陕西和辽宁部分地区羊源媒介蜱类及其携带病原体调查

为了对陕西省和辽宁省部分地区羊源蜱类及其携带病原体的流行情况进行分析,本试验采集了288份羊源蜱样本,采用形态学观察鉴定蜱种;采用分子生物学方法扩增巴贝斯虫18S rRNA、伯氏疏螺旋体16S rRNA、斑点热群立克次体ompA和无形体16S rRNA的基因序列,并结合核苷酸序列分析,确定所收集蜱样本中各类病原体的流行情况。结果显示,经鉴定288份羊源蜱样本均为长角血蜱。陕西省144份长角血蜱中携带巴贝斯虫、斑点热群立克次体和无形体,感染率分别为0.69%、21.53%和77.78%,且巴贝斯虫和斑点热群立克次体均与无形体复合感染;其中长角血蜱中携带Babesia microti、Candidatus Rickettsia longicornis、Uncultured Rickettsia sp. QH-122、Rickettsia endosymbiont of Haemaphysalis longicornis、Uncultured Anaplasma sp. ZJ06/2009、Anaplasma marginale、Anaplasma capra共7种病原体。辽宁省144份长角...     

青海血蜱传斑点热群立克次体带菌率及遗传进化分析

为了明确青海省天峻地区青海血蜱传斑点热群立克次体带菌率及遗传进化特点,根据已发表的斑点热群立克次体外膜蛋白OmpA基因序列设计特异性引物,采用PCR方法对青海省天峻地区青海血蜱进行斑点热群立克次体检测,并对测定序列进行遗传进化分析。结果显示,在316个青海血蜱标本样品池中共检测到31个阳性样品,斑点热群立克次体(SFGR)带菌率为9.8%。遗传进化分析显示,TJ013与立克次体福建分离株FUJ98(AF169629)、黑龙江立克次体HL-93(AF179364)、HL-054(AF179362)及日本立克次体YM(U43795)处于同一分支;SFGR Omp A基因序列同源性分析结果显示,TJ013与立克次体福建分离株FUJ98(AF169629)同源性最高,为95.49%;与黑龙江立克次体绥芬分离株HLJ-054和虎林分离株HL-93的同源性均为91.44%;和日本立克次体YM同源性为90.24%。表明青海省天峻地区青海血蜱传斑点热群立克次体感染较为严重,应尽快制定防制措施,以免危及动物及人类健康。     

青海血蜱传斑点热群立克次体带菌率及遗传进化分析

为了明确青海省天峻地区青海血蜱传斑点热群立克次体带菌率及遗传进化特点,笔者根据已发表的斑点热群立克次体外膜蛋白Omp A基因序列设计特异性引物,采用PCR方法对青海省天峻地区青海血蜱进行斑点热群立克次体检测并对测定序列进行遗传进化分析。结果在316个青海血蜱标本样品池中共检测到31个阳性样品,SFGR带菌率为9.8%。遗传进化分析显示,TJ013与立克次体福建分离株FUJ98(AF169629),黑龙江立克次体HL-93(AF179364),HL-054(AF179362)及日本立克次体YM(U43795)处于同一分支;SFGR Omp A基因序列同源性分析结果显示,TJ013与立克次体福建分离株FUJ98(AF169629)同源性最高,为95.49%;与黑龙江立克次体绥芬分离株HLJ-054和虎林分离株HL-93的同源性均为91.44%;与日本立克次体YM同源性为90.24%。说明青海省天峻地区青海血蜱传斑点热群立克次体感染较为严重,应尽快制定防治措施,以免对动物及人类健康造成威胁。     

蜱传斑点热群立克次体青海分离株分离与遗传进化分析

根据已发表斑点热群立克次体外膜蛋白OmpA基因序列设计特异性引物,对青海省采集的蜱标本进行PCR检测,并对阳性样本进行测序和序列分析,建立分子系统进化树。结果显示在454个蜱样本池中检测到14个SFGR阳性样本,其中9个西伯利亚立克次体阳性样本和5个黑龙江立克次体阳性样本,总感染率3.08%。序列分析结果显示,西伯利亚立克次体青海株HUN与与黑龙江立克次体虎林株HL-93(AF179364)、绥芬株HLJ-054(AF179362)共处一个分支,一致性分别为99.18%和99.51%;黑龙江立克次体青海株GL-1与西伯利亚立克次体前苏联株246(U43807)和北京株BJ-90(AF179365)同处一个分支,序列一致性分别为99.49%和99.67%。说明青海省存在SFGR感染,应尽快制定防控措施,以免危害动物及人类健康。     

