Protection Of Procambarus Clarkii Against White Spot Syndrome Virus With Inactivated WSSV And Polysaccharide

White spot syndrome virus (WSSV), which cause high mortality in many economic shrimp Aquaculture, and is widespread over the world, make the large economic losses to the shrimp farming industry in China. But no very effective treatment against WSSV was found, so more and more study focused on the adaptive immunity of crustacean. Polysaccharides like glucan, chitosan and chitin have been successfully used to enhance resistance of crustacean against bacterial and viral infections (Sung et al.1994; Itami et al.1998; Chang et al.1999; Chang et al.2003; Chotigeat et al.2004).This study was carried out to explore the possibility of protecting Procambarus clarkii from WSSV by oral vaccination with BEI-inactivated WSSV. And different concerntration of chitosan, chitin, glucan (5 mg/g,10 mg/g,15 mg/g) was tested to enhace the relative percent of survival in crayfish when they were challenged with WSSV.1 WSSV inactivation by BEIIM injection experiment showed that WSSV had been inactivated by 2mM BEI completely and the solution was safety for oral vaccination. The cumulative mortalities in the 37℃heated WSSV group indicated WSSV could keep the infectivity even exposed to 37℃over 24h. PCR detection and SDS-PAGE revealed that BEI only destroy the nucleic acid but not destroy envelope proteins in the process of inactivation. Intramuscular injection (IM) experiment showed that WSSV had been inactivated completely and the solution was safety for feeding.2 Protective effects of BEI-inactivated WSSV (BIW) in crayfish by oral deliveryThe potentiality of oral vaccination against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. Efficacy of BEI-inactivated WSSV was tested by oral vaccination followed by oral challenge of crayfish with WSSV. The crayfish fed with the food pelletes coated with BIW showed a resistance to WSSV on the 7th day post-vaccination (dpv). Calculated RPS values were 60%,70%and 75% for the vaccinated once, twice and thrice with BIW. Crayfish vaccinated thrice showed significantly higher RPS values compared to the control groups (P<0.05), the RPS values of 70%,53%,38%and 20%for crayfish challenged at 7,14,21 and 28 days after cessation of oral vaccination. The immunological parameters analyzed revealed that crayfish vaccinated with inactivated WSSV showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD) and lysozyme (LZM) compared to the control groups at 7 days post vaccination (dpv). But all of four enzyme activity decreased at 14,21 and 28 dpv. The result indicated that the resistance induced BEI-inactivated WSSV could persist for 20 days. Crayfish that survived from oral challenges were positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. This find provide a feasible way to control the prevalence of WSSV in prawn breeding through oral administration and perhaps has good effect to control other envelope virus.3 A pilot study on mechanism of protective effects conducted by BIWEfficacy of BIW was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish fed with BIW showed a better survival (P< 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared with the control. Calculated relative percent survival (RPS) values were 60%and 42%on the 7th and 21st dpv for BIW. The crayfish injected with BEI-inactivated WSSV showed a better survival (P< 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared with the control. Calculated relative percent survival (RPS) values were 75%and 60% on the 7th and 21st dpv for BIW. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect crsyfish from WSSV. 4 Protective effects of Polysaccharide (chitosan, chitin andβ-glucan) in crayfishCrayfish, Procambarus clarkii, were fed with chitosan, chitin or p-glucan at 5,10 and 15mg/g for four weeks, and oral challenged with WSSV. The cumulative mortalities in the groups fed with chitosan at 10mg/g was significantly lower than the control (P<0.05) but the other groups was not. The RPS showed that 10mg/g chitosan and 15mg/gβ-glucan provided significantly better protection against WSSV compared to the control (P<0.05). PCR analysis demonstrated that the surviving crayfish were WSSV-negative. The immunological parameters analyzed revealed that the crayfish fed with chitosan and glucan showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD), peroxidase (POD)and lysozyme (LZM) compared to the control groups. The high levels of prophenoloxidase, superoxide dismutase, peroxidase and lysozyme may be responsible for enhancing resistance against WSSV in crayfish fed with chitosan or glucan.IM injection experiment had showed that WSSV had been inactivated by 2mM BEI completely and the solution was safety for oral vaccination. PCR detection and SDS-PAGE revealed that BEI only destroy the nucleic acid but not destroy envelope proteins in the process of inactivation. The immunological parameters analyzed revealed that crayfish vaccinated with inactivated WSSV showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD), and lysozyme (LZM) compared to the control groups at 7 days post vaccination (dpv). Crayfish that survived from oral challenges were positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. This study indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect crsyfish from WSSV.