新疆昭苏地区马梨形虫病病原及抗体检测

马梨形虫病是由马驽巴贝斯虫和马泰勒虫寄生于马属动物的红细胞内所引起的一类血液原虫病,呈全球性分布,尤其在新疆发病率更高,处于逐年上升趋势,对区域性马产业的发展影响极大。为了解2018年新疆昭苏养马区域马梨形虫的感染情况,随机采集昭苏县18个乡镇的马全血及血清各858份,采用PCR和间接ELISA分别进行检测,对两种方法检测的18个地区、不同年龄阶段的马驽巴贝斯虫、马泰勒虫及混合感染情况进行统计学分析。结果显示,PCR检测马驽巴贝斯虫、马泰勒虫及混合感染的阳性率分别为12.12%、13.87%和2.80%;间接ELISA检测马驽巴贝斯虫、马泰勒虫及混合感染的抗体阳性率分别为15.50%、10.14%和2.56%;不同年龄阶段筛查结果显示,在6岁以下的马匹感染马驽巴贝斯虫、马泰勒虫及混合感染的阳性率较高,并且不同地区的不同年龄阶段马匹的马梨形虫感染率存在不同程度的差异。此次获得的昭苏县马梨形虫感染情况的一线数据,可为当地养马区域马梨形虫病的综合防控提供技术支撑。     

阿勒泰富蕴县放牧马驽巴贝斯虫和马泰勒虫抗体的检测初报

马梨形虫病按病程可分为急性、亚急性及慢性,尤其是慢性病例长期携带病原-带虫状态。为了解阿勒泰富蕴县放牧马携带马驽巴贝斯虫和马泰勒虫状况,采用血清学-竞争ELISA方法 (以血液涂片为辅),对放牧马(n=233)进行了马泰勒虫和驽巴贝斯虫2种虫血液循环抗体的检测试验。结果表明:富蕴县放牧马泰勒虫抗体阳性率为13.30%,驽巴贝斯虫抗体阳性率为17.58%,两者混合感染的抗体阳性率为2.15%,说明该县放牧马感染不同程度的马梨形虫病,且出现同一个马场和在同一匹马同时存在2种虫体抗体的情况;在试验抽样病例中几乎未发现有马梨形虫病临床症状,均呈隐性感染状态。因此带虫的放牧马随时有被蜱虫叮咬散播病原的危险,要加强防范。     

云南部分地区马、驴、骡梨形虫感染情况调查

为了解云南地区马驴骡感染马泰勒虫和驽巴贝斯虫情况,采用双抗原夹心ELISA方法对云南8个地区马、驴、骡三种动物（n=969）进行了马泰勒虫和驽巴贝斯虫抗体阳性率的检测。结果显示：梨形虫总抗体阳性率为5.47%,其中马泰勒虫抗体阳性率为4.75%,驽巴贝斯虫抗体阳性率为4.85%,两者混合感染的抗体阳性率为4.13%;通过调查分析发现云南马、驴、骡存在梨形虫感染,且不同地区的不同动物的马梨形虫感染程度不同。本研究结果为马驴骡梨形虫病的流行防控提供了数据参考。     

18S rRNA序列差异在马梨形虫分类学中的应用

为揭示18S rRNA V4高变区在物种分类学的本质意义,及进一步阐述马泰勒虫(之前称马巴贝斯虫)的分类学地位,本试验参考马泰勒虫(DQ287951)和驽巴贝斯虫(FJ209026) 18S rRNA基因序列,在其V4高变区设计引物.将获得的片段克隆至pMD19-T进行测序,正确的结果与其他种梨形虫的相应序列进行分析.系统发生树结果显示,泰勒虫和巴贝斯虫处于明显的两个分支,而称之为马巴贝斯虫的物种和泰勒虫有着较为密切的关系,他们处于同一分支,其亲缘关系较巴贝斯虫更远.序列对齐分析表明,巴贝斯虫核苷酸序列相对于泰勒虫种序列存在多个位点的缺失和突变.而马巴贝斯虫和泰勒虫18S rRNA V4高变区核苷酸序列的相似程度较高,与巴贝斯虫在该序列上的差异较大.以上结果表明,泰勒虫种和巴贝斯虫种18S rRNA V4高变区核苷酸序列间的这种缺失和突变可作为泰勒虫和巴贝斯虫分类依据的本质因素之一.马巴贝斯虫应隶属于泰勒虫科,泰勒虫属.     

