Inhibitory action of phenothiazinium dyes against Neospora caninum

Luiz Miguel Pereira,Ana Patrícia Yatsuda,Cássia Mariana Bronzon Da Costa,Mark Wainwright,Caroline Martins Mota,Tiago Wilson Patriarca Mineo,Luciana Baroni,Jade Cabestre Venancio Brochi,Gilberto Úbida Leite Braga

doi:10.1038/s41598-020-64454-x

Abstract

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9–Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019–1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9–Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9–Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control.

Highlights

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows
Based on the promising effects of Methylene Blue (MB) against Plasmodium spp, the etiologic agent of malaria, our group determined the efficacy of this molecule on N. caninum, either alone or combined with Pyrimethamine (Pyr)[27]
MB was used against Plasmodium until the use of Chloroquine and other drugs, which lack some of the reversible MB side effects[30,31]

Summary

Introduction

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Future assays related to the mechanism of action of dyes in N. caninum will be mandatory to elucidate the lower inhibitory/resistance induction of NMB compared to MB, TBO, and DMMB. The CPRG assay, performed after the fourth cycle, indicated that NMB exhibited a higher N. caninum infection rate when compared to DMMB, demonstrating a diminution of the parasite burden in 68% and 79% respectively (Fig. 3C).

Results

Conclusion