江西口岸及国检监管区鼠类感染钩端螺旋体的检测及基因序列分析

目的为了解江西省口岸及国检监管区鼠类携带的钩端螺旋体情况,2015年7-12月,对江西省2个一类口岸和9个国检监管区捕获的鼠类携带的钩端螺旋体进行了检测。方法采用荧光定量PCR和特异性PCR扩增两种方法对样本进行检测。结果研究发现捕获的107只鼠类中存在12例致病性钩端螺旋体阳性,阳性率为11.2%。包括褐家鼠6只,黄毛鼠3只,黄胸鼠1只,社鼠2只,分布在昌北机场、康替龙国检监管区、南丰国检监管区、鹰潭国检监管区和瑞金国检监管区。同源性对比和系统进化分析显示,12例钩端螺旋体中11例为问号钩端螺旋体,另1例为博氏钩端螺旋体。结论在江西口岸及国检监管区鼠类中存在问号钩端螺旋体和博氏钩端螺旋体感染。两种PCR检测方法均可适用于口岸及国检监管区对钩端螺旋体的检测,防止相应传染病在口岸的爆发与传播,保卫口岸安全。     

1例宏基因二代测序提示钩端螺旋体感染患者的实验室诊断

目的 对一不明原因发热伴呼吸困难患者进行实验室诊断,探讨钩端螺旋体实验室确诊的检测方法。方法 利用宏基因二代测序技术(mNGS)对一不明原因发热伴呼吸困难患者的肺泡灌洗液进行病原体分析,利用实时荧光定量PCR(Real-time PCR)、显微镜凝集试验(MAT)和传统培养方法对患者进行实验室确诊。结果 mNGS对不明原因发热伴呼吸困难患者的肺泡灌洗液进行病原体分析提示患者可能被问号钩端螺旋体感染,Real-time PCR和传统培养方法对患者的肺泡灌洗液、血液和尿液检测均为阴性,血清抗体检测(MAT法)致病性钩端螺旋体澳洲型滴度为1:800。结论 该患者确诊为致病性钩端螺旋体澳洲型感染,mNGS与实验室传统方法的有机结合有助于对不明原因发热伴呼吸困难患者实验室诊断,特别是对钩端螺旋体的实验室诊断具有重要意义。     

广州增江沿岸地区鼠形动物及携带病原体调查

目的 了解广州增江沿岸地区鼠形动物及携带伯氏疏螺旋体、钩端螺旋体、恙虫病东方体、埃立克体、新型布尼亚病毒和汉坦病毒6种病原体的情况。方法 使用笼夜法在广州市增城区捕获鼠形动物,利用荧光定量PCR方法检测鼠形动物脏器中的伯氏疏螺旋体、钩端螺旋体、恙虫病东方体、埃立克体、新型布尼亚病毒和汉坦病毒。采用χ²检验和Fisher确切概率法比较不同种属、雌雄和调查点鼠形动物病原体的感染情况。结果 捕获臭鼩鼱和褐家鼠共69只,鼠形动物密度为13.29%。褐家鼠中检出钩端螺旋体为57.89%、恙虫病东方体为31.58%、汉坦病毒为21.05%和伯氏疏螺旋体为5.26%;臭鼩鼱中仅检出伯氏疏螺旋体为2.00%。埃立克体、新型布尼亚病毒均未检出。此外,褐家鼠中存在4种病原体的双重感染,其双重感染率为36.84%。结论 广州增江沿岸地区鼠形动物以臭鼩鼱和褐家鼠为主且密度较高;褐家鼠中病原体感染率高于臭鼩鼱且存在多种病原体的复合感染。当地应加强对鼠形动物尤其是褐家鼠的监测和控制、降低鼠形动物密度。     

浙江省衢州地区鼠类携带莱姆病螺旋体研究

目的 了解衢州市鼠类携带莱姆病螺旋体状况以及莱姆病螺旋体基因型。方法 采用针对莱姆病螺旋体5S~23S基因间隔区的巢式PCR方法检测鼠脾标本,对阳性样本进行基因测序及分析,构建系统发育树。结果 在378份鼠标本中检出莱姆病螺旋体核酸阳性57份,阳性率15.08%。有Borrelia garinii(B.g)基因型、Borrelia valaisiana(B.v)基因型Borrelia spielmanii(B.sp)基因型。结论 衢州境内鼠类存在至少3个型别莱姆病螺旋体感染,应加强该病原体的检测和莱姆病的防制。     

