【作者】 杨龙圣；

【导师】 胡元亮； 薛家宾；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2008， 博士

【摘要】 兔出血症是家兔的一种烈性传染病,以发病急、传播速度快、死亡率高为特点,并主要感染3月龄以上成年兔,曾给养兔业造成巨大的经济损失。几年以前,通过疫苗注射可较好地控制本病。但是近年来,3月龄以下、甚至2月龄以下幼兔时有发病,给该病的防控提出了新问题。究竟是抗原变异的原因还是免疫程序的问题需要进一步研究。本研究首先对华东三省RHD的病原进行了分子流行病学调查,对三个分离毒株的基因序列、致病性和免疫原性进行了比较,研究了幼兔出血症的免疫程序,并探讨了中药佐剂对疫苗的免疫增强作用和作用机理。本研究的目的,旨在寻找理想的免疫程序和疫苗佐剂,为幼兔出血症的防控提供理论依据。具体内容为以下五个部分:试验Ⅰ、华东三省RHDV的分子流行病学调查为了弄清兔出血症病毒的流行规律,从2005年到2007年对山东、江苏、安徽三个养兔大省的家兔的带毒情况进行了调查。首先建立了RHDV RT-PCR检测方法,按照GenBank上公布的RHDV基因序列设计一对引物,以RHDV发病兔的新鲜兔肝为材料,提取总RNA,进行cDNA的合成和PCR扩增,对扩增产物进行克隆测序,扩增出与预期的269bp大小一致的片段,该产物序列与公布的RHDV基因序列有95.8%～98.5%的同源性。然后对RT-PCR方法的敏感性、特异性和RHDV的脏器分布进行试验。结果表明,RT-PCR方法的敏感性是HA的1×10~4倍,对RHD发病死亡兔,能从除粪便以外的各种脏器中检测到病毒。运用建立的RT-PCR方法对采集的不同日龄段的兔鼻拭样品和出入境兔肉产品进行检测,同时选择数个分离毒株进行测序和序列比较。结果,400份出入境兔肉产品中没有发现阳性样品,512份鼻拭子中有48份阳性样品,表明兔出血症病毒在华东三省广泛存在,不同日龄段兔之间的带毒率存在显著性差异;而不同年份、不同省份兔之间带毒率差异不显著;序列比较结果发现三株不同序列,其与Genbank上已公布序列的同源性为93.7%～99.6%。试验Ⅱ、幼兔RHDV三个分离株的基因序列、致病性和免疫原性比较为了研究幼兔RHDV与疫苗株RHDV的区别,应用RT-PCR技术对幼兔RHDV的3个分离株(HN、SH、DY株)和疫苗对照FY株进行RT-PCR扩增,同时进行VP60序列测定、序列比对和蛋白质同源性分析;用4个毒株对兔只进行攻毒,记录死亡时间和死亡率,比较致病性差异;用4个毒株制成疫苗,免疫家兔,比较其免疫原性。结果,3个分离株的基因序列同源性为95.2%～97.4%,氨基酸同源性为95.8%～99.3%;4个毒株的致死率、致病病程和病毒血凝性没有显著性差异;4个疫苗免疫后产生的抗体为同一血清型,各个疫苗均可以抵抗其它毒株的攻击,表明幼兔RHDV的3个分离株与疫苗株之间的免疫原性无显著差异。试验Ⅲ、免疫日龄对兔病毒性出血症抗体水平产生的影响试验1通过攻毒实验比较了新西兰家兔0、1、2、3和4log2 5种抗体水平对的病毒性出血症(RHD)的保护效果,结果表明,抗体效价高于3 log2时可产生100%的保护,2 log2的保护率仅为50%。试验2测定了30、35、40、45、50、55和60日龄非免疫兔的母源抗体效价和用RHD组织灭活苗免疫后8周内血清抗体效价的动态变化,结果发现,母源抗体与日龄呈负相关,在30日龄时最高,可以产生有效的保护,在40日龄后已无保护作用;免疫日龄与抗体峰值和有效保护时间呈正相关,30日龄免疫,抗体效价不但不升、反而显著下降,无保护作用;35日龄免疫,抗体峰值较低,有效保护时间仅为2周;大于40日龄免疫,抗体峰值较高,有效保护时间延长。试验Ⅳ、幼兔出血症免疫程序的研究试验1选择35、40、45三种日龄新西兰家兔各30只,每个日龄兔随机均分为5组,对照组注射生理盐水,4个试验组分别注射4种剂量(1mL、2mL、3mL、4mL)的兔病毒性出血症疫苗,对照组注射生理盐水,分别于免疫前及免疫后1～7周内采血测定血清抗体效价的动态变化。结果表明,各日龄兔的2mL、3 mL、4 mL剂量组的抗体上升速度和有效抗体维持时间均显著大于同日龄的1 mL剂量组;35日龄兔的2 mL、3 mL、4 mL剂量组的抗体峰值显著高于1 mL剂量组。35日龄一次性注射疫苗2mL,其抗体效价在多数时间点显著高于二次注射1mL疫苗的抗体效价。试验2比较两种免疫程序的免疫效果,结果表明,1次性免疫2mL组的抗体水平在大多数时间点显著高于两次免疫1mL组。以上结果提示35日龄免疫、注射剂量2 mL为最佳免疫程序。试验Ⅴ、中药佐剂对RHD疫苗的免疫增强作用为了增强兔出血症疫苗的免疫效果,选择4中药成分(黄芪多糖,淫羊藿多糖,蜂胶黄酮,人参皂甙)组成2个复方,分别与兔出血症疫苗混合后免疫幼兔,以铝胶苗和无佐剂苗为对照,分别于免疫后7、14、21、28、35、49天采血,用微量血凝法测定血清抗体的动态变化,用MTT法测定外周血T、B淋巴细胞转化的动态变化;于免疫后63天测定免疫器官指数并进行攻毒试验。结果表明,复方1在部分时间点能显著提高血清抗体效价,复方2在多数时间点能显著提高血清抗体效价;两个复方在第7天显著促进T淋巴细胞转化,在第7～21天显著促进B淋巴细胞转化;两个复方显著增大圆小囊、肠淋巴结器官指数,提高保护率。试验Ⅵ、中药佐剂对淋巴细胞的转化及其IFN-γ、IL-10 mRNA表达的影响为了进一步证实两个中药成分复方佐剂的免疫增强作用,并探讨其作用机理,比较了两个复方对培养的兔外周血淋巴细胞增殖的作用,运用荧光定量RT-PCR方法比较了两个复方对兔外周血淋巴细胞IFN-γ、IL-10 mRNA表达的影响。结果表明,两个中药成分复方在一定的浓度下可以显著促进淋巴细胞增殖,提高IFN-γ、IL-10 mRNA的表达。这可能是两个中药佐剂增强免疫的机理之一。更多还原

【Abstract】 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.更多还原