Genome Characterization and Phylogenetic Analysis of Scale Drop Disease Virus Isolated from Asian Seabass (Lates calcarifer).

Scale drop disease virus (SDDV), a double-stranded DNA virus in the family Iridoviridae, has been reported widely in southeast Asian countries as a causative agent of scale drop syndrome (SDS) in Asian seabass. SDS has resulted in high mortality and significant economic losses to the aquaculture industry. This study demonstrated the use of metagenomic methods to investigate bacterial and viral communities present in infected fish tissues and recover a complete genome of the causative agent named SDDV TH7_2019. Characterization of the TH7_2019 genome revealed a genome size of 131 kb with 134 putative ORFs encoding viral proteins potentially associated with host apoptosis manipulation. A comparative genome analysis showed a high degree of amino acid identity across SDDV strains, with variations in number of repeat sequences and mutations within core genes. Phylogenetic analyses indicate a close relationship among SDDV genomes. This research enhances our understanding of the genetic diversity and evolutionary relationship of SDDV, contributing valuable insights for further development of effective control strategies of SDDV.

Protective efficiency and immune responses to single and booster doses of formalin-inactivated scale drop disease virus (SDDV) vaccine in Asian seabass (Lates calcarifer).

BACKGROUND: Scale drop disease virus (SDDV) threatens Asian seabass (Lates calcarifer) aquaculture production by causing scale drop disease (SDD) in Asian seabass. Research on the development of SDDV vaccines is missing an in-depth examination of long-term immunity and the immune reactions it provokes. This study investigated the long-term immune protection and responses elicited by an SDDV vaccine. The research evaluated the effectiveness of a formalin-inactivated SDDV vaccine (SDDV-FIV) using both prime and prime-booster vaccination strategies in Asian seabass. Three groups were used: control (unvaccinated), single-vaccination (prime only), and booster (prime and booster). SDDV-FIV was administered via intraperitoneal route, with a booster dose given 28 days post-initial vaccination. RESULTS: The immune responses in vaccinated fish (single and booster groups) showed that SDDV-FIV triggered both SDDV-specific IgM and total IgM production. SDDV-specific IgM levels were evident until 28 days post-vaccination (dpv) in the single vaccination group, while an elevated antibody response was maintained in the booster group until 70 dpv. The expression of immune-related genes (dcst, mhc2a1, cd4, ighm, cd8, il8, ifng, and mx) in the head kidney and peripheral blood lymphocytes (PBLs) of vaccinated and challenged fish were significantly upregulated within 1-3 dpv and post-SDDV challenge. Fish were challenged with SDDV at 42 dpv (challenge 1) and 70 dpv (challenge 2). In the first challenge, the group that received booster vaccinations demonstrated notably higher survival rates than the control group (60% versus 20%, P < 0.05). However, in the second challenge, while there was an observable trend towards improved survival rates for the booster group compared to controls (42% versus 25%), these differences did not reach statistical significance (P > 0.05). These findings suggest that the SDDV-FIV vaccine effectively stimulates both humoral and cellular immune responses against SDDV. Booster vaccination enhances this response and improves survival rates up to 42 dpv. CONCLUSIONS: This research provides valuable insights into the development of efficient SDDV vaccines and aids in advancing strategies for immune modulation to enhance disease management in the aquaculture of Asian seabass.

Review of current perspectives and future outlook on bacterial disease prevention through vaccination in Asian seabass (Lates calcarifer).

Asian seabass, Lates calcarifer, is an important aquatic species in mariculture. Intensive farming of this species has faced episodes of bacterial diseases, including those due to vibriosis, scale drop, and muscle necrosis disease, big belly disease, photobacteriosis, columnaris, streptococcosis, aeromoniasis, and tenacibaculosis. Vaccination is one of the most efficient, non-antibiotic, and eco-friendly strategies for protecting fish against bacterial diseases, contributing to aquaculture expansion and ensuring food security. As of now, although numerous vaccines have undergone laboratory research, only one commercially available inactivated vaccine, suitable for both immersion and injection administration, is accessible for preventing Streptococcus iniae. Several key challenges in developing vaccines for Asian seabass must be addressed, such as the current limited understanding of immunological responses to vaccines, the costs associated with vaccine production, forms, and routes of vaccine application, and how to increase the adoption of vaccines by farmers. The future of vaccine development for the Asian seabass industry, therefore, is discussed with these key critical issues in mind. The focus is on improving our understanding of Asian seabass immunity, including maternal immunity, immunocompetence, and immune responses post-vaccination, as well as developing tools to assess vaccine effectiveness. The need for an alignment of fish vaccines with state-of-the-art vaccine technologies employed in human and terrestrial animal healthcare is also discussed. This review also discusses the necessity of providing locally-produced autogenous vaccines, especially for immersion and oral vaccines, to benefit small-scale fish farmers, and the potential benefits that might be extended through changes to current husbandry practices such as the vaccination of broodstock and earlier life stages of their off-spring.

