用螨原代培养细胞检测螨体内汉坦病毒和恙虫病东方体复合感染的研究

目的研究汉坦病毒（Hantavirus，HV）和恙虫病东方体（Orientia tsutsugamushi，Ot）能否同时共生于同一媒介小盾纤恙螨（Leptotrombidium scutellare）体内。方法采集HFRS和恙虫病混合感染疫区鼠体恙螨和游离恙螨饲养的幼虫、若虫、成虫，蛋白酶消化后作螨细胞原代培养细胞片，用原位RT-PCR分子杂交和PCR法分别检测HV和Ot在培养螨细胞内的分布和定位。结果HV和Ot在感染的螨细胞中多分布于消化系统的上皮细胞、卵巢组织细胞内，且随传代次数的增加而阳性率增高。结论小盾纤恙螨可同时自然感染HV和Ot。     

新疆部分地区人群及动物感染恙虫病东方体调查

目的调查新疆部分地区人群及动物感染恙虫病东方体（Ot）情况,并对媒介昆虫进行调查。方法采用多种方法捕获可能感染Ot动物取脾,当地牧民静脉取血,从野鼠体表捕获恙螨,用试剂盒提取DNA,应用巢式PCR（nPCR）检测Ot-Sta56基因;接种昆明小白鼠进行Ot病原传代分离。结果共采取人群血液2430份,在6份血液中检测并分离到Ot;捕获动物13种5783个（只）,从5种啮齿动物（小家鼠、灰仓鼠、大砂土鼠、草原兔尾鼠、灰旱獭）、2种鸟类（麻雀、灰腹鹡鸰）的脾中检测并分离到Ot,从绵羊血液中检测到Ot。其他动物体内未检测到Ot。从野鼠体表分离恙螨5种,从博乐纤恙螨体内检测分离到Ot。基因序列分析表明,新疆地区存在恙虫病东方体的基因型以Karp型为主,可能存在Saitama型。结论新疆地区存在人群及动物感染Ot,其基因型以Karp为主,可能存在Saitama型。     

1例恙虫病合并肺部感染及肠梗阻的护理

<正>恙虫病又名丛林斑疹伤寒,是由恙虫病立克次体引起的急性传染病,是一种自然疫源性疾病,鼠类是主要的传染源,恙螨为此病的传播媒介,多发生于夏秋季,6~7月为高峰,人群普遍易感[1]。临床表现多样化,合并症较多[2]。2013年3月我院收治了1例恙虫病合并肺部感染及不完全性肠梗阻患者,经积极治疗及精心护理治愈出院。现将护理体会报告如下。1资料与方法1.1临床资料患者,男,55岁,因进食后恶心、肛门停止排便排气、腹胀、     

晋南地区恙虫疾疫源地的发现及其动物流行病持点

山西省南部的翼城和绛县一带农区存在有恙虫病的自然疫源地，该地区连年有恙虫病病例发生。通过连续数年的调查发现，该地的恙虫病属秋冬型，每年九月下旬至十一月上旬为病例的发生高峰。主要疫源动物为大仓鼠（Ｃｒｉｃｅｔｕｌｕｓ ｔｒｉｔｏｎ），在寄生于鼠类的节肢动物中，姬鼠纤恙螨（Ｌｅｐｔｏｔｒｏｍｂｉｄｉｕｍ ａｐｏｄｅｍｉ）为绝对优势种，其数量约占当地恙螨科种类的９３．２％，其季节消长与当地恙虫病的流行趋     

多聚酶链反应检测恙虫病立克次体核酸

作者设计合成一对恙虫病立克次体ｓｔａ５８基因的ＤＮＡ引物，分别从恙虫病立克次体ｋａｒｐ、Ｇｉｌｌｉａｍ及ＣＭＹ株基因组中扩增出１ｋｂ片段，普氏及莫氏立克次体、西伯利亚立克次体和贝氏柯克斯体则不能，表明该引物为恙虫病立克次体种特异。以含ｓｔａ５８基因片段的重组质粒为模板作敏感性鉴定，表明ＰＣＲ可检测到１０ｐｇ的靶ＤＮＡ。用该引物作ＰＣＲ检测实验感染恙虫病立克次体的小鼠血、腹水及脾标本均获满意结果。     

