Immunofluorescence Study of Wild Rabies Virus and Antibody

A concentrate of wild rabies antibody was prepared from hyperimmune serums of three dogs refractory to wild rabies. The animals resisted repeated intramuscular injections of large doses of wild rabies virus in emulsions of whole brain, in emulsions of submaxillary salivary glands, and in emulsified mixtures of brain and submaxillary glands taken from naturally rabid dogs.

The antibody was conjugated with fluorochrome and then absorbed by a procedure that gave “cell-free” working solutions of fluorescent antibody. The procedure entailed parallel absorption steps with minced pathological canine submaxillary glands from (1) naturally rabid dogs (these glands contained specific, undegraded, natural antigens of live wild rabies virus plus nonspecific substances and antigens) and (2) nonrabid dogs from a rabies endemic region (these glands contained nonspecific substances and antigens).

Extracts from submaxillary glands of the three naturally rabid dogs and one nonrabid dog were stained with a cell-free solution of the fluorescent antibody. The glands of the rabid dogs contained fluorescent aggregates of intense green spherical and filamentous particles. When nonfluorescent canine hyperimmune serum was incubated with rabies-containing submaxillary extract, the rabies antigens were quenched. When nonfluorescent equine fixed virus antiserum was incubated with such extracts, the aggregates still retained bright fluorescence.

     

狂犬病病毒糖蛋白转基因小鼠对该病毒形成免疫耐受

对带有绵羊ＭＴ启动子－狂犬病病毒糖蛋白基因的（ＭＴ－Ｒｇｐ）的ＴｇＮ（ｏＭＴ－Ｒｇｐ）２Ｌｇｅ、ＴｇＮ（ｏＭＴ－Ｒｇｐ）４Ｌｇｅ和ＴｇＮ（ｏＭＴ－Ｒｇｐ）６Ｌｇｅ３株转基因小鼠系（子代）进行了表达检测。ＥＬＩＳＡ结果表明，小鼠肝脏、肾脏中均有糖蛋白表达产物，以肝脏的表达量较高；对肝脏的免疫组化检测结果显示，糖蛋白分布在肝脏实质细胞，主要位于细胞膜上和胞浆内。对８只ＴｇＮ（ｏＭＴ－Ｒｇｐ）２Ｌｇｅ子代鼠一次接种灭活的狂犬病病毒８２０２株，接种前和接种后３周的血清抗体水平变化在转基因鼠和非转基因鼠之间有明显区别，非转基因鼠抗狂犬病病毒抗体水平明显升高，而转基因鼠抗体水平基本保持不变。经统计学分析，转基因鼠的抗体水平变化相差不显著，表明转基因小鼠对该病毒形成了部分免疫耐受性。     

基因重排狂犬病病毒疫苗株免疫效果的初步研究

试验探究了基因重排狂犬病病毒(RV)弱毒疫苗株能否刺激小鼠产生RV抗体免疫球蛋白G(IgG),比较了其不同免疫方式、不同免疫剂量对小鼠抗体水平的影响及安全性评估。选取28只体重(20±3)g、35日龄左右的昆明系小白鼠,随机分为7组:第1组为低剂量口服组:复合佐剂50μL+病毒液50μL;第2组为中等剂量口服组:复合佐剂150μL+病毒液150μL;第3组为高剂量口服组:复合佐剂300μL+病毒液300μL;第4组为低剂量注射组:复合佐剂50μL+病毒液50μL;第5组为中等剂量注射组:复合佐剂150μL+病毒液150μL;第6组为高剂量注射组:复合佐剂300μL+病毒液300μL;第7组为口服对照组:复合佐剂300μL+SRV9亲本毒株300μL,分别于0、7、14d进行免疫,采用ELISA定量检测试剂盒对各免疫组小鼠的血清IgG水平进行检测,免疫期结束后取各免疫组小鼠的肝脏、肾脏、肺脏、脑组织制作病理组织切片对疫苗的安全性进行评估。结果显示,各免疫组均产生了RV抗体IgG;肌内注射免疫产生的抗体速度较口服免疫快,但肌内注射免疫会导致小鼠发病死亡;600μL口服免疫组产生的抗体水平显著高于100、300μL口服免疫组及亲本毒株SRV9对照组(P<0.05),说明适当加大口服免疫剂量会使口服免疫组产生的抗体水平增加,并且不会引起不良反应。病理组织学检查发现,各口服免疫组小鼠的肝脏、肾脏、肺脏、脑组织的形态结构较为正常,未发生病变,表明基因重排RV疫苗株口服免疫副作用小、安全性较好。本试验结果为研制新型口服疫苗开辟了新的方向。     

