C. Venkatesan

Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV)

The protective efficacy of oral delivery of a DNA construct containing the VP28 gene of WSSV encapsulated in chitosan nanoparticles was investigated in black tiger shrimp (Penaeus monodon). The results showed that significant survival was obtained in WSSV-challenged shrimp at 7, 15 and 30 days post-treatment (relative survival, 85%, 65% and 50%, respectively) whereas 100% mortality was observed in the control shrimp fed with feed containing chitosan/pcDNA 3.1 or chitosan/PBS complex. The ability of the chitosan to form a complex with the pVP28 and to stabilize it from endonuclease degradation was studied by agarose gel electrophoresis. Cytotoxicity of chitosan-encapsulated pVP28 was also evaluated by the MTT assay, which showed 90% viability of SISK cells incubated with the pVP28/chitosan complexes. Transcription analysis of the chitosan-encapsulated pVP28 gene in different tissues of DNA-treated shrimp and SISK cell line was confirmed by an RT-PCR reaction. The present study also measured the changes in the level of important immunological parameters such as prophenoloxidase, superoxide dismutase and superoxide anion in hemolymph of chitosan-encapsulated VP28 DNA-treated and controls shrimp. The study also correlated the changes in the level of immunological parameters with the survival percentage and protective efficacy of oral route of DNA construct against WSSV in shrimp.

Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV).

The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus (WSSV) using antiviral plant extract derived from Cyanodon dactylon and the modulation of the shrimp non-specific immunity. To determine the antiviral activity, the shrimp were treated by both in vitro (intramuscular injection) and in vivo (orally with feed) methods at the concentration of 2mg per animal and 2% of the plant extract incorporated with commercially available artificial pellet feed, respectively. The antiviral activity of C. dactylon plant extract was confirmed by PCR, bioassay and Western blot analysis. In the present study, anti-WSSV activity of C. dactylon plant extract by in vivo and in vitro methods showed strong antiviral activity and the immunological parameters such as proPO, O(2)(-), NO, THC and clotting time were all significantly (P<0.05) higher in the WSSV-infected shrimp treated with plant extract when compared to control groups. These results strongly indicate that in vivo and in vitro administration of C. dactylon plant extract enhances immunity of the shrimp. Based on the present data and the advantages of plant extract available at low price, we believe that oral administration of C. dactylon plant extract along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp.

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.

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.

Efficacy of bacterially expressed dsRNA specific to different structural genes of white spot syndrome virus (WSSV) in protection of shrimp from WSSV infection

White spot syndrome virus (WSSV) is responsible for severe economic losses in shrimp culture worldwide. Any practical method to eradicate or inactivate WSSV in culture systems would have enormous practical benefits for shrimp farmers and hatchery operators. Various studies have been carried out on control of WSSV, including neutralization using antiserum (Van Hulten, Witteveldt, Snippe & Vlak 2001), recombinant subunit vaccines (Namikoshi, Wu, Yamashita, Nishizawa, Nishioka, Arimoto & Muroga 2004; Jha, Xu, Shen, Bai, Sun & Wei 2006), DNA vaccines (Rajesh Kumar, Ishaq Ahmed, Sarathi, Nazeer Basha & Sahul Hameed 2008) and antiviral plant extract (Balasubramanian, Sarathi, Rajesh Kumar & Sahul Hameed 2007). However, there is no efficient strategy to control the spread of this very serious disease. RNA interference (RNAi) has recently emerged as a powerful tool for specific gene silencing in antiviral therapy. The efficiency of RNAi in shrimp was demonstrated by in vivo injection of dsRNA corresponding to the gonad inhibiting hormone (GIH) in the sand shrimp, Metapenaeus ensis, which lowered the expression of GIH (Guan, Mak & Chan 2004). Several recent articles have reported that siRNAs or dsRNA synthesized in vitro are potential therapeutic agents for treating white spot syndrome (Robalino, Bartlett, Shepard, Prior, Jaramillo, Scura, Chapman, Gross, Browdy & Warr 2005; Westenberg, Heinhuis, Zuidema & Vlak 2005; Kim, Kosuke, Nam, Kim & Kim 2007; Xu, Han & Zhang 2007). The in vitro production of dsRNA may not be useful for field applications; however, the present study describes the production of bacterially expressed virus specific dsRNAs, which opens the possibility of developing dsRNA based antiviral therapeutics for commercial use. This study reports the use of an RNase III-deficient strain of E. coli HT115(DE3) (Timmons, Court & Fire 2001) to produce bacterially derived dsRNAs specific to WSSV structural genes and their interference with WSSV infection in shrimp. Our previous studies have shown that intramuscular administration of bacterially expressed VP28dsRNA results in the protection of Penaeus monodon against WSSV (Sarathi, Simon, Ishaq Ahmed, Rajesh Kumar & Sahul Hameed 2008a). The present study determined the protective effect of bacterially expressed dsRNA specific to different structural genes of WSSV other than VP28. We targeted the four Journal of Fish Diseases 2010, 33, 603–607 doi:10.1111/j.1365-2761.2010.01157.x

Synthesis and Characterization of Chitosan Tripolyphosphate Nanoparticles and its Encapsulation Efficiency Containing Russell's Viper Snake Venom

Chitosan Tripolyphosphate (CS/TPP) nanoparticle is a biodegradable and nontoxic polysaccharide, used as a carrier for drug delivery. The morphology and particle‐size measurements of the nanoparticles were studied by field emission scanning electron microscopy and Fourier Transform Infrared Spectroscopy (FTIR). This study aims to evaluate the impact of Russells viper venom encapsulation on various factors and loading capacity, in addition to explore the physicochemical structure of nanoparticles. FTIR confirmed that tripolyphosphoric groups of TPP linked with ammonium groups of CS in the nanoparticles. Our results showed that CS can react with TPP to form stable cationic nanoparticles. The results also showed that encapsulation efficiency of venom at different concentrations of 20, 40, 60, 500, and 1000 µg/mL were achieved for CS/TPP nanoparticles at different concentrations of 1.5, 2, and 3 mg/mL. The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT (‐3 (4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide, a tetrazole) assay.

Experimental exposure of Artemia to Hepatopancreatic parvo-like Virus and Subsequent transmission to post-larvae of Penaeus monodon

The different life stages of Artemia franciscana were experimentally exposed to Hepatopancreatic parvo-like virus (HPV), in order to evaluate the possibility of Artemia acting as reservoir or carrier for HPV. All the five developmental stages of Artemia were challenged with HPV both by immersion and oral infection routes. The viral infectivity to Artemia was studied by PCR but not much difference in mortality between control and challenge groups were observed. To confirm the vector status of Artemia for HPV, the HPV exposed Artemia were fed to postlarval forms of Penaeus monodon. Post-larvae of P. monodon were fed with HPV exposed Artemia and could get infected upon feeding on them. Mortality was observed in the post-larvae, which were fed with HPV exposed Artemia, and whereas no mortality was observed in post-larvae fed with Artemia not exposed to HPV and these post-larvae were PCR negative for HPV, as well. Results of this experiment suggest that Artemia might be a possible horizontal transmission pathway for HPV. Further research however is required with histology, immunohistochemistry and transmission electron microcopy to determine whether the Artemia are actually infected with this virus or whether they are simply mechanical carriers. This will enable us to understand better whether Artemia is a carrier of this virus and if so the mechanism involved.

Detection and neutralization of cobra venom using rabbit antiserum in experimental envenomated mice

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra (Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.