Department of Aquaculture biotechnology

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.

A cell line (SICH) was established from heart muscle of Indian major carp (Catla catla), a freshwater fish
cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 15% fetal bovine
serum. These cells have been sub-cultured more than 130 times over a period of two years. The SICH cell line
consists predominantly of fibroblastic-like cells. The cells were able to grow at a wide range of temperatures
from 24 °C to 37 °C with an optimum temperature of 28 °C. The growth rate of heart cells increased as the FBS
proportion increased from 2 to 20% at 28 °C with optimum growth at the concentrations of 10 or 15% FBS. PCR
amplification of mitochondrial 12s rRNA using primers specific to C. catla confirmed the origin of this cell line
from catla. The cells were successfully cryopreserved and revived at passage numbers 20, 35, 70 and 120. The
cell cycle analysis by FACS (Fluorescence Activated Cell Sorter) revealed that most of the cells on the second
day of culture were in S-phase, indicating a high growth rate. When the SICH cells were transfected with
pEGFP using lipofectamine 2000 or chitosan nanoparticle, fluorescent signals were observed, suggesting that
the SICH cell line could be a useful tool for transgenic and genetic manipulation studies. The bacterial
extracellular products from Aeromonas sp., or Vibrio anguillarum were found to be toxic to this cell line. This
cell line was further characterized by immunocytochemistry using confocal laser scanning microscopy. In
addition, an attempt was also made to use this cell line for genotoxicity studies.

Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (Mr NV) and extra small virus (XSV). The insects were screened as potential natural carriers of Mr NV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for Mr NV and XSV in Belostoma sp., Aesohna sp., Cybister sp. and Notonecta sp., and negative results for Nepa sp. An Aedes albopictus mosquito cell line (C6/36) was used for infectivity assays, with viral inoculum prepared from the aquatic insects, since C6/36 cells have recently been shown to be susceptible to infection with Mr NV and XSV. The C6/36 cells were harvested 4 d postchallenge for examination by electron microscopy. This revealed aggregation of viral particles throughout the cytoplasm for cells challenged with inocula from all the insect species except Nepa sp. Our results indicate that several aquatic insect species may present a risk for Mr NV and XSV transmission to M. rosenbergii.

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita, and the brain of catla, Catla catla, respectively. The cell lines were maintained in LeibovitzÕs L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32°C with an optimum of 28°C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28°C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp. were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.

Viral nervous necrosis (VNN) has emerged to become a major problem in the culture of larval and juvenile marine fish worldwide. Bioactive phytochemicals isolated from commonly available medicinal plants are often screened for their efficacy in controlling fish viral diseases. Occurrence of newer viral strains and resistance to existing antiviral drugs are problems currently associated with treatment of VNN, which necessitates looking for alternate sources for effective antiviral drugs. The aim of the present study was to screen antiviral potential of gymnemagenol (C 30 H 50 O 4 ) previously extracted from leaves of Gymnema sylvestre. The fish nodavirus, grouper nervous necrosis virus (GNNV) in infected Sahul Indian Grouper Eye (SIGE) cell lines were used to study the antiviral activity of gymnemagenol under in vitro conditions. The susceptibility of the virus to gymnemagenol was confirmed by measuring the viral titre (TCID 50 mL −1 ) in virus-infected SIGE cells every 24 h. Gymnemagenol at 20 µg mL −1 inhibited the proliferation of GNNV to 53% at the end of the 6th d by inhibiting the proliferation of GNNV-infected SIGE cells. The viable SIGE cells were reduced to 47% as determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The viral titre (TCID 50 mL −1 ) was also reduced to log 2.8 at the end of the 7th d in gymnemagenol-treated SIGE cells after inoculated with GNNV when compared to untreated control SIGE cell viral titre (log 4.1). Based on our results it can be concluded that gymnemagenol could be used as an antiviral agent against GNNV infection.

The protective efficacy of oral delivery of a DNA construct containing the VP28 gene of WSSV encapsulated in chitosan nanoparticles was investigated in black tiger shrimp (Penaeus monodon). The results showed that significant survival was obtained in WSSV-challenged shrimp at 7, 15 and 30 days posttreatment (relative survival, 85%, 65% and 50%, respectively) whereas 100% mortality was observed in the control shrimp fed with feed containing chitosan/pcDNA 3.1 or chitosan/PBS complex. The ability of the chitosan to form a complex with the pVP28 and to stabilize it from endonuclease degradation was studied by agarose gel electrophoresis. Cytotoxicity of chitosan-encapsulated pVP28 was also evaluated by the MTT assay, which showed 90% viability of SISK cells incubated with the pVP28/ chitosan complexes. Transcription analysis of the chitosan-encapsulated pVP28 gene in different tissues of DNA-treated shrimp and SISK cell line was confirmed by an RT-PCR reaction. The present study also measured the changes in the level of important immunological parameters such as prophenoloxidase, superoxide dismutase and superoxide anion in hemolymph of chitosan-encapsulated VP28 DNA-treated and controls shrimp. The study also correlated the changes in the level of immunological parameters with the survival percentage and protective efficacy of oral route of DNA construct against WSSV in shrimp.

