Mediterranean spotted fever: A preliminary tick field study

We studied the prevalence of rickettsia-like organisms infection in different tick species (3154 adults) collected in several areas of the Ebro valley (Spain). The Gimenez stain showed a prevalence of rickettsia-like organisms in 38.6% of the studied ticks. Using the immunofluorescence assay, only two species, Rhipicephalus sanguineus (16.4%) and R. pusillus (19.9%) have shown organisms compatible with spotted fever group rickettsiae.     

斑点热群立克次体HN-98株的分离和初步鉴定

目的 从海南省斑点热疫源地不明热病人体内分离斑点热群立克次体。方法 用鸡胚卵黄囊培养法直接从斑点热疫区一不明热病人血标本中分离立克次体,继之用分子生物学及血清学试验对分离株进行鉴定。结果 从海南省琼中县斑点热疫区一不明热男性民工的血标本中分离到了一株立克次体,命名为ＨＮ－９８株立克次体,西伯利亚种。结论 该结果进一步从病原学上证实海南省琼中县存在有斑点热疫源地。     

我国斑点热立克次体新种检测及其rOmpA基因序列分析

采用斑点热群立克次体特异引物扩增福建南部来源蜱标本和动物标本中的DNA,以发现我国可能存在的新种斑点热群立克次体.以立氏立克次体rOmpA基因序列中的相关序列为引物,扩增标本中rOmpA基因部分片段,用pGEM-T载体及其宿主菌进行阳性克隆筛选,阳性克隆经鉴定之后提取质粒DNA,进行序列测定.测定序列用Blast软件进行序列同源性比较并采集相关序列,用Bootstrip方法随机取样100次,用PHYLIP软件包进行聚类分析.共检测采自福建宁化地区的越原血蜱100只和野鼠类血标本38份.其中从越原血蜱(不同来源)扩增出斑点热立克次体基因片段,阳性率为15.04%(17/113);并从社鼠、黄胸鼠、褐家鼠扩增出阳性片段,以鼠类为单位计算其阳性率为44.73%.对寄生于野鼠的越原血蜱扩增的阳性片段序列分析表明新测序列与其它斑点热群立克次体的同一基因片段不同,其与日本立克次体同源性最高,核苷酸序列同源性为94%,推定的氨基酸序列同源性为89%.从病原学角度首次证实我国福建省存在日本立克次体近缘种立克次体,这对斑点热群立克次体的深入研究有重要意义.     

一组长角血蜱中检出莱姆病螺旋体和斑点热群立克次体复合感染

目的 了解浙江省山区野生动物和蜱中莱姆病、斑点热、埃立克体病(无形体病)的感染情况.方法 采用巢式PCR对采集的鼠、蜱标本进行莱姆病伯氏疏螺旋体、斑点热群立克次体、埃立克体(无形体)特异性核酸片段检测分析.结果 从121份鼠标本和105组蜱标本中检出阳性结果 14份.鼠标本中检出伯氏疏螺旋体5S～23S rDNA间隔区片段1份和埃立克体(无形体)16SrDNA 5'端片段2份.蜱标本中检出阳性11份,包括伯氏疏螺旋体5S～23S rDNA间隔区片段3份和斑点热群立克次体外膜蛋白OmpA基因5'端片段8份.其中1组长角血蜱成虫标本为伯氏疏螺旋体和斑点热群立克次体复合感染,5S～23S rRNA基因间隔区和ompA基因片段均阳性,分别与伯氏疏螺旋体法雷氏基因型和马赛立克次体株等关系较近.结论 在同一组长角血蜱成虫中同时检出莱姆病疏螺旋体和斑点热群立克次体复合感染.     

内蒙通辽市斑点热群立克次体W—88株的分离与鉴定

1988年我们自通辽市一例发热、头痛、临床诊断为"上感"、用青霉素、病毒灵治疗无效的病人,分离出一株斑点热群立克次体W-88株。经动物感染试验、鸡胚卵黄嚢培养和传代,达到立克次体纯培养,用泛影葡胺密度株度离心纯化,制出立克次饵抗原,用我室建立的抗西伯利亚立克次体群和种特异性单克隆抗体进行免疫荧光检测,与斑点热群立克次体7株国际标准株同从中国分离株对比鉴定。结果确定W-88株为西伯利亚立克次体,该例病人所患的疾病属北亚蜱传斑点热,此为我国在城市发现的北亚热病例首次报道。     

Prevalence of antibodies to spotted fever group rickettsiae in the region of Split (southern Croatia)

The prevalence of IgG antibodies to spotted fever group rickettsiae (SFG) was investigated by the indirect immunofluorescence assay in a sample of 1207 healthy residents of the central part of southern Croatia (eastern coast of Adriatic Sea). Sera obtained from subjects living in three different places of residency (urban, suburban and rural environs of the region) were tested. Overall 528 (43.7%) persons had significant antibody titers (1:40 or higher), with a significantly higher prevalence in suburban and rural areas. The results show that inhabitants in the area are clearly being exposed to SFG rickettsiae. Risk factors significantly associated with seropositivity to SFG rickettsiae were: living in suburban and rural settlements (odds ratio [OR] = 1.81; 95% confidence interval [CI] = 1.35–2.4), contact with animals (OR: 1.52; 95% CI: 1.15–1.99), and increase of age (OR: 1.18; 95% CI: 1.09–1.26).     

