Studies On The Cultivation Of Cyprinid Herpesvirus 2 By Microcarrier And The Immune Efficacy Of Immersion With Inactivated Vaccine

Crucian carp(Carassius auratus)is one of the most important fish species in freshwater aquaculture in China.Hematopoietic necrosis of crucian carp is a newly emerged infectious disease caused by cyprinid herpesvirus 2(CyHV-2),which is characterized by wide-range spreading,fast transmission,high mortality rate,and has caused huge economic losses.At present,no effective prevention and treatment methods are available for the control of the disease.Vaccination has been proven to be the most effective method in fish for preventing infectious diseases.The CyHV-2 propagation for inactivated vaccine preparation is mainly dependant on the adherent culture of gibel carp brain cells(GiCB)in bottles,which represents a limiting factor for the large-scale production of the vaccine.The study on the technologies for large-scale cultivation of GiCB cells and cyprinid herpesvirus 2 by microcarrier has potential application.In this study,the cultivation technology of GiCB and CyHV-2 by microcarriersand the immunization efficacy by immersion were investigated.These are essential for designing new vaccination strategies to control the hematopoietic necrosis of crusian carp.The following are the details:1.The technologies for the large-scale cultivation of GiCB cells and CyHV-2 by Cephodex microcarrier in suspension system were investigated.The results showed that the Cephodex is a kind of microcarrier suitable for the growth of anchorage-dependent GiCB cells.The optimal serum concentration for cells attaching to the Cephodex microcarrier was 10%,the microcarrier concentration was 6g/L and the density of cells inoculated was 2.5×105 cells/m L.With an intermittent agitation for 2 min at 35 rpm after 30 min stilling culture,the attachment efficacy reached 90% after 8 h cultivation;while in growing period,the optimal agitation speed was 45 rpm for continuous stirring.After infection of GiCB cells on Cephodex microcarrier with CyHV-2 at a multiplicity of infection(MOI)of 0.2,the typical cytopathic effect(CPE)appeared after 5 days post-infection and the virus titer(TCID50/mL)reached 106.50±0.30.This study established a solid basis for the large-scale preparation of vaccine against the crucian carp hematopoietic necrosis.2.The cyprinid herpesvirus 2(CyHV-2)cultured by microcarrier was inactivated with ?-propiolactone to prepare the inactivated vaccine,then the healthy gibel carp(Carassius gibelio)with a size of 3.20±0.15 cm were immunized by immersion with the prepared vaccine at a dose of 105TCID50/mL and 104TCID50/mL,respectively.At 4 weeks and 8 weeks post the first immunization,a part of the immunized gibel carp were challenged with CyHV-2 for the determination of immune efficacy with different dose.At the same time,another part of the immunized gibel carp were immersed with a booster immunization at 4 weeks post the first immunization as the Immunized-booster groups.On the 2,4,7,14,21 and 28 d post the second immersion immunization,the relative expression levels of IL-11,C3 and IgM genes in fish body were detected by quantitative RT-PCR.The dead fish in challenge test were collected to confirm the CyHV-2 pathogen by PCR assay and for CyHV-2 loading examination by TaqMan real-time PCR.The relative percentage survival(RPS)values of the 105TCID50/mL and 104TCID50/m Ll groups at 4 weeks post the first immunization showed 52% and 48%,respectively.In addition,the RPS at 8 weeks post the first immunization showed 61.6% and 53.9%,and the Immunized-booster groups showed 73.1% and 65.4%,respectively.In the immunized-booster group,the expression levels of IL-11,C3 and IgM mRNAs were significantly up-regulated compared with the control group.All of the dead fish in the challenge test were positive of CyHV-2 by PCR assay.The viral loading in dead fish of the Immunized-booster groups was significantly higher than that in dead fish of other groups(p< 0.01).These results indicated that the immunizatuion dose of the vaccine by immersion is 105TCID50/mL,a booster immunization showed a tendency to enhance the protection against the CyHV-2 infection in gibel carp.This study provides useful information for the application of CyHV-2 inactivated vaccine in practice.