Delivery of viral recombinant VP28 protein using chitosan tripolyphosphate nanoparticles to protect the whiteleg shrimp, Litopenaeus vannamei from white spot syndrome virus infection.

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.

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP-infected whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), in India.

Stunted growth in pond-reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post-challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP-infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP-infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.

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.

Experimental vertical transmission of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) from brooders to progeny in Macrobrachium rosenbergii and Artemia.

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.