Duration of Protection and Humoral Immune Response in Nile Tilapia (Oreochromis niloticus L.) Vaccinated against Streptococcus agalactiae.

Streptococcosis caused by Streptococcus agalactiae (S. agalactiae) is a major bacterial disease affecting the production of Nile tilapia (Oreochromis niloticus L.), causing significant economic losses due to mortality in the growing phase. Vaccination is the most effective method for preventing streptococcosis on Nile tilapia farms. In Brazil, the major tilapia-producing regions have long production cycles (6-10 months) and harvest tilapias weighing over 900 g for fillet production. Thus, data on the duration of the humoral immune response and protection in farmed tilapia have not been reported or are poorly described. Furthermore, the efficiency of serological testing for the long-term monitoring of immune responses induced by vaccination against S. agalactiae has never been addressed. This study evaluated the duration of protection and humoral immune response induced in Nile tilapia vaccinated against S. agalactiae until 300 days post-vaccination (dpv). The immunization trial was composed of two groups: vaccinated (Vac), vaccinated intraperitoneally with a commercial vaccine, and unvaccinated (NonVac) group, injected fish with sterile saline solution. At 15, 30, 150, 180, 210, and 300 dpv, blood sampling was conducted to detect anti-S. agalactiae IgM antibodies using indirect Enzyme-Linked Immunosorbent Assay (ELISA), and the fish were challenged with pathogenic S. agalactiae to determine the duration of vaccine protection through relative percentage survival (RPS). Spearman's rank correlation was performed between the ELISA optical density (OD) of vaccinated tilapia and the duration of vaccine protection (RPS). The mean cumulative mortality in NonVac and Vac groups ranged from 65 to 90% and less than 35%, respectively. The average RPS was 71, 93, 94, 70, 86, and 67% at 15, 30, 150, 180, 210, and 300 dpv, respectively. RPS revealed that the vaccine provided protection from 15 to 300 dpv. The specific anti-S. agalactiae IgM antibody levels were significantly higher in the Vac group than that non-Vac group up to 180 dpv. The vaccinated fish exhibited significant protection for up to 10 months after vaccination. There was a positive correlation between the antibody response and RPS. This study revealed that a single dose of commercial vaccine administered to Nile tilapia can confer long-term protection against S. agalactiae and that indirect ELISA can monitor the duration of the humoral immune response for up to six months following vaccination. Finally, vaccine protection over six months can be associated with other components of the fish immune system beyond the humoral immune response by IgM antibodies.

Characterization of Lactococcus garvieae and Streptococcus agalactiae in cultured red tilapia Oreochromis sp. in Thailand.

OBJECTIVE: Acute mortality with clinical symptoms of streptococcal-like infections was observed in red tilapia Oreochromis sp. cultured in floating cages in Prachin Buri Province, Thailand, during May 2023. Herein, we identified an emerging pathogen, Lactococcus garvieae, as the etiological agent. METHODS: After bacterial isolation from the brain and kidney of diseased fish, identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK 2 system. Sequencing of the 16S ribosomal RNA (rRNA) gene and phylogenetic analysis were applied to confirm bacterial species. Antimicrobial susceptibility testing was conducted. Histopathological findings in the brain, kidney, spleen, liver, and heart were evaluated. RESULT: From 20 fish samples, L. garvieae (n = 18 isolates) and Streptococcus agalactiae (n = 2 isolates) were identified. A phylogenetic tree of the 16S rRNA gene revealed that Thai isolates of either L. garvieae or S. agalactiae clustered with reference piscine isolates from intercontinental locations. Our isolates showed resistance against quinolones while being susceptible to other antimicrobials. Histopathological changes demonstrated severe septicemic conditions, with more invasive lesions-especially in the heart and liver-being apparent in L. garvieae-infected fish compared to S. agalactiae-infected fish. CONCLUSION: This study represents the first reported outbreak of L. garvieae with a concurrent S. agalactiae infection in farmed red tilapia in Thailand.

Immuno-protective response of Asian seabass (Lates calcarifer) to inactivated vaccines against Streptococcus iniae and Vibrio harveyi.

