A self-nanoemulsifying drug delivery system for the treatment of Theileria annulata infections in cattle short tile: treatment of Theileria annulata infections

Recently, an increase in the rate of failure is reported using Buparvaquone (BPQ) for the treatment of Theileria annulata infections, especially those bearing V135A and P253S mutations on the Cytochrome b gene of the parasite mitochondrial genome. To develop and characterize a Self-Nanoemulsifying Drug Delivery System (SNEDDS) loaded with BPQ for the treatment of T. annulata infections in cattle. Two different formulas of BPQ-SNEDDS was prepared and characterized using different physio-chemical techniques. Then, it was used for the treatment of T. annulata infections in cattle. Briefly, 100 suspected cows and calves to be infected with T. annulata in different regions of Sulaimaniyah, were involved, from May 2025 to December 2025. After collection of blood samples, the positive cases were initially confirmed by stained blood smear and later by molecular testing targeting specifically the V4 hypervariable region of the 18 S ribosomal RNA gene (450 bp). Then, 2 positive products were sequenced, confirmed by the NCBI and evolutionary relationships were inferred. After that, protozoan schizont was cultured, propagated and inoculated to animals, followed by 2 treatments with formulated BPQ, 7 and 13-days post-inoculation, respectively. Finally, absolute quantification of target gene was analyzed to find the effect of formulated BPQ on pathogen load and disease progression. Characterizations of developed BPQ formula were within acceptable ranges, as Dynamic light scattering showed the mean particle size, polydispersity index and zeta potential of 117. ± 4.4 nm, 0.48, and − 14.9 mV, respectively. Whereas, the TEM revealed that BPQ-SNEDDS particles were nearly regular and seemly spherical, without aggregation. The FTIR spectrum showed the presence of the OH group, C-H/C-O stretching, and O-H bending of H2O molecules. The UV–Vis spectroscopy showed a maximum absorption at 260 nm with elevated absorbance intensity. There were identical XRD spectra and the broad diffraction peaks at 20°. Loaded nanoemulsions had more diffused X-ray scattering than blank-SNEDDS due to a small crystallite size of the BPQ. Additionally, the entrapment efficiency was 99%, and the drug loading capacity was 7.5%. Then, upon examination of the collected blood samples, 81/100 of samples were confirmed to be T. annulata using qRT-PCR. The 18 S rRNA gene sequences have been confirmed ZZA (PQ394724.1) and ZZA3 strains (PX247965.1) with an evolutionary link within the Middle East and neighboring regions. After animal inoculation and starting treatments, on day 13, the untreated group exhibited significant pathogen proliferation, while therapeutic interventions significantly reduced the infectious burden ( p < 0.0001). BPQ-SNEDDS was successfully prepared and treated Theileria annulata infections in cattle dose dependently.