Systemic and mucosal immune responses in red tilapia (Oreochromis sp.) following immersion vaccination with a chitosan polymer-based nanovaccine against Aeromonas veronii.

A mucoadhesive chitosan polymer-based nanoplatform has been increasingly recognized as an effective mucosal vaccine delivery system for fish. The present study aimed to investigate the effectiveness of immersion vaccination with a chitosan polymer-based nanovaccine to elicit an immune response in serum and mucus of red tilapia and evaluate its protective efficacy after immersion challenge with a heterogenous strain of Aeromonas veronii UDRT09. Six hundred red tilapia (22 ± 1.8 g) were randomly allocated into four experimental groups: control, empty-polymeric nanoparticle (PC), formalin-killed vaccine (FKV), and chitosan polymer-based nanovaccine (CS-NV) in triplicate. The specific IgM antibody levels and their bactericidal activity were assessed in serum and mucus for 28 days after immersion vaccination and followed by immersion challenge with A. veronii. The immersion vaccine was found to be safe for red tilapia, with no mortalities occurring during the vaccination procedure. The specific IgM antibody levels and bactericidal activity against A. veronii in both serum and mucus were significantly higher in red tilapia vaccinated with CS-NV compared to the FKV and control groups at all time points. Furthermore, the serum lysozyme activity, ACH50, and total Ig levels demonstrated a significant elevation in the groups vaccinated with CS-NV compared to the FKV and control groups. Importantly, the Relative Percentage Survival (RPS) value of the CS-NV group (71 %) was significantly higher than that of the FKV (15.12 %) and PC (2.33 %) groups, respectively. This indicates that the chitosan polymer-based nanovaccine platform is an effective delivery system for the immersion vaccination of tilapia.

Efficacy of whole-cell-based monovalent and bivalent vaccines against Streptococcus iniae and Flavobacterium covae in fingerling Asian seabass (Lates calcarifer).

Streptococcosis and columnaris caused by Streptococcus spp. and Flavobacterium spp. have been recognized as critical problems in Asian seabass aquaculture development because they cause severe mortality. In this study, we identified various isolates of S. iniae and F. covae from diseased Asian seabass farmed in Thailand for use as candidates for vaccine development. The efficacy of the vaccines was evaluated by challenge tests and immune parameter analyses in fish that received whole-cell-based monovalent and bivalent vaccines containing S. iniae (Sin) and F. covae (Fco) delivered by top-dressed feed (TD) and intraperitoneal injection (IP). The results showed that all vaccinated groups exhibited increased antibody titers compared with control fish that peaked on day 28 after booster administration with high detection levels in the Sin-IP and Fco-IP groups. Moreover, the immune responses to the injected monovalent vaccines (Sin-IP and Fco-IP) were better than the responses in the other vaccinated groups. The hematological and innate immunological parameters were significantly increased by Sin-IP and Fco-IP, particularly lysozyme activity, nitroblue tetrazolium (NBT) activity, bactericidal activity, and white blood cell numbers, and immune-related genes, including IgM, MHC-IIα, TCRß and CD4, were significantly upregulated in the head kidney, whole blood and spleen (P < 0.05). After experimental challenge, survival in the Sin-IP and Fco-IP groups was significantly higher than that in the Sin-TD, Fco-TD, Sin + Fco-TD, and Sin + Fco-IP groups, with 80.0 % and 60.0 % survival after S. iniae and F. covae infection, respectively. In contrast, survival after bacterial challenge in the control groups was 10 % in each group. Histopathological analysis revealed that Sin-IP- and Fco-IP-vaccinated fish exhibited significantly more goblet cells in the intestines and melanomacrophage centers (MMCs) in the head kidney and spleen than those in the other groups (P < 0.05). Overall, the results of our study indicated that the monovalent vaccines Sin-IP and Fco-IP provoked better vaccine efficacy and immune responses than their orally administered counterparts, and these results are consistent with those from the immunological assays that showed significantly increased responses after immunization.

Dietary administration of yeast (Saccharomyces cerevisiae) hydrolysate from sugar byproducts promotes the growth, survival, immunity, microbial community and disease resistance to VP (AHPND) in Pacific white shrimp (Litopenaeus vannamei).

