V. Parameswaran

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 of a Pluripotent ES-like Cell Line from Asian Sea Bass (Lates calcarifer)—An Oviparous Stem Cell Line Mimicking Viviparous ES Cells

We report a pluripotent embryonic stem cell-like cell line designated as SBES from blastula stage embryos of Asian sea bass (Lates calcarifer), which is an economically important cultivable and edible marine fish species in India. The SBES cells were cultured at 28°C in Leibovitz L-15 medium supplemented with 20% fetal bovine serum without a feeder layer. The ES-like cells were round or polygonal and grew exponentially in culture. The SBES cells exhibited an intense alkaline phosphatase activity and expression of transcription factor Oct 4. The undifferentiated state of these cells was maintained at low seeding densities and the cells formed embryoid bodies when seeded in bacteriological plates. On treatment with all-trans retinoic acid, these cells differentiated into neuron-like cells, muscle cells, and beating cardiomyocytes, indicating their pluripotency. This embryonic ES-like cell line derived from an oviparous fish blastula conserved several peculiar features of viviparous mammalian embryonic stem cell lines. The present study highlights the importance and potential of piscine ES-like cell line for stem cell research without evoking ethical issues and invasive interventions sparing mammalian embryos.