Novel PCR exclusion assay to detect spotted fever group rickettsiae in the lone star tick (Amblyomma americanum)

The lone star tick (Amblyomma americanum) is the most common and abundant human-biting tick in the southeastern United States where spotted fever rickettsioses frequently occur. However, the role of this tick in transmitting and maintaining pathogenic and non-pathogenic spotted fever group rickettsiae (SFGR) remains poorly defined. This is partially due to the high prevalence and abundance of Rickettsia amblyommatis in most populations of A. americanum. Many molecular assays commonly employed to detect rickettsiae use PCR primers that target highly conserved regions in the SFGR so low abundance rickettsia may not be detected when R. amblyommatis is present. It is costly and inefficient to test for low abundance rickettsial agents with multiple individual specific assays even when they are multiplexed, as most samples will be negative. Real time PCR assays may also be hampered by inadequate limits of detection (LODs) for low abundance agents. We exploited the absence of an otherwise relatively SFGR-conserved genome region in R. amblyommatis to design a hemi-nested PCR-assay which has a sensitivity of 10 copies in detecting the presence of most SFGR, but not R. amblyommatis in DNA of infected lone star ticks. This deletion is conserved in 21 isolates of R. amblyommatis obtained from multiple states. We demonstrated the assay’s utility by detecting a pathogenic SFGR, Rickettsia parkeri, in 15/50 (30 %) of field collected A. americanum ticks that were previously screened with conventional assays and found to be positive for R. amblyommatis. These co-infected ticks included 1 questing female, 6 questing nymphs, and 8 attached males. The high prevalence of R. parkeri among host-attached ticks may be due to several variables and does not necessarily reflect the risk of disease transmission from attached ticks to vertebrate hosts. This novel assay can provide accurate estimates of the prevalence of less common SFGR in A. americanum and thus improve our understanding of the role of this tick in the maintenance and transmission of the SFGR commonly responsible for human rickettsioses.     

吉林省长白山区斑点热立克次体自然疫源地调查

目的　了解吉林省长白山区蜱传斑点热的自然疫源地情况。方法　利用立氏立克次体[相对分子质量 (Mr) 190× 10 3 ]外膜蛋白A(R .rOmpA)基因序列设计引物 ,对 6 83只蜱类标本进行聚合酶链反应 (PCR)检测 ,并随机抽取一株森林革蜱阳性扩增产物进行克隆与序列测定。结果　从森林革蜱和嗜群血蜱标本中检测出了斑点热立克次体DNA片段 ,阳性率分别为 5 3.81%和 7.4 1% ;所测序列与前苏联的DnS 14株的同源性为 10 0 .0 0 % ,与DnS 2 8和RpA 4的同源性均为99.0 0 % ;而与国内所检测的BJ 90、HLJ 0 5 4的同源性分别为 88.0 0 %和 86 .0 0 %。结论　长白山区存在斑点热的自然疫源地 ,存在与DnS 14株型别一致的斑点热立克次体 ,在中国系首次发现。     

海南省澄迈县斑与执群立克次体分离株多间隔序列分型

目的 采用多间隔序列分型（MST）对从海南省澄迈县发热患者及野鼠分离的62株斑点热群立克次体进行分型研究。方法 根据文献检索,应用9对成功用于斑点热群立克次体MST分型引物,对实验62株菌进行PCR扩增及测序。校正后序列进行Blast分析。同时,对序列进行Megalign比对分析单核苷酸多态性（SNPs）。将每株细菌成功测序的6段间隔序列按一定顺序拼接并构建进化树。结果 使用6对间隔序列引物扩增实验菌株,结果显示每对引物扩增序列均显示较大的变异性,共获得48个变异型,以h3为代表的主要变异型占21.7%（13/60）,剩余的47个变异型为独立型,分别占1.6%。进化树分析结果显示48个变异型均归为一簇。同其他参考菌株比较发现,黑龙江立克次体与上述菌株关系密切。SNPs分析发现位点变异主要以T-C转换及A-G转换为主。结论 海南地区斑点热群立克次体分离株非编码间隔序列呈高度变异性。MST分型较传统的血清表型分型以及MLST分型更具有鉴别能力,对未来立克次体暴发流行可提供快速有效的流行株追踪。     

