Cloning and Bioinformatics Analysis of P23 Gene from Theileria sergenti

[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.

Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti

[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.

牛中华泰勒虫(Theileria sinensis sp.nov.)新种 1.传统分类学研究

对分离自我国甘肃中部地区土种黄牛体的泰勒虫未定种作了鉴定。与环形泰勒虫 (T .annulata)、瑟氏泰勒虫 (T .sergenti)、小泰勒虫 (T .parva)、突变泰勒虫 (T .mutans)、斑羚泰勒虫 (T .tautotragi)、附膜泰勒虫 (T .velifera)、水牛泰勒虫 (T .buffeli)作了形态学上的比较研究 ,观察到该未定种除了泰勒虫所共有形态外 ,还有其它泰勒虫所没有的且难以描述的特异形态 ,尤其在染虫率高峰期 ,特异形态占虫体总数的 2 0 %左右。该种还具有出芽增殖的特性 ,可在除脾牛体内大量繁殖 ,染虫率可达 5 2 6 9%的高峰 ,试验感染牛出现高烧、极度贫血、精神沉郁、食欲不振等临床症状 ,导致个别牛只死亡 ,剖检观察到某些脏器有严重病变 ,表明具有一定的致病力。媒介蜱尚不清楚 ,但已证实 ,在我国传播家畜泰勒虫和巴贝斯虫的 7种媒介蜱对该种无传播能力。传统分类学研究结果表明 ,该种不同于已知牛泰勒虫有效种 ,系一新种。由于这一新种首次在中国分离到 ,因而被命名为中华泰勒虫 (Theile riasinensissp .nov .) (梨形虫亚目 :泰勒虫科 )。

在我国新发现的一种牛的泰勒虫（Theileria）未定种

对分离自我国西北某地自然感染牛体的一株泰勒虫（Ｔｈｅｉｌｅｒｉａ）未定种，进行了接种试验、形态学观察和部分传播试验。结果表明，该种可在除脾牛体内大量繁殖，寄生物血症达５２．６９％。虫体形态具多形性：有梨子形、圆环形、椭圆形、杆状、三叶形、边虫样、十字架形，还有许多难以形容的不规则形虫体；在同一红细胞内不同数目的边虫样虫体可发育变大，生成的原生质伸延，而后互相连接或交融，重新构成各种不同形态的虫体；有些虫体具有出芽生殖的特性。虫体形态以梨子形和圆环形为主，占所见虫体总数的５１．５％。不能通过长角血蜱和残缘璃眼蜱传播，其病原性、媒介蜱、传播方式、宿主特异性、发育史等尚不清楚。

First molecular detection of Theileria ovis in Rhipicephalus sanguineus tick in Iran

Objective:To determine tick infestation of domestic ruminants and their infection to ovine theileriosis in northern Iran.Methods:About 425 domestic ruminants in Ghaemshahr city in northern Iran were inspected for tick infestations.Twenty tick specimens(13 females and 7 males) of Rhipicephalus sanguineus(R.sanguineus).the most common lick in the study area, were tested by PCR amplification against 18s rRNA genome of Theileria spp using specie specific primers and then the PCR products were sequenced for species identification In comparison with data base available in GenBank.Results:About 323 ticks were collected from 102 animals(88 sheep,12 goats and 2 cattle).The prevalence of ticks infesting animals was R.sanguineus(82.35%), Rhipieeplialus bursa(R.bursa)(0.3%),Ixodes ricinus(I.ricinus)(15.2%),Boophilus annulatus (B.annulalus)(1.2%).Haemaphxsalis punctata(H.punctata)(0.3%) and Haemaphysalis numidiana(H.numidianu)(0.6%).Eleven(55%) tick specimens were PCR positive against genome of Theileria ovis(T.avis).Sequence analysis of the PCR products confirmed presence of T. oris in one R.sanguinus.Conclusions:This is the first report of tick infection to T.oris in Iran. Due to dominant prevalence of R.sanguineus as well as its infection to T.oris,it is postulated this tick is the main vector of ovine theileriosis in northern Iran.