中哈边境艾比湖湿地游离蜱斑点热群立克次体的分子流行病学研究

了解中哈边境新疆艾比湖湿地游离蜱中斑点热群立克次体的感染情况。利用斑点热群立克次体外膜蛋白A(ompA)基因对艾比湖湿地游离亚洲璃眼蜱、边缘革蜱、血红扇头蜱进行PCR检测,并对阳性样本进行测序和BLAST序列分析,并应用Mega5.0软件建立分子系统进化树。结果中哈边境地区艾比湖湿地游离蜱斑点热群立克次体电泳阳性率36.84%(56/152),BLAST分析显示,艾比湖湿地游离蜱斑点热群立克次体基因型为Rickettsia raoultii,与从匈牙利患蜱传染的病人肿大淋巴结中分离获得的R.raoultii关系最近,处于同一分支(Gen Bank登录号为JQ798904)。结论是新疆艾比湖湿地游离蜱斑点热立克次体感染较为严重,存在立克次体自然疫源地,应尽快制定防制措施,以免危机动物及人类健康。     

新疆南疆部分地区蜱种鉴定及斑点热群立克次体流行病学调查

为了解新疆南疆部分地区不同蜱种携带斑点热群立克次体(spotted fever group Rickettsia,SFGR)情况,采用PCR技术扩增16S rDNA基因和SFGR外膜蛋白A(OmpA)基因,进行蜱种鉴定及SFGR检测。对测序阳性样本进行BLAST序列分析,应用DNAStar和Mega 6.0软件构建遗传进化树。结果显示:从新疆南疆9个县市共采集蜱1 340只,其种类包括3属4种,分别为图兰扇头蜱、边缘革蜱、银盾革蜱、亚洲璃眼蜱;在随机挑选的90份蜱DNA样本中共检测出57份SFGR阳性样本,其中阿拉尔市十一团、七连地区的阳性样本均来自犬体表寄生蜱,其余地区的阳性样本均来自绵羊体表寄生蜱。序列分析及遗传进化树显示,此次检测的亚洲璃眼蜱、边缘革蜱携带的SFGR与GenBank公布的SFGR疆内株Candidatus R.barbariae(登录号为MG668832.1)亲缘关系最近,处于同一分支;图兰扇头蜱携带的SFGR也同疆内株R.massiliae、R.conorii、Candidatus R.barbariae(MG668831.1、KP214024.1、MG668832.1)亲缘关系最近,处于同一分支;而银盾革蜱携带的SFGR与国外株R.raoultii(KT805295.1)亲缘关系最近,处于同一分支,并首次于新疆南疆绵羊寄生银盾革蜱中检出饶氏立克次体。本试验为该地区蜱传立克次体病的研究及防控提供重要依据。     

青海省蜱传立克次体流行病学调查

为研究青海省蜱种分布及蜱传立克次体感染情况,试验在青海省20个县市开展蜱传立克次体流行病学本底调查,应用布旗法采集1 294只蜱,采用形态学及分子生物学的方法进行鉴定。结果表明:共有青海血蜱(Haemaphysalis qinghaiensis)674只,草原革蜱(Dermacentor nuttalli)462只,森林革蜱(Dermacentor silvarum)158只。通过扩增gltA和ompA基因筛查立克次体阳性蜱样品,发现青海省蜱传立克次体总体感染率为3.90%。青海地区蜱传立克次体主要包含4种,斯洛瓦立克次体(Rickettsia slovaca,0.72%)、饶氏立克次体(Rickettsia raoultii,0.87%)、西伯利亚立克次体(Rickettsia sibirica,1.97%)、斑点热立克次体(Rickettsia sp. DnS28,0.08%)。说明青海血蜱为青海省优势蜱种,蜱传立克次体在青海省的感染率较高,应做好青海省蜱传立克次体的防治工作。     

四川省攀西地区蜱携带立克次体检测及基因序列分析

为了解攀西地区蜱种类及蜱源立克次体的种类和感染状况,采用形态学和PCR方法,对随机采集自攀枝花和凉山州部分地区牛羊体表的252只蜱进行种类鉴定;利用特异性引物对立克次体ompA、gltA基因片段进行PCR扩增,随机选择阳性样本进行测序,并对测序序列进行分析,构建系统发育树。经种类鉴定,攀西地区存在微小扇头蜱和褐黄血蜱2种蜱种;蜱携带立克次体平均阳性率为29.37%(74/252),存在敬信立克次体暂定种(Candidatus Rickettsia jingxinensis)、立克次体共生菌(Rickettsia endosymbiont of Rhipilephalus microplus)、马赛立克次体(Rickettsia massiliae)共3种立克次体,其中敬信立克次体暂定种为优势种,共检出31份。不同县市的蜱携带立克次体阳性率不同,其中盐源县和昭觉县未检测到立克次体,而布拖县(57.14%)、西昌市(55.26%)阳性率较高;牛、羊体表蜱携带的立克次体阳性率分别为29.55%(26/88)和29.27%(48/164),差异不显著。结果表明,攀西地区存在多种蜱传立克次体,其...     

