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 current experiment was designed to estimate the comparative efficacy of selected phytobiotics Persicaria odorata leaf meal (POLM) and Piper betle leaf meal (PBLM) with halquinol, and tetracycline in broiler chickens. The 150-day-old broiler chickens were randomly assigned to five dietary groups. The dietary supplementation groups were the basal diet (BD), which served as the negative control (NC), and BD + 0.2 g/kg tetracycline, which served as the positive control (PC); BD + 0.03 g/kg halquinol (HAL), BD + 8 g/kg POLM (Po8), and BD + 4 g/kg PBLM (Pb4) were the treatment groups. Growth performance, gut morphology, ileal digestibility, and cecal microbiota composition were measured. On day 21, the body weight gain (BWG) was enhanced (p < 0.05) in the broiler chickens fed on phytobiotics (Po8 and Pb4) relative to the NC group, however, on day 42 and in terms of overall growth performance, BWG was enhanced (p < 0.05 in diets (Po8, Pb4, HAL and PC) in comparison with the NC group. Conversely, feed conversion ratio (FCR) was recorded reduced (p < 0.05) in Pb4, Po8, HAL, and PC group in comparison with the NC group. Supplementation of phytobiotics (Po8 and Pb4), HAL and PC, positively improved the gut morphology compared to the NC group. Furthermore, the maximum (p < 0.05) villus height (VH) in duodenum and jejunum was observed in broilers fed on diet Pb4. Supplementation of phytobiotics, HAL and PC, improved (p < 0.05) the digestibility of dry matter (DM) (except for HAL), organic matter (OM), crude protein (CP), ether extract (EE), and ash compared to the NC group. Dietary supplementation of phytobiotics (Po8 and Pb4), HAL and PC, significantly reduced the E. coli, Salmonella, and Staphylococcus aureus (except for HAL) counts compared to the NC group. However, supplementation of Pb4 resulted in significantly decreased total anaerobic bacteria and Clostridium spp. counts compared to the NC group. In addition, supplementation of phytobiotics significantly increased the Lactobacillus count compared to HAL, PC, and NC groups. In conclusion, dietary supplementation of phytobiotics improved the gut morphology, positively modulated and maintained the dynamics of cecal microbiota with enhanced nutrient digestibility, thus, increased the growth performance. Based on current results, phytobiotics could be used as an alternative to AGPs for sustainable broiler chicken production.

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.

Vibrio harveyi causes vibriosis in various commercial marine fish species. The infection leads to significant economic losses for aquaculture farms, and vaccination is an alternative approach for the prevention and control of fish diseases for aquaculture sustainability. This study describes the use of formalin-killed Vibrio harveyi (FKVh) strain Vh1 as a vaccine candidate to stimulate innate and adaptive immunities against vibriosis in a marine red hybrid tilapia model. Tilapia are fast growing; cheap; resistant to diseases; and tolerant to adverse environmental conditions of fresh water, brackish water, and marine water and because of these advantages, marine red hybrid tilapia is a suitable candidate as a model to study fish diseases and vaccinations against vibriosis. A total of 180 healthy red hybrid tilapias were gradually adapted to the marine environment before being divided into two groups, with 90 fish in each group and were kept in triplicate with 30 fish per tank. Group 1 was vaccinated intraperitoneally with 100 µL of FKVh on week 0, and a booster dose was similarly administered on week 2. Group 2 was similarly injected with PBS. Skin mucus, serum, and gut lavage were collected weekly for enzyme-linked immunosorbent assay (ELISA) and a lysozyme activity assay from a total of 30 fish of each group. On week 4, the remaining 60 fish of Groups 1 and 2 were challenged with 10⁸ cfu/fish of live Vibrio harveyi. The clinical signs were monitored while the survival rate was recorded for 48 h post-challenge. Vaccination with FKVh resulted in a significantly (p < 0.05) higher rate of survival (87%) compared to the control (20%). The IgM antibody titer and lysozyme activities of Group 1 were significantly (p < 0.05) higher than the unvaccinated Groups 2 in most weeks throughout the experiment. Therefore, the intraperitoneal exposure of marine red hybrid tilapia to killed V. harveyi enhanced the resistance and antibody response of the fish against vibriosis.

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.