Inhibition of Neospora caninum activity by niclosamide: Evidence from in vitro and in vivo studies

Neosporosis caused by Neospora caninum (N. caninum) is one of the main causes of bovine miscarriage, but there are currently no effective drugs or vaccines for treatment and prevention. Our previous works have found that NLRP3 inflammasome activation participated in controlling N. caninum proliferation and niclosamide has been regarded as an NLRP3 inflammasome inducer. This study aimed to evaluate the resistance of niclosamide to N. caninum infection. Niclosamide-mediated NLRP3 inflammasome activation was determined by LDH and ELISA measurement of IL-1 beta release as a marker for inflammasome activation in a model of N. caninum-infected macrophages. The in vitro antiparasitic effect of niclosamide was further explored in Vero cells by plaque assays, qPCR, and Giemsa staining. The in vivo effects were investigated in N. caninum-infected mice by measuring parasite burden, histopathology, and survival. Results showed that niclosamide partially enhanced macrophagemediated clearance of N. caninum via the NLRP3 inflammasome activation and displayed direct antiparasitic activity. Plaque assays confirmed significant inhibition of N. caninum growth, and niclosamide effectively reduced cell invasion and intracellular proliferation compared to toltrazuril. In vivo, after niclosamide treatment, the body weight was regained, survival rate was increased, tissue damage was reduced, and parasite burden in tissues was significantly decreased. The numerous vacuole formations were observed in niclosamide-treated N. caninum tachyzoites by electron microscopy. Mitochondrial membrane potential and ATP production of N. caninum tachyzoites were reduced considerably by niclosamide treatment. In conclusion, niclosamide showed strong potential as a therapeutic agent for N. caninum infection, offering a promising treatment option for neosporosis.