Preliminary study of natural reservoirs as sentinels of <i>Anaplasma phagocytophilum</i>and <i>Ehrlichia chaffeensis</i>in Soria, northern Spain

The serum of foxes and red deer from the Province of Soria (northern Spain) was screened in indirect immunofluorescence assays to determine whether these animals could be used as sentinels of the tick-borne pathogens Anaplasma phagocytophilum and Ehrlichia chaffeensis. The results suggest that foxes and red deer would not make good sentinels in epidemiological studies on E. chaffeensis in this region, although red deer could be used as such for the study of A. phagocytophilum transmission.

3株嗜吞噬细胞无形体AnkA蛋白原核表达及免疫原性差异性研究

目的研究分析3株嗜吞噬细胞无形体AnkA蛋白的免疫原性。方法利用原核表达PCR扩增了嗜吞噬细胞无形体Webster株,斯洛文尼亚1 567株,美国96HE58株ankA基因部分序列,构建pET-30a-AnkA表达载体,转化至大肠杆菌BL21中,以IPTG诱导高效表达融合蛋白,经飞行质谱鉴定氨基酸序列,免疫BALB/c小鼠制备多克隆抗体。Western blot分析3株菌AnkA重组蛋白与免疫小鼠血清抗体相互间的免疫反应以及交叉反应性。同时检测3株菌AnkA重组蛋白与我国无形体病人血清抗体反应性。结果表达的重组蛋白主要以可溶性形式存在,小鼠免疫获得高效价抗AnkA IgG抗体,3株菌重组蛋白免疫抗体间存在免疫交叉。3株菌重组蛋白与我国无形体病人血清抗体存在免疫反应。结论成功原核表达并纯化了3株嗜吞噬细胞无形体AnkA蛋白,重组蛋白与我国无形体病人阳性血清存在免疫反应。3株嗜吞噬细胞无形体AnkA重组蛋白与免疫小鼠多克隆抗体间存在交叉反应。

无形体分泌蛋白Ats-1与宿主细胞互作蛋白筛选及其生物信息分析

目的筛选与无形体Ats-1互作的THP-1宿主细胞蛋白,为揭示病原体侵袭宿主的致病机理提供科学数据。方法利用分子克隆法构建p GBKT7-ats-1诱饵质粒并转化酵母Y2HGold,验证诱饵质粒是否有自激活、毒性现象;制备THP-1细胞c DNA并连接到p GADT7-Rec上,转化酵母Y187并鉴定c DNA文库质量;酵母双杂交筛选与Ats-1互作的宿主细胞靶蛋白,通过GO、String生物信息分析细胞靶蛋白可能的生物学过程。结果成功构建诱饵质粒;cDNA文库容量4×10~6克隆,插入片段100~3 000 bp,且无污染,达到建库质控要求;酵母双杂交结果显示,与Ats-1蛋白互作的宿主细胞靶蛋白7个,分别是真核延伸因子1复合体α1亚基(EEF1A1)、TIMP金属蛋白酶抑制子1(TIMP1)、锌指蛋白36,C3H样2 (ZFP36L2)、半乳糖凝集素1(LGALS1)、前胸腺素α(PTMA)、WBSCR22、吸引素(ATRN)。生物信息分析显示,与Ats-1互作的宿主靶蛋白主要参与细胞分化增殖、细胞凋亡、自噬、炎症等生物学过程。结论病原诱饵蛋白与宿主细胞靶蛋白互作是一种细胞-细胞间发生信号联系的分子水平生物学过程,可能影响病原粘附、侵袭及胞内生存,最终导致疾病发生及临床症状出现。

Seroepidemiological Investigation of Lyme Disease and Human Granulocytic Anaplasmosis among People Living in Forest Areas of Eight Provinces in China

