Abstract
Methylene blue, the first fully synthetic drug, has been widely used in medical treatments. A few decades ago,
this drug was used as an antimalarial agent. In this study, the in vitro inhibitory effect of methylene blue on
Babesia bovis, B. bigemina, B. caballi, and Theileria equi (B. equi) and the in vivo inhibitory effect on B. microti
were evaluated. Methylene blue significantly inhibited the growth of B. bovis, B. caballi, and T. equi at a 0.1 µM
concentration, while B. bigemina was significantly inhibited at 0.01 µM, on day 3 of cultivation. The half
maximal inhibitory concentrations (IC50) of methylene blue against B. bovis, B. bigemina, B. caballi, and T. equi
were calculated as 0.83±0.02, 0.68±0.09, 0.54±0.14, and 0.49±0.06 µM, respectively. The subsequent viability
assays, in which drug-free media were used for the cultivation, showed that there were no growths of B. bovis or
B. bigemina that had been previously treated with 10 µM methylene blue. Similarly, B. caballi and T. equi that
had been previously treated with 1 µM methylene blue failed to grow in the viability tests. As for the in vivo
inhibition assay, the high dose of methylene blue showed a low inhibitory effect on the in vivo growth of B.
microti at 50 mg/kg body weight treatment groups as compared with the untreated group. Therefore, methylene
blue might not be used for against Babesia and Theileria parasites.
