副猪嗜血杆菌病四价灭活苗的制备及对豚鼠的免疫效果评价

Preparation of tetravalent inactivated vaccine against Haemophilus parasuis disease and evaluation of its immune efficacy on guinea pigs

为了对一种新型副猪嗜血杆菌病(又称猪革拉瑟氏病,Gl?sser’s disease)四价灭活疫苗的研发提供技术储备,本研究利用筛选的4株副猪嗜血杆菌(HPS)(血清4、5、7和13型,编号分别为AT-2、SX-1P、HF-1P和HQ-2)作为疫苗菌株,经甲醛灭活后,配比ISA 201 VG双向油乳佐剂,制成灭活前活菌分别为1.0×10~9cfu/mL、2.0×10~9cfu/mL、4.0×10~9cfu/mL的四价灭活苗,依次命名为V-1、V-2和V-3。以豚鼠为实验动物模型,将V-1、V-2和V-3与商品化疫苗(血清4、5型HPS,灭活前活菌浓度各为4.0×10~9cfu/mL)分别免疫(依次为I组~IV组,空白对照组注射等量生理盐水)并进行免疫效果评价:通过间接ELISA测定各组豚鼠血清中IgG抗体水平;通过血清杀菌试验(SBT)测定各组豚鼠血清功能性抗体吞噬活性;通过噻唑蓝溴化四唑溶液(MTT)法测定豚鼠脾淋巴细胞的增殖指数;通过双抗夹心ELISA测定各组豚鼠细胞因子(IL-10、IL-4、TNF-β、IFN-γ、MCP-1)含量;对免疫豚鼠腹腔攻毒后检测各组疫苗的保护率;采用平板计数法测定各组豚鼠组织荷菌数以评估攻毒菌株在其中的定植数量;制作攻毒后各组豚鼠的肺、肝、脾和肾脏病理组织切片并观察。结果显示,在整个免疫周期,空白对照组各项检测指标始终无明显变化。I~IV组豚鼠血清IgG抗体效价均为1:12800;与空白对照组相比,I~IV组均能诱导豚鼠血清功能性抗体水平持续升高且无显著差异(P>0.05),表明各疫苗均具有较强的杀菌效果;I~IV组均能刺激豚鼠血清相关细胞因子水平(IL-4、IL-10、INF-γ、TNF-β、MCP-1)持续上升且无显著差异(P>0.05)。腹腔攻毒后,I~IV组总体保护率分别为85%、100%、100%和60%;I~III组豚鼠各脏器(肺、肝、肾、脾)细菌定植量均显著降低且显著低于免疫组IV(P<0.05),且该3组豚鼠各脏器病理变化均较免疫组IV轻微。上述结果表明,V-1、V-2和V-3免疫豚鼠后均能产生良好的体液免疫和细胞免疫反应,其中V-2和V-3能够100%抵抗致死剂量HPS血清4、5、7和13型的攻毒。本研究首次证明V-2和V-3可作为HPS病四价(血清4、5、7和13型)灭活苗的候选疫苗,为HPS病的疫苗研制奠定了基础。

To provide a technical reserve for the development of a new tetravalent inactivated vaccine against Haemophilus parasuis(HPS)disease(Gl?sser’s disease),four strains of HPS(serotypes 4,5,7 and 13,codenamed AT-2,SX-1P,HF-1P and HQ-2)were screened and used as vaccine strains,and three kinds of tetravalent inactivated vaccine(named V-1,V-2 and V-3)were prepared with the concentration of 1.0×10~9cfu/mL,2.0×10~9cfu/mL and 4.0×10~9cfu/mL following by inactivated with formaldehyde and mixed with mineral oil adjuvant ISA 201VG.The guinea pigs were used as experimental animal models and divided into four immune groups(I-IV group)injected with V-1,V-2,V-3 and commercial vaccine(serotypes 4,5),respectively,at dose of 4.0×10~9cfu/mL to evaluate the efficacy.The indirect ELISA was used for detecting the level of IgG antibody in serum,serum bactericidal test(SBT)for detecting the phagocytic activity of functional antibody,MTT assay for detecting the proliferation of spleen lymphocyte,and ELISA for detecting the levels of cytokines(IL-4,IL-10,IFN-γ,TNF-βand MCP-1).The immune protection rate was measured by intraperitoneal challenge,the bacteria load in tissues of guinea pigs in each group was measured by plate counting method to evaluate the colonization number of challenge strains,and pathological sections of lung,liver,spleen and kidney were prepared and observed.The results showed that V-1,V-2 and V-3 fully qualified for physical properties,sterility and safety testing.During the whole immune cycle,there were no significant changes in the detection indexes of the blank control group.The serum IgG antibody titers in immune groups I-IV were 1:12800,the level of IgG increased continuously and there was no significant difference among the four groups(P>0.05),the corresponding serum collected had strong bactericidal effect.The levels of IL-4,IL-10,IFN-γ,TNF-βand MCP-1 in serum increased continuously and had no significant difference among the four groups(P>0.05).The overall protective rate of I-IV was 85%,100%,100%and 60%,respectively,after intraperitoneal challenge;The amount of bacteria colonized in all organs(lung,liver,kidney and spleen)of immune group I-III was significantly lower than that of immune group IV(P<0.05),and the pathological changes of each organ were milder than those in immune group IV.In conclusion,the V-1,V-2 and V-3 could evoke good humoral and cellular immunity in guinea pigs,and among which V-2 and V-3could resist 100%of lethal doses of HPS serotypes 4,5,7 and 13.This study verified for the first time that V-2 and V-3 can be used as candidates of tetravalent inactivated(serotypes 4,5,7 and 13)vaccine against Haemophilus parasuis disease,which lay the foundation for the vaccine development of Gl?sser’s disease.