Objective Lyme disease and Human granulocytic anaplasmosis are tick-borne diseases caused by Borrelia burgdorferi and Anoplasrna phagocytophilum respectively. We have investigated infection and co-infection of the two diseases in the population of forest areas of eight provinces in China by measuring seroprevalence of antibodies against B. burgdorferi and A. phagocytophilum. Methods Forest areas in 8 provinces were chosen for investigation using whole sampling and questionnaire survey methods. 3 669 serum samples from people in the forest areas were tested for the presence of antibodies by indirect immunofluorescent assay （IFA）. Results Seroprevalence against B. burgdorferi was 3% to 15% and against A. phagocytophilum was 2% to 18% in the study sites in the 8 provinces in China. We also found co-infection of B. burgdorferi and A. phagocytophilum in 7 of the 8 provinces （the exception being the Miyun area in Beijing）. The seroprevalence for both B. burgdorferi and A. phagocytophilum was significantly higher among people exposed to ticks than among people who were not exposed to ticks. Conclusion We conclude that both pathogens are endemic in the forest areas in the eight provinces, but the prevalence of B. burgdorferi and A. phagocytophilum differs between the provinces.

Investigation of anaplasmosis in Yiyuan County,Shandong Province,China

Objective:To investigate the situation of anaplasmosis in Yiyuan county.Shandong Province. Methods:A total of 26 blood samples from febrile patients suspected of anaplasmosis,48 blood samples from healthy farmers,8 from dogs,and 10 from goats and 170 ticks were collected in the same area during 2005-2007,and detected by serological and molecular methods.Results: Eight confirmed cases and 6 probable cases were determined using serologic and molecular methods.The seroprevalence of Anaplasma phagocytophilum(A.phagocytophilum) was 26.7% in healthy cases.Nine out of 10 sheep samples and 7 out of 8 dog samples reacted positively to the A.phagocytophilum antigen.PCR amplification and sequencing of the 16SrRNA of,4. phagocytophilum gene showed that some samples from patients,goats and ticks were 100% identical.The seroprevalence of Rickettsia typhi was 22.9%,Orientia tsutsugamushi 6.3%, Rickettsia sibirica 27.1%,Coxiella burnetii 18.8%,Bartonella henselae 31.3%,and Borrelia burgdorferi 41.6%.Conclusions:It is important to make differential diagnosis of febrile patients and to apply treatment with specific antibiotics.It is needed to enforce essential prevention and control measures including tick control and to improve sanitation conditions.

Serological investigation of vector-borne disease in dogs from rural areas of China

Objective:To evaluate the Anaplasma phagocytophilum(A.phagocytophilum),Ehrlichia canis(E.canis,Dirofilaria immitis(D.immitis)(canine heartworm),Borrelia burgdorferi(B.burgdorferi)infections in countryside dogs from Yunnan,Hainan and Anhui provinces.Methods:Serum samples were collected from 26 dogs in Yunnan.Hainan and Anhui provinces.The samples were tested using a commercial ELISA rapid diagnostic assay kit(SNAP^(?)4Dx^(?);IDEXX Laboratories,Inc.U.S.A.).Meaiiwliile,indirect immunofluorescence assay(IFA)recommended by WHO was conducted to delect IgG to A.phagocytophilum.Two methods were analyzed and compared.Results:The number of serologically positive dogs for IgG to A.phagocytophilum was only 2which was from Hainan province and none of the 26 dogs responded positive for E.canu.D.immitis(canine heartworm,and B.burgdorferi by ELISA rapid diagnostic method.The number of serologically positive dogs for IgG to A.phagocytophilum was 13(50%)by IFA method.Data of the two methods were analyzed by statistical software and the difference was statistically significant(P=0.002).Conclusions:It can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method.However,we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including.4.phagocytophilum,E.canis,D.immitis(canine heartworm),and B.burgdorferi which have public health significance.

