Expression of immuno-transcriptome response in red hybrid tilapia (Oreochromis sp.) hindgut following vaccination with feed-based bivalent vaccine.

Streptococcosis and aeromoniasis are the main obstacles to sustainable tilapia production. Vaccination offered an effective method to control microbial infections. Previously, a feed-based bivalent vaccine (FBBV) containing killed whole organisms of Streptococcus agalactiae and Aeromonas hydrophila mixed with 10% palm oil was successfully developed, which provided good protection against streptococcosis and aeromoniasis in Oreochromis sp. However, the mechanisms of immunities in vaccinated fish still need clarification. Here, the hindgut transcriptome of vaccinated and control fish was determined, as the gut displays higher affinity towards antigen uptake and nutrient absorption. The efficacy of FBBV to improve fish immunity was evaluated according to the expression of immune-related genes in the vaccinated fish hindgut throughout the 8-week experimental period using RT-qPCR. The vaccinated fish hindgut at week 6 was further subjected to transcriptomic analysis due to the high expression of immune-related genes and contained killed whole organisms. Results demonstrated the expression of immune-related genes was in correlation with the presence of killed whole organisms in the vaccinated fish hindgut. Transcriptomic analysis has allowed the prediction of robust immune-related pathways, including innate and adaptive immunological responses in vaccinated fish hindgut than control fish. Pathways related to the regulation of lipid metabolism and modulation of the immune system were also significantly enriched (p ≤ .05). Overall, results offer a fundamental study on understanding the immunological response in Oreochromis sp. following vaccination with the FBBV pellet and support further application to prevent bacterial diseases in aquaculture.

Immunogenicity and Efficacy of a Feed-Based Bivalent Vaccine against Streptococcosis and Motile Aeromonad Septicemia in Red Hybrid Tilapia (Oreochromis sp.).

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.

Evaluating the Intestinal Immunity of Asian Seabass (Lates calcarifer, Bloch 1790) following Field Vaccination Using a Feed-Based Oral Vaccine.

This study describes the levels of gut lysozyme and IgM, the number, size and density of gut-associated lymphoid tissue (GALT) regions, and the lymphocyte population in Asian seabass following field oral administration of a feed-based vaccine. Fish in a grow-out farm were selected and divided into two groups; Group 1 was vaccinated at week 0, 2, and 6, while Group 2 was not vaccinated. Samplings were done at 2-week intervals when the fish were observed for clinical signs, and gross lesions were recorded. The intestinal tissue and gut lavage fluid were collected. GALT regions (numbers, size, density and population of lymphocytes) were analyzed. Clinical signs such as abnormal swimming pattern and death, and gross lesions including scale loss, ocular opacity, and skin ulceration were observed in both groups. At the end of the study, the incidence rate between both groups were significantly different (p < 0.05). The gut IgM level and lysozyme activity, lymphocyte population, number, size and density of GALT regions of Group 1 were significantly (p < 0.05) higher than Group 2. Therefore, this study concludes that the feed-based vaccine reduces the incidence of vibriosis by stimulating the gut immunity of the vaccinated fish with an enhanced GALT region, specific IgM production against Vibrio harveyi, and lysozyme responses.

Advances in Aptamer-Based Biosensors and Cell-Internalizing SELEX Technology for Diagnostic and Therapeutic Application.

Aptamers are a group of synthetic single-stranded nucleic acids. They are generated from a random library of single-stranded DNA or RNA by a technology named systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a repetitive process to select and identify suitable aptamers that show high affinity and specificity towards target cells. Great strides have been achieved in the design, construction, and use of aptamers up to this point. However, only a small number of aptamer-based applications have achieved widespread commercial and clinical acceptance. Additionally, finding more effective ways to acquire aptamers with high affinity remains a challenge. Therefore, it is crucial to thoroughly examine the existing dearth and advancement in aptamer-related technologies. This review focuses on aptamers that are generated by SELEX to detect pathogenic microorganisms and mammalian cells, as well as in cell-internalizing SELEX for diagnostic and therapeutic purposes. The development of novel aptamer-based biosensors using optical and electrical methods for microbial detection is reported. The applications and limitations of aptamers are also discussed.

Chemical, Nutrient and Physicochemical Properties of Brown Seaweed, Sargassum polycystum C. Agardh (Phaeophyceae) Collected from Port Dickson, Peninsular Malaysia.