用分子生物学方法检测螨体内汉坦病毒的研究

目的 研究汉坦病毒(HV)在革螨、总螨体内增殖、定位等。方法 采用聚合酶链反应和原位分子杂交法检测革螨、总螨体内HV—RNA。结果 用PCR技术从革螨、总螨体内检测到HV—RNA，最低检测为5只螨组；取革螨和恙螨幼虫、若虫定期测定TCID50／m1滴度，证明HV在革螨、恙螨体内可经期传播并有增殖现象；用原位分子杂交技术在革螨、恙螨的卵巢细胞、支肠细胞等组织中检测到HV—RNA。结论 研究结果证明HV在螨体内可经期传播，并有增殖现象；HV通常定位于螨的卵巢和支肠细胞等组织内。为革螨、恙螨的媒介意义提供了具有分子水平的直接证据。     

晋南地区恙虫疾疫源地的发现及其动物流行病学特点

西省南部的翼城和绛县一带农区存在有恙虫病的自然疫源地,该地区连年有恙虫病病例发生。通过连续数年的调查发现,该地的恙虫病属秋冬型,每年九月下旬至十一月上旬为病例的发生高峰。主要疫源动物为大仓鼠(Cricetulustriton),在寄生于鼠类的节肢动物中,姬鼠纤恙螨(Leptotrombidiumapodemi)为绝对优势种,其数量约占当地恙螨科种类的93.2%,其季节消长与当地恙虫病的流行趋势一致,被认为是该地恙虫病流行的传播媒介,不同于北方地区其它已知的恙虫病自然疫源地     

恙虫病立克次体sta58主要抗原基因片段的克隆及表达

作者设计合成一对ＤＮＡ引物，经ＰＣＲ扩增出Ｋａｒｐ及ＣＭＹ株恙虫病立克次体ｓｔａ５８主要抗原基因片段，分别建立其无性繁殖系，构建了表达质粒ｐＢＶＲＫ５和ｐＢＶＲＣ４５，在国内首次应用大肠杆菌成功表达了恙虫病立克次体抗原。其意义在于为我国恙虫病立克次体研究提供特异性核酸探针，为恙虫病基因工程诊断试剂的研制奠定物质基础。     

山东费县五种恙螨季节消长观察

作者于１９９５．５～１９９６．４对山东费县农田野鼠体外五种恙螨季节消长进行了逐月观察。结果表明，须纤恙螨主要出现在秋冬季（１０月至翌年４月），高峰在１２月份，为冬季优势螨种；临淮岗纤恙螨５月份始现，１１月份消失，高峰在８月份，为夏季优势种；小盾纤恙螨集中出现在秋季及初冬（９～１２月），其高峰在１１月份，是９～１２月间鼠体外绝对优势螨种；泰山纤恙螨主要出现在６～１０月份，该螨种数量起伏较大；太平洋无前恙螨全部采自大仓鼠，主要出现在４～１２月，高峰期在７月份。阐明当地不同季节优势螨种对研究该地恙虫病以及肾综征出血热（ＨＦＲＳ）传播媒介具有十分重要的意义。     

恙虫病临床诊治特点及预防

恙虫病是由恙螨叮咬,感染恙虫病东方体引起的一种自然疫源性传染病。它常常以急性发热、常伴皮疹或焦痂、对抗生素治疗敏感为特征,若救治不及时可能导致严重的多器官衰竭甚至高达70%的死亡率。延迟救治是导致恙虫病患者高死亡率的主要原因。恙虫病往往无典型症状,易与其他地区性疾病混淆,且临床诊断手段相对落后,易被忽视。本文针对恙虫病的病原体、临床诊治特点及防控策略进行文献综述,以加强公众和临床医生对恙虫病的认识,提高该疾病的诊断水平,降低恙虫病的重症率和死亡率。     

