CRISPR-Cas-Based Pen-Side Diagnostic Tests for Anaplasma marginale and Babesia bigemina

Abstract

<i>Anaplasma marginale</i> and <i>Babesia bigemina</i> are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of <i>Babesia bigemina</i> and <i>Anaplasma marginale</i> in infected animals relies primarily on microscopic examination of Giemsa-stained blood or organ smears, which has limited sensitivity. Molecular methods offer higher sensitivity but are costly and impractical in resource-limited settings. Following the development of a pen-side test for detecting <i>Theileria parva</i> infections in cattle, we have created two additional CRISPR-Cas12a assays targeting <i>Anaplasma marginale</i> and <i>Babesia bigemina</i>. The assays target the major surface protein 5 (MSP5) for <i>A. marginale</i> and rhoptry-associated protein 1a (RAP1a) for <i>B. bigemina</i>. These additional tests involve a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a detection with a lateral strip readout. Results demonstrate high specificity, with no cross-reactivity against other tick-borne parasites, and a limit of detection down to 10² DNA copies/µL of each target marker. The findings pave the way for sensitive and user-friendly pen-side tests to diagnose <i>A. marginale</i> and <i>B. bigemina</i> infections.