Streptococcosis and aeromonasis inflicted by Streptococcus iniae and Aeromonas hydrophila, respectively, have affected tilapia industries worldwide. In this study, we investigated antibody responses and explored the mechanisms of protection rendered by an oral bivalent vaccine in red tilapia following challenges with S. iniae and A. hydrophila. The results of specific IgM antibody response revealed that the IgM titers against S. iniae and A. hydrophila in the bivalent incorporated (BI) vaccine group were significantly higher (p < 0.05) than those in the bivalent spray (BS) vaccine fish and unvaccinated control fish throughout the experiment. Real-time qPCR results also showed that the gene expression of CD4, MHC-I, MHC-II, IgT, C-type lysozyme, IL-1β, TNF-α, and TGF-β remained significantly higher (p < 0.05) than that of the controls between 24 and 72 h post-infection (hpi) in both mucosal (hindgut) and systemic (spleen and head–kidney) organs of BI vaccinated fish. Furthermore, the highest relative expression of the TGF-β, C-type lysozyme, and IgT genes in the BI vaccinated group was observed in the challenged fish’s spleen (8.8-fold), head kidney (4.4-fold), and hindgut (19.7-fold) tissues, respectively. The present study suggests that the bivalent incorporated (BI) vaccine could effectively improve the immune function and activate both humoral and cell-mediated immunities in vaccinated red tilapia following the bacterial challenges.

The genus Aeromonas has been recognised as an important pathogenic species in aquaculture that causes motile Aeromonas septicaemia (MAS) or less severe, chronic infections. This study compares the pathogenicity of the different Aeromonas spp. that were previously isolated from freshwater fish with signs of MAS. A total of 124 isolates of Aeromonas spp. were initially screened for the ability to grow on M9 agar with myo-inositol as a sole carbon source, which is a discriminatory phenotype for the hypervirulent A. hydrophila (vAh) pathotype. Subsequently, LD₅₀ of six selected Aeromonas spp_. were determined by intraperitoneal injection of bacterial suspension containing 10³, 10⁵, and 10⁷ CFU/mL of the respective Aeromonas sp. to red hybrid tilapias. The kidneys, livers and spleens of infected moribund fish were examined for histopathological changes. The screening revealed that only A. dhakensis 1P11S3 was able to grow using myo-inositol as a sole carbon source, and no vAh strains were identified. The LD_50–240h of A. dhakensis 1P11S3 was 10⁷ CFU/mL, while the non-myo-inositol utilizing A. dhakensis 4PS2 and A. hydrophila 8TK3 was lower at 10⁵ CFU/mL. Similarly, tilapia challenged with the myo-inositol A. dhakensis 1P11S3 showed significantly (p < 0.05) less severe signs, gross and histopathological lesions, and a lower mortality rate than the non-myo-inositol A. dhakensis 4PS2 and A. hydrophila 8TK3. These findings suggested that myo-inositol utilizing A. dhakensis 1P11S3 was not a hypervirulent Aeromonas sp. under current experimental disease challenge conditions, and that diverse Aeromonas spp. are of concern in aquaculture farmed freshwater fish. Therefore, future study is warranted on genomic level to further elucidate the influence of myo-inositol utilizing ability on the pathogenesis of Aeromonas spp., since this ability correlates with hypervirulence in A. hydrophila strains.

Vibriosis is one of the most common threats to farmed grouper; thus, substantial efforts are underway to control the disease. This study presents an oral vaccination against multiple Vibrio spp. in a marine fish with double booster immunisation. The Vibrio harveyi strain VH1 vaccine candidate was selected from infected groupers Epinephelus sp. in a local farm and was formalin inactivated and combined with commercial feed at a 10% ratio (v/w). A laboratory vaccination trial was conducted for seventy days. The induction of IgM antibody responses in the serum of Asian seabass Lates calcarifer immunised with the oral Vibrio harveyi strain VH1 was significantly (p < 0.05) increased as early as week one post-primary vaccination. Subsequent administration of the first and second booster for 5 consecutive days, starting on days 14 and 42, respectively, improved the specific antibody level and reached a highly significant (p < 0.05) value at days 35 and 49 before slightly decreasing from day 56 onwards. Antibody titres of the control unvaccinated group remained relatively stable and low throughout the experimental period. At the end of the 70-day vaccination trial, 23 days post final boost, an intraperitoneal challenge with a field strain of Vibrio harveyi, V. alginolyticus, and V. parahaemolyticus was carried out. Our challenge study showed that oral Vibrio harveyi strain VH1 vaccine candidate could induce significant protection, with an RPS of 70–80% against different Vibrio species. Thereafter, a field trial was conducted in a mariculture farm to study the effect of field vaccination using the oral Vibrio harveyi strain VH1 vaccine candidate. A total of 3000 hybrid grouper juveniles were divided into two groups in triplicate. Fish of Group 1 were not vaccinated, while Group 2 were vaccinated with the feed-based vaccine. Vaccinations were carried out on days 0, 14, and 42 via feeding the fish with the vaccine at 4% body weight for 5 consecutive days. At the end of the study period, the fish survival rate was 80% for the vaccinated group, significantly (p < 0.05) higher than the 65% seen in the control unvaccinated group. Furthermore, the vaccinated fish showed significantly (p < 0.05) better growth performances. Therefore, the oral Vibrio vaccine from the inactivated Vibrio harveyi strain VH1 is a potential versatile vaccine candidate that could stimulate good immune responses and confer high protection in both Asian seabass, Lates calcarifer, and farm hybrid grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus.

Streptococcosis and motile Aeromonad septicemia (MAS) are the main bacterial diseases in tilapia culture worldwide, causing significant economic losses. Vaccination is an effective method of preventing diseases and contributes to economic sustainability. This study investigated the immuno-protective efficacy of a newly developed feed-based bivalent vaccine against streptococcosis and MAS in red hybrid tilapia. The feed-based bivalent vaccine pellet was developed by incorporating the formalin-killed S. agalactiae and A. hydrophila antigens into a commercial feed pellet with palm oil as the adjuvant. The bivalent vaccine was subjected to feed quality analyses. For immunological analyses, 900 fish (12.94 ± 0.46 g) were divided into two treatment groups in triplicate. Fish in Group 1 were unvaccinated (control), while those in Group 2 were vaccinated with the bivalent vaccine. The bivalent vaccine was delivered orally at 5% of the fish’s body weight for three consecutive days on week 0, followed by boosters on weeks 2 and 6. Lysozyme and enzyme-linked immunosorbent assays (ELISAs) on serum, gut lavage, and skin mucus were performed every week for 16 weeks. Lysozyme activity in vaccinated fish was significantly (p ≤ 0.05) higher than in unvaccinated fish following vaccination. Similarly, the IgM antibody levels of vaccinated fish were significantly (p ≤ 0.05) higher after vaccination. The bivalent vaccine provided high protective efficacy against S. agalactiae (80.00 ± 10.00%) and A. hydrophila (90.00 ± 10.00%) and partial cross-protective efficacy against S. iniae (63.33 ± 5.77%) and A. veronii (60.00 ± 10.00%). During the challenge test, fewer clinical and gross lesions were observed in vaccinated fish compared with unvaccinated fish. Histopathological assessment showed less severe pathological changes in selected organs than the unvaccinated fish. This study showed that vaccination with a feed-based bivalent vaccine improves immunological responses in red hybrid tilapia, and thus protects against streptococcosis and MAS.