Seasonal occurrence of Leptotrombidium deliense (Acari: Trombiculidae) attached to sentinel rodents in an orchard near Bangkok, Thailand

Leptotrombidium deliense Walch that attached to sentinel laboratory mice and the roof rat, Rattus rattus (L.), placed in an orchard habitat near Bangkok, Thailand, were studied between April 1993 and April 1995. A single L. deliense larva was attached to only 1 of 51 laboratory mice placed in the study area between April and September 1993. Overall, 89/202 (44.1%) R. rattus had 1 or more L. deliense larvae attached, and Orientia tsutsugamushi (Hayashi), the etiologic agent for scrub typhus, was isolated from liver/spleen samples of 2/202 (1.0%) rats placed in an endemic area for a single night. A total of 474 L. deliense attached to sentinel R. rattus, of which 314 larvae successfully fed to repletion and were recovered, and 2 (0.6%) of these were naturally infected with O. tsutsugamushi. The occurrence of L. deliense was influenced by rainfall, with more chiggers attached to rodents in the wetter months of the year. The study showed that the risk of exposure to infection with O. tsutsugamushi is greater during the wetter months of the year, and that only a relatively small number of chigger attachments are needed to infect potential hosts.     

Occurrence of Orientia tsutsugamushi in chiggers (Acari: Trombiculidae) and small animals in an orchard near Bangkok, Thailand.

The ecology of Orientia tsutsugamushi (Hayashi) was studied in chiggers and small animals in an orchard near Bangkok, Thailand. Small animals were trapped monthly between July and November 1992 and examined for the presence of O. tsutsugamushi and ectoparasitic chiggers. A total of 40 Rattus rattus (L.) and 16 Tupaia glis (Diard) was trapped. O. tsutsugamushi was isolated from liver and spleen samples of 30.8% of R. rattus and 18.6% of T. glis. Antibodies to O. tsutsugamushi were detected in 95% of R rattus, and IgG antibodies persisted for up to 10 mo after removing rats from potential reinfection in the field. A total of 1,133 chiggers was identified and examined for the presence of O. tsutsugamushi using a direct fluorescent antibody test, and 2.6% Leptotrombidium deliense Walch, 5.1% Aschoshoengastia indica Hirst, 2.6% A. (Laurentella) sp. #2 and 0.9% A. (Laurentella) sp. #4 were infected. Forty-four pools of chiggers from these animals were triturated and injected into mice. Seven pools were obtained from T. glis and 1 was positive for O. tsutsugamushi, and 3 of 37 pools from R. rattus were positive. A proportion of the engorged chiggers collected was reared to the adult stage, and the progeny of these adults tested for the presence of O. tsutsugamushi. The progeny of 186 females was tested, and the progeny of 2 L. deliense was found to be naturally infected with O. tsutsugamushi.     

Vertical transmission of Orientia tsutsugamushi in two lines of naturally infected Leptotrombidium deliense (Acari: Trombiculidae)

Vertical transmission of Orientia tsutsugamushi (Hayashi), the etiologic agent for scrub typhus, was studied in two lines of naturally infected Leptotrombidium deliense Walch. In one line of mites originating from a single adult (V3M), the rate of filial transmission was 100% for the first two laboratory generations, but declined to 86.6% in the third laboratory generation. The vertical infection rate in this line of mites was 100% for the parental generation, but declined to 95.6% for the F1 generation and 88.6% for F2. The transmission of O. tsutsugamushi in another line of L. deliense (V3F) was less efficient than mites originating from V3M. In the initial laboratory generation of V3F a filial transmission rate of 100% was recorded. However, none of the F2 generation of this line transmitted rickettsiae to mice (Mus musculus L.), resulting in a filial transmission rate of 0%. Transmission of O. tsutsugamushi to mice by progeny from cohort larvae originally from the same adult (V3F) was also studied in the laboratory and these were found to be relatively poor transmitters of rickettsiae. The filial infection rate of F2 larvae was 60%, F3 was 88.8%, and F4 was 55.9%. The biology of infected L. deliense was studied and compared with uninfected mites reared under the same laboratory conditions. The results showed that infected female L. deliense laid approximately the same or more eggs as uninfected adults. The rate of development of the progeny of infected L. deliense was not significantly different from uninfected mites.     

