Development of international standard diagnostic method for equine babesiosis

马巴贝斯虫病是由马巴贝斯虫和卡巴贝斯虫引起的，其特征是发烧、贫血、黄疸、肝脾肿大、嗜睡，在某些情况下，会给马业造成巨大的经济损失。受感染的马通常在很长一段时间内仍然是寄生虫的携带者，并且已知通过蜱虫媒介成为随后感染其他马的来源。因此，巴贝虫感染的血清学诊断需要开发高质量的系统。本研究通过免疫血清对巴贝斯虫DNA的基因克隆、重组抗原的制备、酶联免疫吸附试验（ELISA）的评价以及抗原的免疫层析检测，探讨建立具有高特异性和敏感性的国际标准的马巴贝斯虫病诊断方法。新基因Be82、Be158和Bc134分别从马b.b eequi和caballi中克隆得到。利用这些重组抗原进行ELISA检测，对两种巴贝虫感染均有较高的特异性和敏感性。在大肠杆菌EMA-2和BC48中，重组抗原的产生也可以通过构建截断基因或修饰培养或诱导因子来提高。使用这些截断的EMA-2和BC48进行ELISA检测抗体具有较高的特异性和敏感性。建立了一种同时检测caballi和B.equi特异性抗体的免疫色谱检测方法。ELISA对未感染马和实验感染马血清的检测结果显示出较高的敏感性和特异性。利用18S核糖体RNA基因，建立了单轮和多重聚合酶链反应（PCR）方法，同时检测caballi和B.equi。结果表明，EMA-2和BC48分别是马b.a i和卡巴利b.a i的最佳诊断抗原。ELISA和免疫层析检测两种抗原对抗体的检测具有较高的特异性和敏感性。在世界范围内对这些血清学检测和遗传分析进行实验比较和评价，将成为国际公认的马巴贝斯虫病诊断的可行检测方法。少

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