G型产气荚膜梭菌全菌灭活疫苗的研制及其免疫效力的评估

Study of whole strain inactivated Clostridium perfringens type G vaccine and its immune efficacy

目的:以临床分离的G型产气荚膜梭菌(Clostridium perfringens type G,GCP)GCP-D25菌株为对象,研制GCP全菌灭活苗,并评估疫苗免疫效力。方法:利用甲醛灭活法处理GCP-D25菌液,白油佐剂乳化后免疫1日龄雏鸡检测灭活效果;150只1日龄817肉鸡,随机分为5组(G_(1)~G_(5)),即高剂量免疫组、中剂量免疫组、低剂量免疫组、攻菌对照组和空白对照组。G_(1)~G_(3)组分别于1、8、15日龄通过皮下注射不同剂量的GCP-D25乳化灭活疫苗进行免疫,G_(4)~G_(5)组通过皮下注射PBS,其中G_(1)~G_(4)组于26~29日龄每天灌服1次8×10^(8)CFU/mL GCP新鲜菌液,G_(5)组为不攻菌对照组。分别于一免、二免和三免后第7天采集鸡血液并分离血清,采用ELISA方法检测血清抗体及IL-2、INF-γ水平,同时结合攻菌后鸡临床表现及肠道病变计分等指标评估疫苗免疫保护效果。结果:GCP-D25全菌液能被0.5%甲醛完全灭活,灭活时间为36 h。间接ELISA测定抗原(全菌液)的最佳包被浓度为2×10^(8)CFU/mL,血清最佳稀释度为1∶400。肉鸡免疫保护试验显示,攻菌对照组试验鸡的坏死性肠炎病变平均分显著高于各免疫组(P<0.01)。三免后第7天,各免疫组试验鸡血清抗体呈阳性,高剂量与中剂量免疫组试验鸡IL-2与IFN-γ含量均显著高于空白对照组(P<0.05)。结论:GCP-D25全菌灭活疫苗免疫1日龄817肉鸡可诱导机体产生高水平免疫应答,能够在一定程度上预防鸡坏死性肠炎的发生。

Objective:Clostridium perfringens type G(GCP)is a bacterial pathogen,which can cause necrotic enteritis in broilers.GCP inactive whole bacterial vaccine was prepared and its immune efficacy was studied.Methods:The bacterial solution of GCP-D25 was inactivated with 0.5%formaldehyde.One-day-old chicks were immunized with inactivated vaccine to detect the inactivation effect.The antibody,IL-2 and INF-γwere detected by ELISA at 7 days after the first,second and third immunization.The immune effect of the vaccine was evaluated by the levels of serum antibodies,IL-2 and INF-γ,clinical symptoms and intestinal lesion scores after challenge.Results:The whole bacterial solution of GCP-D25 could be completely inactivated by 0.5%formaldehyde for 36 h.The optimal coating concentration of whole bacterial solution by indirect ELISA was 2×10^(8)CFU/mL,and the optimal dilution of serum was 1∶400.The results of immunization trial in vivo showed that the mean lesion scores of necrotizing enteritis in the challenge control group were significantly higher than those in the high,medium,low dose inactivated vaccine groups and the negative control group(P<0.01).On the 7th day after the third immunization,the antibody of the chickens in different immunization groups were all positive compared with the negative control group.The levels of IL-2 and INF-γin high dose group and middle dose group were significantly higher than those in negative control group(P<0.05).Conclusion:The GCP-D25 whole bacteria inactivated vaccine can induce immune response in broilers,which can protect the chickens against necrotizing enteritis.