体外培养牛白细胞中环形泰勒焦虫(Theileria annulata)的增殖、释放与感染

在体外培养的牛外周血白细胞中,环形泰勒焦虫裂殖子与裂殖体寄生于宿主细胞的细胞质中,并且随着宿主细胞的分裂而分到两个子细胞中。焦虫染色质粒的分裂方式为二分裂,随着焦虫颗粒的不断增殖,逐渐发育为成熟的裂殖体。体外培养感染焦虫的牛白细胞可通过伪足与细胞裂解两种途径向培养液中释放焦虫颗粒。释放到培养液中的焦虫颗粒对体外培养的健康牛外周血白细胞具有感染能力,感染细胞能在体外连续传代培养。

Experimental infectivity of Theileria luwenshuni and Theileria uilenbergi in Chinese Kunming mice

Theileria luwenshuni and Theileria uilenbergi are important tick-borne pathogens and cause substantial losses to the sheep industry in China. The improvement in detection techniques has allowed the identification of multi-homing parasitism in Theileria parasites. Herein we evaluated the experimental infectivity of T. luwenshuni and T. uilenbergi in Chinese Kunming mice by screening blood samples of experimentally inoculated mice by microscopic examination（ME） and PCR. T. luwenshuni infected Chinese Kunming mice and 20 mice inoculated with this parasite were positive by ME and PCR. In addition, T. uilenbergi infected mice and 20 mice inoculated with this species were positive by ME and PCR. However, the number of red blood cells and the levels of hemoglobin of 40 infected mice had no obvious changes in the course of infection. Our results demonstrated the multi-homing parasitism of T. luwenshuni and T. uilenbergi, which were believed to be parasites of sheep and goats. This study was the first to demonstrate the infection of T. luwenshuni and T. uilenbergi in Kunming mice.

Inhibitory effects of methanolic Olea europaea and acetonic Acacia laeta on growth of Babesia and Theileria

Objective:To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea(MOE)and acetonic extract of Acacia laeta(AAL)against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia(B.)microti in vivo.Methods:Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments,8-weekold female BALB/c mice were injected intraperitoneally with 1× 107 B.microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts.Results:The half maximal inhibitory concentration(IC50)values of AAL against B.bovis,B.bigemina,B.divergens,B.caballi,and Theileria equi were lower than those of MOE extracts.Toxicity assay on Madin-Darby bovine kidney,mouse embryonic fibroblast(NIH/3T3),and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin-Darby bovine kidney cell line with half maximal effective concentrations(EC50)of(794.7±41.9)and(873.9±17.5)μg/mL,respectively.The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B.microti in mice by 80.4% and 64.4%,respectively.The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy.Conclusions:MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis.Furthermore,the combination therapy of MOE + DA was more potent against B.microti infection in mice than their monotherapies.

Cocktail of Theileria equi antigens for detecting infection in equines

Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain,Theileria equi(T.equi)protein 82(Te 82)and T.equi 104 k Da microneme-rhoptry antigen precursor(Te 43),to diagnose T.equi infection in horses as compared with equi merozoite antigen-2(EMA-2).Methods:In the current study,we applied a cocktail-ELISA containing two antigens(EMA-2+Te 82)to diagnose T.equi infection either in experimentally infected horses or in field infection.Results:Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T.equi infection in horses as compared with Te 82 or Te 43 alone.Conclusions:The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

Milk losses due to bovine tropical theileriosis(Theileria annulata infection) in Algeria

The authors studied the impact of tropical theileriosis onset on milk yield decrease in 10 local bred cows in Skikda(Northern Algeria) during 2015 summer season.The milk yield decrease estimated weekly during two months was 2.76 L/day/cow corresponding to31.92% of the total milk yield.This decrease corresponds to 110.5 Algerian Dinars(1.02US$)/day/diseased cow.The relative variation of milk yield showed a dramatic decrease from 82.72% to 0.76% at Day 21 then became constant.Further studies are needed to improve these estimations of financial losses due to bovine tropical theileriosis in Algeria.

Perforin Expression in <i>Theileria parva</i>Specific Cytotoxic T Cells Correlates with Cytotoxicity

Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.

