猪链球菌3型疫苗候选菌株筛选及3＋9型二价灭活疫苗对小鼠免疫效果评估

doi:10.16431/j.cnki.1671-7236.2022.06.031

摘要/Abstract

摘要： 【目的】筛选猪链球菌血清3型疫苗候选菌株，制备猪链球菌3＋9型二价灭活疫苗并评估其在BALB/c小鼠上的免疫保护效果。【方法】从发病猪病料中分离猪链球菌血清3型菌株，通过蜡螟幼虫和BALB/c小鼠模型筛选出强毒株作为疫苗候选菌株，并对候选菌株进行生物学特性研究。将筛选得到的3型疫苗候选菌株和前期筛选的9型疫苗候选菌株灭活浓缩，使其抗原浓度为2×10¹⁰ CFU/mL，无菌检测后将浓缩抗原液按1：1配比混合，再混合抗原与Summit Poly Solution佐剂按4：1比例混合，制备猪链球菌3＋9型二价灭活疫苗，疫苗中猪链球菌血清3和9型含菌量均为2×10⁹ CFU/mL。将制备的疫苗免疫6周龄BALB/c小鼠，首免后第14天进行二免，二免后第14天进行攻毒。同时设立商品化疫苗免疫组和阴性对照组，评估疫苗的安全性和免疫保护效果。【结果】 PCR鉴定结果显示，23株临床分离株均为血清3型猪链球菌，依次命名为KQ3-1~KQ3-23。分别通过蜡螟和小鼠进行初筛和复筛，筛选到强毒株KQ3-1。毒力基因检测显示，该菌株基因型为gapdh^＋/sly^＋/fbps^-/orf2^-/mrp^-/89K^-/gdh^＋/epf^-；生长曲线显示，该菌株在37 ℃培养8~10 h时生长达到对数生长后期；BALB/c小鼠致病性结果显示，腹腔接种12 h内可引起小鼠精神萎靡、扎堆、毛发耸立、运动迟缓、死亡等临床症状，该菌株的LD₅₀为5.2×10⁷ CFU/只。制备的疫苗免疫小鼠后，小鼠精神状况、采食等均正常，疫苗注射部位无肿胀、硬块等不良反应，无死亡发生，表明该疫苗具有良好的安全性。免疫后进行猪链球菌血清2型菌株ZYS、猪链球菌血清3型菌株KQ3-1和猪链球菌血清9型菌株YT攻毒，对照组死亡率分别为90%、100%和100%，猪链球菌3＋9型二价灭活疫苗免疫组保护率分别为30%、80%和70%，商品化疫苗组的保护效果分别为70%、0和10%。【结论】本研究研制的疫苗能对猪链球菌血清3和9型强毒株提供良好的免疫保护，该疫苗具备疫苗市场开发的潜在价值。

关键词: 猪链球菌; 血清3和9型; 二价灭活疫苗

Abstract: 【Objective】 The study was aimed to screen vaccine candidate strains of Streptococcus suis serotype 3, prepare a bivalent inactivated vaccine of Streptococcus suis serotype 3+9 and evaluate its immunoprotective effect on BALB/c mice.【Method】 In this study, serotype 3 strain of Streptococcus suis isolated from diseased pigs were screened by the larvae of Galleria mellonella and BALB/c mice model and was studied as vaccine candidate strains.Then the biological characteristics of the candidate strains were studied.The serotype 3 vaccine candidate strains and the previously screened serotype 9 candidate vaccine strains were inactivated and concentrated, and the antigen concentration was 2×10¹⁰ CFU/mL.After the sterility test, the concentrated antigen liquid was mixed in a ratio of 1:1, and then mixed with the Summit Poly Solution adjuvant in a ratio of 4:1 to prepare bivalent inactivated vaccine of Streptococcus suis serotype 3+9, the bacterial content of Streptococcus suis serotype 3 and 9 were both 2×10⁹ CFU/mL.The prepared vaccine was inoculated into 6-week-old BABL/c mice.The second immunization was carried out on the 14th day after the first immunization, and the challenge was carried out on the 14th day after the second immunization.At the same time, a commercial vaccine immunization group and a negative control group were established to evaluate the safety and immune protection effect of the vaccine.【Result】 PCR identification result showed that all 23 clinical isolates were Streptococcus suis serotype 3, named KQ3-1 to KQ3-23 in succession.The virulent strain KQ3-1 was screened by primary screening and rescreening of Galleria mellonella and BALB/c mice.Virulence gene testing showed that the genotype of KQ3-1 was gapdh⁺/sly⁺/fbps^-/orf2^-/mrp^-/89K^-/gdh⁺/epf^-.The growth curve showed that the strain reached its peak when cultured at 37 ℃ for 8 to 10 h.The pathogenicity of BALB/c mice showed that intraperitoneal inoculation could cause clinical symptoms such as depression, pile up, hair erection, motor retardation and death within 12 h.The LD₅₀ of the strain was 5.2×10⁷ CFU/piece.After immunizing the mice with the prepared vaccine, the mental status and food intake of the mice were normal, there were no adverse reactions such as swelling and lumps at the injection site of the vaccine, and there was no death, indicating that the vaccine had good safety.After immunization, Streptococcus suis serotype 2 strain ZYS, Streptococcus suis serotype 3 strain KQ3-1 and Streptococcus suis serotype 9 strain YT were challenged.The mortality rate of control group was 90%, 100% and 100%, respectively.The protection rates of Streptococcus suis serotype 3+9 bivalent inactivated vaccine immunization group were 30%, 80% and 70%, respectively.The protection effects of commercial vaccine group were 70%, 0 and 10%, respectively.【Conclusion】 The vaccine developed in this study could provide good immune protection against Streptococcus suis serotype 3 and 9 strains and had potential value of vaccine market development.

