Studies On The Efficacy And Safety Of Transgenic Cyanobacteria Harbouring Vp28 Gene Used For Against WSSV In Macrobrachium Nipponense

White Spot Syndrome Virus(WSSV)has been identified it can infect 40 species of shrimp and 39 species of crabs,which affects as many as 25 countries.It has become the most widespread,the fastest spreading and the highest-mortality virus in the global shrimp industry.Macrobrachium nipponense is widely distributed and available all the year round,but there are few reports on what is its susceptibility to WSSV and how protect M.nipponense from WSSV In this paper,the transgenic cyanobacteria(Anabaena sp.PCC7120)harbouring vp28 gene preparation will be administered to Macrobrachium nipponense to investigate the infection relationship between Macrobrachium nipponense and WSSV,to study the immune protective effect of transgenic cyanobacteria on Macrobrachium nipponense,and to discuss how to prevent transgenesis.Cyanobacteria release to the environment during the pharmaceutical process to ensure that the anti-WSSV cyanobacterial oral agent can be safely and effectively used in aquaculture.The main results are as follows:(1)By means of epidemiological investigation,different means of infection test,the investigation showed that M.nipponense was the natural host of WSSV,and the natural carrying rate was about 8.33%.M.nipponense can be infected with WSSV via oral administration,muscle injection and immersion,and the cumulative infection rate of 10 d was 100%,and the cumulative mortality rates were 100%,75%,and 0%,respectively.The infection of WSSV is fast by muscle injection.The virus content after 5 days injection is 1,000 times higher than that of the first day of infection,and the mortality rate reached 100%after 8 days.The median lethal dose(LD50)measured as the mortality of infected M.nipponense via injection indicated the LD50 in the concentration of WSSV of 2.71 ×105 virions/?L.In shrimp farming,M.nipponense can infect WSSV by ingesting WSSV infected shrimp or dead shrimp,and can also be infected by soaking in WSSV-containing water and thus become a vector,which affecting the spread and pathogenicity of WSSV.(2)Macrobrachium nipponense was immunized with vp28 cyanobacterial oral agent for 7 days,and then was feeding with the muscle tissue of Macrobrachium nipponense infected WSSV to challenge the virus.After 10 days of challenge,the cumulative mortality rates of positive control,wild type,and blank were as high as 81.25%,61.5%,and 87.5%,respectively,in the immunization group 1 was 56.25%,whereas in the immunization group 2 was only 37.5%.Compared with the positive control group,the survival rates of the immune groups 1 and 2 were increased by 25%and 43.75%,respectively.The content of WSSV in the tissues of gills,hepatopancreas and tail muscles of Macrobrachium nipponense on the 4th day after challenge was detected.The results showed that WSSV could be detected in all the macrobrachiums nipponense after challenge,but the content of WSSV in the immunized group was significantly lower than that in the control group.In the same sample infected with Macrobrachium nipponense,virus content>hepatopancreas>muscle;WSSV content in the gills of positive control group,wild-type,blank reached 108 copies/mg,immune group 1 decreased 100-fold,and immune group 2 decreased.10000 times;WSSV content in the hepatopancreas of positive control group,wild-type,blank were 107copies/mg,immune group 1 and immune group 2 were reduced 100 times and 1000 times;WSSV content in the muscle of positive control group,wild Type,blank is low,were 106copies/mg,and the immunization group 1 was reduced 100-fold,while the immunization group 2 virus content was lower than 102copies/mg.WSSV lesions were found in both gills and hepatopancreas cells after challenge.Among the positive control group,the wild type and blank group,there were numerous signs of cell rupture,necrosis,and nuclear shedding in the tendon and hepatopancreas.The lesions appeared in the hepatopancreas tissue and the histological health was significantly better than that of the challenge control group;while the sputum and hepatopancreas tissue cells of the immunization group 2 had no pathological phenomenon.There was no significant difference in muscle cells between Macrobrachium nipponense and negative control muscles after challenge.No WSSV pathological findings were found.This may be due to the unfavorable survival and reproduction of WSSV in muscle cells.After vp28 cyanobacterial oral administration of Macrobrachium nipponense,the immunity of Macrobrachium nipponense against WSSV was improved,and the proliferation of WSSV in vivo was reduced,effectively protecting the tissues of gills and hepatopancreas in Macrobrachium nipponense,which Greatly improved the survival rate of Macrobrachium nipponense.(3)The prerequisite for the preparation of genetically modified drugs is their safety,which includes both human and environmental aspects.For the cyanobacteria we have constructed,people are more concerned about the release of this photosynthetic microorganism to the environment and affect the ecological balance in nature.The transgenic Anabaena sp.was treated with different pH and observed at different times,temperatures and illuminations.The response surface method was used to optimize the treatment conditions.The optimum treatment conditions were as follows:pH?2.5,t?50 min,T>30 ? or pH?12,t?120 min,T?30 ?,and the killed cell rate is up to 100%.The two optimal combinations were as follows:pH 2.12,29.96 ?,33.9 min or pH 12.33,31.75 ?,96.8 min.After the treatment with pH,the transgenic Anabaena sp.was not able to continue to grow on the plate completely.The killed cell rate of the pH method up to 100%which can effectively prevent the release into environment of transgenic Anabaena sp.during the preparation of the oral shrimp drug.The results show that the susceptibility to WSSV is greater than that of South American prawn,which means that the shrimp may have an endogenous anti-WSSV mechanism.The macroscopical mortality,microviral content and pathological biopsy showed that transvp28 cyanobacteria oral agent had immunological effect on the shrimp,which could be used for the control of WSSV In addition,the environmental release of transgenic cyanobacteria during the preparation of WSSV-resistant oral agent was prevented by the pH method,and the safe production of anti-white spot virus was ensured.