The Molecular Survey Of Babesiosis And The High Throughput-screening Of Diagnostic Antigens Of Babesia Microti

Babesiosis is a typical zoonotic, emerging disease caused by a tick-borne intraerythrocytic protozoan infection with Babesia spp., and it also can be transmitted by blood transfusion. Babesiosis imposes an increasing threat to public-health. To the patients with immunocompromised, the clinical manifestations can be more serious, and even having hazards of life-threatening.Object: To carry on the survey on the febrile cases and evaluate the infections of Babesia microti in the areas on China-Myanmar border,and the screening and analysis of candidate molecular of diagnostic antigen were related with babesiosis.Methods: 1)DNA was extracted from 200 blood samples obtained from the febrile patients excluded infections of malaria during June to August, 2014. The molecular survey by Nested PCR applying the primers based on 18 srRNA and the beta-tubulin protein of B.microti were carried out and all these positive samples were confirmed by sequencing and phylogenetic trees were produced. 2) Construction of the native database on these sequences belonging to Signal peptides, Repeated sequences and homologue sequences between B. microti, B. bovis and Plasmodium falciparum from PiroplasmaDB. cDNA of B. microti was extracted from the blood of BALB/c mice infected by B. microti. All of these target gene fragments were amplified from cDNA as a template. The amplification products of target gene fragments were connected with plasmid pEU-His by In-Fusion cloning technology. Finally,the plasmids containing the target gene fragment were identified by cloning PCR and sequencing. Proteins were expressed with wheat germ cell-free protein synthesis system after the recombinant plasmids being extracted by extraction kit. The expressed proteins were identified by western-blot technique. The correctly expressed proteins were screened with sera from BALB/c mice infected by B. microti in different stages by protein microarray.Results: 1)By PCR assay and sequencing, 2(1%) of 200 febrile patients with malaria-like symptoms were confirmed to be infections of B. microti. According to the sequences of 18 srRNA and the beta-tubulin, the clade of B.microti detected in our research were zoonotic. 2) There were 222 target gene fragments, and 215 target gene fragments were amplificated successfully by PCR., One hundred and eighty six recombinant plasmids were constructed by In-Fusion cloning,and there were 167 recombinant proteins expressed successfully by wheat germ cell-free protein synthesis system. Ten recombinant antigens were identified using protein microarray from 167 recombinant proteins with sera from BALB/c mice infected by B. microti. These antigens are BmSP44, BmRS8, BmRS21, BmRS28, BmRS29, BmHP33, BmHP37, BmHP41, BmHP42 and BmHP43.Conclusion: Babesiosis caused by B. microti is emerging in China-Myanmar border areas in Yunnan province, P.R. China, where is the malaria endemic areas as well. Ten recombinant antigens were identified by sera from BALB/c mice infected by B. microti. However, the application of these antigens in immuno-diagnosis in human infections of B.microti remains to be further verified.