Sea Bass Kidney Research Articles

In Vitro Validation of Camphene as a Potential Antiviral Agent Against Betanodavirus Causing Viral Nervous Necrosis in Barramundi

Viral infections are a major threat to the aquaculture industry throughout the world. Betanodavirus is one of the most infectious viruses that cause the highest mortality in larval and juvenile stages of Latescalcarifer commonly known as Barramundi. It is a single-stranded positive- sense RNA virus and causes viral nervous necrosis(VNN).VNN is caused by a RNA virus that gets transmitted both horizontally and vertically so the most effective method against this virus is to vaccinate the fish, however, vaccination becomes difficult since the disease is associated with outbreaks in larval and juvenile stages which are not that much immunocompetent. In our previous in silico studies, we proved the stability of camphene as a better phytochemical agent. In continuation to prove the authenticity of camphene as a potential antiviral agent against betanodavirus, its in vitro validation was performed. Sea bass kidney cell line (SISK) was selected for carrying out the in vitro studies and cytotoxicity studies of camphene in the SISK were done by MTT assay. Based on the analysis of the MTT assay, different dosages of camphene were selected viz.,0.2, 0.5, 1, 1.5, 2, 2.5, 5, 10, 20, 30 μg/ml. The SISK cells were infected with a virus inoculum(200μl). Further, the antiviral activity of camphene on infected SISK cells by Betanodaviruswas elucidated with the help of quantitative Real-time PCR(qPCR) on the 3rd and 5th day of infection. Analysis of results depicted that the dose of camphene 2 to 10 μg/ml is the safest dose against Betanodavirus. Hence this is aptly revealed that camphene can be used as a potential antiviral agent against Betanodavirus.

Analysis of Immune Gene Expression in Seabass (Lates calcarifer) Immunized with Inactivated Vaccine against Similar Damselfish Virus

Background: Control of viral disease outbreaks in aquaculture and minimizing the loss of production can be achieved by development of effective vaccines. Efficacy of these vaccines can be improved by using adjuvants, immunostimulants or vaccine carriers. In this study, inactivated similar damselfish virus (SRDV) vaccine was prepared and expression profiles of immune related genes against virus challenge of the vaccine were investigated in seabass (Lates calcarifer). Methods: Formalin-inactivated virus vaccine was developed to assess its immune responses to SRDV challenge in fish. The immune response was induced by intra-peritoneal injection with inactivated viral vaccine added Quil-A® adjuvant. The transcriptional levels of immune genes IRF-7 and IL-10 were evaluated in the spleen and kidney of seabass from different groups by quantitative real-time RT-PCR assays. Result: Expression profiles of both genes (IRF-7 and IL-10) in the kidney and spleen of seabass immunized with vaccine added adjuvant were up-regulated at 48 hpi of the virus. In comparison, spleen of seabass immunized with vaccine added adjuvant showed highest expression profiles than kidney. The current study provides evidence for the presence of expression profiles of immune-related genes during the SRDV infection. The study also strongly suggests that Quil-A® adjuvant enhances the immune response of the vaccine candidates.

In vitro screening of selected antiviral drugs against betanodavirus

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 μM, 10 μM, 100 μM, 200 μM and 500 μM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 μM, 10 μM and 100 μM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 μM and 100 μM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.

Identification of Nod like receptor C3 (NLRC3) in Asian seabass, Lates calcarifer: Characterisation, ontogeny and expression analysis after experimental infection and ligand stimulation.

