<i>Trypanosoma evansi</i>: A Qualitative and Quantitative Ultrastructural Analysis of the Spleen during Experimental Murine Infections

A murine model is used to study qualitatively and quantitatively the splenic ultrastructural changes induced by two Trypanosoma evansi strains derived from naturally infected local equine hosts (Equusasinus and E. caballus);T. evansi causes ultrastructural modifications in the spleen of the infected mice. The modifications include tissular disorganization, fibrosis, mitochondrial swelling, apoptosis and necrosis. The initial phases of the infection are quite similar, whereas the final phases differ qualitatively depending on the strain’s source. The ultrastructural quantitative changes were studied in the reticular splenocytes covering alterations in the area of the cytoplasm and nucleus. Analysis of the results shows the induction of various splenic alterations caused by local T. evansi strains. Also, it was documented that discriminative time modulation, as well as progressive tissular, cellular and subcellular changes, are more associated with derived infections from E. caballus strain.

Therapeutic Activity of Partially Purified Fractions of Emblica officinalis （Syn, Phyllanthus embfica） Dried Fruits against Trypanosoma evansi

Emblica officinalis （E. oJficinalis） dried fruits were evaluated for its antitrypanosomal activity and cytotoxic effects. Vero cell line maintained in DMEM （Dubecco＇s Modified Eagle Medium） and incubated with Trypanosoma evansi for more than 12 h. MPE was added to the Vero cell culture medium at different concentrations （250-1,000 μg/mL） with trypanosomes concentration （1 × 106 trypanosomes/mL in each ELISA plate well） and incubated at appropriate conditions for 72 h. In-vitro cytotoxieity of MPE of E. officinalis was determined on Vero cells at concentrations （（1.56-100 ~tg/mL）. Acute toxicity and in-vivo infectivity tests were done in mice. Obtained MPE ofE. officinalis underwent process of purification via column chromatography, preparative chromatography and HPLC （higher performance liquid chromatography） with bioassay at different strata on Alsever＇s medium. In-vivo assay for trypanocidal activity, MPE and PPFs （partially purified fractions） of E. officinalis with two sets of mice, each mouse was inoculated with 1 × 104/mL oftrypanosomes and treated （48 h post inoculation） at concentrations （12.5, 25, 50, 100 and 200 mg/kg body weight） were administered at dose rate of 100 [tL per mouse via intraperitoneal route （in treating parassitemic mice） to different groups of mice, 6 mice per concentration. HPLC of partially purified fractions ofE. officinalis was carried out with mobile phase ofacetonitdle： water （40：60） in gradient mode. In vitro, MPE induced immobilization and killing of the parasites in concentration-time dependent manner. Significant reduction of trypanosomes counts from concentration of 250μg/mL and complete killing of trypanosomes at 5th hour of observation, which was statistically equivalent to 4th hour of Diminazine Aceturate （Berenil）, standard reference drug used. HPLC of the partially purified fractions revealed two major prominent peaks at retention time of 1-4 min. In vivo, both MPE and PPFs of test material did prolong lives of mice by 6-9 days but could not cure them. At concentration of 2,000 kg/kg body weight of MPE in acute test, all mice survived. For in-vivo infectivity test, mice injected with immobilized trypanosomes developed parasitemia and died while, the other group survived. MPE, PPFs and Diminazine Aceturate were toxic to Vero cells at all concentrations exception of 1.56, 1.56-3.13 and 1.56-6.25 μg/mL, respectively. From this report, PPFs ofE. officinalis dried fruits demonstrated potential pathway for a new development oftrypanocide in near future if additional investigations are put in place.

