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1.
目的 了解江西省赣州市蜱的种类及其携带的病原体,为蜱传疾病的防控提供科学依据方法 在野外用布旗法和在动物体表采集蜱,进行种类鉴定。用PCR法扩增立克次体(Rickettsia sp.)gltA基因和埃立克体(Ehrlichia sp.)16S rRNA基因片段并测序,进行系统进化分析结果 江西省赣州市4县采集的395只蜱的种类包括2属4种,分别为血蜱属的长角血蜱(Haemaphysalislongicornis)、嗜群血蜱(H. concinna)、具角血蜱(H. cornigera)和扇头蜱属的微小扇头蜱(Rhipicephalus microplus)。395只蜱分为43批检测立克次体和埃立克体,共有18批阳性。系统进化分析显示,龙南县22批蜱中检测到16批阳性,来源于具角血蜱的LN1615株与扇头蜱立克次体(R. rhipicephali)处于同一分支,来源于微小扇头蜱的LN1620株与马赛立克次体(R. massiliae)处于同一分支,其余的12株来源于具角血蜱和2株来源于微小扇头蜱,与日本立克次体(R. japonica)处于同一分支。崇义县CY1602株来源于嗜群血蜱与R. raoultii处于同一分支。于都县YD1606株为来源于长角血蜱的埃立克体,与2010年Yonaguni206(HQ697589)、Yonaguni138(HQ697588)和2009年HLAE178(GU075695)处于同一分支。安远县微小扇头蜱检测均为阴性结论 本文对江西省赣州市采集的蜱进行了鉴定,首次在江西省赣州市的蜱中检测到日本立克次体、扇头蜱立克次体、马赛立克次体、R. raoultii和埃立克体,为江西省蜱种类的调查及其传播疾病的预防控制提供根据。  相似文献   
2.
Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis. In this study, we developed Etf-1–specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti–Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1–GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Human monocytic ehrlichiosis (HME), one of the most prevalent, life-threatening, and emerging tick-borne diseases in the United States (1, 2) is caused by infection with Ehrlichia chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales. E. chaffeensis replicates within human monocytes-macrophages and causes severe flu-like symptoms accompanied by hematologic abnormalities and hepatitis. Currently, the only HME therapy is the broad-spectrum antibiotic doxycycline, which is effective only if initiated early because delayed initiation (e.g., because of misdiagnosis can lead to severe complications or death). In addition, doxycycline is contraindicated for pregnant women and children or those with drug allergies. The presence of underlying illness or injury, immunosuppression, and coinfection with other tick-borne pathogens can similarly lead to severe complications or death (3). No vaccine exists for HME. Tick-borne diseases have risen dramatically in the past 20 y and continue to rise, underscoring the importance of developing new therapeutic approaches and preventive measures (4).The type IV secretion system (T4SS) is conserved among all rickettsial organisms. The recent elucidation of critical roles of T4SS for E. chaffeensis and Anaplasma phagocytophilum infection (5) may provide potential targets for new approaches against rickettsial diseases. For example, the T4SS effectors Ehrlichial translocated factors 1 and 2 (Etf-1 and Etf-2) are critical E. chaffeensis proteins secreted via T4SS into the host cell cytoplasm, as knockdown of Etf-1 or Etf-2 by transfection of E. chaffeensis with specific antisense peptide nucleic acids significantly inhibits E. chaffeensis infection (6, 7). Secreted Etf-1 localizes to mitochondria and blocks mitochondria-mediated host cell apoptosis to keep the infected host cell alive for bacterial intracellular replication (8). A subpopulation of Etf-1 molecules that are not localized to mitochondria interacts with Beclin 1 (ATG6) and active Rab5 (Rab5-GTP), and induces Rab5-regulated autophagy for E. chaffeensis to acquire catabolites as nutrients (9). Etf-2 directly binds Rab5-GTP on Ehrlichia-containing inclusion membranes and blocks Rab5 GTPase activating protein (RabGAP-5) engagement with Rab5-GTP to prevent Ehrlichia-containing inclusions from maturing into late endosomes and fusing with lysosomes (7).Camelidae produce two types of antibodies: conventional antibodies and heavy-chain–only antibodies (10). The variable domain of the heavy chain of heavy-chain–only antibodies (VHHs) of camelids is the smallest (11 to 15 kDa) antigen-binding fragment relative to conventional antibodies. VHHs are soluble and display long surface loops, which are often larger than those of conventional murine and human antibodies (11, 12). The VHHs can be cloned into bacterial or mammalian expression plasmids (13) to produce a nanobody (Nb), a monomeric variable antibody. VHHs cloned into mammalian expression vectors can produce intracellular Nbs within mammalian cells that are superior to conventional antibodies for modulating intracellular functions because they can operate in the reducing intracellular environment, are proteolytically stable, can target subcellular sites, can penetrate cavities in target antigens, and can bind efficiently to antigens, such as enzyme catalytic