Nod like receptors (NLRs) are a large group of cytoplasmic PRRs believed to play an important role in bacterial recognition in higher vertebrates. In this study, a novel Nod like receptor C3 (AsNLRC3) has been identified, cloned and characterised from Asian seabass, Lates calcarifer. The full-length AsNLRC3 transcript composed of a 4142bp nucleic acid sequence encode for a protein of 1134 deduced amino acids. Three signature domains identified are conserved NACHT-domain, C-terminal LLR domain and N-terminal CARD effector domain. From the domain architecture and phylogenetic analysis, it was quite evident that AsNLRC3 is different from the NLR subfamily C of other teleosts. AsNLRC3 expressed in all the 11 tissues tested but highly expressed in tissues facing external environment such as gill, hindgut and midgut. The ontogenic expression profile of this receptor showed constitutive expression throughout the embryonic and larval developmental stages, which could be an innate immune strategy against different marine pathogens for larval survival. Infection with Vibrio alginolyticus and poly I:C induction showed an alteration of expression pattern in different tissues but did not show significant alteration in expression with Staphylococcus aureus infection. Invitro study in Asian seabass kidney cell line (SISK) stimulated with different ligands such as LPS, PGN and poly I:C showed considerable up-regulation at some of the time-points tested. These results suggest that AsNLRC3 can be a pivotal cytosolic innate immune receptor for recognizing wide array of pathogens in a euryhaline teleost model like Asian seabass in diverse environmental conditions.

A Novel Virus Causes Scale Drop Disease in Lates calcarifer.

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Delivery of DNA vaccine using chitosan–tripolyphosphate (CS/TPP) nanoparticles in Asian sea bass, Lates calcarifer (Bloch, 1790) for protection against nodavirus infection

Abstract The present study examines the efficacy of DNA vaccine against nodavirus through oral route using CS/TPP (chitosan–tripolyphosphate) nanoparticles encapsulation. The RNA2 capsid protein gene of nodavirus was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pFNCPE. The size of the RNA2 capsid protein gene was 42 kDa and it was named as pFNCPE42. The CS/TPP nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expressions of FNCP (fish nodavirus capsid protein) gene were observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of CS/TPP–pFNCPE42 DNA nanoparticles was evaluated by MTT assay using fish cell line. Distribution of DNA vaccines in different tissues was studied in fish fed with the pFNCPE42-DNA encapsulated in CS/TPP nanoparticles and the expression of the gene was confirmed by PCR, RT-PCR, immunohistochemistry and ELISA analysis. The results indicate that DNA vaccine can be easily delivered into fish by feeding with CS/TPP nanoparticles. After oral vaccination Asian sea bass were challenged with nodavirus by intramuscular injection. A relative percent survival (RPS) rate of 60.0% was recorded. This study suggested that CS/TPP nanoparticles were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by oral approach.

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV)

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6–90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass ( Lates calcarifer) to protect from Vibrio ( Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass ( Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

Protective efficiency of DNA vaccination in Asian seabass ( Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass ( Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 μg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924 bp cDNA of sea bass caspase-9 was obtained, consisting of 1308 bp open reading frame coding for 435 amino acids, 199 bp of the 5′-UTR and 417 bp of the 3′-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp 121, Asp 325 and Asp 343. The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida ( Phdp) strain PP3, showed increased expression from 0 to 12 h returning to control levels at 24 h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48 h post-infection, when the fish were with advanced septicaemia.

Establishment and characterization of India's first marine fish cell line (SISK) from the kidney of sea bass (Lates calcarifer)

A continuous cell line (SISK) from kidney of sea bass, Lates calcarifer, has been established and characterized. The cell line was maintained in Leibovitz' L-15 supplemented with 15% fetal bovine serum. This cell line has been subcultured more than 100 times over a period of 2 years. The SISK cell line consists of predominantly of epithelial-like cells. These cells showed strong positive for epithelial markers such as cytokeratin 19 and pancytokeratin. The cells were able to grow at temperature between 25 and 32 °C with optimum temperature of 28 °C. The growth rate of sea bass kidney cells increased as the FBS proportion increased from 2% to 20% at 28 °C with optimum growth at the concentrations of 15% or 20% FBS. The distribution of chromosome number was 30 to 56 with a modal peak at 48 chromosomes. Polymerase chain reaction products were obtained from SISK cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. Five fish viruses were tested on this cell line to determine its susceptibility to these viruses and this was found to be susceptible to MABV NC1 and nodavirus, and the infection was confirmed by RT-PCR and CPE. This suggests that the SISK cell line has good potential for the isolation of various fish viruses. This cell line has been shown to be susceptible to bacterial extracellular products. The SISK cell line is the India's first marine fish cell line.