广东省牛体寄生蜱种及携带病原情况调查

为了解广东地区家畜体表寄生蜱的种类及其携带病原情况,评估蜱媒病在广东省的传播风险,对该地区3个生境点(湛江、汕头、清远)的牛体寄生蜱进行了调查。2012年10月—2013年10月采用体表检视法和布旗法分别采集广东省不同地区的牛体表寄生蜱和牛舍环境周围游离蜱样本共109只,并进行实验室鉴定、检测。通过特异PCR方法分别检测无形体、莱姆病、巴贝西原虫、立克次体和Q热5种主要病原携带情况。结果显示:采集到的109只蜱虫样本均为微小牛蜱,其中湛江样品中检出6份无形体阳性样品,阳性率为5.5%(6/109);莱姆病、巴贝西原虫、立克次体和Q热均检测阴性。研究表明,广东省牛体寄生蜱种类相对单一为微小牛蜱,携带有阳性蜱传病原(嗜吞噬细胞无形体)。提示:广东省极有可能存在该病的自然疫源地,需要进一步调查并进行更深入的生态与分子流行病学研究。     

新疆维吾尔自治区阿克苏地区牛羊体表蜱携带斑点热群立克次体调查

[目的]对新疆维吾尔自治区阿克苏地区牛羊体表蜱携带斑点热群(spotted fever group Rickettsia,SFGR)立克次体情况进行调查。[方法 ]从阿克苏地区阿克苏市(n=344)、温宿县(n=93)、沙雅县(n=98)、乌什县(n=39)随机采集牛羊体表蜱574只,以立克次体外膜蛋白B基因(ompB)和细胞表面抗原1基因(sca1)为靶基因,运用PCR方法检测立克次体携带情况,确定所属的SFGR基因型。选择2个基因的强阳性样本进行测序,将获得的序列与GenBank数据库中已登录的相关基因序列进行同源性比对,并构建系统发育树。比较4个县(市)蜱的立克次体感染率差异。[结果]对牛羊体表蜱携带的立克次体进行检测,发现存在3种SFGR基因型,分别为暂定巴布瑞立克次体(Candidatus Rickettisa barbariae)、西伯利亚立克次体(Rickettisa sibirica)、马赛立克次体(Rickettisa massiliae)。蜱样本中立克次体的总体阳性率为15.16%(87/574),温宿县的立克次体阳性率最高,为23.66%(22/93)。在检出的3...     

Detection of the new Ehrlichia species closely related to Ehrlichia ewingii from Haemaphysalis longicornis in Yonaguni Island, Okinawa, Japan

We collected a total of 206 Haemaphysalis longicornis ticks by flagging in pastures in Yonaguni Island, Okinawa, Japan, in April 2008. Four of the 206 tick DNA samples tested were positive in a polymerase chain reaction (PCR) screening for the 16SrRNA gene of Anaplasmataceae. Partial sequences of 4 PCR products were identical to each other. Longer sequences of the 16SrRNA gene were successfully determined in 2 of the 4 tick samples, and the obtained 1,392 bp and 1,300 bp sequences revealed high similarity to the 16SrRNA gene sequences of the validated Ehrlichia species, including Ehrlichia ewingii, E. chaffeensis, and E. canis (98.3-98.6%). We also sequenced 1,304 bp of the groEL gene from the 2 tick samples, and found that these had the highest similarity to sequences of E. ewingii (94.0-94.4%) in the validated ehrlichial species. Based on the 16SrRNA and groEL gene sequences, the ehrlichial agents detected in this study were similar to the Ehrlichia species detected in Asia and may compose a new Ehrlichia species with other Ehrlichia species detected in Asia.     

Prevalence,molecular characterization and risk factor analysis of Ehrlichia canis and Anaplasma platys in domestic dogs from Paraguay

This is the first study to investigate the prevalence and risk factors associated with Ehrlichia canis and Anaplasma platys positivity in dogs from Paraguay. Conventional PCR assays for the E. canis 16SrRNA gene and A. platys p44 gene were carried out in blood samples from 384 dogs from Asunción city, Paraguay. Sequencing and phylogenetic analysis were performed in selected positive E. canis and (16SrRNA gene) and A. platys (16S and p44 genes) samples. The overall prevalence of E. canis and A. platys in dogs in Paraguay was 10.41% (40/384) and 10.67% (41/384), respectively. Older dogs without veterinary care had higher odds for E. canis positivity and a higher number of dogs in the same household, as well as absence of anti-tick treatment were considered risk factors for A. platys. Ehrlichia canis and A. platys circulate in the dog population from Asunción, and are described for the first time in Paraguay.     

