Phylogeography of Rickettsia rickettsii Genotypes Associated with Fatal Rocky Mountain Spotted Fever

Rocky Mountain spotted fever (RMSF), a tick-borne zoonosis caused by Rickettsia rickettsii, is among the deadliest of all infectious diseases. To identify the distribution of various genotypes of R. rickettsii associated with fatal RMSF, we applied molecular typing methods to samples of DNA extracted from formalin-fixed, paraffin-embedded tissue specimens obtained at autopsy from 103 case-patients from seven countries who died of RMSF. Complete sequences of one or more intergenic regions were amplified from tissues of 30 (29%) case-patients and revealed a distribution of genotypes consisting of four distinct clades, including the Hlp clade, regarded previously as a non-pathogenic strain of R. rickettsii. Distinct phylogeographic patterns were identified when composite case-patient and reference strain data were mapped to the state and country of origin. The phylogeography of R. rickettsii is likely determined by ecological and environmental factors that exist independently of the distribution of a particular tick vector.     

斑点热群立克次体新种──虎林－93株的分离和鉴定

本文用鸡胚卵黄囊培养法及实验动物感染法从黑龙江省虎林县境内采集的嗜群血蜱中分离到了一株立克次体。命名为ＨＬ－９３株立克次体。经形态学及血清学试验证实为斑点热群立克次体。分别用微量免疫荧光法、十二烷基硫酸钠－聚丙烯酰胺凝胶不连续电泳法、单克隆抗体免疫酶斑点法、单克隆和多克隆抗体免疫印迹法、聚合酶链反应和限制性片段长度多态性分析法对该分离株进行鉴定并同已知国际标准株、国际及国内参考株进行比较。结果表明;ＨＬ－９３株立克次体无论在抗原性多肽上,还是在基因水平上在斑点热群立克次体中都是独特的。根据目前立克次体分类法,ＨＬ－９３株立克次体可以考虑是斑点热群立克次体的新种。     

中国斑点热群立克次体媒介和宿主的初步研究

斑点热群立克次体（ＳｐｏｔｔｅｄＦｅｖｅｒＧｒｏｕｐＲｉｃｋｅｔｔｓｉａ,ＳＦＧＲ）是引起斑点热的一组病原体。该组病原体是一组经蜱或螨叮咬传播的专性细胞内寄生菌且具有明显的自然疫源性特征。目前被认为的斑点热有：立氏立克次体引起的落矶山斑点热;西伯利亚立克次体引起的北亚热;康氏立克次体引起的钮扣热;澳大利亚立克次体引起的昆士兰斑点热;小蛛立克次体引起的立克次体痘;日本立克次体引起的红疹热等。自７０年代后,一方面由于人们生产、生活等实践活动的扩大和资源的开发利用及旅游业的发展等因素导致了斑点热发病率…     

斑点热群立克次体内蒙W-88蛛89102抗原的蛋白免疫印迹分析

蛋白免疫印迹试验表明从内蒙通辽病人旺某血标本分离的斑点热群立克次体W-88株与西伯利亚立克次体(232株及246株)的蛋白图谱完全相同,具有2个分子量为130KD及181KD的主要抗原性多肽及1个反应较弱的分子量为110KD的抗原性多肽,此与其它斑点热群立克次体不同。说明W-88株为西伯利亚立克次体。斑点热群立克次体中西伯利亚立克次体、立氏立克次体、康氏立克次体及派氏立克次体相互间抗原有较强的交叉反应。小蛛立克次体、澳大利亚立克次体与上述立克次体仅有微弱的交叉反应。说明斑点热群立克次体主要抗原分子有相同的决定簇。     

内蒙阿盟斑点热群立克次体Ha—91株的分离与鉴定

我们于1991年从内蒙阿盟亚东璃眼蜱分离到一株斑点热群(SFG)立克次体,命名为Ha-91株。免疫荧光法及蛋白免疫印迹分析表明Ha-91株立克次体与西伯利亚立克次体抗原性相近,但不完全相同;明显不同于其它种班点热群立克次体。这是首次证实亚东璃眼蜱为SFG立克次体的贮存宿主,也是首次从内蒙发现抗原性与西伯利亚立克次体不完全相同的SFG立克次体。     