黑龙江省口岸地区蜱类感染嗜吞噬细胞无形体Anaplasma phagocytophila的初步调查

为调查黑龙江省口岸蜱类携带嗜吞噬细胞无形体Anaplasma phagocytophila感染情况,运用聚合酶链式反应方法扩增嗜吞噬细胞无形体柠檬酸合成酶（gltA）基因片段,对黑龙江省各口岸地区采集的蜱标本进行检测,并对阳性结果进行序列分析.共检测蜱1 609只,29只阳性,阳性率为1.80%,结果表明,黑龙江省口岸蜱类除日本血蜱外均有自然感染嗜吞噬细胞无形体的现象.所感染的嗜吞噬细胞无形体gltA 基因与GenBank 中登录的嗜吞噬无形体gltA基因片段,相似性为82%～98%.因此,我国黑龙江省口岸地区存在蜱粒细胞无形体感染.

Serological survey on some pathogens in wild brown hares(Lepus europaeus) in Central Italy

Objective: To determine the exposure of wild brown hares [Lepus europaeus(L. europaeus), pallas] to Anaplasma phagocytophilum(A. phagocytophilum), Borrelia burgdorferi(B. burgdorferi) sensu lato, Encephalitozoon cuniculi(E. cuniculi), Leishmania sp., Neospora caninum(N. caninum) and Toxoplasma gondii(T. gondii). Methods: Two hundred twentytwo blood serum samples of wild brown hares captured in protected areas of the province of Pisa(Central Italy) were tested to detect antibodies against the reported pathogens. Results: Thirty one(14.0%) animals resulted positive for at least one tested agent, with antibody titres ranging from 1:20 to 1:320. In particular, 13(5.8%) samples were positive to B. burgdorferis.l., 11(4.9%) to N. caninum, 3(1.3%) to T. gondii, 2(0.9%) to A. phagocytophilum and 2(0.9%) to Leishmania sp. No samples scored positive to E. cuniculi. Four animals(14.8%) resulted coinfected with 2 different pathogens. Conclusion: The obtained results showed that B. burgdorferi s.l. N. caninum, T. gondii, A. phagocytophilum and Leishmania sp circulate in wild brown hares in Central Italy, suggesting a possible role of L. europaeus as reservoir of these pathogens. The obtained results showed that autochthonous wild brown hares living in Central Italy have been exposed to several pathogens circulating in this area, suggesting a possible role of L. europaeus as reservoir.

Tick-Borne Rickettsial Pathogens in Rodents from Mexico

Tick-Borne Rickettsial Diseases (TBRD) are emerging zoonotic diseases, and a problem of human health and veterinary medication. The distribution of these diseases is related to the distribution of vector. The presence of pathogens in the host is a risk indicator of population exposure to these areas. A total of 478 tissues samples from rodents, A. phagocytophilum 18 (3.7%), E. canis 47 (9.8%), Rickettsia rickettsii 18 (3.7%) and E. chaffeensis 19 (3.9%) were detected using species-specific PCR assay. It is the first report in Mexico the presence of rodents infected with A. phagocytophilum and E. chaffeensis. The rodent Peromyscus spp. were the most commonly prevalent host of infection for all the bacteria’s. We have to consider as host of TBRD transmitter and provide a useful contribution to understanding their epidemiology. The health sector should be considered all the fevers of unknown causes in humans and animals in Mexico as infections by these vector-borne rickettsial pathogens.

2022年安徽省铜陵市鼠形动物中4种病原体的检测分析

目的通过对铜陵市鼠形动物病原体感染情况的调查,为本地鼠媒传染病控制提供科学依据.方法2022年1月至12月,在安徽省铜陵市枞阳县、义安区、铜官区和郊区进行鼠类监测和标本采集,利用荧光定量PCR方法对其脾、肝、脾、肺组织中汉坦病毒、大别班达病毒、嗜吞噬细胞无形体和致病性钩端螺旋体等4种病原体进行检测、分析.结果共捕获234只鼠形动物,病原总检出率为4.27%,汉坦病毒和致病性钩端螺旋体检出率均为2.14%;汉坦病毒在黄胸鼠和褐家鼠中检出率为3.77%和1.59%,在不同生境中,重点行业鼠形动物中汉坦病毒检出率为6.67%,农村居民区为2.15%;致病性钩端螺旋体在北社鼠、褐家鼠和黄胸鼠中检出率依次为8.33%、4.76%和0.94%,在不同生境中,农田耕地鼠形动物中致病性钩端螺旋体检出率为6.25%、农村居民区为2.15%.结论铜陵市鼠形动物中存在病原体感染,需加强鼠类防制及鼠媒传染病控制,以降低人群感染风险.

