RESUMO
The Asian seabass, Lates calcarifer, is a key species in Asian aquaculture due to its nutritional value and adaptability. However, disease outbreaks, particularly viral and bacterial infections, pose significant challenges to its production. Immunostimulants offer promising solutions but raise safety concerns. Paraprobiotics and postbiotics (CPP) emerge as safer alternatives, exerting health benefits without live microorganisms. This study investigated the potential of probiotic paraprobiotic and postbiotic supplements derived from Bacillus subtilis to enhance the immune response and antioxidant capacity of Asian seabass and improve their resistance to Streptococcus iniae infection. Analysis of antioxidant activity and lipid peroxidation revealed significant improvements in fish supplemented with CPP, indicating their effectiveness in mitigating oxidative stress. Immunological assays demonstrated enhanced growth performance and serum immunity, including increased alternative complement activity, immunoglobulin levels, and phagocytic activity, in supplemented fish. Furthermore, upregulated expression of proinflammatory cytokines (TNF-α, IL-6, IL-1ß) and pattern recognition receptors (NLRC3, TLR22, MDA5) in immune tissues. Fish supplemented with CPP exhibited higher resistance and survival rates against S. iniae infection challenge compared to control groups. The study elucidates the mechanisms underlying the immunomodulatory effects of CPP, shedding light on their potential applications in aquaculture.
Assuntos
Ração Animal , Bacillus subtilis , Dieta , Doenças dos Peixes , Imunidade Inata , Probióticos , Infecções Estreptocócicas , Streptococcus iniae , Animais , Doenças dos Peixes/imunologia , Probióticos/farmacologia , Probióticos/administração & dosagem , Infecções Estreptocócicas/veterinária , Infecções Estreptocócicas/imunologia , Ração Animal/análise , Imunidade Inata/efeitos dos fármacos , Bacillus subtilis/química , Dieta/veterinária , Streptococcus iniae/fisiologia , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Receptores de Reconhecimento de Padrão/genética , Suplementos Nutricionais/análise , Transdução de Sinais , Perciformes/imunologia , Bass/imunologiaRESUMO
IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Dourada , Infecções Estreptocócicas , Streptococcus iniae , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Infecções Estreptocócicas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Dourada/genética , Dourada/imunologia , Dourada/microbiologia , Streptococcus iniae/fisiologia , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Regulação da Expressão Gênica , Imunidade Inata/genéticaRESUMO
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.
Assuntos
Doenças dos Peixes , Vibrioses , Vibrio , Animais , Streptococcus iniae , Vacinas de Produtos Inativados , Vibrioses/prevenção & controle , Vibrioses/veterinária , Vacinas Bacterianas , Doenças dos Peixes/microbiologiaRESUMO
Streptococcus iniae is a fish pathogen that can also infect mammals including dolphins and humans. Its prevalence in farmed fish, particularly tilapia, provides potential for zoonotic infections, as documented by multiple case reports. Systematic clinical data beyond cellulitis for S. iniae infection in humans, including antimicrobial susceptibility data, are unfortunately rare. Here, we present a case of cellulitis progressing to bacteremia caused by Streptococcus iniae in a functionally immunocompromised patient based on CDK4/CDK6 inhibitor and endocrine therapy, and we discuss risk factors, identification, and antimicrobial susceptibility of this rare pathogen.
Assuntos
Anti-Infecciosos , Bacteriemia , Infecções Estreptocócicas , Animais , Humanos , Bacteriemia/diagnóstico , Bacteriemia/tratamento farmacológico , Celulite (Flegmão)/diagnóstico , Celulite (Flegmão)/tratamento farmacológico , Peixes , Mamíferos , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/epidemiologia , Streptococcus , Streptococcus iniae , Zoonoses/diagnósticoRESUMO
Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.
Assuntos
Vacinas Bacterianas , Doenças dos Peixes , Nanotubos de Carbono , Infecções Estreptocócicas , Tilápia , Animais , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Imersão , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae , Streptococcus iniaeRESUMO
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.