2017-2019北京地区犬钩端螺旋体病流行病学调查

目的 了解北京地区犬钩端螺旋体的感染情况。方法 采用快速实时荧光快速聚合酶链式反应(Rapid real-time fluorescence quantitative polymerase chain reaction, qPCR)对2017年1月至2019年2月从宠物医院、犬舍、流浪动物基地收集到的1 600份血液和尿液样本进行分子学检测,以及风险因素分析。并对qPCR阳性病例样本进行血清学和PCR鉴定。结果 qPCR检出阳性样本17份,检出率为1.01%(95%CI 0.6- 1.7)。其中尿液阳性检出率2.00%(16/800)显著高于血液检出率0.13%(1/800)(χ²=11.653,P<0.01)。地区(χ²=17.828,P<0.05)、季节(χ²=10.916,P<0.05)、样本来源(χ²=9.432,P<0.01)是犬钩体感染的风险因素,但不同年龄(χ²=1.103, P=0.613)、性别(χ²=1.124, P=0.223)、品种(χ²=0.427, P=0.448)的犬感染钩体的差异无统计学意义。显微凝集试验显示12份qPCR阳性病犬血清样本存在澳洲群和拜伦群抗体;菌株经PCR鉴定均为问号钩端螺旋体。结论 北京部分地区存在犬钩体病的流行,应加强疾病监测和宠物犬定期疫苗接种以降低其向人类传播的风险。     

北京市鼠形动物感染巴尔通体的调查

目的 调查北京市鼠形动物巴尔通体感染状况和遗传特征。方法 采用夹夜法在北京市16个区捕获鼠形动物,使用PCR方法检测巴尔通体gltA基因,测序后进行系统发育树分析。结果 共采集7属9种鼠形动物样本448份,检测到巴尔通体阳性样本46份,总阳性率为10.3%,且阳性率在鼠种间、地区间和生境间的差异均具有统计学意义(X²_鼠种=114.980,X²_地区=22.133,X²_生境=96.466,P均<0.001)。系统发育树分析显示,40份样本序列位于进化树上的8个不同分支,提示北京市鼠形动物巴尔通体感染存在遗传多样性。结论 巴尔通体在北京市鼠形动物中广泛存在,并具有遗传多样性,本地人群存在一定的感染风险。     

广东省鼠形动物携带巴尔通体的调查和基因特征分析

目的 了解巴尔通体在广东省鼠形动物中的流行分布情况及其基因型特征,为巴尔通体的研究和自然疫源性疾病的防控提供科学依据。方法 在广东省惠来县、惠东县、潮安县、罗定县和高州市5个地区进行捕鼠,采集鼠肝脏标本提取总核酸,利用聚合酶连反应(PCR)检测巴尔通体病原,并对其进行分离培养,阳性产物进行测序,并根据序列进行系统进化分析,统计分析巴尔通体的分布情况和特征。结果在广东省5个地区共捕鼠375只,检出巴尔通体阳性标本73份,阳性率为19.47%。不同鼠种(χ²=6.361)、不同地区(χ²=7.778)、不同性别(χ²=0.292)、不同生境间(χ²=0.621),阳性率差异均没有统计学意义(P>0.05)。73份样本中,10份成功分离培养出巴尔通体。系统进化分析表明广东鼠形动物携带的巴尔通体存在6种基因型:Bartonella elizabethae、 B.phoceensis、 B.japonica、 B.henselae、 B.rochalimae、B.tribocorum。结论 巴尔通体在广东省鼠形动物中广泛存在,且基因型呈多样性,其中4种基因型对人类有致病性,对人类健康具有潜在威胁。     

2016-2018年江西省钩端螺旋体病鼠类动物流行病学调查与分离菌株鉴定

目的 初步了解江西省钩端螺旋体病主要宿主动物鼠类种类构成、带菌率及血清学和分子流行病学特征,为钩体病的防控提供科学依据。方法 2016-2018年选取江西省钩体病4个国家级监测点上饶、上高、上犹及浮梁县作为现场监测地点,开展鼠类种类调查、钩体分离培养工作。利用暗视野显微镜凝集试验和MLST技术对分离菌株进行血清学鉴定和MLST基因分型。结果 共计捕鼠14 387只,平均鼠密度为9.76%,分离菌株64株,鼠类钩体分离率为4.56%。鼠种构成包括黑线姬鼠、褐家鼠、黄毛鼠、黄胸鼠及社鼠5个种类。4个监测点优势鼠种均以黑线姬鼠和黄毛鼠为主。血清群鉴定结果显示:64株菌株隶属于3个血清群,其中黄疸出血群为主要优势血清群,占81.25%,其次是爪哇群和致热群。MLST研究显示:64株菌株隶属于6个ST型别,分别为ST1、ST17、ST92、ST143、ST216和ST224,其中ST1为主要基因型,占76.56%。BioNumerics软件分析显示:64株菌株隶属于6个Clusters,分别对应6个ST型,血清学、MLST基因型分布具有明显地域差异。结论 江西省以黑线姬鼠和黄毛鼠为优势鼠种,黄疸出血群和ST1为江西省钩体病主要流行型别,充分了解江西省钩体病可能的宿主动物以及血清学和分子流行病学特征,对钩体病的预防控制有一定的指导意义。     

