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。     

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。     

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。     

读者来信

《中国畜禽传染病》编辑部:在贵刊1990年第二期封三上见到湖南省安江农校邓治邦的文章“牛感染布氏锥虫病的诊治”,先是感到极大兴趣,当读完之后又大失所望。我是一个研究家畜锥虫病的科研工作者,从我们收集到的中国锥虫虫种(包括从湖南牛体内的虫种)中从未发现过布氏锥虫,文献上也未见中国有布氏锥虫的报道,如郑策平等曾对我国13个地理宿主株的锥虫进行分类鉴定,最终确定都是伊氏锥虫.伊氏锥虫的传播媒介为虻及吸血蝇,而布氏锥虫则为采采蝇,这种蝇在非洲、南美洲、西亚较多,我国尚未见报道.邓治邦文章中提到的布氏锥虫的症状,病原检查和血清学诊断均不足以说明是布氏锥     

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对布氏锥虫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，而各限制酶对伊氏锥虫ＫＮＤＡ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。     

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。     

伊氏锥虫安锥赛抗药性相关基因-TbTA1的分析

为探讨伊氏锥虫对安锥赛抗药性的分子机理，以布氏锥虫对砷剂药物抗药性相关的TbTA1基因设计引物，以55℃、57℃、60℃、63℃和65℃不同退火温度，分别从伊氏锥虫敏感株的cDNA和基因组DNA中扩增出TbTA1基因的全长序列，但是从安锥赛抗药株伊氏锥虫的cDNA和基因组DNA中都未扩增出TbTA1基因，这表明基因TbTA1可能与伊氏锥虫安锥赛抗药性的产生有关。伊氏锥虫与布氏锥虫的，TbTA1基因序列比较，它们有10个碱基不同，即第88位的G（T.e）-A（T.b）、第144位的T（T．e）-C（T．b）、第224位的C（T．e）-T（T．b）、第471位的C（T.e）-T（T.b）、第472位的A（T．e）-G（T.b）、第549位的G（T．e）-T（T．b）、第1008位的C（T．e）-T（T.b）、第1033位的A（T．e）-G（T．b）、第1293位的A（T．e）-G（T．b）和第1384位的T（T．e）-C（T．b），其中有5个碱基所编码的氨基酸不同，即Val^30（T.e）Ile（T．b）、Ala^75（T．e）-Val（T．b）、Ile^158（T．e）-Val（T．b）、Thr^345（T．e）-Ala（T．b）和Ser^462（T.e）-Pro（T．b），这表明伊氏锥虫与布氏锥虫的TbTA1差异可能是它们的种间差异。     

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     

伊氏锥虫不同株的表面变异糖蛋白提取和重要特性比较

抗原变异是锥虫的重要特征，主要表现于锥虫表面变异糖蛋白（VariableSurfaceGlycoprotein，VSG）的变化，而且锥虫的VSG变异非常频繁，国外对布氏锥虫的VSG研究较多，对中国伊氏锥虫的VSG，杨汉春［1］、李国清等［2］曾作过分离和生化分析，但对中国不同地理宿主株VSG的分离比较尚无报道。而我国北方新疆骆驼伊氏锥虫和南方牛、马伊氏锥虫存在来源、地理、宿主和生化特性的变异[3,4]，我们对这两个虫株的VSG进行了分离、纯化和特性比较研究，以期为伊氏锥虫的抗原变异和伊氏锥虫病的防治研究提供依据。1材料与方法1．1材料和试剂…     

抗安锥赛克隆伊氏锥虫的生物特性

为了解对安锥赛有抗药性的伊氏锥虫的特性和危害，将伊氏锥虫浙江株克隆，然后人工诱导其对安锥赛产生抗药性，观察抗药性锥虫的生物特性。克隆繁殖后的伊氏锥虫分为3组C(0)0为原种对照组；C(15)0为不接触安锥赛的同步繁殖对照组；第3组为连续经环磷酰胺免疫抑制小鼠人工诱导对安锥赛产生的5种不同程度抗药性锥虫C(1)6、C(3)16、C(6)43、C(9)83和C(15)199。采用无细胞培养技术分别测定C(0)0、C(15)0、C(1)6、C(3)16、C(6)43、C(9)83和C(15)199的IC50，结果依次为0.01550、0.01346、0.0263、0.10237、1.40929、1.92290和9.92330。将这7种锥虫各自按1.0×104条/只经腹腔感染5只小鼠，镜检它们在小鼠尾血中的出虫时间，结果依次为69.6、67.2、76.8、86.4、93.6、98.4、96.0h；各组感染小鼠存活时间依次为154、142、194、207、205、198、202h；各组死亡率均为100%。结果表明，伊氏锥虫经安锥赛作用于小鼠体内传15代后，其生长繁殖和对小鼠的毒性均发生变化，随着伊氏锥虫对安锥赛的抗药性程度升高至IC50为1.92290，抗药锥虫的生长繁殖速度降低，对小鼠的毒性减弱，但毒性并不随锥虫抗药性程度的提高而进一步减弱。这些结果显示，伊氏锥虫在安锥赛作用下，以生长、繁殖、毒力的代谢发生变化来适应安锥赛的作用而生存。     

