用Vero—E6细胞直接从野鼠脏器中分离恙虫病立克次体

近年来流行病学和血清学调查显示恙虫病分布较广、疫源地不断扩大、长江以北逐年增多。由于传统的病原分离和增殖方法有一定的限制，以及对弱毒株又无法从动物分离方法中获得，至使有的毒株丢掉。近年国内用L929细胞从病人血液分高立克次体已被证实。本文报告从野外疫区捕获的大林姬鼠等9种野鼠，用Vero－E6细胞直接分离到恙虫病立克次体9株。结果报告如下。材料与方法一、试验材料（一）标本来源：1992～1994年的5～6月间，在吉林省珲春市、黑龙江省密山市、辽宁省宽甸县调查点上，采用d笼法捕获活野鼠，带回实验室，以同生境，同种鼠3～…     

应用NPCR发现我国Kawasaki型恙虫病立克次体

本文报告用恙虫病立克次体表面蛋白５６ＫＤａ型特异抗原基因编码区的引物，采用嵌合式聚合酶链反应鉴定江苏地区的２株恙虫病立克次体。结果该２株恙虫病立克次体与ＧｉｌｌｉａｍＫａｒｐ，Ｋａｔｏ和Ｋｕｒｏｋｉ型特异引物无任何ＤＮＡ扩增带，而与日本Ｋａｗａｓａｋｉ株型特异引物扩增后有５２３ｂｐ的ＤＮＡ扩增带，表明我国存在Ｋａｗａｓａｋｉ型恙虫病立克次体。     

云南省云龙县恙虫病立克次体生物学特性研究

通过对云南省云龙县恙虫病立克次体分离株对实验动物的致病性研究表明，各分离株对小白鼠和豚鼠均有较强的致病性，对家兔亦有一定的致病性，而恒河猴则不敏感；经毒力测定，７个分离株中，有５株ＬＤ５０≥８．５，为强毒株。该县恙虫病立克次体分离株抗原型以Ｇｉｌｌｉａｍ型为主，其次为Ｋａｒｐ型，不同的抗原型株间有很强的交互保护力。     

恙虫病立克次体弱毒株分离方法的研究

江苏省于1986年发现秋冬型恙虫病的流行,并从病原学和血清学得到证实。但用病人、鼠、螨标本接种小白鼠,鼠的症状和病变不明显,且难以检出恙虫病立克次体。1990年以来,我们在分离恙虫病立克次体方法上作了以下改进:多次用环磷酰胺和稀释液处理接种标本的小白鼠;增加稀释液的成分;把握好小白鼠的传代时机等。结果从病人外周血、鼠和小盾纤恙螨中均分离到恙虫病立克次体,说明该方法对分离恙虫病立克次体弱毒株是有效的。     

恙螨体内恙虫病立克次体经精胞传递的实验研究

本文报道地里纤恙螨雄虫人工接种恙虫病立克次体后与雌虫配对培养和传代，应用幼虫叮咬小鼠分离立克次体和PCR结合核酸杂交（核酸杂交检测PCR产物）检测子代体内立克次体的结果。幼虫叮咬小鼠分离立克农作检查第3子代幼虫体内立克次体阳性。PCR结合核酸杂交检测佛山市的病人恙虫病立克次休（未定株）接种雄虫和健康雌虫配对的第1、2、3子代成虫体内立克次体DNA阳性。Karp株接种雄虫的第1子代幼虫体内立克次体的幼虫叮咬小鼠法和PCR结合核酸杂交均呈阳性。     

我国南亚热带地区(南澳县)恙虫病疫源地的证实

目的　我国广东省南澳县恙虫病发病率近年明显增高 ,而国内关于此疫源地的记录极少。本研究对该地区恙虫病疫源地进行全面研究。方法　疫源地流行病学调查 ,病原分离鉴定与分子生物学研究 ,预防措施的制定。结果　该地区为南亚热带岛屿疫源地 ,主要宿主为褐家鼠 ,主要媒介为地里纤恙螨 ,褐家鼠与地里纤恙螨的季节消长与发病均一致。从宿主与媒介分离到恙虫病立克次体经鉴定为 Karp株。分子生物学结果与鉴定结果基本一致同时还证明存在 Kato株与韩国地方株(Yonchon)。血清流行病学调查表明该地区居民与部队人群均存在恙虫病立克次体抗体。在流行季节前应用综合预防措施当年无病例发生。结论　我国南澳县是恙虫病疫源地     