鸭疫里默氏杆菌PCR快速检测方法的建立

在鸭疫里默氏杆菌外膜蛋白保守区设计了一对特异性引物,建立PCR方法,对5株已知血清型的鸭疫里默氏杆菌纯培养菌和6株临床分离未定型的里默氏杆菌菌株以及病死鸭病料组织进行检测,结果表明不同样品都可扩增出592bp的特异目的条带,而对鸭源大肠杆菌、鸭源多杀性巴氏杆菌、鸭源沙门氏菌的扩增结果均为阴性,说明该PCR法具有较好的特异性,可用于快速鉴定鸭疫里默氏杆菌,也适用于病料的直接检测.     

云南鸭疫里默氏杆菌外膜蛋白A 及16S rRNA序列分析

为了探讨鸭疫里默氏杆菌(Riemerella anatipestifer, RA)云南流行株的外膜蛋白A (OmpA)的基因序列差异及其与16S rRNA序列的相关性,PCR扩增18株云南流行株鸭疫里默氏杆菌OmpA基因及16S rRNA核苷酸序列,分别构建其系统进化树,分析其系统进化关系。结果表明,18株鸭疫里默氏杆菌OmpA基因分为2个群,其同源性分别为86%~99.2%和92.6%~100%。18株鸭疫里默氏杆菌16S rRNA基因同属1个群,同源性高达96.1%~100%。 RA-1、RA-2、RA-11和RA-39 4株分离株的OmpA基因位于进化树的同一个亚群,其16S rRNA基因也位于进化树的同一亚群,两者呈现出明显的相关关系,其他14株分离株的OmpA基因系统进化树与16S rRNA基因系统进化树无明显的相关关系。     

Seroprevalence of spotted fever group rickettsiae in canines along the United States–Mexico border

Portions of northern Mexico are experiencing a re‐emergence of Rocky Mountain spotted fever (RMSF), a tickborne disease caused by Rickettsia rickettsii, a member of the spotted fever group of rickettsiae (SFGR). Infection with R. rickettsii can result in serious and life‐threatening illness in people and dogs. Canine seroprevalence has been used as a sentinel for human RMSF in previous studies. This study aims to quantify SFGR seroprevalence in canines in three northern Mexican states and identify risk factors associated with seropositivity. A total of 1,136 serum samples and 942 ticks were obtained from dogs participating in government sterilization campaigns and from animal control facilities in 14 Mexican cities in three states. SFGR antibodies were detected using indirect immunofluorescence antibody assays at titre values ≥1/64. Six per cent (69 dogs) showed antibodies to SFGR, with the highest seroprevalence reported in Baja California (12%), Coahuila (4%) and Sonora (4%). Dogs from Baja California had three times higher odds of having SFGR antibodies compared to dogs from Sonora (OR = 3.38, 95% CI, 1.81–6.37). Roughly one quarter (25%) of surveyed dogs were parasitized by ticks (Rhipicephalus sanguineus sensu lato) at the time of sample collection. A portion of collected ticks were tested for rickettsial DNA using polymerase chain reaction. Positive samples were then sequenced, showing evidence of SFGR including R. massiliae, R. parkeri and R. rickettsii. Dogs that spent the majority of time on the street, such as free‐roaming or community‐owned dogs, showed a greater risk of tick infestation, seropositivity, bearing seropositive ticks, and may play a pivotal role in the spread of SFGR among communities. Estimating the seroprevalence of SFGR in the canine population can help public health campaigns target high‐risk communities for interventions to reduce human RMSF cases.     

