Immunization with the Neospora caninum TKL1-deficient Strain Protects C57BL/6 Mice against Neosporosis

Neospora caninum is an apicomplexan protozoan that causes reproductive disorders in cattle and sheep, resulting in serious economic losses worldwide. However, there is still a lack of effective drugs and commercial vaccines for the prevention and treatment of neosporosis. Gaining insights into the identification and characterization of virulence gene-encoded proteins of N. caninum is critical for vaccine development. Tyrosine kinase-like proteins (TKL) are serine/threonine kinases. Toxoplasma gondii TKL1 (TgTKL1) has been shown to be associated with parasite growth, adhesion, and invasion, but the function of N. caninum tyrosine kinase-like protein 1 (NcTKL1) remains unclear. In this study, NcTKL1 was found to be located in the nucleus of N. caninum tachyzoites. Using the CRISPR-Cas9 gene editing system, an NcTKL1-deficient strain (ΔNcTKL1) was constructed, and its phenotypic characteristics and immune protection potential were investigated. Compared with the wild-type Nc-1 strain, the NcTKL1-deficient strain exhibited impaired growth, invasion, and replication abilities. ΔNcTKL1-infected mice showed 100% survival, reduced secretion of cytokines (IL-6, TNF-α, and IFN-γ), lower parasite burdens, and alleviated pathological damage. Furthermore, mice were immunized with ΔNcTKL1 tachyzoites for 10, 20, 30, and 40 days, followed by challenge with the wild-type Nc-1 strain to evaluate protective efficacy against N. caninum infection. The greatest reduction in parasite burden and the highest levels of IL-6, IL-12/IL-23 (p40), TNF-α, and IFN-γ production were observed at 10 days post-immunization. At this time point, the survival rate reached 100%, and liver and lung pathological damage were significantly alleviated in infected mice. In conclusion, these results indicate that the absence of NcTKL1 affects the growth, invasion and replication of the parasite. Moreover, immunizing with the ΔNcTKL1 strain confers effective protection against N. caninum infection, highlighting its potential as a live-attenuated vaccine candidate. • NcTKL1 was identified as a nuclear-localized protein in Neospora caninum tachyzoites, lacking both a signal peptide and transmembrane domain. • The ΔNcTKL1 strain was generated using CRISPR-Cas9, confirming NcTKL1 as a key virulence factor regulating parasite growth, invasion, replication. • A live-attenuated ΔNcTKL1 vaccine was developed, and immunization with ΔNcTKL1 tachyzoites conferred protective immunity against Neospora caninum infection.