Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja).

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits.

Clearance of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) and immunological changes in experimentally injected Macrobrachium rosenbergii.

Macrobrachium rosenbergii was experimentally challenged with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) to study the clearance of these viruses and consequent changes in various immunological parameters. The healthy animals were injected MrNV and XSV intramuscularly and various organ samples such as gill tissue, head soft tissue, pleopods and intestine were collected at different time intervals of 3, 5, 10, 15, 25, 50, 75 and 100d post-infection (p.i.) to study the viral clearance. Tissue tropism and clearing of MrNV and XSV were confirmed by RT-PCR, nested RT-PCR and bioassay. These 2 viruses failed to cause mortality or clinical signs of disease in injected adult prawns during the experimental period of 100 days. The result of RT-PCR analysis revealed that all the organs showed positive for both viruses by single step RT-PCR on 3, 5 and 10 d p.i., positive by nested RT-PCR on 15 and 20 d p.i. and all the organs became negative at 25 d p.i. onwards. The viral inoculum prepared from the tissue of MrNV and XSV-injected M. rosenbergii at 3, 5, 10, 15 and 20 d p.i. caused 100% mortality in post-larvae of M. rosenbergii at 9, 8, 7, 10 and 10 d p.i., respectively whereas the inoculum prepared at 25, 50 and 100 d p.i. failed to cause significant mortality in post-larvae of prawn. Immunological parameters such as proPO, superoxide anion, SOD, THC, clotting time and oxyhemocyanin were determined in MrNV and XSV-injected prawns and significant differences in some of the immunological parameters were found in the early days p.i. and became insignificant in the later days p.i.

Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii.

A time course experimental challenge of WSSV was carried out to examine the clearance of WSSV in Macrobrachium rosenbergii and the consequent immunological changes. The experimental animals were injected with WSSV and the samples of gills, pleopods, head soft tissue and hemolymph were collected at different intervals of 1, 3, 5, 10, 25, 50, 75 and 100 days post infection (p.i.). WSSV infection and clearing were confirmed by single step PCR, nested PCR and bioassay. At 3 days p.i., M. rosenbergii became lethargic and stopped feeding in contrast to the control prawns that behaved and fed normally. However, the WSSV-injected prawns suffered no mortality during the experimental period and recovered without any further gross signs of disease or any mortality over a period of 100 days p.i. The single step PCR analysis showed positive at 1, 3 and 5 days p.i. in gills, head soft tissue, pleopods and hemolymph, and all the organs showed negative at 10 days p.i. onwards. The nested PCR results showed that all organs were positive for WSSV from 3 days p.i. and extended up to 25 days p.i. At 50 days p.i, head soft tissue sample alone showed WSSV-positive while all other organs were negative by nested PCR. All the organs at 75 and 100 days p.i. showed nested PCR negative for WSSV as observed in the control prawn. The hemolymph collected from experimentally infected M. rosenbergii at 1, 3 and 5 days p.i. caused 100% mortality at 40 h p.i., 55 h p.i. and 72 h p.i, respectively in Penaeus monodon whereas hemolymph collected at 10, 25, 50, 75 and 100 days p.i. failed to cause mortality in shrimp. The moribund shrimp showed WSSV-positive and surviving shrimp showed negative by PCR. Immunological parameters such as proPO, O(2)(-) and clotting time in WSSV-injected M. rosenbergii were found to be significantly higher than those of the control groups, whereas THC and superoxide dismutase were significantly lower when compared to control groups.

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV).

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6-90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.