A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan.

Immersion vaccination, albeit easier to administer than immunization by injection, sometimes has challenges with antigen uptake, resulting in sub-optimal protection. In this research, a new strategy to enhance antigen uptake of a heat-inactivated Vibrio harveyi vaccine in Asian seabass (Lates calcarifer) using oxygen nanobubble-enriched water (ONB) and positively charged chitosan (CS) was explored. Antigen uptake in fish gills was assessed, as was the antibody response and vaccine efficacy of four different combinations of vaccine with ONB and CS, and two control groups. Pre-mixing of ONB and CS before introducing the vaccine, referred to as (ONB + CS) + Vac, resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. The integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) within a vaccine trial experiment set out to further evaluate how survival rates post exposure to V. harveyi might be improved. Antibody responses were measured over 42 days, and vaccine efficacy was assessed through an experimental challenge with V. harveyi. The expression of immune-related genes IL1ß, TNFα, CD4, CD8, IgT and antibody levels were assessed at 1, 3, and 7-day(s) post challenge (dpc). The results revealed that antibody levels in the group (ONB + CS) + Vac were consistently higher than the other groups post immersion immunization and oral booster, along with elevated expression of immune-related genes after challenge with V. harveyi. Ultimately, this group demonstrated a significantly higher relative percent survival (RPS) of 63 % ± 10.5 %, showcasing the potential of the ONB-CS-Vac complex as a promising immersion vaccination strategy for enhancing antigen uptake, stimulating immunological responses, and improving survival of Asian seabass against vibriosis.

Factors Influencing Disease Dynamics in Small-Scale Carp Polyculture in Bangladesh.

Small-scale carp polyculture plays a key role in food supply in Bangladesh. However, factors including water pollution, limited infrastructure, and inadequate disease management hinder its sustainability. This paper reports on a survey of 231 farmers across the six major carp producing regions in Bangladesh, analyzing factors including farmers' social aspects, farm characteristics, information on disease and approaches adopted to combat them, and biosecurity practices. Almost half (46.8%) of the farms surveyed experienced disease in carp species, with clear regional variations. Eighty-four percent of farms reported carp mortalities during disease outbreaks, with an average mortality level of 10.23 ± 11.81%. Clinical signs during outbreaks lasted between a week and a month, with a peak in disease outbreaks occurring in two seasonal periods between June and July and October and December. Disease incidence was related to a range of factors including the farmer's experience, ponds/farm type, stocked species, and biosecurity practice. A combination of disinfecting measures during pond preparation and measures during stocking, including discarding fingerling transport water away from the farm, fingerling disinfection, and checking the health of fingerlings before stocking, significantly reduced disease occurrence. Treatments involving antibiotics, ciprofloxacin, erythromycin, and azithromycin were reported as ineffective, raising concerns about their non-prudent use, inadequate dosing (perhaps without appropriate veterinary guidance), and the potential for driving antimicrobial resistance in the environment. The research unveils a concerning pattern of high disease incidence across small-scale carp farms in Bangladesh, and the significant potential for disease spread highlights the need for responsible disposal practices. The study emphasizes the need for improving training and awareness programs for addressing biosecurity and disease management challenges, ensuring sustainable aquaculture and community well-being.

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.

Immersion prime and oral boost vaccination with an inactivated Vibrio harveyi vaccine confers a specific immune response and protection in Asian seabass (Lates calcarifer).