BACKGROUND: In this study, the protective immunity and immunogenicity of the monovalent and bivalent Streptococcus iniae and Vibrio harveyi vaccine were evaluated in Asian seabass. To analyze immune responses, 1200 Asian seabass with an average weight of 132.6 ± 25.4 g were divided into eight treatments in triplicates (50 fish per tank) as follows: S. iniae immunized by injection (SI), V. harveyi immunized by injection (VI), bivalent S. iniae and V. harveyi (SVI) immunized by injection, S. iniae immunized by immersion (SIM), V. harveyi (VIM) immunized by immersion, bivalent S. iniae and V. harvei (SVIM) immunized by immersion, phosphate-buffered saline (PBS) by injection (PBSI) and control group without vaccine administration (CTRL). Blood and serum samples were taken at the end of the 30th and 60th days. Then the vaccinated groups were challenged with two bacteria (S. iniae) and (V. harveyi) separately and mortality was recorded for 14 days. RESULTS: This study reveals that there is no significant difference in the hematological parameters on the 30th and 60th days of the experiment in the vaccine-immunized groups compared to the CTRL group (P > 0.05). Meanwhile, there was no significant difference in the amount of serum albumin level, respiratory burst activity, and serum bactericidal activity in the vaccine-immunized groups compared to the CTRL group on the 30th and 60th days of the experiment (P > 0.05). Total protein on the 60th day (in the VI and SVI groups), globulin on the 30th day (in the VI and SVI groups) and the 60th day (in the VI group) compared to the CTRL and PBSI groups had a significant increase (P < 0.05). Complement activity (in the VI and SVI groups) and lysozyme (in the SI and SVI groups) increased significantly compared to the control group (P < 0.05). Serum antibody titer against S. iniae had a significant increase in the SI, VI, SVI and SVIM groups compared to the CTRL and PBSI groups (P < 0.05). Serum antibody titer against V. harveyi had a significant increase in the groups immunized with the vaccine compared to the CTRL and PBSI groups (P < 0.05). A significant increase in the relative percentage survival (RPS) following challenge with S. iniae in the SVI (86.6%), SI (83.3%,) and VI (73.3%) groups were observed compared to the CTRL (43.3%) and PBSI (40%) groups (P < 0.05). Also, a significant increase in the RPS after challenge with V. harveyi in the SVI group, VI 86.6%, SVI 83.3%, VIM 80% and SVIM 76.6% were observed compared to the CTRL (46.6%) and PBSI (50%) groups (P < 0.05). CONCLUSION: Overall, the results demonstrated that the bivalent vaccine of S. iniae and V. harveywas able to produce significant immunogenicity and RPS in Asian seabass.

Exosomes derived from olive flounders infected with Streptococcus parauberis: Proteomic analysis, immunomodulation, and disease resistance capacity.

Multidrug-resistant Streptococcus parauberis causes high fish mortality in aquaculture, necessitating an urgent need for innovative control strategies. This study aimed to develop an immunizing agent against S. parauberis using exosomes isolated from the plasma of olive flounders infected experimentally with S. parauberis (Sp-Exo). Initially, we tested the in vitro immunomodulatory effect of Sp-Exo in murine macrophage RAW264.7 cells and compared it to that of exosomes isolated from naïve fish (PBS-Exo-treated). Notably, Sp-Exo treatment significantly (p < 0.05) upregulated pro-and anti-inflammatory cytokines (Il1ß, Tnfα, and Il10), antimicrobial peptide, defensin isoforms (Def-rs2 and Def-ps1), and antiviral (Ifnß1 and Isg15) genes. In vivo studies in larval and adult zebrafish revealed similar patterns of immunomodulation. Furthermore, larval and adult zebrafish exhibited significantly (p < 0.05) enhanced resistance to S. parauberis infection following treatment with Sp-Exo compared to that with PBS-Exo. Proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) approach revealed the presence of 77 upregulated and 94 downregulated differentially expressed proteins (DEPs) in Sp-Exo, with 22 and 37 significantly (p < 0.05) upregulated and downregulated DEPs, respectively. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins analyses revealed that these genes are associated with key pathways, such as innate immune responses, complement system, acute phase responses, phospholipid efflux, and chylomicron remodeling. In conclusion, Sp-Exo demonstrated superior immunomodulatory activity and significant resistance against S. parauberis infection relative to that on treatment with PBS-Exo. Proteomic analysis further verified that most DEPs in Sp-Exo were associated with immune induction or modulation. These findings highlight the potential of Sp-Exo as a promising vaccine candidate against S. parauberis and other bacterial infections in olive flounder.