Achillea fragrantissima,rich in flavonoids and tannins,potentiates the activity of diminazine aceturate against Trypanosoma evansi in rats

Objective:To evaluate activity of methanol extract of Achillea fragrantissima(meth)(A.fragrantissima) alone or in combination with diminazine aceturate(DA) against Trypanosoma evansi in experimentally infected rats.Methods:Sixty adult male Wister albino rats were divided equally into 6 groups(A-F).Rats in groups A-E were experimentally infected with T.evansi and those in group F were uninfected.The groups were treated respectively as follows:group A- with 3.5 mg/kg DA;group B- with 1 000 mg/kg meth,A.fragrantissima;group C-3.5mg/kg DA plus 500 mg/kg meth A.fragrantissima;group D-3.5 mg/kg DA plus 1 000 mg/kg meth A.fragrantissima.Group E was left untreated.Parasitaemia,survivability,packed cell volume,hemoglobin concentration,total leucocytes count,lymphocyte count,and serum malondialdehyde and reduced glutathione(GSH) levels were estimated.Phytochemical screening of meth A.fragrantissima was also performed.Results:The phytochemical analysis of the meth A.fragrantissima indicated a higher content from polyphenols tannins and non tannins and flavonoids.The efficacy percentage against trypanosomiasis in groups A to E was respectively as follows 80,40,90.100,0.The administration of meth-A.fragrantissima(1000)mg/kg b.wt.) produced a moderate efficacy against trypanosomiasis.Untreated rats in group E died between 25 and 30 d post infection.The rats given DA and meth A.fragrantissima combinations(C and D) showed faster and higher recovery rates than the uninfected control and groups A and B.The initial reduction in packed cell volume,hemoglobin,total leucocytes count,increases in serum malondialdehyde and decreases in GSH levels were reversed by the treatments.C onclusions:The administration of the methanol extracts of A.fragrantissima and DA combination therapy was more effective than each product alone in the treatment of rats infected with Trypanosoma evansi and further studies are required to isolate more active ingredients.

Hematological and serum biochemical aspects associated with a camel(Camelus dromedarius)naturally infected by Trypanosoma evansi with severe parasitemia in Semnan,Iran

Objective:To determine the presence of Trypanosoma eransi(T.evansi) and the effect of trypanosomosis on hemato-biochemical profile of dromedary camels in Semnan,Iran,which has not been reported yet.Methods:To perform this project,blood samples were collected by venipuncture into plain and EDTA-K2-containing vacutainer tubes from 21 dromedary camels(12 males and 9 females) aged3—18 years,from 4 different regions of Semnan.Results:Microscopic examination of stained thin blood smears revealed the presence of T.evansi in one of the samples.However,it should be noted that this sample showed a very high parasitemia(more than 5 trypomastigote were visible per microscopic field with MGG,1000×.This heavy parasitemia was associated with an 18-year-old female camel that showed symptoms of corneal opacity,intense emaciation and pale mucous membranes.Comparison of hematological and serum biochemical profiles between the camel infected by T.eransi and uninfected camels indicated anemia,leukocytosis,hyperproteinemia.hypoalbuminemia,hyperglobulinemia,reduction A/G ratio,increased a,,p and globulins and decreased of a,globulins and increased the concentration of gumma-glutamyl transferase enzyme.Conclusions:Results of the present study revealed that trypanosomosis was present in dromedary camels of Semnan,Iran(infection rate is 4.76%) and hemato-biochemical parameters were markedly affected by camel trypanosomosis.

Detection of Kinetoplast DNA From Trypanosoma evansi by PCR

Trypanosoma evansi, an economically important haemoflagellate, infects many domestic and wild animals such as horses, camels, buffalo and deer, causing the trypanosomiasis called surra in these animals. This parasite has an extremely wide geographical distribution in the continents of Asia, Africa and South America. Surra is also one of the most important endemic diseases of animals in China, especially

