Abstract
BackgroundBabesiosis 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.ResultsThis 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.ConclusionsThe 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.
Highlights
Babesiosis is caused by the invasion of erythrocytes by parasites of the Babesia spp
PCR analysis and specific antibody level in BALB/c mice infected by B. microti All 10 inoculated mice were infected successfully with B. microti
After 20 dpi, few parasites were detected into the chronic stage of infection, and after 60 dpi, no parasitaemia was observed up to when the experiment was ended at 270 dpi (Table 1)
Summary
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. The zoonotic parasitic disease, babesiosis, is caused by the intraerythrocytic protozoans Babesia spp. Babesia spp. are usually transmitted by Ixodes ticks, blood transfusions or from mother to child transplacentally [1,2,3]. Babesia microti has been implicated in the majority of clinical cases in the USA; including both tick-borne and transfusion-transmitted babesiosis (TTB) [8]. This is reflected in a survey of TTB over the period of 1979-2009, where B. microti was attributed as the infectious species [8]. Since there is a lack of a Food and Drug Administration (FDA) approved blood screening, B. microti has become a high-risk pathogen that is transmitted by blood transfusions in the USA [6]
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