Susceptibility Profile and Epidemiological Cut-Off Values Are Influenced by Serotype in Fish Pathogenic Streptococcus agalactiae.

Streptococcus agalactiae is a major health concern in tilapia farming worldwide. In contrast to the availability of susceptibility profile results, interpretative criteria for disk diffusion assays and the influence of serotypes on resistance profiles are not available. To address this, sixty isolates (thirty of each serotype, Ib and III) were evaluated using the disk diffusion assay against six antibiotics, and the epidemiological cut-off value (ECV) was calculated. All the isolates were classified as non-wild type (NWT) for sulfamethoxazole (SUT) and norfloxacin (NOR). The inhibition zones for oxytetracycline (OXY) and doxycycline (DOX) were largely distinct; all serotype Ib and III isolates were classified as wild-type (WT) and NWT, respectively. The results for serotype III of fish group B Streptococcus (GBS) were comparable to the NWT tetracycline profile of human GBS available in EUCAST, suggesting the presence of resistance mechanisms in these fish isolates. The calculation of the cut-off wild type (COWT) values for OXY and DOX was appropriate for both serotypes. Differences between the distribution of florfenicol (FLO) and amoxicillin (AMO) were found, and we attribute this to the faster growth rate of serotype III, which promotes smaller inhibition zones. Therefore, using separate COWT for each serotype is necessary. In conclusion, the serotype of fish GBS affects its susceptibility profile, and it is recommended to use serotype-specific COWT values as interpretative criteria for disk diffusion assays against FLO and AMO.

Main Causes of Death in Piglets from Different Brazilian Nursery Farms Based on Clinical, Microbiological, and Pathological Aspects.

Necropsies can reveal herd problems or comorbidities that can lead to management corrections, improvements in animal performance, and better decision making. Furthermore, the pattern and causes of mortality might differ when different systems are evaluated. The present study was conducted to establish the main causes of death in nursery pigs from different systems in Brazil, as well as the clinical, microbiological, and pathological aspects of these mortalities. Eighteen nurseries were analyzed (a total of 120,243 housed piglets), and 557 necropsies were performed. Streptococcus suis infection was the most prevalent cause of death (21.2%), followed by bacterial polyserositis (16.7%), chronic atrophic enteritis (13.5%), salmonellosis (8.8%), pneumonia (8.6%), and colibacillosis (6.1%). The increase in mortality rate in individual nurseries and, consequently, in the diagnoses was commonly associated with disease outbreaks. Infectious diseases constituted the largest portion of the diagnoses, making a great opportunity for improving production rates in herds. Moreover, the extensive range of observed diagnoses highlights the importance of conducting preliminary diagnostic investigations based on necropsy to determine the causes of death. This approach allows for the direction of complementary tests, which can diagnose agents with greater specificity. As a result, this allows for the implementation of more effective prevention and control strategies.

Improving the diagnosis of Streptococcus iniae using a novel probe-based qPCR assay combined with an enrichment step.

Streptococcus iniae is a bacterial pathogen that causes streptococcosis, leading to significant losses in fish aquaculture globally. This study reported a newly developed probe-based quantitative polymerase chain reaction (qPCR) method for the detection of S. iniae. The primers and probes were designed to target the lactate oxidase gene. The optimized method demonstrated a detection limit of 20 copies per reaction and was specific to S. iniae, as evidenced by no cross-reactivity when assayed against genetic materials extracted from 23 known aquatic animal pathogens, and fish samples infected with Streptococcus agalactiae or Streptococcus dysgalactiae. To validate the newly developed qPCR protocol with field samples, fish specimens were systematically investigated following the Food and Agriculture Organization of the United Nations & Network of Aquaculture Centres in Asia-Pacific three diagnostic levels approach, which integrated basic and advanced techniques for disease diagnosis, including observation of gross signs (level I), bacterial isolation (level II), qPCR and 16S rDNA sequencing (level III). The result showed that 7/7 affected farms (three Asian seabass farms and four tilapia farms) experiencing clinical signs of streptococcosis were diagnosed positive for S. iniae. qPCR assays using DNA extracted directly from fish tissue detected S. iniae in 11 out of 36 fish samples (30.6%), while 24 out of 36 samples (66.7%) tested positive after an enrichment step, including apparently healthy fish from affected farms. Bacterial isolation of S. iniae was only successful in a proportion of clinically diseased fish but not in healthy-looking fish from the same farm. Overall, the newly developed qPCR protocol combined with enrichment would be a useful tool for the diagnosis and surveillance of S. iniae infections in fish populations, thereby aiding in the disease control and prevention.