Assuntos
Doenças dos Peixes , Perciformes , Infecções Estreptocócicas , Animais , Streptococcus iniae , Flavobacterium , Vacinas Combinadas , Streptococcus , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterinária , Vacinas BacterianasRESUMO
Streptococcus iniae, a zoonotic Gram-positive pathogen, poses a threat to finfish aquaculture, causing streptococcosis with an annual economic impact exceeding $150 million globally. As aquaculture trends shift towards recirculating systems, the potential for horizontal transmission of S. iniae among fish intensifies. Current vaccine development provides only short-term protection, driving the widespread use of antibiotics like florfenicol. However, this practice raises environmental concerns and potentially contributes to antibiotic resistance. Thus, alternative strategies are urgently needed. Endolysin therapy, derived from bacteriophages, employs hydrolytic endolysin enzymes that target bacterial peptidoglycan cell walls. This study assesses three synthetic endolysins (PlyGBS 90-1, PlyGBS 90-8, and ClyX-2) alongside the antibiotic carbenicillin in treating S. iniae-infected hybrid striped bass (HSB). Results demonstrate that ClyX-2 exhibits remarkable bacteriolytic potency, with lytic activity detected at concentrations as low as â¼15 µg/mL, approximately 8-fold more potent than the PlyGBS derivatives. In therapeutic effectiveness assessments, both carbenicillin and ClyX-2 treatments achieved significantly higher survival rates (85 % and 95 %, respectively) compared to placebo and PlyGBS-based endolysin treatments. Importantly, no statistical differences were observed between ClyX-2 and carbenicillin treatments. This highlights ClyX-2 as a promising alternative for combating S. iniae infections in aquaculture, offering potent bacteriolytic activity and high survival rates.
Assuntos
Bass , Endopeptidases , Doenças dos Peixes , Infecções Estreptocócicas , Animais , Bass/microbiologia , Streptococcus , Streptococcus iniae , Antibacterianos , CarbenicilinaRESUMO
BACKGROUND: Streptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24. RESULTS: The results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24. CONCLUSION: Comparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.
Assuntos
Infecções Estreptocócicas , Streptococcus iniae , Animais , Streptococcus iniae/genética , Virulência/genética , Streptococcus/genética , Infecções Estreptocócicas/veterinária , Antibacterianos/farmacologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Farmacorresistência Bacteriana/genética , Peixes/genética , GenômicaRESUMO
Vibriosis and Streptococcosis are the most important bacterial diseases that infect Asian seabass (Lates calcarifer) in various stages of its life cycle. Vaccination is a cost-effective strategy to prevent the occurrence of infectious diseases and increase sustainability in the aquaculture industry. This study was aimed to develop and evaluate a killed polyvalent vaccine against Vibrio harveyi, V. alginolyticus and Streptococcus iniae, delivered by intraperitoneal injection in Asian seabass. The fish were divided into three groups with 60 fish in triplicate: I) a control group injected with phosphate-buffered saline (PBS), II) a group vaccinated by polyvalent vaccine (V. alginolyticus + V. harveyi + S. iniae) and III) a group vaccinated with the same polyvalent vaccine plus an oral booster. Immunological parameters and antibody titer were measured before and at three, five-, and eight-weeks post-vaccination. The efficacy of the killed vaccine was assessed five weeks post-vaccination by challenging with each isolate separately. The vaccinated groups had higher survival rate than control group. The highest relative percentage survival rate, 85.71 ± 3.57 % was observed in group III when challenged with V. harveyi. The vaccinated fish produced significantly higher antibody titers against V. alginolyticus, V. harveyi and S. iniae than the control group (P < 0.05). Non-specific immune parameters were significantly enhanced in the vaccinated groups, especially group III, compared to the control. The results demonstrated that the administration of a killed polyvalent vaccine can effectively protect Asian seabass against V. alginolyticus, V. harveyi and S. iniae.