马泰勒虫病致死孕马报道

<正>马泰勒虫(Theileia equi,旧称马巴贝斯虫)和马驽巴贝斯虫(Babesia caballi)是寄生于马属动物红细胞内,是马梨形虫病的主要病原。据报道马泰勒虫病比驽巴贝斯虫病有更强的致病性和传播能力[1]。马泰勒虫(Theileria equi)隶属于泰勒科(Theileriidae Levine)泰勒属(Theileria),是寄生于马属动物红细胞体内,临床以稽留热、贫血、黄疸等为     

马梨形虫病双重PCR检测方法的建立

为了建立马梨形虫病双重PCR检测方法,试验根据Gen Bank发表的驽巴贝斯虫和马泰勒虫18S rRNA保守基因序列分别合成2对特异性引物,以核酸混合物为模板,优化反应条件建立马梨形虫病双重PCR检测方法,并检验该方法的特异性和敏感性。同时用该双重PCR检测方法对采自昭苏种马场的马疑似病例全血进行检测,并与镜检法和单重PCR法进行比较。结果表明:该双重PCR检测方法能对驽巴贝斯虫和马泰勒虫核酸扩增出大小为529 bp和789 bp特异性目的片段,而对双芽巴贝斯虫、环形泰勒虫、羊泰勒虫、牛巴贝斯虫核酸的扩增均为阴性;驽巴贝斯虫和马泰勒虫阳性DNA被稀释1×108倍时均能检出其相应目的片段;对采集的46份马疑似病例血样进行PCR诊断,其中驽巴贝斯虫感染率为30.4%(14/46),马泰勒虫感染率为41.3%(19/46);双重PCR检测方法与单重PCR方法的符合率为100%。说明建立的双重PCR检测方法具有一定的特异性和敏感性,可用于驽巴贝斯虫和马泰勒虫临床感染隐性病例的联合检测与鉴别诊断。     

马驽巴贝斯虫Bc48蛋白的纯化及其应用

为了解新疆南北疆部分疫区马匹携带驽巴贝斯虫(Babesia caballi)抗体情况,运用实验室已构建保存的地方虫株重组Bc48为靶抗原,将重组原核表达质粒pGEX4T-1-Bc48转化至BL21表达系统中,诱导表达出36 KDa的重组蛋白。经KCl法切胶纯化重组蛋白后作为rELISA包被抗原,对阿克苏、伊犁、和硕等7个区域的随机采集样品(n=256)进行马驽巴贝斯虫抗体检测。结果显示,Bc48重组抗原能够精确识别马驽巴贝斯虫标准阳性和阴性血清;所检测的临床样品中,驽巴贝斯虫抗体阳性率为19.5%,其中,南疆2个地区(和硕、阿克苏)马驽巴贝斯虫病阳性率分别为30%、9%;北疆5个地区(昭苏种马场、伊犁卡拉苏、洪纳海乡、阿尕什敖包乡和富蕴县区域)马驽巴贝斯虫抗体阳性率分别为17%、12.5%、17%、5%和35%。本次调查结果为新疆驽巴贝斯虫防控措施及疫苗和驱虫药的使用提供必要的理论依据。     

新疆部分焉耆马场马梨形虫病病原学检测及序列分析

为了解新疆地区焉耆马场马梨形虫病病原感染情况，促进当地马梨形虫病的综合防控和马产业的健康发展，选取5个焉耆马场作为试验点，随机采集156匹焉耆马全血进行马梨形虫病病原DNA检测，并使用SPSS Statistics 17.0、Megalign与MEGA 11.0软件分析不同采样点、不同年龄、不同性别马匹的感染情况差异，同时进行病原核酸的同源性比较和系统进化树构建。结果显示：156匹焉耆马全血样品中，检出马梨形虫病病原核酸阳性82份，其中马泰勒虫、马驽巴贝斯虫和两者混合感染阳性率分别为33.3%(52/156)、26.9%(42/156)和7.7%(12/156)。不同采样点、不同年龄、不同性别焉耆马的马梨形虫病病原阳性率均无显著差异（P> 0.05）。通过Megalign与MEGA11.0软件分析发现：测序得到的马泰勒虫18S rRNA基因序列与选取的参考虫株同源性为95.5%～100%，马驽巴贝斯虫BC-18S rRNA基因序列与选取的参考虫株同源性为98.6%～99.6%。马泰勒虫18S rRNA基因与马泰勒虫伊犁株（OL589505.1）位于同一分支上，进化关系较近，与其他...     

