Theses and Dissertations

ORCID

https://orcid.org/0000-0002-6853-5568

Advisor

Lawrence, Mark

Committee Member

Petrie-Hanson, Lora

Committee Member

Abdelhamed, Hossam

Committee Member

Pharr, G.

Date of Degree

5-15-2026

Original embargo terms

Embargo 1 year

Document Type

Dissertation - Open Access

Major

Veterinary & Biomedical Science (Infectious Disease)

Degree Name

Doctor of Philosophy (Ph.D.)

College

College of Veterinary Medicine

Department

Department of Comparative Biomedical Sciences

Abstract

Aquaculture plays an essential role in global food security, with catfish farming leading the U.S. commercial production. Since 2009, virulent Aeromonas hydrophila (vAh) has caused catastrophic outbreaks of motile Aeromonas septicemia (MAS), leading to mortalities reaching 60-100% and economic losses exceeding USD 12 million per cycle. This dissertation identified the repeat-in-toxin (RTX) cytotoxin as a central virulence determinant of vAh and translated that insight into a practical live-attenuated vaccine for channel catfish. Through systematic construction of seven in-frame deletion mutants targeting virulence genes (chiA, rtxA, rtxC, rtxA-C, ent, col, and sia), phenotypic characterization demonstrated that RTX toxin is required for vAh virulence. Deletion of rtxA, rtxC, or full rtxA-C operon strongly attenuated virulence in catfish (2.5-15% mortality versus 100% in wild-type vAh infections), while mutants lacking chitinase, enterotoxin, collagenase, or sialidase retained near-wild-type mortality rates. Importantly, fish vaccinated with RTX cytotoxin-deficient mutants developed robust protective immunity, becoming 100% protected against subsequent wild-type challenge. Cell-based assays revealed that RTX cytotoxin-deficient strains invaded epithelial barriers as efficiently as wild-type vAh but exhibited significantly reduced cytotoxicity, establishing RTX cytotoxin as a principal effector of host epithelial cell and macrophage destruction. Optimization of immersion vaccination demonstrated exposure-dependent protection, with 4-hour immersion achieving ~90% survival during subsequent wild-type vAh infection. Critically, culturing the ΔrtxA-C vaccine strain under iron-limited conditions with deferoxamine (DFO) showed that 1- hour immersion delivered 100% protection against wild-type challenge with only ~5% vaccination mortality. Transcriptome profiling uncovered an iron-RTX cytotoxin-T6SS regulatory axis potentially contributing to vAh virulence under iron limitation. Both wild-type and RTX cytotoxindeficient strains upregulated iron-acquisition systems in response to DFO; however, only wildtype vAh significantly induced type VI secretion system (hcp1), whereas the RTX cytotoxindeficient strain showed hcp1 downregulation. Structural analysis revealed that vAh RtxA contains functional domains for heme utilization (FhaB) and actin cross-linking (ACD), positioning RTX cytotoxin as a multifunctional virulence protein coupling iron acquisition to cytotoxicity. This work establishes RTX cytotoxin as a critical virulence determinant in vAh and provides a scalable live-attenuated vaccine with potential for protecting U.S. catfish aquaculture against MAS.

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