Key words: Streptococcus suis; serotype 3 and 9; bivalent inactivated vaccine

中图分类号:

S852.61⁺1

李倩倩, 龙云志, 梁巩, 金清, 刘锦锦, 杨柳, 黄英, 余道兵, 宋文博, 黄超, 汤细彪. 猪链球菌3型疫苗候选菌株筛选及3＋9型二价灭活疫苗对小鼠免疫效果评估[J]. 中国畜牧兽医, 2022, 49(6): 2307-2317.

LI Qianqian, LONG Yunzhi, LIANG Gong, JIN Qing, LIU Jinjin, YANG Liu, HUANG Ying, YU Daobing, SONG Wenbo, HUANG Chao, TANG Xibiao. Screening of Vaccine Candidate Strains of Streptococcus suis Serotype 3 and Evaluation of the Immune Effect of the Bivalent Inactivated Vaccine Streptococcus suis Serotype 3+9 on Mice[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2307-2317.

参考文献

[1] HAAS B, GRENIER D.Understanding the virulence of Streptococcus suis:A veterinary, medical, and economic challenge[J].Medicine et Maladies Infectieuses, 2018, 48(3):159-166.
[2] SEGURA M. Streptococcus suis vaccines:Candidate antigens and progress[J].Expert Review of Vaccines, 2015, 14(12):1587-1608.
[3] GOYETTE-DESJARDINS G, AUGER J P, XU J, et al.Streptococcus suis, an important pig pathogen and emerging zoonotic agent an update on the worldwide distribution based on serotyping and sequence typing[J].Emerging Microbes & Infections, 2014, 3(6):e45.
[4] SEGURE M. Streptococcus suis research:Progress and challenges[J].Pathogens, 2020, 9(9):707.
[5] 赵顾.2017-2018年安徽部分地区猪链球菌的分离鉴定及其生物学特性研究[D].合肥:安徽农业大学, 2019. ZHAO G.Isolation, identification and biological characteristics of Streptococcus suis in parts of Anhui from 2017 to 2018[D].Hefei:Anhui Agricultural University, 2019.(in Chinese)
[6] 王娟, 刘琪, 周如月, 等.广东地区猪群中猪链球菌流行病学调查分析[J].中国预防兽医学报, 2017, 39(8):621-623. WANG J, LIU Q, ZHOU R Y, et al.The Streptococcus suis epidemiological analysis of swine in Guangdong province[J].Chinese Journal of Preventive Veterinary Medicine, 2017, 39(8):621-623.(in Chinese)
[7] 王巍.四川地区猪链球菌流行调查及其病原特性研究[D].雅安:四川农业大学, 2015. WANG W.Prevalence and pathogen research of Streptococcus suis in Sichuan[D].Ya'an:Sichuan Agricultural University, 2015.(in Chinese)
[8] ZHANG B, KU X, YU X, et al.Prevalence and antimicrobial susceptibilities of bacterial pathogens in Chinese pig farms from 2013 to 2017[J].Scientific Reports, 2019, 9(1):9908.
[9] LIU Z, ZHENG H, MARCELO G, et al.Development of multiplex PCR assays for the identification of the 33 serotypes of Streptococcus suis[J].PLoS One, 2013, 8(8):e72070.
[10] 易雪丽, 袁朝原, 陈晓颖, 等.人源猪链球菌19株分离鉴定及毒力基因检测[J].中国感染与化疗杂志, 2020, 20(2):153-156. YI X L, YUAN Z Y, CHEN X Y, et al.Identification and virulence analysis of 19Streptococcus suis strains isolated from human subjects[J].Chinese Journal of Infection and Chemotherapy, 2020, 20(2):153-156.(in Chinese)