问号钩端螺旋体溶血素基因特征分析

目的　预测问号钩端螺旋体黄疸出血型赖株致病因子———溶血素基因 ,并对其编码蛋白结构特征进行深入分析。方法　使用NCBI ,Swissprot/TrEMBL ,ProDom ,Pfam ,Jpred2 ,TMpred ,SignalP ,ClustW对问号钩端螺旋体黄疸出血型赖株溶血素基因诠释 ,所编码蛋白进行在线分析 ,并使用Bioedit软件进行氨基酸同源性比较分析。结果　确定了问号钩端螺旋体黄疸出血型赖株 9个溶血素基因 ,分为两大类 :鞘磷脂酶类溶血素基因和非鞘磷脂酶类溶血素基因。并对溶血素基因编码蛋白二级结构、结构域、跨膜区域、信号肽及进化等进行了分析。结论　多种类型多条溶血素基因同时存在于钩端螺旋体菌中 ,暗示溶血素基因和钩端螺旋体致病性之间有密切的关系 ,可能在致病过程中起重要作用     

东北边境地区啮齿动物中新埃立克体感染调查及groEL基因序列分析

目的 了解东北地区啮齿动物新埃立克体(Candidatus Neoehrlichia mikurensis,Nm)感染及基因型分布情况。方法 采用巢式PCR检测从辽宁省宽甸、吉林省集安林区及黑龙江省密山耕地捕获的啮齿动物中Nm groEL DNA, 对阳性扩增产物进行测序和遗传进化分析。结果 PCR检测鼠脾标本共181份, 阳性5份,阳性率为2.76%。不同地区及不同鼠种Nm检出阳性率无明显差异。遗传进化分析显示,从宽甸、集安黑线姬鼠和大林姬鼠检出的5份Nm groEL基因序列与俄罗斯亚洲地区以及我国东北地区Nm同源性最高,基因型为ClusterⅠ型。结论 我国东北东部边境地区鼠类存在Nm感染,可能存在Nm自然疫源地。     

Rattus norvegicus acting as reservoir of leptospira interrogans in the Middle Black Sea region of Turkey, as evidenced by PCR and presence of serum antibodies to Leptospira strain

Leptospirosis, a common form of zoonosis, especially in rainy countries, is caused by Leptospira interrogans. In our region of Turkey this type of disease has often been encountered in connection with rice harvesting and we therefore attempted to evaluate the prevalence of L. interrogans in wild rats in our region. Fifty-nine Rattus norvegicus rats were trapped alive in different areas of an approximately 100 km stretch of seashore in the Middle Black Sea region of Turkey. L. interrogans was determined by PCR in sera, kidney and brain tissue. Sixteen (27.1%) kidney samples and 10 brain tissue samples (16.9%) were positive for L. interrogans. No PCR positivity was seen in sera samples. Five sera were positive by microagglutination test. A large proportion of wild rats in our region were found to be carriers of L. interrogans. We conclude that people who are exposed to rat urine in their daily life are at risk of acquiring L. interrogans.     

福建省鼠感染钩端螺旋体调查和分离株基因种的鉴定北大核心CSCD

目的了解福建省鼠感染钩端螺旋体(钩体)现状及分离株基因种的特征,为钩体病防控提供科学依据。方法2015-2019年根据疫情选取14个调查点,采用笼夜法捕鼠并采集心血或肾脏标本进行钩体培养,测定血清抗体;对鼠中分离保存的23株钩体菌株提取DNA,PCR扩增16S rRNA基因、PCR产物纯化测序,与GenBank数据库收录的钩体17种基因型进行比对确定基因种,并用Mega 6.0软件构建系统进化树并进行序列分析。结果福建省鼠种以黄毛鼠为主要优势鼠种,占40.87%(499/1221)、其次依次是黄胸鼠19.57%(239/1221)、针毛鼠16.79%(205/1221)和褐家鼠8.85%(108/1221),钩体血清抗体阳性率为29.32%(207/706),以黄毛鼠最高,为42.16%(121/287),不同地区鼠种构成及鼠钩体感染率不同。23株菌株均扩增出16S rRNA基因的目标条带1484 bp,测序及序列分析属于致病性的基因种有13株,其中L.borgpetersenii(伯氏)占8株,L.interrogans(问号)占5株,属于非致病的基因种10株。钩体菌株分离自福建省的优势鼠种(黄毛鼠、黄胸鼠、针毛鼠)及不同地区(闽东、闽南、闽西和闽北)。结论福建省鼠类感染钩体普遍,以致病性基因种(L.borgpetersenii和L.interrogans)为主,也携带非致病性基因种,需加强钩体病传染源啮齿动物的监测工作。     