南澎列岛恙虫病立克次体分离株的PCR/RFLP分析

目的　利用ＰＣＲ／ＲＦＬＰ方法证实南澎列岛是恙虫病的疫源地并对恙虫病立克次体初步分型。方法　采用套式聚合酶链反应（ＮＰＣＲ）扩增恙虫病立克次体５６ｋＤ蛋白基因片段,阳性标本的扩增产物进行限制性片段长度多态性（ＲＦＬＰ）分型。结果　南澎列岛的８ 株恙螨／野鼠分离株中７ 株与南澳岛的１ 株扩增出阳性带;ＲＦＬＰ图谱分析表明南澎列岛存在三个型别的立克次体感染,其中一型酶切图谱与Ｋａｒｐ 株相同,一型图谱与Ｋａｔｏ 相同,还有一型既不同于Ｋａｒｐ,也不同于Ｋａｔｏ、Ｇｉｌｌｉａｍ 。并且７株当中有５株为混合感染。南澳岛分离株酶切图谱与Ｋａｒｐ 株相同。结论　本文结果证实了南澎列岛和南澳岛是恙虫病疫源地。南澎列岛上存在三个型别的立克次体,不同型别的立克次体可形成双重感染     

恙虫病立克次体sta56基因片段的克隆

应用半套式PCR技术从Karp株恙虫病立克次体基因组中扩增出872hpDNA片段。该片段经Pstl消化后，与经PStl酶解的pUC18连接，转化大肠杆菌，电泳初筛得pUCRL3等重组质粒。pUCRL3经PCR及酶切证实含约294hp恙虫病立克次体sta56基因片段，可作为检测恙虫病立克次休的特异核酸探针。     

恙虫病的实验室诊断

恙虫病的实验室诊断刘登菊，王均利，田立华，王玉琴恙虫病实验室诊断早期多采用病原体分离，外一斐反应、补体结合试验、中和试验等方法。但因恙虫病立克次体的抗原性比较复杂，株间变异大，上述试验技术在特异性，敏感性及早期诊断方面都显示出不同程度的缺陷。近年来，...     

用Vero-E6细胞大量增殖恙虫病立克次体的研究

目的　为了探讨建立一种简便而可靠的恙虫病立克次体增殖方法, 以得到较大量的恙虫病立克次体。方法　采用了以 Ｖｅｒｏ － Ｅ６ 细胞增殖恙虫病立克次, 并且对 Ｖｅｒｏ － Ｅ６ 感染细胞的培养条件进行了改进。结果　成功地使恙虫病立克次体在 Ｖｅｒｏ － Ｅ６ 细胞中得到大量增殖。结论　改进后的 Ｖｅｒｏ － Ｅ６ 细胞培养法可以获得大量的恙虫病立克次体, 该方法既经济又适用。     

Clinical and immunologic responses of silvered leaf monkeys (Presbytis cristatus) to experimental reinfection with Rickettsia tsutsugamushi

Silvered leaf monkeys (Presbytis cristatus) that had recovered from active Rickettsia tsutsugamushi infections 14 months previously became rickettsemic when inoculated with homologous, related, or unrelated strains of R. tsutsugamushi. In contrast to the results after the initial infection that produced disease, no signs were observed after the subsequent infections. Most animals responded with detectable titers of antibody to the Karp antigen regardless of the inoculated strains. Strains recovered during rickettsemia usually had antigens related only to the challenge strains, but in three silvered leaf monkeys, antigens characteristic of both the original and the challenge strains were detected.     

Virulence of Malaysian isolates of Orientia tsutsugamushi in mice

The pathogenicity of Malaysian isolates of Orientia tsutsugamushi was investigated by a mouse virulence assay. The isolates could be differentiated as low (4 isolates), moderately (3 isolates) and highly virulent (2 isolates) based on the different responses in infected mice. No direct correlation between severity of human scrub typhus infections and virulence of the O. tsutsugamushi in mice was observed. Mice infected with virulent strains of O. tsutsugamushi showed splenomegaly, ascitis accumulation and enlargement of kidneys and livers whereas avirulent O. tsutsugamushi strains were asymptomatic and exhibited ruffled fur for a short period after infection. There was low antibody response in mice infected with isolates of low pathogenicity as compared with those of highly virulent isolates. Upon dissection of the infected mice, enlargement of mouse organs such as spleen, kidney and liver was noted. Presence of rickettsemia in mice was confirmed by the growth of O. tsutsugamushi in the L929 cells when inoculated with blood from infected mice. O. tsutsugamushi was also cultured from the peritoneal exudates of the infected mice. However, DNA of O. tsutsugamushi was only detected in the peritoneal exudates (by PCR) and blood (by cell culture) and not from other tissue samples.     

应用裂解气相色谱法鉴定恙虫病立克次体的探讨

本文报道用两组仪器系统对3株恙虫病立克次体的15份兔睾丸细胞培养物进行裂解鉴定的初步结果。通过对裂解色谱图的特征峰的目视分析,观察到不同毒株的色谱图存在差别;通过与常规方法鉴定的结果相比较,初步探讨了恙虫病立克次体裂解色谱图与其毒力强弱之间的关系。此外,还探讨了不同培养时间及不同批次培养物对色谱图的影响。初步结果表明,裂解气相色谱法用于不同毒株恙虫病立克次体的鉴定是有可能的。     

Orientia tsutsugamushi in peripheral white blood cells of patients with acute scrub typhus.