Pyronaridine combined with diminazene aceturate inhibits Babesia in vitro and in vivo

Objective:To evaluate the combination therapy of pyronaridine tetraphosphate and diminazene aceturate against Babesia in vitro and in vivo.Methods:Bioinformatic analysis was performed using atom pair fingerprints.An in vitro combination test was performed against Babesia bovis and Theileria equi.Moreover,the in vivo chemotherapeutic efficacy of pyronaridine tetraphosphate in combination with diminazene aceturate was investigated against the growth of Babesia microti in mice using a fluorescence inhibitory assay.Results:Pyronaridine tetraphosphate and diminazene aceturate exhibited nearly similar molecular weights.The in vitro combination of pyronaridine tetraphosphate and diminazene aceturate was synergistic on Babesia bovis and additive on Theileria equi.In addition,5 mg/kg pyronaridine tetraphosphate combined with 10 mg/kg diminazene aceturate inhibited Babesia microti growth significantly compared with those observed after treatment with 25 mg/kg diminazene aceturate alone from day 6 post treatment to day 12 post treatment.The combination therapy also normalized the hematological parameters of infected mice.Conclusions:An oral dose of pyronaridine tetraphosphate combined with a subcutaneous dose of diminazene aceturate inhibits Babesia in vitro and in mice,suggesting it might be a new paradigm for the treatment of babesiosis.

新疆和田地区于田县牛源环形泰勒虫种群基因型及遗传多样性分析

为探究新疆和田地区于田县牛源环形泰勒虫种群的基因型分布及遗传多样性,试验首先从该县10个乡镇(阿羌乡、喀尔克尔乡、科克亚乡、木尕拉镇、斯也克乡、先拜巴扎镇、英巴格乡、加依乡、奥依托格拉克乡、阿日希乡)采集疑似环形泰勒虫病患牛血液样本共443份,提取基因组DNA后对牛源环形泰勒虫Tams1基因进行PCR检测,统计10个乡镇牛源环形泰勒虫的阳性样本数及阳性率;然后在发现阳性样本的乡镇中,每个乡镇随机选取2个阳性样本进行测序,将测序结果进行同源性分析并与参考序列共同构建NJ系统发育树,分析基因型;最后对于田县环形泰勒虫种群进行遗传多样性分析。结果表明:在443份牛血液样本中,有93份样本环形泰勒虫检测结果为阳性,阳性率为20.99%;10个乡镇中有9个乡镇牛源环形泰勒虫检测结果为阳性,阳性率在10.45%~45.45%之间,其中阿羌乡阳性率最高,加依乡未检出。18株环形泰勒虫Tams1基因序列的核苷酸相似性为76.0%~100%,同乡镇的2个阳性样本之间的核苷酸相似性为91.0%~100%;这18株牛源环形泰勒虫在NJ系统发育树中未全部聚为一支,一些虫株甚至与其他国内外参考毒株表现出了更近的亲缘关系,但均为G1基因型。于田县G1基因型环形泰勒虫种群多态性位点数为119个,核苷酸多样性为0.053,平均核苷酸差异数为28.95个,单倍型数为12个,单倍型多样性为0.895,Tajima’s D值和Fu and Li’s F值均小于0。说明于田县G1基因型环形泰勒虫种群具有较高的遗传多样性且正在发生扩张。

河北省牛梨形虫流行病学调查及遗传进化分析

为了解我国河北省牛梨形虫的种类、感染率、遗传进化及季节流行性情况,本研究共收集河北省7个地级市的牛血液样品564份,以提取的牛全血液基因组DNA为模板,采用套式PCR对牛梨形虫18S r RNA基因进行扩增检测,并鉴定虫种,选取10份不同地区牛犁形虫阳性样品测序,并构建其18S r RNA基因系统进化树分析其遗传进化特征。结果显示,我国河北省牛梨形虫总感染率为18.6%(105/564),其中张家口地区牛梨形虫感染率最高,为83.3%(20/24),极显著高于河北省其他地区(P<0.01),唐山、沧州、石家庄及承德的牛梨形虫感染率分别为27.4%(32/117)、22.1%(17/77)、25.0%(20/80)和19.0%(16/84),秦皇岛和衡水地区牛梨形虫感染率为0。感染梨形虫的虫种主要为双芽巴贝斯虫、牛巴贝斯虫和瑟氏泰勒虫,感染率最高的为双芽巴贝斯虫(12.2%,69/564)。18S r RNA基因的进化树分析结果显示,10份牛梨形虫18S r RNA基因均呈地区性聚集;统计学分析结果显示,8月份河北省牛梨形虫感染率最高(29.7%),显著高于其他月份(P<0.05),4月~8月间牛梨形虫感染率呈逐渐上升的趋势,8月份达到顶峰后感染率逐渐下降。本研究首次对河北省牛梨形虫进行了流行病学调查,并分析了其遗传进化特征,结果表明河北省存在牛梨形虫的感染,尤其是张家口地区,该地区应在夏季应加强对牛梨形虫的重点防控。本研究为河北省牛梨形虫的防制提供了科学依据。