多聚酶链反应技术在斑点热群立克次体流行病学调查中的应用

作者首次用来自立氏立克次体（Ｒ．ｒｉｃｋｅｔｔｓｉｉ，Ｒｒ）１９０ＫＤａ蛋白抗原基因序列设计的一对引物扩增从黑龙江、河北、海南、北京采集的蜱、蜱卵、幼蜱、蜱粪及啮齿动物脏器中斑点热群立克次体ＤＮＡ。结果从上述标本中均检测出了斑点热群立克次特异的５３２ｂｐ大小的ＤＮＡ片段，该结果部分与同期进行的病原分离结果一致。故认为，ＰＣＲ技术是一种快速、敏感、特异的斑点热群立克次体流行病学调查方法。     

Serologic response to rickettsial antigens in patients with Astrakhan fever

Astrakhan fever is a new spotted fever group (SFG) rickettsiosis. Sera of patients with Astrakhan fever have been examined by microimmunofluorescense and western immunoblotting to determine the serologic responses to the Astrakhan strain and toR. conorii M-1 strain and the Israelian isolate of SFG rickettsiae. The serologic response to specific rickettsial agent and to Israelian isolate has been found to be similar, but was different of that toR. conorii. Immunoglobulin G (IgG) and IgM antibodies were detected in most sera and were directed against the lipopolysaccharide. Only one of tested sera contained IgG antibodies which also recognized high molecular weight proteins.     

浙江省蜱标本中斑点热群立克次体rOmpA和gltA基因检测

目的了解浙江省部分山区蜱中感染斑点热群立克次体的状况,探讨以外膜蛋白A（rOmpA）和柠檬酸合成酶基因（gltA）为靶基因的PCR方法可行性。方法利用PCR方法,检测天台县左溪镇和临安县西天目地区共46组蜱类标本中rOmpA和gltA基因特异片段。对所检测到的阳性结果进行克隆与序列测定,并进行聚类分析。结果从46组蜱标本中检测发现2组长角血蜱中斑点热群立克次体的rOmpA和gltA基因片段均为阳性,核酸序列基本一致,但推测斑点热群立克次体种的进化位置存在差异。结论浙江省部分山区存在蜱中感染斑点热群立克次体的状况。利用rOmpA和gltA基因能从标本中检测到斑点热群立克次体,但从2个结果推测立克次体的分类位置存在差异。     

Antigens of Rickettsia conorii recognized by seropositive healthy people from Salamanca (Central-West Spain)

The antigenic reactivity in Western immunoblotting assay of individual Rickettsia conorii components with sera of healthy people living in Salamanca Province, an endemic zone of Mediterranean spotted fever, is evaluated. Polypeptides of molecular weights 100 kDa (92.7%), 135 kDa (75.6%), 160 kDa (70.7%) and 115 kDa (48.8%) were recognized by a higher proportion of sera with indirect immunofluorescent antibody test titers 1:80. Reaction with apparent rickettsial lipopolysaccharide was found in 15 (36.6%) of these samples. The involvement of different rickettsial strains, atypical routes of inoculation, varying content of the inoculum, and host factors may be determinants of the clinical expression of the spotted fever group rickettsial infection in people who produce antibodies reactive with Rickettsia conorii antigens.     

Evidence of the presence of spotted fever group rickettsiae in dogs and dog ticks of the central provinces in Spain

To assess canine exposure to spotted group rickettsiae in the central provinces of Spain, ticks removed from dogs were studied by immunofluorescence (IF) staining. Twenty-eight out of 65 (43.0%) samples from ticks were positive. Sera from 58 dogs were also collected and the presence of antibodies to Rickettsia conorii studied. Thirty-four (58.6%) dogs presented significant titers by IF. Surveillance data from these provinces show the presence of Boutonneuse fever cases during recent years, evidence that dogs could serve as an indicator of rickettsial activity in these areas.Corresponding author.     

斑点热群立克次体实时荧光定量PCR检测方法的 建立及应用

摘要:目的　探讨实时荧光定量PCR快速检测斑点热群立克次体标本的应用价值。方法　根据斑点热群立克次体外膜蛋白A（OmpA）基因保守序列设计TaqMan特异性探针和引物,进行实时荧光定量PCR方法学评估,并同时运用该法和普通PCR法对对海南省2012年不明原因发热病人157份血标本进行斑点热群立克次体的检测及结果分析。结果　本研究建立的定量标准曲线循环阈值（Ct值）和模板拷贝数呈良好的线性关系（R2值为0.998）。该法能检出斑点热群立克次体阳性标准品—西伯利亚立克次体（R.sibirica）最小浓度为102 copies/μl,灵敏度比普通PCR高100倍。用该法检测斑点热群立克次体DNA,结果为阳性,检测金黄色葡萄球菌、铜绿假单胞菌、致泻性大肠埃希菌和副溶血弧菌等其他病原细菌DNA结果均为阴性。采用实时荧光定量PCR法对实验室库存的发热病人血标本DNA进行回顾性检测,结果示阳性2例,检出率为1.2%（2/157）,而普通PCR检测结果均为阴性。结论　实时荧光定量PCR法具有快速、特异和灵敏的优点,在处理突发公共卫生事件中可发挥快速检测的优势,在斑点热诊断中具有重要的临床意义。     