伊氏锥虫感染的昆明小鼠血常规变化和病理组织学观察

为了解伊氏锥虫感染昆明小鼠的血常规和病理组织学变化,人工感染昆明小鼠、制作血液涂片、检测血常规、分子生物学(PCR)鉴定、病理组织切片,结果显示伊氏锥虫对小鼠具有较高的致病性,随着感染天数的增加,白细胞(WBC)、中性粒细胞计数(NEU)、红细胞压积(HCT)、平均红细胞血红蛋白量(MCH)、血小板计数(PLT)值逐渐降低,淋巴细胞百分数(LYM%)、红细胞平均体积(MCV)、红细胞分布宽度的标准差(RDWR-SD)、血小板平均容积(MPV)值在死亡时明显升高,健康小鼠血常规正常。对比试验和对照小鼠各器官大小,感染小鼠脾脏、肝脏,心脏均明显变大,肺脏及肾脏变化较小。PCR鉴定在799 bp处出现目的条带,与预期片段大小相符。病理组织学观察,试验小鼠脾脏淤血,髓质间可见大量红细胞,出现较多形态改变的淋巴滤泡;肝脏中央静脉、小叶间静脉淤血,肝细胞肿胀,肝窦间隙变窄;肺脏肺泡肿胀较明显,肺泡壁增宽,支气管出现纤维化,间质可见炎症细胞;心脏中心肌纤维稍肿胀,颗粒变性,颜色加深,并伴有较多的伊氏锥虫虫体;肾小囊间隙变窄,肾小管上皮细胞肿胀,管腔变窄;对照组小鼠均未出现病理变化。研究结果可为伊氏锥虫病的诊断与预防提供理论依据。

新疆部分养马区域马匹血源性和虻源性伊氏锥虫PCR检测

为了解新疆部分养马区域马伊氏锥虫感染情况,保障当地马伊氏锥虫病的综合防控和马产业的健康发展,论文采集了不同区域马体表吸血虻(n=157)、疑似血液样本(n=238)进行PCR检测,并将结果分析及防疫建议提供给马场。通过对虻的形态学鉴定及其特异性基因(COI)和携带病原ISG 65基因扩增、测序、同源性比对,鉴定和检测了采样区域放牧马体表吸血虻及其携带病原情况。结果显示,伊犁、阿勒泰、塔城等地马血源性伊氏锥虫PCR阳性率分别为29.6%(16/54)、12.8%(17/133)、19.6%(10/51);马虻鉴定为突额瘤虻,其虻源性伊氏锥虫PCR携带率为35%。研究结果表明马锥虫具有一定的感染率,对该病进行病原监测和综合防控十分重要。

Saponins-rich fraction of Calotropis procera leaves elicit no antitrypanosomal activityin a rat model

Objective:To examine thein vitroandin vivoanti-Trypanosoma evansi(T.evansi)activity ofsaponins-rich fraction ofCalotropis procera(cpsf)leaves as well as the effect of the fraction onthe parasite-induced anemia.Methods:A 60-minutes time course experiment was conductedwith various concentrations of the fraction using a 96-well microtiter plate technique,andsubsequently used to treat experimentallyT.evansiinfected rats at 100 and 200 mg/kg bodyweight.Index of anemia was analyzed in all animals during the experiment.Results:The cpsfdid not demonstrate anin vitroantitrypanosomal activity.Further,the cpsf treatments did notsignificantly(P>0.05)keep the parasites lower than the infected untreated groups.At the end ofthe experiment,allT.evansiinfected rats developed anemia whose severity was not significantly(P>0.05)ameliorated by the cpsf treatment.Conclusions:It was concluded that saponins derivedfromCalotropis proceraleaves could not elicitin vitroandin vivoactivities againstT.evansi.

