通辽市家畜感染无形体属病原体的调查

目的 调查内蒙古通辽地区绵羊和牛的无形体感染情况。方法 采用PCR方法检测内蒙古通辽市扎鲁特旗及科左中旗散放76只绵羊和20头牛的血样本,扩增嗜吞噬细胞无形体(Anaplasma phagocytophilum)、中央无形体(A. centrale)和牛无形体(A. bovis)的16S rRNA基因和羊无形体(A. capra)的gltA基因。结果 76只绵羊血样本中37份扩增到嗜吞噬细胞无形体目的基因,阳性率为 48.7%;76只绵羊血样本中16份扩增到中央无形体目的基因,阳性率为21.1%。所有绵羊的血样本均未扩增到羊无形体和牛无形体的目的基因,20头牛的血样本均未扩增到4种无形体的目的基因。结论 通辽地区羊存在嗜吞噬细胞无形体和中央无形体感染,提示该地区存在无形体病的自然疫源地和可能存在人无形体病。     

云南西北部地区家畜无形体感染的分子流行病学调查

目的 了解云南省西北部地区家畜无形体感染状况,为人无形体病的防控提供科学依据。方法 在云南省西北部地区15个县市采集部分家畜血液样本进行DNA提取,PCR扩增无形体16S rRNA基因片段;测定扩得基因片段DNA序列并对其做同源性比对,以确定无形体种类。结果 共采集1 791头牛、羊、犬、马、驴的血样, PCR扩增阳性503份,其中牛、羊和犬的阳性率分别为41.08%(228/555)、38.10%(240/630)、6.29%(35/556)。测定PCR扩增16S rRNA 基因片段序列,序列同源性分析显示73株序列(14.51%)属嗜吞噬细胞无形体(Anaplasma phagocytophilum), 226株序列(44.93%)属绵羊无形体(A.ovis), 137株序列(27.24%)属边缘无形体(A.marginale), 48株序列(9.54%)属血小板无形体(A.platys), 16株序列(3.18%)属牛无形体(A.bovis), 3株序列(0.60%)属山羊无形体(A.capra)。其中牛样本检出6种无形体,以边缘无形体为主;羊样本检出5种无形体,以绵羊无形体为主,未检测到山羊无形体;犬样本仅检出嗜吞噬细胞无形体。在15个县市中有12个县市的家畜样本检出无形体基因,盈江县阳性率最高(95.65%),主要是边缘无形体。对人致病的嗜吞噬细胞无形体基因分别从牛(30份)、羊(8份)、犬(35份)样本检出,对人致病的山羊无形体从3份牛样本检出,这2种人兽共患病原体感染的家畜主要分布于滇西北的德钦和香格里拉地区。结论 云南省西北部地区部分县(市)无形体感染的家畜主要是牛、羊,其次是犬,无形体种类丰富,需加强当地人无形体病的防控。     

北京东北部山区人群嗜吞噬细胞无形体血清流行病学调查

目的 了解北京东北部山区人群嗜吞噬细胞无形体的感染状况,为制定相应的防控策略提供依据。方法 在北京密云与怀柔区采集人群血清,采用间接免疫荧光法检测嗜吞噬细胞无形体IgG抗体,进行血清流行病学调查。结果 801份血清中嗜吞噬细胞无形体IgG抗体阳性者106份,阳性率13.23%,其中密云区为13.48%,怀柔区为12.70%,差异无统计学意义(P>0.05)。结论 北京密云与怀柔区正常人群中均有嗜吞噬细胞无形体感染的存在,应加强人粒细胞无形体病的监测和防控工作。     

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16S rRNA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16S rRNA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。     