微波微量免疫荧光检测斑点热立克次体方法的建立

目的　建立微波免疫荧光检测斑点热立克次体的方法。方法　通过微波促免疫荧光染色最佳时间、最佳火力的选择及微波免疫荧光方法与常规免疫荧光方法敏感性的比较。结果表明 :微波法反应速度快 ,只需 15min就可以观察结果 ,大大缩短了检测时间 ,提高了工作效率 ,并且两种方法在特异性上没有显著性差别。结论　我们认为微波免疫荧光方法是一种简便的、实用的快速检测立克次体的方法。     

Spotted Fever Group Rickettsia in Small Rodents from Areas of Low Endemicity for Brazilian Spotted Fever in the Eastern Region of Minas Gerais State,Brazil

We investigated the humoral immune response against different species of Rickettsia in serum samples from small rodents collected in two areas of a silent focus for Brazilian spotted fever in the eastern region of Minas Gerais State, Brazil. Sera samples were analyzed by indirect immunofluorescence assay using antigens from Rickettsia species of the spotted fever, ancestral, and transition groups. Titers ≥ 1:64 were considered positive. In Santa Cruz do Escalvado, 94% (30 of 32) of the samples collected from Rattus rattus, 22% (5 of 23) from Nectomys squamipes, and 80% (4 of 5) from Akodon sp., reacted by indirect immunofluorescence assay with Rickettsia antigens of the spotted fever group. In the municipality of Pingo D''Água, 84% (26 of 31) of the samples collected from R. rattus, 86% (6 of 7) of the samples from Oryzomys subflavus, 86% (6 of 7) from N. squamipes, and 100% (1 of 1) from Bolomys sp. contained antibodies that reacted with rickettsial antigens of the spotted fever group. These results demonstrated the previous exposure of small rodents to spotted fever group Rickettsia, suggesting the participation of these animals in the natural history of these rickettsiae in this region.     

Seroepidemiological Study of Outdoor Recreationists' Exposure to Spotted Fever Group Rickettsia in Western Australia

Bushland activity has previously been linked to rickettsial exposure in eastern and central regions of Australia, whereas little is known about the risks in Western Australia. The isolation of Rickettsia gravesii sp. nov. from Amblyomma triguttatum ticks and anecdotal reports of low-grade illness among bush recreationists raised the possibility of rickettsial transmission in the State. This study investigated rickettsial seroprevalence and potential risk of exposure to the spotted fever group rickettsiae in rogainers. Our results showed that rogainers active in the bush had a significantly higher risk of seropositivity (immunofluorescence total antibody titer ≥ 128) for the spotted fever group Rickettsia (odds ratio [OR] = 14.02, 95% confidence interval [CI] = 1.38–142.07) compared with a reference population, the overall seroprevalence in the rogainer group being 23.1%.     

Rickettsia rickettsii and Rickettsia montana from Ixodid ticks in Connecticut

Dermacentor variabilis, infected with spotted fever group rickettsiae, parasitized 8 of 70 raccoons captured in Newtown, Connecticut. The spotted fever agent, Rickettsia rickettsii, was isolated and identified from 4 adult D. variabilis and from 1 nymphal Ixodes texanus removed from raccoons. This verifies the presence of this etiologic agent in ticks in an area where 6 people had clinical signs and symptoms of Rocky Mountain spotted fever (RMSF) and antibodies to R. rickettsii. These are the first isolations of R. rickettsii from D. variabilis in southern New England and the first identified rickettsiae from I. texanus. No rickettsiae were isolated from Ixodes muris or I. cookei. Rickettsia montana was recovered in Vero cell culture from a D. variabilis collected in East Haddam, Connecticut where RMSF is not known to be prevalent.     