Assuntos
Doenças dos Peixes , Perciformes , Infecções Estreptocócicas , Vibrioses , Animais , Streptococcus iniae , Vibrioses/prevenção & controle , Vibrioses/veterinária , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterinária , Vacinas Combinadas , Vacinas BacterianasRESUMO
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.
Assuntos
Doenças dos Peixes , Infecções Estreptocócicas , Tilápia , Animais , Streptococcus iniae , Doenças dos Peixes/microbiologia , Streptococcus agalactiae/genética , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/veterinária , Infecções Estreptocócicas/microbiologia , Tilápia/microbiologiaRESUMO
Streptococcal disease has severely restricted the development of global tilapia industry, which is mainly caused by Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae). Vaccination has been proved to be a potential strategy to control it. In this study, a multi-epitope subunit vaccine Sip-Srr (SS) was prepared based on the B-cell antigenic epitopes prediction and multiple sequence alignment analysis of Sip and Srr sequences. Furthermore, the BNC-rSS nanocarrier vaccine system was constructed by connecting the rSS protein with modified bacterial nanocellulose (BNCs) and characterized by Fourier Transform Infrared Spectroscopy and Scanning Electron Microscope, the immersion immune effect against S. agalactiae and S. iniae infection was evaluated. The results showed that compared with the control group, BNC-rSS significantly enhanced serum antibody production, related enzyme activities and immune-related genes expression. It was noteworthy that BNC-rSS vaccine improved immune protection of tilapia, with survival rates of 66.67 % (S. agalactiae) and 60.00 % (S. iniae), respectively, compared with those of rSS vaccine (30 % and 33.33 %, respectively). Our study indicated that the BNC-rSS nanovaccine could elicit robust immune responses in tilapia by immersion immunization, and had the potential to offer cross-protection against S. agalactiae and S. iniae infection in tilapia.
Assuntos
Ciclídeos , Doenças dos Peixes , Infecções Estreptocócicas , Tilápia , Animais , Streptococcus agalactiae , Streptococcus iniae , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterinária , Vacinas BacterianasRESUMO
Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1ß, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.
Assuntos
Ciclídeos , Doenças dos Peixes , Probióticos , Infecções Estreptocócicas , Animais , Streptococcus iniae , Fator de Necrose Tumoral alfa , Interleucina-6 , Doenças dos Peixes/prevenção & controle , Suplementos Nutricionais , Dieta/veterinária , Ração Animal/análise , Resistência à Doença , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterináriaRESUMO
Interferon regulatory factor 7 (IRF7) regulates type I interferon (IFN) genes via combining to the ISRE region in the immune response against bacteria. Streptococcus iniae is one of the dominant pathogenic bacteria of yellowfin seabream, Acanthopagrus latus. However, the regulatory mechanisms of A. latus IRF7 (AlIRF7) mediated by the type I IFN signalling pathway against S. iniae was ambiguously. In the present study, IRF7, and two IFNa3s (IFNa3 and IFNa3-like) were authenticated from A. latus. The total length of AlIRF7 cDNA is 2142 bp, containing a 1314 bp open reading frame (ORF) encoding an inferred 437 amino acids (aa). Three typical regions, a serine-rich domain (SRD), a DNA-binding domain (DBD), and an IRF association domain (IAD), are conserved in AlIRF7. Furthermore, AlIRF7 is fundamentally expressed in various kinds of organs, with high levels in the spleen and liver. Additionally, S. iniae challenge promoted AlIRF7 expression in the spleen, liver, kidney, and brain. AlIRF7 is confirmed to be located at the nucleus and cytoplasm by overexpression of AlIRF7. Moreover, truncation mutation analyses shows that the regions, -821 bp to +192 bp and -928 bp to +196 bp, were known as core promoters from AlIFNa3 and AlIFNa3-like, respectively. The point mutation analyses and electrophoretic mobile shift assay (EMSA) verified that AlIFNa3 and AlIFNa3-like transcriptions are depended on the M2/5 and M2/3/4 binding sites with AlIRF7 regulation, respectively. Additionally, an overexpression experiment showed that AlIRF7 can dramatically decrease the mRNA levels of two AlIFNa3s and interferon signalling molecules. These results suggest that two IFNa3s may mediate the regulation of AlIRF7 in the immune responses of A. latus against S. iniae infection.
