Development and Evaluation of Multiple Vaccination Strategies Against Flavobacterium covae in Channel Catfish (Ictalurus punctatus)

Abstract

Columnaris disease, caused by Flavobacterium covae, is a principal cause of mortality in the catfish industry that can lead to several million dollars in annual production losses and is the most prevalent columnaris-causing bacteria in channel catfish. As aquaculture production continues to increase, the need for cost-effective prophylactics also increases. Recent work has identified the upregulation of specific F. covae biofilm genes after stimulation with channel catfish mucus and demonstrated the effectiveness of using a F. covae recombinant protein for bath immersion vaccination of catfish to protect against columnaris disease. To build upon this work, two additional F. covae recombinant proteins, catalase and DNA starvation/stationary phase protein (DPS), were evaluated for their efficacy as vaccine formulations delivered by bath immersion. Different groups of fish were immunized with the recombinant protein(s) mixed with an immersion adjuvant or sham immunized with adjuvant alone. Nine weeks post-immunization, all groups were challenged by bath immersion with F. covae at a calculated dose of 1.54 x 107 CFU/mL and the vaccinated groups showed significant increases in survival (13–45 %) when compared to the adjuvant only control group. F. covae catalase and DPS were further evaluated as orally-delivered vaccines in their efficacy in preventing columnaris disease. Fish were fed varying doses of either catalase or DPS for two weeks before laboratory challenged six weeks post immunization with F. covae at a calculated dose of 1.0 x 107 CFU/mL. At six weeks post vaccination, catalase was protective at 20 µg/g of feed while DPS was protective with as little as 1 µg/g of feed. Additionally, Bacillus velezensis AP193 has been shown to reduce mortality associated with aquatic pathogens. DPS at 20 µg/g of feed was further evaluated for its potential synergy with B. velezensis AP193 but no increase in protection was observed at one week and nine weeks post immunization. Flavobacterium covae is an opportunistic pathogen that manifests as a biofilm on the host's mucosal surfaces as the disease progresses. The quorum sensing gene luxR was reported to be upregulated in F. covae biofilm-grown cells stimulated with channel catfish mucus. To understand LuxR’s contributions to F. covae virulence, a F. covae C#2 luxR deletion mutant (ΔluxR) was created along with a complemented luxR (ΔluxR + pCE1) by site-directed mutagenesis. Channel catfish fingerlings challenged with the wild-type or ΔluxR + pCE1 were observed to have 100% or 90% mortality, respectively; in contrast, only 2% mortality was observed for catfish fingerlings challenged with ΔluxR, indicating an important role for LuxR-regulated genes in F. covae virulence. Since the attenuated F. covae ΔluxR was avirulent in channel catfish, it became an ideal whole cell inactivated vaccine candidate. Pickering emulsions are oil-water mixtures that are stabilized by organic or inorganic particles and have been widely used in both biomedical and agricultural settings. Using orange peel powder, a common agricultural waste, F. covae ΔluxR and parent strain C#2 were evaluated as Pickering emulsified vaccines against columnaris disease. Different groups of channel catfish were immunized with either F. covae ΔluxR or parent strain C#2 Pickering emulsions with or without 0.5% orange peel powder. Six weeks post-immunization, all groups were challenged with an industry-relevant isolate of F. covae. None of the vaccinated groups showed significant increases in survival when compared to the naïve control group, but the F. covae C#2 Pickering emulsion with orange peel powder did increase survival compared to the Pickering emulsion without the stabilizing particle. No antigen specific serum or gill mucosal IgM antibodies were detected via ELISA. Herein are data presenting two alternative forms of columnaris vaccine design and their efficacy in preventing F. covae infection in channel catfish: recombinant protein vaccines and whole cell bacterin vaccines.