斑点热群立克次体内蒙W-88株全细胞脂肪酸气相色谱-质谱联用系统的分析

本文报告用毛细管柱气相色谱-质谱联用系统,分析研究了斑点热群立克次体内蒙W-88株(人株)的全细胞脂肪酸。结果表明W-88株与国际标准株西伯利亚立克次体232株(人株)的脂肪酸气相色谱图和其主要色谱峰的质谱图基本一致,从而认为W-88株属于斑点热群中的西伯利亚立克次体(Rickettsia sibirica)。     

Multispacer Typing (MST) of Spotted Fever Group Rickettsiae Isolated from Humans and Rats in Chengmai County,Hainan Province,China

Spotted fever caused by spotted fever group rickettsiae (SFGR) is found throughout China. During 2007–2008, 28 human SFGR isolates and 34 rat SFGR isolates including 15 isolates from Rattus fulvescens, 5 isolates from R. edwardsi, 7 isolates from Callosciurus erythraeus roberti and 7 isolates from Dremomys rufigenis) were obtained from L929 cell culture. Previous research indicated that the 62 strains of SFGR mentioned above shared not only the same serophenotype but also 100% of identity sequences of 16S rRNA, gltA, ompA, groEL and 17KD, which enabled us to apply multispacer typing (MST) to the 62 SFGR isolates in the study. Six primer pairs, which were used for typing of Rickettsia rickettsii and Rickettsia conorii, were chosen, and the results exhibited greater nucleotide polymorphisms among the 62 isolates tested. A total of 48 distinct genotypes were identified. The dominant genotype, represented by h3 isolates, accounted for 21.7% (13/60) of the isolates tested, and the remaining 47 genotypes were all unique. Phylogenetic analysis showed that all the 48 genotypes could be classified in the same clade, while the genetically related strain, R. heilongjiangensis, was close but not the same as the cluster. We concluded that the genetically diverse of spotted fever group rickettsiae strains are endemic in Chengmai County, Hainan Province, China.     

黑龙江立克次体054株单克隆抗体对斑点热群立克次体抗原结合位点及特性的分析

本文利用三株黑龙江立克欢你054株（HLJ－054）的单克隆抗体对四株斑点热群立克次体西伯利亚种（R．s．）246株、精河株（JH－74）,立氏立克次体（R.r．）R株及HLJ-054株进行了血清学反应,对其蛋白组成及单抗结合抗原特性等方面进行了研究。结果表明：JH-74林与246株在上述三个方面完全一致,不同于HLJ－054及R．r．R株;HLJ－054株与R．r．R株与三株单抗血清学反应高度交叉．但二者在蛋白组成及与单抗反应的抗原组成上均存在差异。该结果支持黑龙江立克次体054株为斑点热群立党次作的一个新成员。单抗F7针对的抗原结合表位为蛋白多肽;而单抗2E1、4G2结合的抗原表位可能为精蛋白。     

四株斑点热群立克次体DNA同源性的初步研究

用缺口翻译法将斑点热群立克次体西伯利亚 246 株 DNA 和康氏 Simko株DNA标记~(32)P。分别与中国分离株黑龙江 054株和新疆精河株 DNA进行斑点杂交,以对国内两分离株基因组与北亚、康氏基因组同源性进行初步研究。结果显示,新疆精河株 DNA与西伯利亚246株DNA高度同源。与血清分类实验结果相吻合。黑龙江 054株DNA 与西伯利亚 246株 DNA和康氏 Simko 株 DNA的杂交阳性最低稀释度均低于阳性对照100倍以上。提示054株基因组与西伯利亚立克次体、康氏立克次体 Simko 株基因组存在较明显之差别。     

Epidemiology of Spotted Fever Group and Typhus Group Rickettsial Infection in the Amazon Basin of Peru

A seroprevalence study for IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) Rickettsia among humans and domestic pets was conducted in the city of Iquitos, located in the Amazon basin of Peru. Of 1,195 human sera analyzed, 521 (43.6%) and 123 (10.3%) were positive for SFGR and TGR antibodies, respectively. District of residence and participant age were associated with antibody positivity for both groups, whereas rodent sightings in the home were associated with TGR antibody positivity. Of the 71 canines tested, 42 (59.2%) were positive for SFGR antibodies, and two (2.8%) were positive for TGR antibodies; one active SFGR infection was detected by polymerase chain reaction. An uncharacterized SFGR species was detected in 95.9% (71/74) of Ctenocephalides felis pools collected from domestic pets. These data suggest that rickettsial transmission is widespread in Iquitos. Rickettsia species should be further explored as potential causes of acute febrile illnesses in the region.     

