羊瘙痒因子263K感染的金黄仓鼠脑中PrP~(Sc)的动态沉积特点

目的　观察羊瘙痒因子 2 63K感染的金黄仓鼠脑中PrPSc的沉积特点。方法　颅内接种羊瘙痒因子 2 63K感染金黄仓鼠 ,分别于接种后第 2 0天、40天、50天、60天和 70天取脑组织 ,用免疫组化法检测PrPSc的沉积 ,HE染色观察病理变化 ,Westernblot检测蛋白酶抗性PrPSc。结果　颅内接种 2 63K后第 2 0天 ,脑组织中即可检测到耐热和耐蛋白酶K的PrPSc的沉积 ,呈散在分布 ;第 40天可见细胞内PrPSc的沉积 ,第 50天脑组织中出现灶性、颗粒状、细胞内PrPSc的沉积 ,累及部位扩大 ,第60 - 70天可见弥漫性呈桑葚状、斑块状沉积物。PrPSc主要沉积于大脑皮质Ⅲ、Ⅳ、Ⅴ层 ,海马锥体细胞层 ,小脑皮质颗粒层。HE染色结果显示大脑皮质海绵样病变在接种后 40天出现 ,随着接种时间的延长而加剧。Westernblot检测显示PrPSc在接种后 40天即可检出 ,PrP总量和PrPSc含量随着接种时间的延长而增加。结论　随着接种时间延长 ,羊瘙痒因子 2 63K感染的金黄仓鼠脑中PrPSc沉积累及的范围、程度扩大 ,IHC检测脑组织中PrPSc的沉积早于临床症状的出现 ,甚至其它检测方法的检出。     

羊瘙痒病小鼠适应株ME7可突破种属屏障颅内感染金黄地鼠

目的　羊瘙痒病感染因子在哺乳动物种属间的传播认为受种属屏障的限制 ,研究羊瘙痒病小鼠适应株ME7在仓鼠中的发病特征。方法　ME7毒株颅内接种至金黄地鼠 ,观测疾病发生的潜伏期、临床病程和主要症状 ;Westernblot和电子显微镜动态观测脑组织中PrPSc和痒病相关纤维的出现和分子特征。结果　与以往报道的金黄地鼠适应株 2 6 3K和小鼠适应株 139A不同 ,在接种后 4 6 0～ 5 30d后 ,感染动物出现明显的症状 ,主要为消瘦、嗜睡、运动迟缓 ;感染动物脑组织中存在有羊瘙痒病相关纤维和PrP -res;羊瘙痒病相关纤维和PrP -res的出现明显早于临床发病 ;比较三种毒株在仓鼠和小鼠脑组织中出现的PrP -res发现虽然在电泳移动位置上无差异 ,但ME7和 139A的无糖分子比例明显降低。结论　小鼠适应株ME7可突破种属屏障感染金黄地鼠 ;不同毒株在潜伏期、主要临床症状和临床病程等方面有明显差异 ;PrP -res的形成不仅受接种毒株的影响 ,而且受宿主PrP分子特征的影响     

