猪弓形虫代谢分泌抗原免疫应答的研究
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摘要
弓形虫(Toxoplasma gondii)为专性细胞内寄生原虫,呈全球性分布,引起的弓形虫病是一种危害十分严重的人兽共患寄生虫病,对人类健康、优生优育和畜牧业发展造成严重威胁。因此,弓形虫病疫苗的研制已成为该病防治的当务之急。
为阐明弓形虫代谢分泌抗原(E/SA)对猪的免疫效应机制,将猪随机分为6组,每组5只,分别为E/SA组、E/SA+CpG(未乳化)组、E/SA+CpG组、E/SA +IL-2组、E/SA+IL-2+CpG组及空白对照组。共免疫2次,间隔2周,在第2次免疫后2周用弓形虫GJS株速殖子攻击感染。分别于免疫前、免疫后2周、免疫后4周、攻毒后1周及攻毒后2周采集仔猪外周血,用IHA检测试剂检测血清中抗弓形虫抗体水平;用ELISA方法检测血清中IL-2、IL-4、IFN-γ及TNF-α的含量;用流式细胞仪检测外周血中CD4+及CD8+ T淋巴细胞亚群的动态变化规律。结果表明,免疫后各免疫组抗弓形虫IHA抗体水平大部分升至1:1024;血清中IL-2、IL-4、IFN-γ的含量均不同程度的升高,其中E/SA组IL-2、IL-4、IFN-γ含量显著提高,其含量分别由免疫前的105.05、105.33、67.50 pg/mL升高到免疫后的194.03、342.50、296.50 pg/mL,与对照组差异显著(P<0.05);TNF-α含量变化不明显。免疫组外周血CD4+、CD8+T淋巴细胞在免疫后升高,其中E/SA组升高最为明显,CD4+、CD8+T细胞的水平分别由免疫前的21.51%,23.9%升高到免疫后的35.72%,32.8%。用弓形虫GJS强毒攻击后,免疫组和对照组猪体温差异明显,免疫组猪呈一过性低烧,食欲正常;对照组仔猪呈稽留高温,食欲不振,并且伴有拉稀症状。
研究表明,用E/SA免疫猪后可刺激机体抗弓形虫抗体产生,促进IL-2、IL-4、IFN-γ的分泌,诱导机体CD4+、CD8+T淋巴细胞大量增殖,产生较强的体液免疫和细胞免疫应答,激发宿主抗弓形虫感染的免疫应答反应。
Toxoplasma gondii is an obligate intracellular protozoan parasite and widespread in the world. Toxoplasmsis caused by T.gondii is a serious parasitic zoonosis and threats human health and the farming industry. So development of Toxoplasma gondii vaccine is an urgent task for toxoplasmsis control.
To elucidate the immune mechanism of Toxoplasma gondii excreted/secreted antigens in swine,piglets were randomly divided into 6 groups of 5 each, named E/SA group, E/SA~+CpG group, E/SA~+CpG(unemulsification)group, E/SA~+IL-2 group, E/SA~+IL-2~+CpG group and control group. The animals were immunized for 2 times at 2 weeks interval and challenged with T. gondii GJS 2 weeks after the second vaccination. Blood samples were collected pre-immunization,week 2 and week 4 post- immunization, week 1 and week 2 post-challenge , respectively. The antibodies were tested by IHA ,and the IL-2, IL-4, IFN-γand TNF-αwere detected by ELISA.The dynamic changes of the CD4 ~+ and CD8 ~+ T lymphocyte subpopulations in peripheral blood were analyzed by the flow cytometry. The results showed that the levels of specific antibody in most piglets of immmunized group increased to 1:1024. The level of IL-2, IL-4 and IFN-γin serum of immunized groups increased , with the E/SA group increased significantly from 105.05, 105.33,67.50 pg/mL to 194.03,342.50,296.50 pg/mL,respectively.These were significantly different from the control group (P<0.05). The level of TNF-αdid not change significantly at all points time. The level of peripheral blood CD4 ~+, CD8 ~+ T lymphocyte in immuned group significantly increased compared with the control group,and the E/SA group increased significantly, from 21.51%,23.9% to 35.72%,32.8%, respectively. After challeng with T.gondii GJS strain, the immuned group and the control group had significant difference temperature, the piglets of immuned group had transient low-grade fever and normal appetite,the piglets of the control group had higher fever, loss of appetite and symptoms associated with diarrhea.