Asian seabass (Lates calcarifer) holds significant economic value in fish farming in the Asia-Pacific region. Vibriosis caused by Vibrio harveyi (Vh) is a severe infectious disease affecting intensive farming of this species, for which prevention strategies by vaccination have been developed. This study investigated an alternative approach to injectable vaccination to prevent vibriosis in Asian seabass juveniles. The strategy begins with an immersion prime vaccination with a heat-inactivated Vh vaccine, followed by two oral booster doses administered at 14- and 28-days post-vaccination (dpv). Expression of five immune genes TNFα, IL1ß, CD4, CD8, and IgM in the head kidney and spleen, along with investigation of anti-Vh antibody response (IgM) in both systemic and mucosal systems, was conducted on a weekly basis. The efficacy of the vaccines was assessed by a laboratory challenge test at 43 dpv. The results showed that the immunized fish displayed higher levels of mRNA transcripts of the immune genes after the immersion prime and the first oral booster dose compared to the control group. The expression levels peaked at 14 and 28 dpv and then declined to baseline at 35 and 42 dpv. Serum specific IgM antibodies were detected as early as 7 dpv (the first time point investigated) and exhibited a steady increase, reaching the first peak at 21 dpv, and a second peak at 35 dpv. Although the antibody levels gradually declined over subsequent weeks, they remained significantly higher than the control group throughout the experiment. A similar antibody response pattern was also observed in the mucosal compartment. The laboratory challenge test demonstrated high protection by injection with 1.65 × 104 CFU/fish, with a relative percent of survival (RPS) of 72.22 ± 7.86 %. In conclusion, our findings highlight the potential of an immersion prime-oral booster vaccination strategy as a promising approach for preventing vibriosis in Asian seabass.

Effects of Mangosteen (Garcinia mangostana) Peel Extract Loaded in Nanoemulsion on Growth Performance, Immune Response, and Disease Resistance of Nile Tilapia (Oreochromis niloticus) against Aeromonas veronii Infection.

Nanotechnology can enhance nutrient delivery and bioavailability; hence, it has recently been considered the most practical alternative technology for nutritional supplements and disease control in fish farming. The present study was designed to evaluate the effects of mangosteen peel extract loaded in nanoemulsion (MSNE) on the inhibition of A. veronii (in vitro) and in vivo growth performance, serum biochemical parameters, the immune response, and the disease resistance of Nile tilapia (Oreochromis niloticus) against A. veronii challenge. The particle size, polydispersity index, and particle surface charge of MSNE were 151.9 ± 1.4 nm, >0.3, and -30 mV, respectively. Furthermore, MSNE, mangosteen peel extract (MPE), and nanoemulsion (NE) improved the antimicrobial activity against A. veronii. Fish fed MSNE, MPE, and NE-supplemented diets had a significantly lower (p < 0.05) feed conversion ratio (FCR) and higher specific growth rate (SGR) than fish fed the control diet. Furthermore, the MSNE had significantly higher serum glucose and protein levels than the control group in Nile tilapia. Total immunoglobulin, serum lysozyme, alternative complement activity, and survival of Nile tilapia fed with MSNE were significantly higher (p < 0.05) than the control diet. Therefore, MSNE has the potential to be employed as a supplement in sustainable Nile tilapia farming.

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.

Oral delivery of a Streptococcus agalactiae vaccine to Nile tilapia (Oreochromis niloticus) using a novel cationic-based nanoemulsion containing bile salts.