Recent increased interest in seaweed is motivated by attention generated in their bioactive components that have potential applications in the functional food and nutraceutical industries. In the present study, nutritional composition, metabolite profiles, phytochemical screening and physicochemical properties of freeze-dried brown seaweed, Sargassum polycystum were evaluated. Results showed that the S. polycystum had protein content of 8.65 ± 1.06%, lipid of 3.42 ± 0.01%, carbohydrate of 36.55 ± 1.09% and total dietary fibre content of 2.75 ± 0.58% on dry weight basis. The mineral content of S. polycystum including Na, K, Ca, Mg Fe, Se and Mn were 8876.45 ± 0.47, 1711.05 ± 0.07, 1079.75 ± 0.30, 213.85 ± 0.02, 277.6 ± 0.12, 4.70 ± 0.00 and 4.45 ± 0.00 mg 100/g DW, respectively. Total carotenoid, chlorophyll a and b content in S. polycystum were detected at 45.28 ± 1.77, 141.98 ± 1.18 and 111.29 µg/g respectively. The total amino acid content was 74.90 ± 1.45%. The study revealed various secondary metabolites and major constituents of S. polycystum fibre to include fucose, mannose, galactose, xylose and rhamnose. The metabolites extracted from the seaweeds comprised n-hexadecanoic acid, 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester, benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy- methyl ester, 1-dodecanol, 3,7,11-trimethyl-, which were the most abundant. The physicochemical properties of S. polycystum such as water-holding and swelling capacity were comparable to several commercial fibre-rich products. In conclusion, results of this study indicate that S. polycystum is a potential candidate as functional food sources for human consumption and its cultivation needs to be encouraged.

Vaccine Efficacy of a Newly Developed Feed-Based Whole-Cell Polyvalent Vaccine against Vibriosis, Streptococcosis and Motile Aeromonad Septicemia in Asian Seabass, Lates calcarifer.

Multiple infections of several bacterial species are often observed under natural farm conditions. The infections would cause a much more significant loss compared to a single infectious agent. Vaccination is an essential strategy to prevent diseases in aquaculture, and oral vaccination has been proposed as a promising technique since it requires no handling of the fish and is easy to perform. This research attempts to develop and evaluate a potential feed-based polyvalent vaccine that can be used to treat multiple infections by Vibrios spp., Streptococcus agalactiae, and Aeromonas hydrophila, simultaneously. The oral polyvalent vaccine was prepared by mixing formalin-killed vaccine of V. harveyi, S. agalactiae, and A. hydrophila strains with commercial feed pellet, and palm oil as an adjuvant was added to improve their antigenicity. Thereafter, a vaccinated feed pellet was tested for feed quality analysis in terms of feed stability in water, proximate nutrient analysis, and palatability, safety, and growth performance using Asian seabass, Lates calcarifer as a fish host model. For immune response analysis, a total of 300 Asian seabass juveniles (15.8 ± 2.6 g) were divided into two groups in triplicate. Fish of group 1 were not vaccinated, while group 2 was vaccinated with the feed-based polyvalent vaccine. Vaccinations were carried out on days 0 and 14 with oral administration of the feed containing the bacterin at 5% body weight. Samples of serum for antibody and lysozyme study and the spleen and gut for gene expression analysis were collected at 7-day intervals for 6 weeks. Its efficacy in protecting fish was evaluated in aquarium challenge. Following vaccination by the polyvalent feed-based vaccine, IgM antibody levels showed a significant (p < 0.05) increase in serum against Vibrio harveyi, Aeromonas hydrophila, and Streptococcus agalactiae and reached the peak at week 3, 5, and 6, respectively. The high-stimulated antibody in the serum remained significantly higher than the control (p < 0.05) at the end of the 6 weeks vaccination trial. Not only that, but the serum lysozyme level was also increased significantly at week 4 (p < 0.05) as compared to the control treatment. The immune-related gene, dendritic cells, C3, Chemokine ligand 4 (CCL4), and major histocompatibility complex class I (MHC I) showed significantly higher expression (p < 0.05) after the fish were vaccinated with the oral vaccine. In the aquarium challenge, the vaccine provided a relative percentage survival of 75 ± 7.1%, 80 ± 0.0%, and 80 ± 0.0% after challenge with V. harveyi, A. hydrophila, and S. agalactiae, respectively. Combining our results demonstrate that the feed-based polyvalent vaccine could elicit significant innate and adaptive immunological responses, and this offers an opportunity for a comprehensive immunization against vibriosis, streptococcosis, and motile aeromonad septicemia in Asian seabass, Lates calcarifer. Nevertheless, this newly developed feed-based polyvalent vaccination can be a promising technique for effective and large-scale fish immunization in the aquaculture industry shortly.