This study investigated the effects of yeast hydrolysate (YH) from sugar byproducts on various parameters in Pacific white shrimp (Litopenaeus vannamei). The study found no significant differences in water quality parameters across all treatment tanks, ensuring that the observed effects were not due to environmental variations. There were no significant differences in growth parameters between the control group and groups receiving YH at different dosages. However, the group given YH at 10.0 g/kg feed exhibited a notably higher survival rate and higher expression of growth-related genes (IGF-2 and RAP-2A) in various shrimp tissues. YH was associated with enhanced immune responses, including lysozyme activity, NBT dye reduction, bactericidal activity, and phagocytic activity. Notably, the 10.0 g/kg feed group displayed the highest phagocytic index, indicating a dose-dependent immune response. Expression of immune-related genes (ALF, LYZ, ProPO, and SOD) was upregulated in various shrimp tissues. This upregulation was particularly significant in the gills, hepatopancreas, intestine, and hemocytes. While total Vibrio counts remained consistent, a reduction in green Vibrio colonies was observed in the intestine of shrimp treated with YH. YH, especially at 5.0 and 10.0 g/kg feed dosages, significantly increased survival rates and RPS values in response to AHPND infection. The findings of this study suggest that incorporating additives derived from yeast byproducts with possible prebiotic properties obtained from sugar byproducts can lead to positive results in terms of enhancing growth performance, immunity, histological improvements, and resistance to V. parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND).

First Investigation of the Optimal Timing of Vaccination of Nile Tilapia (Oreochromis niloticus) Larvae against Streptococcus agalactiae.

To investigate early immune responses and explore the optimal vaccination periods, Nile tilapia at 1, 7, 14, 21, 28, 35, and 42 days after yolk sac collapse (DAYC) were immersed in formalin-killed Streptococcus agalactiae vaccine (FKV-SA). A specific IgM was first detected via ELISA in the 21 DAYC larvae (0.108 g) at 336 h after vaccination (hav), whereas in the 28-42 DAYC larvae (0.330-0.580 g), the specific IgM could be initially detected at 24 hav. qRT-PCR analysis of the TCRß, CD4, MHCIIα, IgHM, IgHT, and IgHD genes in 21-42 DAYC larvae immunized with the FKV-SA immersion route for 24, 168, and 336 hav revealed that the levels of most immune-related genes were significantly higher in the vaccinated larvae at all DAYCs than in the control larvae (p < 0.05) at 336 hav. Immunohistochemistry demonstrated stronger IgM signals in the gills, head kidney, and intestine tissues at 21, 28, and 35 DAYC in all vaccinated larvae compared with the control. Interestingly, at all DAYCs, FKV-SA larvae exhibited significantly higher survival rates and an increased relative percent survival (RPS) than the control after challenge with viable S. agalactiae, particularly in larvae that were immunized with FKV-SA at 168 and 336 hav (p < 0.05).

Effects of the combination of chitosan and Acinetobacter KU011TH on the growth and health performances and disease resistance of juvenile hybrid catfish (Clarias gariepinus × C. macrocephalus).

Aquatic animal health management has become a crucial component in the goal of increasing catfish aquaculture productivity. Additionally, hybrid catfish (Clarias gariepinus × C. macrocephalus) has been promoted as a highly profitable freshwater fish in Asia. Interestingly, the crucial diseases induced by Aeromonas hydrophila have been reported to greatly impede catfish production. To overcome this challenge, the aim was to investigate the effects of the oral administration of potentially synbiotic chitosan (CH) and Acinetobacter KU011TH (AK) on the growth performance, immunological responses, and disease resistance of hybrid catfish against A. hydrophila. The control group was fed a basal diet (A), the diet fed to treatment group B was supplemented with 20 mL of CH/kg diet (B), and the experimental feed fed to groups C-D was mixed with 1 × 108, 1 × 109 and 1 × 1010 CFU/mL AK coated with 20 mL of CH/kg diet. Five different groups of juvenile hybrid catfish were continuously fed the 5 formulated feeds for 4 weeks. The results revealed that all tested feeds did not significantly enhance the hybrid catfish's average daily gain, specific growth rate, feed conversion ratio, hematocrit and erythrocyte counts. Interestingly, the application of CH and AK significantly increased the leukocyte counts, respiratory burst, lysozyme activity, alternative complement pathway hemolytic activity, and bactericidal activity (P < 0.05). The expression levels of the immune-related genes in the whole blood, head kidney, and spleen were significantly increased after CH-AK application (P < 0.05), but this finding was not observed in the liver (P > 0.05). Additionally, after 14 days of A. hydrophila peritoneal injection, the fish in group C showed significantly higher survival rates of approximately 70.0 % compared with the control fish in groups B, D, and E (52.5 %, 40.0 %, 45.0 %, and 45.0 %, respectively) (P < 0.05). These results collectively suggest that short-term application of the diet fed to group C effectively boosted the immune responses and disease resistance of hybrid catfish against A. hydrophila.

