CR用于诊断人类埃立克体病方法的建立

「目的」建立一个快速、简便的检测埃立克体感染的实验方法,用于人类埃立克体病的临床诊断及分子流行病学调查。「方法」用查菲埃立克体分离株（９１ＨＥ１７）感染犬巨噬细胞（ＤＨ８２细胞）,感染４０天后收集ＤＨ８２细胞。根据查菲埃立克体１６ＳｒＲＮＡ基因序列设计引物,特生扩增查菲埃立克体ＤＮＡ。运用限制性片段长度多态性分析对ＰＣＲ扩增产物进行鉴定。「结果」扩增产物经过１．５％琼脂糖凝胶电泳和５％聚丙烯酰胺凝     

Cutaneous Plasmacytosis Associated with Ehrlichiosis in an African-American Patient

Cutaneous plasmacytosis is a rare disease that presents clinically with multiple red-brown papules and plaques with minimal to no epidermal change. Histopathologic findings include a perivascular dermal infiltration of polyclonal plasma cells. The etiology of cutaneous plasmacytosis is unknown, but hypothesized to be due to persistent or repeated antigenic stimulation. Ehrlichia represents a family of obligate intracellular bacteria that have been associated with the development of plasma cell dyscrasias in the veterinary literature. We present a case of a 67-year-old male patient with the development of progressively worsening cutaneous plasmacytosis following prolonged hospitalization secondary to ehrlichiosis sepsis. The patient initially presented with isolated cutaneous involvement and normal laboratory findings that eventually progressed to include multiple laboratory abnormalities, including anemia, hyperproteinemia, and elevated serum creatinine. Further diagnostic workup was declined by the patient despite evidence of progression to systemic plasmacytosis or multiple myeloma.     

黑龙江全沟硬蜱中检测出类似人粒细胞埃利希体的病原体DNA

目的：寻找我国蜱中人粒细胞埃利希体感染存在的病原学证据。方法：应用从人粒细胞埃列希体16 rRNA基因构建的特异引物进行半套式PCR,检测蜱标本中埃利希体DNA。然后对PCR扩增产物进行克隆和序列测定,与已知序列进行同源性比较。结果：从黑龙江采集的全沟硬蜱（Ixodes persulcatus）中扩增出特异DNA片段,计算最小阳性率为0.8％。对919bp的扩增产物进行序列分析,证实为埃利希体DNA,与美国人粒细胞埃利希体分离株对应序列比较,相差4个核苷酸。结论：这是首次证明我国有类似人粒细胞埃利希体的病原体存在。表明我国北方林区可能存在人粒细胞埃利希体感染垢自然疫源地。     

查菲埃立克体120kDa抗原蛋白基因的克隆与表达

目的 克隆查菲埃立克体120kDa膜表面免疫决定性蛋白基因,并使之在原核表达系统中表达。方法 查菲埃立克体分离株（91HE17）感染DH82细胞40天后收集DH82细胞。根据查菲埃立克体P120蛋白基因序列设计特异性引物,套式PCR扩增查菲埃立允体P120蛋白基因部分片段,将PCR扩增产物用BamHⅠ和EcoRⅠ限制性内切酶双酶切后与pUC18载体连接,在获得阳性克隆子后用限制性内切酶将克隆片段切下,定向手稿原核表达载体pProEX HTb构建pProEX HTb/P120重组质粒。将重组质粒转化E.coli DH5α,并使之在IPTG的诱导下进行蛋白质表达。结果 裁短成1080bp的查菲埃立克体P120蛋白基因克隆到pUC18载体,用IPTG诱导pProEX HTb/P120重组质粒转化的E.coli DH5α,表达一分子量大小约为47kDa的融合蛋白。结论 查菲埃立克体120kDa抗原蛋白基因能在原核表达系统中表达,为诊断试剂盒的研制及其它研究工作提供了基础。     

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease,Ehrlichia ruminantium,from the Panola Mountain Ehrlichia

Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick‐borne disease of cattle, sheep and goats responsible for stock losses in sub‐Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross‐reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP‐1B peptides for ER and PME, we tested the cross‐reactivity of this assay using sera from infected livestock. The MAP‐1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual‐plex Taqman^™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease‐causing organism.     

Frequency and Distribution of Rickettsiae,Borreliae, and Ehrlichiae Detected in Human-Parasitizing Ticks,Texas, USA

To describe the presence and distribution of tickborne bacteria and their vectors in Texas, USA, we screened ticks collected from humans during 2008–2014 for Rickettsia, Borrelia, and Ehrlichia spp. Thirteen tick species were identified, and 23% of ticks carried bacterial DNA from at least 1 of the 3 genera tested.     