羊瘙痒因子263K经多种途径感染仓鼠的脑外组织PrP~(Sc)分子和沉积特点研究

目的　比较研究实验仓鼠经不同途径感染羊瘙痒病 (Scrapie)毒株 2 6 3K的终末期病鼠脑外组织内PrPSc分子和沉积特点。方法　以Scrapie 2 6 3K毒株经颅内、腹腔、心内、肌肉注射及灌胃等途径接种金黄地鼠 ,在终末期取脑、脊髓、脾、肾、舌、肌肉、小肠回盲部和胃组织 ,用HE染色观察病理变化 ,免疫组化法检测PrPSc的组织沉积特点 ,Westernblot检测PrPSc分子特征。结果　五种感染方式均可引起动物发病 ,在脑和脊髓组织中观察到典型的病理改变 ;PrPSc免疫组化检测显示外周途径感染动物脊髓白质内有大量沿纤维走行的PrPSc沉积 ,灰质前后角内围绕空泡点状或网状沉积 ,而颅内感染主要在中央管附近和后角出现大量的点状PrPSc沉积 ;脾脏、肾、小肠回盲部、胃组织中均观察到点状PrPSc的沉积 ;WesternBlot检测发现不同感染途径动物脊髓提取物均出现可抵抗蛋白酶消化的PrPSc条带 ,与脑提取物中PrPSc电泳性状完全一致 ;外周组织仅在脾脏检测到抵抗蛋白酶消化的PrPSc,但与正常对照比较 ,各种组织中PrP的总量明显增高 ,同时呈现与中枢神经组织不同的PrP电泳特征。结论　在TSE感染发病过程中 ,多种组织细胞成分可能参与了TSE感染因子的向中枢神经系统传递过程 ,PrP蛋白在中枢神经组织和其他外周组织细胞中的翻译后修饰及     

2005年中国人兽共患病杂志总目次

·述评·WHO和我国发出警告:防止马尔堡病毒病传入…………………………………………刘岱伟,于恩庶(8·641)严防猪链球菌感染人…………于恩庶,刘岱伟,李友松(9·737)布鲁氏菌17·3 kDa蛋白的表达及其免疫学评价………………………………曾政,邓小红,王希良(10·835)·论著·日本血吸虫SIEA26-28kDa的抗雌虫生殖免疫作用———HGPRT是否是主要的靶抗原(英文)………………………………李文凯,汪世平,吕志跃等(1·1)羊瘙痒因子263K经多种途径感染仓鼠的脑外组织PrPSc分子和沉积特点研究………张瑾,陈岚,肖新莉等(1·5)流感病毒血凝素…     

脂质体葡糖胺锑用于利什曼病时原虫毒力对治疗的影响

作者使用以脂质体为载体的葡糖胺锑(MA)治疗实验感染的金色仓鼠,并观察了原虫毒力对药物治疗的影响。试验方法是将已感染杜氏利什曼的仓鼠肝内的原虫给体重50～70g的仓鼠心内注射接种,每鼠10~7个原虫,感染后70天,以心脏注射给药法,用脂质体葡糖胺锑进行单剂或分剂治疗,连续给药     

脑型疟发病中肿瘤坏死因子α与一氧化氮的作用机制研究

目的 研究肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)与一氧化氮(nitric oxide,NO)在脑型疟(cerebral malaria,CM)发病中的作用. 方法首先建市C57BL/6J小鼠的CM动物模型.然后,取雌性(C57BL/6J 小鼠100只,随机分为:正常对照组,腹腔注射生理盐水;感染对照组,接种约氏疟原虫By265;CM模型组,腹腔注射感染们氏疟原虫K173;地塞米松处理组,于感染伯氏疟原虫K173前一天在饮水中加入地塞米松,浓度为10 ms/L;L-硝基精氰酸(L-NNA)处理组,从感染伯氏疟原虫K173当天开始每只每大腹腔注射25 g/L的L-NNA溶液0.2 ml.每组均为20只.CM 模型组鼠于CM发病时取脑组织,其他各组小鼠于感染后第10天取脑组织匀浆.观察脑组织TNF-α、NO及氧化氮合酶(nitric oxide synthase,NOS)在CM发病过程中的含量变化;同时观察TNF-α合成抑制剂地塞米松及NO合成抑制剂L-NNA对脑组织中TNF-α和NO浓度的影响. 结果 (1)CM模型组小鼠均发牛CM,地塞米松组、L-NNA 组及感染对照组小鼠至感染后第10天均末发生CM.(2)小鼠CM发病时脑组织TNF-α、NO、NOS均高于感染第5天(P＜0.01).(3)小鼠CM发病时和感染后第5天脑组织FNF-α、NO、NOS均高于感染对照组和止常对照组(P＜0.01).(4)地塞米松组小鼠感染后第10天、第5天与同时间CM模型组比较,脑组织TNF-α均明显下降(P＜0.01).(5)L-NNA组与CM模型组比较,脑组织NO、NOS均显著性降低(P＜0.01). 结论 (1)成功地建立起较理想的CM动物模型.(2)伯氏疟原虫感染小鼠给予地塞米松或L-NNA后,可有效地预防CM的发病.     