Streptococcus agalactiae is one of Thailand's most important pathogens in tilapia aquaculture. Vaccination is a very effective method for protecting fish against disease in aquaculture. Oral vaccination is an interesting route for vaccine delivery as it mimics the pathogenesis of S. agalactiae and provides convenient administration for mass vaccination of fish. Moreover, gut mucosal immunity is associated with a mucus layer on the gastrointestinal tract. Therefore, this study aimed to develop a novel cationic-based nanoemulsion vaccine containing bile salts (NEB) coated by chitosan (CS) and determined its physicochemical characterization, morphology, in vitro mucoadhesive property, permeability, and acid-base tolerance. In addition, the efficacy of NEB-CS as an oral vaccination for Nile tilapia was evaluated in order to investigate the innate immune response and protection against S. agalactiae. The groups of fish consisted of: (1) deionized water as a non-vaccinated control (Control); (2) an inactivated vaccine formulated from formalin-killed bacteria (IB); and (3) a novel cationic-based nanoemulsion vaccine containing bile salts (NEB) coated by chitosan (CS). The control, IB, and NEB-CS were incorporated into commercial feed pellets and fed to Nile tilapia. In addition, we evaluated the serum bactericidal activity (SBA) for 14 days post-vaccination (dpv) and protective efficacy for 10 days post-challenge, respectively. The mucoadhesiveness, permeability, and absorption within the tilapia intestine were also assessed in vivo. The NEB-CS vaccine appeared spherical, with the nanoparticles having a size of 454.37 nm and a positive charge (+47.6 mV). The NEB-CS vaccine had higher levels of mucoadhesiveness and permeability than the NEB (p < 0.05). The relative percent survival (RPS) of IB and NEB-CS, when administered orally to fish, was 48% and 96%, respectively. Enhanced SBA was noted in the NEB-CS and IB vaccine groups compared to the control group. The results demonstrate that a feed-based NEB-CS can improve the mucoadhesiveness, permeability, and protective efficacy of the vaccine, and appear to be a promising approach to protecting tilapia in aquaculture against streptococcosis.

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.

Pathological effects due to metacercariae of Clinostomum piscidium migration in snakeskin gourami (Trichopodus pectoralis) in Thailand.

Clinostomum spp. is a fish-borne pathogen and a digenetic trematode with a global range. Despite its zoonotic relevance, the pathogenic impact of the parasite in Thai aquaculture is currently unclear. The present study deals with the pathogenic changes that fluke causes in their host, Trichopodus pectoralis, and the molecular confirmation of the Clinostomum piscidium by targeting 18 s rDNA and ITS gene. The metacercariae of C. piscidium were discovered in the body cavity of infected fish. The gross pathological examination revealed a few white migratory tracks on the surface of the liver and spleen. The migratory track showed histologically as a primary hemorrhage and necrosis of hepatic cells surrounded by a layer of macrophages and epithelioid cells, inflammatory cells, and eosinophilic granular cells in the cytoplasm of liver cells and close to the epithelial cells of the intestine. Also, the migratory track in the spleen appeared as a marked decrease of Red Blood Cell (RBC) count and changes in the necrotic tissue. Infection with this metacercaria produced hepatic tissue injury, which disrupted hepatic metabolism and decreased body weight in the fish hosts. The findings of the study suggest that the pathological effect of C. piscidium on farm T. pectoralis can cause significant economic loss by stunting fish development and predisposing fish to opportunistic pathogens in the environment. Hence, the treatment and control of C. piscidium infections are crucial for the viability of the aquaculture sector since this parasite has been found to cause pathological damage to the vital organs of fish.

Risk factors of Bartonella spp. infection and the association between Bartonella spp. and T-lymphocyte subset alteration in asymptomatic retrovirus-infected cats in Bangkok Metropolitan, Thailand.

Background and Aim: Cats are a reservoir for Bartonella spp. infection in humans. Human bartonellosis causes disseminated inflammation to develop in immunocompromised patients, such as those infected with human immunodeficiency virus. However, the associated risks of Bartonella spp. infection in immunocompromised retroviral-infected cats have been inconclusive. This study aimed to evaluate the associated risks of Bartonella spp. infection with the alteration of T-lymphocyte subsets of retroviral-infected cats. Materials and Methods: We collected blood samples from 161 client-owned cats at veterinary clinics and hospitals throughout the Bangkok Metropolitan area from 2017 to 2020. The samples underwent hematological biochemical tests, feline retroviral status evaluation, Bartonella spp. polymerase chain reaction assay, immunofluorescence assay, and CD4+ and CD8+ lymphocyte counts. Risk factors associated with Bartonella spp. infection were determined by odds ratio (OR). Hematological and biochemical parameters were compared using independent t-tests. CD4+ and CD8+ lymphocyte counts and the CD4+/CD8+ ratio were compared among groups classified according to their retroviral and Bartonella spp. infection status. Results: The prevalence of Bartonella spp. in our study cohort was 16.1%, and the seroprevalence was 94.9%. Cats aged >1 year were at a higher risk of seropositivity than cats aged <1 year (OR: 4.296, 95% confidence interval: 1.010-18.275). The CD8+ percentage was significantly higher in seropositive cats (p = 0.026). There was a significant reduction in the CD4+/CD8+ ratio between cats negative for both retrovirus and Bartonella spp. infection and cats with concurrent retrovirus and Bartonella spp. infection (p = 0.041). Conclusion: In endemic countries or areas, cat owners must be made aware of the risk of exposure to Bartonella spp. due to the high rate of bacteremia and seroprevalence. Retrovirus-infected cats with concurrent Bartonella spp. infection also showed a significant, inverted CD4+/CD8+ ratio, which may be used as a novel marker in bartonellosis. Similar studies focusing on the different stages of retrovirus infection should be undertaken further to elucidate the effect of retrovirus infection on Bartonella spp. infection.

