Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs

Six surra negative piglets (6-week-old) were infected with Trypanosoma evansi and two uninfected piglets were used as negative controls. Detection performances of various diagnostic tests (LAMP, PCR and parasitological tests) were compared by analysing blood samples collected weekly over a period of 11 weeks. With a two by two analysis without a gold standard, all methods were 100% specific. MI had the highest sensitivity of 65%, while LAMP, PCR, MHCT and TBS had sensitivities of 45, 33, 38 and 24%, respectively. However, when the analysis was done using MI as a gold standard, the sensitivity of MHCT was the highest at 53% followed by LAMP, PCR and TBS at 49, 44 and 35%, respectively. All methods gave high specificity above 60%. This study validates LAMP as an alternative method for the diagnosis of surra.     

秉志锥虫的重描述及其系统发育分析

为丰富鱼类锥虫的物种资源,了解养殖鱼类锥虫的感染情况,通过调查湖北地区鱼类锥虫的感染情况,在武汉市江夏区牛山湖渔场养殖的鲫血液中分离到一种锥虫,并采用形态学和分子生物学数据对该锥虫进行描述。通过形态特征和衍征的比较与分析,发现该锥虫与秉志锥虫(Trypanosoma pingi)形态一致,故鉴定为秉志锥虫。扩增并获得秉志锥虫的18SrDNA分子序列,通过序列比对分析,发现其与黄颡锥虫(Trypanosoma pseudobagri)18SrDNA序列相似性最高,为94.62%,分子系统发育树分析显示秉志锥虫落于淡水鱼类锥虫支类,与黄颡锥虫(Trypanosoma pseudobagri)形成姊妹群。     

家兔免疫伊氏锥虫弱毒疫苗后的T细胞,IgG和IgM动态变化

分别以3Ｈ—ＴｄＲ掺入的淋巴细胞转化反应和ＢＡ—ＥＬＩＳＡ方法检测家兔免疫后及攻虫后Ｔ细胞及特异性抗体ＩｇＧ和ＩｇＭ的动态变化。发现免疫后家兔外周血Ｔ淋巴细胞增殖反应于２４ｄ达到高峰,第３１天开始迅速降低。攻虫后特异性Ｔ细胞增殖反应强度未见升高,而特异性抗体Ｉｇｈ、ＩｇＭ均于第２１天达到高峰,攻虫后ＩｇＧ、ＩｇＭ分别在第６天和第９天又达到高峰。结果表明,在这种抗锥虫免疫中Ｔ细胞没有起主导作用,而特异性抗体ＩｇＧ和ＩｇＭ起主导作用。     

伊氏锥虫病原性受体纯化的研究

以伊氏锥虫表膜蛋白做配基辛和层析，从小鼠淋巴细胞、巨噬细胞和肾上皮细胞上分别纯化出了分子量相同的一种蛋白质。该蛋白经ＰＡＧＥ电泳分析，是表观分子量约为１２０．１ｋＤ的单一蛋白质，在ＳＤＳ－ＰＡＧＥ电泳系统中显示为分子量分别是７５．１ｋＤ和３５．６ｋＤ的两种蛋白质，从淋巴细胞上纯化出来的该蛋白质具有免疫球蛋白的抗原性，但来源于肾上皮细胞的物质却无此活性。用淋巴细胞膜上的该受体做阻断试验发现，某一浓度下的该受体不仅不能阻断，反而会促进固定的小鼠细胞与锥虫的粘附结合。     

体外培养对布氏锥虫伊氏亚种药敏性的影响

用小鼠治疗试验和体外药敏试验观察了4个布氏锥虫伊氏亚种虫株在长期体外培养条件下药敏性的稳定性.各虫株的原始群体、连续培养30d和90d的群体对贝尼尔、苏拉灭、安锥赛和硫胂聚氰胺的敏感性基本相同,说明连续培养90d各虫株对上述4种抗锥虫药的敏感性无明显改变.     

Differences in cloning and sub-cloning success rates in four stocks of Trypanosoma evansi and variation in suramin resistance of the clones

Four Trypanosoma evansi stocks with sensitivity to suramin in mice ranging from 0.05 to 160 mg kg⁻¹ were cloned and sub-cloned and the sensitivity of the clones determined. The results suggest that it is easier to clone and sub-clone trypanosome stocks which are sensitive to suramin than those that are resistant to the action of the drug. The clones obtained from the four stocks had sensitivities to suramin which were similar to or different from the parent stocks. These results are important in view of the development of resistance for, in the presence of suramin, these resistant yet heterogeneous populations would provide the material from which selective processes could operate. These observations also suggest that the maintenance and spread of suramin-resistant trypanosomes might be curtailed by their comparative inability to establish themselves in a new host.     

家畜伊氏锥虫弱毒株免疫家兔抗体的消长规律及保护作用

以家畜伊氏锥虫弱毒株免疫家兔12只,然后进行攻虫试验。其中11只被保护,1只发病死亡。以BA-ELISA检测家兔免疫后及攻虫后特异性IgG、IgM的变化,发现免疫后IgG、IgM均于第21d达到峰值;攻虫后IgM于第6d达到峰值,IgG于第9d达到峰值。攻虫后,随着免疫组家兔体内特异性IgG、IgM的不断上升,其血液内的锥虫也同时被清除。体外中和试验也表明,特异性抗体对锥虫的感染力具有明显的抑制作用。     

伊氏锥虫新疆株在动物体内抗原变异程序的观察

为了探明伊氏锥虫在动物体内抗原变异,以伊氏锥虫新疆株克隆感染兔,每３天采兔血１次,共采血５次,并用环磷酰胺处理的小白鼠对各分离期锥虫予以克隆,获得５个锥虫克隆群体,经ＡＢＣ－酶标试验和间接免疫荧光试验,对各克隆锥虫抗原进行鉴定,结果：获得５个表面抗原性互不相同的克隆群体,说明伊氏锥虫新疆株第１次抗原变异发生在４－６日内,以后每３天变异１次,本研究为进一步探明伊氏锥虫抗原变异规律打下了基础。     

Chemotherapy of surra in horses and mules with diminazene aceturate

During June–July 2000, an outbreak of surra occurred on an equine breeding farm in Khonkaen Province, Thailand. Forty-two percent of pregnant mares aborted or gave stillbirth and 40% (19/47) of horses and 10% (1/10) of mules died from surra. In August 2000 Trypanosoma evansi were detected in the remaining animals (28 horses and nine mules) on the farm by blood smear and/or the haematocrit centrifuge technique. All animals were treated with diminazene aceturate at 3.5 mg/kg body weight by intramuscular injection on days 0 and 41 of the study. Blood samples of eight randomly selected horses and mules were collected on days 0, 1 and once a week until day 56 and examined for T. evansi by various parasitological techniques. The sera were tested for antibodies against T. evansi using an indirect enzyme linked immunosorbent assay (ELISA).

The results revealed that diminazene aceturate at 3.5 mg/kg appeared to be effective in the first treatment of horses and mules infected with T. evansi. Parasites were cleared from the peripheral blood of horses on days 1 and 7 and mules on days 1 and 14. Thereafter the number of positive animals increased. After the second treatment, 50% of horses and 25% of mules were still positive to surra 24 h after treatment demonstrating that diminazene had no protective effect. Mild to severe toxicity of diminazene was seen in the horses and mules after injection.