Assuntos
Interferon Tipo I , Perciformes , Dourada , Animais , Fator Regulador 7 de Interferon/genética , Dourada/genética , Regulação da Expressão Gênica , Streptococcus iniae/genética , Proteínas de Peixes/química , Sequência de Bases , Sequência de Aminoácidos , Perciformes/genética , Interferon Tipo I/genéticaRESUMO
Early disease prevention by vaccination requires understanding when fry fish develop specific immunity to a given pathogen. In this research, we explored the immune responses of Asian seabass (Lates calcarifer) at the stages of 35- and 42- days post-hatching (dph) to an immersive heat-killed Streptococcus iniae (Si) vaccine to determine whether fish can produce specific antibodies against the pathogen. The vaccinated fish of each stage (V35 and V42) were immersed with the Si vaccine at 107 CFU/ml for 3 h, whereas the control groups (C35 and C42) were immersed with tryptic soy broth (TSB) in the same manner. Specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA) before and post-immunization (i.e., 0, 7, and 14 days post-immunization, dpi). Expression of innate (TNFα and IL-1ß) and adaptive (MHCI, MHCII, CD4, CD8, IgM-like, IgT-like, and IgD-like) immune-related genes were evaluated at the same time points with the addition of 1 dpi. The results showed that a subset of immunized fish from both V35 and V42 fry could elicit specific antibodies (IgM) against Si at 14 dpi. All tested innate and adaptive immune genes upregulated at 7 dpi among fish in V35 group. Interestingly, 42 dph fish appeared to respond to the Si vaccine faster than that of 35 dph, as a significant increase in transcripts was observed in CD4, IL-1ß, IgM-like, and IgD-like at 1 dpi; and specific antibody titers of some fish, although not all, were higher than a threshold (p = 0.05) since 7 dpi. In conclusion, this study reveals that 35-42 dph Asian seabass fry can elicit specific immunity to Si immersion vaccine, suggesting that early vaccination of 35 dph fry Asian seabass is feasible.
Assuntos
Doenças dos Peixes , Perciformes , Animais , Streptococcus iniae , Vacinas de Produtos Inativados , Temperatura Alta , Imersão , Imunização , Vacinação/veterinária , Vacinas Bacterianas , Imunoglobulina M , Doenças dos Peixes/prevenção & controleRESUMO
Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. Emerging evidence suggests the immune regulatory roles of CSTC in antigen presentation, the release of different inflammatory mediators, and apoptosis in various pathophysiologies. In this study, the 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse (Hippocampus abdominalis) was cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in ovaries. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 14.29-kDa protein of recombinant HaCSTC (rHaCSTC) was expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, HaCSTC overexpression strongly decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability. Our findings imply the profound role of HaCSTC against pathogen infections by modulating fish immune responses.
