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。     

Isolation and PCR detection of rickettsiae from clinical and rodent samples in Malaysia

Isolation of rickettsiae from patients' blood samples and organ samples of wild rodents from areas with high seroprevalence of rickettsial infections was attempted using cell culture assay and animal passages. L929 mouse fibroblast cells grown in 24 well tissue culture plate were inoculated with buffy coat of febrile patients and examined for the growth of rickettsiae by Giemsa, Gimenez staining and direct immunofluorescence assay. No rickettsiae were isolated from 48 patients' blood samples. No symptomatic infections were noted in mice or guinea pigs infected with 50 organ samples of wild rodents. There was no rickettsial DNA amplified from these samples using various PCR detection systems for Orientia tsutsugamushi, typhus and spotted fever group rickettsiae.     

Development and evaluation of a latex agglutination test for the rapid diagnosis of scrub typhus

The purpose of this research was to develop a simple and rapid diagnostic test for scrub typhus using a latex agglutination test (LAT) to detect antibodies against Orientia tsutsugamushi. Five strains of O. tsutsugamushi were propagated in L929 cells. The rickettsiae were purified and concentrated with percoll density gradient centrifugation. A suitable concentration of O. tsutsugamushi soluble antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity, and accuracy of the latex antigen were assessed. The LAT, indirect immunofluorescent antibody test (IFA), and Weil-Felix agglutination test (WF) were compared by testing 109 acute febrile illness cases and 100 confirmed non-scrub typhus cases (50 other febrile disease cases and 50 healthy controls). By using the IFA as the standard reference method, the overall sensitivity, specificity, and accuracy of the LAT were 89.1, 98.2, and 93.6%, respectively. By contrast, the sensitivity of the WF, compared with the IFA, was only 47.3%, while the specificity and accuracy were 92.6 and 69.7%, respectively. Thus, the LAT described here is another important alternative test for the diagnosis of scrub typhus.     

Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected, laboratory-reared chiggers

The strains of Rickettsia tsutsugamushi found in naturally infected, laboratory-reared Leptotrombidium (Leptotrombidium) arenicola and L. (L.) fletcheri chiggers were characterized by direct immunofluorescence (FA) and by mouse and monkey virulence tests. The strains existing in the L. (L.) arenicola chiggers consisted of different combinations of TA716, TA763, TA686, Karp, and Kato. In addition to these five strains, Gilliam was found in the L. (L.) fletcheri chiggers. Results indicate that individual chiggers can be simultaneously infected with several antigenic strains of R. tsutsugamushi. Although these antigens appear to remain stable within familial lines when several generations were viewed, the antigenic patterns observed in two succeeding generations did not always correlate. This variable expression of antigens was considered to be due to a quantitative fluctuation from one generation to the next in the strains of rickettsiae combined with a lack of sensitivity of the direct FA test in detecting small numbers of antigenically different rickettsiae. Phenotypic variation was considered to be a less probable explanation. Morbidity and mortality were minimal in ICR mice fed upon by individual chiggers of either species, but infection rates were 85-99%. Tissue suspensions prepared from mice infected by L. (L.) arenicola produced higher mortality and longer duration of illness in mice than those prepared from L. (L.) fletcheri-infected mice. Silvered leaf and cynomolgus monkeys were fed upon by the two species of chiggers or inoculated with the mouse tissue suspensions. In both cases, minimal clinical responses were observed.     

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.     

Development and persistence of antibodies to Orientia tsutsugamushi in the roof rat, Rattus rattus and laboratory mice following attachment of naturally infected Leptotrombidium deliense

The development and persistence of antibodies to Orientia tsutsugamushi in Rattus rattus and laboratory mice following infection from the bite of naturally infected Leptotrombidium deliense is reported. Antibodies in R. rattus were first detected using an indirect immunoperoxidase method 2 weeks after attachment of an infected mite. The IgM antibody response was mild, and was detected 2, 4 and 6 weeks after infection, while the IgG response was stronger and was detected from 2 to 19 weeks after infection, when the tests ceased. Active rickettsiae were isolated from R. rattus 1-8 weeks after attachment of infected L. deliense, but rats were not adversely affected by infection and appeared to behave in the same way as uninfected rats. In contrast to rats, laboratory mice were killed by O. tsutsugamushi following infection by attachment of infected L. deliense. The development of antibodies in mice was not detected before gross symptoms of infection were observed. Antibodies were first detected 14 days after attachment of infected mites, when mice were close to death.     