云南省梁河县鼠疫疫源地野外鼠形动物嗜吞噬细胞无形体感染调查

目的 了解云南省梁河县鼠疫疫源地野外鼠形动物嗜吞噬细胞无形体自然感染情况。方法 应用单纯随机抽样法,从665份野外鼠形动物肝脾标本中抽取407份,采用巢式PCR对样本进行嗜吞噬细胞无形体16SrRNA基因检测,并对扩增产物测序,应用生物软件MEGA7.0将所测序列与GenBank注册的其它无形体属种株进行序列对比分析。结果 检测野外鼠形动物3目5科12属17种407只,阳性率为3.19%(13/407),其中大足鼠、黄胸鼠和斯氏家鼠阳性率分别为21.43%(3/14)、6.00%(6/100)和4.94%(4/81),其它鼠形动物均未检测出阳性;不同海拔梯度(P=0.219)、不同性别(χ²=0.441,P=0.506)及不同生境间(χ²=0.386,P=0.534)鼠形动物嗜吞噬细胞无形体感染率差异无统计学意义;不同季节野外鼠形动物嗜吞噬细胞无形体感染率差异具有统计学意义(P=0.044);序列对比分析显示所有样本基因序列与GenBank中收录的国内外部分嗜吞噬细胞无形体16SrRNA基因序列同源性达100%,进化分析显示与国内外部分嗜吞噬细胞无形体处同一个分支。结论 云南省梁河县鼠疫疫源地野外鼠形动物存在嗜吞噬细胞无形体低度感染。     

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16S rRNA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.     

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16S rRNA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16S rRNA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。     

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16SrRNA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16SrRNA基因（1442bp）与GenBank中收录的部分嗜吞噬细胞无形体16srRNA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。     

内蒙古部分地区草原革蜱携带无形体检测与进化分析

目的 探究内蒙古地区草原革蜱无形体感染情况,鉴定蜱传无形体种属并进行系统进化分析。方法 基于无形体16S rRNA基因、groEL基因和MSP4基因合成特异引物,对内蒙古地区采集的蜱虫标本进行PCR扩增,将阳性样本进行测序和分析并构建系统发育进化树。结果 将采集的905只蜱虫经形态学鉴定后将168只饱血蜱与737只未饱血蜱分为220个蜱虫混合样本,经检测有41个无形体阳性样本均来自于单只饱血蜱样本,总阳性检出率为4.53%(41/905),其中呼伦贝尔地区阳性率为1.03%(3/292);赤峰地区阳性率为3.13%(17/543);鄂尔多斯地区阳性率为30%(21/70),Blast比对分析结果显示与绵羊无形体同源性高达99%以上。系统发育进化树和同源性比对分析结果显示内蒙古地区16S rRNA基因的序列与新疆绵羊无形体xjmns03(JN400674)同源性最高,为99.49%;groEL基因序列与苏丹青尼罗州绵羊无形体G-BN-40(MG383903)同源性最高,为98.77%;MSP4基因序列与与陕西绵羊无形体D/SX786(MN307493)同源性最高,为100.00%。结论 内蒙古地区存在蜱传绵羊无形体,鄂尔多斯地区阳性率最高,为避免蜱传疾病对动物及人类健康造成危害,应重点对鄂尔多斯地区进行科学防控。     

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。     

A newly discovered Anaplasma phagocytophilum variant in rodents from southeastern China

Anaplasma phagocytophilum was detected by polymerase chain reaction in 13 (14.1%) of 92 rodents captured from a mountainous area of Zhejiang Province in southeastern China. The nucleotide sequences of 1442-bp, nearly entire 16S rRNA gene amplified from these rodents, had 100% identity, but varied from all known corresponding sequences of A. phagocytophilum deposited in GenBank. To further identify and classify the variant, fragments of 357-bp partial citrate synthase gene (gltA), 849-bp major surface protein 4 gene (msp4), and 443-bp groESL heat-shock operon gene, were amplified and analyzed. The nucleotide sequences of the partial gltA gene amplified from the rodents were identical to each other, but distinct from previously reported A. phagocytophilum sequences,as were msp4 and groESL. These findings indicate that the newly discovered agent represents a novel A. phagocytophilum variant.     

