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。     

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。     

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。     

应用L929细胞从患者血液分离立克次体

作者收集临床疑似立克次体病患者血液标本２０份，应用Ｌ９２９细胞分离立克次体，用ｍＩＦ法鉴定出恙虫病立克次体３株，斑疹伤寒立克次体２株，斑点热立克次体７株。恙虫病立克次体感染的细胞悬液用ＰＣＲ技术均检出恙虫病立克次体ＤＮＡ。在国内首次应用细胞培养方法从患者血液直接分离立克次体成功。     

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。     

恙虫病立克次体蛋白抗原及其编码基因研究进展

恙虫病立克次体（Rickettsiatsutsugamushi）是恙虫病（Scrubtphus）的病原体,专性活细胞内寄生,革兰氏阴性,恙螨是其传播媒介。在分类地位上,恙虫病立克次体原属于立克次体科立克次体属,但由于其在许多生物学和遗传特性方面不同与其他立克次体,1995年将其另立一属一东方体属（Orientia）,称为恙虫病东方体（Orientiatsutsugamushi）[1]。近十多年来,新的技术方法尤其分子生物学方法不断引入恙虫病立克次体的研究,使病原学研究更加系统深入广泛。现对其蛋白抗原与其编码基因的研究综述如下。1恙由病立克次体的多胜组成纯化的恙虫…     

国外恙虫病立克次体研究进展

<正> 1975年日本发现流行株与古典型不同的新型恙虫病立克次体[Rickettsia tsutsugamushi,Rt]。新型Rt主要流行于秋冬,对小白鼠致病力弱或不致病,从而给分离病原带来难度。Kabayashi 采用 Tachibana 建立的CPA处理小白鼠后分离立克次体的方法,成功地从病人外周血中分离到弱毒株Rt,促进了弱毒株Rt的研究。本文对近几年Rt研究情况作一综述。     

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     

我国人畜共患无形体病防治面临的挑战——诊断与经验治疗

【摘要】 立克次体可分为立克次体科及无形体科,前者引起恙虫病、斑疹伤寒等传统立克次体病,后者引起人粒细胞无形体病、埃立克体病等新发立克次体病。本文着重对无形体病防治所要面对的挑战即诊断困难和原因进行阐述,提出在临床上高度怀疑本病时,应使用特效药物--四环素类抗生素进行经验治疗。     

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.     

实验性恙虫病立克次体感染或免疫鼠的ADCC活性和cAMP水平变化

本文用~(61)Cr释放试验和~3H-cAMP蛋白竞争结合分析法研究了实验性恙虫病立克次体感染或免疫鼠的ADCC活性和cAMP水平变化。结果发现强毒株(B株)和减毒株(49株)接种后均能导致鼠脾细胞的ADCC水平增高及cAMP水平降低,但灭活立克次体接种后对鼠脾细胞的ADCC活性及cAMP水平则无明显影响。     

恙虫病东方体CMY株sta56基因片段序列分析

目的　了解恙虫病东方体 Ｃ Ｍ Ｙ 株与其它株的关系。方法　测定 Ｃ Ｍ Ｙ 株 ｓｔａ５６ 基因片段的核苷酸序列并与其它株相应序列进行比较。结果　在测定的３２４ｂｐ 序列中, Ｇ Ｃ 含量为４３５ ％ ; 与 Ｋａｒｐ 、 Ｇｉｌｌｉａｍ 、 Ｋａｔｏ 、ｋｕｒｏｋｉ、 Ｋａｗａｓａｋｉ 及 Ｓｈｉｍｏｋｏｓｈｉ 株相应序列的同源性分别为９１０ ％ 、８７３ ％ 、８７０ ％ 、８７３ ％ 、８６１ ％ 及７３８ ％ 。结论　 Ｃ Ｍ Ｙ 株与已报道的恙虫病东方体菌株不同, 与 Ｋａｒｐ 株有很高的同源性。     

山西恙虫病立克次体分型及其56—kD蛋白基因序列分析

作者在我国新发现的山西恙虫病疫区病人血液中分离出3株恙虫病立克次体，SXh951，Sxh952，Sxh953。为明确其分类地位．对其进行了血清型，基因型和56－kD蛋白基因的部分序列分析。用免疫荧光抗体染色法将病人血清抗体与Karp,Gilliam．Kato株抗原反应．结果显示山西株与Gilliam型一致；用来自56－kD蛋白基因的引物扩增后，分别用限制性核酸内切酶Hinfl，HaeⅢ．Hhal消化DNA片段的多态性（PCR／RFLP）显示山西3株基因型一致，但均与Karp，Kato，Gilliam株的电泳图谱不同，显示为一独有的PCR／RFLP基因型；对Sxh951株56-kD蛋白基因片段扩增产物（1．2kb）进行DNA序列分析后．根据所推导氨基酸序列，比较Sxh951株与三个标准株氨基酸序列的同源性，发现Sxh951株与Karp、Kato和Gilliam株均有明显差异。对此，本实验室正在进一步研究，以便进一步确定其分类地位。     

