Screening for biomarkers reflecting the progression of Babesia microti infection

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Babesia microti; Cell free expression system; Expression and evaluation of proteins; Mice model; Protein microarray; Screening biomarker; Strain ATCC®PRA-99TM.

Fig. 1

Dynamics of parasitaemia, PCR analysis and antibody level in BALB/c mice with B. microti model. Each point represents the mean ± SD

Fig. 2

Two-dimensional western blotting. Immune blot patterns of B. microti crude proteins were recognized by 7 dpi mice plasma and 30 dpi mice plasma. a and d provide the results of two-dimensional western blotting (2D-western blotting) incubated by normal mice plasma; b and e show hybridized membranes incubated by 7 and 30 dpi mice plasma, respectively; c and f represent the images of silver gel to identify of the crude proteins. Blue circles represent the different areas compared with negative control for B. microti crude proteins

Fig. 3

Western blot analysis of the expression levels of B. microti proteins. M represents protein size marker, Target proteins are marked with red arrows

Fig. 4

Immunoreactivity patterns of B. microti proteins. a and b show that a total 87 antigens exhibit IgG and IgM, respectively, antibody responses to different period plasma samples isolated by mice inoculation (0, 3, 7, 14, 21, 30, 60, 120, 150 and 270 dpi). Immunoreactivity profiles were clustered according to fluorescence intensity values (M-values). On the heatmap, the scale is from 0 to 3500 and 0 to 2000 in IgG and IgM antibody responses, respectively

Fig. 5

Expression, purification and evaluation of a specific antibody against the recombinant proteins. Lane M: protein size marker; Lane 1: recombinant clone before induction; Lane 2: recombinant clone after induction; Lane 3: purified recombinant protein. Recombinant proteins were evaluated using different periods plasma samples from mice inoculated B. microti (0, 3, 7, 14, 21, 30, 60, 120, 150 and 270 dpi) by ELISA

Fig. 6

Comparison of the sensitivity and specificity of B. microti recombinant antigens. a ELISA reactivity of B. microti recombinant antigens and multi-antigens to babesiosis patient plasma (n = 8). b ELISA reactivity of B. microti recombinant antigens to P. vivax and P. falciparum patient plasma (n = 10)

Fig. 7

The evaluation and effect of immunization with Bm2D41 and Bm7. a The levels of IgG immunized with Bm2D41 and Bm7 in BALB/c mice. b The parasitaemia and antibody levels of BABL/c mice after challenge infection with B. microti. Asterisks indicate statistically significant differences [*P < 0.05, **P < 0.005, ***P < 0.0001 (compared to either the PBS-immunized or non-immunized BABL/c mice)]. The solid and dashed lines denote parasitaemia and antibody titter, respectively