环形泰勒虫亚端粒编码可变分泌蛋白家族部分基因的研究进展

环形泰勒虫作为一种转化型泰勒虫,可感染牛单核巨噬细胞,并使其发生转化。目前,仅知环形泰勒虫通过劫持宿主细胞信号通路完成转化,但其转化机制尚未阐明。环形泰勒虫亚端粒编码可变分泌蛋白(SVSP)家族作为环形泰勒虫基因组的最大多基因家族,其在虫体-宿主相互作用中的功能尚不清楚。因此,本文通过对环形泰勒虫SVSP家族中部分基因的结构、表达特征、与宿主相互作用和可能发挥的功能进行总结,以期为环形泰勒虫-宿主相互作用机制和致病机理研究提供思路,同时也为癌症基础生物学研究提供借鉴。

牛环形泰勒虫烯醇化酶重组蛋白对小鼠免疫应答的研究

为了评估牛环形泰勒虫(Theileria annulata)烯醇化酶(enolase)原核重组蛋白对实验动物体液免疫水平和细胞免疫水平的影响,用His纯化柱和超滤管对pET-32a-enolase进行纯化和浓缩,应用Western blot分析蛋白的反应原性;另外利用牛环形泰勒虫烯醇化酶重组蛋白(r Ta-enolase)免疫Balb/c鼠制备多克隆抗体,经间接ELISA检测血清中重组蛋白的特异性抗体水平。免疫后,取小鼠脾脏制备淋巴细胞悬液,用流式细胞术检测脾脏T细胞亚群情况;用MTT试剂盒检测体外刺激后T淋巴细胞增殖情况;用Spss ANOVA单因素检验进行差异显著性分析。Western blot表明,enolase重组蛋白可与阳性血清发生特异性反应。ELISA检测免疫鼠特异性IgG/IgG1和IgG2a抗体均与对照组差异极显著(P<0.01);流式细胞术检测表明,免疫组(2.53±0.11)与对照组(1.85±0.11)CD4^(+)/CD8^(+)T细胞的比值差异极显著(P<0.01);用r Ta-enolase重组蛋白刺激小鼠脾脏淋巴细胞后明显促进其增殖(P<0.05)。研究结果表明r Ta-enolase免疫小鼠能够诱导小鼠产生极高的体液免疫水平以及细胞免疫应答水平,细胞免疫应答偏向于CD4^(+)T细胞,为后续其在疫苗中的免疫功能提供数据资料。

新疆生产建设兵团第九师肉牛感染泰勒原虫的PCR检测与种类鉴定

[目的]掌握新疆生产建设兵团第九师(以下称第九师)肉牛泰勒原虫的感染情况。[方法]从第九师161团、163团、164团和165团规模化肉牛养殖场或养殖户,分别采集肉牛抗凝血48、22、25、23份,其中,规模化养殖肉牛48份,散养肉牛70份,提取血液全基因组DNA。利用设计的特异性引物,采用PCR法对肉牛全血DNA样本分别进行环形泰勒虫、东方泰勒虫、中华泰勒虫检测,通过序列比对鉴定泰勒原虫种类,构建系统发育树进行种类遗传进化分析。采用χ^(2)检验法分析不同采样地点、不同饲养模式下肉牛泰勒原虫的感染率差异。[结果]PCR检测结果显示,在118份肉牛血液DNA样本中,检测出泰勒原虫阳性样本82份,感染率为69.49%(82/118);检测到的阳性样本均为东方泰勒虫,未检测到环形泰勒虫和中华泰勒虫。第九师161团、163团、164团和165团肉牛的泰勒原虫感染率分别为29.17%(14/48)、95.45%(21/22)、96.00%(24/25)和100%(23/23);不同采样地点肉牛泰勒原虫的感染率统计学差异极显著(χ^(2)=62.195,P<0.01)。散养条件下肉牛泰勒原虫感染率较高,为97.14%(68/70),规模化养殖条件下肉牛泰勒虫感染率较低,为29.17%(14/48);不同饲养模式下肉牛泰勒原虫的感染率差异极显著(χ^(2)=62.061,P<0.01)。在获得的82条序列中,有78条与我国新疆维吾尔自治区牛源东方泰勒虫MPSP基因序列(GenBank登录号:ON462019)同源性为100%,序列型命名为TSE1;有4条与我国新疆维吾尔自治区牛源东方泰勒虫MPSP基因序列(GenBank登录号:ON462018)同源性为100%,序列型命名为TSE2。种系发育分析结果显示,鉴定的东方泰勒虫TSE1和TSE2序列均与我国新疆维吾尔自治区牛源东方泰勒虫聚成一支。[结论]新疆生产建设兵团第九师肉牛感染的泰勒原虫种类为东方泰勒虫,散养条件下感染率更高,应加强对散养肉牛泰勒原虫的检测。