部分省区啮齿动物中斑点热群立克次体的检测

目的了解我国部分省区啮齿动物中斑点热群立克次体的自然感染情况，比较南北地区不同鼠种带菌率的差异。方法在黑龙江、内蒙古、新疆、浙江和贵州5省（自治区）共捕获鼠473只，用聚合酶链反应检测鼠脾脏中的斑点热群立克次体DNA序列片段。结果共检测鼠473只，69只阳性，阳性率为14．6％；在黑龙江、内蒙古、新疆、浙江和贵州省（自治区）都检测出阳性标本，阳性率分别为3．1％、21．6％、30．0％、10．8％和21．4％。结论我国黑龙江、内蒙古、新疆、浙江和贵州省（自治区）的鼠中都存在斑点热群立克次体的感染，南北地区鼠的带菌率差异无统计学意义（P〉0．05）。     

二连浩特口岸鼠体寄生蜱感染病原体调查

目的 调查二连浩特口岸鼠体寄生蜱种群组成及自然感染病原体情况.方法 从捕获的鼠体上收集寄生蜱,提取单只蜱DNA基因组及总RNA,采用PCR或RT-PCR方法检测莱姆病螺旋体、Q热立克次体、巴贝西原虫、斑点热立克次体、无形体、森林脑炎病毒、布尼亚病毒、克里米亚-刚果出血热病毒.结果 共采集鼠体寄生蜱152只,分别为草原血蜱、银盾革蜱和亚东璃眼蜱,其中草原血蜱为优势种,占捕获总数的98.03％.从草原血蜱、银盾革蜱和亚东璃眼蜱中检测到斑点热立克次体19份,检出率为12.50％,其他病原体检测均为阴性.结论 二连浩特口岸地区存在斑点热立克次体自然疫源地.     

Detection of chicken antibodies to mosquito salivary gland antigens by enzyme immunoassay

Sentinel chickens are used to detect western equine encephalomyelitis, St. Louis encephalitis, and West Nile virus activity. Flocks that receive high mosquito exposure will be most effective for surveillance purposes. However, mosquito population indices at the flock sites may only provide an indirect measure of potential exposure. Therefore, we developed an indirect enzyme immunoassay to detect chicken antibodies to salivary gland antigens (SGAs) from Culex tarsalis, the primary arbovirus vector in California. Chickens fed upon by Cx. tarsalis developed an antibody response that was proportional to the amount of exposure. Cross-reactivity between sera from Cx. tarsalis-exposed chickens and SGAs from Culex pipiens quinquefasciatus, Culex pipiens pipiens, Ochlerotatus melanimon, and Ochlerotatus sierrensis was likely due to shared SGAs among these species. This serologic assay for mosquito exposure could be used to evaluate the sensitivity of sentinel flocks for detecting arboviral activity.     

非洲猪瘟媒介软蜱在中国潜在的适生性分析

目的非洲猪瘟病毒可引起家猪的出血性致死性疫病,其一旦发生,很难根治。对此病传入我国的危险性进行评价,预测其生物传播媒介在我国的潜在分布区,对于防控工作具有重要意义。方法运用适生性分析软件Climex预测软蜱在我国的潜在分布区。结果根据软蜱的生物学数据和软件自带的模板参数,得出软蜱的调试参数;经Climex分析,软蜱在我国云南、贵州、四川东部、重庆、陕西南部、湖南、湖北、江西东北部、安徽、河南、浙江、江苏、山东、河北南部等地的生态气候指数值〉20,为高适宜潜在发生区。结论我国部分地区可能成为非洲猪瘟的自然疫源地。提示应将这些地区列为监测和防控的重点。     

Isolation on « VERO » cells of a Spotted Fever Group Rickettsia « Mortilli strain » from Rhipicephalus sanguineus

The isolation of a Spotted Fever Group (SFG) Rickettsia « Mortilli strain » from brown dog ticks Rhipicephalu.s sanguineus removed from dogs associated with a confirmed case of Boutonneuse Fever (BF) in a site of a Western Sicily is reported. The need is stressed for isolation and adaption on cell cultures of SFG rickettsial strains for analysis of antigenic structure employing modern methodology.Corresponding author