R. Sudhakaran

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.

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

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 Leibovitzs 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 degrees C with an optimum of 28 degrees C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 degrees 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.

Natural aquatic insect carriers of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV)

Five different species of aquatic insects were collected from nursery ponds containing the freshwater prawn Macrobrachium rosenbergii infected with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). The insects were screened as potential natural carriers of MrNV and XSV. RT-PCR (reverse transcription polymerase chain reaction) analysis gave positive results for MrNV 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 MrNV and XSV. The C6/36 cells were harvested 4 d post-challenge 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 MrNV and XSV transmission to M. rosenbergii.