Adsorption of Wild Rabies Virus and Neurotropic-Adapted Viruses by Activated Attapulgite

The adsorption by attapulgite (a naturally occurring clay) of wild rabies virus in naturally rabid dogs was investigated. Attapulgite selectively absorbed rabies virus in the brain tissue of naturally rabid dogs and rejected rabies virus in extracts from the submaxillary salivary glands of the rabid dogs. Attapulgite rejected the neurotropic-adapted viruses lymphocytic choriomeningitis, Eastern equine and Western equine encephalitis, Japanese B encephalitis, and St. Louis encephalitis in the brain tissue of suckling mice.     

流浪犬狂犬病口服疫苗免疫效果评估

为了解狂犬病病毒CAV2-ΔE3-CGS口服疫苗在流浪犬体内产生的抗体水平,探索抗体产生规律,选取上海市收容的12只流浪犬开展口服疫苗免疫试验,按照免疫程序采用间接ELISA方法测定狂犬病病毒免疫抗体.结果显示:CAV2-ΔE3-CGS口服疫苗一免和二免后平均抗体水平分别达到0.23和0.81 IU/mL;口服疫苗平均...     

新疆部分地区散养犬狂犬病免疫抗体监测与效果评估

[目的]了解、评估新疆散养犬近几年狂犬病抗体水平和疫苗免疫效果。[方法] 2010—2014年,在新疆部分地区开展免疫犬狂犬病摸底调查工作,采集犬血895份,用间接ELISA方法进行狂犬病免疫抗体检测。[结果] 895份检测样品中,免疫抗体合格数497份,合格率为55.53%。[结论] 五年来散养犬狂犬病抗体水平忽高忽低,免疫合格率处于较低水平,今后要加大对散养犬的狂犬病免疫密度及抗体监测。     

Antigenic Characterization of Rabies Virus Isolates from Vaccinated Dogs in Plateau State,Nigeria

Rabies isolates (genotype 1 lyssaviruses) from vaccinated dogs that died of rabies infection in the Plateau area of Nigeria were characterized using monoclonal antibodies (MAbs). The isolates were examined for rabies (genotype 1) and rabies-related (genotypes 2, 3 and 4) viruses by the indirect fluorescent antibody test carried out with MAb 502-2, which recognizes the nucleocapsid protein of all known lyssaviruses, and with MAb 422-5, which identifies only rabies-related viruses. All three isolates showed positive immunofluorescence with MAb 502-2 and were negative with MAb 422-5, indicating that they were all rabies (genotype 1) viruses.Characterization with a panel of 36 anti-nucleocapsid monoclonal antibodies showed that all three isolates reacted positively with 35 of the anti-nucleocapsid MAbs, including MAb 102-27 and MAb 377-7. Characterization using a panel of 44 anti-glycoprotein MAbs differentiated the isolates sharply from LEP Flury and PM vaccine viruses. The pattern of anti-glycoprotein reactivity of the isolates showed them to belong to one distinct viral subtype, except for a minor variation in one isolate that was not neutralized by MAb 1101-3. None of the three isolates was identified as the Flury low egg passage (LEP) vaccine strain used for vaccinating dogs in Nigeria. In fact, all the three isolates had the typical pattern of reactivity of isolates from unvaccinated dogs, including MASS 83, a rabies virus isolated in Nigeria and characterized at the Wister Institute before this study.     