Efficacy of Feed-Based Formalin-Killed Vaccine of Streptococcus iniae Stimulates the Gut-Associated Lymphoid Tissues and Immune Response of Red Hybrid Tilapia.

Red hybrid tilapia were fed a formalin-killed oral Streptococcus iniae vaccine (FKV) in the present study was assessed. Three hundred Red hybrid tilapia 80 ± 10 g were divided into five groups (1A, 1B, 2A, 2B, and Cx), each consisting of 60 fish. Fish from Groups 1A, 1B, 2A, and 2B were fed with FKV over different periods of administration, while Group 2B was the only group of fish to receive an oral booster vaccination on day 14- and 21-days post-vaccination (dpv). Group Cx was fed with normal pellets containing no vaccine as a control group. At four weeks post-vaccination (wpv), all fish were experimentally infected with S. iniae. Groups 2A and 2B had the lowest level of mortalities following vaccination (45% and 30%, respectively) compared to Groups 1A and 1B (80% and 55%, respectively), while the level of mortalities in Group Cx was 100%. All vaccinated groups showed a significant increase in anti-S. iniae IgM levels (p < 0.05) in serum, mucus, and gut-lavage, while Group Cx did not (p > 0.05) and all fish in this group died by five weeks post-infection. In conclusion, fish fed with the S. iniae FKV had a greater level of protection against S. iniae, with increased specific antibody response to the vaccine and there was also evidence of GALT stimulation by the vaccine.

Efficacy of Whole Cell Inactivated Vibrio harveyi Vaccine against Vibriosis in a Marine Red Hybrid Tilapia (Oreochromis niloticus × O. mossambicus) Model.

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.

Co-Infections of Tilapia Lake Virus, Aeromonas hydrophila and Streptococcus agalactiae in Farmed Red Hybrid Tilapia.

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.

Elucidating the Efficacy of Vaccination against Vibriosis in Lates calcarifer Using Two Recombinant Protein Vaccines Containing the Outer Membrane Protein K (r-OmpK) of Vibrio alginolyticus and the DNA Chaperone J (r-DnaJ) of Vibrio harveyi.

Recombinant cell vaccines expressing the OmpK and DnaJ of Vibrio were developed and subsequently, a vaccination efficacy trial was carried out on juvenile seabass (~5 cm; ~20 g). The fish were divided into 5 groups of 50 fish per group, kept in triplicate. Groups 1 and 2 were injected with 107 CFU/mL of the inactivated recombinant cells vaccines, the pET-32/LIC-OmpK and pET-32/LIC-DnaJ, respectively. Group 3 was similarly injected with 107 CFU/mL of inactivated E. coli BL21 (DE3), Group 4 with 107 CFU/mL of formalin killed whole cells V. harveyi, and Group 5 with PBS solution. Serum, mucus, and gut lavage were used to determine the antibody levels before all fish were challenged with V. harveyi, V. alginolyticus, and V. parahemolyticus, respectively on day 15 post-vaccination. There was significant increase in the serum and gut lavage antibody titers in the juvenile seabass vaccinated with r-OmpK vaccine. In addition, there was an up-regulation for TLR2, MyD88, and MHCI genes in the kidney and intestinal tissues of r-OmpK vaccinated fish. At the same time, r-OmpK triggered higher expression level of interleukin IL-10, IL-8, IL-1ß in the spleen, intestine, and kidney compared to r-DnaJ. Overall, r-OmpK and r-DnaJ triggered protection by curbing inflammation and strengthening the adaptive immune response. Vaccinated fish also demonstrated strong cross protection against heterologous of Vibrio isolates, the V. harveyi, V. alginolyticus, and V. parahaemolyticus. The fish vaccinated with r-OmpK protein were completely protected with a relative per cent of survival (RPS) of 90 percent against V. harveyi and 100 percent against V. alginolyticus and V. parahaemolyticus. A semi-quantitative PCR detection of Vibrio spp. from the seawater containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. In conclusion, our results suggest r-OmpK as a candidate vaccine molecule against multiple Vibrio strain to prevent vibriosis in marine fish.

Study on Streptococcus agalactiae infection in Javanese medaka (Oryzias javanicus Bleeker, 1854) model.

