Screening, Cloning And Expression Of Diagnostic Antigens For Babesia Microti Infection

Since the discovery of parasitic inclusions in the erythrocytes of cattle in Romania by Victor Babes in the end of the19th century, newly recognised babesial pathogens continue to emerge around the world causing the babesiosis and posing substantial public health impact on livestock and human. The babesia parasite is transmitted by ixodid ticks and causes a host-mediated pathology and erythrocyte lysis, resulting in anemia, hyperbilirubinuria, hemoglobinuria, and possibly organ failure. There are seven Babesia spp. infecting human worldwide, among them, B. microti and Babesia divergens is the most widespread and severe species.Up to now, extensive studies on the sero-diagnosis of babesia infection in veterinary medicine have been reported. However, the diagnosis of human babeisia infection is still mainly based on the microscopy identification, combined with the clinical manifestations and epidemiology information. Little has been seen of the study on the serological and molecular diagnostic methods for the human infection. The morphology identification method is less sensitive and the babesia may be confused with malaria parasite, which may often result in clinical misdiagnosis and delay of right treatment. Therefore, it is urged to develop quick and effective diagnostic reagents for human babesiosis. To this end, identification of diagnostic antigen, particularly, recombinant antigen for B. microti infection is critical.Objective This study aims at the identification of specific and sensitive antigens for immunodiagnosis of B. microti infection through cloning known immunogenic proteins, immunoscreening the cDNA library and proteome analysis.Methods1) Synthesis of selected gene sequences and primers of B. microti potential diagnostic antigens based on the information search, and cloning and expression of the proteins;2) construction of a cDNA-Iibrary of B. microti (a standard strain:Babesia microti ATCC(?)PRA-99TM) for immunoscreening with the pooled sera of mice infected with B. microti to obtain immuno-reactive clones;3) evaluation of the recombinant proteins obtained in serological detection of B. microti infection of mice and examination of the cross reaction with malaria patient sera by ELISA test;4) extraction of B. microti crude antigen to perform a two dimension-western blotting probed with the mice sera collected at7day and30day post-infection; matching the specific spots on the nitrocellulose membrane with the silver stained gel and identifying immuno-reactive antigens by mass spectrum identification.Results1) The gene sequences of9proteins from8Babesia spp. previously reported were synthesized and cloned, they are:BmSA1(from B. microti), thrombospondin-related anonymous protein (from B. gibsoni), rhoptry-associated protein1(from B. divergens), rhoptry-associated protein1(from B. divergens, B. bovis, B. bigemina and B. ovis), merozoite antigen1(from B. equi) and BoP29(from B. orientalis), among them,6proteins were expressed;2) A cDNA library of B. microti was constructed, of which the titer was5.2×105plaque forming unit (pfu)/ml. The inserted fragment length of the library ranged from500to3000bp with a recombination efficiency91.0%. Six positive clones, Bm2, Bm4, Bm6, Bm7, Bm9and Bm15were found and their inserted fragment length were about633,614,1073,890,1001and1032bp, respectively. Sequence analysis revealed that all the6clones contained open reading frames. The deduced amino acid sequences of the6clones contained159,100,254,217,60and244amino acid residues, with Mr of17900,11400,28600,24000,6800and28900respectively. Of the6clones, Bm4, Bm6, Bm7, Bm15were expressed.3) Evaluated by ELISA, the sensitivity of Bm4, Bm6, Bm7, Bm15and BmSA1in examining the sera of infected mice was15%,55%,100%,80%,100%, respectively, while the specificity was100%,100%,100%,90%,100%, respectively. It was noted that the recombinant proteins of other5Babesia spp. that we cloned were cross reactive with the sera of B. microti infected mice showing a cross reaction rate from40%to90%. Whereas, when a panel of malaria patient serum (15falciparum,15vivax and10normal for control) was tested with Bm4、Bm6、 Bm7. Bm15and BmSAl, the specificity was found to be87.7%-100%.4) In the proteome analysis,8proteins spots were recognized by the7d post-infection serum and51spots by30d post-infection serum of mice, while the mass spectrum analysis identified8and79proteins respectively, which remain to be further investigated.Conclusion Through three different approaches, this study obtained several potential immuno-diagnostic antigens of babeisia, two of them, the recombinant Bm7and Bm15, were highly sensitive and specific in detection of B. microti infected mice serum. However, the application of the antigens in immuno-diagnosis of human infection of B. microti remains to be further verified.