用免疫抑制小鼠快速培育抗苏拉明伊氏锥虫

将伊氏锥虫克隆ＪＧｍｃ５用环磷酰胺处理的免疫抑制小鼠频繁地连续传代，并予以亚治疗剂量的苏拉明治疗，连续传代１４次，历时８８日，即获得对苏拉明具有高水平抗药性的伊氏锥虫群体。在用免疫活性正常的健康小鼠测定抗药性时，此抗苏拉明锥虫群体仍保持高水平的抗药性，ＣＤ１００＞４００ｍｇ／ｋｇ。在以体外药敏试验测定时，其ＩＣ５０为１２３．２μｇ／ｍｌ。实验结果表明，宿主免疫系统的损害，在实验条件下，可导致伊氏锥虫迅速产生抗药性。在野外条件下，机体免疫系统的损害对锥虫抗药性的产生可能也起一定作用。     

中国伊氏锥虫kDNA小环的克隆及kDNA探针的建立

本研究以质粒ｐＧＥＭ－７Ｚｆ（＋）为载体，对我国北方和南方疫区伊氏锥虫动基体株的ｋＤＮＡ小环进行了克隆，并以其中之ｐＴＫ０１１－Ｃ＿１为探针对不同地理区的动基体株和无动基体株的不同ＤＮＡ进行杂交，结果表明，我国北、南两大疫区动基体株的ｋＤＮＡ小环大小均约为１ｋｂ，且均属Ａ型，ｐＴＫ０１１·Ｃ＿１只与动基体株的ｋＤＮＡ及ｔＤＮＡ杂交，不与其ｎＤＮＡ杂交，也不与无动基体株的任何ＤＮＡ及黄牛白细胞ＤＮＡ杂交。说明具有特异性，可以用于诊断。ｔＤＮＡ的杂交信号与ｋＤＮＡ相当，诊断中可以代替ｋＮＤＡ作为检测对象。     

伊氏锥虫HGPRT基因cDNA的克隆及其在E.coli中的表达

根据引物设计的一般原则，参照伊氏锥虫HGPRT基因的核苷酸序列设计、合成一对引物，PCR扩增伊氏锥虫HGPRT基因cDNA。低熔点琼脂糖回收：PCR产物，并将其克隆至pGEM—T Easy载体中，经酶切、PCR鉴定和序列分析，获得重组中间质粒pGEM／HGPRT。重组中间质粒pGEM／HGPRT经Nco I和Sal I双酶切，回收目的片段HGPRT，以非融合形式插入原核表达质粒pBV220构建表达质粒pBV／HGPRT，转化大肠杆菌DH5a，42℃诱导表达，聚丙烯酰胺凝胶电泳和薄层扫描分析，表达产物HGPRT的分子量约为23kD，与理论推算值相符，表达率占总菌体蛋白的19％，经间接ELISA检测，表达产物能被伊氏锥虫阳性血清所识别。     

接种泰氏锥虫及其抗原使家兔和小鼠产生抗伊氏锥虫的交叉免疫力

用培养的泰氏锥虫制备的死、活两种虫体抗原,接种于家兔和小鼠,一定时间后用伊氏锥虫强毒株攻击,观察其血清抗体的变化及交叉免疫保护能力。试验表明,泰氏锥虫虫体抗原可刺激机体产生较高的体液抗体;免疫动物有一定的抵御伊氏锥虫攻击的能力,与对照组相比,免疫动物血液中虫体出现的时间较迟,最初的数量少,临床症状轻。死虫抗原组的交叉免疫保护能力较活虫抗原组强。     

锥虫长期超低温保藏方法的研究

作者观测不同保护剂、稀释液、ＰＨ值、降温方法、复苏温度、冻融次数、液相气相交替以及解冻后在普通冰瓶中存放时间对伊氏锥虫浙江虫浙江虫株的感染性及致病力的影响。在此试验基础上，又对伊氏锥虫的６个不同虫株、媾疫锥虫、铡果锥虫、布氏锥虫等４个种的９个早株，进行了超低温保藏试验和长期保藏效果观察。已测定的有效保藏期伊氏锥虫达５７４－３２００天，媾疫锥虫达２８６６天，则果锥虫达７６３天，布氏锥虫７８３天。通过     

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。     

Comparison of various tests for the serological diagnosis of Trypanosoma equiperdum infection in the horse

Comparative tests such as FAT, ELISA, RIA, IEO and CF in the diagnosis of dourine in the horse have proved a satisfactory concordance ratio of the ELISA with CF, which seems to be the most reliable test. Discrepancies have been observed as to the other tests which appear less sensitive than CF test.     

