应用质粒PTK探针鉴定锥虫的初步研究

用^32P标记质粒探针PTK1、PTK1.1和PTK1．2，对12株中国伊氏锥虫的斑点杂交试验显示，3个探针均能与8株具有正常动基体的伊氏锥虫杂交，而不与其余4株异常动基体伊氏锥虫杂交，对正常动基体株的敏感度为10^2虫体。探针PTK1亦能与马媾疫锥虫杂交，敏感度为10^2个虫体。但PTK1与布氏锥虫仅发生微弱的杂交反应．敏感度为10^5个虫体。试验表明伊氏锥虫株之间的kDNA微环是同源的，伊氏锥虫与马媾疫锥虫和布氏锥虫的kDNA微环存在着共同序列。     

锥虫不同分离株克隆及其等电聚焦分析

将两个伊氏锥虫及一个马媾疫锥虫的不同分离株分别接种用球磷酰胺处理的小鼠，获得五个克隆，用等电聚焦比较其蛋白质差异。结果表明，三个分离株之间有明显区别；马媾疫锥虫显著不同于伊氏锥虫；分离株不同克隆间区别较小，其中广东水牛株两克隆间带型相同，安徽水牛两克隆间带型略有区别。证明伊氏锥虫克隆间变异较小，锥虫不同分离株间变异较大，马媾疫锥虫与伊氏锥虫之间变异最大。     

伊氏锥虫、马媾疫锥虫、布氏锥虫、刚果锥虫的18S rDNA部分序列测定与系统发育关系

对伊氏锥虫（Trypanosoma evansi）：新疆株（XJCA）、湖北株（HBM）、云南株（YNB）、广东株（GDB2）；马媾疫锥虫（Trypanosoma equiperdum）、布氏锥虫（Trypanosoma brucei）、刚果锥虫（Trypanosoma congolense）提取基因组DNA，根据已报道的伊氏锥虫株18SrDNA基因序列设计合成引物，用PCR扩增了锥虫虫株基因组DNA,伊氏锥虫新疆株、湖北株、云南株、广东株、布氏锥虫、刚果锥虫均为373bp的片段；马媾疫锥虫为372bp的片段，PCR产物经电泳鉴定后用试剂盒回收纯化，纯化后PCR产物经连接、转化后测序，将测得的序列用DNAMAN软件分析并与国外已发表的相应序列进行了同源性比较，并绘制了系统发育进化树。结果与国外AJ009153、AJ223564、D89527株同源性达到99％～100％，与另外11株同源性75％。本研究为锥虫分子流行病学研究及分类研究打下基础。     

检测伊氏锥虫循环抗原的McAb-RPHA试验

用一株抗伊氏锥虫、马媾疫锥虫和布氏锥虫共同抗原(与泰氏锥虫等抗原无交叉反应)的单克隆抗体建立了检测伊氏锥虫循环抗原(TcA)的反向间接血凝试验。用以检测人工感染兔,于感染后8～10天TcA转阴;如不治疗直至观察期结束持续阳性;治疗后一周即转阴。用以检测疫区36份虫血症阳性水牛血清,25份阳性(69.44％);16份虫血症阴性、IHA阳性水牛血清,3份阳性;25份虫血症和IHA阴性水牛血清,全部阴性。     

伊氏锥虫补反抗原与媾疫锥虫补反抗原诊断马媾疫病的比较

<正> 用补体结合反应诊断马媾疫,所用的抗原有伊氏锥虫补反抗原和媾疫锥虫补反抗原,为了探讨两种抗原对马媾疫的检出率,我们做了对比试验,现将试验情况分述如下: 试验抗原一、伊氏锥虫补反抗原,成都兽医生物药品制造厂出品。二、媾疫锥虫补反抗原,陕西省兽医研究所制造。     

媾疫锥虫、伊氏锥虫差异抗原的分离鉴定

以50％饱和硫酸铵沉淀的兔抗伊氏锥虫高免血清球蛋白制备琼脂糖-4B免疫吸附柱,对马媾疫锥虫超声全虫可溶性抗原进行反亲和层析免疫吸附。当抗原量在抗体吸附范围内时,最先洗脱下来的成分即为媾疫锥虫特异性抗原(差异抗原)。该抗原与媾疫锥虫血清抗体反应较强,与伊氏锥虫血清抗体反应较弱,经SDS-PAGE检测,其为分子量大于68000的大分子蛋白。     

伊氏锥虫和媾疫锥虫蛋白组分的电泳分析

应用SDS—PAGE和IEF电泳对伊氏锥虫和媾疫锥虫的蛋白组分进行了分析.在SDS—PAGE中,伊氏锥虫有21条带.媾疫锥虫有19条带,两种虫体的蛋白质区带在分子量40000～90000之间存在差异,尤其表现在表面糖蛋白上,伊氏锥虫为43000,媾疫锥虫为60000.在IEF电泳中,伊氏锥虫出现26条带,媾疫锥虫出现33条带,两种虫体的蛋白质区带在等电点4.8～5.6之间相同,而在5.6～7.0之间存在差异.本实验还对马媾疫锥虫的蛋白质进行了双相电泳分析,显示出86个多肽斑点.     

