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.

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。

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。

羊泰勒虫与嗜吞噬细胞无浆体双重PCR检测方法的建立

为建立一种能快速对羊泰勒虫(ovine and caprine theileria)和嗜吞噬细胞无浆体(A.phagocytoph-ilum)同时进行检测的双重PCR方法。根据GenBank已报道的羊泰勒虫MPSP基因和羊嗜吞噬细胞无浆体16S rRNA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。双重PCR可特异扩增出羊泰勒虫和嗜吞噬细胞无浆体目的条带,片段大小分别为875bp和394bp。该方法具有较好的特异性,对羊泰勒虫和嗜吞噬细胞无浆体的最低检出浓度为16fg/μL和1fg/μL。对采集的60份血液样本进行双重PCR检测,羊泰勒虫阳性率为46.67%(28/60),嗜吞噬细胞无浆体阳性率为13.33%(8/60),混合感染率为10%(6/60)。结果表明,双重PCR方法可用于羊泰勒虫和嗜吞噬细胞无浆体的快速诊断和流行病学调查。

新疆一例牛环形泰勒虫、念珠菌和绵羊无浆体混合感染的病例初报

2021年8月17日,新疆托克逊县一头西门塔尔母牛出现高热、瘤胃胀气、肌肉震颤、呼吸急促、结膜黄染和血红蛋白尿等临床症状。为确认发病原因,采集该牛全血和体表蜱虫样品进行光学显微镜观察、血常规检测和聚合酶链式反应(PCR)检测。血涂片镜检结果显示,患牛疑似牛环形泰勒虫、念珠菌和绵羊无浆体混合感染;血常规检测显示,血红蛋白、血红细胞压积、平均血红蛋白浓度和中性粒细胞等指标下降,血小板和淋巴细胞百分比等指标上升,提示该牛机体存在贫血和炎症反应;血液样品PCR结果显示,牛环形泰勒虫、念珠菌和绵羊无浆体检测均呈阳性;蜱虫样品PCR结果显示,牛环形泰勒虫检测为阳性。综上,患牛最终被诊断为牛环形泰勒虫、念珠菌和绵羊无浆体混合感染。目前,此3种病原共同感染牛的案例极为少见。本研究为今后牛混合感染牛环形泰勒虫、念珠菌和绵羊无浆体的临床诊断提供了参考。

First molecular identification of multiple tick‑borne pathogens in livestock within Kassena‑Nankana,Ghana

The risk of pathogen transmission continues to increase significantly in the presence of tick vectors due to the trade of livestock across countries.In Ghana,there is a lack of data on the incidence of tick-borne pathogens that are of zoonotic and veterinary importance.This study,therefore,aimed to determine the prevalence of such pathogens in livestock using molecular approaches.A total of 276 dry blood spots were collected from cattle(100),sheep(95)and goats(81)in the Kassena-Nankana Districts.The samples were analyzed using Polymerase Chain Reaction(qPCR)and conventional assays and Sanger sequencing that targeted pathogens including Rickettsia,Coxiella,Babesia,Theileria,Ehrlichia and Anaplasma.An overall prevalence of 36.96%was recorded from the livestock DBS,with mixed infections seen in 7.97%samples.Furthermore,the prevalence of infections in livestock was recorded to be 19.21%in sheep,14.13%in cattle,and 3.62%in goats.The pathogens identified were Rickettsia spp.(3.26%),Babesia sp.Lintan(8.70%),Theileria orientalis(2.17%),Theileria parva(0.36%),Anaplasma capra(18.48%),Anaplasma phagocytophilum(1.81%),Anaplasma marginale(3.26%)and Anaplasma ovis(7.25%).This study reports the first molecular identification of the above-mentioned pathogens in livestock in Ghana and highlights the use of dry blood spots in resource-limited settings.In addition,this research provides an update on tick-borne pathogens in Ghana,suggesting risks to livestock production and human health.Further studies will be essential to establish the distribution and epidemiology of these pathogens in Ghana.