巴泰病毒云南株生物学性状研究

摘要： 目的 掌握巴泰（Batai）病毒生物学特性,为巴泰病毒的监测和调查研究提供科学依据。方法 用分离自云南省菲律宾按蚊（Anopheles philippinensis）的巴泰病毒LC92-4株,采用细胞培养、小白鼠感染、血凝抑制、间接免疫荧光、空斑试验以及病理检测和电镜观察等方法进行生物学特性试验。结果 巴泰病毒可引起＜4周龄小白鼠发病死亡。感染鼠脑病毒颅内、皮下和腹腔接种2周龄小白鼠的LD50分别为5.67logLD50/0.025mL、5.3logLD50/0.03mL和4.5logLD50/0.03mL。感染病毒鼠的心、肝、脾、肺、肾病毒悬液颅内接种2周龄小白鼠,LD50分别为3.75、4.25、2.25、3.0、2.25 logLD50/0.025mL。感染发病小白鼠脑、心、肝、脾、肺、肾均发现病理改变。感染鼠的脑组织经电镜检测发现圆形有包膜病毒颗粒,大小约82nm。巴泰病毒能在BHK21和Vero细胞产生明显病变,并可产生空斑;在BHK21细胞上,空斑滴度为6.04logPFU/mL;在C6/36细胞上不产生病变。感染病毒鼠脑接种BHK21和Vero细胞,TCID50分别为 5.50logTCID50/0.025mL和5.00 logTCID50/0.025mL。在pH5.75～7.0条件下,巴泰病毒不具有与鸽、鹅、鸡、鸭、人O型和绵羊红血球发生凝集的特性;空斑减少中和试验检测云南省357份人血清,阳性2份,中和抗体滴度均为1:10。结论 乳小白鼠和幼年小白鼠对巴泰病毒高度易感。BHK21和Vero细胞对巴泰病毒敏感,可用于病毒分离培养和空斑测定。建立的免疫荧光和空斑减少中和试验具有特异性,可用于巴泰病毒抗体检测。应进一步开展该病毒的分布及其与疾病关系的研究。     

γ-干扰素联合微卡免疫治疗结核病小鼠的实验研究

目的研究γ-干扰素联合微卡对结核分枝杆菌感染小鼠的免疫治疗作用及其免疫学机制。方法60只BALB/c小鼠用随机数字表法随机分为正常组、模型组、γ-干扰素组、微卡组及联合组,每组12只。除正常组不造模外,其余4组小鼠经尾静脉注射结核分枝杆菌(H37Rv,106个菌/只)。微卡组于攻毒后10 d腹腔内注射微卡22.5μg/只。γ-干扰素组攻毒后10天腹腔注射γ-干扰素(1万U/只,1次/d)5 d,间隔2 d,继续腹腔注射5 d。联合组2者均注射。感染6周后处死30只小鼠(每组6只),称取小鼠体质量和肺脏及脾脏质量。取肺、脾组织进行结核菌培养和菌落计数,观察肺脏病理改变,同时用ELISA法检测血清IFN-γ、IL-4水平和用RT-PCR法测定肺组织IFN-γmRNAI、L-4 mRNA的表达。其余30只小鼠观察60 d,计算其存活时间。结果模型组肺组织病理改变以渗出为主,病变广泛,增生改变不明显;3个用药组病变范围局限,并可见类上皮细胞、纤维母细胞。模型组观察期结束无一小鼠存活;正常组和联合组在观察期内无一死亡,γ-干扰素组和微卡组在观察期内仅有1只小鼠死亡。感染后6周,γ-干扰素组肺脾菌荷量(×106CFU/g...     