Anaplasma phagocytophilum infection in domestic animals in ten provinces/cities of China

A nationwide epidemiologic investigation of domestic animal infections has been conducted in nine provinces and one city during 2007-2010. Serum samples from a total of 707 goats, 433 cattle, and 219 dogs were collected for detecting Anaplasma phagocytophilum IgG antibody by immunofluorescence assays and the average seroprevalences were 10.05% for dogs, 3.82% for goats, and 0.69% for cattle, respectively. A total of 472 goats, 201 cattle, 102 dog blood clots, and 1,580 ticks were collected for polymerase chain reaction (PCR) amplifying A. phagocytophilum 16S rRNA genes and the PCR-positive rates were 26.69% for goats, 23.38% for cattle, and 10.89% for dogs. Six species were identified and the average PCR-positive rates were 58.3% for Dermacentor silvarum, 43.9% for Haemaphysalis longicornis, 12.5% for Ixodes persulcatus, 7.5% (3 of 40) for Boophilus microplus, and 5.2% for Rhipicephalus sanguineus, respectively. The evidence in the study indicated the zoonotic Rickettsia is highly prevalent in China.     

Prevalence of Anaplasma sp. in goats and sheep in Cyprus

A seroprevalence study of Anaplasma infection was conducted in a stratified random sample of goats and sheep in Cyprus in which the sample locations were recorded using a geographical information system (GIS). The aim of the study was to estimate the prevalence of Anaplasma phagocytophilum and other Anaplasma species in sheep and goats, and to identify high-risk regions. A total of 689 serum samples (343 from sheep and 346 from goats) were randomly collected and tested for the detection of antibodies against A. phagocytophilum antigen using an indirect immunofluorescent assay. The polymerase chain reaction followed by sequencing analysis was used for the detection and molecular characterization of Anaplasma sp DNA in the blood samples. The prevalence of IgG antibodies against A. phagocytophilum antigen was 18% for goats, and 31% for sheep. Six new genotypes were detected in goats and sheep; by sequence analysis one was identified as A. phagocytophilum, one as Anaplasma platys and the remaining four as Anaplasma species. The results provide evidence for the presence of A. phagocytophilum and Anaplasma species in sheep and goats in Cyprus.     

Potential vertebrate reservoir hosts and invertebrate vectors of Anaplasma marginale and A. phagocytophilum in central Spain

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, A. marginale, causes bovine anaplasmosis and only infects ticks and ruminants. A. phagocytophilum causes human and animal granulocytic anaplasmosis, and genetically closely related strains show a wide host range, including ticks, ruminants, rodents, equids, canids, birds, and humans. Recent reports demonstrated that A. marginale and A. phagocytophilum co-exist in geographic areas and that concurrent infections occur in ruminants and ticks. In this study, we characterized A. marginale and A. phagocytophilum infections in wild and domestic animals, and ticks collected in central Spain by serology, PCR, and sequence of 16S rRNA genotypes. Species tested included humans, cattle, dogs, rodents, Iberian red deer, European wild boar, birds, and ticks. Species of hematophagous Diptera were analyzed as potential mechanical vectors of Anaplasma spp. A. marginale was detected in tabanids, ticks, cattle, and deer, while A. phagocytophilum was detected in ticks, deer, cattle, and birds. Concurrent infections of the two Anaplasma were found in cattle and deer. These results illustrate the complexity of the epizootiology of A. marginale and A. phagocytophilum in regions where both pathogens co-exist and share common reservoir hosts and vectors. The increasing contact between wildlife, domestic animals, and human populations increases the risk of outbreaks of human and bovine anaplasmosis, and the difficulty of implementing surveillance and control measures.     

Q fever in humans and animals in the United States

Coxiella burnetii, the etiologic agent of Q fever, is a worldwide zoonotic pathogen. Although Q fever is present in the United States, little is known about its current incidence or geographic distribution in either humans or animals. Published reports of national disease surveillance, individual cases, outbreak investigations, and serologic surveys were reviewed to better characterize Q fever epidemiology in the United States. In national disease surveillance reports for 1948-1986, 1,396 human cases were reported from almost every state. Among published individual case reports and outbreak investigations, occupational exposures (research facilities, farm environments, slaughterhouses) were commonly reported, and sheep were most frequently implicated as a possible source of infection. In studies conducted on specific groups, livestock handlers had a significantly higher prevalence of antibodies to C. burnetii than did persons with no known risk. Animal studies showed wide variation in seroprevalence, with goats having a significantly higher average seroprevalence (41.6%) than sheep (16.5%) or cattle (3.4%). Evidence of antibody to C. burnetii was reported among various wild-animal species, including coyotes, foxes, rodents, skunks, raccoons, rabbits, deer, and birds. This literature review suggests that C. burnetii is enzootic among ruminants and wild animals throughout much of the United States and that there is widespread human exposure to this pathogen. Sheep and goats appear to be a more important risk for human infection in the United States than cattle or wild animals, and research studies examining the natural history and transmission risk of Q fever in sheep and goats in this country should be encouraged.     