These data indicated that T.gondii E/SA vaccine can promote the secretion of specific antibody and IL-2、IL-4、IFN-γin pigs , improve the percentage of peripheral CD4 ~+ and CD8~+ T lymphocytes , elevate the cellular immunity and the humoral immunity of swine,elicit the anti-T gondii immune response.
为阐明弓形虫代谢分泌抗原(E/SA)对猪的免疫效应机制,将猪随机分为6组,每组5只,分别为E/SA组、E/SA+CpG(未乳化)组、E/SA+CpG组、E/SA +IL-2组、E/SA+IL-2+CpG组及空白对照组。共免疫2次,间隔2周,在第2次免疫后2周用弓形虫GJS株速殖子攻击感染。分别于免疫前、免疫后2周、免疫后4周、攻毒后1周及攻毒后2周采集仔猪外周血,用IHA检测试剂检测血清中抗弓形虫抗体水平;用ELISA方法检测血清中IL-2、IL-4、IFN-γ及TNF-α的含量;用流式细胞仪检测外周血中CD4+及CD8+ T淋巴细胞亚群的动态变化规律。结果表明,免疫后各免疫组抗弓形虫IHA抗体水平大部分升至1:1024;血清中IL-2、IL-4、IFN-γ的含量均不同程度的升高,其中E/SA组IL-2、IL-4、IFN-γ含量显著提高,其含量分别由免疫前的105.05、105.33、67.50 pg/mL升高到免疫后的194.03、342.50、296.50 pg/mL,与对照组差异显著(P<0.05);TNF-α含量变化不明显。免疫组外周血CD4+、CD8+T淋巴细胞在免疫后升高,其中E/SA组升高最为明显,CD4+、CD8+T细胞的水平分别由免疫前的21.51%,23.9%升高到免疫后的35.72%,32.8%。用弓形虫GJS强毒攻击后,免疫组和对照组猪体温差异明显,免疫组猪呈一过性低烧,食欲正常;对照组仔猪呈稽留高温,食欲不振,并且伴有拉稀症状。
研究表明,用E/SA免疫猪后可刺激机体抗弓形虫抗体产生,促进IL-2、IL-4、IFN-γ的分泌,诱导机体CD4+、CD8+T淋巴细胞大量增殖,产生较强的体液免疫和细胞免疫应答,激发宿主抗弓形虫感染的免疫应答反应。
Toxoplasma gondii is an obligate intracellular protozoan parasite and widespread in the world. Toxoplasmsis caused by T.gondii is a serious parasitic zoonosis and threats human health and the farming industry. So development of Toxoplasma gondii vaccine is an urgent task for toxoplasmsis control.
To elucidate the immune mechanism of Toxoplasma gondii excreted/secreted antigens in swine,piglets were randomly divided into 6 groups of 5 each, named E/SA group, E/SA~+CpG group, E/SA~+CpG(unemulsification)group, E/SA~+IL-2 group, E/SA~+IL-2~+CpG group and control group. The animals were immunized for 2 times at 2 weeks interval and challenged with T. gondii GJS 2 weeks after the second vaccination. Blood samples were collected pre-immunization,week 2 and week 4 post- immunization, week 1 and week 2 post-challenge , respectively. The antibodies were tested by IHA ,and the IL-2, IL-4, IFN-γand TNF-αwere detected by ELISA.The dynamic changes of the CD4 ~+ and CD8 ~+ T lymphocyte subpopulations in peripheral blood were analyzed by the flow cytometry. The results showed that the levels of specific antibody in most piglets of immmunized group increased to 1:1024. The level of IL-2, IL-4 and IFN-γin serum of immunized groups increased , with the E/SA group increased significantly from 105.05, 105.33,67.50 pg/mL to 194.03,342.50,296.50 pg/mL,respectively.These were significantly different from the control group (P<0.05). The level of TNF-αdid not change significantly at all points time. The level of peripheral blood CD4 ~+, CD8 ~+ T lymphocyte in immuned group significantly increased compared with the control group,and the E/SA group increased significantly, from 21.51%,23.9% to 35.72%,32.8%, respectively. After challeng with T.gondii GJS strain, the immuned group and the control group had significant difference temperature, the piglets of immuned group had transient low-grade fever and normal appetite,the piglets of the control group had higher fever, loss of appetite and symptoms associated with diarrhea.
These data indicated that T.gondii E/SA vaccine can promote the secretion of specific antibody and IL-2、IL-4、IFN-γin pigs , improve the percentage of peripheral CD4 ~+ and CD8~+ T lymphocytes , elevate the cellular immunity and the humoral immunity of swine,elicit the anti-T gondii immune response.
引文
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