伊氏锥虫伊犁株扩繁及其PFR基因克隆表达和生物信息学分析

【目的】本研究旨在研究锥虫入侵宿主细胞的机制并为其检测方法的建立奠定基础。【方法】采用已分离保存的伊氏锥虫在昆明白小鼠体内进行培养繁殖,对其鞭毛束旁棒(PFR)基因进行克隆,并构建系统发育树。通过生物信息学方法分析和预测PFR蛋白的理化特性、亲疏水性、跨膜区结构、二级结构和三级结构;构建原核表达载体pET28a-PFR,用Western blotting检测PFR重组蛋白的反应原性。【结果】在昆明白小鼠体内成功扩繁伊氏锥虫伊犁株,第5天染虫率达到最高;PFR基因PCR扩增片段大小为834 bp,与冈比亚布氏锥虫PFR基因(XP_011775815.1)相似性为99.52%,基于PFR蛋白氨基酸序列的进化树显示,该虫株与冈比亚布氏锥虫的亲缘关系也最近。PFR蛋白分子式为C_(1416)H_(2286)N_(416)O_(442)S_(11),理论等电点为5.74,是一种碱性、亲水性及不稳定蛋白,没有跨膜区和信号肽,有10个潜在抗原表位;主要定位于细胞质中;PFR蛋白二级结构主要由α-螺旋组成(占92.34%),三级结构与二级结构一致。成功构建伊氏锥虫PFR基因表达质粒pET28a-PFR,经诱导时间、温度和IPTG浓度等条件优化后发现,浓度为1 mmol/L IPTG于37℃诱导重组菌液6 h时,PFR蛋白表达量最高,并且以包涵体的形式表达;Western blotting结果显示,重组蛋白与伊氏锥虫阳性血清反应为阳性。【结论】本试验成功在昆明白小鼠体内扩繁出伊氏锥虫,克隆了马伊氏锥虫PFR基因,构建了PFR原核表达载体,诱导表达出PFR重组蛋白,该蛋白具有良好的反应原性,为后续该蛋白功能和马伊氏锥虫的致病相关机制研究提供了理论依据。

梨形虫和伊氏锥虫双重PCR检测方法的建立

为了建立梨形虫(Piroplasmida)和伊氏锥虫(Trypanosoma evansi)的双重PCR检测方法,试验根据梨形虫的18S rRNA基因和伊氏锥虫的kDNA基因设计两对特异性引物,以吕氏泰勒虫和伊氏锥虫阳性样品DNA为模板进行单重PCR和双重PCR,并对双重PCR的退火温度进行优化,对本研究建立并优化的双重PCR方法进行特异性试验、敏感性试验及临床样品的检测。结果表明:本研究建立的双重PCR方法能够扩增出350 bp和600 bp的特异性目的条带,而无浆体、肝簇虫、弓形虫和埃立克体样品均呈阴性,梨形虫和伊氏锥虫最低检测浓度分别为54.70 fg/μL和14.30 fg/μL,用本试验建立的方法对临床样品的检测结果与对照方法(巢式PCR)检测结果一致。说明本研究成功建立了灵敏、快速、简便、特异性高的双重PCR检测方法,能够用于梨形虫和伊氏锥虫的临床检测及流行病学调查。

伊氏锥虫体外培养株的建立及其HGPRT活性测定

用带虫培养法和带虫传代法建立了伊氏锥虫体外培养株，无论有、无饲养细胞，虫体均能快速增殖。培养７０ｄ以上，虫体仍保持原有生物学特性，对小鼠有感染性，活力旺盛，在体外能连续培养传代。虫数最高达２．４８×１０６／ｍＬ，群体增倍时间为８．８６～１０．８ｈ。液氮保存、复苏后的虫体可在饲养细胞上立即增殖。培养中发现由巨噬细胞转化的巨核母细胞对虫体生长有促进作用，经胰酶消化能与虫体一道连续传代。通过ＨＡＴ选择性培养液测定，伊氏锥虫对氨基喋呤不敏感，试验组虫体与对照一样生长良好，证明伊氏锥虫具有次黄嘌呤鸟嘌呤磷酸核糖转化酶（ＨＧＰＲＴ）

水牛伊氏锥虫病免疫胶体金试纸条的研制及初步应用

为建立一种快速、简便、适应现场应用的水牛伊氏锥虫病诊断方法,采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗伊氏锥虫VSG抗原的OB6单克隆抗体,在硝酸纤维素膜上包被纯化的抗伊氏锥虫VSG抗原的TB7单克隆抗体和兔抗小鼠IgG,作为检测带和质控带,然后优化条件,研制成检测伊氏锥虫病的免疫胶体金试纸条。通过交叉试验表明该试纸条与衣原体、弓形虫、旋毛虫、巴贝斯焦虫和大片吸虫无交叉反应。该试纸条最低可以检出1∶3 200倍稀释的伊氏锥虫VSG抗原(浓度为3.11mg.mL-1)。应用该试纸条对230份水牛血清样本进行初步检测,同时用ELISA做平行试验,阳性符合率为88.2%。结果证明该试纸条是一种快速、灵敏、特异的水牛伊氏锥虫病的检测方法,为水牛伊氏锥虫病的现场检测诊断和预控提供了有效的方法。

