应用TaqMan探针实时荧光定量PCR法调查北京市部分城郊区鼠传病原体流行状况
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摘要
目的调查北京市部分城郊区6种鼠传病原体流行状况。方法将已报道的TaqMan探针实时荧光定量PCR(qPCR)检测方法在本实验室进行评估,制备质粒标准品和绘制标准曲线,依据重新评估的数据确定结果的检测阈值。2017年1-6月应用夹夜法在北京市朝阳、怀柔、门头沟和平谷4个地区捕获啮齿动物,采集组织样品提取核酸,应用上述qPCR方法检测病原体。结果共捕获啮齿动物160只,其中褐家鼠59只、小家鼠69只、朝鲜姬鼠4只、北社鼠25只和鼩鼱3只。检测动物脾组织结果显示,除鼩鼱外其他啮齿动物均分别感染莫氏立克次体(8.1%)、恙虫病东方体(1.3%)、嗜吞噬细胞无形体(5.6%)、钩端螺旋体(18.7%)和巴尔通体(5.0%);所有动物中均未检测到土拉弗朗西斯菌。褐家鼠和小家鼠中存在钩端螺旋体、嗜吞噬细胞无形体等多种病原体复合感染情况。结论北京地区的啮齿动物可携带多种常见、新发及再发传染病病原体,应关注并加强该地区这些病原体动物宿主和传播媒介的分布调查及相关传染病的防制工作。
Objective To investigate the prevalence of 6 rodent-borne pathogens, in some suburban areas of Beijing,China. Methods TaqMan fluorescent probe-based quantitative real-time PCR(qPCR) was used for laboratory evaluation.Plasmid standards were prepared and standard curves were plotted. Detection thresholds were determined according to the reevaluated data. From January to June, 2017, the trap-at-night method was used to capture rodents in Chaoyang, Huairou,Mentougou, and Pinggu districts of Beijing, tissue samples were collected to extract nucleic acids, and qPCR was used to detect pathogens. Results A total of 160 rodents were captured, among which there were 59 Rattus norvegicus rodents, 69 Mus musculus rodents, 4 Apodemus peninsulae rodents, 25 Niviventer confucianus, and 3 Sorex spp.. The examination of spleen tissues showed that all rodents except Sorex were infected with Rickettsia typhi(8.1%), Orientia tsutsugamushi(1.3%), Anaplasma phagocytophilum(5.6%), Leptospira interrogans(18.7%), or Bartonella spp.(5.0%). No Francisella tularensis was detected in all rodents. There were co-infections with various pathogens including Leptospira interrogans and A. phagocytophilum in R. norvegicus and M. musculus. Conclusion Rodents in Beijing carry the pathogens of common,emerging and reemerging infectious diseases. The distribution investigation of animal hosts and vectors of these pathogens in these areas and prevention and treatment of related infectious diseases should be strengthened.
Objective To investigate the prevalence of 6 rodent-borne pathogens, in some suburban areas of Beijing,China. Methods TaqMan fluorescent probe-based quantitative real-time PCR(qPCR) was used for laboratory evaluation.Plasmid standards were prepared and standard curves were plotted. Detection thresholds were determined according to the reevaluated data. From January to June, 2017, the trap-at-night method was used to capture rodents in Chaoyang, Huairou,Mentougou, and Pinggu districts of Beijing, tissue samples were collected to extract nucleic acids, and qPCR was used to detect pathogens. Results A total of 160 rodents were captured, among which there were 59 Rattus norvegicus rodents, 69 Mus musculus rodents, 4 Apodemus peninsulae rodents, 25 Niviventer confucianus, and 3 Sorex spp.. The examination of spleen tissues showed that all rodents except Sorex were infected with Rickettsia typhi(8.1%), Orientia tsutsugamushi(1.3%), Anaplasma phagocytophilum(5.6%), Leptospira interrogans(18.7%), or Bartonella spp.(5.0%). No Francisella tularensis was detected in all rodents. There were co-infections with various pathogens including Leptospira interrogans and A. phagocytophilum in R. norvegicus and M. musculus. Conclusion Rodents in Beijing carry the pathogens of common,emerging and reemerging infectious diseases. The distribution investigation of animal hosts and vectors of these pathogens in these areas and prevention and treatment of related infectious diseases should be strengthened.