1975年新疆出血热现场病原学工作总结

1975年3月下旬到7月初在新疆阿克苏农一师三团哈拉库勒地区进行了新疆出血热的防治工作,于此期间从急性期患者血液中分离到3株病原体,现场初步鉴定结果及羊只感染结果报告于下。1材料和方法1.1病毒的分离与初步鉴定:标本包括急性期患者血液、疫区绵羊血液,野生动物(如子午沙鼠、鸟类等)血液和脏器,以及流行季节原因不明发热患者的血液。标本处理基本按本实验室常规进行。病人的血液以无菌手续取静脉血液不加抗凝剂(或者取血块研磨后加等量稀释剂)立即接种1~2日龄新生乳鼠脑内0.01mL和腹腔0.02mL,接种后的乳鼠一般观察14天,如此期间出现可疑发病症状,则解剖取脑组织制成10%悬液传代,如无症状,则于接种7~10天左右,任取乳鼠2只育传一代,第2代阴性者弃去。发病乳鼠传代按常规进行,稀释液采用10%灭活正常兔血清磷酸缓冲盐水(pH7.4),制成10%悬液传代。第2代发病典型的乳鼠取脑制成乙醚抗原以备鉴定用。1.1.1不同途径敏感性试验:取第3~7代发病乳鼠脑组织制成10%悬液经2000~2500rpm/min,20~30min离心沉淀后取上清液连续10倍稀释至10-7,然后取不同稀释度接种1~3日龄乳鼠脑内,腹腔、皮下各...     

PRU株弓形虫感染小鼠后脑组织免疫病理的研究

目的了解弓形虫PRU株感染小鼠后脑内局部细胞因子对脑组织的免疫病理作用。方法 51只ICR小鼠分为感染组(33只)和对照组(18只),感染组经腹腔注射弓形虫PRU株10个包囊/鼠,对照组腹腔注射同等量的PBS。于感染后第5d、10d、15d、20d、30d和90d,剖杀感染组小鼠5只和对照组小鼠3只,取脑组织,部分作病理切片;部分提取其RNA,检测IFN-γ、IL-4、IL-6和TNF-αmRNA;部分获取脑组织上清液,用ELISA法测定上述细胞因子。结果相对于正常对照组,感染小鼠脑组织中IFN-γ于感染后第5d开始升高,第10d时下降达到最低,之后开始上升;TNF-α在感染第10d或15d时明显降低,接着上升并于第30d时达到高峰;IL-6在感染第5d时开始升高,感染第10d时降至最低,之后缓慢上升,并于第30d后达到高峰;IL-4未见明显变化。结论弓形虫PRU感染ICR小鼠的过程中,脑组织中IFN-γ、IL-6和TNF-α细胞因子在感染第10d或15d的低表达有利于弓形虫逃避宿主的免疫杀伤作用,导致速殖子在脑组织中存活;同时细胞因子的分泌不平衡,导致小鼠脑组织中出现弓形虫包囊与病理变化共存的隐性感染状态。     

Autocatalytic self-propagation of misfolded prion protein

Prions are thought to replicate in an autocatalytic process that converts cellular prion protein (PrP(C)) to the disease-associated misfolded PrP isoform (PrP(Sc)). Our study scrutinizes this hypothesis by in vitro protein misfolding cyclic amplification (PMCA). In serial transmission PMCA experiments, PrP(Sc) was inoculated into healthy hamster brain homogenate containing PrP(C). Misfolded PrP was amplified by rounds of sonication and incubation and reinoculated into fresh brain homogenate every 10 PMCA rounds. The amplification depended on PrP(C) substrate and could be inhibited by recombinant hamster PrP. In serial dilution experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed the structural conversion of PrP(C) as efficiently as PrP(Sc) from brain of scrapie (263K)-infected hamsters, yielding an approximately 300-fold total amplification of PrPres after 100 rounds, which confirms an autocatalytic PrP-misfolding cascade as postulated by the prion hypothesis. PrPres formation was not paralleled by replication of biological infectivity, which appears to require factors additional to PrP-misfolding autocatalysis.     