伊氏锥虫在兔和豚鼠体内抗原变异研究

为了解伊氏锥虫在动物体内抗原变异情况，用伊氏锥虫安徽株单虫克隆ＳｈＴａｔｌ分别感染兔和豚鼠。在兔体３０ｄ中，每三天分离兔血中锥虫并克隆，获得１０个克隆锥虫，经间接免疫荧光试验和生物素抗生物素酶标试验鉴定，为９个抗原性互不相同的抗原变异体ＳｈＴａｔ１．１～１．９。同种方法在４只兔体内３０ｄ获得同样９个变异体，并且出现顺序一致。该克隆在豚鼠体内１５ｄ所获得的５个变异体，经鉴定均在兔体出现过，但变异顺序不同，显示克隆锥虫在兔和豚鼠体内均发生抗原变异。在感染早期，同种不同个体宿主体内，抗原变异体出现顺序一致，而不同种宿主体内变异体出现顺序不同。

直接PCR法检测血液中伊氏锥虫感染

为建立伊氏锥虫动基体DNA(KinetoplastDNA,kDNA)的PCR扩增技术,血液直接PCR法检测伊氏锥虫的感染,本文以伊氏锥虫kDNA片段为靶扩增DNA设计引物,确定最适PCR反应条件,建立kDNA片段的PCR扩增技术,应用直接扩增缓冲液(Ampdirect)从感染小鼠的滤纸血标本直接PCR检测伊氏锥虫。结果发现,PCR扩增伊氏锥虫kDNA片段分子量为364bp,能检测的最小DNA量是0·06pg,与布氏锥虫、活动锥虫没有交叉反应。应用直接扩增缓冲液不需进行样本的DNA抽提,直接PCR能检测感染小鼠的早期滤纸血样本,敏感性高于镜检法。本实验建立的直接PCR法检测伊氏锥虫具有较高的敏感性和特异性,方法快捷,可进一步应用于伊氏锥虫病的早期诊断以及现场调查。

体外培养伊氏锥虫变异抗原型的鉴定

用云南株伊氏锥虫克隆后体外培养，培养后于第１８～６０ｄ之间，每间隔一周通过免疫抑制鼠（ＣＹ鼠）克隆，建立了７个克隆群体（代号为０１８，０２５，０３２，０３９，０４６，０５３，０６０），并将其分别与７种克隆特异性抗血清（ＡＳ０１８～０６０）进行免疫溶解和中和试验。结果如下：①０１８和０２５能被ＡＳ０１８和ＡＳ０２５所溶解或中和；②０３２，０３９，０４６，０５３能被ＡＳ０３２，ＡＳ０３９，ＡＳ０４６，ＡＳ０５３所溶解或中和；③０６０只能被ＡＳ０６０所溶解或中和。根据上述结果，将７个克隆群体鉴定为３种变异抗原型（ＶＡＴ），按国际ＶＡＴ命名法，分别命名为ＹｕＴａｔ１·１，ＹｕＴａｔ１·２和ＹｕＴａｔ１·３。又以针对３个ＶＡＴ的抗血清ＡＳ０２５，ＡＳ０３９和ＡＳ０６０与超低温保存的、培养不同天数的克隆前群体进行免疫溶解试验，结果表明；体外培养的伊氏锥虫由混合的ＶＡＴ所组成，随着培养时间的不同，不同ＶＡＴ的比例发生改变。

伊氏锥虫抗原变异型及其优势代表变异体的分离鉴定

以连续克隆分离的方法,从1株水牛伊氏锥虫中分离到40个克隆群体,从中分离鉴定出18个抗原变异型。经间接免疫荧光试验检测,发现其中2个抗原变异型(即HbTat1.18和HbTat1.15)分别能与约80%和60%克隆群体的血清发生较强的阳性反应,初步确定这2个抗原变异型为该虫株的优势代表变异体。这为伊氏锥虫免疫预防、免疫诊断、分子诊断以及遗传进化等的研究奠定了良好的基础。

