A high death rate among red hybrid tilapias was observed in a farm in Selangor, Malaysia, in January 2020. The affected fish appeared lethargic, isolated from schooling group, showed loss of appetite, red and haemorrhagic skin, exophthalmia and enlarged gall bladders. Histopathological assessment revealed deformation of kidney tubules, and severe congestion with infiltrations of inflammatory cells in the brains and kidneys. Syncytial cells and intracytoplasmic inclusion bodies were occasionally observed in the liver and brain sections. Tilapia Lake Virus (TiLV), Aeromonas hydrophila and Streptococcus agalactiae were identified in the affected fish, either through isolation or through PCR and sequencing analysis. The phylogenetic tree analysis revealed that the TiLV strain in this study was closely related to the previously reported Malaysian strain that was isolated in 2019. On the other hand, A. hydrophila and S. agalactiae were closer to Algerian and Brazilian strains, respectively. The multiple antibiotic resistance index for A. hydrophila and S. agalactiae was 0.50 and 0.25, respectively. Co-infections of virus and bacteria in cultured tilapia is a new threat for the tilapia industry.

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 LD50–240h 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.

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.