Advancing disease surveillance in rhinoceroses: A multiplex real-time PCR assay for detecting Theileria bicornis and Babesia bicornis

Black (Diceros bicornis) and white (Ceratotherium simum) rhinoceroses in Southern Africa face multiple threats, including poaching, habitat loss, and translocation stress. Infections with Theileria bicornis (in both rhino species) and Babesia bicornis (confirmed only in black rhinos) add further health risks, with stressors such as translocation potentially increasing susceptibility. Effective management requires sensitive molecular diagnostic assays for accurate detection and surveillance. To address this, we developed a multiplex qPCR assay (MqTbBb) using species-specific TaqMan™ minor groove binder (MGB) probes for the simultaneous detection of T. bicornis and B. bicornis. The assay targets 18S rRNA gene regions, amplifying an 87 bp fragment for T. bicornis and a 51 bp fragment for B. bicornis, with efficiencies of 100 % and 98 %, respectively. Probit analysis determined a 95 % Limit of detection of 1.00 × 10⁻⁶ % and 6.27 × 10⁻⁶ % equivalent parasitized erythrocytes for T. bicornis and B. bicornis, respectively. No cross-reactivity was observed with other related protozoa tested. A total of 223 field samples from rhinos (101 black and 122 white) in Mpumalanga province were screened using both the MqTbBb and Reverse Line Blot (RLB) hybridization assays. The MqTbBb detected T. bicornis in 57 % of black and 99 % of white rhinos, with co-infections in 40 % of black rhinos. RLB detected T. bicornis in 96 % of black and 95 % of white rhinos, with a Babesia catch-all probe signal in 75 % and 32 %, respectively. B. bicornis was not detected by RLB and was never detected as a single infection by qPCR. These findings highlight high T. bicornis prevalence and rare B. bicornis infections (co-infections). The MqTbBb assay strengthens detection, surveillance, and conservation efforts.