Addressing Nanovaccine Strategies for Tilapia.

Tilapia is the world's most extensively farmed species after carp. It is an attractive species for aquaculture as it grows quickly, reaching harvest size within six to seven months of production, and provides an important source of food and revenue for many low-income families, especially in low- to middle-income countries. The expansion of tilapia aquaculture has resulted in an intensification of farming systems, and this has been associated with increased disease outbreaks caused by various pathogens, mostly bacterial and viral agents. Vaccination is routinely used to control disease in higher-value finfish species, such as Atlantic salmon. At the same time, many tilapia farmers are often unwilling to vaccinate their fish by injection once the fish have been moved to their grow-out site. Alternative vaccination strategies are needed to help tilapia farmers accept and use vaccines. There is increasing interest in nanoparticle-based vaccines as alternative methods for delivering vaccines to fish, especially for oral and immersion administration. They can potentially improve vaccine efficacy through the controlled release of antigens, protecting antigens from premature proteolytic degradation in the gastric tract, and facilitating antigen uptake and processing by antigen-presenting cells. They can also allow targeted delivery of the vaccine at mucosal sites. This review provides a brief overview of the bacterial and viral diseases affecting tilapia aquaculture and vaccine strategies for farmed tilapia. It focuses on the use of nanovaccines to improve the acceptance and uptake of vaccines by tilapia farmers.

Effects of ribonucleotide supplementation in modulating the growth of probiotic Bacillus subtilis and the synergistic benefits for improving the health performance of Asian seabass (Lates calcarifer).

In aquaculture, due to the requirements for high-density culture, the diseases caused by bacterial pathogens have become a serious issue. To solve this problem, we performed synbiotic application of RNA and Bacillus subtilis as a sustainable and eco-friendly approach to improve the health and immunity of Asian seabass (Lates calcarifer) during cultivation without using any harmful antibiotics or chemicals. Among various forms of nucleic acids, such as mononucleotides and DNA, RNA was found to be most effective in promoting the growth performance of probiotic B. subtilis in all the tested minimal medium conditions. Accordingly, we used the synbiotic combination of B. subtilis and RNA for Asian seabass cultivation. After feed supplementation for fourteen days, the fish that received the combination treatment exhibited a significant increase in innate cellular and humoral immune parameters, including phagocytic activity, phagocytic index, respiratory burst, serum lysozyme and bactericidal activities, as well as upregulated expression of immune-related genes, including HEPC1, A2M, C3, CC, CLEC, LYS, HSP70, and HSP90. Furthermore, significant increases were observed in the ileal villus height and goblet cell numbers in the intestinal villi in all fish treatment groups. The combination treatment did not cause histopathological abnormalities in the intestine and liver, suggesting that the synbiotic treatment is safe for use in fish. The treated Asian seabass also exhibited a significantly increased survival rate after Aeromonas hydrophila challenge. These results indicate that the synbiotic mixture of B. subtilis and RNA can be considered a beneficial feed additive and immunostimulant for Asian seabass cultivation.

Long-Term Application of a Synbiotic Chitosan and Acinetobacter KU011TH Mixture on the Growth Performance, Health Status, and Disease Resistance of Hybrid Catfish (Clarias gariepinus × C. macrocephalus) during Winter.

The effects of potential synbiotic chitosan and Acinetobacter KU011TH mixture on growth performance, immune response, and A. hydrophila resistance were investigated for the first time. The control group was fed a basal diet (A), and group B was given the formula B diet that was supplemented with chitosan at 20 mL/kg diet via top dressing. The other synbiotic groups, C, D, and E, were top-dressed with the target probiotics at 1 × 108, 1 × 109, and 1 × 1010 CFU/kg diet, respectively, and coated with the same concentration of chitosan. Fish were continuously fed the five different feeds for 16 weeks during winter. At the end of the trial, the growth parameters of the test groups did not significantly differ from those of the control (p > 0.05). All the symbiotic-chitosan treatments significantly increased various hematological and serum immune parameters. Moreover, the expression levels of immune-related genes were strongly elevated in the head kidney and spleen, whereas upregulated expression was observed in the liver and whole blood (p < 0.05). Survival analysis indicated that fish in groups B and C showed significantly higher survival (84.33 ± 2.21 and 79.50 ± 6.34%) than those in groups A, D and E (55.33 ± 8.82%-74.00 ± 6.50) (p < 0.05) after injection with A. hydrophila for 14 days.

