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Development of Bioluminescent Virulent Aeromonas hydrophila for Understanding Pathogenicity
Eda Ozdemir1, Hossam Abdelhamed1, Ozan Ozdemir1
1Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA.
Abstract:
Virulent Aeromonas hydrophila (vAh) strains that cause motile Aeromonas septicemia (MAS) in farmed channel catfish (Ictalurus punctatus) have been an important problem for more than a decade. However, the routes of infection of vAh in catfish are not well understood. Therefore, it is critical to study the pathogenicity of vAh in catfish. To this goal, a new bioluminescence expression plasmid (pAKgfplux3) with the chloramphenicol acetyltransferase (cat) gene was constructed and mobilized into vAh strain ML09-119, yielding bioluminescent vAh (BvAh). After determining optimal chloramphenicol concentration, plasmid stability, bacteria number-bioluminescence relationship, and growth kinetics, the catfish were challenged with BvAh, and bioluminescent imaging (BLI) was conducted. Results showed that 5 to 10 µg/mL chloramphenicol was suitable for stable bioluminescence expression in vAh, with some growth reduction. In the absence of chloramphenicol, vAh could not maintain pAKgfplux3 stably, with the half-life being 16 h. Intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) challenges of catfish with BvAh and BLI showed that MAS progressed faster in the injection group, followed by the modified immersion and immersion groups. BvAh was detected around the anterior mouth, barbels, fin bases, fin epithelia, injured skin areas, and gills after experimental challenges. BLI revealed that skin breaks and gills are potential attachment and entry portals for vAh. Once vAh breaches the skin or epithelial surfaces, it can cause a systemic infection rapidly, spreading to all internal organs. To our best knowledge, this is the first study that reports the development of a bioluminescent vAh and provides visual evidence for catfish-vAh interactions. Findings are expected to provide a better understanding of vAh pathogenicity in catfish.
Insights
This study developed bioluminescent Aeromonas hydrophila (vAh) to track infections in channel catfish. Findings reveal skin breaks and gills as key entry points for vAh, leading to rapid systemic disease.
Area of Science:
- Aquatic Animal Health
- Microbiology
- Fish Pathology
Background:
- Motile Aeromonas septicemia (MAS), caused by virulent Aeromonas hydrophila (vAh), is a significant economic problem in farmed channel catfish.
- The infection routes and pathogenicity of vAh in catfish remain poorly understood, hindering effective disease management.
Purpose of the Study:
- To develop a bioluminescent strain of vAh (BvAh) for visualizing infection dynamics in channel catfish.
- To investigate the routes of vAh infection and its pathogenicity in channel catfish using bioluminescent imaging (BLI).
Main Methods:
- Construction of a novel bioluminescence expression plasmid (pAKgfplux3) and its introduction into vAh strain ML09-119 to create BvAh.
- Optimization of conditions for stable bioluminescence expression, including chloramphenicol concentration and plasmid stability.
- Challenge experiments using intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) in catfish, followed by BLI to track BvAh dissemination.
Main Results:
- Stable bioluminescence expression in BvAh was achieved with 5-10 µg/mL chloramphenicol, though it slightly reduced growth.
- BvAh lost plasmid stability without chloramphenicol (half-life of 16 hours).
- BLI demonstrated that MAS progressed fastest via intraperitoneal injection, followed by modified immersion and immersion routes. BvAh was detected at the anterior mouth, barbels, fin bases, fin epithelia, injured skin, and gills, indicating these as potential entry sites.
Conclusions:
- Skin breaks and gills serve as critical entry points for vAh, facilitating rapid systemic infection in channel catfish.
- The developed BvAh and BLI technique provide valuable visual evidence of vAh-catfish interactions and pathogenicity.
- This study enhances understanding of vAh virulence mechanisms and infection pathways in channel catfish, aiding future disease control strategies.
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