[11] 宦海霞, 黄莉莉, 张科.猪链球菌多样性及其致病因子研究进展[J].中国畜牧兽医, 2014, 41(2):218-223. HUAN H X, HUANG L L, ZHANG K.Advance on diversity and virulent factors of Streptococcus suis[J].China Animal Husbandry & Veterinary Medicine, 2014, 41(2):218-223.(in Chinese)
[12] ROODSANT T, PUTTEN B, TAMMINGA S, et al.Identification of Streptococcus suis putative zoonotic virulence factors:A systematic review and genomic meta-analysis[J].Virulence, 2021, 12(1):2787-2797.
[13] 魏凤, 张文通, 李峰, 等.商品化猪链球菌疫苗分类及特点[J].猪业科学, 2018, 35(11):103-104. WEI F, ZHANG W T, LI F, et al.Classification and characteristics of commercial Streptococcus suis vaccines[J].Swine Science, 2018, 35(11):103-104.(in Chinese)
[14] 刘琪, 王娟, 周如月, 等.广东地区健康猪群和发病猪群猪链球菌流行病学调查分析[J].中国畜牧兽医, 2017, 44(6):1825-1831. LIU Q, WANG J, ZHOU R Y, et al.The Streptococcus suis epidemiological analysis of healthy and infected swine in Guangdong province[J].China Animal Husbandry & Veterinary Medicine, 2017, 44(6):1825-1831.(in Chinese)
[15] 石大丽, 葛晨玲, 朱远致, 等.广西部分地区猪链球菌血清型及其毒力因子流行性调查分析[J].中国畜牧兽医, 2019, 46(4):1116-1126. SHI D L, GE C L, ZHU Y Z, et al.Investigation on the prevalence of serotypes and virulence factors of Streptococcus suis in some areas of Guangxi[J].China Animal Husbandry & Veterinary Medicine, 2019, 46(4):1116-1126.(in Chinese)
[16] 黄秦.猪链球菌2型疫苗候选株的生物学特性研究[D].南京:南京农业大学, 2009. HUANG Q.Study on biological characteristics of candidate strain of Streptococcus suis type 2 vaccine[D].Nanjing:Nanjing Agricultural University, 2009.
[17] 吴华健.猪链球菌2型疫苗株的筛选及其培养条件的优化[D].合肥:安徽农业大学, 2018. WU H J.The screening of Streptococcus suis type 2 vaccine strains and optimization of their culture conditions[D].Hefei:Anhui Agricultural University, 2018.
[18] VELIKOVA N, KAVANAGH K, WELLS J M.Evaluation of Galleria mellonella larvae for studying the virulence of Streptococcus suis[J].BMC Microbiology, 2016, 16(1):291.
[19] VECHT U, WISSELINK H J, JELLEMA M L, et al.Identification of two proteins associated with virulence of Streptococcus suis type 2[J].Infection & Immunity, 1991, 59(9):3156-3162.
[20] 吴超.猪链球菌流行病学特征和hp0197基因多态性研究[D].武汉:华中农业大学, 2013. WU C.Epidemiological characteristics of Streptococcus suis and hp0197 gene polymorphism[D].Wuhan:Huazhong Agricultural University, 2013.(in Chinese)
[21] 张荣, 李菁, 武永淑, 等.动物疫苗佐剂应用进展概述[J].甘肃畜牧兽医, 2021, 51(4):5-9. ZHANG R, LI J, WU Y S, et al.Overview of the application progress of animal vaccine adjuvants[J].Gansu Animal Husbandry and Veterinary, 2021, 51(4):5-9.(in Chinese)