Aquaculture is one of the fastest-growing food-producing sectors in the world. However, its growth is hampered by various disease problems due to infectious microorganisms, including Gram-negative bacteria in finfish aquaculture. Disease control in aquaculture by use of antibiotics is not recommended as it leads to antibiotic residues in the final product, selection, and spread of antibiotic resistance in the environment. Therefore, focus is on disease prevention by vaccination. All Gram-negative bacteria possess surface-associated outer membrane proteins (OMPs), some of which have long been recognized as potential vaccine candidates. OMPs are essential for maintaining the integrity and selective permeability of the bacterial membrane and play a key role in adaptive responses of bacteria such as solute and ion uptake, iron acquisition, antimicrobial resistance, serum resistance, and bile salt resistance and some adhesins have virulence attributes. Antigenic diversity among bacterial strains even within the same bacterial species has constrained vaccine developments, but OMPs that are conserved across serotypes could be used as potential candidates in vaccine development, and several studies have demonstrated their efficacy and potential as vaccine candidates. In this review, we will look into the application of OMPs for the design of vaccines based on recombinant proteins, subunit vaccines, chimeric proteins, and DNA vaccines as new-generation vaccine candidates for major bacterial pathogens of fish for sustainable aquaculture.

Aeromonas hydrophila is a Gram-negative bacterium that causes high mortality in different fish species and at different growth stages. Although vaccination has significantly contributed to the decline of disease outbreaks in aquaculture, the use of oral vaccines has lagged behind the injectable vaccines due to lack of proven efficacy, that being from primary immunization or by use of boost protocols. In this study, the outer membrane protein W (OmpW) of A. hydrophila was cloned, purified, and encapsulated in poly d,l-lactide-co-glycolic acid (PLGA) nanoparticles (NPs) for oral vaccination of rohu (Labeo rohita Hamilton). The physical properties of PLGA NPs encapsulating the recombinant OmpW (rOmpW) was characterized as having a diameter of 370–375 nm, encapsulation efficiency of 53% and −19.3 mV zeta potential. In vitro release of rOmpW was estimated at 34% within 48 h of incubation in phosphate-buffered saline. To evaluate the efficacy of the NP-rOmpW oral vaccine, two antigen doses were orally administered in rohu with a high antigen (HiAg) dose that had twice the amount of antigens compared to the low antigen (LoAg) dose. Antibody levels obtained after vaccination showed an antigen dose dependency in which fish from the HiAg group had higher antibody levels than those from the LoAg group. The antibody levels corresponded with post challenge survival proportions (PCSPs) and relative percent survival (RPS) in which the HiAg group had a higher PCSP and RPS than the LoAg group. Likewise, the ability to inhibit A. hydrophila growth on trypticase soy agar (TSA) by sera obtained from the HiAg group was higher than that from the LoAg group. Overall, data presented here shows that OmpW orally administered using PLGA NPs is protective against A. hydrophila infection with the level of protective immunity induced by oral vaccination being antigen dose-dependent. Future studies should seek to optimize the antigen dose and duration of oral immunization in rohu in order to induce the highest protection in vaccinated fish.

The use of oral vaccination in finfish has lagged behind injectable vaccines for a long time as oral vaccines fall short of injection vaccines in conferring protective immunity. Biodegradable polymeric nanoparticles (NPs) have shown potential to serve as antigen delivery systems for oral vaccines. In this study the recombinant outer membrane protein A (rOmpA) of Edwardsiella tarda was encapsulated in chitosan NPs (NP-rOmpA) and used for oral vaccination of Labeo fimbriatus. The rOmpA purity was 85%, nanodiameter <500 nm, encapsulation efficiency 60.6%, zeta potential +19.05 mV, and there was an in vitro release of 49% of encapsulated antigen within 48 h post incubation in phosphate-buffered saline. Empty NPs and a non-formulated, inactivated whole cell E. tarda (IWC-ET) vaccine were used as controls. Post-vaccination antibody levels were significantly (p = 0.0458) higher in the NP-rOmpA vaccinated fish (Mean OD₄₅₀ = 2.430) than in fish vaccinated with inactivated whole cell E. tarda (IWC-ET) vaccine (Mean OD₄₅₀ = 1.735), which corresponded with post-challenge survival proportions (PCSP) of 73.3% and 48.28% for the NP-rOmpA and IWC-ET groups, respectively. Serum samples from NP-rOmpA-vaccinated fish had a higher inhibition rate for E. tarda growth on tryptic soy agar (TSA) than the IWC-ET group. There was no significant difference (p = 0.989) in PCSPs between fish vaccinated with empty NPs and the unvaccinated control fish, while serum from both groups showed no detectable antibodies against E. tarda. Overall, these data show that the NP-rOmpA vaccine produced higher antibody levels and had superior protection over the IWC-ET vaccine, showing that encapsulating OmpA in chitosan NPs confer improved protection against E. tarda mortality in L. fimbriatus. There is a need to elucidate the possible adjuvant effects of chitosan NPs and the immunological mechanisms of protective immunity induced by OMPs administered orally to fish.