Prevalence and genetic divergence of Leptospira interrogans and Leptospira borgpetersenii in house rats(Rattus rattus) from Peninsular Malaysia

Objective: To evaluate the prevalence and divergence of genetically identified Leptospira spp. in the population of Rattus rattus.Methods: A total of 130 rats were used in this study. The infection within the rats were screened using polymerase chain reaction(PCR)-based diagnosis, with Leptospira genusspecific 16 S r RNA primer and pathogenic Leptospira spp. specific Lip L32 primer, on both kidney and liver tissues of Rattus rattus to detect the presence of potential Leptospira spp.Results: Out of 130 rats studied, 51(39.23%) individuals were positive for leptospiral DNA. Basic Local Alignment Search Tool(BLAST) and phylogenetic analysis revealed that both pathogenic Leptospira interrogans and Leptospira borgpetersenii were predominantly identified. Phylogenetically, both genes disclosed similar clustering patterns of tree topologies between the two species. Although both genes were conserved, Lip L32 gene portrayed higher nucleotide divergence(5.80%) compared to the 16 S r RNA gene(0.60%). Minimum-spanning network displayed several haplotypes that are unique to each species, suggesting a higher degree of subdivision between both species. As for prevalence surveillance, both adult and subadult rats were susceptible to the infection, in which males were the most susceptible. Kidney was notable as the favourable organ for colonisation of leptospires. Rats captured from fresh markets were highly infected with Leptospira spp.(54.28%) compared to those from housing areas(26.47%).Conclusions: Rattus rattus represents an important asymptomatic transmitter of pathogenic leptospires, and hence is of public health concerns.     

致病性钩端螺旋体的多位点序列分型研究

目的 通过对致病性钩端螺旋体(简称钩体)多位点序列分型(MLST)分析,了解国内与全球致病性钩体种群结构及其分子进化.方法 应用16SrRNA基因测序和MLST分析方法对不同来源、不同时期国内钩体分离菌株与国际参考菌株进行分析,并与国内7株疫苗株结果平行比较,同时收集已公开的来自不同国家和不同时期1 238株致病性钩体...     

Leptospira autumnalis isolated from a human case from Avadi, India, and the serovar's predominance in local rat and bandicoot populations

Leptospira were successfully isolated from the urine of an Indian patient who had been clinically diagnosed as having leptospirosis. In an attempt to determine the source of this infection, 28 rats (Rattus rattus) and 58 bandicoots (Bandicota bengalensis) living in the vicinity of the patient's home in Avadi, a suburban area of the city of Chennai (Madras), India, were then investigated. Each animal was checked for infection by microscopical examination of fresh and stained urine, serological analysis of serum, and the culture of urine and kidney samples. Direct, dark-field, observation of fresh urine samples and examination of urine samples after Fontana's silver staining were found to be the least sensitive of the tests used. The results of the serological microscopic agglutination test (MAT) indicated that four (14.3%) of the rats and nine (16.1%) of the bandicoots had significant agglutinins, predominantly for the serogroups icterohaemorrhagiae and autumnalis. Leptospira were isolated from at least one culture of samples from one rat and each of four bandicoots. Each of these rodent isolates and the human isolate were typed as Leptospira interrogans serovar autumnalis.     

首次自褐家鼠粪便标本中检出香港海鸥菌

目的了解褐家鼠粪便标本中是否携带香港海鸥菌,并分析其耐药特征及分子分型。方法2015年6月-2016年5月,利用笼捕法于广州市南方医院及其周边居民区捕获褐家鼠,其粪便样本经增菌后接种改良头孢哌酮MacConkey琼脂(CMA)平板培养分离可疑菌株,经生化试验及16SrRNA测序确认。采用K-B法进行药物敏感性测定,并运用多位点序列分型进行分子分型分析。结果共捕获褐家鼠191只,并在2份褐家鼠粪便标本中检出香港海鸥菌,检出率为1.05%。两株菌16SrRNA的测序结果与香港海鸥菌标准株HKU1的一致性达100%。药敏试验显示两株分离株均对头孢菌素类抗生素及利福平耐药。多位点序列分型结果显示两株菌分别为2个新的ST型:ST-163和ST-164。结论褐家鼠粪便中存在香港海鸥菌污染。褐家鼠与人类生活关系密切,可能为人类感染的的另一潜在来源。     