Development of a bivalent mucoadhesive nanovaccine to prevent francisellosis and columnaris diseases in Nile tilapia (Oreochromis niloticus).

The occurrence of francisellosis caused by Francisella orientalis sp. nov. (Fo) and columnaris disease caused by Flavobacterium oreochromis (For) is negatively impacting Nile tilapia (Oreochromis niloticus) production, especially when high stocking densities are used. A new and innovative bivalent mucoadhesive nanovaccine was developed in this study for immersion vaccination of tilapia against francisellosis and columnaris disease. It was shown to have the potential to improve both innate and adaptive immunity in vaccinated Nile tilapia. It increased innate immune parameters, such as lysozyme activity, bactericidal activity, phagocytosis, phagocytic index, and total serum IgM antibody levels. Additionally, the vaccine was effective in elevating specific adaptive immune responses, including IgM antibody levels against Fo and For vaccine antigens and upregulating immune-related genes IgM, IgT, CD4+, MHCIIα, and TCRß in the head kidney, spleen, peripheral blood leukocytes, and gills of vaccinated fish. Furthermore, fish vaccinated with the mucoadhesive nanovaccine showed higher survival rates and relative percent survival after being challenged with either single or combined infections of Fo and For. This vaccine is anticipated to be beneficial for large-scale immersion vaccination of tilapia and may be a strategy for shortening vaccination times and increasing immune protection against francisellosis and columnaris diseases in tilapia aquaculture.

Immune responses and histopathological analyses of giant river prawn (Macrobrachium rosenbergii, De Man 1879) challenged with a sub-lethal dose of decapod iridescent virus 1 (DIV1) and chemical control investigation.

Decapod iridescent virus 1 (DIV1) is a lethal virus that has a significant influence on the shrimp and prawn culture industries. The mechanism through which infected prawns respond to the DIV1 virus is currently unknown. Here, we examined in detail the clinical signs, histopathology, and humoral, cellular, and immune-related gene responses after a sub-lethal dose of DIV1 during the acute infection period of 0-120 h post infection (hpi). Interestingly, at the end of the experiment, DIV1-infected prawns had black lesions on several external regions. The DIV1-infected prawns also exhibited few karyopyknotic nuclei in the gills and intestine tissues and exhibited increasing immunological responses, as revealed by significant increases in all examined parameters, including total hemocytes, phagocytosis, lysozyme, and overall bactericidal activity, from 6 to 48 hpi. In addition, between 72 and 120 hpi, all immune response activities of DIV1-infected prawn were impaired compared with those of normal prawns, indicating negative impacts on immunological parameters. A viral load analysis of various tissues by qPCR indicated that hemocytes were the dominant initial viral target tissues, followed by the gills and hepatopancreas. An expression analysis of crucial immune-related genes by qRT‒PCR revealed various expression patterns in response to DIV1 infection; in particular, fold changes in the relative expression of anti-lipopolysaccharide factors (ALFs), prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) were observed. Additionally, five common chemicals, calcium hypochlorite [Ca(OCl)2] at 16.25-130 ppm, hydrogen peroxide (H2O2) at 8.75-70 ppm, povidone iodine (PVP-I) at 3-24 ppm, benzalkonium chloride (BKC) at 20-160 ppm, and formalin at 25-200 ppm, had a significant effect on the killing of DIV1 particles in vitro within 24 h after exposure. These data will be helpful for determining the health status and immune defense mechanisms of giant river prawns during DIV1 infection periods. The study performed the first application of very common disinfectants, and the obtained information will be useful for implementing effective strategies to prevent and control DIV1 infection in both hatchery and grow-out ponds.

The first description of the blue swimming crab (Portunus pelagicus) transcriptome and immunological defense mechanism in response to white spot syndrome virus (WSSV).