Infrequency of Rickettsia rickettsii in Dermacentor variabilis removed from humans, with comments on the role of other human-biting ticks associated with spotted fever group Rickettsiae in the United States

From 1997 to 2009, the Tick-Borne Disease Laboratory of the U.S. Army Public Health Command (USAPHC) (formerly the U.S. Army Center for Health Promotion and Preventive Medicine) screened 5286 Dermacentor variabilis ticks removed from Department of Defense (DOD) personnel, their dependents, and DOD civilian personnel for spotted fever group rickettsiae using polymerase chain reaction and restriction fragment length polymorphism analysis. Rickettsia montanensis (171/5286 = 3.2%) and Rickettsia amblyommii (7/5286 = 0.1%) were detected in a small number of samples, but no ticks were found positive for Rickettsia rickettsii, the agent of Rocky Mountain spotted fever (RMSF) until May 2009, when it was detected in one D. variabilis male removed from a child in Maryland. This result was confirmed by nucleotide sequence analysis of the rickettsial isolate and of the positive control used in the polymerase chain reaction, which was different from the isolate. Lethal effects of rickettsiostatic proteins of D. variabilis on R. rickettsii and lethal effects of R. rickettsii infection on tick hosts may account for this extremely low prevalence. Recent reports of R. rickettsii in species Rhipicephalus sanguineus and Amblyomma americanum ticks suggest their involvement in transmission of RMSF, and other pathogenic rickettsiae have been detected in Amblyomma maculatum. The areas of the U.S. endemic for RMSF are also those where D. variabilis exist in sympatry with populations of A. americanum and A. maculatum. Interactions among the sympatric species of ticks may be involved in the development of a focus of RMSF transmission. On the other hand, the overlap of foci of RMSF cases and areas of A. americanum and A. maculatum populations might indicate the misdiagnosis as RMSF of diseases actually caused by other rickettsiae vectored by these ticks. Further studies on tick vectors are needed to elucidate the etiology of RMSF.     

Spotted fever: isolation of Rickettsia from a skin biopsy sample]

A 2 years old child living in an area of the State of S?o Paulo, known in the past as endemic for rickettsiosis developed clinical evidences of spotted fever after a tick bite. Rickettsiae were isolated from guinea pigs inoculated with a skin homogenate. In sera tested by indirect immunofluorescence with Rickettsia rickettsii standard antigen, IgG specific antibody titers raised from 1:512 in the first sample to 1:2048 in the third one; IgM specific antibody titer was 1:128 in the three samples. Also positive were sera obtained from the inoculated guinea pigs. In the last 20 years no other case of rickettsial spotted fever has been confirmed by isolation of the agent in Brasil. To our knowledge, there are no previous reports of isolation of Rickettsiae through inoculation of skin biopsy homogenates.     

Expanding Range of Amblyomma americanum and Simultaneous Changes in the Epidemiology of Spotted Fever Group Rickettsiosis in the United States

Spotted fever group (SFG) Rickettsia species are etiologic agents of a wide range of human infections from asymptomatic or mild infections to severe, life-threatening disease. In the United States, recent passive surveillance for SFG rickettsiosis shows an increased incidence and decreased severity of reported cases. The reasons for this are not well understood; however, we hypothesize that less pathogenic rickettsiae are causing more human infections, while the incidence of disease caused by more pathogenic rickettsiae, particularly Rickettsia rickettsii, is relatively stable. During the same period, the range of Amblyomma americanum has expanded. Amblyomma americanum is frequently infected with Candidatus Rickettsia amblyommii, a SFG Rickettsia of unknown pathogenicity. We tested our hypothesis by modeling incidence rates from 1993 to 2013, hospitalization rates from 1981 to 2013, and case fatality rates from 1981 to 2013 regressed against the presence of A. americanum, the decade of onset of symptoms, and the county of residence. Our results support the hypothesis, and we show that the expanding range of A. americanum is associated with changes in epidemiology reported through passive surveillance. We believe epidemiological and acarological data collected on individual cases from enhanced surveillance may further elucidate the reasons for the changing epidemiology of SFG rickettsiosis.