Questioning inbreeding: Could outbreeding affect productivity in the North African catfish in Thailand?

The North African catfish (Clarias gariepinus) is a significant species in aquaculture, which is crucial for ensuring food and nutrition security. Their high adaptability to diverse environments has led to an increase in the number of farms that are available for their production. However, long-term closed breeding adversely affects their reproductive performance, leading to a decrease in production efficiency. This is possibly caused by inbreeding depression. To investigate the root cause of this issue, the genetic diversity of captive North African catfish populations was assessed in this study. Microsatellite genotyping and mitochondrial DNA D-loop sequencing were applied to 136 catfish specimens, collected from three populations captured for breeding in Thailand. Interestingly, extremely low inbreeding coefficients were obtained within each population, and distinct genetic diversity was observed among the three populations, indicating that their genetic origins are markedly different. This suggests that outbreeding depression by genetic admixture among currently captured populations of different origins may account for the low productivity of the North African catfish in Thailand. Genetic improvement of the North African catfish populations is required by introducing new populations whose origins are clearly known. This strategy should be systematically integrated into breeding programs to establish an ideal founder stock for selective breeding.

Identification, Expression and Antimicrobial Functional Analysis of Interleukin-8 (IL-8) in Response to Streptococcus iniae and Flavobacterium covae in Asian Seabass (Lates calcarifer Bloch, 1790).

In this research, the proinflammatory cytokine interleukin-8 (IL-8) was shown to play a key role in inflammatory responses in fish. This study involved the cloning of the gene that encodes IL-8 in Asian seabass (Lates calcarifer) as well as analyses of its expression and function in this fish. The expression levels of LcIL-8 indicated that it was broadly expressed in most analyzed tissues, with the most predominant expression in the whole blood 6 to 24 h after infection with S. iniae at concentrations of 105 colony-forming units (CFU)/fish (p < 0.05). After fish were immersed in F. covae, the LcIL-8 transcript was upregulated in the gills, liver and intestine, and the highest expression level was observed in the gills. However, LcIL-8 was downregulated in all the tested tissues at 48 and 96 h after infection with the two pathogenic strains, indicating that Lc-IL8 has a short half-life during the early immune responses to pathogens. Moreover, the MIC of the rLcIL-8 protein against S. iniae was 10.42 ± 3.61 µg/mL. Furthermore, functional analyses clearly demonstrated that 10 and 100 µg of the rLcIL-8 protein efficiently enhanced the phagocytic activity of Asian seabass phagocytes in vitro (p < 0.05). Additionally, in vivo injection of S. iniae following the rLcIL-8 protein indicated that 50 and 100 µg of rLc-IL-8 were highly effective in protecting fish from this pathogen (p < 0.001). The obtained results demonstrate that rLcIL-8 possesses a biological function in the defense against bacterial infections in Asian seabass.

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.

Overcoming taxonomic challenges in DNA barcoding for improvement of identification and preservation of clariid catfish species.

DNA barcoding without assessing reliability and validity causes taxonomic errors of species identification, which is responsible for disruptions of their conservation and aquaculture industry. Although DNA barcoding facilitates molecular identification and phylogenetic analysis of species, its availability in clariid catfish lineage remains uncertain. In this study, DNA barcoding was developed and validated for clariid catfish. 2,970 barcode sequences from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes and D-loop sequences were analyzed for 37 clariid catfish species. The highest intraspecific nearest neighbor distances were 85.47%, 98.03%, and 89.10% for COI, Cytb, and D-loop sequences, respectively. This suggests that the Cytb gene is the most appropriate for identifying clariid catfish and can serve as a standard region for DNA barcoding. A positive barcoding gap between interspecific and intraspecific sequence divergence was observed in the Cytb dataset but not in the COI and D-loop datasets. Intraspecific variation was typically less than 4.4%, whereas interspecific variation was generally more than 66.9%. However, a species complex was detected in walking catfish and significant intraspecific sequence divergence was observed in North African catfish. These findings suggest the need to focus on developing a DNA barcoding system for classifying clariid catfish properly and to validate its efficacy for a wider range of clariid catfish. With an enriched database of multiple sequences from a target species and its genus, species identification can be more accurate and biodiversity assessment of the species can be facilitated.

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.

Genomics-driven prophylactic measures to increase streptococcosis resistance in tilapia.