Effects of Jerusalem Artichoke (Helianthus tuberosus) as a Prebiotic Supplement in the Diet of Red Tilapia (Oreochromis spp.).

The aim of the present study was to evaluate the effects of a Jerusalem artichoke-supplemented diet on the blood chemistry, growth performance, intestinal morphology, expression of antioxidant-related genes, and disease resistance against Aeromonas veronii challenge in juvenile red tilapia. A completely randomized design (CRD) was followed to feed red tilapias with three experimental diets: control, 5.0 g/kg JA-supplemented (JA5), or 10.0 g/kg JA-supplemented (JA10) diets in triplicates for 4 weeks. The results revealed that the growth performance, weight gain (WG), specific growth rate (SGR), and average daily gain (ADG) of fish fed diets JA5 and JA10 were significantly higher (p < 0.05) than those of fish fed the control diet. Fish fed the control diet had significantly higher T-bilirubin, D-bilirubin, and ALT in blood serum than fish fed JA5 and JA10, as well as higher BUN than fish fed JA5. The number of goblet cells in the proximal and distal parts of the intestine revealed that the number of acid, neutral, and double-staining mucous cells of fish fed diets JA5 and JA10 was significantly higher (p < 0.05) than in fish fed the control diet. The diets including the prebiotic (JA5 and JA10) were associated with a significant increase in the expression of gpx1 and gst antioxidant-related genes and disease resistance against A. veronii in juvenile red tilapia. Therefore, JA5 and JA10 can be employed as promising prebiotics for sustainable red tilapia farming.

Chitosan-polymer based nanovaccine as promising immersion vaccine against Aeromonas veronii challenge in red tilapia (Oreochromis sp.).

Red tilapia (Oreochromis sp.), one of the important freshwater fish species in fish farming in Thailand, has for long been suffering from a serious bacterial disease named epizootic ulcerative syndrome and hemorrhagic septicemia. The disease is mainly caused by Aeromonas veronii. Vaccine is proposed to be a major impact tool for sustainable control and prevention strategies. Vaccination by immersion has many benefits over injection. However, the conventional immersion method suffers from a low potency due to the inefficient uptake of antigens across mucosal tissue. Here, we developed a chitosan-polymer based nanovaccine together with an efficient delivery vehicle to enhance the immunogenicity of immersion vaccination, increasing bioavailability and inducing local immune responses during transit to mucosal inductive immune sites. The physiochemical properties of nanovaccine, which was modified on surface particle by using a mucoadhesive polymer, were assessed for size, zeta potential, and particle distribution. Our study demonstrated by SEM image and microscopic fluorescence image that nanovaccine greatly increased the binding and penetrating ability into gills when compared with formalin killed vaccine. The nano-sized particles were well dispersed in water and trapped in core nanoparticle as confirmed by TEM image. The efficacy of vaccine was performed by immersion challenge with virulent A.veronii after 30 days post vaccination in tilapia. The result revealed a high level of mortality in the control, empty-polymeric nanovaccine and formalin killed bacterin vaccine groups. A high relative percentage survival (RPS) of vaccinated fish was noted with chitosan-polymer based nanovaccine. Our studies indicated that this chitosan-polymer based nanovaccine derived from cell fragments and supernatant was the improved version of the conventional formalin killed vaccine. The chitosan polymer based particle could increase the efficacy of nanovaccine toward the target mucosal membrane and enhance protection against A. veronii infection in red tilapia.

