Development of international standard diagnostic method for equine babesiosis

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Equine babesiosis, caused by Babesia equi and Babesia caballi, is characterized by fever, anemia, icterus, hepatosplenomegaly, lethargy, and, in some cases, leading to great economic losses in the horse industry. The infected horses often remain carriers of the parasites for a long period and are known to act as sources for subsequent infections to other horses via tick vectors. Therefore, the development of a high-quality system has been required for the serological diagnosis of babesial infection. In the present study, development of international standard diagnostic methods with high specificity and sensitivity for equine babesiosis was examined by gene cloning of Babesia DNA with immune sera, production of recombinant antigens, evaluation of ELISA and imunochromatographic test using those antigens. Novel genes, Be82, Be158 and Bc134, were cloned from B.equi and B.caballi, respectively. ELISA using these recombinant antigens showed the high specificity and sensitivity for the detect … More ion of antibodies against both babesial infections. Productions of recombinant antigens in Escherichia coli, EMA-2 and BC48, were also improved by the construction of truncated genes or modification of culture or induction factors. ELISA using these truncated EMA-2 and BC48 showed high specificity and sensitivity for the detection of antibodies. An immunochromotographic test for simultaneous detection of both B.caballi and B.equi specific antibodies was developed. Evaluation in the detection of sera from uninfected horses and experimentally infected horses showed high sensitivity and specificity as shown in ELISA. A differential single-round and multiplex polymerase chain reaction (PCR) method was also developed for the simultaneous detection of B.caballi and B.equi, by targeting 18S ribosomal RNA genes. In conclusion, EMA-2 for B.equi and BC48 for B.caballi were most excellent as antigens for the diagnosis. ELISA and immunochromatographic test using both antigens have shown to have high specificity and sensitivity for the detection of antibodies. The worldwide experimental comparison and evaluation of these serological tests and genetic analysis will would be feasible tests for internationally recognized diagnosis for equine babesiosis in the world. Less