2021年蚌埠市4种鼠传病原体检测分析

目的掌握蚌埠市鼠传病原体的分布情况,为鼠传疾病的防控工作提供科学依据。方法在蚌埠市禹会区、蚌山区、龙子湖区和淮上区野外捕鼠,采用实时荧光定量PCR法对4种鼠传病原体,包括新型布尼亚病毒、汉坦病毒、致病性钩端螺旋体和嗜吞噬细胞无形体,进行核酸检测。结果共检测200只鼠,1份样本汉坦病毒核酸检测阳性,阳性率为0.50%;18份样本致病性钩端螺旋体核酸检测阳性,阳性率为9.00%;未检出新型布尼亚病毒和嗜吞噬细胞无形体。结论2021年蚌埠市鼠类致病性钩端螺旋体感染水平较高,应做好防控工作。

新疆阿克苏地区蜱和羊感染无浆体的分子流行病学调查

[目的]了解新疆阿克苏地区蜱与羊感染无浆体的情况。[方法]共采集192只蜱和477份羊抗凝血样品;以线粒体16S rDNA和ITS-2为靶基因,采用PCR法对经过形态学鉴定的蜱虫进行分子生物学鉴定;利用特异性引物,分别采用普通PCR法及巢式PCR法检测绵羊无浆体(Ananlasma ovis,A.ovis)和嗜吞噬细胞无浆体(Ananlasma phagocytophilum,A.phagocytophilum)在蜱和羊抗凝血样品中的携带情况;采用统计学方法分析不同种类、性别蜱虫和不同区域、不同饲养模式下羊感染上述2种无浆体的情况。[结果]采集到的192只蜱虫包括雄蜱79只、雌蜱113只,共有2属3种,分别为扇头蜱属的图兰扇头蜱(Rhipicephalus turanicus,R.turanicus)以及璃眼蜱属的亚洲璃眼蜱(Hyalomma asiaticm,H.asiaticm)和小亚璃眼蜱(Hyalomma anatolicm,H.anatolicm),所占比例分别为52.60%、45.83%、1.56%。蜱样品中,感染2种无浆体的总体阳性率为7.29%,未检测到A.ovis和A.phagocytophilum混合感染;R.turanicus、H.asiaticm、H.anatolicm感染无浆体的阳性率分别为8.91%、5.68%、0,三者差异有统计学意义(χ^(2)=3.20,P<0.05);雄蜱和雌蜱感染无浆体的阳性率分别为6.33%、7.96%,二者差异无统计学意义(χ2=0.30,P>0.05)。羊抗凝血样品中,感染2种无浆体的总体阳性率为51.15%,与蜱样品的总体阳性率差异有统计学意义(χ^(2)=111.35,P<0.05);A.ovis与A.phagocytophilum混合感染率为16.56%;舍饲羊和散养羊感染无浆体阳性率分别为28.16%和83.00%,二者差异有统计学意义(χ^(2)=139.81,P<0.05);库车市和沙雅县羊无浆体阳性率最低,为5.00%,温宿县阳性率最高,为64.83%,二者差异有统计学意义(χ^(2)=27.72,P<0.05)。[结论]阿克苏地区蜱和羊普遍感染无浆体,应该加强对无浆体病的防治工作。