Scrub typhus, caused by Orientia tsutsugamushi, is an acute illness that occurs in many parts of Asia. Clinical manifestations range from inapparent to organ failure. Organisms disseminate from the skin to target organs, suggesting that they may enter the peripheral circulation. Here, peripheral blood cell smears from patients with acute scrub typhus were obtained before treatment and for 2 days after treatment and reacted with antibodies specific for O. tsutsugamushi. White blood cells from 3 of 7 patients with acute scrub typhus stained positively for O. tsutsugamushi. Cells containing O. tsutsugamushi were mononuclear and were detected on each day of sampling. The presence of O. tsutsugamushi in peripheral white blood cells of patients with acute scrub typhus is a new finding with clinical and pathogenic implications.     

用PCR检测现场单个小盾纤恙螨体内恙虫病立克次体的研究

恙虫病立克次体表面蛋白５６ＫＤａ基因编码区构建的群特异引物，采用嵌合式聚合酶链反应检测现场捕获的单个小盾纤恙螨幼虫体内Ｒ．ｔ的ＤＮＡ。共检测江苏省恙虫病疫区小盾纤恙螨幼虫６１只，结果２只幼虫提取的ＤＮＡ经扩增后见４８１￣５０７ｂｐ的ＤＮＡ扩增带，表明这２只幼虫携带有Ｒ．ｔ，其该种螨Ｒ．ｔ携带率为３．２７％，证明该法可用于单个恙螨幼虫体的Ｒ．ｔ的检测，对恙虫病疫区媒介恙螨的流行病学调查具有实用价值。     

Identification of strain-specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi

Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.     

In vitro demonstration of the hemolytic,cytotoxic activities and induction of apoptosis in Orientia tsutsugamushi infected L929 mouse fibroblast cells

Using cultured mouse fibroblast L929 cells, this study demonstrated the hemolytic and cytotoxic activities and induction of apoptosis in cells infected with Orientia tsutsugamushi. Low levels of hemolytic activity were detected using heavily infected cells. No hemolysin or cytotoxin were detected in the infected culture fluid regardless of the pathogenicity of the O. tsutsugamushi strains in mice. Using propidium iodide uptake assay, acridine orange/ethidium bromide staining and terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labeling assay, apoptosis was observed in L929 cells infected with Karp and Gilliam strains.     

江西省啮齿动物携带立克次体分子流行病学研究

目的 调查江西省部分县区啮齿动物携带的立克次体。方法 在江西省南丰县、铜鼓县、上犹县、浮梁县、上高县、上饶市广信区捕获啮齿动物,运用形态学结合分子生物学方法鉴定其种类。采用巢式PCR方法检测捕获的啮齿动物的脾脏组织样本中的柯克斯体属、斑点热群、东方体属和无形体属立克次体。结果 捕获啮齿动物6种共393只,包括黑线姬鼠、褐家鼠、黄毛鼠、黄胸鼠、社鼠和臭鼩鼱,黑线姬鼠为优势鼠种(311/393)。从19只黑线姬鼠检出柯克斯体(6.11%),从2只黑线姬鼠检出东方体(0.64%),从1只黑线姬鼠检出斑点热群立克次体(0.32%)。对扩增阳性基因片段测序和对获得基因序列做同源性比对,结果浮梁县黑线姬鼠检出新塔拉萨维奇立克次体、恙虫病东方体、贝氏柯克斯体;铜鼓县和上饶市广信区黑线姬鼠检出贝氏柯克斯体;南丰县黑线姬鼠检出恙虫病东方体。除黑线姬鼠外,其它5种啮齿动物均未检出立克次体。结论 江西省部分地区的黑线姬鼠携带贝氏柯克斯体、新塔拉萨维奇立克次体、恙虫病东方体等立克次体病原体,人群存在相关立克次体感染的风险,疾控和医疗机构应加强立克次体病的监测。     

Analysis of immunological characteristics of newly isolated strains of Rickettsia tsutsugamushi using monoclonal antibodies

Since 1975, there has been an increase in the number of patients with tsutsugamushi disease in Japan, and marked antigenic heterogeneity has been found among newly isolated strains of Rickettsia tsutsugamushi. For antigenic analysis of these strains, we produced monoclonal antibodies against the Irie strain isolated in 1971, and the Hirano and Shimokoshi strains isolated in 1980. In all, 34 monoclonal antibodies were produced and their reactivities were determined by the immunofluorescent antibody test. The serological reactivity of the antibodies against these three strains and classic representative strains (Gilliam, Karp and Kato) showed varied reactive characteristics, i.e., serotype-specific, species-specific and intermediate reactivities. It was revealed that these strains are antigenically different from the classic ones. Moreover, by using the serotype-specific monoclonal antibodies, nine strains newly isolated in Miyazaki Prefecture were classified into the Irie and the Hirano types. The antigenicity of the Shimokoshi strain differed from those of the other strains used in this study. From these results, the strains of R. tsutsugamushi used in this study fell into six serotypes including the classic strains. SDS-PAGE and immunoblotting were performed to determine the molecular sizes of the antigenic polypeptides. The results revealed that the serotype-specific antigens belong to the 60-kDa class whereas the species-specific antigens belong to the 61-kDa, 60-kDa or 44-kDa class.