套式PCR用于恙虫病的早期诊断及恙螨幼虫体内立克次体的检测

从恙虫病立克次体（Ｒｔ）Ｋａｒｐ株的群特异性抗原基因序列设计两对引物建立套式ＰＣＲ检测现场鼠体采集的恙螨幼虫体内及恙虫病现症病人外周血单核细胞内的ＲｔＤＮＡ。除一份已用过药病人标本外，其余９份均可检见８８ｂｐＤＮＡ扩增产物；检测结果表明：现场鼠体采集的地里纤恙螨（Ｌｅｐｔｏｔｒｏｍｂｉｄｉｕｍｄｅｌｉｅｎｓｅ）幼虫的Ｒｔ携带率是１３．３％，证实套式ＰＣＲ可用于急性期恙虫病的诊断和媒介恙螨流行病学的调查。     

恙虫病立克次体山东株在野鼠内脏分布研究

目前对鼠体内恙虫立克次体（Ｒｔ）的分离，一般采用取鼠肝、脾、肾混合匀浆后接种小白鼠腹腔传代［１，２］，但一些学者单独采用脾匀浆［３，４］，或脾与肾混合匀浆［５，６］接种小白鼠亦能成功地分离出Ｒｔ，于恩庶等报告从鼠（沟鼠、屋顶鼠、田鼠）脑、脾和肾脏内分离Ｒｔ结果并不一致，以肾为分离材料为阳性率为最高，脑、脾次之［７］。作者采用Ｒｔ分离及脏器组织印片检查等方法分别从黑线姬鼠、大仓鼠的脾、肝或肾中分离到Ｒｔ，现将结果报告如下。１　材料和方法１１　鼠标本　于１９９７年１０月在秋冬型恙虫病重疫区山东费县…     

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。     

新疆博乐地区恙虫病东方体自然疫源地调查报告

目的　调查新疆博乐地区恙虫病东方体（Ot）自然疫源地情况。方法　采用多种方法捕鼠取脾,当地牧民静脉取血、从鼠体表捕获恙螨,用试剂盒提取DNA,应用巢式PCR(nPCR)检测Ot-Sta56基因;阳性标本脾制成悬液,接种昆明小白鼠进行Ot病原进行传代分离。结果　在博乐地区平原湿地、农田和居民区捕鼠126只,其中7只nPCR检测Ot阳性,阳性率5.55%;从当地牧民血液中检测到Ot阳性,阳性率1.87%;从野鼠体表捕获博乐纤恙螨,带螨率为8.4%,检测并分离到Ot病原。DNA序列分析表明,鼠类、人血及恙螨中检测并分离到的Ot碱基序列相同,与Karp株相应DNA片段的碱基序列同源性最高,均为99％,应属于Karp型。在山地草原捕获鼠58只,nPCR检测无Ot阳性,捕获旱獭26只,nPCR检测无Ot阳性,从鼠体表未捕获到恙螨。结论　新疆博乐地区平原湿地可能存在恙虫病东方体自然疫源地,宿主动物可能为小家鼠、普通仓鼠、大沙土鼠,传播媒介可能为博乐纤恙螨     

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。     

恙虫病立克次体实验感染地里纤恙螨经卵传递的研究

地里纤恙螨成虫腹内人工接种恙虫病立克次体Karp株首次获得成功。在接种立克次体悬液量极微(0.1μl以下)的成虫感染率达41.2％;接种后的恙螨成虫4个月后其活存率为83.7％;成虫在第一年内能保持较好的生殖能力。经人工接种的恙虫病立克次体Karp株,能在恙螨体内增殖,并经卵传递至子代。     

安徽三界地区啮齿动物感染恙虫病东方体调查

目的调查安徽三界地区啮齿动物自然感染恙虫病东方体分离株,并确定其基因型别。方法流行季节在野外用鼠笼捕鼠并鉴定鼠种、携螨率;巢式PCR对鼠脏器进行恙虫病东方体56kD基因片段检测;将鼠脏器接种小自鼠进行病原体分离,PCR扩增分离株0t56kD基因片段,并将目的片段进行测序,基因序列同源性及进化分析。结果捕获啮齿动物数及其携螨率,黑线姬鼠60％（15／25）;褐家鼠45．5％（5／11）;东方田鼠和购睛各2只,均阴性。40只野鼠脏器有3只恙虫病东方体PCR扩增阳性,阳性率为7．5％;从黑线姬鼠标本中分离到1株恙虫病东方体Sanjie,56kD基因片段测定该株与台湾TT071la和NT0511b分离株同源性最高（98％）,系统发生树分析也显示与Kawasaki型共处于同一分支。结论安徽三界地区存在鼠类恙虫病东方体自然感染,黑线姬鼠为其主要宿主,当地恙虫病东方体属Kawasaki基因型。     

华南沼泽田鼠自然感染恙虫病立克次体的发现

<正> 1967—1968年在湖南湘西古丈县发现恙虫病以后,1981年开始进行疫源地调查,从恙螨和鼠类均分离出恙虫病立克次体,证明该地确有疫源地存在。 1983年6—10月捕到华南沼泽田鼠14只,从鼠体收到恙螨664只,主要是纤恙螨属604只,其他属60只,鼠染螨率为100％,平均恙螨指数为47.5,纤恙螨指     