Vaccination with bacterial ghosts of Streptococcus iniae and Lactococcus garvieae originated from outbreak of marine fish streptococcosis, induce potential protection against the disease in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758).

Streptococcosis is an important bacterial disease affects fresh, brackish and marine fish. The disease caused annual severe economic losses in Egyptian Mari-culture. S. iniae and L. garvieae usually the main causative agents isolated. The presented study conducted to prepare bacterial ghost vaccine (BGV) candidates from isolated strains of marine streptococcosis outbreaks using NaOH chemical approach. Selected strains confirmed as pathogenic for Nile tilapia, therefore the fish selected as an experimental model. In such respect, the re-isolated S. iniae and L. garvieae were used for ghost preparations, BGVs evaluation and fish challenges. Apart of four, three fish groups namely, A, B, C designated for BGVs evaluations, while the fourth one (D) designated as control. Vaccination experiments performed via intra-peritoneal injection with 0.1 mL (1.5 × 108 CFU/mL/fish) of their corresponding BGVs twice with 2 weeks' interval; however, control fish received 0.1 mL of fish saline instead. Blood, serum, and tissue samples collected from all groups at 2 and 4 weeks post immunization (PI) for estimation of hematological, innate, and specific immune parameters. At the end, all remained fish challenged with appropriated pathogen (s) and the relative percentage of survival (RPS) calculated. Three BGVs generated namely, SiG, in addition to, novel contributions of LgG and SiLgG. Ghosts were corresponding to S. iniae, L. garvieae and their both ghost mixtures, respectively. Fish groups immunized with prepared BGVs revealed variable significant increases in PCV, GLB, PP, SOD, CAT, C5, IL-ß1, LZM, specific antibody titers and CD4 expression 2 and 4 weeks PI. MDA decreased in all vaccinated groups that was significantly with group C. Expression of MHC-II showed elevations 2 weeks PI, however, it significantly decreased at 4 weeks. The RPS recorded 90, 88.89 and 95.46% in immunized groups A, B and C, respectively. At all levels tested, obtained results proposed SiG, LgG and SiLgG as innovative vaccine candidates, which can protect cultured fish from being attacked by S. iniae, and/or L. garvieae.

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.

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

Streptococcosis and motile Aeromonad septicemia (MAS) are the main bacterial diseases in tilapia culture worldwide, causing significant economic losses. Vaccination is an effective method of preventing diseases and contributes to economic sustainability. This study investigated the immuno-protective efficacy of a newly developed feed-based bivalent vaccine against streptococcosis and MAS in red hybrid tilapia. The feed-based bivalent vaccine pellet was developed by incorporating the formalin-killed S. agalactiae and A. hydrophila antigens into a commercial feed pellet with palm oil as the adjuvant. The bivalent vaccine was subjected to feed quality analyses. For immunological analyses, 900 fish (12.94 ± 0.46 g) were divided into two treatment groups in triplicate. Fish in Group 1 were unvaccinated (control), while those in Group 2 were vaccinated with the bivalent vaccine. The bivalent vaccine was delivered orally at 5% of the fish's body weight for three consecutive days on week 0, followed by boosters on weeks 2 and 6. Lysozyme and enzyme-linked immunosorbent assays (ELISAs) on serum, gut lavage, and skin mucus were performed every week for 16 weeks. Lysozyme activity in vaccinated fish was significantly (p ≤ 0.05) higher than in unvaccinated fish following vaccination. Similarly, the IgM antibody levels of vaccinated fish were significantly (p ≤ 0.05) higher after vaccination. The bivalent vaccine provided high protective efficacy against S. agalactiae (80.00 ± 10.00%) and A. hydrophila (90.00 ± 10.00%) and partial cross-protective efficacy against S. iniae (63.33 ± 5.77%) and A. veronii (60.00 ± 10.00%). During the challenge test, fewer clinical and gross lesions were observed in vaccinated fish compared with unvaccinated fish. Histopathological assessment showed less severe pathological changes in selected organs than the unvaccinated fish. This study showed that vaccination with a feed-based bivalent vaccine improves immunological responses in red hybrid tilapia, and thus protects against streptococcosis and MAS.