引文
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[22]张鑫,邢海英,张凤革,等.北京市平谷区恙虫病134例临床特征分析[J].浙江临床医学,2014,16(7):1079-1080.
[23]董翠珍,李文靖.昌平县钩端螺旋体病感染状况调查[J].中国人兽共患病杂志,1995,15(5):133.
[24]齐海霞,张海云,邓小雨,等.犬、猫钩端螺旋体病的流行病学调查[J].中国畜牧兽医,2012,39(11):203-206. DOI:10.3969/j.issn.1671-7236.2012.11.047.
[25]肖园,吴海燕,孙艳争,等.犬钩端螺旋体病3例报告[J].中国人兽共患病学报,2017,33(5):469-471. DOI:10.3969/j.issn.1002-2694.2017.05.016.
[26]王雅琴,张海云,唐艳荣,等. 2016年朝阳区动物诊疗机构犬狂犬病和A型流感的流行病学调查报告[J].中国畜牧兽医文摘,2017,33(11):124-126.
[27] Diaz JH. Recognition and management of rodent-borne infectious disease outbreaks after heavy rainfall and flooding[J].J La State Med Soc,2014,166(5):186-192.
[28] Villette P,Afonso E,Couval G,et al. Consequences of organ choice in describing bacterial pathogen assemblages in a rodent population[J]. Epidemiol Infect,2017,145(14):3070-3075.DOI:10.1017/S0950268817001893.
[29] Galan M,Razzauti M,Bard E,et al. 16S rRNA amplicon sequencing for epidemiological surveys of bacteria in wildlife[J].mSystems,2016,1(4):e00032-16. DOI:10.1128/mSystems.00032-16.
[30] World Health Organization. 2018 annual review of diseases prioritized under the research and development blueprint[R].Geneva:WHO,2018:2,9.
[2] Meerburg BG,Singleton GR,Kijlstra A. Rodent-borne diseases and their risks for public health[J]. CRC Crit Rev Microbiol,2009,35(3):221-270. DOI:10.1080/10408410902989837.
[3] Bordes F,Blasdell K,Morand S. Transmission ecology of rodentborne diseases:new frontiers[J]. Integr Zool,2015,10(5):424-435. DOI:10.1111/1749-4877.12149.
[4] Wolfe ND,Dunavan CP,Diamond J. Origins of major human infectious diseases[J]. Nature,2007,447(7142):279-283.DOI:10.1038/nature05775.
[5] Bakken JS,Dumler JS. Human granulocytic anaplasmosis[J].Infect Dis Clin North Am,2015,29(2):341-355. DOI:10.1016/j.idc.2015.02.007.
[6] Failloux AB,Moutailler S. Zoonotic aspects of vector-borne infections[J]. Rev Sci Tech,2015,34(1):165-283.
[7]李贵昌,栗冬梅,李焱,等. 2006-2016年我国恙虫病流行特征分析[J].疾病监测,2018,33(2):139-143. DOI:10.3784/j.issn.1003-9961.2018.02.007.
[8]孙印旗,董妥,姜霞,等. 2009-2012年河北鼠型斑疹伤寒流行概况及实验室调查分析[J].中国人兽共患病学报,2015,31(6):552-555. DOI:10.3969/cjz.j.issn.1002-2694.2015.06.012.
[9] Bossi P,Bricaire F. Tularemia,a potential bioterrorism weapon[J]. Presse Med,2003,32(24):1126-1130.
[10]贾延库,高永荣,陈长安,等.北京地区啮齿动物种类和地理分布调查[J].中国媒介生物学及控制杂志,1999,10(2):87-90. DOI:10.3969/j.issn.1003-4692.1999.02.004.
[11]杨晓,陈梅玲,温博海,等.实时荧光定量PCR检测莫氏立克次体[J].解放军医学杂志,2007,32(10):1054-1056. DOI:10.3321/j.issn:0577-7402.2007.10.016.