In the global shellfish farming industry, white spot syndrome virus (WSSV) is a major cause of mortality and a significant factor in economic losses. However, information on molecular immune responses to WSSV in blue swimming crabs (Portunus pelagicus) has never been reported. First, viral loads were measured in the gills, hepatopancreas, intestines, subcuticular epithelium and hemocytes of blue swimming crabs (50 ± 10 g) (n = 4) after WSSV induction at 0, 24, 48 and 96 h post injection (hpi). A significant increase in WSSV particles was observed in gills at 48 and 96 hpi, as supported by histopathology. To further investigate the acute immune response to WSSV, total RNA from the same gill tissues at 0, 24, and 96 hpi was used to construct 16 high-quality RNA-seq cDNA libraries. In summary, 162,740 unigenes were discovered in these transcriptomic libraries analyzed with the GO, KO, KOG, NR, NT, PFAM and SwissProt databases. Intensive sequence analysis against control crabs using three major categories of gene oncology (GO) of DEGs, biological processes (BPs), molecular functions (MFs), and cellular components (CCs), indicated that induction of WSSV in blue swimming crabs strongly affected the immune responses of the target animals significantly during the early stages of infection from 24 to 96 hpi. Furthermore, KEGG identified approximately twenty biological pathways of gene expression that were both downregulated and upregulated. Interestingly, at 24 and 96 hpi, several immune-related genes involved in virus defense in the blue swimming crab, particularly crustin 2, chitinase, anti-lipopolysaccharide, proteinase inhibitor, and lysozyme, were highly expressed during the WSSV early infection stages. At the same time, viral mRNA transcripts, including WSV289, WSV343, WSV306, deoxyuridine 5' triphosphate nucleohydrolase, RING finger containing E3 ubiquitin-protein ligase WSV403 and WSV404, were recorded in the top twenty upregulated genes. Moreover, some immune-responsive genes related to growth development, such as chitinase, tubulin alpha and beta chains, trypsin, and the cathepsin family, were also differentially expressed during these periods. Expression validation of 20 upregulated and 11 downregulated immune-related genes using qRT‒PCR showed similar patterns with transcriptome information. Overall, the data showed that during WSSV infection, a number of immune-, metabolism-, and growth-related pathways were activated, and several of the pathways involved differed depending on the stage of virus invasion. These findings could effectively help us better understand the impact of WSSV on the physiology of blue swimming crabs and serve as a valuable reference for future research on the immune system and disease control in this target species.

Development of Immersion and Oral Bivalent Nanovaccines for Streptococcosis and Columnaris Disease Prevention in Fry and Fingerling Asian Seabass (Lates calcarifer) Nursery Farms.

In the present study, chitosan-based bivalent nanovaccines of S. iniae and F. covae were administered by immersion vaccination at 30 and 40 days after hatching (DAH), and the third vaccination was orally administered by feeding at 50 DAH. ELISA revealed that the levels of total IgM and specific IgM to S. iniae and F. covae were significantly elevated in all vaccinated groups at 10, 20, and 30 days after vaccination (DAV). A qRT-PCR analysis of immune-related genes revealed significantly higher IgT expression in the vaccinated groups compared to the control group, as revealed by 44-100-fold changes in the vaccinated groups compared to the control (p < 0.001) at every tested time point after vaccination. All vaccinated groups expressed IgM, MHCIIα, and TCRα at significantly higher levels than the control group at 10 and/or 20 DAV (p < 0.05). In the S. iniae challenge tests, the survival of vaccinated groups ranged from 62.15 ± 2.11 to 75.70 ± 3.36%, which significantly differed from that of the control group (44.44 ± 1.92%). Similarly, all vaccinated groups showed higher survival rates of 68.89 ± 3.85 to 77.78 ± 5.09% during F. covae challenge than the control groups (50.00 ± 3.33%) (p < 0.05).

Mystifying Molecular Structure, Expression and Repertoire Diversity of IgM Heavy Chain Genes (Ighµ) in Clarias Catfish and Hybrids: Two Novel Transcripts in Vertebrates.

Two novel immunoglobulin heavy chain (Ighµ) transcripts encoding membrane-bound forms of IgM (mIgM) were discovered in bighead catfish, Clarias macrocephalus. The first transcript contains four constant and two transmembrane domains [Cµ1-Cµ2-Cµ3-Cµ4-TM1-TM2] that have never been reported in teleosts, and the second transcript is an unusual mIgM that has never been identified in any vertebrate [Cµ1-(Cδ2-Cδ3-Cδ4-Cδ5)-Cµ2-Cµ3-TM1-TM2]. Fluorescence in situ hybridization (FISH) in bighead catfish, North African catfish (C. gariepinus) and hybrid catfish revealed a single copy of Ighµ in individual parent catfish, while two gene copies were found in diploid hybrid catfish. Intensive sequence analysis demonstrated multiple distinct structural variabilities in the VH domain in Clarias, and hybrid catfish were defined and used to generate diversity with various mechanisms. Expression analysis of Ighµ in Aeromonas hydrophila infection of the head kidney, peripheral blood leukocytes and spleen revealed significantly higher levels in North African catfish and hybrid catfish than in bighead catfish.

