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媒介和宿主中伯氏疏螺旋体感染调查及基因分型研究
Survey on the Prevalence and Genotyping of Borrelia Burgdorferi Sensu Lato in Vectors and Hosts
【作者】 褚宸一;
【导师】 曹务春;
【作者基本信息】 中国人民解放军军事医学科学院 , 流行病与卫生统计学, 2009, 博士
【摘要】 伯氏疏螺旋体是莱姆病的病原体,它主要在媒介和宿主中循环传播,人被感染病原体的媒介叮咬而感染。不同基因型伯氏疏螺旋体感染人引起疾病的临床症状及严重程度不同,而不同基因型伯氏疏螺旋体的地理分布与媒介和宿主分布相关,因此开展媒介和宿主中伯氏疏螺旋体感染调查及基因分型研究对预测不同地区莱姆病发生的危险有非常重大的现实意义。为了解我国不同地区莱姆病的潜在威胁及宿主动物在伯氏疏螺旋体进化和莱姆病发生或流行中作用,我们于2004-2008年在我国内蒙古大兴安岭林区、黑龙江长白山林区、新疆伊犁草原、贵州荔波林区和浙江金华和天台林区5个已知莱姆病自然疫源地,及西藏阿里草原和云南横断山脉林区2个莱姆病的潜在自然疫源地进行分子流行病学调查研究,调查潜在媒介和宿主中伯氏疏螺旋体的感染情况,确定伯氏疏螺旋体的基因型及其地理分布,探索致病性不明确的伯氏疏螺旋体对人类的致病性,分析生态类型和气候状况等地理环境因素,及媒介、宿主动物感染和伯氏疏螺旋体基因型分布等生物因素对莱姆病发生的潜在影响。本研究采用现场调查和实验研究相结合的方法进行。首先进行现场调查和标本采集,进而进行巢式PCR检测。结果显示,在除新疆外的其它调查点采集的标本中均检测到伯氏疏螺旋体存在,这一结果提供了莱姆病自然疫源地依然存在于多数调查地区的分子流行病学证据。检测的38种鼠中11种阳性,并且气候温暖的浙江和贵州林区鼠中伯氏疏螺旋体的感染率(分别为8.00%,7.14%)显著高于气温较低的内蒙古和黑龙江林区及云南高原林区(分别为3.45%,4.84%,0.84%) (χ2=21.255,df = 5, P=0.001),提示气候温暖的地区伯氏疏螺旋体的传播循环可能比气候寒冷的地区更易于维持。并证实棕背鼠平是东北林区伯氏疏螺旋体的主要储存宿主,提示社鼠可能是浙江林区伯氏疏螺旋体的主要宿主动物。还发现短耳兔和长尾鼩携带伯氏疏螺旋体,提示在我国作为伯氏疏螺旋体潜在储存宿主的小动物种类较多。采自大兴安岭林区的3种蜱中,全沟硬蜱的感染率(21.93%)显著高于森林革蜱(4.17%) (χ2=62.965,df = 2, P=0.000),未检测出嗜群血蜱有带菌情况,全沟硬蜱是当地林区优势蜱种,证实全沟硬蜱依然是该林区伯氏疏螺旋体的主要传播媒介。采自西藏阿里地区寄生于羊体表的羊蜱蝇存在伯氏疏螺旋体的感染(33/916, 3.6%),提示伯氏疏螺旋体可能通过羊蜱蝇的吸血活动在牲畜间传播,因此该牧区牲畜可能面临发生莱姆病的危险。对标本中伯氏疏螺旋体进行了RFLP分析,结果显示,我国存在3种基因型(群)伯氏疏螺旋体:Borrelia garinii、Borrelia afzelii和Borrelia valaisiana-like group。致病基因型B. garinii普遍存在于各调查地区,因此各地区均有必要采取防控措施预防莱姆病的发生或流行,B. valaisiana-like group只在南方地区发现,其确切的分类学地位和致病性尚不明确。同时发现单只蜱复合感染B. garinii和B. afzelii,这些蜱叮咬人可能使人发生复合感染,人感染这两种伯氏疏螺旋体表现的临床症状不同,因此临床医生在诊治表现复杂症状的疑似莱姆病病人时应考虑这种情况的存在,并采取相应的措施。此外,还发现B. garinii和B. valaisiana-like group及表现不同RFLP带型的B. valaisiana-like group同时存在于单只羊蜱蝇中,这种现象有利于伯氏疏螺旋体间的重组,从而促进变异体的产生,加速病原体的进化;可能是伯氏疏螺旋体遗传多样性出现的原因之一。对RFLP分析不能确定分类学地位的B. valaisiana-like group,进行了16S rRNA和fla基因片段的系统发育分析,结果表明B. valaisiana-like group与B. valaisiana在系统发育上接近,但又存在差别。进一步的MLSA分析表明B. valaisiana-like group菌株与B. valaisiana、B. afzelii及其它基因型伯氏疏螺旋体菌株7个位点的串联序列相似性低于判别基因型别的截点值,证明B. valaisiana-like group是一种新基因型伯氏疏螺旋体,并建议命名为Borrelia yangtze sp.,这一结果解释了B. valaisiana-like group与B. valaisiana在表型和传播循环等方面的差异。为探索B. valaisiana-like group对人类的致病性,选取3株菌株感染模型动物C3H/HeN实验小鼠,并用对C3H/HeN小鼠致病性明确的B. garinii菌株JW1作对照,病理结果显示B. valaisiana-like group菌株与JW1引起小鼠病理改变的器官相同,病变的程度相当或更严重,病变出现的时间和变化趋势相近。B. valaisiana-like group菌株对小鼠肾脏的病理损伤较为严重,而且呈持续加重的趋势;并引起小鼠心肌纤维细胞坏死,且病变也呈加重趋势;引起小鼠关节和脾脏的一过性病理改变;对脑组织病理改变在感染后第4周最为严重,此后间歇性复发并呈减轻趋势;对小鼠肝脏没有损伤。推测B. valaisiana-like group可能感染人类引起多器官不同程度的损伤。为进一步了解宿主动物在伯氏疏螺旋体扩散及莱姆病发生和流行中的作用,对广泛分布于欧亚地区的B. garinii和B. afzelii进行了基于8个看家基因的多位点序列分析,结果表明B. garinii的遗传多样性高于B. afzelii,并且对人致病性较强的OspA血清型4的B. garinii广泛分布于欧亚地区,该血清型的B. garinii以鼠为主要宿主,并被提议命名为一个新基因型B. bavariensis sp;序列相似性分析及聚类分析表明,我国东北部与日本岛的B. bavariensis遗传距离较近,而两者与欧洲该基因型伯氏疏螺旋体遗传距离稍远;同样以鼠为主要宿主的B. afzelii,欧洲菌株和与欧洲接壤的我国西北部菌株遗传距离较近,而两者与我国东北部该基因型伯氏疏螺旋体遗传距离均稍远;而以鸟为宿主的B. garinii,我国东北部分离株与欧洲分离株遗传距离较近。提示宿主的活动(迁移或迁徙)可能引起伯氏疏螺旋体在欧亚两地间播散,同时这些结果也进一步证实了伯氏疏螺旋体的进化和地理分布受其宿主分布的影响。本研究明确了目前我国不同地区媒介和宿主感染伯氏疏螺旋体的情况和伯氏疏螺旋体的基因型,确定了B. valaisiana-like group为一种新基因型伯氏疏螺旋体,并对其致病性作了进一步探讨,分析我国与相邻地区B. garinii和B. afzelii遗传特征的区别和联系,不仅为我国莱姆病的防治提供了理论依据,而且为我国莱姆病的深入研究奠定了基础。