Evaluation of the card agglutination test (CATT/T. evansi) for detection of Trypanosoma evansi infection in water buffaloes (Bubalus bubalis) in Egypt

A card agglutination test (CATT/T. evansi) was evaluated for detection of antibodies against Trypanosoma evansi (T. evansi) in experimentally and naturally infected buffaloes. Four calves were inoculated with a strain of T. evansi isolated from a dromedary camel. Parasitological examination of the calves revealed trypanosomes in the blood from days 4 to 9 post-inoculation (PI). General emaciation appeared from day 26 PI and aggravated until the end of the experiment (day 88 PI). Antibodies against T. evansi were detectable from day 8 PI till the end of the experiment. Parasitological examination of 200 water buffalo blood samples obtained from slaughterhouses revealed negative results. Serological examination of these animals showed that 48 (24%) water buffaloes had anti-T. evansi antibodies.     

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/T. evansi), an indirect card agglutination test (LATEX/T. evansi) and a newly developed antibody detection ELISA (ELISA/T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals (n=8) were positive in all tests. Diagnostic specificity was highest in CATT/T. evansi (98%) followed by the ELISA/T. evansi (95%) and the LATEX/T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1-8%) false positive results were actually due to a specific reactions, and that LATEX/T. evansi and ELISA/T. evansi detected more immune trypanolysis positives than the CATT/T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/T. evansi with a 30% positivity cut-off and LATEX/T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/T. evansi with a 50% positivity cut-off and the CATT/T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.     

不同浓度甘油和pH对DEAE-纤维素柱分离伊氏锥虫效果的影响

在不同ｐＨ条件下以不同浓度甘油破坏鼠红细胞后,用ＤＥＡＥ－５２纤维素柱层析分离纯化伊氏锥虫（Ｔｒｙｐａｎｏｓｏｍａｅｖａｎｓｉ）,结果表明在ｐＨ７．４时,２０％甘油能溶解大约９５％的大、小鼠红细胞;经此处理后,对Ｔ．ｅｖａｎｓｉ的回收率和活力无不良影响,可明显提高其分离纯化效果。作者还观察了渗透压和ｐＨ条件改变对Ｔ．ｅｖａｎｓｉ的影响。     

奶牛附红细胞体和伊氏锥虫二重PCR诊断方法的建立

为建立奶牛附红细胞体和伊氏锥虫两种病原诊断方法并探索二者之间在奶牛感染中的关系,本研究针对两病原分别设计两对特异性引物,建立了奶牛附红细胞体和伊氏锥虫感染的二重PCR诊断方法,其扩增片段大小分别为415bp和237bp。敏感性试验和特异性试验表明,附红细胞体和伊氏锥虫的DNA的最低检测量为0.154pg和0.105pg;与猪肺炎支原体、大肠杆菌、葡萄球菌、鸡艾美耳球虫、牛双芽巴贝斯虫无交叉反应。35份临床血样检测结果为:奶牛附红细胞体阳性率22.89%,伊氏锥虫阳性率8.89%,其中共感染率为2.89%。临床试验表明,该方法可用于奶牛附红细胞体和伊氏锥虫的诊断,特别适用于早期诊断。     

Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya

Camel trypanosomosis (Surra) causes high morbidity and is an impediment to the camel husbandry in Kenya. The lack of a sensitive diagnostic test has hindered the collection of accurate epidemiological data and institution of control programmes. A cross-sectional study was conducted in three districts of Kenya to estimate the prevalence of Trypanosoma evansi (T. evansi) and to compare four diagnostic tests: polymerase chain reaction (PCR), card agglutination test (CATT/T. evansi), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI). A total of 549 camels were randomly sampled. The overall prevalence of Surra was 5.3% using MHCT, 26.6% using PCR and 45.9% using CATT/T.evansi. There was a significant difference (P < 0.001) between PCR and CATT/T.evansi test, MHCT and MI in detection of T. evansi. The prevalence of T. evansi was 39.8% in Samburu, 24.7% in Nanyuki and 14.4% in Isiolo districts using PCR. A male camel was 2.6 times more likely to be infected with T. evansi compared to a female camel (OR = 3.0% CI: 1.6, 4.1), while an adult camel was 2.2 times more likely to be infected compared to non-adults (OR = 2.2; 95% CI: 1.2, 5.0). There was a poor association between the presence of the published clinical signs and seropositivity (kappa = 0.12), PCR (kappa = 0.11) and MHCT (kappa = 0.05). However, there was a higher agreement between farmers' classification of disease with the PCR test (kappa = 0.5, n = 61). The mean PCV varied with age, presence of infection, locality and gender, with the lowest mean PCV being recorded in MHCT-positive animals (20.97 +/- 0.5) and from infected calves (19.5 +/- 1.2). This study shows that PCR was more sensitive in detecting T. evansi than other tests used. Further, the prevalence of T. evansi in the camel herds sampled is higher than that previously reported in Kenya, and that the judgment by camel keepers may be a reliable "pen-side" diagnostic test for Surra. Considering the low sensitivity of parasitological techniques in detection of chronic T. evansi infection and high cost of PCR, development of a sensitive pen side diagnostic test, with a low cost is still a priority.     