山东恙虫病立克次体生物学及免疫学性质的研究

本文报告了山东恙虫病立克次体人株生物学及免疫学性质的初步研究。所研究3株中的1株感染小鼠发病不典型,极少致死;另两株感染小鼠发病典型,小鼠呈规律性的死亡,易适应于鸡胚,对小鼠毒力LD_(50)为3.4。感染小鼠之IgG抗体出现于接种后的第13天,于接种后的第3～4周抗体水平达峰值。上述分离株与Karp毒株间存在较强的交叉保护作用。酶免疫染色血清学反应结果指出,与Gilliam株很一致,可能属Gilliam同一血清型。     

实验室保存的恙虫病东方体Karp株与福建省分离株的sta56基因的序列分析

目的比较实验室保存的恙虫病东方体Karp株、福建省两个分离株的sta 56基因序列的差异。方法提取感染各株恙虫病东方体的Vero细胞的DNA,扩增出sta56的ORF。对不同株的ORF进行测序,对15株恙虫病东方体的sta56构建进化树并进行分析。结果实验室保存的Karp株的sta56基因序列有98%与参考株一致;FQ株和NA株仅有两个碱基的差异,有96%的序列与Karp株一致。结论实验室保存的Karp株sta56基因在长期传代中可发生变异,FQ株和NA株均属于Karp株。     

Antigenic types of Rickettsia tsutsugamushi isolated from patients with tsutsugamushi fever and their distribution in Miyazaki Prefecture

Rickettsia tsutsugamushi (Rt) isolated from patients with tsutsugamushi fever were examined for their antigenicity. This was done by indirect immunofluorescence (IIF) with guinea pig antisera against three standard strains (Karp, Kato and Gilliam) and two local strains (Kawasaki and Kuroki) isolated in 1981, and with mouse monoclonal antibodies against the three standard strains. In the meantime, antibodies in sera from 317 out of 442 patients registered during 1985 to 1988 were titrated by IIF with those five Rt strains. 1) Local isolates, Kawasaki and Kuroki strains, reacted most effectively with the homologous antiserum, respectively, showing four fold lower IIF titers against the heterologous antisera. 2) Kawasaki strain reacted with none of the monoclonal antibodies, whereas Kuroki strain showed a slight reaction with anti-Karp and anti-Kato, but not anti-Gilliam, monoclonal antibodies. 3) Seventeen out of 27 strains isolated in 1985 resembled the Kawasaki strain in their reaction patterns with the antisera and monoclonal antibodies, and the other 10 strains showed reactivity similar to the Kuroki strain. 4) Sera of 233 (74%) out of 317 patients showed the highest antibody titers against the Kawasaki strain and 69 (22%) of 317 against the Kuroki strain. It is thus evident that Kawasaki and Kuroki strains are antigenically different from the standard strains, and Kawasaki and Kuroki strains also differ from each other. It is suggested that two antigenic types (Kawasaki and Kuroki) of Rt were distributed in Miyazaki Prefecture, Rt of the Kawasaki type slightly dominates Rt of the Kuroki type, and recent tsutsugamushi fever has been caused by either one or the other type of Rt.     

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.     

Detection of surface antigen in Rickettsia tsutsugamushi infected mouse reticuloendothelial cells

Antibody produced by immunizing CBA/CaJ mice with RE cells from C57B1/6J mice infected 14 days earlier with R. tsutsugamushi Gilliam strain bound readily to Gilliam strain non-cell associated rickettsiae and less readily to the periphery of infected RE cells. Conversely, antibody produced by immunizing with RE cells infected 21 days earlier did not bind to Gilliam rickettsiae but bound to the surface of RE cells from mice infected 21 days earlier. This binding was not related to alloantibodies because these were absorbed prior to testing. The demonstration of rickettsial antibody staining of infected cell associated antigen(s) in this assay system provides a new method for the detection of R. tsutsugamushi infection.     

Characterization of Orientia tsutsugamushi strains isolated in Shandong Province, China by immunofluorescence and restriction fragment length polymorphism (RFLP) analyses

In order to identify the characteristics of the Sta56 gene of the 23 isolates of Orientia (O.) tsutsugamushi isolated in Shandong Province, indirect immunofluorescence assay (IFA) was used to identify the gene type of 23 strains O. tsutsugamushi isolated from scrub typhus patients, chigger mites, and rodents. Restriction fragment length polymorphism (RFLP) analysis was also used to analyze the restriction profiles of the Sta56 gene PCR amplification products of the 23 isolated strains of the O. tsutsugamushi; the results were compared with those acquired by nested PCR. By IFA, 21 of the 23 isolates belonged to the Gilliam type, and 2 to the Karp type. Using RFLP analysis, 21 strains had similar restriction profiles to the Japan Kawasaki strain, but they had no restriction site Hha I, and thus had some difference in gene sequence compared with the Japan Kawasaki strain. The other 2 strains had similar restriction profiles to Karp. These results were identical to that acquired by nested-PCR. In Shandong Province, the gene types of epidemic O. tsutsugamushi strains were similar to the Japan Kawasaki type, but had some differences in gene sequence. In addition, Karp also existed.