【Abstract】 The spirochetes of the Borrelia burgdorferi sensu lato complex, the causative agents of Lyme borreliosis, are maintained in natural foci by circulation between the vector ticks in the Ixodes ricinus complex and animal hosts in various vertebrate taxa. This complex comprises at least 13 species, among which 4 species have been believed to be pathogenic to humans, including Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii and Borrelia spielmanii. Some of species have a wide and overlapping distribution while other species are distributed in different geographical regions. Different species are associated with distinct ecologic features, levels of pathogenicity, and clinical symptoms in patients.In order to find out underlying risk of Borrelia infection in the different regions of China’s mainland, and to infer the role of hosts that shape the evolution and epidemiology of B. garnii and B. afzelii, which were widely distributed in Eurasia, surveys were conducted in endemic foci (i.e. Heilongjiang province, Inner Mongolia autonomous region, Zhejiang province, Guizhou province, Xinjiang autonomous region) and potential endemic foci (i.e. Yunan province and Tibet autonomous region) in peak seasons of ticks during 2004 to 2008. Samples were collected, and Borrelia strains were isolated, and further PCR-RFLP analysis of 5S-23S rRNA intergenic spacer was conducted to investigate the prevalence and genospecies of B. burgdorferi s.l. in vectors and hosts. For samples which genospecies were not be clearly identified by using PCR-RFLP analysis, phylogenetic analyses of single gene (i.e. 16S rRNA,fla) and multilocus sequence analysis (MLSA) were performed to clarify their taxonomic status. Moreover, three B. valaisiana-like strains were inoculated into C3H/HeN mice in order to examine their pathogenic potential to humans. Besides, we analyzed the phylogeographic population structure of B. burgdorferi sensu lato in Eurasia by using a recently developed multilocus sequence typing (MLST) scheme based on eight chromosomally located housekeeping genes.A total of 1632 unfed adult ticks (i.e. Ixodes persulcatus, Dermacento silvarum, Haemaphysalis concinna and Haemaphysalis tibetensis) and 929 small animals (i.e. rodent, hare and animals of insectivora) and 916 Melophagus ovinus were collected and detected by nested PCR targeting 5S-23S rRNA intergenic spacer and RFLP analysis, with additional sequence analysis when necessary. B. burgdorferi s.l. was found in other six study areas except for Xinjiang autonomous region. This finding suggested that the endemic foci of Lyme disease might be still maintained in most of survery areas in mainland.Among 38 rodent species detected, 11 species were positive. Significant difference in prevalence of B. burgdorferi