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。     

抑制 RhoA 基因表达诱导乳腺癌细胞凋亡的实验研究

目的：探讨抑制RhoA基因表达诱导乳腺癌（ BC）细胞凋亡的作用。方法通过已建立的RhoA腺病毒载体系统,感染BC MCF -7细胞。经过细胞培养、病毒滴度等,将收集的样本分别用Western blot、RT-PCR技术检测RhoA 蛋白表达和基因表达变化;对感染Adsi RNA-RhoA的BC 细胞进行MTT检测及细胞内DNA片段化检测。结果腺病毒siRNA-RhoA感染BC细胞能明显抑制RhoA基因表达（抑制率为75．64％）,降低RhoA基因的mRNA转录水平69．44％。 MTT检测结果表明,感染腺病毒Adsi RNA-RhoA组的肿瘤细胞生长、增殖被明显抑制。 TUNEL检测显示,抑制RhoA基因表达能明显导致BC细胞凋亡。结论利用siRNA抑制BC细胞中RhoA基因表达,能诱导BC细胞凋亡。     

拮抗型抗hTNF-α单克隆抗体的筛选和活性鉴定

目的 制备和筛选拮抗型人肿瘤坏死因子-α（hTNF-α）单克隆抗体（McAb）并检测其活性,为临床靶向治疗提供理论依据.方法 用经典免疫方法获得抗hTNF-α McAb,经接种于小鼠腹腔后获得腹水,采用硫酸铵沉淀和蛋白G亲和层析法纯化得到纯度〉95%的抗hTNF-α McAb,采用ELISA方法测定效价、抗体亚型和亲和力,MTT法测定抗体阻断hTNF-α对L929细胞的细胞毒作用和筛选拮抗型抗hTNF-α McAb,流式细胞仪法测定抗体阻断hTNF-α诱导L929细胞凋亡的作用.结果 获得1株能稳定分泌抗hTNF-α McAb的杂交瘤细胞株XB10,分泌的抗体亚型为IgG2b;该抗体能高亲和性与hTNF-α结合,特异性阻断hTNF-α对L929细胞的细胞毒作用和抑制细胞凋亡,并呈一定剂量依赖性.结论 成功制备能稳定分泌拮抗型抗hTNF-α的McAb的杂交瘤细胞株,其分泌的抗体对hTNF-α有特异性拮抗作用,为今后研制临床治疗型抗hTNF-α的基因工程抗体奠定了实验基础.     

Molecular detection and characterization of spotted fever group rickettsiae in Taiwan

Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.     

Identification and antigenic typing of Rickettsia tsutsugamushi in naturally infected chiggers (Acarina: Trombiculidae) by direct immunofluorescence.

Rickettsia tsutsugamushi organisms were detected and typed antigenically by direct immunofluorescence in mites from laboratory-maintained infected colonies of Leptotrombidium (Leptotrombidium) fletcheri and L. (L.) arenicola. Rickettsiae were identified most readily in unengorged larvae, but were also discernable in engorged larvae and all post-larval stages of the vectors.     

Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi

Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5552 copies/microL of mouse blood, 14448-86012 copies/microL of mouse liver/spleen homogenate, and 3-21 copies/microL of monkey blood.     

