Inhibitory effects of pepstatin A and mefloquine on the growth of Babesia parasites

Abstract

We evaluated the inhibitory effects of pepstatin A and mefloquine on the in vitro and in vivo growths of Babesia parasites. The in vitro growth of Babesia bovis, B. bigemina, B. caballi, and B. equi was significantly inhibited (P < 0.05) by micromolar concentrations of pepstatin A (50% inhibitory concentrations = 38.5, 36.5, 17.6, and 18.1 μM, respectively) and mefloquine (50% inhibitory concentrations = 59.7, 56.7, 20.7, and 4 μM, respectively). Furthermore, both reagents either alone at a concentration of 5 mg/kg or in combinations (2.5/2.5 and 5/5 mg/kg) for 10 days significantly inhibited the in vivo growth of B. microti in mice. Mefloquine treatment was highly effective and the combination treatments were less effective than other treatments. Therefore, mefloquine may antagonize the actions of pepstatin A against babesiosis and aspartic proteases may play an important role in the asexual growth cycle of Babesia parasites.

Figure 1.

Inhibitory effect of different concentrations of pepstatin A on the in vitro growth of Babesia bovis (A), B. bigemina (B), B. caballi (C), and B. equi (D). Diminazene aceturate (Di) and tetracycline hydrochloride (T) were used as positive controls. Each value represents the mean ± SD for experiments performed in triplicate. Curves represent results of one representative experiment of three separate replicates. Asterisks, (*) and (**), indicate statistically significant differences (P < 0.05, by Student's t-test) between 50 and 25 μM drug-treated cultures and the control cultures, respectively. DMSO = dimethylsulfoxide.

Figure 2.

Light micrographs of pepstatin A-treated bovine and equine Babesia parasites in an in vitro culture. Micrographs were taken on day 3 of the experiment. B. bovis: control (A) and 25 μM pepstatin A (B). B. bigemina: control (C) and 50 μM pepstatin A (D). B. caballi: control (E) and 5 μM pepstatin A (F). B. equi: control (G) and 5 μM pepstatin A (H). Drug-treated cultures showed a higher number of degenerated parasites than control cultures. Scale bars = 10 μm.

Figure 3.

Inhibitory effects of different concentrations of mefloquine on the in vitro growth of Babesia bovis (A), B. bigemina (B), B. caballi (C), and B. equi (D). Diminazene aceturate (Di) and tetracycline hydrochloride (T) were used as positive controls. Each value represents the mean ± SD for experiments performed in triplicate. Curves represent the results of one representative experiment of three separate replicates. *Statistically significant differences (P < 0.05, by Student's t-test) between the drug-treated cultures and control cultures. DMSO = dimethylsulfoxide.

Figure 4.

Light micrographs of mefloquine-treated bovine Babesia parasites in an in vitro culture. Micrographs were taken on day 3 of the experiment. B. bovis: control (A) and 1 μM mefloquine (B). B. bigemina: control (C) and 1 μM mefloquine (D). B. caballi: control (E) and 1 μM mefloquine (F). B. equi: control (G) and 1 μM mefloquine (H). Drug-treated cultures showed a higher number of degenerated parasites than control cultures. Scale bars = 10 μm.

Figure 5.

Inhibitory effect of combinations of pepstatin A and mefloquine (2.5 mg/kg of each and 5 mg/kg of each), pepstatin A (5 mg/kg), and mefloquine (5 mg/kg) on the in vivo growth of Babesia based on observations obtained for four mice per experimental group. Each value represents the mean ± SD. * = statistically significant differences (P < 0.05, by Student's t-test) between the treated groups and the control group. ↔ = time of inoculation or control reagent (dimethlysulfoxide) application of intraperitoneal pepstatin A, mefloquine, or a combination of pepstatin A and mefloquine.