Studies On Attenuation Of Cell-cloned Strains Cultivation Of Very Virulent Infectious Bursal Disease Virus And Immunogenicity Of Attenuated Strains

Infectious bursal disease(IBD) is caused by infectious bursal disease virus, this virus strain can cause highly contagious and killed lymphocyte of chickens immuno-suppression.In the late of 1980s,Vaccination failure often happened because of the appearance of vvIBDV strains and the variant strains.In order to control the vvIBDV infection,many scholars succeeded in cultivation of the vvIBDV strain and the attenuated variant strain(Rosenberger, Kibenge, Tsai , Hassan etal). Additionally,many attenuated virus strains have also acquired in BGM-70,Vero,CEF et al cell,for example variant Del-A, MD, GLS.But immunogenicityof these attenuate strains is not ideal.In order to discuss the chickens infection of vvIBDV,we attenuate the local vvIBDV,and also make a research on kimmunogenicity of this attenuated strain.The experiment divide two parts:1. Selection and cultivation of the attenuated virus strains of cell-cloned strain of vvIBDV and study on its genetic stability:This experimentation use the cell-cloned strains GX-IBDVE10C25Cl5 as the researched object, this strains come from GX8/99 of vvIBDV isolated in our country ,We let this strains passage on CEF for 25 generations.Through testing TCID50 (/0.1ml) of clone-cell 1,5, 10, 15, 20, 25 passaged virus, the result was that from 1 to 15clone-cell-passage the TCID50 is gradually decreasing (105.40 , 104.80,104.67,103.76) ,from15 to 25clone-cell-passage theTCID50 is stable (103.76,103.80,103.67)。 This results demonstrate the virulence of clone-cell-passaged is already stable;The result of pathogenicity experiment of inoculating 41-days-SPF chickens showed that between 1, 5 passage and negative comparative chickens,the difference of the bursa index, spleen index and thymus index is very distinct(P<0.05),the 5 passage virus still has very strong pathogenicity,mortality is 10%;but 10,15,20,25 passage is not remarkalbe (P>0.05) ,from 15 passage this clone-cess-passaged has no pathogenicity for 41-days-SPF chickens.So we can say this strain has already become a stable attenuated strain.The 25-cell-passaged virus strains of GX-IBDVE10C25Cl5 was successed in SPF chickens for 5 times.The results showed that this strain were attenuated after passage for 25 passages.The virulence of this strain is a comparatively stable virus.this result prove that clone-cell-passaged strain has been cultivated attenuated virus.With the passage increasing,the transmissibility is stable. We expect that this attenuated strains can offer a new vaccine strain. 2. Studies on Immunogenicity of attenuated virus strains of vvIBDV:We cultivate the 25th GX-IBDVE10C25Cl5 strain on well CEF,after CPE of cell reach to 80%,we collect cell liquid and test its TCID5o(103.67/0.1ml),then making attenuated vaccine and oil inactivated vaccine.The attenuated vaccine were first inoculated 14-day chickens at three different doses of 5000TCID50, 10000TCID50 and 20000TCID50 by intranasal and oral and drop eyes. Inactivated vaccine were first inoculated 14-day chickens at three different dose of 0.1 mh 0.3ml and 0.5ml by subcutaneous and intramuscular injection,and take 15 chickens from every groups for second immunization at 28-days.All groups was challenged with vvIBDV GX 8/99 strain on 7,14,26 days after first immunization and on 12 days after second immunization。 The lab challenge results demonstrated with vvIBDV GX8/99 challenging, 10000TCID50 immune dose can provide 95% protection,5000TCID50 and 20000TCID50 can provide 75% and 70%protection,So the immune doses of 10000TCID50 is the best immune dose of live attenuated vaccine;0.3ml immune dose can provide 90% protection,0.1ml and 0.5ml can provide 70% and 85% protection,so 0.3ml is the best immune dose of dead vaccine.The neutralizing antibody ebb and flow results demonstrated that the least protection neutralizing antibody titer of attenuated vaccine and inactivated vaccine is 212.For 10000TCID50 immune dose of attenuated vaccine,the full duration of immunity is at least 110 days,but 0.3ml immune dose of dead vaccine is 66 days.The best immune dose of dead vaccine just induced neutralizing antibody titer of chickens very slowly,and the maintenance of high antibody titer is shorter;But the attenuated virus vaccine may induced high neutralizing antibody titer,and can completely resist vvIBDV GX8/99 challenge,so attenuated vaccine has good immuno-genicity.Draw the conclusion,we cultivate the attenuated strains has good immunogenicity,and this attenuated strains using as a vaccine strain is suitable.