Assuntos
Smegmamorpha , Animais , Cistatina C/genética , Papaína/genética , Streptococcus iniae/fisiologia , Poli I-C/farmacologia , Proteínas de Peixes/química , FilogeniaRESUMO
Infectious diseases caused by Streptococcus iniae lead to massive death of fish, compose a serious threat to the global aquaculture industry, and constitute a risk to humans who deal with raw fish. In order to realize the early diagnosis of S. iniae, and control the outbreak and spread of disease, it is of great significance to establish fast, sensitive, and convenient detection methods for S. iniae. In the present study, two methods of real-time MIRA (multienzyme isothermal rapid amplification, MIRA) and MIRA-LFD (combining MIRA with lateral flow dipsticks (LFD)) for the simA gene of S. iniae were established, which could complete amplification at a constant temperature of 42 °C within 20 min. Real-time MIRA and MIRA-LFD assays showed high sensitivity (97 fg/µL or 7.6 × 102 CFU/mL), which were consistent with the sensitivity of real-time PCR and 10 times higher than that of PCR with strong specificity, repeatability simplicity, and rapidity for S. iniae originating from Trachinotus ovatus. In summary, real-time MIRA and MIRA-LFD provide effective ways for early diagnosis of S. iniae in aquaculture, especially for units in poor conditions.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Streptococcus iniae , Animais , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Peixes , Reação em Cadeia da Polimerase em Tempo Real , Aquicultura , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Aeromonas , Doenças dos Peixes , Oxitetraciclina , Pericardite , Animais , Aeromonas veronii/genética , Streptococcus iniae/genética , Doxiciclina , Enrofloxacina , RNA Ribossômico 16S/genética , Doenças dos Peixes/microbiologia , Peixes/genética , Amoxicilina , Eritromicina , Sulfametoxazol , Trimetoprima , Tailândia , Aeromonas/genéticaRESUMO
Streptococcus iniae is a Gram-positive bacterium and is considered a harmful aquaculture pathogen worldwide. In this study, S. iniae strains were isolated from East Asian fourfinger threadfin fish (Eleutheronema tetradactylum) reared on a farm in Taiwan. A transcriptome analysis of the head kidney and spleen was performed in the fourfinger threadfin fish 1 day after infection using the Illumina HiSeq™ 4000 platform for RNA-seq to demonstrate the host immune mechanism against S. iniae. A total of 7333 genes based on the KEGG database were obtained after the de novo assembly of transcripts and functional annotations. Differentially expressed genes (DEGs) (2-fold difference) were calculated by comparing the S. iniae infection and phosphate-buffered saline control group gene expression levels in each tissue sample. We identified 1584 and 1981 differentially expressed genes in the head kidney and spleen, respectively. Based on Venn diagrams, 769 DEGs were commonly identified in both the head kidney and spleen, and 815 and 1212 DEGs were specific to the head kidney and spleen, respectively. The head-kidney-specific DEGs were enriched in ribosome biogenesis. The spleen-specific and common DEGs were found to be significantly enriched in immune-related pathways such as phagosome, Th1, and Th2 cell differentiation; complement and coagulation cascades; hematopoietic cell lineage; antigen processing and presentation; and cytokine-cytokine receptor interactions, based on the KEGG database. These pathways contribute to immune responses against S. iniae infection. Inflammatory cytokines (IL-1ß, IL-6, IL-11, IL-12, IL-35, and TNF) and chemokines (CXCL8 and CXCL13) were upregulated in the head kidney and spleen. Neutrophil-related genes, including phagosomes, were upregulated post-infection in the spleen. Our results could offer a strategy for the treatment and prevention of S. iniae infection in fourfinger threadfin fish.
Assuntos
Doenças dos Peixes , Infecções Estreptocócicas , Animais , Peixes , Rim Cefálico , Baço , Streptococcus iniaeRESUMO
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.
Assuntos
Anguilla , Doenças dos Peixes , Animais , Streptococcus iniae , Letargia/veterinária , Europa (Continente)/epidemiologia , Itália/epidemiologiaRESUMO
Streptococcus iniae is a worldwide fish pathogen that cause tremendous economic losses to the global aquaculture industry. Vaccination is regarded as the most effective and safe way to control fish diseases. In our study, we developed a formalin-inactivated vaccine against S. iniae and evaluated its effect in golden pompano (Trachinotus ovatus). In addition, in order to clarify the molecular mechanisms underlying the vaccine protection, we compared the spleen transcriptomes of vaccinated and unvaccinated golden pompano at 1, 2 and 7 d post vaccination using the RNA-seq technology. The relative percentage survival (RPS) reached 71.1% at 28 days post-vaccination which suggested that the vaccine provided highly protection against S. iniae. KEGG pathway analysis revealed that phagosome, cytokine-cytokine receptor interaction, MAPK signaling pathway, and CAMs were activated by the vaccine. The most of strongly up-regulated genes in golden pompano spleen are involving in innate immunity. For adaptive immunity, the vaccine evoked a CD8+ CTL-mediated response by MHC â pathway to achieve immune protection.