| Summary: | <i>Theileria equi</i> (<i>T. equi</i>) and <i>Babesia caballi</i> (<i>B. caballi</i>) are the causative pathogens of Equine piroplasmosis (EP), a disease that has brought huge economic losses and great restrictions to the global equine industry. Rapid and accurate diagnostic methods are critical for the effective monitoring of the disease. In this study, we developed novel competitive ELISA methods and western blot assays based on the EMA1 or Bc48 proteins to detect antibodies against <i>T. equi</i> or <i>B. caballi</i>, respectively. In the novel cELISA, horseradish peroxidase (HRP)-labeled monoclonal antibodies are used in place of enzyme-conjugated secondary antibodies, in order to speed up the entire procedure. These methods have high sensitivity and no cross-reactivity with antibodies against other equine diseases. In the newly developed western blot assays, we optimized the dilution of <i>T. equi</i> or <i>B. caballi</i> positive serum samples to 1:200. Compared with the commercially available kit, both the novel cELISA assay and the western blot assay showed high coincidence rates in detecting antibodies against <i>T. equi</i> and <i>B. caballi</i>. Taken together, the novel cELISA and the western blot assays for detecting antibodies against <i>T. equi</i> or <i>B. caballi</i> have the potential to rapidly test for <i>T. equi</i> or <i>B. caballi</i> and to contribute to the surveillance and control of this disease.
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