Contagious agalactia (CA) is a serious disease notifiable to the World Organisation for Animal Health (OIE) causing severe economic losses to sheep and goat producers worldwide. Mycoplasma agalactiae, considered as its main etiological agent, inflicts a variety of symptoms in infected animals, including keratoconjunctivitis, mastitis, arthritis, ankylosis, abortions, stillbirths and granular vulvovaginitis. Despite its significance, developing a successful vaccine remains elusive, mostly due to the lack of knowledge about M. agalactiae’s pathogenicity factors and pathogenic mechanisms, including its "core" antigens. The aim of this study was to identify, characterize and express antigenic proteins of M. agalactiae as potential vaccine candidates. Predicted proteins of type strain PG2 were analyzed using bioinformatic algorithms to assess their cellular localization and to identify their linear and conformational epitopes for B cells. Out of a total of 156 predicted membrane proteins, three were shortlisted as potential antigenic surface proteins, namely [MAG_1560 (WP_011949336.1), MAG_6130 (WP_011949770.1) and P40 (WP_011949418.1)]. These proteins were expressed in recombinant Escherichia coli strains. Purified proteins were evaluated for their antigenicity using Western blot and ELISA using sera of M. agalactiae-naturally infected and non-infected sheep and goats. All 3 proteins were specifically recognized by the tested sera of M. agalactiae-infected animals. Also, specific rabbit antisera raised against each of these 3 proteins confirm their membrane localization using TritonX-114 phase partioning, Western and colony immunoblotting. In conclusion, our study successfully identified P40 (as proof of concept and validation) and two novel antigenic M. agalactiae proteins as potential candidates for developing effective CA vaccines.

传染性无乳（CA）是世界动物卫生组织（OIE）应通报的严重疾病，对全世界绵羊和山羊生产者造成了严重的经济损失。无乳支原体被认为是其主要病原体，在被感染的动物中引起多种症状，包括角膜结膜炎，乳腺炎，关节炎，强直性，流产，死产和颗粒状阴道炎。尽管具有重要意义，但开发成功的疫苗仍然难以实现，这主要是由于缺乏对无乳分枝杆菌的致病因子和致病机制（包括其“核心”抗原）的了解。本研究的目的是鉴定，表征和表达无乳分枝杆菌的抗原蛋白。作为潜在的疫苗候选者。使用生物信息学算法分析了PG2型菌株的预测蛋白，以评估其细胞定位并鉴定B细胞的线性和构象表位。在总共156种预测的膜蛋白中，有3种被选为潜在的抗原表面蛋白，即[MAG_1560（WP_011949336.1），MAG_6130（WP_011949770.1）和P40（WP_011949418.1）]。这些蛋白质在重组大肠杆菌菌株中表达。使用蛋白质印迹法和天然感染的无乳分枝杆菌的血清，通过蛋白质印迹和ELISA评估纯化的蛋白质的抗原性。无乳分枝杆菌的血清可特异性识别所有3种蛋白质感染的动物。同样，针对这3种蛋白质中的每一种产生的特异性兔抗血清也可以通过TritonX-114相分配，Western和菌落免疫印迹来确认它们的膜定位。总之，我们的研究成功地鉴定了P40（作为概念验证和验证）和两种新型抗原性无乳支原体蛋白作为开发有效CA疫苗的潜在候选者。