This study determines the median lethal dose, and describes the clinico-pathological changes and disease development following Streptococcus agalactiae infection in Javanese medaka model. Javanese medakas were infected with S. agalactiae via intraperitoneal (IP) from 104 to 108 CFU/mL, and immersion (IM) route from 103 to 107 CFU/mL. The LD50-240h and clinico-pathological changes of the fish was determined until 240 h post infection (hpi). Next, the disease development was determined for 96 hpi in the fish following IP and IM infection at 103 CFU/mL and 104 CFU/mL, respectively. The LD50-240h of S. agalactiae in Javanese medaka was lower following IP injection (4.5 × 102 CFU/mL), compared to IM route (3.5 × 103 CFU/mL). The clinical signs included separating from the schooling group, swimming at the surface of water column, lethargy, erratic swimming pattern, corneal opacity and exophthalmia. Histopathological examinations revealed generalized congestion in almost all internal organs, particularly in liver and brain, while the kidney displayed tubular necrosis. Both IP and IM routes showed significant positive correlation (p < 0.05) between the CFU/g of S. agalactiae in the fish tissue and fish deaths. Moreover, the lesions for histopathological scoring in selected organs following IP and IM challenges were also reflecting the CFU/g and fish deaths. This study indicates the capability of Javanese medaka as a model organism in study of streptococcosis development.

Environmental Factors Associated with the Presence of Vibrionaceae in Tropical Cage-Cultured Marine Fishes.

This study investigated the environmental factors associated with the presence of Vibrionaceae in economically important cage-cultured tropical marine fishes: the Asian Seabass Lates calcarifer, snapper Lutjanus sp., and hybrid grouper Epinephelus sp. Fish sampling was conducted at monthly intervals between December 2016 and August 2017. The body weight and length of individual fish were measured, and the skin, eye, liver, and kidney were sampled for bacterial isolation and identification. Water physicochemical parameters during the sampling activities were determined, and the enumeration of total Vibrionaceae count was also conducted from water and sediment samples. Nine species of Vibrio were identified, including V. alginolyticus, V. diabolicus, V. harveyi, V. campbellii, V. parahaemolyticus, V. rotiferianus, V. furnissii, V. fluvialis, and V. vulnificus. Photobacterium damselae subsp. damselae was also identified. A total of 73% of the isolated Vibrio belonged to the Harveyi clade, followed by the Vulnificus clade (5.5%) and Cholera clade (0.6%). Highest occurrence of Vibrio spp. and P. damselae subsp. damselae was found in hybrid grouper (72%), followed by Asian Seabass (48%) and snapper (36%). The associations of Vibrio spp. and P. damselae subsp. damselae with the host fish were not species specific. However, fish mortality and fish size showed strong associations with the presence of some Vibrio spp. On average, 60% of the infected cultured fish exhibited at least one clinical sign. Nevertheless, inconsistent associations were observed between the pathogens and water quality. The yearlong occurrence and abundance of Vibrionaceae in the environmental components indicate that they might serve as reservoirs of these pathogens.

Natural Concurrent Infection of Vibrio harveyi and V. alginolyticus in Cultured Hybrid Groupers in Malaysia.

In September 2016, a marine fish farm operator in Selangor, Malaysia, reported a disease outbreak affecting juvenile hybrid groupers (Camouflage Grouper Epinephelus polyphekadion × Tiger Grouper E. fuscoguttatus). The average daily mortality was 120 fish, resulting in a cumulative mortality rate of 29% within 10 d. The affected hybrid groupers displayed lethargy, excessive mucus production, rotten fins, congestion of livers and kidneys, and enlargement of spleens. Microscopically, general congestion of the brains and internal organs was evident. Vibrio harveyi and V. alginolyticus were successfully isolated from the diseased fish. The isolated pathogens were found to be sensitive to oxytetracycline and tetracycline, but resistant towards ampicillin and vancomycin. Experimental infections using the isolated V. harveyi (108  CFU/mL), V. alginolyticus (108  CFU/mL), and concurrent infection by V. harveyi (108  CFU/mL) and V. alginolyticus (108  CFU/mL) in juvenile Asian Seabass Lates calcarifer resulted in 60, 100, and 100% mortality, respectively, within 240 h postinfection. The experimentally infected Asian Seabass demonstrated similar clinical signs and histopathological changes as the naturally infected hybrid groupers. However, concurrently infected fish demonstrated severe clinical signs and histopathological changes compared with single infections. These results suggest that both isolates of Vibrio are pathogenic to fish and responsible for the disease outbreak. However, concurrent infection involving V. alginolyticus and V. harveyi leads to a more devastating impact to the cultured fish. This is the first report of concurrent Vibrio infection in cultured marine fish in Malaysia.