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.

Distribution of Vibrionaceae in farmed Asian sea bass, Lates calcarifer in Thailand and their high prevalence of antimicrobial resistance.

This study describes the etiological agent of Vibriosis along with its distribution and antimicrobial resistance profiles among farmed Asian sea bass (Lates calcarifer) in Thailand. The study isolated 283 Vibrionaceae from 15 Asian sea bass farms located around the provinces of the Andaman Sea and Gulf of Thailand coasts to uncover the distribution and antimicrobial resistance profiles. Bacterial identification based on a combination of the biochemical characteristics, Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, and the species-specific PCR demonstrated the predominant Vibrionaceae were Vibrio harveyi (n = 56), Photobacterium damselae (n = 35), and V. vulnificus (n = 31), respectively. According to a laboratory challenge experiment, among the six isolates, only V. harveyi was found to cause clinical signs of muscle necrosis and scale loss in Asian sea bass. Antibiotics resistance test results exhibited high resistance to antibiotics such as metronidazole (100%), streptomycin (97%), clindamycin (96%), colistin sulphate (70%) and amoxicillin (59%). Remarkably, 100% of Vibrionaceae isolates are susceptible to florfenicol. The 28 of 29 resistance profiles were multidrug resistances (MDR), with V. vulnificus having the highest MAR value (0.66). The findings of this study advise that a surveillance program, as well as preventive and control measures, be developed for Vibrionaceae to reduce production loss, pathogen proliferation, and antibiotic abuse, whereas AMR data indicate substantial health problems for aquatic animals and humans.

Expression of Antimicrobial Peptide Genes in the Canine Amniotic Membrane.

The human amniotic membrane has been successfully used in human ocular reconstruction. Several studies have demonstrated its properties, including antimicrobial features. As a result of the restricted availability of human amniotic membrane for veterinary use, canine amniotic membrane has become an attractive alternative. Clinical studies of the application of canine amniotic membrane in animals and the understanding of its biological properties are limited. This study aimed to determine the expression of peptide genes of natural antimicrobials in canine amniotic membrane. Expressions of canine ß-defensin 1, 102, and 103, and canine Elafin were determined in healthy puppies by real-time quantitative polymerase chain reaction. Canine ß-defensin 1, 103, and Elafin were expressed in all samples, possibly suggesting a role in the innate immune system of normal canine amniotic membrane. Further investigations of protein expression and localization are recommended.

Diversity and antimicrobial susceptibility profiles of Aeromonas spp. isolated from diseased freshwater fishes in Thailand.

Motile Aeromonas septicemia (MAS), a disease caused by Aeromonas spp., is recognized as a major disease in freshwater aquaculture. This study aimed to investigate the distribution and diversity of Aeromonas spp. and their antimicrobial susceptibility patterns. A total of 86 isolates of Aeromonas spp. were recovered from diseased freshwater fishes from 13 farms in Thailand. All isolates were identified using biochemical characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), polymerase chain reaction assays, and the gyrB gene sequence analysis. The result of MALDI-TOF MS showed 100% (86 isolates) accuracy at genus-level identification, and 88.4% (76 isolates) accuracy at species-level identification. Six species of Aeromonas were confirmed through nucleotide sequencing and phylogenetic analysis of the gyrB gene Aeromonas veronii (72.1%), Aeromonas jandaei (11.6%), Aeromonas schubertii (9.3%), Aeromonas diversa (3.5%), Aeromonas hydrophila (2.3%), and Aeromonas punctata (1.2%). Antimicrobial susceptibility tests for all isolates revealed resistance against amoxicillin (99%), ampicillin (98%), oxolinic acid (81.4%), oxytetracycline (77%), trimethoprim-sulfamethoxazole (24%), and enrofloxacin (21%). The multiple antibiotic resistance (MAR) index varied between 0.14 and 0.86, with MAR values more than 0.2 in 99% of isolates. Furthermore, four diverse multidrug-resistant (MDR) patterns were found among Aeromonas isolates. Our finding show that A. veronii is the most abundant species in Thai cultured freshwater fish with the highest MDR patterns.