A Sero-cpidemiological Survey And Preliminary Research Of Glideosome Associated Protein Genes Of Chinese Babesia Motasi For Small Ruminants

Ovine babesiosis is an economically important tick-borne haemo-protozoosis of small ruminants. Thisdisease is generally characterized by fever, anaemia, jaundice, haemoglobinuria clinical symptoms andeven death in seriously infected animals. It’s widespread, consistently with distribution of its vectorticks, in tropical, subtropical and temperate areas of the world and causes serious problems for thesustainable and healthy development of sheep industry. The research of the disease in China is late andits pathogens have their characteristics. The primary studies were performed for sero-epidemiologicalstatus and glideosome associated protein genes of Chinese Babesia motasi infective for small ruminantsin the present study.The enzyme-linked immunosorbent assay (ELISA) developed by VVBD laboratory using solublemerozoite antigens from in vitro culture is specific for Chinese B. motasi with low pathogenicity forsmall ruminants, such as Babesia sp. BQ1(Lintan) and Babesia sp. Tianzhu. Specificity of the ELISAwas95.5%. In this study, a total of3204serum samples from small ruminants in44prefectures of22provinces located in different districts of China were tested for antibodies against the parasites. Theresults indicated that there were infected animals by the parasites in almost all of the flocks ofsheep/goats. The positive rates of investigated provinces were significantly various from1.6%to91.0%,and the average positive rate was43.5%.Oligonucleotide primers for rapid amplification of cDNA ends (RACE) were designed in the conservedregions of7component protein gens of glideosome, such as gap45, gap50, myosin A, mlc, actin,aldolase and trap based on the gene sequences of apicomplexan parasites submitted to GenBank. Thefull-length sequences of these genes were amplified by RACE and the sequence characteristics wereanalyzed using bioinformatics methods. The partial or complete open reading frames (ORF) of thosegenes were further inserted into prokaryotic expression vector and then transformed into BL21(DE3)pLys to express by inducing with IPTG. The purified fusion proteins, with MagneGST ProteinPurification System, were injected into rabbits to produce polyclonal antibodies. Specificities and titersof the polyclonal antibodies were determined by ELISA and Western blot assay. The recognizations ofnative proteins in B. motasi merozoites by prepared polyconal antibodies were evaluated by westernblot. The results indicated that the full-length cDNA of gap45, gap50, myosin A, mlc, actin, aldolaseand trap, were664bp,1391bp,2538bp,773bp,1110bp,1199bp and2254bp, and open reading frame(ORP) were567bp,1194bp,2514bp,606bp,1005bp,1074bp and2100bp in size, respectively. Thesimilarities of sequences with these of apicomplexan parasites were various, ranging from28%to98%.Titers of the polyclonal antibodies were more than1:25600, and the polyclonal antibodies against toGAP45, GAP50, Myo A and Aldolase could specifically recognize the native proteins in B. motasimerozoites.This is the first large-scale sero-epidemiological survey for B. motasi infection in sheep and goats,which would provide the firsthand comprehensive epidemiological data during performing a nationalprogramme for control ovine babeisiosis in future. In addition, during the study on glideosome associated protein genes, we obtained some of fundamental biological resources for further exploringmechanisms of Babesia motion and invasion host cells.