sites (1316). Although the therapeutic potential of Nbs has been investigated for several infectious diseases (14, 1719), the use of Nbs as a therapeutic agent against intracellular bacteria such as E. chaffeensis has not been reported. In the present study, we developed an intracellular Nb approach to block T4SS effectors within mammalian cells, thereby inhibiting intracellular pathogen infection.Progress in developing effective therapy and investigative approach for obligatory intracellular pathogens has been hindered by many factors, not the least of which is the lack of safe and efficient intracellular delivery methods of macromolecules. Although cyclic peptides are generally unable to cross the cell membrane, some naturally occurring cyclic peptides (e.g., cyclosporine A) possess the unusual ability of crossing the cell membrane by passive diffusion and are orally bioavailable (20). Cyclized Arg-rich cell-permeable peptides (CPPs)—such as cyclo(FΦRRRRQ) or cFΦR4, where Φ is l-2-naphthylalanine—or newer and more effective CPPs, such as CPP9 and CPP12, that include d-arginine or d-phenylalanine, provide rapid and efficient cytosolic delivery of their linked cargo proteins into >95% of cells (2123). They are not cytotoxic at effective concentrations and have oral and intravenous bioavailability based on preliminary pharmacokinetics in mice (22). The cyclic CPPs (and the CPP-cargo conjugates) bind directly to plasma membrane phospholipids and enter cells by endocytosis (22). They then efficiently escape from the early endosome into the cytosol unlike Tat, which escapes only from late endosomes (2224).In the present study, we have developed anti–Etf-1 Nbs. We obtained a Nb that blocks Etf-1 functions and demonstrated its effectiveness in combination with a cyclic CPP for inhibition of E. chaffeensis infection in cell culture and in a mouse model. These findings represent a significant advance in developing therapeutic and investigative strategy of obligatory intracellular pathogens.  相似文献   
3.
We present a case of possible transfusion‐transmitted Ehrlichia chaffeensis infection in a heavily transfused cord blood transplant recipient, resulting in severe infection and graft loss. Transfusion‐transmitted, vector‐borne infections in immunocompromised individuals can have severe consequences, and should be considered in hospitalized patients receiving blood products with unexplained fever or sepsis.  相似文献   
4.
5.
目的 调查新疆部分地区鼠类携带无形体和埃立克体的状况.方法 对新疆博乐、石河子、乌鲁木齐3个地区捕获的鼠类(沙鼠、褐家鼠、田鼠)401份,采集肺脏、脾脏提取总DNA,通过巢式PCR扩增无形体和埃立克体16SrRNA片段并与GenBank中相应基因序列进行比对分析.结果 在沙鼠样本中扩增到无形体和埃立克体16SrRNA片段,而在其它两种鼠类未扩增到目的片段,扩增片段经测序、比对后确定为Anaplasma phagocytophilum和Erhlichia chaffeenisis.在312份沙鼠检测样本中,检出无形体17份,占5.45%;埃立克体48份,占15.38%;无形体和埃立克体均检出12份,占3.84%.结论新疆地区存在无形体和埃立克体病原,其主要存在与荒漠和半荒漠地带生存的沙鼠中,新疆存在这两种病原的自然疫源地,而且两种病原可以共同存在于同一宿主动物.  相似文献   
6.
Twenty‐three free‐ranging white‐tailed deer (WTD; Odocoileus virginianus) and six mule deer (MD; Odocoileus hemionus) from south‐central British Columbia, Canada, were tested for Anaplasma marginale by msp5 gene‐specific PCR and Ehrlichia spp. by 16S rRNA or citrate synthase (gltA) gene‐specific PCR, as well as by PCR with universal 16S rRNA primers detecting a wide range of bacteria. No deer tested positive for A. marginale. Amplification with universal 16S rRNA primers followed by sequencing of cloned fragments detected an Anaplasma sp. in one of 23 (4.3%) WTD and six of six (100%) MD and Bartonella sp. in four of 23 (17.4%) WTD. The Anaplasma sp. was genetically distinct from A. marginale and all other recognized members of the genus. Four of six (66.7%) MD and 0 of 23 (0%) WTD were Ehrlichia positive by PCR with primers for 16S rRNA and gltA genes. The sequences of gltA PCR fragments were identical to each other and to the respective region of the gltA gene of an Ehrlichia sp. which we detected previously in naturally infected cattle from the same area, suggesting the possibility of biological transmission of this rickettsia between cattle and wild cervids. Antibodies reactive with the MSP5 protein of A. marginale were detected using a competitive enzyme‐linked immunosorbent assay in two of six (33.3%) MD, but not in WTD. The two seropositive MD were PCR positive for both the Anaplasma sp. and Ehrlichia sp. detected in this study, suggesting a reaction of antibodies against one or both of these rickettsias with the MSP5 antigen.  相似文献   
7.