Genetic variation of Leptospira isolated from rats catched in Yogyakarta Indonesia

Objective: To detect genetic variations among pathogenic Leptospira isolated from rats using 16 S r RNA gen as chronometer. Methods: This is an observational study with cross sectional design. Rats saples were taken in Yogyakarta Special Region of Indonesia. Leptospira in the rats was detected by two methods ie. real time PCR(q PCR) by using primers correspond to16 S r RNA gene of Leptospira, and standard PCR by using dif erent set of primer correspond to the 16 S r RNA gene of Leptospira. The standard PCR amplicon then subjected for DNA sequencing. Analysis genetic variation was performed using MEGA 6.2. Software. Results:There were 99 DNA samples from rats included in this study. Detection of Leptospira by using q PCR revealed 25 samples positive for pathogenic Leptospira, while only 6 samples were able to be detected using standard PCR. The new primer set correspond to 16 S r RNA gene was able to detect specii cally pathogenic Leptospira in the rats. Sequencing analysis of 6 PCR amplicons showed that the Leptospira which infect the rats catched in Yogyakarta genetically close related with pathogenic Leptospira which were isolated from human, animal, rodents, and environment. Conclusions: It can be considered that rats are the most important vector and reservoir of Leptospira.     

Cross-species surveillance of Leptospira in domestic and peri-domestic animals in Mahalla City, Gharbeya Governorate, Egypt

A survey of 179 animals (black rats, dogs, sheep, buffaloes, cattle, donkeys, weasels, and cats) for Leptospira infection was conducted in Mahalla City (Lower Egypt). Blood, urine, and kidney were collected and tested by culture, microscopic agglutination test (MAT), and/or polymerase chain reaction (PCR). Among rats, 26% were positive by PCR, including 7% that were also positive by culture for L. interrogans serovars Grippotyphosa, Pyrogenes, and Icterohaemorrhagiae. L. borpetersenii serovar Polonica was isolated for the first time in Egypt in three rats. MAT titers ≥ 1:800 were observed in 11% of rats and 12% of dogs. L. interrogans serovar Grippotyphosa was detected in one cat. Sheep and donkeys were negative for leptospirosis by all methods. Buffaloes and cattle were seropositive in 20% and 44% of animals, respectively. Data indicate that several pathogenic serovars are circulating in the animals, which may pose exposure risks and account for high rates of acute febrile illness.     

云南省泸西县啮齿动物携带立克次体调查

目的 调查云南省泸西县啮齿动物携带恙虫病东方体、无形体和埃立克体的状况,了解该类病原体在当地自然界中的保存状况和基因特征。方法 用鼠笼和鼠夹在云南省泸西县捕鼠,将捕获的动物种类鉴定后解剖取脾脏,活鼠取血。采用巢式PCR扩增脾脏的恙虫病东方体groEL基因,无形体和埃立克体的16S rRNA基因特异片段;测定PCR扩增阳性产物的DNA序列,对获得序列进行序列比对和系统进化分析。IFA法检测鼠血清中恙虫病东方体IgG抗体。结果 在泸西县共捕获啮齿动物10种225只。其中黄胸鼠36.89%(83/225)、大绒鼠35.11%(79/225)和中华姬鼠13.78%(31/225)为优势鼠种。获得鼠血清85份。鼠脾脏中检测到5株东方体groEL基因阳性标本,带毒鼠种为黄胸鼠2.41%(2/83)和大绒鼠3.80%(3/79)。同源性比较显示,这5株东方体的相似性在99.02%~100%之间,他们分别与GenBank中已知立克次体序列的相似性在98.75%~100%。系统发生树显示,5株OT与来自日本、泰国和中国安徽的菌株位于同一分支。3份16S rRNA阳性标本,其中1份埃立克体阳性,来源于大绒鼠;1份沃尔巴克氏体和1份巴尔通体阳性均来源于黄胸鼠。无形体均为阴性。埃立克体株序列比对显示与来自美国、中国和巴西的埃立克体基因同源性为98.0%~100%,并与分离自美国野外工作者皮肤的伊文氏埃立克体在同一分支。鼠血清恙虫病IgG抗体阳性7份,阳性率8.24%(7/85)。结论 该地区存在以黄胸鼠和大绒鼠为主要宿主的恙虫病自然疫源地。埃立克体、巴尔通体和沃尔巴克氏体在啮齿动物中也存在感染,需注意防控。