The Efficacy Of Cross-protection Of NCLIV₀19770Protein Against Neospora And Toxoplasma

Neosporiasis is a new Apicomplexan disease caused by Neospora caninum, which invades the host cells, and at least40%of cattle abortions all over the world are caused by this disease. Thus Neosporiasis is considered one of the major causes of cattle abortions worldwide. Since positive serum test results appeared in patients, it is possible that Neosporiasis could cause parasitic zoonoses. However, no effective drugs or vaccines are available until now. Toxoplasma gondii is a major opportunistic protozoon and widely distributed in humans and animal tissues and cells causing serious diseases. Both Neospora caninum and Toxoplasma gondii are protozoan parasites of the phylum Apicomplexan. In addition, these two parasites are similar in structures and biological characteristics and distribute widely in the world causing widespread infections, including mammals, such as cattle, dogs, cats, etc. Their clinical symptoms are similar, such as livestock breeding dysfunction and dam miscarriage, stillbirth or weak fetus. The host of Neosporiasis and Toxoplasma can be infected with both at the same time. Thus, it is necessary to develop a new cross protection vaccine against both Neospora caninum and Toxoplasma gondii.The gene NCLIV₀19770used in the study is obtained by screening a Neospora caninum cDNA expression library using anti-Neospora polyclonal antibody and NCLIV₀19770protein was obtained by prokaryotic expression and purified by Ni affinity chromatography. We got the NCLIV₀19770recombinant protein finally. The production levels of IFN-γ, IL-4as well as antibody in serum in mice vaccinated three times with NCLIV₀19770recombinant protein were evaluated. The efficacy of cross protection was evaluated in immunized mice after being challenged with a lethal dose of Neosporiasis and Toxoplasma respectively by the living time and the number of cysts in tissues. 1. Bioinformatics analysis suggested that NCLIV₀19770gene is homogenous with Neospora caninum hypothetical protein gene NCLIV₀19770and Toxoplasma gondii AY223538.1gene with the max identity of99%,78%respectively. The protein sequence is also homogenous with Neospora caninum hypothetical protein with the same max identity of99%, however, the identity is67%with Toxoplasma gondii AY223538.1is67%.2.The recombinant protein NCLIV₀19770（34kDa） existed mainly in inclusion body and could be recognized by an anti-Neospora tachyzoite polyclonal antibody. Western blot results showed that this protein has cross reaction with Toxoplasma.3.The antibody titer in mice serum increased gradually and had a significant increase compared to control groups after receiving the recombinant protein. The levels of IFN-Y in serum increased significantly compared to that in controls, however, the levels of IL-4did not increase significantly compared to that in controls. The mice brain real-time PCR results suggested that there was a significant decrease in the number of Neospora caninum compared to mice in controls after the lethal dose challenges of Neospora caninum and Toxoplasma. The number of parasites decreased41.4%with a live parasite rate of31.7%. There were no dead mice in all groups in40days.4. After the lethal challenge of Toxoplasma, mice vaccinated with recombinant NCLIV₀19770protein survived longer. In all, the recombinant protein NCLIV₀19770triggered strong humoral and Thl cellular responses and had protecting effects against both Neospora caninum and Toxoplasma gondii to certain degree.