Novel development of cationic surfactant-based mucoadhesive nanovaccine for direct immersion vaccination against Francisella noatunensis subsp. orientalis in red tilapia (Oreochromis sp.).

Francisella noatunensis subsp. orientalis (Fno) is one of the infectious diseases that causes economic losses associated with tilapia mortality. Even though direct immersion administration of vaccines is more practicable for small fish and fry compared with oral and injection vaccination in the fields, the efficacy is still insufficient due to lower potency of antigen uptake. Herein, we accomplished the development of a mucoadhesive nanovaccine platform using cetyltrimethylammonium bromide (CTAB), a cationic surfactant, to improve the efficiency of immersion vaccination against Fno in tilapia. Cationic Fno nanovaccine (CAT-Fno-NV) was prepared though emulsification using an ultrasonic method. In our investigation, the CAT-Fno-NV increased the opportunity of Fno vaccine uptake by extending the contact time between vaccine and mucosal surface of fish gills and enhancing the protective efficacy against Fno infection. Fish were vaccinated with the CAT-Fno-NV by a direct immersion protocol. The challenge trial by Fno injection revealed that CAT-Fno-NV at the concentration 1:100 ratio (approximately 1 × 106 cfu/mL) had the highest efficacy to protect fish from Fno infection at day 30 after post challenge period according to the total number of Fno detected in head kidney, spleen and liver. A significant upregulation of IgM gene was observed in gills, skin, head kidney, serum and peripheral blood lymphocytes (PBLs) and spleen tissues treated with WC and CAT-Fno-NV (1:100) vaccines, while IgT gene was highly expressed in only gills and skin tissues for treated WC and CAT-Fno-NV (1:100) groups. We anticipate that the cationic surfactant-based nanovaccine developed in this study could become an efficient alternative for direct immersion vaccination to induce humoral immune responses against Fno in vaccinated tilapia.

Mucoadhesive cationic lipid-based Flavobacterium oreochromis nanoencapsulation enhanced the efficacy of mucoadhesive immersion vaccination against columnaris disease and strengthened immunity in Asian sea bass (Lates calcarifer).

Columnaris is a bacterial disease, found in freshwater fish, caused by Flavobacterium oreochromis. The disease has a devastating impact on a range of cultured and wild freshwater fish species e.g. Lates calcarifer (Asian sea bass), which is a serious economic losses to the freshwater aquaculture in Thailand. The disease can be prevented by an efficacious vaccine, however, no licensed effective vaccine is available to date. Current study was based on the development of a novel mucoadhesive nano-encapsulated vaccine (EncapFlavoNP++), where, cationic lipid-based nanoparticles were combined with an antigen obtained from F. oreochromis. Various parameters including transmission electron microscopy (TEM), physiochemical properties; zeta potential, and polydispersity index were determined. The TEM results depicted well-formed circular-shaped nano-encapsulates complexed with cationic lipid surfactants. The average diameter of the molecules was 200 nm, having a zeta potential of 31.82 mV, while, the polydispersity index (PDI) was 0.31. The in vivo study lasted for 8 weeks, the immunologic and protective potentials of the prepared molecules were determined by challenging the fish for 8 weeks. The most effective dilutions of EncapFlavoNP++ solution were 1:100 and 1:200, which significantly improved the efficacy of the immunity by increasing the level of antibody specific to F. oreochromis. A trend of upregulation was found in the immune-related genes including immunoglobulin M heavy chain (IgM), major histocompatibility complex class IIα molecules (MHC-IIα), and dendritic cell specific transcript (DCs) in gills, skin, liver, peripheral blood lymphocytes (PBLs), head kidneys, and spleen as compared to the control group (P < 0.05 and P < 0.01). Upon immunization with EncapFlavoNP++ solution at the dilution of 1:100 and 1:200, the significant increase in survival rate (SR) and relative percent survival (RPS) were found in fish challenged with virulent F. oreochromis bacterium (SR 72.50% and RPS 62.07) and (SR 65.83% and RPS 52.87), respectively as compared to the control group (P < 0.05). It can be concluded that immunization with EncapFlavoNP++ solution has significant immunologic and protective effects against Columnaris disease. Furthermore, the prepared vaccine candidate has more potential as compared to whole-cell immersion vaccination (FK-WC). It can be used on a large scale in the freshwater aquaculture industry to boost immunity against Columnaris disease.