New studies on the heat resistance of hamster-adapted scrapie agent: threshold survival after ashing at 600 degrees C suggests an inorganic template of replication

One-gram samples from a pool of crude brain tissue from hamsters infected with the 263K strain of hamster-adapted scrapie agent were placed in covered quartz-glass crucibles and exposed for either 5 or 15 min to dry heat at temperatures ranging from 150 degrees C to 1,000 degrees C. Residual infectivity in the treated samples was assayed by the intracerebral inoculation of dilution series into healthy weanling hamsters, which were observed for 10 months; disease transmissions were verified by Western blot testing for proteinase-resistant protein in brains from clinically positive hamsters. Unheated control tissue contained 9.9 log(10)LD(50)/g tissue; after exposure to 150 degrees C, titers equaled or exceeded 6 log(10)LD(50)/g, and after exposure to 300 degrees C, titers equaled or exceeded 4 log(10)LD(50)/g. Exposure to 600 degrees C completely ashed the brain samples, which, when reconstituted with saline to their original weights, transmitted disease to 5 of 35 inoculated hamsters. No transmissions occurred after exposure to 1, 000 degrees C. These results suggest that an inorganic molecular template with a decomposition point near 600 degrees C is capable of nucleating the biological replication of the scrapie agent.     

Pall leukotrap affinity prion-reduction filter removes exogenous infectious prions and endogenous infectivity from red cell concentrates

BACKGROUND AND OBJECTIVES: Three recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In this study, we investigated the performance of a new filter for reducing the levels of infectious prions (PrP(Sc)) from red cell concentrates (RCC). MATERIALS AND METHODS: Endogenous Infectivity: A pool of 500 ml of whole blood was collected from 263K-strain scrapie-infected hamsters into an anticoagulant, processed into non-leucoreduced RCC (NL-RCC), and then passed through a prion-reduction filter. Pre- and postfiltration samples were tested for PrP(Sc) by Western blot and infectivity by inoculation of healthy hamsters. Results of the endogenous infectivity study after 200 days post-inoculation are discussed. Exogenous (Spiking) Study: Scrapie-infected hamster brain homogenates containing PrP(Sc) were added to human RCC and then filtered. Levels of PrP(Sc) were determined by Western blot assay. The effect of prior leucodepletion of 'spiked' RCC on PrP(Sc) removal by the prion-removal filter was also assessed. RESULTS: In the endogenous infectivity study, at 200-day observation time, the prefiltered RCC transmitted disease to six of the 187 hamsters, whereas the filtered RCC did not transmit disease to any of 413 animals, P = 0.001. The prion filter also significantly reduced the concentration of leucocytes in the RCC by about 4 logs, P < 0.05. In the exogenous (spiking) study, the level of PrP(res) was significantly reduced in RCC P < 0.05. Prior leucodepletion of the RCC with a leucoreduction filter did not significantly reduce the concentration of exogenously spiked PrP(Sc), P > 0.05. CONCLUSION: The use of this new prion-reduction filter should reduce the risk of vCJD transmission through transfusion of RCC, the most widely transfused blood component.     

Tetracyclines affect prion infectivity

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrP(Sc) are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Based on previous findings that tetracyclines revert abnormal physicochemical properties and abolish neurotoxicity of PrP peptides in vitro, we tested the ability of these compounds to interact with PrP(Sc) from patients with the new variant of Creutzfeldt-Jakob disease (vCJD) and cattle with bovine spongiform encephalopathy (BSE). The incubation with tetracycline hydrochloride or doxycycline hyclate at concentrations ranging from 10 microM to 1 mM resulted in a dose-dependent decrease in protease resistance of PrP(Sc). This finding prompted us to investigate whether tetracyclines affect prion infectivity by using an animal model of disease. Syrian hamsters were injected intracerebrally with 263K scrapie-infected brain homogenate that was coincubated with 1 mM tetracycline hydrochloride, 1 mM doxycycline hyclate, or vehicle solution before inoculation. Hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. These effects were paralleled by a delay in the appearance of magnetic-resonance abnormalities in the thalamus, neuropathological changes, and PrP(Sc) accumulation. When tetracycline was preincubated with highly diluted scrapie-infected inoculum, one third of hamsters did not develop disease. Our data suggest that these well characterized antibiotics reduce prion infectivity through a direct interaction with PrP(Sc) and are potentially useful for inactivation of BSE- or vCJD-contaminated products and prevention strategies.     

