| Rabbit hemorrhagic disease is an acute disease, It is associated with hepatocellular necrosis and disseminated intravascular coagulation, and causes epidemics with high mortality rates, it mainly infected adult rabbits at 3 to 4 months of age, tremendously economic losses to rabbitry were caused by the disease. It was demonstrated that the virus could be controlled by either an inactivated vaccine or a recombinant vaccine, However, Rabbit hemorrhagic disease (RHD) still exists in some areas. It has been reported recently that young rabbits, at the age of one to two months, or shortly after weaning, are likely to be infected by RHD, which aroused a new question for the prevention and control of the disease, was it the reason of variation of antigen or the immune program which induce the loss of immunization? This study investigated the epidemiology of RHDV in three eastern provinces, the gene sequences, pathogenicity and antigenicity of three young rabbit hemorrhagic disease virus were compared, the immune program of young rabbit hemorrhagic disease was studied, at last, the immune enhancement and mechanism on vaccine of cCHMIs were discussed. The purpose of this study was to find perfect immune program and immune adjuvant, provide academic proof for the prevention and control of RHD in young rabbits. The details are divided into five parts as follows:Test 1 The molecular epidemiology of RHDV in three eastern provinces of China In order to study the epidemiology of RHDV, the A pair of primers were designed according to the gene sequences of rabbit hemorrhagic disease virus (RHDV) published in Genbank, total RNA of RHDV was extracted from the fresh liver of RHD-rabbits for synthesis of cDNA and amplification of PCR, then the PCR product was cloned and sequenced. The result showed that an expected 269bp segment was amplified and the homology was 95.8 %～98.5 % compared with the reference sequence published in Genbank. Then the sensitivity test, speciality test as well as virus distribution test were carried out to confirm the RT-PCR method. In order to investigate the prevalence of RHDV, 512 nasal secretion samples from rabbits in three provinces of China from 2005 to 2007 were collected and tested by RT-PCR, several positive samples were sequenced and compared. The results showed that the RT-PCR method had good specificity as it did not detect two other RNA viruses of rabbits, the sensitivity was 1×10~4 times higher than that of the hemagglutination assay (HA) , The RT-PCR method was able to detect RHDV in all viscera, but not in feces. There were 48 positive samples of 512 samples, our epidemiological investigation further suggest that RHDV is widely distributed in eastern districts of China. There were significant differences in the prevalence of RHDV in rabbits of different ages, but there were no significant differences among different provinces and years. The homology between three newly discovered isolates and isolates from different countries that have been recorded in Genbank ranged between 93.7% and 99.6%.Test 2 The comparison of gene sequences, pathogenicity, antigenicity of three young rabbit hemorrhagic disease virus isolates In order to study the difference between young RHD virus and vaccinal RHDV, three young RHD virus isolates and one vaccinal RHDV were amplified by RT-PCR, then the gene sequences of VP60 were sequenced and compared, as well as the analysis of the homology of VP60 amino acid; to compare the difference of pathogenicity, those four RHDV were disposed to attack rabbits by comparing time and rates of death. At last, four vaccines were produced by the four RHDV to compare antigenticity. The results showed that homology of gene sequences was 95.2%～97.4% and amino acid was 95.8%～99.3%; there was no significant difference of pathogenicity; test of antigenicity showed that the antibody which were produced by four RHDV vaccines was one serological type. The antibody could effectively protect the rabbits from different RHDV, it was indicated that there were no significant differences of antigenicity among the four RHDV.Test 3 The effect of vaccinated age on antibody titers against RHD In experiment 1, the protective effects of 5 serial antibody titers, 0, 1, 2, 3 and 4 log2 on rabbit hemorrhagic disease (RHD) were compared by virus-attacking experiment in New Zealand rabbits, the results showed that the protective rate was 100% when antibody titer was more than 3 log2, 50%, 2 log2. In experiment 2, the dynamic changes of antibody titers in mother-source and vaccinated with inactivated rabbit hemorrhagic disease (RHD) vaccine within 8 weeks were determined at 30-, 35-, 40-, 45-, 50-, 55- and 60-day-old rabbits, there was a negative relationship between mother-source antibody titer and age of rabbit. The antibody titer of 30-day-old rabbits was highest, which could protect effectively, elder than 40-day-old, no protection. While between immunization age and antibody peak value, together with effective protecting time, there was a positive relationship. When 30-day-old rabbit was vaccinated, the antibody titers reduced significantly as compared with the mother-source, without protective effect. Vaccination at 35 days old, the antibody peak value was lower and effective protecting time lasted for only 2 weeks. While at 40 days old or more, peak values were higher and effective protecting times were longer.Test 4 The study of immune program of RHD vaccine In experiment 1, 35 day-old, 40 day-old and 45 day-old young New Zealand rabbits, 30 per age, were chosen and divided randomly into five equal groups. The rabbits in control group were injected with physiological saline, in 4 trial groups, vaccinated with four dosages of RHD vaccine respectively. The antibody titer before and 1 to 7 weeks after vaccination were measured, the rising speed antibody and last time of effective antibody in dosage 2,3, 4mL group was notably more and longer than it in dosage 1mL group. In 35 day-old rabbits, the antibody peak value in dosage 2, 3,4 mL group was significantly higher than those in dosage 1mL group. In experiment II, two vaccinated programs were compared. In most time points, the antibody titers of once vaccinated 2mL group were significantly larger than that twice vaccinated 1mL group. It was indicated that the best vaccinated program was 35 day-old rabbit vaccinated at dosage 2mL.Test 5 Immune enhancement of two Chinese medicinal adjuvants on Rabbit hemorrhagic disease (RHD) vaccine In order to enhance the immune enhancement of RHD vaccine, two Chinese medicinal adjuvants (CMAs) were prepared with four Chinese herbal medicinal ingredients (CHMI), Astragalus polysaccharide (APS), Epimedium polysaccharide (EPS), Propolis flavone (PF), Ginsenosides (GS). The two CMAs were mixed respectively with RHD vaccine virus to vaccinate rabbits with aluminum and non-adjuvant vaccine as controls. On the days of 7, 14, 21, 35, and 49 after vaccination, the dynamic changes of peripheral lymphocyte proliferation and antibody titers were tested by MTT method and HI method, on the 63 th day, the immune organ index was calculated, the rest rabbits were challenged with RHD virus. The results showed that the two CMAs could significantly enhance lymphocyte proliferation and serum antibody titers, stimulate the organ index of Sacculus Rotundus and intestinal lymph node, all rabbits vaccinated with CMAs vaccine were protected from viral challenge.Test 6 Effecting of Two Chinese medicinal adjuvants on lymphocyte proliferation, mRNA expression of IFN-γand IL-10 In order to evaluate the immune enhancement of two CMAs. The effects of the two CMAs on lymphocyte proliferation and mRNA expression of IFN-γand IL-10 were determined by MTT method and fluorescent real-time PCR method. The results showed that The two CMAs could significantly enhance lymphocyte proliferation at certain concentration, raise mRNA expression of IFN-γand IL-10, which might be one of the immune enhancing mechanisms of two CMAs.
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