Four novel cell lines from tissues of eye, gill, kidney and brain of Etroplus suratensis were developed
and characterized. The cell lines of eye, gill, kidney and brain were sub-cultured for 245, 185, 170
and 90 passages, respectively, since 2008. These cell lines showed predominantly epithelial-like
cells. Effects of temperature and foetal bovine serum concentration on the growth of these cell lines
were examined and optimum growth was found at the temperature of 28◦ C with 20% foetal bovine
serum. All the four cell lines were successfully cryopreserved and revived at different passage
levels. Cell-cycle analysis of these cell lines was carried out by fluorescence-activated cell sorting.
Polymerase chain reaction (PCR) products obtained from the cells and tissues of E. suratensis with
primers specific to the conserved region of 16S ribosomal RNA and cytochrome oxidase I genes
of E. suratensis revealed the origin of cell lines from E. suratensis. Antibodies raised against the
tissues and cells of eye, kidney and gill were highly cross reacted to their specific tissue and cells
of E. suratensis. Chromosomal analysis revealed that E. suratensis cells have a normal diploid
karyotype with 2n = 48. The cells of these cell lines were successfully transfected with pEGFP
vector DNA. The eye (IEE), gill (IEG) and kidney (IEK) cell lines were found to be susceptible to
nodavirus but resistant to infectious pancreatic necrosis virus (IPNV). The cells of gill, kidney and
eye were applied to test the cytotoxicity of tannery effluents.

Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC50 values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r2 = 0.95–0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC50 and the in vivo LC50 data, were highly significant p < 0.001 with r2 = 0.977, 0.968 and 0.906 for AB50, NR50, and CB50, respectively.

This work examines the potential use of chitosan tripolyphosphate (CS/TPP) nanoparticles for gene delivery in different tissues of fish through oral route. The porin gene of Vibrio anguillarum was used to construct a DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP. The CS/TPP nanoparticles were synthesized by ionic gelation process and these nanoparticles particles were characterized. The morphology and particle size measurements of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The pVAOMP was encapsulated by CS/TPP nanoparticles by mixing of equal volume of heated CS/TPP and pVAOMP DNA solutions. The encapsulation efficiency of CS/TPP nanoparticles was found to be 79.9% of DNA binding with CS/TPP nanoparticles. The stability of plasmid DNA was also determined after encapsulation using DNase I and chitosanase. The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by immunohistochemistry, ELISA and real-time PCR analyses. The results indicate that DNA can be easily delivered into fish by feeding with CS/TPP nanoparticles.

The antiviral activity of furan-2-yl acetate (C 6 H 6 O 3 ) extracted from Streptomyces spp. VITSDK1 was studied in cultured SIGE (Sahul Indian Grouper Eye) cells infected with fish nodavirus (FNV). The susceptibility of SIGE cells for nodavirus infection was confirmed by cytopathic effect (CPE) and reverse transcriptase-polymerase chain reaction (RT-PCR). The antiviral activity of furan-2-yl acetate was assessed by cytopathic effect, as well as reduction in nodaviral titre (TCID 50 /ml) in the cultured SIGE cells under in vitro conditions. Furan-2-yl acetate (20 g/ml) effectively inhibited the replication of FNV infected in the SIGE cell lines and the viral titre was reduced to 2.45 log TCID 50 ml −1 when compared to 4.3 log TCID 50 ml −1 for control. Also, furan-2-yl acetate (20 g/ml) treated SIGE cell survival was found to be 90% as determined by MTT assay. The results suggest that the furan-2-yl acetate suppressed FNV replication in cultured fish cells, providing a potential approach for the control of nodaviral diseases in marine fishes.

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most important viruses which mainly infects the larva of marine and freshwater fish with high mortality and affects the fishery industry worldwide. Currently, there are no effective vaccines available for the fish larva infected with NNV. Immunoglobulin yolk (IgY) origin of oviparous animals is passed from the blood serum and concentrated in the egg yolk. With the advantages of high yield, cost-effectiveness, and high stability, IgY can be widely used in passive immunization, especially in young animals in which adaptive immunity is not fully developed. In this study, we have cloned and expressed the recombinant capsid protein of RGNNV in Escherichia coli and used as an immunogen for generating specific anti-RGNNV IgY antibody in laying hens. Water-soluble fractions (WSF) of the specific IgY were isolated from egg yolk and purified by two-step precipitation with saturated ammonium sulfate salting. By Enzyme linked immunosorbent assay (ELISA), the titer of the IgY reached a peak at the 6th week post of immunization and had a strong stability at a wide range of temperature, pH, and pepsin enzyme digestion. The purified IgY was competent to neutralize and completely inhibited the RGNNV replication in the grouper fin cell line (GF-1), indicating that it was highly specific and effectively recognized RGNNV. The results will pave a new way for the prevention of RGNNV infection.