National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology; Key Laboratory of Parasite and Vector Biology, National Health and Family Planning Commission, Shanghai, People's Republic of China.
Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.
Parasit Vectors. 2018 Jul 3;11(1):379. doi: 10.1186/s13071-018-2951-0.
Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp. Babesia microti is one of the primary causative agents of human babesiosis. To better understand the status of the disease, discovering key biomarkers of the different infection stages is crucial.
This study investigated B. microti infection in the mouse model from 0 to 270 days post-infection (dpi), using blood smears, PCR assays and ELISA. PCR assays showed a higher sensitivity when compared to microscopic examination. Specific IgG antibodies could be detected from 7 days to 270 dpi. Two-dimensional electrophoresis was combined with western blotting and mass spectrometric analysis to screen for specific reactive antigens during both the peak parasitaemia period (7 dpi) and IgG antibody response peak period (30 dpi) by the infected mice plasma. The 87 positive reactive proteins were identified and then expressed with the wheat germ cell-free system. Protein microarrays of all 87 targeted proteins were produced and hybridized with the serial plasma of infected mice model. Based on the antigen reaction profile during the infection procedure, 6 antigens were selected and expressed in Escherichia coli. Due to an early response to IgM, lower immunoreactivity levels of IgG after two months and higher immunoreactivity level IgG during nine months, four recombinant proteins were selected for further characterization, namely rBm2D97(CCF75281.1), rBm2D33(CCF74637.1), rBm2D41(CCF75408.1) and rBm7(CCF73510.1). The diagnostic efficacy of the four recombinant protein candidates was evaluated in a clinical setting using babesiosis patient plasma. The rBm2D33 showed the highest sensitivity with a positive rate of 62.5%. Additional characterization of the two candidate proteins using a mouse vaccination assay, demonstrated that rBm2D41 could reduce peak parasitaemia by 37.4%, indicating its efficacy in preventing severe babesiosis.
The detection technologies of microscopic examination, PCR assays and antibody tests showed different sensitivities and accuracy during the different stages of B. microti infection. Antibody detection has a unique significance for B. microti infection in the asymptomatic stages. Using immunoreactivity profiles, biomarkers for disease progression were identified and represent useful information for future the diagnosis and vaccine development for this serious disease of public health significance.
巴贝斯虫病是由巴贝斯虫属寄生虫侵入红细胞引起的。微小巴贝斯虫是引起人类巴贝斯虫病的主要病原体之一。为了更好地了解疾病的现状，发现不同感染阶段的关键生物标志物至关重要。
本研究通过血液涂片、PCR 检测和 ELISA 检测，在感染后 0 至 270 天（dpi）的小鼠模型中研究了微小巴贝斯虫感染。PCR 检测比显微镜检查具有更高的灵敏度。特异性 IgG 抗体可在 7 天至 270 dpi 时检测到。通过感染小鼠的血浆，用二维电泳结合 Western 印迹和质谱分析筛选出在高峰虫血症期（7dpi）和 IgG 抗体反应高峰期（30dpi）的特异性反应抗原。鉴定出 87 个阳性反应蛋白，并在小麦无细胞体系中表达。用所有 87 个靶向蛋白的蛋白微阵列与感染小鼠模型的系列血浆杂交。根据感染过程中的抗原反应谱，选择 6 种抗原并在大肠杆菌中表达。由于对 IgM 的早期反应，两个月后 IgG 的免疫反应性水平较低，九个月后 IgG 的免疫反应性水平较高，因此选择了四个重组蛋白进一步表征，即 rBm2D97（CCF75281.1）、rBm2D33（CCF74637.1）、rBm2D41（CCF75408.1）和 rBm7（CCF73510.1）。使用巴贝斯虫病患者血浆在临床环境中评估了这四种重组蛋白候选物的诊断效果。rBm2D33 的阳性率最高，为 62.5%。使用小鼠疫苗接种试验对两种候选蛋白进行进一步表征，结果表明 rBm2D41 可使峰值寄生虫血症减少 37.4%，表明其在预防严重巴贝斯虫病方面的有效性。
在微小巴贝斯虫感染的不同阶段，显微镜检查、PCR 检测和抗体检测技术的敏感性和准确性不同。抗体检测对无症状阶段的微小巴贝斯虫感染具有独特的意义。通过免疫反应性谱，确定了疾病进展的生物标志物，为这种严重的公共卫生疾病的诊断和疫苗开发提供了有用的信息。
