| dc.description.abstract | Columnaris disease, caused by columnaris-causing bacteria (CCB), is a constant threat to farmed fish species. Several types of columnaris vaccines have shown various levels of protection in multiple fish species; however, it is hypothesized that due to CCB host associations, development of a vaccine that targets specific host species could elicit a strong immune response and provide protection. The first study of this dissertation investigated rifampicin-resistant CCB strains as potential live-attenuated vaccine candidates in channel catfish (Ictalurus punctatus) and Nile tilapia (Oreochromis niloticus). Five Flavobacterium covae (ALG-00-530, FBCC-CC-12K, ML-17-22A, IPRS-15-49, and ALG-15-231) and two Flavobacterium oreochromis (Costa Rica 04-02-TNT and R18-27) mutant strains demonstrated attenuation in channel catfish and Nile tilapia, respectively. Comparative genomic analysis of parent and mutant strains predicted deleterious rpoB mutations within 8/10 rifampicin-resistant mutant genomes. Other mutations affecting gene transcription, nutrient acquisition, and essential physiological pathways were also predicted as deleterious, suggesting attenuation is multifactorial. The second study assessed the efficacy of the five F. covae mutant isolates generated from the first study as live-attenuated vaccines in channel catfish. The vaccines were administered to fish via a static immersion for 30 minutes, then the fish were maintained for up to 8 weeks post-vaccination. Fish were then exposed to corresponding virulent parent strains and cumulative percent mortality and relative percent survival was determined. Two mutant strains (FBCC-CC-12K and ALG-15-231), exhibited variable protection; however, serum IgM antibody titers did not correlate with protection observed in channel catfish, and vaccinated fish antibody titers were comparable to sham-vaccinated fish. Protein profiles revealed conserved antigenic patterns between the parent and mutant strains. This study demonstrated protective efficacy of F. covae mutants is strain-specific and trial dependent, with F. covae FBCC-CC-12K and ALG-15-231 mutants warranting further investigation and optimization as live-attenuated vaccines. The third study involved virulence screening of yellow-pigmented bacteria recovered from diseased farmed catfish. Whole genome sequencing of isolates revealed the identities of one Chryseobacterium gleum, four C. mucoviscodosis, one C. cucumeris, one Empedobacter brevis, and one Flavobacterium saliperosum. The E. brevis isolate demonstrated high mortality and clinical signs of disease following intraperitoneal injection in channel catfish. Koch’s postulates were fulfilled in a replicated study, and the median lethal dose was estimated. A co-infection study with E. brevis and F. covae demonstrated significant mortality in the co-infection group compared to the single infection group of F. covae, but no significant difference in mortality between the co-infection group and single infection of E. brevis. This study demonstrated the exacerbated effect on mortality and disease following co-infection with E. brevis and F. covae. The final study assessed growth and proteomic responses of F. covae (Fc), virulent Aeromonas hydrophila (vAh), and Edwardsiella ictaluri (Ei) in co-culture. Growth metrics and secreted extracellular protein responses were assessed for three bacterial co-culture combinations. Growth metric analysis revealed cooperative growth in co-culture of Fc + Ei, while competitive growth was observed in co-cultures of vAh + Ei and Fc + vAh. Differential abundance of virulence-associated secreted proteins provided insight into species-specific alterations in co-culture. Collectively, these studies advance the understanding of host-specific live-attenuated vaccine development against columnaris disease, identify other yellow-pigmented bacterial pathogens affecting farmed catfish, and provide insight to mechanisms that influence bacterial virulence during co-infection. | en_US |