[12]张晶波,温博海,陈梅玲,等.荧光定量PCR检测嗜吞噬细胞无形体[J].中国人兽共患病学报,2006,22(4):289-293.DOI:10.3969/j.issn.1002-2694.2006.04.001.
[13]付秀萍,贺金荣,张景山,等. TaqMan-MGB探针实时荧光定量PCR检测恙虫病东方体方法的建立[J].中国媒介生物学及控制杂志,2012,23(2):108-110.
[14]史清海,曲识,周冬生,等.应用TaqMan荧光定量PCR检测土拉弗朗西斯菌[J].生物技术通讯,2009,20(6):806-809.
[15] Diaz MH,Bai Y,Malania L,et al. Development of a novel genusspecific real-time PCR assay for detection and differentiation of Bartonella species and Genotypes[J]. J Clin Microbiol,2012,50(5):1645-1649.
[16]张翠彩,李秀文,聂一新,等.致病性钩端螺旋体TaqMan Realtime PCR检测技术的建立及其应用[J].中华流行病学杂志,2011,32(10):1018-1021. DOI:10.3760/cma.j.issn.0254-6450.2011.10.015.
[17] Bustin SA,Benes V,Garson JA,et al. The MIQE guidelines:Minimum/information for publication of quantitative real-time PCR experiments[J]. Clin Chem,2009,55(4):611-622. DOI:10.1373/clinchem.2008.112797.
[18] Conlan JW. Early host-pathogen interactions in the liver and spleen during systemic murine listeriosis:an overview[J].Immunobiology,1999,201(2):178-187. DOI:10.1016/S0171-2985(99)80057-6.
[19] Maurin M. Real-time PCR as a diagnostic tool for bacterial diseases[J]. Expert Rev Mol Diagn,2012,12(7):731-754.DOI:10.1586/erm.12.53.
[20] Telfer S,Lambin X,Birtles R,et al. Species interactions in a parasite community drive infection risk in a wildlife population[J]. Science,2010,330(6001):243-246. DOI:10.1126/science.1190333.
[21] Schmidt S,Essbauer SS,Mayer-Scholl A,et al. Multiple infections of rodents with zoonotic pathogens in Austria[J].Vector Borne Zoonotic Dis,2014,14(7):467-475. DOI:10.1089/vbz.2013.1504.
[22]张鑫,邢海英,张凤革,等.北京市平谷区恙虫病134例临床特征分析[J].浙江临床医学,2014,16(7):1079-1080.
[23]董翠珍,李文靖.昌平县钩端螺旋体病感染状况调查[J].中国人兽共患病杂志,1995,15(5):133.
[24]齐海霞,张海云,邓小雨,等.犬、猫钩端螺旋体病的流行病学调查[J].中国畜牧兽医,2012,39(11):203-206. DOI:10.3969/j.issn.1671-7236.2012.11.047.
[25]肖园,吴海燕,孙艳争,等.犬钩端螺旋体病3例报告[J].中国人兽共患病学报,2017,33(5):469-471. DOI:10.3969/j.issn.1002-2694.2017.05.016.
[26]王雅琴,张海云,唐艳荣,等. 2016年朝阳区动物诊疗机构犬狂犬病和A型流感的流行病学调查报告[J].中国畜牧兽医文摘,2017,33(11):124-126.
[27] Diaz JH. Recognition and management of rodent-borne infectious disease outbreaks after heavy rainfall and flooding[J].J La State Med Soc,2014,166(5):186-192.
[28] Villette P,Afonso E,Couval G,et al. Consequences of organ choice in describing bacterial pathogen assemblages in a rodent population[J]. Epidemiol Infect,2017,145(14):3070-3075.DOI:10.1017/S0950268817001893.
[29] Galan M,Razzauti M,Bard E,et al. 16S rRNA amplicon sequencing for epidemiological surveys of bacteria in wildlife[J].mSystems,2016,1(4):e00032-16. DOI:10.1128/mSystems.00032-16.
[30] World Health Organization. 2018 annual review of diseases prioritized under the research and development blueprint[R].Geneva:WHO,2018:2,9.