Genetic and ecologic characteristics of Bartonella communities in rodents in southern China

Ecologic and bacteriologic observations of small mammals captured in Yunnan Province in the People's Republic of China indicated that Bartonella infections occurred at a high prevalence among some rodent species. Sequence analyses of the citrate synthase genes of these Bartonella demonstrated that rodents in this region harbored a diverse assemblage of strains. The Bartonella isolates obtained from Apodemus, Eothenomys, and Rattus typically clustered separately by genus of rodent host. Cultures obtained from Rattus rats were genetically related to Bartonella elizabethae, a recognized human pathogen. The finding of Bartonella species in a high proportion of the rodent samples from Yunnan suggests the need to investigate whether these agents might be responsible for cases of febrile illnesses of unknown etiology in southern China and elsewhere in southeastern Asia.     

Identification of Ehrlichia chaffeensis,Anaplasma phagocytophilum,and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea

A total of 1,467 tick (1,463 of Haemaphysalis longicornis, three of Ixodes persulcatus and one of I. turdus) collected from nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia and Anaplasma species. One set of primers and a probe were designed for detection of all of the Ehrlichia and Anaplasma species. Template DNAs (total 803) were prepared either from pools of larvae, nymphs, adult males and females, or from the salivary gland and midgut of adult ticks. Only DNAs positive in TaqMan PCR were examined for A. phagocytophilum with nested PCR and for E. chaffeensis with PCR. Four A. phagocytophilum 16S rRNA gene PCR products were sequenced for comparison with sequences previously reported. Amplification of a 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (45.3% of the total). Of these 364 positive ticks, species-specific PCRs confirmed that 35 H. longicornis and one I. persulcatus were positive for A. phagocytophilum and one I. persulcatus was positive in E. chaffeensis. Except for one (AB-GGHL, GenBank accession number [GAN] AF470698), three of the four 16S rRNA gene fragment sequences of the A. phagocytophilum-positive samples were similar or identical to the sequences of variants of A. phagocytophilum deposited in GenBank. The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma (Ehrlichia) bovis 16S rRNA (GAN U03775). The identities of the Anaplasmataceae genus and species DNA in the 327 ticks that could not be confirmed infected with either E. chaffeensis, A. phagocytophilum, or A. bovis are not known. This study is the first to demonstrate the presence of E. chaffeensis, A. phagocytophilum and A. bovis in Korean ticks.     

New genetic variants of Anaplasma phagocytophilum and Anaplasma bovis from Korean water deer (Hydropotes inermis argyropus)

Wild deer are one of the important natural reservoir hosts of Anaplasma species, which cause granulocytic anaplasmosis in equines, canines, and humans. The objective of the present study was to determine whether and what species of Anaplasma naturally infect Korean water deer (KWD) in the Republic of Korea. A total of 66 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife in Korea between March 2008 and May 2009. Polymerase chain reaction (PCR) was performed using 16S ribosomal (r)RNA, with ankA, groEL, and msp2 gene primers to amplify the genes of Anaplasma and Ehrlichia. Using 16S rRNA-based nested PCR, Anaplasma phagocytophilum and Anaplasma bovis were detected in 42 (63.6%) and 23 (34.8%) of 66 KWD spleens, respectively. The 42 A. phagocytophilum were classified into five genotypes and the 23 A. bovis were classified into two genotypes by sequence analysis. By ankA-, groEL-, and msp2-based nested PCR, A. phagocytophilum was detected in 1 (1.5%), 7 (10.6%), and 3 (4.6%) of 66 samples, respectively. These gene sequences had only one genotype. Five of seven obtained 16S rRNA gene sequences have never been identified. The ankA, groEL, and msp2 obtained gene sequences represented new genotypes. This is the first report of A. phagocytophilum and A. bovis in KWD, suggesting that they may act as reservoirs for anaplasmosis zoonotic pathogens.