马泰勒虫病PCR检测方法的建立和应用

为寻求一种快速、有效的马泰勒虫 (Theileria equi,T.equi) PCR检测方法。基于马泰勒虫18S rRNA基因序列,在其V4高变区设计特异性引物Te-18F、Te-18R,通过PCR技术获得了531 bp的核酸片段。用该引物对马泰勒虫、马泰勒虫和驽巴贝斯虫混合样本、驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫基因组模板进行特异性试验。同时对马泰勒虫基因组模板进行不同浓度稀释后扩增,以便于确定试验的敏感性。用本试验建立的方法与常规显微镜镜检方法对45份马属动物血样进行检测。特异性试验显示,在被检测的9个样本中,只有马泰勒虫及马泰勒虫和驽巴贝斯虫混合模板中扩增出了符合大小的特异核酸片段。驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫的扩增结果均为阴性。灵敏度试验结果表明,PCR对马泰勒虫的扩增效率可达到10^-13。对本试验建立的PCR检测马泰勒虫方法评估结果显示,PCR对马泰勒虫的检出率为17.78%(8/45),显微镜镜检结果只有8.89%(4/45)两者的符合率为100%。本试验建立的马泰勒虫PCR检测方法不失,为一种好的检测方法。     

甘肃省河西区域牛羊梨形虫病病原及媒介蜱分子流行病学调查

巴贝斯虫（Babesia spp.）和泰勒虫（Theileria spp.）是世界范围内流行的蜱传播梨形虫病病原体。为评价梨形虫病传播情况,为甘肃省河西区域梨形虫病防治提供流行病学资料,采集该区域部分县区的牛羊抗凝血样品和环境游离蜱虫进行梨形虫病病原检测,分析样品中病原体的存在和分布情况,利用MEGA 6.06软件和NCBI GenBank数据库的BLASTn工具,对阳性样品中的巴贝斯虫和泰勒虫18S rRNA基因进行序列分析和遗传进化树构建。通过基于18S rRNA的巢氏PCR方法,检出梨形虫病病原1目2科9种,包括莫氏巴贝斯虫、双芽巴贝斯虫和隐藏巴贝斯虫3种巴贝斯虫,东方泰勒虫、中华泰勒虫、分离泰勒虫、吕氏泰勒虫、狍泰勒虫和环形泰勒虫6种泰勒虫。牛羊抗凝血样品梨形虫病病原总感染率为8.84%（16/181）,阳性样品分布于武威市（感染率14.94%）和张掖市（感染率3.45%）;检出携带梨形虫病病原的蜱9只,蜱病原携带率为2.52%（9/357）。检出的梨形虫病病原分属泰勒虫属和巴贝斯虫属2大类,每种病原跟国内外检出虫株的同源性均较高,处于各虫株相应分支上,提交序列相似率达99%～...     

Prokaryotic Expression of the Cysteine Proteinase Gene of Xinjiang Strain of Theileria equi/Babesia caballi and Its Bioinformatics Analysis

This study aimed to clone and express cysteine proteinase (CP) gene of Theileria equi and Babesia caballi. The CP proteins were analyzed using bioinformatics tools and online databases. The total DNA of T. equi and B. caballi as the template sequence for PCR. The CP genes were cloned, expressed using the cloning technique and prokaryotic expression system, respectively. Then the recombinant CP (rCP) proteins were purified by Urea dialysis. Finally the specific reaction of polyclonal horse anti-CP serum with rCPs was analyzed through Dot blot method. The CPs were predicted and analyzed by the software MAGA, Prot Param, TMHMM, SignalP-5.0, Antibody Epitope Prediction, SYFPEITHⅡ, ProPred and EzMol^2.1 respectively. The CP genes were successfully amplified from DNA of T. equi and B. caballi and expressed in the inclusion body fractions, Dot blot assay indicated that the recombinant protein could react with polyclonal horse anti - CP serum. Compared with T. equi-CP and B. caballi-CP, different protozoon CP genes were highly conserved in evolution, and phylogenetic analysis results were consistent with their protozoon taxonomy. The bioinformatics analysis revealed that two CPs are the hydrophilic protein instead of a transmembrane one, no transmembrane domain, no Th cell epitope was found, and more localized in the cytoplasm, mitochondria and nuclei. The relative molecular weight (MW) of T. equi-CP was 29 948.60, the theoretical isoelectric point (pI) was 5.53, stability coefficient was 22.50; B. caballi-CP’MW was 14 603.62, the theoretical pI was 8.54, stability coefficient was 47.69, but signal peptide was contained. The CPs have good reactionogenicity, as the hydrophilic extracellular proteins with no Th cell epitope, and more localized in the cytoplasm, mitochondria and nuclei, which helped T. equi and B. caballi avoid host immune response outside the cell membrane and the CPs involved in proliferation, differentiation and programmed cell death. In conclusion, a theoretical basis was provided for the study on CP’s functions and the pathogenesis of T. equi and B. caballi.     