Development of a Monoclonal Antibody Specific to the IgM Heavy Chain of Bighead Catfish (Clarias macrocephalus): A Biomolecular Tool for the Detection and Quantification of IgM Molecules and IgM+ Cells in Clarias Catfish.

Catfish is a commonly-cultivated freshwater fish in Thailand and many Southeast Asian countries. The molecular data obtained for the IgM heavy chain (IgMH) of catfish have been useful for distinguishing monoclonal antibodies (mAbs). A mAb specific to Cµ1 of the IgMH of catfish (IgMHCµ1 mAb) was developed in a rabbit model using sequence information from bighead catfish (Clarias macrocephalus). The IgMHCµ1 mAb strongly recognized the IgM heavy chain of the tested catfish, namely, bighead catfish, African catfish (Clarias gariepinus) and their hybrid (C. macrocephalus × C. gariepinus), in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCµ1 mAb successfully recognized IgM+ cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCµ1 mAb was further used to quantify the percentage of IgM+ cells among PBLs through flow cytophotometry. The IgM+ cell percentages of healthy bighead catfish, African catfish and their hybrid were 38.0-39.9%, 45.6-53.2%, and 58.7-60.0%, respectively. Furthermore, the IgMHCµ1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish.

Acinetobacter Strain KUO11TH, a Unique Organism Related to Acinetobacter pittii and Isolated from the Skin Mucus of Healthy Bighead Catfish and Its Efficacy Against Several Fish Pathogens.

The bacterial strain KU011TH was isolated from the skin mucus of healthy bighead catfish. The strain is a Gram-negative coccobacillus that is nonmotile, aerobic, catalase positive, oxidase negative, and nonhemolytic. Sequence analyses of the housekeeping genes 16S rRNA, gyrB and rpoB indicate that this strain is a new member of the Acb complex of the genus Acinetobacter and is closely related to Acinetobacter pittii and Acinetobacter lactucae. In addition, the genome relatedness-associated ANIb (<95-96%) and in silico DDH (<70%) values clearly supported the new member of the genus Acinetobacter and the Acb complex. The genome of the strain KU011TH was approximately 3.79 Mbp in size, comprising 3619 predicted genes, and the DNA G+C content was 38.56 mol%. The major cellular fatty acids were C18:1ω9c, C16:0, C16:1, C20:2, C18:2ω6c and C18:1ω9t. The whole-genome sequences and phenotypic, phylogenetic, and chemotaxonomic data clearly support the classification of the strain KU011TH as a new member in the genus Acinetobacter which is closest to A. pittii. Additionally, the new bacterial strain exhibited strong activity against a broad range of freshwater fish pathogens in vitro.

Probiotic Effects of a Novel Strain, Acinetobacter KU011TH, on the Growth Performance, Immune Responses, and Resistance against Aeromonas hydrophila of Bighead Catfish (Clarias macrocephalus Günther, 1864).

In the present study, the novel probiotic strain Acinetobacter KU011TH with an evident lack of pathogenicity in catfish was experimented. Three practical administration routes, namely, feed additive (FD), water-soluble additive (SOL), and a combination route (FD+SOL), were applied in two sizes of catfish. After 120 days of FD+SOL administration, catfish fingerlings (15 g) exhibited a significant improvement in all tested growth performance parameters. For 15- and 30-day applications at the juvenile stage (150 g), phagocytic activity, phagocytic index, lysozyme activity, respiratory burst activity, alternative complement pathway, and bactericidal activity were significantly increased. Furthermore, probiotic-administered bighead catfish exhibited an upregulated expression of several immune-related genes in tested organs. Significant colonization by Acinetobacter KU011TH in rearing water and on skin and gills was observed among experimental groups. Histological analysis clearly indicated enhanced physical characteristics of skin mucosal immunity in the treated groups. No histopathological changes in the gills, skin, intestine or liver were observed among the fish groups. Interestingly, after challenge with Aeromonas hydrophila, the survival rates of the treated groups were significantly higher than those of the controls. In conclusion, the novel probiont Acinetobacter KU011TH provides a potent strategy for improvement in growth and disease resistance, which is an important steppingstone for sustaining catfish aquaculture.