Streptococcosis caused by Streptococcus agalactiae and S. iniae is a significant problem that affects the success of tilapia aquaculture industries worldwide. In this critical review, we summarize the applicable practical strategies which may effectively enhance the world tilapia aquaculture development. Recently, the effect of vaccination and selective breeding programmes has been recognized as valuable tools to control the target disease and other consequent negative impacts caused by chemical and drug application. Advances in sequencing and molecular technologies are vital helpful factors with which to develop robust vaccines and increase the selective breeding programme's precision against streptococcosis. The genomic selection for streptococcosis-resistant tilapia strains and crucial genomic application for genomics' contribution to the development of novel Streptococcus vaccine, comparative genomics approach identifying vaccine candidates by reverse vaccinology, and next-generation vaccine design were described. Information from our review is encouraging for practical implementation of the development of vaccination and genomic selection in tilapia for streptococcosis resistance, which may be vital factors to sustain the world tilapia aquaculture industry effectively.

Comparative study of the effects of Montanide™ ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is regarded as a major bacterial pathogen of farmed fish, with outbreaks in Nile tilapia causing significant losses. Vaccination is considered the most suitable method for disease control in aquaculture, with the potential to prevent such outbreaks if highly efficacious vaccines are available for use. Several vaccines have been produced to protect against S. agalactiae infection in tilapia, including inactivated vaccines, live attenuated vaccines, and subunit vaccines, with variable levels of protection seen. Two commercial adjuvants, Montanide™ ISA 763A VG and ISA 763B VG, have been developed recently and designed to improve the safety and efficacy of oil-based emulsions delivered by intraperitoneal injection. In particular, their mode of action may help identify and stimulate particular immunological pathways linked to the intended protective response, which is an important tool for future vaccine development. Therefore, this study aimed to characterize the potential of two adjuvanted-bacterial vaccines against S. agalactiae (SAIV) comparatively, to determine their usefulness for improving protection and to analyse the immune mechanisms involved. Nile tilapia were divided into four groups: 1) fish injected with PBS as a control, 2) fish injected with the SAIV alone, 3) fish injected with the SAIV + Montanide™ ISA 763A VG, and 4) fish injected with the SAIV + Montanide™ ISA 763B VG. Following immunization selected innate immune parameters were analysed, including serum lysozyme, myeloperoxidase, and bactericidal activity, with significantly increased levels seen after immunization. Cytokines associated with innate and adaptive immunity were also studied, with expression levels of several genes showing significant up-regulation, indicating good induction of cell-mediated immune responses. Additionally, the specific IgM antibody response against S. agalactiae was determined and found to be significantly induced post-vaccination, with higher levels seen in the presence of the adjuvants. In comparison to the protection seen with the unadjuvanted vaccine (61.29% RPS), both Montanide™ ISA 763A VG and Montanide™ ISA 763B VG improved the RPS, to 77.42% and 74.19% respectively. In conclusion, Montanide™ ISA 763A VG and Montanide™ ISA 763B VG have shown potential for use as adjuvants for fish vaccines against streptococcosis, as evidenced by the enhanced immunoprotection seen when given in combination with the SAIV vaccine employed in this study.

Pharmacokinetics, optimal dosages and withdrawal time of florfenicol in Asian seabass (Lates calcarifer) after oral administration via medicated feed.

Asian seabass (Lates calcarifer) is an economically important fish in Asian and Australian markets, but few pharmacokinetic (PK) data of antimicrobial drugs in this species is available. The present study investigated the PK behaviour of florfenicol (FF) through medicated feed in Asian seabass cultured at 25°C. The serum and muscle/skin concentrations of FF and its metabolite florfenicol amine (FFA) were determined by the HPLC-FLD method and analysed by one-compartmental model. The optimal dosages were determined by pharmacokinetic-pharmacodynamic (PK-PD) approach and the linear regression analysis was used to determine the withdrawal time (WDT). The PK study following a single oral administration of 15 mg/kg FF via medicated feed revealed that the absorption half-life (t1/2Ka ), elimination half-life (t1/2K ), peak concentration (Cmax ), area under the concentration-time curve (AUC), volume of distribution (Vd/F) and clearance (CL/F) were 1.47 h, 8.07 h, 8.61 µg/ml, 146.41 h·µg/ml, 1.19 L/kg and 0.102 L/kg/h, respectively. The muscle/skin concentration-time profile was similar to that of the serum, suggesting well distribution but only a small fraction of FF was metabolized to FFA. The optimal dosage for a minimum inhibitory concentration of 2 µg/ml was calculated as 13.38 mg/kg/day. The appropriate WDT after multiple oral medications with 15 mg/kg FF once daily for 7 days was determined as 8 days. Information obtained from the current study can potentially be applied for the treatment of bacterial diseases in farming Asian seabass.

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).

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.

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.

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.