成都市区犬猫狂犬病病毒和弓形虫感染的调查

为了解四川省成都市区家养犬、猫狂犬病病毒及弓形虫的感染情况,采用商品化狂犬病病毒和弓形虫抗原快速检测试纸卡,对2010年8～10月间来自成都市区的103份家养犬以及75份家猫的唾液和血液样品进行检测;同时采用文献报道的PCR方法对家养犬血液样品中的弓形虫核酸进行检测。结果显示,103份家养犬唾液样品狂犬病病毒抗原均为阴性,而75份家猫唾液样品中,检出阳性样品5份(阳性率6.7%),可疑样品7份;103份家养犬血液样品弓形虫抗原阳性样品33份(32.0%),可疑样品22份,75份家猫血液样品中,检出阳性样品2份(阳性率2.7%),可疑样品3份。弓形虫核酸PCR检测结果显示,96份家养犬血液样品弓形虫核酸阳性样品57份(阳性率59.4%),与弓形虫抗原阳性和可疑样品总和所占比例基本一致(53.4%)。提示应重视源于家猫的狂犬病病毒和家养犬弓形虫对人的威胁。     

肌注狂犬病病毒糖蛋白cDNA表达载体诱导的小鼠体液免疫反应

将狂犬病病毒糖蛋白ｃＤＮＡＢｇｌⅡ（１．６７ｋｂ）片段分别正向插入ｐｍＭＴ－１ＢｇｌⅡ位点和ｐＭＴ０１０／Ａ＋ＢａｍＨＩ位点，构建重组质粒ｐｍＭＴ－Ｒｇｐ和ｐＭＴ０１０／Ａ＋－Ｒｇｐ，通过磷酸钙共沉淀法转染ＢＨＫ－２１细胞，经ＰＣＲ、打点杂交及ＥＬＩＳＡ检测，证明糖蛋白基因发生了整合并获得表达。以该表达载体给小鼠肌肉内注射２～３次，采其注射前、后双份血清，经ＥＬＩＳＡ检测证明，注射了表达载体的小鼠，血清中病毒特异性抗体明显升高，表明狂犬病病毒糖蛋白ｃＤＮＡ在小鼠体内表达后，刺激机体产生了抗该糖蛋白特异性抗体     

狂犬病毒的增殖及保藏效果的初步研究

狂犬病是人和家畜的一各生中枢神经系统的疾病，严重地威胁人类和家畜的生命。狂犬病街毒的收集，增殖及对这些毒株进行妥当的保藏，有利于人们充分保护这些病毒资源，并为今后利用该病毒开展治病防病工作提供实验材料。     

鹿源狂犬病野毒8202株基因型的研究

为了从基因水平确定鹿源狂犬病野毒8202株与其它狂犬病病毒的进化关系,应用RT-半嵌套式PCR技术,利用3条引物对病毒的核蛋白基因进行扩增、克隆及序列测定,并与已发表的狂犬病病毒株3aG、PV、CVS、HEP-Flury、RC-HL、Nishigahara、SADB19的核蛋白基因进行了比较分析。结果表明,8202株与其它7个病毒株均来源于同一个进化枝,属于基因Ⅰ型。     

Electrophoresis Study of Extracts of Submaxillary Salivary Glands from Naturally Rabid Dogs

Rabies virus in submaxillary salivary glands from naturally infected dogs was investigated by a paper electrophoresis technique, and the virus activity was quantitated by intracerebral titration in mice. Extracts from these salivary glands were found to contain (a) 10⁴ to 10⁶ mouse ICLD ₅₀ units of wild rabies seed and (b) a protein complex that migrated electrophoretically in the albumin band and more conspicuously in the beta and gamma bands of normal horse serum. The protein complex was interpreted to comprise aggregates of neutralizing antirabies antibody.     