新疆部分地区牛和骆驼常见蜱传原虫病流行病学调查

研究旨在调查新疆阿克苏地区、阿勒泰地区、昌吉州、哈密市、和田地区、伊犁州等地方的部分试验区域牛和骆驼蜱携带病原情况。通过PCR方法对所采集的507份牛和骆驼血样进行病原检测(巴贝斯虫、环形泰勒虫、立克次体、无浆体),并采用MAGA11.0软件进行系统进化分析。结果显示,3个地区(阿克苏地区、阿勒泰地区、哈密市)的环形泰勒虫病总阳性率为9.86%(50/507),其中牛的阳性率为7.81%(19/243),骆驼的阳性率为11.74%(31/264);巴贝斯虫、立克次体和无形体未检出。进化树显示,骆驼环形泰勒虫的阳性样本与中国株亲缘关系较近(登录号:OM140998),相似性为99.31%;牛环形泰勒虫与沙特阿拉伯株亲缘关系较近(登录号:OM273908),相似性为99.60%。试验对新疆6个地区牛和骆驼的蜱传疾病的感染情况进行了调查,并首次在骆驼体内检出环形泰勒虫,结果可为新疆牛和骆驼蜱传疾病的综合防控提供技术支撑。

基于TashHN基因的牛环形泰勒虫病PCR诊断方法的建立

根据Genbank已发表的环形泰勒虫TashHN基因序列设计合成引物,以此建立基于环形泰勒虫TashHN基因的PCR检测方法,对该方法的特异性、敏感性、稳定性进行验证,并用建立的方法对宁夏固原地区各县共计135份牛血样进行检测。结果表明:建立的基于TashHN的PCR检测方法与中华泰勒虫、东方泰勒虫、牛巴贝斯虫、双芽巴贝斯虫均无交叉反应;可检测的最低模板浓度为1.64×10^(2) copies/μL;环形泰勒虫的DNA在-20℃冰箱放置8个月后仍可检测出目的条带;该方法利用已有文献中的PCR方法进行一致性检测,从135份牛血样中检测出5份阳性,说明该方法准确可靠。因此,试验建立的基于环形泰勒虫TashHN基因的PCR检测方法具有特异性强、敏感性高、稳定性强的特点,适用于临床上环形泰勒虫早期感染检测以及流行病学的调查。

卵形巴贝斯虫与中华泰勒虫双重PCR检测方法的建立

为建立一种能快速对卵形巴贝斯虫(Babesia ovata)和中华泰勒虫(Theileria sinensis)同时进行检测的双重PCR方法。根据GenBank已报道的卵形巴贝斯虫AMA1基因和中华泰勒虫MPSP基因设计合成了2对特异性引物,通过条件优化,建立了双重PCR检测方法。结果显示:双重PCR可特异扩增出卵形巴贝斯虫和中华泰勒虫目的条带,片段大小分别为500 bp和986 bp。该方法具有较好的特异性,对卵形巴贝斯虫和中华泰勒虫的最低检出浓度为16 fg/μL。对采集的90份牛血液样本进行双重PCR检测,卵形巴贝斯虫阳性率为30%(27/90),中华泰勒虫阳性率为16.67%(15/90),混合感染率为10%(9/90)。结果表明,双重PCR方法可用于卵形巴贝斯虫和中华泰勒虫的快速诊断和流行病学调查。