RESUMO
The VP28 gene of white spot syndrome virus was amplified by PCR using gene specific primer set and cloned into pRSET B vector to produce recombinant VP28 (r-VP28) in E. coli GJ1158. The chitosan tripolyphosphate nanoparticles (CS/TPP) were prepared by ionic gelation process and characterized. The purified r-VP28 protein was encapsulated by CS/TPP nanoparticles. The encapsulation efficiency of CS/TPP nanoparticles was found to be 84.8% for r-VP28 protein binding with CS/TPP nanoparticles. The in vitro release profile of encapsulated r-VP28 was determined after treating with protease and chitosanase. The different types of feed were formulated and named as normal feed with PBS, Feed A coated with crude r-VP28, Feed B with purified r-VP28 and Feed C with CS/TPP encapsulated r-VP28 (Purified). Tissue distribution and clearance of r-VP28 at different time intervals were examined in shrimp fed with different types of feed by ELISA and the results showed the presence of r-VP28 protein in different organs. Various immunological parameters were assessed in experimental shrimp. The mRNA expression of five immune-related genes was analysed by qPCR in order to investigate their response to all types of feed in shrimp. A cumulative percentage mortality was also recorded in treated shrimp challenged with WSSV.
Assuntos
Quitosana/química , Nanopartículas/química , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genética , Animais , Quitosana/farmacologia , Escherichia coli/genética , Géis/química , Penaeidae/genética , Penaeidae/virologia , Proteínas Recombinantes/genética , Proteínas do Envelope Viral/química , Vírus da Síndrome da Mancha Branca 1/patogenicidadeRESUMO
White tail disease (WTD) is a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India. Experiments were carried out to determine the possibility of vertical transmission of M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) in M. rosenbergii and Artemia. Prawn broodstock inoculated with MrNV and XSV by oral or immersion challenge survived without any clinical signs of WTD. The brooders spawned 5-7 days after inoculation and the eggs hatched. The survival rate of larvae gradually decreased, and 100% mortality was observed at the post-larvae (PL) stage. Whitish muscle, the typical sign of WTD, was seen in advanced larval developmental stages. The ovarian tissue and fertilized eggs were found to be positive for MrNV/XSV by reverse transcriptase-polymerase chain reaction (RT-PCR) whereas the larval stages showed positive by RT nested PCR (nRT-PCR). In Artemia, reproductive cysts and nauplii derived from challenged brooders were normal and survival rates were within the expected range for normal rearing conditions. The reproductive cysts were found to be positive for MrNV/XSV by RT-PCR whereas the nauplii showed MrNV/XSV-positive by nRT-PCR. The PL of M. rosenbergii fed nauplii derived from challenged Artemia brooders died at 9 days post-inoculum with clinical signs of WTD.