Concurrent infections of Streptococcus iniae and Aeromonas veronii in farmed Giant snakehead (Channa micropeltes).

The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram-positive cocci and cream-coloured, round and convex colonies of rod-shaped gram-negative bacteria. Additional biochemical and species-specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole-trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole-trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies.

Immune Activation Following Vaccination of Streptococcus iniae Bacterin in Asian Seabass (Lates calcarifer, Bloch 1790).

Juvenile Asian seabass (Lates calcarifer) (body weight 10 ± 0.7 g) were intraperitoneally injected with 1012 CFU fish-1 of formalin-killed Streptococcus iniae. The protective efficacy of the vaccine on survival and infection rate was assessed upon challenge at 4, 8, 12, 20, and 28 weeks post-vaccination. The results revealed that the challenged vaccinated fish showed no mortality at all time points, and the control fish presented 10-43.33% mortality. The infection rate at 2 weeks post-challenge was 0-13.33% in the vaccinated fish and 30-82.35% in the control group. At 8 weeks post-vaccination, the vaccinated fish showed comparable ELISA antibody levels with the control; however, the antibody levels of the vaccinated fish increased significantly after the challenge (p < 0.05), suggesting the presence of an adaptive response. Innate immune genes, including MHC I, MHC II, IL-1ß, IL-4/13B, and IL-10, were significantly upregulated at 12 h post-challenge in the vaccinated fish but not in the control. In summary, vaccination with S. iniae bacterin provided substantial protection by stimulating the innate and specific immune responses of Asian seabass against S. iniae infection.

Efficacy of polyvalent vaccine on immune response and disease resistance against streptococcosis/lactococcosis and yersiniosis in rainbow trout (Oncorhynchus mykiss).

Diseases are the most significant challenge in the development and stability of aquaculture. In this study, the immunogenic efficiency of polyvalent streptococcosis/lactococcosis and yersiniosis vaccines was evaluated by injection and immersion methods in rainbow trout.. The 450 fish with an average weight of 50 ± 5 g were divided into three treatments and three replications as follows: injection vaccine treatment, immersion vaccine treatment and control group without vaccine administration. Fish were kept for 74 days and sampling was done on days 20, 40 and 60. Then, from the 60th to the 74th day, the immunized groups were challenged with three bacteria Streptococcus iniae (S. iniae), Lactococcus garvieae (L. garvieae) and Yersinia ruckeri (Y. ruckeri) separately. A significant difference was observed in the weight gained (WG) in the immunized groups compared to the control group (P < 0.05). The relative survival percentage (RPS) after 14 days of challenge with S. iniae, L. garvieae and Y. ruckeri in the injection group compared to the control group increased respectively (60%, 60% and 70%), (P < 0.05). Also, RPS in the immersion group had an increase respectively (30%, 40% and 50%) after the challenge with S. iniae, L.garvieae and Y. ruckeri compared to the control group. Immune indicators such as antibody titer, complement and lysozyme activity significantly increased in comparison to the control group (P < 0.05). In general, it can be concluded that applying three vaccines by injection and immersion method has significant effects on immune protection and survival rate. However, the injection method is more effective and more suitable than the immersion method.

Mortality in farmed European eel (Anguilla anguilla) in Italy due to Streptococcus iniae.