Distribution of PrP(Sc) in cattle with bovine spongiform encephalopathy slaughtered at abattoirs in Japan

Three 80- to 95-month-old Holstein dairy cattle infected naturally with the agent of bovine spongiform encephalopathy (BSE) and slaughtered at abattoirs in Japan were examined for the distribution of disease-specific and protease-resistant prion protein (PrP(Sc)) by immunohistochemistry (IHC) and Western blot (WB) analyses. The cattle showed no clinical signs or symptoms relevant to BSE but were screened as positive by enzyme-linked immunosorbent assay, a rapid test for BSE. This positive result was confirmed by IHC or WB in a specimen of the medulla oblongata. Histopathologically, these cattle showed no vacuolation in tissue sections from the central nervous system except for the medulla oblongata. Both IHC and WB analyses revealed PrP(Sc) accumulation in the brain, spinal cord, satellite and ganglionic cells of the dorsal root ganglia, and the myenteric plexus of the distal ileum. In addition, small amounts of PrP(Sc) were detected in the peripheral nerves of 2 cattle by WB. No PrP(Sc) was demonstrated by either method in the Peyer's patches of the distal ileum; lymphoid tissues including the palatine tonsils, lymph nodes, and spleen; or other tissues. The distribution of PrP(Sc) accumulation in the preclinical stage was different between naturally infected cattle and cattle inoculated experimentally with the BSE agent.     

Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins.

Scrapie-associated fibrils (SAF) and protease-resistant proteins (PrP) were isolated from spleens and brains of clinical animals (mice and hamsters) from three scrapie agent-host strain combinations, and their concentrations were compared with infectivity levels. The spleens of infected animals contained lower levels of infectivity, PrP, and SAF than did brains. Regardless of the route of infection, both SAF and infectivity were detected in spleen before brain. Infectivity increased in brains and spleens of 139A-infected mice before the detection and increase in SAF, suggesting that the synthesis of SAF and PrP may not be the limiting factor in agent replication. In contrast to those in ME7- and 263K-infected animals, the Western blot profiles for PrP from brain and spleen of 139A-infected mice exhibited distinct differences. Results indicate that SAF and PrP found in the spleens are both organ- and scrapie strain-specific.     

Prion infectivity in variant Creutzfeldt-Jakob disease rectum

BACKGROUND: Disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues in variant Creutzfeldt-Jakob disease (vCJD), but not in other forms of human prion disease. This distinctive pathogenesis, with the unknown population prevalence of asymptomatic vCJD infection, has led to significant concerns that secondary transmission of vCJD prions will occur through a wide range of surgical procedures. To date PrP(Sc):prion infectivity ratios have not been determined in vCJD, and it is unknown whether vCJD prions are similar to experimental rodent prions, where PrP(Sc) concentration typically reflects infectious prion titre. AIM: To investigate prion infectivity in vCJD tissue containing barely detectable levels of PrP(Sc). METHODS: Transgenic mice expressing only human PrP (Tg(HuPrP129M(+/+)Prnp(o/o))-35 and Tg(HuPrP129M(+/+)Prnp(o/o))-45 mice) were inoculated with brain or rectal tissue from a previously characterised patient with vCJD. These tissues contain the maximum and minimum levels of detectable PrP(Sc) that have been observed in vCJD. RESULTS: Efficient transmission of prion infection was observed in transgenic mice inoculated with vCJD rectal tissue containing PrP(Sc) at a concentration of 10(4.7)-fold lower than that in vCJD brain. CONCLUSIONS: These data confirm the potential risks for secondary transmission of vCJD prions via gastrointestinal procedures and support the use of PrP(Sc) as a quantitative marker of prion infectivity in vCJD tissues.