吉林省林区蜱粒细胞无形体感染的调查

目的调查吉林省蜱粒细胞无形体感染。方法运用聚合酶链反应方法对吉林延边地区采集的蜱标本粒细胞无形体16S rRNA和gltA基因片段进行扩增及序列分析,将扩增序列与GenBank注册的基因序列进行比较,构建粒细胞无形体gltA基因进化树。结果共检测游离蜱427只,其中全沟硬蜱100只,森林革蜱327只,粒细胞无形体感染阳性率分别为4.00%和0.00%。寄生蜱感染阳性率2.9%。寄生蜱与游离蜱感染率差别无显著性。16S rRNA序列与我国已在GenBank注册的AF205140序列一致,与国外粒细胞无形体16S rRNA序列存在不同程度的差异,相似性为97%～99%;gltA基因与GenBank的粒细胞无形体gltA基因片段比较,相似性为87%～97%,推导的氨基酸序列相似性为84%～99%。结论我国吉林省林区存在蜱粒细胞无形体感染。

湖北省随州市羊携带嗜吞噬细胞无形体的调查

目的调查湖北省随州市羊携带嗜吞噬细胞无形体的状况。方法运用聚合酶链反应方法对湖北随州市采集的羊血标本进行嗜吞噬细胞无形体16S rRNA基因片段进行扩增和序列分析,将扩增序列与GenBank相应基因序列进行同源性比较和进化分析。结果共检测羊血标本29份,阳性标本25份,嗜吞噬细胞无形体阳性感染率为86.2%;所检出的嗜吞噬细胞无形体16S rRNA基因(1 442 bp)与GenBank中收录的部分嗜吞噬细胞无形体16s rRNA基因序列的同源性高达97.1%～99.8%,进化分析显示与嗜吞噬细胞无形体同在一个分支上。结论湖北随州地区山羊存在嗜吞噬细胞无形体的感染。

浙江磐安地区啮齿动物嗜吞噬细胞无形体感染研究

目的调查浙江省磐安地区啮齿动物无形体属嗜吞噬细胞无形体的自然感染状况,了解其分子生物学特性。方法在浙江省磐安地区低山田地里捕获多种啮齿动物230只(黄胸鼠、社鼠、褐家鼠、小林姬鼠、黄毛鼠、青毛鼠、大林姬鼠、白腹巨鼠、针毛鼠、黑线姬鼠和松鼠);解剖取肝脾;提取DNA,用巢式PCR扩增无形体属16SrRNA片段;PCR产物测序,并进行Blast同源性比较,最后采用MEGA4.0软件与GenBank登录的其它无形体属种株进行系统发生分析。结果 230只啮齿动物中,1只黄胸鼠、1只褐家鼠和2只白腹巨鼠肝脾标本检出无形体属16SrRNA片段,磐安地区野鼠无形体属立克次体的自然感染率为1.7%。3份阳性标本PCR产物测序,序列比对分析表明,从褐家鼠同时检出分别与嗜吞噬细胞无形体和浙江无形体株ZJ05/2009完全一致的序列,黄胸鼠和1只白腹巨鼠的序列与浙江无形体株ZJ05/2009完全一致。结论浙江省磐安地区野鼠检出致病性无形体属核酸,存在无形体属不同种株的复合感染感染。提示可能存在无形体病自然疫源地,需要进一步密切关注致病性无形体的人群感染情况。

湖北省长角血蜱携带嗜吞噬细胞无形体的调查

目的调查湖北省长角血蜱是否携带嗜吞噬细胞无形体。方法运用聚合酶链反应方法对湖北随州地区采集的蜱标本进行嗜吞噬细胞无形体16SrRNA基因片段进行扩增和序列分析,将扩增序列与GenBank注册的基因序列进行比较。结果共检测游离蜱72只,蜱种鉴定均为长角血蜱,嗜吞噬细胞无形体最小感染率为4.22%;所检出的嗜吞噬细胞无形体16SrRNA基因与我国已在GenBank注册的24个相对应序列同源性100%。结论湖北省长角血蜱携带嗜吞噬细胞无形体。