Genetic Variation of An Esterase System in Sera of Dogs

Sera from 7 dog families comprising 20 parents with 48 offspring and 70 unrelated dogs of various breeds have been studied with regard to polymorphism of esterases. Isoelectric focusing in Polyacrylamide gels of pH ranges 4.2–4.9 and 4–6 revealed great variation of phenotypes. These appeared as complex band patterns always with more than 8 zones. Treatment with an organophosphorous compound and ions of heavy metals strongly indicate that the majority of zones represent arylesterase (ArE) whereas some of the bands are Cholinesterase. ArE phenotypes in families together with the appearances of band patterns in the 70 unrelated dogs are in agreement with a genetic theory of 1 locus with 5 codominant alleles. These have temporarily been named ArE^D, ArE^H, ArE^K, ArE^Q and ArE^W with ArED being the most anodal phenotype and the others in logical order. The ArE^K and ArE^Q were most common but phenotypes representing the ArE^D, ArE^H and ArE^W alleles were each observed in several animals.     

Antibodies Against Helicobacter felis in Sera of Cats and Dogs

Serum samples from 61 dogs and 49 cats were screened for circulating antibodies against Helicobacter felis by an enzyme-linked immunosorbent assay (ELISA) using sonicated bacteria as an antigen. To improve the specificity of the ELISA, sera were absorbed with Campylobacter jejuni subsp. jejuni H. pylori as well as H. felis. Sera from 26 dogs (43%) and 19 cats (39%) revealed clear positive absorbance readings determined as an optical density (OD) that was statistically significant above the OD mean value [P < 0.025 (one-tailed); log₁₀]. The absorbance pattern of ELISA-positive sera corresponded to results obtained with bovine and human reference sera. Furthermore, a correlation between the immune response and results from histopathological examination of gastric specimens from 22 dogs was demonstrated. It could be shown that antibodies against H. felis in sera of cats and dogs can easily be detected using an ELISA. The diagnostic value of this test must be evaluated in further investigations.     

湖南部分地区犬狂犬病病毒的监测研究

对湖南部分地域的8个县(市、区)的8个城区、乡镇和农村的健康犬抽样采集脑组织标本142份,应用RT-PCR和直接荧光抗体试验(FAT)检测,检测结果142份犬脑样中均不含狂犬病病毒。调查上述监测点及周边近三年狂犬病的免疫情况,免疫率均超过70%。表明湖南部分犬免疫工作较好的地区,通过加大群免疫密度,可以使犬狂犬病病毒携带率降低,这可能与形成免疫屏障有关。     

紫锥菊增强犬瘟热疫苗和犬细小病毒疫苗免疫效果的研究进展

详细阐述了紫锥菊的药理活性、安全药理学、临床应用以及毒理学研究等内容。紫锥菊可作为免疫增强剂,提高犬瘟热疫苗和犬细小病毒疫苗的免疫效果,本文为其研究与开发提供一定的参考。     

表达狂犬病病毒糖蛋白的重组鸡痘病毒的构建和鉴定

将 RVG基因插入到鸡痘病毒表达载体 p UTA- 2 Sm a 位点获得重组转移载体 p U TA- RVG,利用脂质体转染已感染中国鸡痘病毒疫苗株 2 82 E4株 2～ 3h的鸡胚成纤维 (CEF)细胞 ,收获病毒后 ,用 Brd U法进行加压筛选重组病毒 ,PCR检测到重组病毒 RVG基因 ,Western blot检测出 RVG,从而证实已成功构建了表达 RVG的重组鸡痘病毒     

Nested PCR检测狂犬病病毒方法的建立及病毒在小鼠体内的移行动态

应用建立的Nested PCR特异地检出狂犬病病毒株CVC、HEP-Flury、ERA、RC-HL、1008、Komatsug-awa的RNA,但对类狂犬病病毒Lagos bat、Duvenhage、Mokola及水泡性口膜炎病毒、轮状病毒、犬瘟热病毒均为阴性。该法敏感性很高,能检出3 TCID50或0.8pg的狂犬病病毒RNA。用该法测定了小鼠脑内感染CVS株后的病毒增殖和移行动态,对感染小鼠的主要内脏器官进行了病毒RNA检测,结果发现小鼠脑内感染CVS 5 d以后在其心、肝、脾、肺等内脏器官均检出了病毒RNA。