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. Neospora caninum, an obligate intracellular protozoan, belongs to Apicomplexa phylum, such as Plasmodium spp and Toxoplasma gondii. It causes neosporosis, a disease strongly correlated to abortion and decreased fertility in cattle, costing billions of dollars worldwide 1-3. There is no effective commercial treatment for neosporosis, despite the efforts of several chemotherapy-focused groups 4-6. For example, Artemisinins 7-10 , Miltefosine 11 , plant extracts 12-14 , Diamidines 15,16 , Buparvaquone 4,17 , organometallic ruthenium complexes 18 , Thiazolides 19,20 were evaluated in in vitro and in vivo models with encouraging results. Moreover, polyether ionophore antibiotics 21 , Triazinones 22-24 , bumped kinase inhibitors 25 have also been tested in farm ruminants 26. 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, a phenothiazinium dye, was the first synthetic drug described to cure a patient affected by malaria in the XIX century, pioneering work performed by Paul Ehrlich 28,29. Indeed, MB was used against Plasmodium until the use of Chloroquine and other drugs (massively utilized after the Second World War), which lack some of the reversible MB side effects (blue urine and sclera) 30,31. During the later 20 th /early 21 st Centuries, the intense use of Chloroquine, Artemisinin, and Pyrimethamine has led to proven cases of Plasmodium resistance, demanding novel therapeutic candidates 32. Interestingly, no reports of Plasmodium resistance hav...

Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.

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