野外采集标本中人埃立克体16S rRNA基因扩增方法及应用   总被引:3,自引:0,他引:3  
本文应用特异引物,通过多对半巢式PCR从采自内蒙古大兴安岭的蜱标本中扩增出人粒细胞埃立克体和查菲埃立克体的16SrRNA全基因,一方面提供了这2种埃立克体存在于中国的证据,另一方面为今后进行埃立克体病的病原、媒介和宿主的调查研究提供了一个可行的方法.并从一只大林姬鼠的脏器中检测到EC16SrRNA基因片段.  相似文献   
8.
The aims of this study are to investigate the hematology, blood chemistry, pathological study, including macroscopic and microscopic lesions, of experimentally induced canine monocytic erhlichiosis in Thailand and to demonstrate the distribution of Ehrlichia canis in target organs by nested polymerase chain reaction (PCR). Five experimental healthy dogs were inoculated with 5 ml of whole blood (estimated number of E. canis morulae 15 × 10–5% per monocytes) from the splenectomized dog via the saphenous vein. Two healthy dogs served as a negative control. Hematology revealed nonregenerative normocytic normochromic anemia, thrombocytopenia and mild leukopenia. Blood chemistry revealed an increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AP), hypoproteinemia, hypoalbuminemia, and hyperglobulinemia by day 66 post-inoculation. Pathology revealed anemia, ascites, jaundice, interstitial pneumonia, splenomegaly, generalized lymphadenopathy, and severe fatty liver. The detection of E. canis was performed using tissue embedded in paraffin wax by nested PCR showing positive in all target organs. This study concluded that acute induced experimental canine monocytic ehrlichiosis can cause significant clinical and pathological lesions.  相似文献   
9.
Canine monocytic ehrlichiosis is an important tick-borne disease worldwide. No commercial vaccine for the disease is currently available and tick control is the main preventive measure against the disease. The aim of this study was to evaluate the potential of a multi-passaged attenuated strain of Ehrlichia canis to serve as a vaccine for canine monocytic ehrlichiosis, and to assess the use of azithromycin in the treatment of acute ehrlichiosis. Twelve beagle dogs were divided into 3 groups of 4 dogs. Groups 1 and 2 were inoculated (vaccinated) with an attenuated strain of E. canis (#611A) twice or once, respectively. The third group consisted of naïve dogs which served as controls. All 3 groups were challenged with a wild virulent strain of E. canis by administering infected dog-blood intravenously. Transient thrombocytopenia was the only hematological abnormality observed following inoculation of dogs with the attenuated strain. Challenge with the virulent strain resulted in severe disease in all 4 control dogs while only 3 of 8 vaccinated dogs presented mild transient fever. Furthermore, the mean blood rickettsial load was significantly higher in the control group (27–92-folds higher during days 14–19 post challenge with the wild the strain) as compared to the vaccinated dogs. The use of azithromycin was assessed as a therapeutic agent for the acute disease. Four days treatment resulted in further deterioration of the clinical condition of the dogs. Molecular comparison of 4 genes known to express immunoreactive proteins and virulence factors (p30, gp19, VirB4 and VirB9) between the attenuated strain and the challenge wild strain revealed no genetic differences between the strains. The results of this study indicate that the attenuated E. canis strain may serve as an effective and secure future vaccine for canine ehrlichiosis.  相似文献   
10.
浙江省在鼠中检测到埃立克体及无形体DNA片段并测序分析   总被引:1,自引:0,他引:1  
目的:了解鼠中自然感染埃立克体及无形体的状况。方法:用巢式PCR扩增鼠中埃立克体属及嗜粒细胞无形体属16S rDNA的5′末端片段,对阳性产物克隆后测序。结果:在浙江省金华市金东区捕到鼠128只。在黄毛鼠中检到阳性4份,阳性率为3.125%,并对其进行克隆后测序。与Genbank上比对分析,三份与牛埃立克体相同基因区最为接近,属无形体科,无形体属。一份与反刍动物埃立克体相同基因区最为接近,属无形体科,埃立克体属。均为国内首次报道。结论:初步认为浙江省存在埃立克体及无形体病原。黄毛鼠可能为埃立克体及无形体宿主,尚需进一步调查。  相似文献   
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