| Rabbit viral hemorrhagic disease,commonly known as rabbit fever,is an acute,lethal and highly contagious disease caused by rabbit hemorrhagic virus that can cause morbidity and mortality in many different breeds of rabbits.Although there is only one serotype of rabbit haemorrhagic virus,several different genotypes have evolved genetically,which has led to changes in pathogenicity and antigenicity.The danger of rabbit fever is becoming more and more serious,and the economic losses caused by the disease to the rabbit industry can be described as incalculable,and it seriously affects the healthy development of the rabbit industry,which is relatively serious.2020,the first case of RHDV2 was reported in China.Currently,there is no vaccine for the prevention and control of RHDV2 in China,and there is a lack of vaccines that can resist both RHDV1 and RHDV2 infections.Therefore,the aim of this study was to prepare a safe and efficient vaccine against both genotypes of RHDV and to evaluate its safety as well as immune efficacy.We inoculated healthy susceptible rabbits with RHDV1 LQ strain and RHDV2 JT strain,respectively,and compared the immunogenicity of viral tissues harvested at different times after grinding and inactivation.According to the experimental results,we used 24 to 96 hours after virus inoculation as the time of tissue harvesting.For the screening of inactivation conditions,the ground tissue virus solution was inactivated by adding different concentrations of formaldehyde solution at 37 °C,and samples were taken at different times after inactivation for inactivation tests,and the final concentration of 0.4 %formaldehyde solution,acting at 37 °C for 48 hours,was finally determined as the inactivation condition.In the study of the dilution times of the viral solution,the viral tissues within the harvest time were inactivated after grinding with the dilution solution at different dilution ratios,and the final dilution ratio of the viral tissues to the dilution solution was determined to be 1:9 according to the results.in the selection of antigen agglutination values,the inactivated suspensions of tissues prepared with different agglutination antigens were subjected to immune attack protection experiments,and the antigen agglutination values of the ligated vaccine for the inactivated vaccine were determined to be ≥ 1:256 according to the results.In the ratio study of inactivated vaccine components,the inactivated virus tissue suspensions were made into bivalent inactivated vaccines in different ratios and tested for immune attack protection,and the final ratio of RHDV1 inactivated tissue suspension to RHDV2 inactivated tissue suspension was determined to be 1:1.The inactivated vaccines were tested for efficacy after passing the sterility test,formaldehyde residue determination and safety test.The results showed that the protection rates of RHDV1 and RHDV2 could reach 100 % and 90 %,respectively.We developed a bivalent tissue inactivated vaccine for RHDV1 and RHDV2 based on the LQ strain of RHDV1 and the JT strain of RHDV2,screened by tissue harvesting time,inactivation conditions and dilution multiples.In the safety study of the inactivated vaccine,single single dose,single dose repeated and single overdose vaccination were performed,and the results showed that the feeding,drinking and mental condition of the animals after vaccination were normal and there were no adverse clinical reactions,no redness,inflammation and other reactions at the injection site,and no abnormal lesions in all tissues and organs,indicating that the vaccine has good safety.Experiments were conducted to further evaluate the immune effect of the inactivated vaccine.The results of the minimum immunization dose experiment showed that the inactivated vaccine provided complete protection in rabbits vaccinated with a dose of not less than 0.5m L/animal,so the minimum immunization dose was determined as0.5m L/animal,but the recommended immunization dose was 1m L/animal to ensure the clinical immune protection.The results of the correlation study between serological potency test and immune attack protection showed that when HI antibodies to RHDV1 and RHDV2 were ≥1:32,protection was provided to immunized rabbits and the protection rate was up to 100%.The results of offspring passive immunity and duration of immunity tests by maternal antibodies showed that maternal antibodies could provide some protection until weaning(around 25 days of age)of the rabbits.The results of the antibody dissipation pattern and duration of immunity test showed that the antibody level reached the highest after 21 days of immunization and remained at a high level after 180 days,and the duration of immunity of the inactivated vaccine was tentatively determined to be 6 months.The results of cross-attack protection experiments showed that the cross-protection rate of both was low and could not provide effective protection.In conclusion,this study developed a bivalent tissue-inactivated vaccine against RHDV1 and RHDV2,and confirmed that it has good safety and immune efficacy,and the vaccine can provide good immune protection to rabbits,laying the foundation for the development of a commercial bivalent vaccine against rabbit viral haemorrhagic disease. |
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