猪伪狂犬病灭活苗和弱毒苗联合免疫小鼠的免疫效果研究

Protective efficacy of combined immunization of porcine pseudorabies inactivated vaccine and attenuated vaccine in mice

为了优化猪伪狂犬病(PR)免疫程序,本研究将SPF级昆明雌鼠分为JZ-45组(单免灭活苗)、HB-98组(单免弱毒苗)、JZ-45/JZ-45组(两次均免灭活苗)、JZ-45/HB-98组(首免灭活苗,二免弱毒苗)、HB-98/JZ-45组(首免弱毒苗,二免灭活苗)、HB-98/HB-98组(两次均免弱毒苗)和对照组,共7组,采用猪伪狂犬病病毒(PRV)JZ-45分离株灭活苗和商品化HB-98弱毒苗,以上述相应组免疫物对小鼠免疫,在免疫后0、7 d、14 d、21 d、28 d、35 d和42 d分别采血,分离血清利用间接ELISA和中和试验对小鼠血清PRV特异性和中和抗体水平进行检测;并在免疫后42 d利用PRV JZ-45株对小鼠攻毒(攻毒剂量:100 LD_(50),LD_(50)=10^(2.681)/0.1 mL,100μL/只),观察其临床症状,计算小鼠存活率,并对各组小鼠剖检观察各脏器剖检病变。取小鼠脑、脾脏制备病理切片,观察各组小鼠各组织的病变。结果显示:免疫组小鼠血清PRV抗体及中和抗体水平在0~42 d均呈持续上升趋势,且HB-98/JZ-45组小鼠血清PRV抗体水平在各时间点均高于其它组。攻毒结果显示:单免组小鼠存活率均为80%,其余免疫组小鼠均无明显临床症状,对照组小鼠全部死亡。组织病理切片结果显示:除HB-98/JZ-45免疫组小鼠脾脏、脑部无明显异常外,其他免疫组小鼠脾脏组织均有少量中性粒细胞浸润,对照组小鼠脾脏白髓灶性坏死,有大量中性粒细胞浸润,脑部组织出现炎性细胞浸润,胞质空泡化等。结果表明,在首次免疫HB-98弱毒苗的基础上加强免疫1次JZ-45灭活苗,可对小鼠获得更好的免疫保护效果。本实验比较了不同的免疫程序对小鼠免疫保护效果的差异,为后续PRV免疫程序的改进提供指导,也为更好地控制PR提供参考。

In order to optimize the immunization procedure for porcine pseudorabies,this experiment used pseudorabies virus(PRV)JZ-45 isolate inactivated vaccine and commercial HB-98 attenuated vaccine to immunize SPF grade Kunming female mice with different immunization procedures.Briefly,SPF Kunming female mice were divided into JZ-45(inactivated vaccine immunization once),HB-98(attenuated vaccine immunization once),JZ-45/JZ-45(inactivated vaccine immunization twice),JZ-45/HB-98(inactivated vaccine immunization and then attenuated vaccine immunization),HB-98/JZ-45(attenuated vaccine immunization and then inactivated vaccine immunization),HB-98/HB-98(attenuated vaccine immunization twice),and the control group.Subsequently,blood samples were collected at 0,7,14,21,28,35,and 42 days post-immunization(dpi),and the PRV antibody level in mouse serum was detected by using indirect ELISA and neutralization test.On the 42 dpi,mice were challenged with PRV JZ-45 strain to observe their clinical symptoms and calculate the mortality rate.Mice in each group were dissected to examine organ lesions(challenge dose:100 LD_(50),LD_(50)=10^(2.681)/0.1mL,100μL/piece).The pathological sections of brain and spleen of mice were prepared to observe the pathological changes of each group.The results showed that the level of serum PRV antibody and neutralizing antibody in immunized mice had a continuous upward trend from 0 to 42 days,and the level of serum PRV antibody in HB-98/JZ-45 group was higher than that in other groups.The results of challenge showed that the survival rate of mice in the single immunization group was 80%,whereas the other immunized groups had no obvious clinical symptom and the mice in the control group all died.Pathological sections showed that there was no obvious abnormality in the spleen and brain of HB-98/JZ-45 immunized mice,and there was a small amount of neutrophil infiltration in the spleen and brain of other immunized mice.In the control group,the white pulp of the spleen showed focal necrosis with a large number of neutrophils infiltrating,and inflammatory cell infiltration and cytoplasmic vacuolization occurred in brain tissue.The overall results showed that a better immune protection effect could be obtained by strengthening immunization with JZ-45 inactivated vaccine once on the basis of HB-98 attenuated vaccine.This experiment provides guidance for the improvement of the subsequent PRV immunization program and also provides a reference for better control of pig pseudorabies disease by understanding the differences in the protective effects of different immunization programs in mice.