Theses and Dissertations

Issuing Body

Mississippi State University

Advisor

Gaunt, Patricia S.

Committee Member

Hanson, Larry

Committee Member

Petrie-Hanson, Lora

Committee Member

Greenway, Terrence E.

Date of Degree

8-15-2014

Document Type

Dissertation - Open Access

Major

Veterinary Medical Science

Degree Name

Doctor of Philosophy

College

College of Veterinary Medicine

Department

Veterinary Medical Science Program

Abstract

Visceral toxicosis of catfish (VTC) is a sporadic, often devastating disease in catfish aquaculture, caused by botulinum neurotoxin serotype/E (BoNT/E). The median lethal dose of BoNT/E for channel catfish (Ictalurus punctatus) fingerlings is 13.7 pg/fish. The diagnosis of VTC is suspected if clinical signs and lesions are produced when affected serum is injected into sentinel-catfish and confirmed if this effect is neutralized with BoNT/E antitoxin. However, the assay is complicated in diagnostic cases by small serum samples from affected fish, lack of standardization and availability of small catfish (~10g). Therefore a zebrafish (~0.4g) bioassay for VTC diagnosis was tested and validated. Susceptibility was tested with other serotypes of toxin to help establish zebrafish as a diagnostic model for BoNTs. BoNT/E consist of100kD heavy chain (HC) and 50kD light chain (LC) linked by a disulfide bond. The HC transports the LC into the cytosol of the neuron, where LC (Zn2+-endoprotease) cleaves the SNAP-25 protein thereby blocking the signal transduction at the neuromuscular junction. The HC-based vaccines can induce protective immunity in mammals. To evaluate HC immunogenicity in catfish, rBoNT/E/HC vaccine produced by USAMRIID was tested; this vaccine did not induce a robust antibody response, but western blot analysis demonstrated specific antibody production in 3 of 11 vaccinated fish. We then developed four channel catfish virus (Ictaluridherpesvirus 1, CCV) recombinants expressing synthetic BoNT/E/HC using our established Gateway CCV recombination system to determine if the virus vector could improve the response. Catfish were vaccinated with these recombinants or with a control vector that expressed Escherichia coli beta-galactosidase (CCV-lacZ). No significant protective immunity or BoNT/E antibodies were observed but CCV-lacZ induced a strong antibody response. These results suggest that BoNT/E/HC has low immunogenicity in channel catfish and deviates from the high immunogenicity observed in mammals. To develop a protective vaccine for VTC, it will be necessary to enhance the BoNT/E /HC immunogenicity in channel catfish. Development of recombinant animals which are resistant to BoNT/E was explored as another potential way to prevent VTC. However, the attempts to modify SNAP-25 of zebrafish by genome editing using Transcription Activator-Like Effector Nucleases were unsuccessful.

URI

https://hdl.handle.net/11668/20167

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