云南省曲靖市啮齿动物的立克次体血清学和分子流行病学调查

目的调查云南省曲靖市啮齿动物感染立克次体状况。方法用鼠笼法捕鼠,采集鼠血清和脾脏。用间接免疫荧光试验(IFA)检测鼠血清中7种常见立克次体的IgG抗体;用巢氏PCR方法检测鼠脾中嗜单核细胞埃立克体和嗜吞噬细胞无形体16SrRNA基因片段部份序列。结果 2012年7-9月在曲靖市捕获啮齿动物3种592只,褐家鼠(Rattus norvegicus)和黄胸鼠(Rattus flavipectus)构成比分别为61.49%和35.47%。鼠血清中贝氏柯克斯体(Coxiella burentii)、莫氏立克次体(Rickettsia typhi)、恙虫病东方体(Orientia tsutsugamushi)、西伯利亚立克次体(Rickettsia sibirica)、嗜吞噬细胞无形体(Anaplasma phagocytophilum)和嗜单核细胞埃立克体(Ehrlichia chaffeensis)的抗体阳性率依次为21.45%、7.60%、7.09%、3.38%、1.18%和0.51%;未检测到猫立克次体(Rickettsia felis)抗体。从5份褐家鼠脾脏标本中检测到16SrRNA基因序列,同源性和进化分析表明,1株为嗜吞噬细胞无形体,4株为莫氏立克次体。结论曲靖市啮齿动物中存在莫氏立克次体和人粒细胞无形体流行,褐家鼠可能是主要宿主;同时还存在恙虫病东方体、贝氏柯克斯体、斑点热立克次体和埃立克体的感染。当地疾控和医疗机构应加强立克次体病的监测。

福建部分地区人和动物嗜吞噬细胞无形体基因检测和序列分析

目的了解福建省人和动物嗜吞噬细胞无形体的感染状况以及基因特征。方法收集永春县和上杭县高危人群、家畜的全血,采用巢式PCR扩增无形体16SrRNA片段,PCR产物纯化测序并与序列比对分析。结果永春县牛的无形体感染率最高为53.85%,羊、狗无形体感染率分别为24.71%和12.50%。上杭县羊的无形体感染率为53.06%,高危人群的无形体感染率为8.33%。无形体基因序列与相应的参考序列同源性均达97%~100%。结论福建省人和动物存在嗜吞噬细胞无形体感染。

人粒细胞无形体病临床流行病学与防治研究现状

人粒细胞无形体病是由染疫的蜱叮咬将嗜吞噬细胞无形体注入人体引起的一种新发人兽共患疾病,人感染了人粒细胞无形体病,除有发热、不适、头痛、肌痛等临床特征外,常伴多脏器损害,从报告的病例数量呈逐年增加,分布范围呈逐年扩大,已构成世界性公共卫生问题。为我国人粒细胞无形体病的深入研究和临床诊治提供科学依据,本文从病原的分类地位与基本形态、基因特征与结构、理化特性和生物学特性记述了人粒细胞无形体病的病原学;从传染源、传播途径、易感人群和流行特征描述了人粒细胞无形体病的流行病学;从发病机理与病理改变、临床表现与并发症、诊断与鉴别诊断和临床治疗等方面阐述了人粒细胞无形体病的临床学;从血常规与生化检查、血清学检测和病原学检测等方面报告了人粒细胞无形体病的实验室检査技术;从控制传染源、切断传播途径和保护易感人群等论述了人粒细胞无形体病的预防控制。基于上述较系统地概述了我国人粒细胞无形体病临床流行病学的研究进展。

嗜吞噬细胞无形体的致病机制的研究进展

嗜吞噬细胞无形体(Anaplasma phagocytophilum,APH)是重要人兽共患病——人粒细胞无形体病(HGA)的病原体。APH专性侵染哺乳动物中性粒细胞,入侵后在细胞内形成包涵体逃逸溶酶体酶的杀伤,APH同时破坏宿主细胞的凋亡和自噬,并抑制宿主细胞的氧化反应等杀菌活性。APH通过一系列毒力分子表达影响宿主细胞,使其在宿主细胞内有效生存和繁殖,最终导致机体感染的发生。