Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627. Pampulha, CEP 31270-901, Belo Horizonte, Minas Gerais, Brazil.
Departamento de Medicina Veterinária, Universidade Federal de Lavras, Campus Universitário, Caixa Postal 3037, CEP 37200-000, Lavras, Minas Gerais, Brazil.
Microb Pathog. 2020 Oct;147:104345. doi: 10.1016/j.micpath.2020.104345. Epub 2020 Jun 13.
Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.
布氏杆菌病血清学诊断仍然是一个挑战，而疫苗接种是控制牛布鲁氏菌病的主要措施，目前使用最广泛的疫苗是 S19 和 RB51。因此，为了有助于控制布鲁氏菌病，我们使用二维（2D）免疫印迹方法来寻找可用于血清学诊断的免疫原性蛋白，特别是具有用于区分感染动物和接种疫苗动物（DIVA）策略的能力。使用 S19 接种动物、RB51 接种动物、自然感染布鲁氏菌病动物和未接种血清阴性动物的混合血清在 2D 免疫印迹中分析布鲁氏菌 abortus 2308 的免疫蛋白组图谱。对针对各组血清具有不同免疫反应性的抗原进行评估，显示出三种特别重要的蛋白质：S19 接种动物免疫反应性的 MDH（苹果酸脱氢酶）、感染动物反应性的 SOD（超氧化物歧化酶）和针对接种动物（S19 和 RB51）血清反应的 ABC 转运蛋白（多种糖 ABC 转运蛋白）。这三种蛋白质在大肠杆菌中进行了表达和测试，并在间接 ELISA（I-ELISA）中进行了测试。对于 MDH，在 I-ELISA 中，比较接种动物（与使用的疫苗无关）与血清阳性和血清阴性动物，结果显示存在显著差异。关于使用 SOD 的 I-ELISA 的数据表明，与所有其他组相比，未接种自然感染动物的血清在 OD 值上存在显著差异。然而，在 I-ELISA 中使用 ABC 转运蛋白作为抗原时，接种动物（S19 和 RB51）和未接种自然感染动物的血清在 OD 值上没有显著差异，但它们与未接种血清阴性动物均存在显著差异。综上所述，2D 免疫印迹分析和初步 I-ELISA 结果表明，MDH 和 SOD 的联合使用可能成功地应用于基于 LPS 自由蛋白的血清学诊断方法，以检测牛布鲁氏菌病，并区分接种疫苗动物和自然感染动物，特别是在接种疫苗后的早期阶段。