Demonstration of the natural foci of tsutsugamushi disease in Nan Peng Lie Islands in China

In recent years, the incidence of tsutsugamushi disease has increased in Nan Peng Lie Islands in China, and the disease has not been recorded in this region. The natural foci of tsutsugamushi disease were investigated in this paper. Isolation of Orientia tsutsugamushi and the study of preventive measures were also performed. The region was the island natural foci of south subtropical zone. The main host and vector were Rattus norvegicus and Leptotrombidium (L.) deliens respectively. The seasonal quantity trends of Rattus norvegicus and Leptotrombidium (L.) deliense were consistent with the incidence of human infection in the region. The strains of O. tsutsugamushi were isolated from Rattus norvegicus and Leptotrombidium (L.) deliense. The identification showed that most strains were Karp. The seroepidemiology showed a high prevalence of antibody against O. tsutsugamushi. After preventive measures were implemented, the incidence was descent. So Nan Peng Lie Islands were the natural foci of tsutsugamushi disease.     

原花青素处理去细胞牛心包细胞的相容性研究

目的考察原花青素处理去细胞牛心包的细胞相容性。方法采用L929细胞经原花青素处理去细胞牛心包浸提液培养后,噻唑蓝试验（MTT）法测定其相对增值率;将L929细胞与原花青素处理去细胞牛心包直接接触培养,逐日观察细胞生长状态;将L929细胞种植于原花青素处理的去细胞牛心包表面,扫描电镜观察其生长情况。结果原花青素处理去细胞牛心包性质稳定,细胞毒性程度为0～1级。L929细胞与心包材料直接接触培养生长良好,形态学无明显改变。结论原花青素处理去细胞牛心包细胞相容性好。     

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.     

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.     

云南省泸西县啮齿动物携带立克次体调查

目的 调查云南省泸西县啮齿动物携带恙虫病东方体、无形体和埃立克体的状况,了解该类病原体在当地自然界中的保存状况和基因特征。方法 用鼠笼和鼠夹在云南省泸西县捕鼠,将捕获的动物种类鉴定后解剖取脾脏,活鼠取血。采用巢式PCR扩增脾脏的恙虫病东方体groEL基因,无形体和埃立克体的16S rRNA基因特异片段;测定PCR扩增阳性产物的DNA序列,对获得序列进行序列比对和系统进化分析。IFA法检测鼠血清中恙虫病东方体IgG抗体。结果 在泸西县共捕获啮齿动物10种225只。其中黄胸鼠36.89%(83/225)、大绒鼠35.11%(79/225)和中华姬鼠13.78%(31/225)为优势鼠种。获得鼠血清85份。鼠脾脏中检测到5株东方体groEL基因阳性标本,带毒鼠种为黄胸鼠2.41%(2/83)和大绒鼠3.80%(3/79)。同源性比较显示,这5株东方体的相似性在99.02%~100%之间,他们分别与GenBank中已知立克次体序列的相似性在98.75%~100%。系统发生树显示,5株OT与来自日本、泰国和中国安徽的菌株位于同一分支。3份16S rRNA阳性标本,其中1份埃立克体阳性,来源于大绒鼠;1份沃尔巴克氏体和1份巴尔通体阳性均来源于黄胸鼠。无形体均为阴性。埃立克体株序列比对显示与来自美国、中国和巴西的埃立克体基因同源性为98.0%~100%,并与分离自美国野外工作者皮肤的伊文氏埃立克体在同一分支。鼠血清恙虫病IgG抗体阳性7份,阳性率8.24%(7/85)。结论 该地区存在以黄胸鼠和大绒鼠为主要宿主的恙虫病自然疫源地。埃立克体、巴尔通体和沃尔巴克氏体在啮齿动物中也存在感染,需注意防控。     

腺病毒介导多基因对肝癌细胞凋亡的诱导作用

目的 研究腺病毒介导的多基因（ｐ５３、Ｂ７－１、ＧＭ－ＣＳＦ、ＩＬ－２）对肝癌细胞系调亡的诱导,及对肝癌细胞系裸鼠体内成瘤性改变的影响。方法 应用光镜、电镜和ＴＵＮＥＬ法,检测肝癌细胞系感染腺病毒介导的多基因后凋亡的发生,检测ＨｅｐＧ２细胞导入腺病毒多基因后裸鼠体内的成瘤性改变。结果 肝癌细胞系导入多基因后发生凋亡,并对化疗药物顺铂的敏感性增加,册时给予１０ｍｇ／Ｌ的顺铂,可使近３０％的肝癌细胞发     

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。