Development Of An ELISA For Diagnosis Of Babesia Bovis Infection

Babesiosis is a tick-borne hematic protozoosis, formerly known as piroplasmosis, which is causedby parasites of Babesia genus parasiting in host red blood cell. The disease is widely popular all overthe world and bring serious harm to the livestock industry with the main clinical features as fever,anemia, jaundice and hemoglobinuria. There are six Babesia spp. parasitizing in cattle, B. bigemina, B.bovis, B. major, B. ovata, Babesia U. sp and B. orientalis in China, and B. bovis and B. bigemina are ofthe highest incidence and mortality. Both of them share the same vector tick–Rhipicephalus microplusand clinically showed as mixed infection. In this study RAP-1C-terminal fragment of B. bovis wasprokaryotic expressed and an indirect ELISA specific for B. bovis was developed using the recombinantprotein. Then the ELISA was applied evaluate the prevalence of infection with B. bovis in someprovinces. The major works were presented as following:1. The coding sequence of rhoptry-associated protein (RAP-1)of bovis Babesia was analyzed andits similarity alignment with other Babesia and Theileria species was performed with bioinformaticsmethod in order to screen specific and sensitive C-terminal fragment. The RAP-1gene was amplifiedwith specific primers and cloned and then prokaryotic expressed. The reactionogenicity and specificityof recombinant protein was analyzed by Western-blot. The expressed product was confirmed to be about51ku and soluble in supernatant by SDS-PAGE, its expression level was1.51mg/ml. Itsreactionogenicity was identified and there was not cross-reactivity between recombinant protein andpositive sera of other Babesia、Theileria and Anaplasma species. The results demonstrate that therecombinant RAP-1might be a useful antigen for the detection of antibodies to B. bovis and establisheda platform for development of diagnostic method.2. The recombinant protein–BORCT was Prokaryotic expressed, and obtained high purity of therecombinant protein using bead affinity purification method. An indirect ELISA specific for B. boviswas established with the purified recombinant protein. The results showed that the specificity andsensitivity of this method were96.5%and94.2%respectively, when29.6%of antibody rate was chosenas positive threshold via analyzing the data of86negative sera and69positive sera with MedCalcsoftware. And there is no cross-reaction between BORCT and positive serum of other babesia andtheilaria species. Therefore the method were available for distinguish-ing the infection with B.bovis and B.bigemina.3. The ELISA was applied to1472sera collected from13provinces to evaluate the prevalence ofinfection with B. bovis. The result showed the positive rate was between6.40%—68.49%and positivesamples present in every procinces. Associating the natural geographic distribution of ticks, it issuggested that B. bovis was widely distributed in our country, and an effective and efficient methodshould be established to control the spread of the disease prevalence.