论马媾疫锥虫与伊氏锥虫分类性状之异同

马媾疫锥虫(Trypanosoma equiperdum Doflein,1901)1894年在阿尔及利亚发现,是马属动物通过交配经生殖器粘膜感染的一种慢性原虫病的病原。伊氏锥虫[T.evansi(Steel,1885)Balbiani 1888]1880年发现于印度的马和骆驼,是由吸血昆虫虻类等传播的马、牛和骆驼锥虫病的病原。两种锥虫同属锥虫科锥虫属布氏组。迄今为止,各种教材和专著中,都认为这两种锥虫在形态上无区别,但其生物学特性则彼此不同。本文援引有关文献资料,结合笔者的第一手材料,对马媾疫锥虫与伊氏锥虫分类性状诸问题,进行如下对比性分析。     

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。     

伊氏锥虫同工梅,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、蛋白质的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成形态上不能区分的布氏锥虫区别开来，亦可将伊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分成五个同多肽群（株）。本研究结果表明：中国伊氏锥虫遗传传 变异程度较低，是一个相对稳定的种群。     

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.     

泰氏锥虫抗原制备及实验室诊断研究

应用体外培养的泰氏锥虫制备可溶性抗原Ⅰ、抗原Ⅱ和代谢抗原.经测定,其蛋白含量每毫升分别为6.5mg、7.4mg和7.1mg.薄层等电聚焦电泳测定结果表明,抗原Ⅰ出现22条区带,抗原Ⅱ21条区带,代谢抗原28条区带,对照的伊氏锥虫琼脂免疫扩散抗原14条区带.经分析,泰氏锥虫抗原和伊氏锥虫抗原有4条区带在同一迁移率上.琼脂免疫扩散反应中,3种泰氏锥虫抗原均与相应免疫兔血清发生沉淀反应,抗原Ⅰ出现1条致密沉淀线,抗原Ⅱ和代谢抗原出现2～3条沉淀线,抗原效价为1:4～16.免疫电泳显示了类似的结果,抗原Ⅰ与免疫兔血清出现1条弧形沉淀线,抗原Ⅱ和代谢抗原与免疫兔血清出现了3条弧形沉淀线.间接血凝试验结果表明,泰氏锥虫自然感染牛血清效价为1:20～40,免疫兔血清为1:1280～5120.所制泰氏锥虫抗原对伊氏锥虫和媾疫锥虫血清也能很好地发生交叉反应,3份伊氏锥虫病马血清和3份伊氏锥虫人工感染兔血清血凝效价分别为1:10～40和1:8～1024;5份媾疫马血清有4份血凝效价为1:20～320.4份环形泰勒虫病牛血清均为阴性.     

The transmission of mixed Trypanosoma brucei brucei/T. congolense infections by tsetse (Glossina morsitans morsitans)

Laboratory experiments and field observations clearly show that tsetse flies can be carriers of mixed trypanosome infections. Question remains how easy it is for the tsetse fly to acquire such a mixed infection during the first bloodmeal. This is of particular importance in the epidemiology of Trypanosoma brucei s.l., often a cryptic infection and difficult to transmit to non-teneral tsetse flies. To determine the transmission rate of T. brucei as part of a mixed infection, teneral Glossina morsitans morsitans were fed once on cattle with a mixed (Trypanosoma brucei brucei/Trypanosoma congolense) or single (T. brucei) infection. Of the 140 flies fed on animals with a mixed infection and examined 30 days later, 4 had a metacylic T. brucei infection, 29 a T. congolense infection and 13 a mixed T. brucei/T. congolense infection. There was no significant difference between the transmission rate of T. brucei as a single or as part of a mixed infection. The high proportion of mixed T.b. brucei/T. congolense infections was explained best by a model implying that if a fly is refractory to T. congolense, it is also refractory to T.b. brucei and vice versa. Hence, results suggest that the transmission of T.b. brucei is affected mainly by the vectorial capacity of flies and not by concurrent trypanosome infections in the host.     