Heritability of immunity traits and disease resistance of bighead catfish, Clarias macrocephalus Günther, 1864.

Disease outbreak is a major obstruction for intensive aquaculture worldwide. One of the promising solutions is genetic improvement by selective breeding, providing that a sufficient proportion of additive genetic variance (measured by heritability-h2) of disease resistance traits exists. In addition, immunity traits are of interest as potential indirect targeted traits for disease resistance. In this study, the genetic parameters of resistance to Aeromonas hydrophila were reported for the first time in the bighead catfish, Clarias macrocephalus Günther, 1864 which is an important parental species for the production of the commercially important hybrid C. macrocephalus × C. gariepinus. The analyses were performed on 736 data records obtained from 74 full-sib families (31 half-sib families) produced by factorial mating design. The results showed that the heritability of survival rate after disease (Aeromonas hydrophila) challenge (intraperitoneal injection with 0.1 ml containing 1 × 106 CFU/ml of A. hydrophila) was low to moderate (0.05 ±â€¯0.02-0.27 ±â€¯0.15). The immune traits (bactericidal activity-BA, lysozyme activity-LA, and alternative complement activity-ACH50) had low to moderate heritability (h2BA = 0.05 ±â€¯0.02; h2LA = 0.16 ±â€¯0.04; h2ACH50 = 0.31 ±â€¯0.06) while heritability of hematocrit (Hct) was also low (h2Hct = 0.17 ±â€¯0.04). The results suggested the possibility to improve resistance to A. hydrophila by selection, while the possibility to use immunity traits as indirect selection criteria for disease resistance is still unclear.

Monascus purpureus-fermented Thai glutinous rice reduces blood and hepatic cholesterol and hepatic steatosis concentrations in diet-induced hypercholesterolemic rats.

BACKGROUND: Red yeast rice (RYR) is a fermented product used as a food supplement to promote blood circulation and lower blood cholesterol levels in eastern Asia. Interestingly, monacolin K is the most active compound in RYR that proved to inhibit HMG-CoA reductase in the cholesterol biosynthesis pathway. METHODS: The hypocholesterolemic effects of oral administration of Thai RYR, produced by fermentation of Thai glutinous rice (Oryza sativa L. var. Niaw San-pah-tawng) with Monascus purpureus CMU 002U, were determined in normal and hypercholesterolemic rats. The rats were divided into six groups, and fed two different kinds of diet. Groups I-II, normal rats fed with a normal diet (SP-diet), were treated with distilled water (SP-control) and 2.0 g/kg/day of RYR extract (SP-2 g). In Groups III-VI, the rats were rendered hypercholesterolemic by feeding them a high fat and cholesterol diet (HFC-diet), and were treated with distilled water (HFC-control), 1.0 g/kg/day (HFC-1 g), 2.0 g/kg/day (HFC-2 g) of RYR extract, and 5.0 mg/kg/day of rosuvastatin (HFC-rosuvastatin) for 30 days, respectively. RESULTS: The RYR extract significantly decreased the concentrations of serum total cholesterol and low density lipoprotein cholesterol (LDL-C), atherosclerotic index, LDL-C/HDL-C ratio and hepatic cholesterol levels in both HFC-1 g and HFC-2 g groups (p < 0.05) as compared with the HFC-control group, and with no significant change in high density lipoprotein cholesterol (HDL-C) concentrations among all six groups. The reduction of serum TC and LDL-C also paralleled the observed changes in mRNA expressions of the genes involved in cholesterol biosynthesis and homeostasis in the liver. The hypercholesterolemic rats treated with RYR extract were significantly higher in LDLR and HMGR expression, but lower in CYP7A1 expression when compared to the untreated hypercholesterolemic rats (HFC-control) (p < 0.05). The hepatic injuries in hypercholesterolemic rats were also obviously alleviated by RYR extract. CONCLUSIONS: The extract of Thai RYR possessed potent hypocholesterolemic and anti-atherogenic activities in diet-induced hypercholesterolemic rats. The possible mechanism involving cholesterol-lowering potential of the extract might contribute to its ability to increase LDL-C endocytosis in hepatocyte and to competitively inhibit HMG-CoA reductase, a key enzyme for cholesterol biosynthesis in liver.