马泰勒虫棒状体颈部蛋白4基因的克隆与原核表达分析

【目的】克隆马泰勒虫新疆株棒状体颈部蛋白4基因(RON4)并原核表达RON4蛋白。【方法】参考美国马泰勒虫WA株基因组数据及其他顶复门原虫RON4基因信息,设计特异性引物,以马泰勒虫新疆株DNA为模板克隆RON4基因片段,构建pET-32a-RON4原核表达载体后进行原核表达与鉴定,并对RON4基因编码蛋白进行生物信息学分析。【结果】获得马泰勒虫新疆株RON4基因(NCBI登录号为:ON254212)。马泰勒虫新疆株与马泰勒虫WA株亲缘关系最近,RON4基因的核苷酸和氨基酸相似性分别为99.4%和99.0%;RON4基因在种间高度保守,其氨基酸序列与其他顶复门原虫具有高度相似的保守区域。成功构建了RON4蛋白原核表达载体,诱导表达获得分子质量为50 ku的重组包涵体蛋白;重组蛋白可与马泰勒虫阳性马血清特异性反应。生物信息学分析表明,马泰勒虫新疆株RON4蛋白的亲水性、柔韧性以及表面可及性较弱,B细胞表面抗原表位区域较少,形成表面抗原表位的结构基础条件不足。【结论】克隆获得了马泰勒虫新疆株RON4基因,并完成了原核表达及鉴定,该基因高度保守,在不同物种间可能具有相似的功能。

马泰勒虫RON2基因真核表达及互作蛋白预测分析

为探究马泰勒虫入侵宿主细胞关键蛋白RON2的功能及其互作蛋白,并尝试解析马泰勒虫入侵关键结构--移动连接体,以马泰勒虫新疆分离株为研究对象,在课题组前期获得马泰勒虫RON 2基因的基础上,设计特异性引物,构建重组蛋白真核表达载体pmcherry-TeRON2。融合表达重组蛋白,制备His-TeRON2多克隆抗体,通过间接免疫荧光和Western blot鉴定蛋白的表达情况,最后对RON 2基因编码蛋白的互作蛋白进行预测分析。成功构建pmcherry-TeRON2真核表达质粒,获得的马泰勒虫TeRON2重组蛋白分子质量为37 ku,间接免疫荧光和Western blot结果显示,重组蛋白能够被抗体特异性识别,大小正确。间接ELISA结果显示,当抗原包被量为2μg/mL时,His-TeRON2小鼠多克隆抗体效价为1∶409600,多克隆抗体真核重组蛋白发生特异性反应。TeRON2蛋白互作网络预测显示,共有10种蛋白与马泰勒虫TeRON2蛋白发生相互作用关系,大部分蛋白都与虫体入侵宿主细胞的关键结构移动连接体相关,包括顶膜抗原1、棒状体颈部蛋白4及棒状体颈部蛋白11、棒状体相关蛋白和滑行相关蛋白等,另外与鸟苷酸环化酶和钙依赖激酶也有相互作用关系。推测TeRON2蛋白可能与入侵关键结构移动连接体(MJ)形成有关,参与虫体对宿主细胞的入侵过程,为进一步深入研究马泰勒虫TeRON2蛋白作为疫苗候选抗原以及在虫体入侵宿主过程中的作用提供了理论依据。

环形泰勒虫TA13815蛋白的原核表达及反应原性分析

为研究环形泰勒虫(Theileria annulata)TA13815蛋白功能,对环形泰勒虫虫体蛋白TA13815基因的核苷酸序列及编码蛋白的氨基酸序列进行生物信息学分析,构建pET30a-TA13815重组表达载体,并对其进行原核表达及反应原性分析。SDS-PAGE结果显示,蛋白的分子质量大约为29.663 ku,主要以包涵体形式表达,条带位置显示蛋白的分子质量比预期大,蛋白可能存在翻译后修饰。通过qPCR分析环形泰勒虫3个不同发育阶段,结果显示TA13815 mRNA在3个阶段均有表达,且在裂殖体阶段表达量最高。本试验成功表达了环形泰勒虫TA13815重组蛋白,该重组蛋白有较好的反应原性,且能与抗His标签蛋白的单克隆抗体和环形泰勒虫阳性血清发生特异性反应,而与健康牛血清无交叉反应。TA13815蛋白与东方泰勒虫的核苷酸和蛋白同源性均小于68%,表明该重组蛋白可作为间接ELISA诊断方法的备选抗原。