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～…     

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。     

聚合酶链反应用于恙虫病立克次体检测和分型的研究

本文报道聚合酶链反应用于恙虫病立克次本（Ｒｔ）检测和分型研究结果，以构建的Ｒｔ外膜主要蛋白５６ｋＤａ型特异抗原（ｔｓａ５６ｋＤａ）基因编码区的群型特异引物，采用１步法ＰＣＲ分别为Ｇｉｌｌｉａｎ，Ｋａｒｐ，Ｋａｔｏ，Ｋａｗａｓａｋｉ和Ｋｕｒｏｋｉ５株标准株Ｒｔ江苏地区５份Ｒｔ阳性标本，斑点热和莫氏立次体以及５份正常输血员全血标本进行检测和分型，研究结果证明所构建的Ｒｔ群，型引物具有Ｒｔ的特异性，型特     

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.     

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。     

实时荧光定量PCR检测恙虫病东方体

目的建立检测恙虫病东方体的实时荧光定量PCR(quantitative real-ti me PCR)方法。方法根据恙虫病东方体56kD外膜蛋白基因序列设计引物和探针,以克隆的56kD基因片段作DNA模板,建立实时荧光定量PCR检测方法。结果建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.999)。荧光定量PCR检测恙虫病东方体的灵敏度约为套式PCR的100倍,并且具有良好的重复性。用该定量PCR检测其它相关立克次体和病原菌DNA样本,检出结果均为0。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肺脏、肝脏标本,结果脾脏中东方体出现最早和检出量最多,肝脏和肺脏次之。血中的恙虫病东方体量较低。结论本研究建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于东方体感染早期血样本的快速检测作恙虫病感染早期诊断,并且可以定量分析评价恙虫病东方体感染的程度。     

A case of tsutsugamushi disease in the urban area of Komatsu City

Most of patients with tsutsugamushi disease are diagnosed by their clinical histories suggesting the opportunities of Rickettsia infection in a rural region. We reported a 76-year-old female patient, who was considered to be infected in her house in the urban area of Komatsu City. She has shown typical clinical manifestations of tsutsugamushi disease, and was remitted successfully by oral administration of minocycline. Although specific antibodies to Rickettsia tsutsugamushi could not be detected in her serum by the complement fixation (CF) method during her clinical course, their significant elevation was confirmed by the indirect immunofluorescence (IF) method.     

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.     

Immunological studies of experimental tsutsugamushi disease in congenitally athymic (nude) mice

Athymic mice were taken ill and died from infection with the high virulence as well as the low virulence strains of Rickettsia tsutsugamushi, and they did not improve in spite of tetracycline therapy. Moreover, neither 7S nor IgM antibody was detected by immunofluorescent antibody method in serum samples of athymic mice infected with the high virulence strain. Although immune serum-transfer exhibited some protective effect in athymic mice infected with the high virulence strain, it was far lower than in euthymic mice. Although both athymic and euthymic mice having received non-immune T-lymphocytes were taken ill and died, the mice having received immune T-lymphocytes survived infection with the high virulence strain. This protective capacity of T-lymphocytes was weak by 10 days after immunization of donor mice, became firm after a month and lasted as long as 12 months without decay. For athymic mice infected with the low virulence strain, not only immune but also non-immune T-lymphocytes from euthymic mice exhibited significant protective effect. By treatment of immune T-lymphocytes with anti-Thy-1.2 or anti-Lyt-1.2 alloserum, the protective capacity was lost entirely, and considerably diminished by treatment with anti-Lyt-2.2 alloserum in a homologous system using the high virulence strain. The results show that the inhibition of progress of tsutsugamushi disease is principally dependent on cellular immune mechanism(s) and that the production of antibody against R. tsutsugamushi is thymus-dependent.     

Determination and geographical distribution of Orientia tsutsugamushi serotypes in Korea by nested polymerase chain reaction.

Field rodents and chigger mites were collected at 30 locations in Korea in October and November 1997-1999 to determine the serotypes of Orientia tsutsugamushi and their geographical distribution. A nested polymerase chain reaction was performed with the spleen tissues from 546 field-striped mice (Apodemus agrarius) and 104 pools of chigger mites. The positivity rate of O. tsutsugamushi was 45.6% in A. agrarius and 39.4% in the chigger mite pools. Two serotypes, Boryong and Karp, were found in these samples; the former was predominant (78.3% in the mice and 82.9% in the chigger mite pools), with wide distribution throughout the country, including Cheju-do. The latter was confined to the middle of the Korean peninsula, with positivity rates of 15.7% in the mice and 12.2% in the chigger mite pools. The double infection of Karp and Boryong serotypes was found in 15 (6.0%) A. agrarius mice. Gilliam serotype was not detected at any of the study locations. The Boryong and Kuroki serotypes were identical in amino acid sequence of the 56-kDa protein, although they differed in virulence to BALB/c mice.