Effect of Clofibrate, a Peroxisome Proliferator, in Sea Bass (Dicentrarchus labrax), a Marine Fish

The effects of 35 or 70 mg/kg clofibrate on some peroxisomal, mitochondrial, and microsomal enzymes (markers of peroxisome proliferation) in liver, gill, and kidney of sea bass following 2 weeks i.p. treatment have been studied. Induction of lauric acid hydroxylase, UDP-glucuronyl transferase, palmitoyl-CoA oxidase, carnitine-palmitoyl-transferase,p-nitrophenylacetate hydrolase, and benzaldehyde and propionaldehyde dehydrogenase activities was not observed in any case. The clofibrate administration at the dose of 70 mg/kg induced in the liver the glutathioneS-tranferase and reduced epoxide hydrolase activities. These results demonstrate that a marine fish species, such as sea bass, is rafractary to peroxisome proliferation.

Cloning of CYP4F7, a Kidney-Specific P450 in the Sea BassDicentrarchus labrax

A cDNA sequence coding for a cytochrome P450 of the CYP4F subfamily was isolated from total RNA of sea bass kidney by rapid amplification of cDNA ends. The full length sequence coded for a protein of 526 amino acids. The amino acid sequence shared 39% to 56% residue identities with the mammalian CYP4F sequences, and thus was named CYP4F7 (accession number AF123541). RNA blot analysis using CYP4F7 cDNA as a probe indicated that the corresponding mRNA was only detected in kidney. Expression in the kidney was constitutive, and no induction of this mRNA was detected in this or other tissues, with any of the inducers tested, including peroxisome proliferators.

(Na + + K +)- and Na +-stimulated Mg 2+-dependent ATPase activities in kidney of sea bass ( Dicentrarchus labrax L.)

1. 1. Sea bass kidney microsomal preparations contain two Mg 2+ dependent ATPase activities: the ouabain-sensitive (Na + + K +)-ATPase and an ouabain-insensitive Na +-ATPase, requiring different assay conditions. The (Na + + K +)-ATPase under the optimal conditions of pH 7.0, 100 mM Na +, 25 mM K +, 10 mM Mg 2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 μmol P i/mg protein per hr whereas the Na +-ATPase under the conditions of pH 6.0, 40 mM Na +, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 μmol P i/mg protein per hr. 2. 2. The (Na + + K +)-ATPase is specifically inhibited by ouabain and vanadate; the Na +-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca 2+. 3. 3. The (Na + + K +)-ATPase is specific for ATP and Na +, whereas the Na +-ATPase hydrolyzes other substrates in the efficiency order ATP > GTP > CTP > UTP and can be activated also by K +, NH 4 + or Li +. 4. 4. Minor differences between the two activities lie in the affinity for Na 1, Mg 2+, ATP and in the thermosensitivity. 5. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na + transport and, on the other, a distinct modulation in vitro in different tissues.

Distribution of GSH and GSH cycle enzymes in black sea bass ( Centropristis striata)

1. 1. Tissue extracts were prepared from black sea bass kidney, liver, brain, red blood cells and muscle; each of the extracts was analyzed for the following glutathione (GSH)-dependent enzyme activities: Se-dependent and Se-independent GSH-peroxidases, GSH reductase, γ-glutamyl cysteine synthetase, GSH-S-transferase and γ-glutamyl transpeptidase. 2. 2. Each of the black sea bass tissues possessed an active complement of GSH-metabolizing enzymes in a relative distribution similar to that reported for other species including mammals. 3. 3. Tissue GSH concentrations were of the same magnitude as those reported for mammalian tissues.