Investigation of Molecular Etiology of Theileria equi Infection in Three Herds

To explore the prevalence of Theileria equi (T.equi) infection in horse in Guizhou province, the antibody level and 18S rRNA gene were detected from blood samples of Guizhou pony, Southwest horse and Yili horse using competitive ELISA and PCR methods.Giemsa-stained blood smear was prepared to observe T.equi in red blood cells.Intact protozoans of T.equi were observed in red blood cells of horses at Giemsa-stained slide smears with a detection rate of 12.5%.The 18S rRNA gene fragment of T.equi was detected in Guizhou pony, Southwest horse and Yili horse, and the consistent rates with the known nucleotide sequence were 97% to 100%.The PCR result indicated that the positive rates of T.equi in Guizhou pony (76.62%) and Yili horse (73.81%) were similar, which were higher than that in Southwest horse (33.33%).Furthermore, the antibody levels against T.equi in Guizhou pony (24.68%) and Southwest horse (12.12%) were lower than that in Yili horse (31.71%).A weak correlation between the antibody level and the blood physicochemical indexes was calculated from Guizhou pony and Southwest horse, including weak positive correlations with neutrophils numbers, gamma-glutamyl transferase and creatine kinase levels, and weak negative correlations with the numbers of red blood cell, white blood cell, platelet and lymphocyte and contents of hemoglobin.It suggested that a higher proportion of T.equi infection present in three herds.     

3个马群感染马泰勒虫的分子病原学调查

试验采用显微镜观察、PCR和竞争性酶联免疫吸附试验(competitive enzyme-linked immunosorbentassay,cELISA)等方法对贵州矮马、西南马和伊犁马的马泰勒虫病的感染状况进行研究。结果显示,从32份新鲜的血液涂片中,观察到形态完整的马泰勒虫(Theileria equi)虫体,检出率12.5%。从贵州矮马、西南马及伊犁马3个马群血液总DNA中都检测到马泰勒虫的18S rRNA基因片段,与已知序列的同源性为97%~100%;相比之下,贵州矮马与伊犁马的阳性率相近,分别为76.62%和73.81%,西南马较低,仅为33.33%。另外,经cELISA检测,与伊犁马(31.71%)相比,贵州矮马和西南马血液中抗马泰勒虫抗体的阳性率较低,分别为24.68%和12.12%,并与两个马群的血液理化指标存在一定的联系:与中性细胞数量、γ-谷氨酰转移酶和肌酸激酶的含量呈弱正相关;与红细胞、白细胞、血小板、淋巴细胞数量及血红蛋白含量呈弱负相关。这些研究结果提示3个马群中均存在较高比例的马泰勒虫感染。     

A mixed infection of babesia equi and babesia caballiin a racing colt: A report from iran

Babesiosis was diagnosed in a three-year-old racingThoroughbred colt with recurrent, intermittent fever, poor performance, malaise, and one occasion of diarrhea. Other signs included congestion of mucous membranes and petechial hemorrhages of conjunctival mucous membranes. Babesia caballi and B. equi were identified on blood smear, when the colt had fever. The colt was treated with Imidocarb (4 mg/kg four times at 72 intervals) and returned to regular exercise and competition four months after treatment. The colt and two other horses which showed babesial infection had been transported from Turkmen district to Mashhad city.     

Antibody levels to Brucella abortus, toxoplasma gondii, and Leptospira serogroups, in sera collected from healthy people in Fiji

A collection of 300 sera from a predominantly rural community on the island of Viti Levu in Fiji were studied for the presence of antibodies to B. abortus, T. gondii and Leptospira serogroups. Significant levels of immunity were found to B. abortus and T. gondii and over half the population had diagnostic leptospiral antibody levels.     

