Studies On The Cultivation Of Grass Carp Reovirus By Microcarrier And The Immune Effect Of Immersion With Killed Vaccine

Grass carp (Ctenopharyngodon idellus) is the major species of freshwateraquaculture in China, which has numerous advantages including rapid growth rate,various food sources and delicious taste, etc. Grass carp hemorrhage is the most severeinfectious disease affecting grass carp fingerlings caused by grass carp reovirus(GCRV), and leads to huge economic loss in the industry of grass carp culture. Thegrass carp hemorrhage in a wide range of areas with a long period and and causes highmortality. At present, immunization and natural plant anti-virus drugs therapy are themajor methods to prevent the disease. The GCRV propagation for inactivated vaccinepreparation is mainly dependant on the adherent culture of CIK cells on bottles, whichrepresents a limiting factor for the large-scale production of the vaccine. Currently,there are three major methods for fish vaccination, including intraperitoneal injection,immersion and oral immunization. Although the vaccination by immersion is a simpleand convenient method, the mechanism of immune response and the immune efficacyare needed to be investigated in-depth. In this paper, the microcarrier culture technologyof grass carp cell and reovirus, mechanism of immunization by immersion and theefficacy were investigated. These are essential for designing new vaccination strategiesto control the hemorrhage of grass carp. The following are the details:1. Microcarrier culture for grass carp CIK cells and GCRV. In this part theconditions for the large-scale culture of grass carp kidney cell (CIK) and GCRV bymicrocarrier were studied and optimized. The result showed that the Cephodex is a kindof microcarrier suitable for the growth of CIK cells. The cells grew well observed byinverted microscope and scanning electron microscope (SEM). The attachment efficacyreached to90%after8h cultivation with an intermittent agitation for2min at35rpmafter45min stilling culture. The optimal serum concentration for cells attaching to theCephodex microcarrier is5%. The optimal growth result could be achieved while the cell seeding concentration is2.5×105cells/ml and the microcarrier concentration is10mg/ml, and continuous stirring speed of40rpm, respectively. After infection of theCIK cells on Cephodex microcarrier with GCRV at0.1of multiplicity of infection(MOI), the typical cytopathic effect appeared at day3post infection, the GCRV titer(LgTCID50/ml) reached about8.5.2. The immune response and efficacy of immersion immunization withinactivated GCRV vaccine. The grass carp fingerlings were immersed in killed GCRVvaccine achieved by using microcarrier CIK cell culture. The blood samples werecollected from caudal vein of fish in control group and immunized group on the Day7，14，21and28post immunization. The changes of immune parameters, such ashaematocyte number, the phagocytic activity of phagocytes, SOD activity in serum andserum antibody titer were determined. The expressions of IgM and Mx genes in liver,spleen and head kidney of grass carp were monitored by RT-PCR. The results showedthat, vaccinated fish by immersion could increase the number of erythrocytes andleucocytes in peripheral blood，the phagocytic activity of leukocytes and SOD activityin serum. The serum antibody titers gradually increased in immunized fish and reachedthe highest value on day21.The expression level of IgM and Mx gene from three tissueswere all up-regulated after immersion vaccination. The gene expression of IgM in livershowed no significant difference between vaccinated an control fish, while the level inspleen and head kidney were significantly enhanced compared with the control group,and reached the highest value on the14d and21d respectively. Significant higher geneexpression of Mx was observed at7and14in liver of vaccinated fish when comparedwith control fish. The level was significantly enhanced in spleen and head kidneycompared with the control group, and reached the peak value on14d in two tissues. Thegene expression levels of IgM and Mx were higher in spleen and head kidney than thatin liver. The result of challenge test with live GCRV showed that grass carp immunizedhad a good RPS reached76%. All the results stated above suggested that immersioninoculation could enhance grass carp immunity against the grass carp hemorrhage.