BACKGROUND: Streptococcal infections are one of the main causes of fish disease. During the last decade, Streptococcus iniae has become one of the most important aquatic pathogens worldwide, causing high losses in marine and freshwater finfish. Clinical signs in farmed fish include loss of appetite, lethargy and grouping at the bottom of the tank. Gross changes comprise darkening of the skin and haemorrhage at the basis of fins and opercula. To date, S. iniae has been isolated from several wild and farmed fish species but never in the European eel (Anguilla anguilla). In Europe, eel production from aquaculture is around 4500 tonnes and Italy is the third largest producer. This communication represents the first report of an outbreak of S. iniae infection in European eels. CASE PRESENTATION: The outbreak occurred at an eel farm in northern Italy between May 2021 and September 2021. The outbreak caused about 2% mortality per month, resulting in the loss of about 10% of the farmed fish. The diseased eels showed apathy, lethargy, inactivity and inappetence. In July 2021, three eels were necropsied. Necropsy revealed skin and branchial hyperaemia, a few skin ulcers, and diffuse peritoneal congestion with a few haemorrhagic-like spot lesions. Swab samples for bacteriology were taken from the kidneys, liver, spleen, and brain. Additionally, four eels were opened and swap samples as above were taken. All the investigated eels were found dead. Bacteriological examination revealed growth of Streptococcus spp. from all samples. Identification of S. iniae was done by biochemical characterization, the API20STREP microsystem, 16S rDNA sequencing, and MALDI-TOF. Antimicrobial therapy (oxytetracycline and erythromycin) was ineffective. CONCLUSIONS: This is the first report of S. iniae infection in the European eel. Although this may be an isolated outbreak, it is of concern due to the losses associated with this pathogen in fish worldwide and because the European eel is an endangered species. Due to the difficulties of controlling the disease with antimicrobials, it is advisable to plan other effective control measures, such as improving water quality and the environmental conditions, reducing fish density, improving biosecurity, and by using immunostimulants and, when possible, vaccines.

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.

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).

Suppression of Streptococcosis and Modulation of the Gut Bacteriome in Nile Tilapia (Oreochromis niloticus) by the Marine Sediment Bacteria Bacillus haynesii and Advenella mimigardefordensis.

Streptococcosis is one of the major threats to Nile tilapia (Oreochromis niloticus) in most regions of the world. Recently, Enterococcus faecalis has been widely reported to be involved in streptococcosis in O. niloticus in Asia and Africa. This study aimed to isolate beneficial marine bacteria to evaluate their effects on growth, hematological parameters, nonspecific immunity, the gut bacteriome, and streptococcosis prevention efficacy in O. niloticus. A total of 36 marine soil bacteria were isolated, and in vitro screening was conducted to determine their antibacterial activities against fish pathogens. Two antagonistic bacteria were identified based on 16S rRNA gene sequencing, Bacillus haynesii CD223 and Advenella mimigardefordensis SM421. These bacteria were incorporated into fish feed and fed to O. niloticus for 90 days. The application of these strains via incorporation into fish feed significantly promoted growth, improved hematological parameters and immunoglobulin M (IgM) levels, modulated the gut bacteriome by reducing the load of pathogenic Enterococcus spp., and developed disease prevention efficacy in O. niloticus. Furthermore, in vivo assays revealed that the inclusion of extracellular products (ECPs) (at 250 µg mL-1) of CD223 and SM421 with feed significantly enhanced the rate of survival (100%) of O. niloticus from streptococcosis compared to the controls (only 30%). The ECPs of these bacteria also prevented 90 to 100% of fish from developing streptococcosis. These strains could be promising for safe use in O. niloticus farming to prevent and control the emergence of streptococcosis caused by E. faecalis. IMPORTANCE Nile tilapia (Oreochromis niloticus) is one of the most economically important cultured fish species throughout the world. Streptococcosis is a significant threat to global Nile tilapia farming. Enterococcus faecalis has recently emerged as an important pathogen of streptococcosis in Asia and Africa. The application of antibiotics and probiotics and vaccination are the major ways to combat streptococcosis. However, the extensive use of antibiotics leads to the development of antibiotic resistance in pathogenic as well as environmental bacteria, which is a great threat to public health. There is no study on preventing streptococcosis caused by E. faecalis using beneficial bacteria. For the first time, the present study demonstrated that two marine bacteria, Bacillus haynesii strain CD223 and Advenella mimigardefordensis strain SM421, have great potential for controlling streptococcosis in Nile tilapia. These bacteria also enhanced the growth, improved hematological parameters and IgM levels, and positively modulated the gut bacteriome of Nile tilapia.

Efficacy of a recombinant M-like protein, SimA as a subunit vaccine candidate against Streptococcus parauberis infection in olive flounder, Paralichthys olivaceus.