伊氏锥虫同工酶、蛋白质和抗原组分的比较研究

本文采用生化技术对八个中国伊氏锥虫株及一个布氏锥虫株的同工酶、蛋白质和抗原组分进行比较研究。根据同工酶电泳结果,可将伊氏锥虫和与其形态上不能区分的布氏锥虫区别开来,亦可将伊氏锥虫分为两个酶株群(Z1、Z2)。根据SDS-聚丙烯酰胺凝胶电泳、等电聚焦电泳和免疫印迹试验的结果,可将酶株群Z1分成五个不同多肽群(株)。本研究结果表明,中国伊氏锥虫遗传变异程度较低,是一个相对稳定的种群。

牛羊四种寄生虫病联合诊治技术的研究与应用

首次建立的酶标SPADot ELISA联检牛羊血矛线虫、血吸虫、伊氏锥虫和肝片吸虫病技术(简称四联 ) ,已在浙江、湖北、四川、安徽等省 6 2个县市试用 56万余头 ,据 50个县市对其中 194 90头份与酶标SPADot ELISA检测牛羊血吸虫、伊氏锥虫和肝片吸虫病技术 (简称三联 )进行比较结果表明 ,四联与三联法 ,其血吸虫病、伊氏锥虫病和肝片吸虫病阳性符合率依次达 99.13% ,99.0 5%和 99.2 1%。同时 ,对四联法血清学阳性的 4 6 6 0头份与查病原体法进行比较结果 ,血矛线虫病、血吸虫病、伊氏锥虫病和肝片吸虫病阳性符合率分别为 97.59% ,99.2 8% ,95.17%和 98.10 %。对查出血清学阳性的牛羊用研配的广谱、高效、系列抗虫剂进行防治。据不完全统计 ,牛羊经防治 ,其血矛线虫病、血吸虫病、伊氏锥虫病和肝片吸虫病阳性率分别由 1998年的 13.5%～ 37.9% ,0 .3%～ 34.0 % ,9.4 %～ 55.2 %和 17.2 %～ 6 9.6 % ,依次降至 1999年的 0 .9%～ 6 .7% ,0 %～ 6 .1% ,3 0 %～ 10 .7%和 4 .3%～ 13.1% ,分别下降 12 .6～ 31.2个 ,0 .3～ 2 7.9个 ,6 .4～ 4 4 .5个和 13.0～ 56 .5个百分点 ;

二重PCR快速检测牛伊氏锥虫和牛巴贝斯虫的研究

根据伊氏锥虫ITS序列(FJ416612)和GenBank报道的牛巴贝斯虫棒状结合蛋白1序列(FJ588013),设计合成2对特异性诊断引物,建立了牛伊氏锥虫和牛巴贝斯虫的二重PCR诊断方法。结果表明,建立的二重PCR诊断方法可扩增出牛伊氏锥虫和牛巴贝斯虫的特异性条带,大小分别为213bp和360bp,最低检出量分别为4.00pg和0.38pg。但对牛双芽巴贝斯虫、分歧巴贝斯虫、环形泰勒虫、附红细胞体、巴氏杆菌不敏感。用该二重PCR方法对95份水牛样品和134份奶牛样品进行检测,结果发现,水牛伊氏锥虫的感染率为13.7%(13/95)、牛巴贝斯虫的感染率为7.4%(7/95),无混合感染;奶牛伊氏锥虫的感染率为3.7%(5/134)、牛巴贝斯虫的感染率为0(0/134)。说明该二重PCR方法具有敏感、快速、特异的特点,适用于牛伊氏锥虫和牛巴贝斯虫的流行病学调查。

伊氏锥虫和布氏锥虫动基体DNA酶切电泳比较

限制性内切酶ＭｂｏＩ、ＤｄｅＩ、ＨｉｎｆＩ和ＴａｑＩ对市氏锥虫ＫＤＮＡ进行酶切后，电泳中均显示出多条ＤＮＡ区带，其总Ｋｂ数约等于２０ｋｂ，内各限制酶对伊氏锥虫ＫＤＮＡ消化后均显示出１至２条区带，总和约１ｋｂ，明显区别于布氏锥虫。