体外培育抗苏拉灭伊氏锥虫克隆

将３个苏拉灭敏感的伊氏锥虫原种的克隆连续培养于改良Ｂａｌｔｚ无细胞培养系统中，通过逐步提高培养基中苏拉灭的含量，培育了３个抗苏拉灭的伊氏锥虫克隆─ＪＧｃ１－１６０、ＪＸ－１ｃ１－１６０和ＺＪｃ１－１４０。它们体外药敏试验的ＩＣ５０依次为３５８．５、４１２．３和２４６．４μｇ／ｍＬ，是各自亲本克隆的１２９２．５、１８７４．１和１７６０．ｏ倍；小鼠治疗试验的ＣＤ１００，对免疫功能正常小鼠依次为８０、１２０和３０ｍｇ／ｋｇ，为各自亲本克隆的５．３、８．０和３．０倍，对免疫抑制小鼠为２５０、３００和１００ｍｇ／ｋｇ，分别是相应免疫正常小鼠的３．１、２．５和３．３倍。试验结果表明，抗锥虫药治疗剂量不足和宿主免疫功能不全是导致产生抗药虫株的重要因素，各自既可单独发挥作用，又可相互协同。本文报道了体外培育伊氏锥虫抗药虫株的方法，这一方法对研究锥虫抗药性具有重要作用。     

Enzyme treatment of Trypanosoma danilewskyi (Laveran and Mesnil) increases its susceptibility to lysis by the alternative complement pathway of goldfish, Carassius auratus (L.)

This study examined whether in vitro-cultured Trypanosoma danilewskyi were susceptible to lysis in the presence or absence of anti-parasite antibodies and complement. Cultured trypanosomes were resistant to lysis by either immune or non-immune goldfish serum. However, trypanosomes treated with the proteolytic enzyme trypsin, which destroys surface proteins of the parasites, became susceptible to lysis when exposed to either immune or non-immune goldfish serum. The lysis by goldfish serum was dependent on the presence of heat-labile factors and occurred at 4 and 20 degrees C. The lysis was also dependent on the presence of Mg(2+) ions but not Ca(2+) ions. Furthermore, treatment of the parasites with different sialidases did not enhance their susceptibility to lysis by goldfish serum. Trypsinized parasites regained resistance to lysis after at least 6-h cultivation in the absence of trypsin and the restoration of full resistance was observed after 24-h cultivation. The resistance to lysis was abrogated when the protein synthesis inhibitor, puromycin, was added to the cultures. These results suggest that trypsinized trypanosomes were susceptible to lysis by goldfish complement (alternative pathway) and that protective surface proteins of the parasite were required for the resistance of normal trypanosomes to lysis.     

Preliminary evaluation of diagnostic tests using horses experimentally infected with trypanosoma evansi

Seven surra negative horses were intravenously inoculated with 3 x 10(6)Trypanosoma evansi parasites derived from a camel. One horse was maintained as an uninfected negative control. Three antigen and three antibody detection tests were evaluated for diagnosis of infection in horses. The microhaematocrit centrifugation test (MHCT) was the most sensitive, first detecting parasites between one and three days (x 2.4) post infection (p.i.). The antigen (ag)-ELISA detected antigen between three and ten days (x 6.6) p.i. The latex agglutination test (LAT) first gave positive results on day 3 (x 3.0) p.i. Following the treatment of horses with trypanocidal drugs, the MCHT and the mouse inoculation test (MIT) became negative. Antigen levels using LAT declined and reached pre-infection levels in five out of six horses during the period of observation (92-279 days). Antigen levels using the ag-ELISA declined as well but did not reach pre-infection levels in any of the six horses.Three antibody detection techniques, ab-ELISA, card agglutination test (CATT), and immunofluorescent antibody test (IFAT) detected antibodies in the blood of all seven infected horses but not in the uninfected control. However, the ab-ELISA did not discriminate clearly between sera from infected and uninfected horses because unacceptably high ELISA background readings were detected in 15% of the surra negative horses shipped to the UAE from the UK. The ELISA antibody increased above pre-infection levels in the six horses experimentally infected, but not in one horse. In this horse the ELISA antibody level exceeded the cut-off level only after the reoccurrence of the T. evansi infection. The IFAT detected antibodies 15.7 days p.i. in all infected horses.