Neospora caninum, an obligate intracellular protozoan, is the major cause for neosporosis and brings serious economic losses to cattle breeding industries worldwide. After invasion, dense granules proteins are abundantly secreted and being important components of parasitophorous vacuole and intravacuolar network where N. caninum survives and replicates. The aim of the present study was to evaluate the protective immunity induced by DNA vaccines with genes encoding dense granules proteins 1 (GRA1), GRA4, GRA9, GRA14, GRA17, and GRA23 against N. caninum tachyzoites in BALB/C mice. Eukaryotic expressing plasmids of pcNcGRAs were constructed and the mice were intramuscularly immunized with pcNcGRAs followed by challenging infection with lethal doses of N. caninum. Immune responses were evaluated through monitoring the levels of serum antibodies, measurement of lymphocyte proliferation, and secretion of cytokines. Immune protection assays were carried out through monitoring survival time, body weight, and parasite burden in the brains. Results showed that all the pcNcGRA DNA vaccines could trigger remarkably specific humoral and cellular responses, with higher levels of IgG and IgG2a antibodies as well as obviously increased secretion of Th1-type IFN-γ cytokines. The immune protective efficacy revealed that pcNcGRA4, pcNcGRA14, and pcNcGRA17 DNA vaccines could individually increase the survival rate to 50, 37.5, and 25% in comparison with 0% in the control group; prolong the survival time more than 20.88 ± 11.12, 18.88 ± 10.83, and 16.63 ± 10.66 days compared with the control group of 4 ± 1.31 days; and decrease parasite burden in the brains to 297.63 ± 83.77, 471.5 ± 110.74, and 592.13 ± 102.2 parasites/100 ng comparing with 1221.36 ± 269.59 parasites/100 ng in the control group. These findings indicated that NcGRA4, NcGRA14, and NcGRA17 are potential vaccine candidates; NcGRA4 displayed better performance in immune protective efficacy and could be further combined with other advantageous antigens applied to the development of safe and effective DNA vaccines against N. caninum.

新孢子虫，一种专一的细胞内原生动物，是新孢子虫病的主要原因，给全世界的牛养殖业带来了严重的经济损失。入侵后，致密颗粒蛋白大量分泌，是寄生虫液泡和真空内网的重要组成部分，犬新孢子虫生存并复制。本研究的目的是评估由具有致密颗粒蛋白1（GRA1），GRA4，GRA9，GRA14，GRA17和GRA23的基因编码的DNA疫苗诱导的保护性免疫犬新孢子虫BALB / C小鼠中的速殖子。构建了pcNcGRAs的真核表达质粒，并用pcNcGRAs对小鼠进行了肌肉免疫，然后用致死剂量的PCNcGRA挑战感染。犬新孢子虫。通过监测血清抗体水平，测量淋巴细胞增殖和细胞因子分泌来评估免疫反应。通过监测存活时间，体重和大脑中的寄生虫负担来进行免疫保护测定。结果表明，所有pcNcGRA DNA疫苗均可以引发明显的特异性体液和细胞反应，其中IgG和IgG2a抗体的含量更高，并且Th1型IFN-γ的分泌明显增加。γ细胞因子。免疫保护作用表明，pcNcGRA4，pcNcGRA14和pcNcGRA17 DNA疫苗可分别将存活率提高到50％，37.5％和25％，而对照组为0％。与对照组的4±1.31天相比，延长了生存时间超过20.88±11.12、18.88±10.83和16.63±10.66天；并将脑部的寄生虫负担降低至297.63±83.77、471.5±110.74和592.13±102.2寄生虫/ 100 ng，而对照组为1221.36±269.59寄生虫/ 100 ng。这些发现表明，NcGRA4，NcGRA14和NcGRA17是潜在的候选疫苗。NcGRA4在免疫保护功效方面表现出更好的性能，并且可以与其他有益抗原进一步结合，用于开发安全有效的抗DNA疫苗犬新孢子虫。