Establishment and Optimisation of Anti-Babesia microti Drug Efficacy Evaluation Method

Abstract

BackgroundBabesiosis, an infectious zoonotic parasitic disease that occurs globally, is most commonly caused by Babesia microti. For a severe infection, a combination of exchange transfusion and support-therapy is used and, for a mild infection, single antibiotics, or a combination of antibiotics and antiprotozoal drugs, are used for 7–10 days for the treatment of babesiosis; however, as these treatments have problems, a new therapy is needed. Although aetiological and molecular biology methods are often used in drug screening, these methods have disadvantages and there is no standard anti-B. microti drug screening method.MethodThis study was consisted of basal test, drug-suppressive test and drug-therapeutic test. BALB/c mice were used as the animal model and robenidine hydrochloride (ROBH) was used as a positive drug. By modifying Peter’s 4-day suppression method, immunosuppressive and subpassage tests were conducted, in combination with microscopy and real-time fluorescent quantitative PCR (qPCR), the current anti-B. microti drug screening method was optimised. SPSS was used for data analysis.Result1) In the basal test, there were significant differences in the erythrocyte infection rate (EIR) and relative value of the target gene (P = 0.011 and 0.012, respectively) among days. The blood of infected mice 28, 35, and 37 days post-infection (dpi) could infect healthy second-generation mice, even if no parasites could be observed under a light microscope. Resurgence occurred when the immunity of an infected mouse decreased to a certain extent after using an immunosuppressant.2) In the drug-suppressive test, no B. microti was observed under the light microscope in any ROBH group after drug administration. Significant differences were observed in the EIR and relative value of the target gene (both P < 0.001) between the control group and ROBH groups. No B. microti was observed after the administration of an immunosuppressant or in second-generation mice in ROBH-treated groups; however, B. microti was observed in control and proguanil hydrochloride-treated groups.3) In the drug-therapeutic test, there were significant differences in the EIR and relative value of the target gene between the 100 mg/kg ROBH groups and the control group (P = 0.049 and < 0.001, respectively) but not between ROBH groups and atovaquone-azithromycin group (P = 0.587 and 0.418, respectively). None of the second-generation mice in any drug-treated group were infected, whereas all the second-generation mice in the control group were infected; parasites could be observed in the control group from the 2nd day after the administration of an immunosuppressant, several parasites were observed in the atovaquone-azithromycin group from the 10th day after the administration of an immunosuppressant, and no parasites were observed in ROBH groups. ConclusionROBH can be used as a positive drug in anti-B. microti drug screening trials. qPCR results can be used as a judgment standard in preliminary screening tests and the combination of immunosuppressive and subpassage experiments can be used to validate drug efficacy.