybrid catfish, PGD still occurs in hybrid production systems. In a previous metagenomic analysis of clinical PGD cases, numerous other myxozoans were detected within affected gill tissues in addition to H. ictaluri. The objective of this work was to develop and partially validate Henneguya species-specific in situ hybridization assays using RNAscope® technology and to investigate the development and pathologic contributions of H. ictaluri and other myxozoans in natural and experimentally induced PGD infections. Natural PGD infections were sourced from diagnostic case submissions in 2019. Experimental challenges involved channel and hybrid catfish exposed to water from a pond with an active PGD outbreak and were sampled at 1, 7, 10, 12, 14, 16, 18 and 20 weeks post-challenge. Nine, unique ISH probes were designed targeting a diagnostic variable region of the 18S rRNA gene of select myxozoan taxa identified in previous research to be present in clinical PGD cases. Partial validation using tissues from pure infections of H. ictaluri, H. adiposa, H. postexilis and H. exilis illustrated species-specific labelling and no cross reactivity between myxozoans or with the catfish host tissues. In the experimental challenge, plasmodia of H. ictaluri and H. postexilis formed in channel catfish but no mature plasmodia formed in hybrid catfish, suggesting impaired development of multiple myxozoans. ISH investigations of both experimental and naturally infected tissues confirmed the presence of mixed infections with characteristic PGD lesions associated with H. ictaluri. Developmental stages of other myxozoans were associated with secondary lesions and, rarely, PGD-like lesions. These findings suggest other Henneguya spp. may contribute to PGD lesions and supports previous research implying hybrid catfish are a dead-end host in the H. ictaluri life cycle.