Camel trypanosomiasis and its vectors in Somalia

Blood samples from 3000 Somali camels (Camelus dromedarius) were examined for trypanosome infection. Of these, 160 (5.33%) were infected with Trypanosoma evansi, one (0.03%) with T. congolense and one (0.03%) with T. brucei. Camel trypanosomiasis occurred in most areas of tabanid infestation throughout the country. The tabanids Philoliche zonata and P. magretti are incriminated as the major vectors of the disease.     

Performance of enzyme-linked immunosorbent assays for detection of antibodies against T. congolense and T. vivax in goats

Indirect ELISAs using denatured antigen preparations of Trypanosoma (T.) congolense (TcAGd) and T. vivax (TvAGd) for detection of anti-trypanosome antibodies in bovine serum (I-TAB ELISAs), were adapted for serodiagnosis in goats. The diagnostic proficiency, the cross-reactivity with sera from heterologous trypanosome infections and the operational performance of the assays were evaluated on experimentally trypanosome-infected goats. The I-TAB ELISA (TcAGd) detected antibodies in all T. congolense infected goats (100% overall sensitivity) from 2 to 4 weeks post-infection (p.i.) until the end of the experiments. Specificity tested on 92 uninfected goats was 96.7%. Extensive cross-reactions of I-TAB ELISA (TcAGd) with sera from T. vivax or T. brucei infected goats were observed. The I-TAB ELISA (TvAGd) detected antibodies in 5 of the 6 T. vivax infected goats, specificity tested on uninfected goats was 100%. Cross-reactivity with sera from T. congolense or T. brucei infected goats remained limited. Infecting species identification based on the highest percent positivity (PP) in both systems, correctly identified all T. congolense infections, but misidentified in 2/19 occasions a T. vivax infection as a T. congolense infection. In the absence of T. brucei specific antigen coated plates, T. brucei infections were identified in, respectively, 7/9 and 2/9 occasions as T. congolense or T. vivax infections. Acceptable inter-plate repeatability was observed. The implications of results and technical requirements for ongoing applied research are discussed.     

Trypanosome infection rate in cattle at Nguruman, Kenya

Trypanosome infection rate in cattle at Nguruman was investigated in a study conducted in 1984-1986. Shifting pastoralism significantly reduced trypanosome infections in cattle. The cattle were more heavily infected with Trypanosoma congolense (16.5%) than Trypanosoma vivax (4.95%) and Trypanosoma brucei (0.19%). Trypanosoma theileri was observed only once among the cattle examined. Mixed trypanosome infections in cattle were observed to be 2.75% and 0.014% for T. congolense/T. vivax and T. congolense/T. brucei, respectively. The duration of infection in the cattle was 55 days for T. congolense and 79 days for T. vivax. High infections in cattle were observed 2 months after the rains, which were concomitant with high tsetse densities.     

Changes in properties of adenosine transporters in Trypanosoma evansi and modes of selection of resistance to the melaminophenyl arsenical drug, Mel Cy.

Resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake. The transport of melaminophenyl arsenicals into Trypanosoma brucei was shown to be mediated by an unusual adenosine nucleoside transporter, P2 (Carter and Fairlamb, 1993), and the loss of this transporter is associated with resistance to melaminophenyl arsenicals in these parasites. To further understand the mechanisms of arsenical resistance, we generated several lines of Mel Cy-resistant T. evansi from a drug-sensitive isolate using both in vivo and in vitro selection methods. Uptake of the melaminophenyl arsenical, Mel Cy on the P2 transporter was studied in the drug-sensitive as well as Mel Cy-resistant parasites, by means of inhibition of Mel Cy-induced lysis of trypanosomes, in an in vitro lysis assay. Adenosine uptake was also investigated using competition inhibition assays.Our study shows that T. evansi, TREU 1840, possesses the P1/P2 adenosine transport system as reported in T. brucei and T. equiperdum. However, in T. evansi, the P2 transporter is the larger transport process instead of the P1. The P2 transporter in T. evansi mediated the uptake of Mel Cy in the drug-sensitive parasites. The P2 was retained in all the arsenical-resistant T. evansi lines studied. However, the activity of the transporter was reduced to different extents in the different-resistant lines. The residual P2 activity related well to the levels of drug resistance in each line, suggesting that P2 activity could be an important marker for arsenical resistance. Furthermore, important differences were observed between the in vivo- and the in vitro-selected arsenical-resistant parasites suggesting that there may be differences in resistance phenotypes selected on the field.