Streptococcus parauberis, a gram-positive cocci, causes bacterial disease in farmed fish. The recent increase in S. parauberis infection in aquatic farms in South Korea has justified the importance of vaccine development for the prevention of this disease. In this study, we evaluated the effect of subunit vaccines prepared from recombinant M-like protein (SimA) and fibrinogen-binding protein (FBP) candidates with an aluminum hydroxide adjuvant against S. parauberis infection in olive flounder Paralichthys olivaceus. For the in vivo experiment, fish (average length, 7.18 cm; average weight, 3.5 g) were injected intraperitoneally with: phosphate buffer saline (PBS, group 1), PBS/aluminum hydroxide (group 2), FBP/aluminum hydroxide (group 3), SimA/aluminum hydroxide (group 4), and SimA/FBP/aluminum hydroxide (group 5). After 3 weeks, the fish in each group were boosted using PBS (group 1 and 2), FBP (group 3), SimA (group 4), and SimA/FBP (group 5) without adjuvant. We found that the relative percent survival of fish after S. parauberis exposure in group 2, 3, 4, and 5 was 6.25%, 18.75%, 50%, and 12.5%, respectively, whereas the mortality in groups 1 was 80%, respectively. We performed Western blot, ELISA, and quantitative real time RT-PCR (qRT-PCR) after vaccination to investigate the further efficacy of the vaccine. Western blot and ELISA of vaccinated fish serum confirmed the production of specific antibodies against SimA and FBP. Furthermore, results of qRT-PCR showed that recombinant protein SimA induced a remarkably specific-antibody response compared with that in FBP or control and increased the expression of various immune response-related genes including interleukin-8 (IL-8), toll-like receptor 2 (TLR2), tumor necrosis factor-α (TNF-α), CD4-1, and MHC II. Thus, these results indicate that SimA is a potent vaccine candidate for protection against S. parauberis infection.

Trial Evaluation of Protection and Immunogenicity of Piscine Bivalent Streptococcal Vaccine: From the Lab to the Farms.

Streptococcosis is one of the major diseases that causes devastation to farmed fish, leading to significant economic losses all around the world. Currently, two serotypes of Streptococcus agalactiae, serotype Ia and III, have been identified as virulent strains and major causative agents of the disease in farmed Nile tilapia (Oreochromis niloticus Linn.) in Thailand. Upon inactivated vaccine development, monovalent inactivated whole-cell vaccines demonstrated high specific antibody production against homologous serotypes and limited production with heterologous serotypes. However, for higher efficacy, a bivalent streptococcal vaccine was designed to maximize protective immunity to both serotypes. Interestingly, our bivalent vaccine could successfully induce specific antibody production against both serotypes with similar levels, and the response could extend over the 8 weeks of the experimental period. Evaluation of vaccines in the laboratory scale revealed relative percent survival (RPS) of vaccinated tilapia to serotype Ia (81.2 ± 9.4%) and serotype III (72.2 ± 4.8%), respectively. The efficacy of the bivalent vaccine showed significant RPS higher than the monovalent vaccine (p < 0.05) at 30 days, and the protection of all those vaccines was reduced thereafter. Evaluation of the vaccine in a farm trial in different locations in Thailand revealed the efficacy of the bivalent vaccine in increasing the production yield by greater than 80% in all tested farms in 2015 and 2021. Taken together, this study affirms the efficacy of the bivalent streptococcal vaccine in the prevention of streptococcus disease in Nile tilapia, which could be used in different areas. This vaccine development could be effectively applied in the tilapia culture industry.

Streptococcosis a Re-Emerging Disease in Aquaculture: Significance and Phytotherapy.

Streptococcosis, particularly that caused by S. iniae and S. agalactiae, is a major re-emerging bacterial disease seriously affecting the global sustainability of aquaculture development. Despite a wide spread of the disease in aquaculture, few studies have been directed at assessing the in vitro antagonistic activity and in vivo efficacy of medicinal herbs and other plants against streptococcal agents. Most in vitro studies of plant extractives against S. iniae and S. agalactiae have found antibacterial activity, but essential oils, especially those containing eugenol, carvacrol or thymol, are more effective. Although essential oils have shown better anti-streptococcal activity in in vitro assays, in vivo bioassays require more attention. The extracts examined under in vivo conditions show moderate efficacy, increasing the survival rate of infected fish, probably through the enhancement of immunity before challenge bioassays. The available data, however, lack dosage optimization, toxicity and bioavailability assays of a specific plant or its bioactive compound in fish organs; hence, it is difficult to judge the validation of clinical efficacy for the prevention or treatment of fish streptococcosis. Despite the known bioactive compounds of many tested plants, few data are available on their mode of action towards streptococcal agents. This review addresses the efficacy of medicinal plants to fish streptococcosis and discusses the current gaps.