内蒙古中西部草原蜱媒病原体多样性及基因型分析

目的了解内蒙古中西部草原蜱类的群落结构、携带病原体多样性及基因型。方法于2016-2019年春夏季,在内蒙古中西部草原,采用动物体表搜集法采集蜱标本,进行蜱种鉴定。解剖摘取蜱的唾液腺并提取基因组DNA,以斑点热立克次体柠檬酸合成酶A (gltA)、疏螺旋体以鞭毛蛋白B (flaB)、埃立克体属以外膜蛋白质1 (omp1)、无形体属主要表面蛋白2 (msp2)为靶基因进行PCR扩增初筛。立克次体gltA初筛阳性样品经限制性片段长度多态性(RFLP)分类,再根据蜱种和地区每类选20~30个代表性样品进行gltA、立克次体外膜蛋白A (rOmpA)基因测序。扩增序列测序后用BLAST、 Clustal W和MEGA 7.0软件进行同源性分析,以邻接法构建系统进化树。结果共采集成蜱3 822只,经形态学特征和特异性18S r RNA基因分型法鉴定,隶属于2属3种,分别为草原革蜱、亚东璃眼蜱和边缘璃眼蜱,其中草原革蜱占55.7%(2 129/3 822)、亚东璃眼蜱占30.0%(1 147/3 822),为该地区的优势蜱种。PCR检测结果显示,立克次体gltA基因阳性蜱1 899只,阳性率为49.7%(1 899/3 822), gltA基因阳性样品根据RFLP结果分为两类,两类样品的gltA基因序列均为581 bp,与R. raoultii (DQ365804)或R. aeschlimanni (KT873466)的同源性为100%;两类样品的rOmpA基因均长367 bp,与R. raoultii (AH015610)或R. aeschlimanni (U83466)的同源性为100%,与gltA基因的结果相符。3 822只蜱中,R. raoultii和R. aeschlimanni的阳性率分别为37.2%(1 422/3 822)和12.5%(477/3 822),其中草原革蜱中分别为58.5%(1 245/2 129)和11.1%(477/2 129);亚东璃眼蜱中分别为15.4%(177/1 147)和0;边缘璃眼蜱中分别为0和44.0%(240/546)。疏螺旋体flaB基因阳性蜱28只,阳性率为0.7%(28/3 822),其中草原革蜱中为0.8%(16/2 129),亚东璃眼蜱为1.0%(12/1 147)。共获得疏螺旋体flaB基因序列10条,与莱姆病主要病原体B. garinii (AB035602)和B. afzelii PKo (NC008277)的同源性分别为90.6%~100%和95.6%~100%。亚东璃眼蜱中B. garinii和B. afzelii的阳性率分别为0.9%(10/1 147)和0.2%(2/1 147),草原革蜱中均为0.4%(8/2 129)。3 822只蜱中omp1基因阳性1只,TA克隆后获得8个氨基酸序列相同的克隆和3个氨基酸序列存在差异的克隆,11个克隆的氨基酸序列与E. muris的同源性最高,但仅为65%~69%。3 822只蜱中均未检出无形体属菌群。系统进化树分析结果显示,3种蜱感染的立克次体均与R. raoultii和R. aeschlimanni聚在一簇。在获得的10条疏螺旋体菌群flaB基因序列中,源于草原革蜱和亚东璃眼蜱的1条序列与B. garinii聚在一簇,草原革蜱的另1条序列与B. afzelii聚在一簇,其余8条序列与B. garinii和B. afzelii的flaB基因序列处在不同的分支。草原革蜱感染的埃立克体属菌与目前已知的埃立克体属菌群关系较远,形成独自的聚类。结论内蒙古中西部草原存在草原革蜱、亚东璃眼蜱和边缘璃眼蜱,蜱类中广泛存在斑点热立克次体和莱姆病螺旋体的感染,是R. raoultii、R. aeschlimanni、 B. garinii和B. afzelii潜在的自然疫源地。有必要加强该地区蜱媒传染病的预防控制工作。     

Efficiency of inactivated vaccines against heartwater in Burkina Faso: Impact of Ehrlichia ruminantium genetic diversity

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N = 153, p = 0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N = 173, p < 0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.     

Absence of Myelofibrosis in Dogs with Myelosuppression Induced by Ehrlichia canis Infection

Bone marrow (BM) pathology was assessed in 10 dogs with Ehrlichia canis-induced aplastic pancytopenia. BM core biopsy sections were stained with haematoxylin and eosin and with haematoxylin/van Gieson and Gordon and Sweets' reticulin stain for the detection of collagen and reticulin fibres, respectively. Iron stores were assessed by Perls' Prussian blue staining. There was no significant deposition of collagen or reticulin in any sample, but in seven dogs the BM was depleted of haemosiderin. These findings suggest that myelofibrosis does not play a significant role in the development of BM failure in canine monocytic ehrlichiosis and that iron deficiency may exacerbate the anaemia in the myelosuppressive phase of the disease.