Evaluation of antigen and antibody rapid detection tests for Trypanosoma evansi infection in camels in Kenya

The card agglutination test for Trypanosoma evansi (CATT/T. evansi) for the detection of antibodies, and Suratex for the detection of circulating antigens were compared in a cross-sectional study involving camels in eastern and central parts of Kenya. Of the 2227 camels screened, 2038 were owned by nomadic pastoralists in T. evansi endemic areas in eastern Kenya. A herd of 86 camels were from a ranch in Mugwoni. In Athi River area, 35 camels belonged to Kenya Trypanosomiasis Research Institute, and 68 were slaughter animals. Diagnostic sensitivity estimates were obtained by testing sera from 51 camels that had been found to be parasitologically positive by the haematocrit centrifugation technique, buffy-coat technique and mouse inoculation. Diagnostic specificity was estimated by testing sera from 35 camels known to be trypanosome-free. Positive and negative predictive values (NPVs) were calculated using a range of prevalence values. The sensitivity of CATT/T. evansi (68.6%) was higher than that of Suratex (58.8%), but not significantly. Both tests had equally high specificity (100%). The overall prevalence was 2.3% (51 out of 2227) by parasite detection, 32.2% (327 out of 1017) by CATT/T. evansi and 19.6% (188 out of 961) by Suratex. Overall, there was a positive association between CATT/T. evansi and Suratex though the strength of association was low (McNemar's test=46.12, P=0.001; kappa=0.26, CI: 0.20-0.33). Parasite prevalence ranged from 0% in several herds to 27.8% in a herd in Isiolo. Prevalence was highest in Isiolo with 2.5% (51 out of 2030) by parasitological detection, 38.8% (321 out of 828) by CATT/T. evansi and 21.9% (169 out of 772) by Suratex. In Mugwoni prevalence was 7 and 18% by CATT/T. evansi and Suratex, respectively, and no parasites were detected. In Athi River Suratex detected 2.9% (3 out of 103) positive while CATT/T. evansi and parasitological methods gave negative results. At prevalence values between 10 and 100%, CATT/T. evansi as well as Suratex had infinitely high positive predictive values, whereas Suratex had a lower NPV than CATT/T. evansi. In conclusion, results of this study showed that CATT/T. evansi and Suratex were able to detect aparasitaemic infections rapidly and were more sensitive than parasitological methods in revealing the true extent of trypanosomosis in a herd. The tests effectively complemented parasitological methods in the detection of T. evansi infections in camels.     

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.     

The development and validation of an antibody-ELISA to detect Trypanosoma evansi infection in cattle in Australia and Papua New Guinea

Trypanosoma evansi is exotic to Australia and Papua New Guinea (PNG). However, it might have been introduced to Papua (Indonesia); thus, there is a risk of it entering PNG and thence Australia. Because of logistical difficulties in PNG and northern Australia, surveillance for T. evansi must rely on serological tests. The accuracy of an Ab-ELISA using a detergent extract of T. evansi and three antigen fractions purified from the detergent extract using stepwise precipitation with saturated ammonium sulphate (AS) were compared. The ELISA using the AS 40-50% fraction had greater discriminatory power compared to the ELISA using the other antigen fractions. This ELISA then was compared with two commercial tests: the Card Agglutination Test for trypanosomiasis/T. evansi (CATT) and Suratex. CATT/T. evansi at 1/4 serum dilution has higher sensitivity and the ELISA has higher specificity. There is no likely benefit in combining antibody detection tests to improve the accuracy of diagnosis. Furthermore, the combination of Suratex (which was independent of the antibody tests) with the CATT or the ELISA did not improve the sensitivity. None of the tests was sufficiently sensitive to be used confidently to determine freedom from infection in animals imported into Australia from countries where T. evansi infection is endemic.