Efficiency of Leptotrombidium chiggers (Acari: Trombiculidae) at transmitting Orientia tsutsugamushi to laboratory mice

Thirteen different laboratory colonies of Leptotrombidim chiggers [L. chiangraiensis Tanskul & Linthicum, L. deliense Walch and L. imphalum (Vercammen-Grandjean &Langston)] were evaluated for their ability to transmit Orientia tsutsugamushi (Hyashi) to mice. Of 4,372 transmission attempts using individual chiggers from all 13 colonies, 75% (n = 3,275) successfully infected mice. Transmission rates for the individual chigger colonies ranged from 7 to 80%. Increasing the number of chiggers that fed on a given mouse generally increased transmission rates. Transmission of O. tsutsugamushi to mice by different generations (F1-F11) of certain chigger colonies was stable; however, transmission rates varied greatly in other colonies. Transmission rates (both vertical and horizontal) of several L. changraiensis colonies and the L. deliense colony were the highest, suggesting that these colonies may be useful for the development of a chigger-challenge model that can be used to evaluate the efficacy of candidate scrub typhus vaccines or therapeutic agents in laboratory mice.     

Description of six new species of the genus Leptotrombidium from Mindanao Island and notes on the medically important genera of chiggers (Acari: Trombiculidae) of the Philippine Islands.

In the Philippine Islands, the genus Leptotrombidium currently consists of members of the subgenera Leptotrombidium and Trombiculindus. The following species are described as new: L. (L.) macacaphilus, L. (L.) longimedian, L. (L.) mindanensis, L. (L.) urogale, L. (L.) oculascutum, L. (T.) roseannleilaniae. The distribution of, and comments on, the known vectors of Rickettsia tsutsugamushi, L. (L.) deliense and L. (L.) fletcheri and the previously reported L. (L.) sandfordi, are given. Information on species of the potential scrub itch genera Blankaartia, Eutrombicula, and Schoengastia and a key to the genera and species of medically important trombiculids is provided.     

Indirect immunofluorescence antibodies in natural and acquired Rickettsia tsutsugamushi infections of Philippine rodents.

Antibodies against Rickettsia tsutsugamushi detected by the indirect fluorescent-antibody test (IFAT) were present in most rats trapped from a human focus of scrub typhus in the Philippines. Rickettsiae were isolated only from rats with positive IFATs. Naturally acquired antibodies persisted for at least 11 months, and antibodies resulting from experimental infections of rats persisted for at least 7 months. A common Philippine rodent, Rattus mindanensis, tolerated experimental infections with both local and standard Karp strains of R. tsutsugamushi, and such infections always produced a positive IFAT.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

Metabolism of Rickettsia tsutsugamushi and Rickettsia rickettsi in Irradiated Host Cells

The metabolism of Rickettsia tsutsugamushi (Gilliam strain) multiplying in irradiated L cells was investigated by methods involving the use of (14)C-labeled substrates and cycloheximide, an inhibitor of eukaryotic metabolism. Cycloheximide-resistant amino acid and adenine incorporations were appreciably higher in infected than in uninfected cultures during the period from 3 to 5 or 6 days postinoculation. The metabolism of R. rickettsi was similarly studied in primary duck embryo cells, which are more susceptible to infection with this rickettsia than are L cells. A difference in cycloheximide-resistant activity between infected and uninfected cultures was also noted, but was small. This finding is attributed to the more limited growth of R. rickettsi.     

Analysis of antigenic characteristics of Rickettsia tsutsugamushi Boryong strain and antigenic heterogeneity of Rickettsia tsutsugamushi using monoclonal antibodies.

Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.