In vitro induction of the sexual stage of Babesia ovata enhances infection efficiency of Haemaphysalis longicornis ticks in a semi-artificial feeding model.

Haemaphysalis longicornis is an important tick species that transmits multiple pathogens to animals and humans, and its global distribution is expanding. Understanding tickpathogen interaction requires efficient tick infection models. However, a cost-effective, rapid, and easy-to-operate model remains lacking. This study aimed to improve the infection efficiency of a semi-artificial feeding model by in vitro induction of Babesia ovata using dithiothreitol. Induction was confirmed by morphological observation, reverse transcription-quantitative PCR analysis of sexual-stage marker genes (HAP2, 6CYS, CCP2, and CCP3), and immunofluorescence detection of HAP2 and CCP3 proteins. Ticks were subsequently fed blood containing induced or non-induced B. ovata (at 1-2% or 4-5% parasitemia) or red blood cell controls. Infection rates were assessed by nested PCR detection of B. ovata in tick embryo samples. Embryos derived from ticks that fed sexually induced parasites exhibited significantly higher infection rates compared to those from ticks that fed non-induced parasites (18.87% (10/53) vs. 1.92% (1/52), p = 0.0080). The highest infection rate (22.22% (6/27)) occurred in the induced 1-2% parasitemia group, with no infection detected in controls. These findings demonstrate that in vitro induction of B. ovata sexual stage enhances tick infection efficiency. Incorporating sexual-stage induction into the existing semi-artificial feeding method may further enhance the efficiency of establishing B. ovata infection in ticks, providing a valuable approach for studies of tick-pathogen interactions.