The application of nanotechnology in aquaculture for developing efficient vaccines has shown great potential in recent years. Nanovaccination, which involves encapsulating antigens of fish pathogens in various polymeric materials and nanoparticles, can afford protection to the antigens and a sustained release of the molecule. Oral administration of nanoparticles would be a convenient and cost-effective method for delivering vaccines in aquaculture while eliminating the need for stressful, labour-intensive injectables. The small size of nanoparticles allows them to overcome the degradative digestive enzymes and help deliver antigens to the target site of the fish more effectively. This targeted-delivery approach would help trigger cellular and humoral immune responses more efficiently, thereby enhancing the protective efficacy of vaccines. This is particularly relevant for combating diseases caused by pathogens like Aeromonas hydrophila, a major fish pathogen responsible for significant morbidity and mortality in the aquaculture sector. While the use of nanoparticle-based vaccines in aquaculture has shown promise, concerns exist about the potential toxicity associated with certain types of nanoparticles. Some nanoparticles have been found to exhibit varying degrees of toxicity, and their safety profiles need to be thoroughly assessed before widespread application. The introduction of nanovaccines has opened new vistas for improving aquaculture healthcare, but must be evaluated for potential toxicity before aquaculture applications. Details of nanovaccines and their mode of action, with a focus on protecting fish from infections and outbreaks caused by the ubiquitous opportunistic pathogen A. hydrophila, are reviewed here.

Tilapia is the second most farmed fish species after carp in the world. However, the production has come under threat due to emerging diseases such as tilapia lake virus (TiLV) that causes massive mortalities with high economic losses. It is largely unknown whether different tilapia strains are equally susceptible to TiLV infection. In the present study we compared the susceptibility of gray (Oreochromis niloticus x O. aureus) and red tilapia (Oreochromis spp.) to experimental TiLV infection. Virus was injected intraperitoneally at a concentration of 10⁴ TCID₅₀/mL. Our findings show that gray tilapia had a lower mortality, 86.44%, but statistically not significantly different (p = 0.068) from red tilapia (100%). The duration of the mortality period from onset to cessation was similar for the two species, starting at 2–3 days post challenge (dpc) with a median at 10–11 dpi and ending on 20–22 dpi. In addition, there was no difference between species in mean viral loads in brain, liver and headkidney from fish collected soon after death. As for host response, expression levels of IL-1β and TNFα were equally high in brain and headkidney samples while levels in liver samples were low for both red and gray tilapia, which coincides with lower viral loads in liver compared to brain and headkidney for both species. We find that red and gray tilapia were equally susceptible to TiLV infection with similar post challenge mortality levels, equal virus concentration in target organs and similar proinflammatory cytokine responses in target and lymphoid organs at time of death. Nonetheless, we advocate that the search for less susceptible tilapia strains should continue with the view to reduce losses from TiLV infection in aquaculture.

Bacteriocins are emerging as a viable alternative to antibiotics due to their ability to inhibit growth or kill antibiotic resistant pathogens. Herein, we evaluated the ability of the bacteriocin Garvicin KS (GarKS) produced by Lactococcus garvieae KS1546 isolated from cow milk to inhibit the growth of fish and foodborne bacterial pathogens. We found that GarKS inhibited the growth of five fish L. garvieae strains isolated from infected trout and eels. Among fish pathogens, GarKS inhibited the growth of Streptococcus agalactiae serotypes Ia and Ib, and Aeromonas hydrophila but did not inhibit the growth of Edwardsiella tarda. In addition, it inhibited the growth of A. salmonicida strain 6421 but not A. salmonicida strain 6422 and Yersinia ruckeri. There was no inhibition of three foodborne bacterial species, namely Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli. In vitro cytotoxicity tests using different GarKS concentrations showed that the highest concentration of 33 µg/mL exhibited low cytotoxicity, while concentrations ≤3.3 µg/mL had no cytotoxicity on CHSE-214 and RTG-2 cells. In vivo tests showed that zebrafish larvae treated with 33 µg/mL and 3.3 µg/mL GarKS prior to challenge had 53% and 48% survival, respectively, while concentrations ≤0.33 µg/mL were nonprotective. Altogether, these data show that GarKS has a broad inhibitory spectrum against Gram positive and negative bacteria and that it has potential applications as a therapeutic agent for a wide range of bacterial pathogens. Thus, future studies should include clinical trials to test the efficacy of GarKS against various bacterial pathogens in farmed fish.