s.l. in rodents was observed among various regions (χ2=21.255,df = 5, P=0.001), with the highest in southern areas, which might be caused by the difference of climate. This finding suggested that the transmission cycle of B. burgdorferi s.l. might be easily maintained in the region with the warmer climate than that with the colder climate. In addition, B. burgdorferi s.l. was also detected in 3 Chinese hares (Lepus sinensis Gray) and 1 Hodgson’s brown-toothed shrew (Soriculus caudatus), which suggested that there were much species of animals serving as potential reservoir host of B. burgdorferi s.l. in China’s mainland.In Inner Mongolia autonomous region, the infection rate of I. persulcatus was significantly higher than that of D. silvarum, and H. concinna was free of B. burgdorferi s.l. (χ2=62.965,df = 2, P=0.000). I. persulcatus is the principal species of ticks in this forest area. These results indicated that I. persulcatus was still the major vector of B. burgdorferi s.l. in this area. However, D. silvarum and H. concinna usually inhabit grassland. This suggested that the threat of Lyme disease to humans might be less in grassland than that in forested area. Additionally, 3.6% (33/916) of M. ovinus samples was positive in Tibet, which indicated that some sheep in this pasture might be infected with B. burgdorferi s.l. or in risk of Lyme disease.Three genospecies (or group) were identified as B. garinii, B. afzelii and B. valaisiana-like group according to PCR-RFLP analysis. B. garinii being pathogenic to humans was the most prevalent genospecies and widely distributed in five regions except for Xinjiang, followed by B. afzelii. This finding suggested that the risk of Lyme disease to humans and livestock existed far and wide in China’s mainland. Multiple infections of different genospecies were found in single tick from Inner Mongolia and in single M. ovinus sample from Tibet. Multiple infections of different genospecies might also occur in humans by the tick bite. Since different genospecies of B. burgdorferi s.l. are related with distinct clinical manifestation of human Lyme disease, clinical symptom of patient being multiply infected with different Borrelia genospecies may be untypical and even complex, which should be noticed in diagnosis. B. valaisiana-like group was only found in Zhejiang, Guizhou, Yunnan and Tibet of south China, and its taxonomic status and pathogenic potential to humans remain unclear.Phylogenetic analyses of 16S rRNA gene and fla gene indicated that B. valaisiana-like strains were closely related to B. valaisiana. Meanwhile, difference was observed between two species. Further study based on multilocus sequence analysis (MLSA) indicated that the concatenated sequences of B. valaisiana-like strains used in this study were 98.4-99.8% identical with each other, whereas they were 96.4-96.9% related to those of B. valaisiana strains and only 92.5-94.9% related to those of other B. burgdorferi s.l. on the basis of the sequence identity matrix. According to MLSA sequence identity cut-off of 97.9% to differentiate B. burgdorferi s.l. species, B. valaisiana-like strains deserve a species status and are proposed to be named Borrelia yangtze sp., because of its distribution usually in the valley of Yangtze River. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-like strains from Eastern Asia.Three low-passage B. valaisiana-like strains as well as positive control B. garinii isolate JW1 were inoculated into C3H/HeN mice and disease was monitored by histopathology at 4 weeks, 8 weeks and 12 weeks after spirochaete challenge. Joint, heart, kidney, spleen, brain and liver were examined. B. valaisiana-like strains induced similar or more serious lesions in the joints, hearts, kidneys, spleens, brains of infected mice with B. garinii isolate JW1. No lesions were noted in the livers with inoculation of with B. valaisiana-like strains or B. garinii isolate JW1. But the pathogeneicity of these B. valaisiana-like strains to humans remains to be further clarified by the data of the infected patients.Based on sequence analyses of multiple housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG and rplB), B. garinii represented the most diverse species in Eurasia, followed by B. burgdorferi sensu stricto, B. afzelii and B. valaisiana. B. garinii OspA serotype 4 strains, which were seriously pathogenic to humans and proposed to be classified as a new species Borrelia bavariensis, were also widely distributed in Eurasia. Phylogenetic analyses on basis of the concatenated sequence analyses of 8 housekeeping genes showed that B. bavariensis strains from Europe fell into a cluster, which was separated from that of Asian B. bavariensis strains. B. afzelii strains from Europe branched into one cluster with one strains XY1 from Northwest China, which was separated from the cluster of B. afzelii strains from Northeast China. The form of geographical cluster of Borrelia strains might be caused by the highly parapatric distribution of their reservoir host of rodents. Moreover, B. garinii strains from Europe also clustered with those from Northeast China. Unfortunately, B. garinii strains from Northwest China was absent in this study. Further study is needed to clarify whether the high genetic similarity of B. garinii strains from Europe and Northeast China was associated with the migration of their reservoir host of birds. Above all, this study captured the genetic diversity and phylogeographic population structure of B. garinii and B. afzelii in Eurasia, which were related to the species and distribution of their reservoir host.
【Key words】 Borrelia burgdorferi sensu lato; Tick; Rodent; Genospecies; Restriction fragment length polymorphism analysis; Multilocus sequence analysis;
- 【网络出版投稿人】 中国人民解放军军事医学科学院 【网络出版年期】2009年 09期
- 【分类号】R181.3
- 【被引频次】7
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