Cloning and Sequence Analysis of Toxoplasma gondii GRA25 Gene

In this study,sequence variation of GRA25 genes among 22 Toxoplasma gondii (T.gondii) strains from different hosts and geographical locations were examined.The complete GRA25 genes from 22 T.gondii isolates were amplified,sequenced,and nucleotide variations were determined.Phylogenetic analysis among the different T.gondii isolates were conducted using maximum parsimony (MP) and maximum likelihood (ML) methods.The biological characteristics of the protein GRA25 of the T.gondii RH strain were predicted using bioinformatics software.The sequences of all the examined T.gondii strains were 939 or 948 bp in length.Sequence comparison of all 22 GRA25 sequences identified 82 variable nucleotide positions (0 to 4.4%).The results of phylogenetic analysis showed that strains belonging to the classical typeⅠand Ⅱ could not group into their own branches based on the GRA25 sequences.Bioinformatics analysis revealed that the protein GRA25 contained 7 hydrophobicity regions,10 alpha regions,3 beta sheets,8 random coils and 8 linear B-cell epitopes.These results suggested that the GRA25 gene was not an ideal genetic marker for population genetic study of T.gondii strains,but it might represent a good vaccine candidate against toxoplasmosis.     

刚地弓形虫GRA25基因的克隆及序列分析

本研究旨在对来源于不同宿主和地理分布的22株弓形虫的GRA25基因进行PCR扩增并测序,对获得的GRA25基因序列进行比对,利用MP和ML两种方法对不同分离株的GRA25基因构建系统进化树。利用生物信息学软件将弓形虫RH株的GRA25基因翻译成氨基酸序列,对其编码蛋白的生物学特征进行分析。结果显示,弓形虫GRA25基因序列有2种长度,分别为939和948 bp。序列比对结果显示,GRA25基因在不同虫株间有82个核苷酸变异位点,变异率为0~4.4%。进化分析结果显示,用GRA25基因不能区分弓形虫基因Ⅰ型和Ⅱ型虫株。生物信息学分析预测弓形虫GRA25蛋白含有7个亲水区域,10个α-螺旋,3个β-折叠,8个无规则卷曲和8个潜在的线性B淋巴细胞抗原表位。本研究结果表明,GRA25基因不能作为标记分子区分不同基因型的弓形虫虫株,但可能作为疫苗候选分子研制新型抗弓形虫基因疫苗或表位肽疫苗。     

Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

白羊草NAC转录因子家族生物信息学分析

NAC转录因子家族是植物特有的一类重要转录因子,广泛参与植物生长、发育以及器官的建成、逆境胁迫应答等过程。为进一步研究白羊草（Bothriochloa ischaemum） NAC转录因子家族,本研究利用生物信息学方法对白羊草NAC基因所编码的蛋白理化性质、二级结构、保守基序、亚细胞定位、潜在磷酸化位点及系统进化关系进行了分析。结果显示,具有NAC结构域的21条白羊草氨基酸序列共分为10个亚家族,蛋白二级结构以无规则卷曲为主要组成部分,含有5个保守基序,大多数NAC蛋白定位于细胞核,均含有潜在的丝氨酸（Ser）、苏氨酸（Thr）、和酪氨酸（Tyr）磷酸化位点。本研究将为白羊草NAC基因家族的进一步功能分析奠定重要的研究基础。     

臂形草14品种在滇南的适应性及其评价

2006-2007年通过对14个臂形草品种的生产性能和品质特性的研究,旨在筛选出适宜于热带、亚热带地区种植的高产、优质牧草品种,为热带水土保持和畜牧业的持续发展提供依据,结果表明,1)所引种牧草的存活率、越冬率都在97%以上,说明所引种牧草的适应性强;除MG-4珊状臂形草感染了叶锈病以外,其他牧草的抗病虫害能力强。2)种植第1年Mulato 1杂交臂形草干物质产量最高为(34.84±3.98) t/hm² ,与刚果臂形草、Mekong珊状臂形草、杂交臂形草、MG-5×Araes珊状臂形草、MG-5 Vitoria珊状臂形草、MG-4珊状臂形草、Mulato 2杂交臂形草之间差异不显著。3)相关性分析表明,丛径、叶长、叶宽、株高、叶/茎、种子产量与地上生物量呈正相关,对地上生物量的影响大小为:叶宽＞种子产量＞叶/茎＞株高＞叶长＞丛径。4)选择干草产量、粗蛋白、适口性、抗逆性作为综合评价供试品种优劣的指标,用灰色关联法评价牧草,结果表明,杂交旗草、Mulato 1杂交臂形草、Mekong珊状臂形草、Mulato 2杂交臂形草、MG-5×Araes珊状臂形草、刚果臂形草、MG-5 Vitoria珊状臂形草居前7位,它们属高产、质优、适口性好、抗性强的高产优质牧草品种。