多杀性巴氏杆菌OmpA基因的原核表达及生物信息学分析

本试验旨在探究羊源多杀性巴氏杆菌OmpA基因的原核表达及其生物信息学特征。以羊源多杀性巴氏杆菌HN-01株基因组为模板,设计特异性引物扩增OmpA基因;构建pET-28a （+）-OmpA重组质粒后转化大肠杆菌BL21（DE3）感受态细胞,将鉴定正确的重组菌经IPTG诱导表达;通过SDS-PAGE及Western blotting分析表达蛋白的特征,并运用生物信息学工具对OmpA基因序列进行分析。结果显示,羊源多杀性巴氏杆菌OmpA基因大小约为1 044 bp,该基因序列与HN-06株的同源性达89.72%。通过诱导后发现,pET-28a （+）-OmpA重组菌最佳诱导条件为1 mmol/L IPTG 37℃诱导6 h,表达的重组蛋白大小约为40 ku,以包涵体的形式存在。Western blotting结果显示,约40 ku的重组蛋白携带His标签。经生物信息学分析,OmpA分子式为C₁₆₈₄H₂₆₁₉N₄₅₇O₅₀₅S₃,属碱性疏水蛋白,其多肽链的1-21位氨基酸为信号肽区域,并具有多种结构。综上所述,OmpA可能具有特殊结构,与众多外膜蛋白结构特点相似。本研究构建了多杀性巴氏杆菌OmpA基因原核表达系统,优化诱导条件后能稳定获得OmpA重组蛋白,为进一步探究巴氏杆菌的致病机理提供理论依据。     

Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal

Fecal samples from 291 calves and 176 adult cattle in Northern Portugal were screened for Cryptosporidium and Giardia using a formalin-ethyl acetate concentration method. Acid-fast staining techniques for Cryptosporidium oocyst identification and direct microscopic observation of fecal smears for Giardia cyst identification were performed so as immunofluorescence microscopy examination. Polymerase chain reaction methods were employed to determine the genotype of each isolate. Molecular characterization was performed using amplification and sequencing of the hsp70 and 18SrRNA genes of Cryptosporidium and beta-giardin gene and glutamate dehydrogenase for assemblage determination of Giardia duodenalis. Seventy-four out of 291 calves (25.4%) and 8 out of 176 adult bovines (4.5%) were positive for Cryptosporidium. Forty-one out of 291 calf samples (14.1%) and 1 out of 176 adults samples (0.57%) were positive for Giardia. From the Cryptosporidium positive samples we obtained 63 isolates from calves samples and 7 isolates from adult samples. Additionally, Giardia was isolated in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia from the positive adult sample. Molecular characterization of the Cryptosporidium and Giardia isolates showed us that C. parvum and G. duodenalis assemblage E were the prevalent species. C. parvum may infect humans, representing a potential public health risk. On the other hand, the assemblages B and A2 of Giardia, previously described in humans, were here identified in cattle. Further studies will be needed for determine the importance of cattle as carrier of zoonotic assemblages of G. duodenalis.     

Detection of Ehrlichia platys in dogs in Australia

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.     

Greyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents

Greyhound meningoencephalitis is currently classified as a breed-associated idiopathic central nervous system inflammatory disorder. The non-suppurative inflammatory response can be distinguished from the other breed-associated disorders based on histopathology and lesion topography, however the nature of the response primarily suggests a viral infection. In the present study PCR and RT-PCR technologies were employed on frozen cerebral tissue from confirmed cases of meningoencephalitis to target specific viruses and protozoa likely to be implicated and to exclude the presence of bacterial 16SrRNA. Secondly, degenerate primers were used to detect viruses of the herpesvirus and flavivirus families. In addition cerebral tissues were probed for West Nile Virus. Viral nucleic acid sequences to Borna disease virus, to louping ill, tick borne encephalitis, West Nile and other flaviviruses were not detected. Canine distemper virus was detected in one animal with 97% homology to strain A75/15. Degenerate PCR for herpesviruses detected viral amplification products in one animal with 90% homology to canine herpesvirus DNA polymerase gene. Protozoal amplification products were only detected in a single dog with pathological confirmation of a combination of lesions of greyhound meningoencephalitis and a protozoal encephalomyelitis. Neospora was confirmed with sequence homology to Austrian strain 1. Bacterial 16SrRNA was not detected. The present study supports previous observations that many of the known microbial causes of canine meningoencephalitis are not involved. Findings could reflect that the causal agent was not specifically targeted for detection, or that the agent is at undetectable levels or has been eliminated from brain tissue. The potential roles of genetics and of molecular mimicry also cannot be discounted.     

宁夏吴忠地区奶牛临床型乳房炎主要病原菌的分离鉴定及耐药性分析

为了解宁夏吴忠地区奶牛乳房炎的主要病原菌及其耐药情况,采集56份罹患乳房炎的奶牛乳样进行病原菌分离,并对分离菌进行了生化鉴定、16S rRNA基因序列分析、药敏试验以及耐药基因检测.结果表明,该地区的主要病原菌为大肠埃希氏菌和无乳链球菌,检出率分别为23.2％和14.3％.药敏试验结果表明,大肠埃希氏菌主要对头孢他啶、...