Development of international standard diagnostic method for equine babesiosis

马巴贝斯虫病国际标准诊断方法的制定

• 批准号: 13356007
• 负责人: IGARASHI Ikuo
• 金额: $ 28.29万
• 依托单位: Obihiro University of Agriculture and Veterinary Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13356007/
• 关键词: Equine babesiosis; Babesia equi; Babesia caballi; Serological diagnosis; Recombinant antigen; ELISA; Immunochromatographic test; International standard method; 血清診断; 金コロイド粒子; ディップスティック法; 国際標準診断法; ウマバベシア; ラテックス凝集反応; 国際標準

Equine babesiosis, caused by Babesia equi and Babesia caballi, is characterized by fever, anemia, icterus, hepatosplenomegaly, lethargy, and, in some cases, leading to great economic losses in the horse industry. The infected horses often remain carriers of the parasites for a long period and are known to act as sources for subsequent infections to other horses via tick vectors. Therefore, the development of a high-quality system has been required for the serological diagnosis of babesial infection. In the present study, development of international standard diagnostic methods with high specificity and sensitivity for equine babesiosis was examined by gene cloning of Babesia DNA with immune sera, production of recombinant antigens, evaluation of ELISA and imunochromatographic test using those antigens. Novel genes, Be82, Be158 and Bc134, were cloned from B.equi and B.caballi, respectively. ELISA using these recombinant antigens showed the high specificity and sensitivity for the detect … More ion of antibodies against both babesial infections. Productions of recombinant antigens in Escherichia coli, EMA-2 and BC48, were also improved by the construction of truncated genes or modification of culture or induction factors. ELISA using these truncated EMA-2 and BC48 showed high specificity and sensitivity for the detection of antibodies. An immunochromotographic test for simultaneous detection of both B.caballi and B.equi specific antibodies was developed. Evaluation in the detection of sera from uninfected horses and experimentally infected horses showed high sensitivity and specificity as shown in ELISA. A differential single-round and multiplex polymerase chain reaction (PCR) method was also developed for the simultaneous detection of B.caballi and B.equi, by targeting 18S ribosomal RNA genes. In conclusion, EMA-2 for B.equi and BC48 for B.caballi were most excellent as antigens for the diagnosis. ELISA and immunochromatographic test using both antigens have shown to have high specificity and sensitivity for the detection of antibodies. The worldwide experimental comparison and evaluation of these serological tests and genetic analysis will would be feasible tests for internationally recognized diagnosis for equine babesiosis in the world. Less

马巴贝斯虫病由马巴贝斯虫和卡巴贝斯虫引起，以发热、贫血、黄疸、肝脾肿大、嗜睡为特征，在某些情况下，会给养马业造成巨大的经济损失。被感染的马通常在很长一段时间内仍然是寄生虫的携带者，并被认为是随后通过扁虱媒介感染其他马的来源。因此，建立一套高质量的巴贝斯体感染血清学诊断系统是非常必要的。本研究通过免疫血清克隆马巴贝斯虫病DNA基因、制备重组抗原、酶联免疫吸附试验和免疫层析试验，探讨了马巴贝斯虫病国际标准诊断方法的发展。新基因Be82、Be158和Bc134分别是从马和菜豆中克隆得到的。用这些重组抗原进行ELISA检测…具有较高的特异性和敏感性。针对这两种感染的抗体离子更多。在大肠杆菌EMA-2和BC48中，通过构建截短基因或对培养物或诱导因子进行修饰，也可提高重组抗原的产量。用这些截短的EMA-2和BC48进行的ELISA法检测抗体具有很高的特异性和敏感性。建立了一种同时检测卡巴氏杆菌和马巴氏杆菌特异性抗体的免疫层析试验。对未感染马和实验感染马的血清检测结果表明，该方法具有较高的敏感性和特异性。建立了以18S核糖体RNA基因为靶点的差示单轮多重聚合酶链式反应同时检测卡巴氏杆菌和马巴氏杆菌的方法。综上所述，EMA-2和BC48是诊断马巴氏杆菌和卡巴氏杆菌的最佳抗原。使用这两种抗原的酶联免疫吸附试验和免疫层析试验对检测抗体具有很高的特异性和敏感性。这些血清学检测和遗传分析在世界范围内的实验比较和评估将是国际公认的马巴贝斯虫病诊断的可行测试。较少

项目成果

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens

• DOI: 10.1292/jvms.63.1159
• 发表时间: 2001-10-01
• 期刊: JOURNAL OF VETERINARY MEDICAL SCIENCE
• 影响因子: 1.2
• 作者: Xuan, X;Nagai, A;Fujisaki, K
• 通讯作者: Fujisaki, K

Cloning of a truncated Babesia equi gene encoding an 82-kilodalton protein and its potential use in an enzyme-linked immunosorbent assay.

编码 82 千道尔顿蛋白质的截短马巴贝虫基因的克隆及其在酶联免疫吸附测定中的潜在用途。

• 发表时间: 2002
• 期刊: J Clin Microbiol. 40
• 作者: Hirata H;Ikadai H;Yokoyama N;Xuan X;Fujisaki K;Suzuki N;Mikami T;Igarashi I.
• 通讯作者: Igarashi I.

Tamaki Y, Hirata H, Takabatake N, Bork S, Yokoyama N, Xuan X, Fujisaki K et al.: "Molecular cloning of a Babesia caballi gene encoding the 134-kilodalton protein and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay"Clin Diagn

Tamaki Y、Hirata H、Takabatake N、Bork S、Yokoyama N、Xuan X、Fujisaki K 等人：“编码 134 千道尔顿蛋白质的巴贝虫基因的分子克隆及其在酶联免疫吸附剂中的诊断潜力的评估

Ikadai, I., Martin MD, Nagasawa, N., et al.: "Analysisi sof growth-promoting factor for Babesia caballi cultivation"J. Parasitol. 87(6). 1484-1486 (2001)

Ikadai, I.、Martin MD、Nagasawa, N. 等人：“Babesia caballi 培养的生长促进因子分析”J.

Xuan, X., Larsen, A., Ikadai, H., Tanaka, T., Igarashi, I. Et al.: "Expression of Babesia equi merozoite antigen-1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay"J. Clin. Micor

Xu, X., Larsen, A., Ikadai, H., Tanaka, T., Igarashi, I. 等人：“通过重组杆状病毒在昆虫细胞中表达马贝斯虫裂殖子抗原 1 并评估其诊断潜力