副猪嗜血杆菌的分离鉴定与三价灭活疫苗的初步研制

发布时间：2018-04-10 06:02

本文选题：副猪嗜血杆菌　切入点：血清分型　出处：《扬州大学》2015年硕士论文

【摘要】：副猪嗜血杆菌常存在于健康猪的上呼吸道,属于巴斯德菌科嗜血杆菌属,在临床上主要引起猪的多发性浆膜炎和关节炎。近年来国内养猪场副猪嗜血杆菌病的日益严峻,对各养殖场造成严重的损失。本研究主要对江苏省和安徽省养猪场的病死猪和健康猪进行副猪嗜血杆菌的分离鉴定,为副猪嗜血杆菌的防治提供依据。同时,本室初步研制副猪嗜血杆菌血清4型、5型和12型三价灭活疫苗,用BALB/c小鼠对其进行免疫保护力的评价。1副猪嗜血杆菌的分离鉴定在扬州大学动物医院和扬州某屠宰场进行样品采集,将其接种到巧克力培养基上培养,通过PCR检测副猪嗜血杆菌的16 SrRNA。对PCR检测阳性的菌株进行纯化并保存。通过PCR鉴定之后,从病死猪中分离到8株副猪嗜血杆菌；从健康猪中分离到26株副猪嗜血杆菌,其分离率为7.12%。对其中的三株副猪嗜血杆菌进行基因测序,其测序结果与国内外分离株16 SrRNA基因序列同源性均能达到99%以上。对所有分离株用玻板凝集进行血清分型。在病死猪的分离株中血清5型3株、血清12型2株、血清14型1株及不可定型2株。在健康猪的分离株中血清4型4株(15.38%)、5型1株(3.85%)、11型3株(11.54%)、12型10株(38.46%)、13型1株(3.85%)及未定型菌株7株(26.92%)。2副猪嗜血杆菌三价灭活疫苗的初步研制用副猪嗜血杆菌血清4型、5型和12型分离株分别对豚鼠进行腹腔攻毒。结果血清5型和12型菌株攻毒豚鼠全部死亡；血清4型菌株攻毒豚鼠死亡1只(攻毒2只)。对死亡豚鼠进行剖检发现腹腔积液和胸腔积液及少量纤维素渗出,与副猪嗜血杆菌在病猪的症状相似。用三个不同剂量的副猪嗜血杆菌血清4型、5型和12型分离株分别对BALB/c小鼠进行腹腔攻毒。结果三个血清型的菌株攻毒剂量达到5.0×109CFU时,BALB/c小鼠均死亡。而攻毒剂量为1.0×109CFU时,血清4型和5型菌株攻毒小鼠均死亡1只(攻毒2只),而血清12型菌株攻毒小鼠未死亡。用三价灭活疫苗1(含3个血清型菌株各1.0×109CFU)、三价灭活疫苗2(含3个血清型菌株各2.0×109 CFU)、三价灭活疫苗3(含3个血清型菌株各4.0×109CFU)及商品灭活疫苗分别对BALB/c小鼠进行皮下免疫2次,二次免疫后14天,对各小鼠进行腹腔攻毒。结果三价灭活苗免疫组1和2与商品疫苗免疫组对4型和5型菌株的抵抗力相当,保护率为40%左右。但在对12型菌株的抵抗力上,三价灭活疫苗免疫组1和2的保护率达到60%,而商品疫苗的保护率只有20%。三价灭活疫苗免疫组3对4型和5型菌株的免疫保护率要比商品疫苗免疫组保护率高20%,保护率为60%。在对12型菌株的抵抗力上,三价灭活疫苗免疫组3的保护率要比商品疫苗免疫组的保护率高60%,达到80%。综上所述,本研究初步研制的副猪嗜血杆菌三价灭活疫苗的免疫保护效果要优于商品灭活疫苗的免疫保护效果,对副猪嗜血杆菌血清4型、5型及12型具有较好的免疫保护能力。
[Abstract]:Haemophilus parasuis is commonly found in the upper respiratory tract of healthy pigs and belongs to Haemophilus Pasteulidae. It mainly causes polyserositis and arthritis in pigs clinically.In recent years, Haemophilus paraswine disease in domestic pig farms is becoming more and more serious, causing serious losses to various farms.In this study, Haemophilus parasuis was isolated and identified from diseased and dead pigs and healthy pigs in Jiangsu and Anhui pig farms in order to provide the basis for the prevention and control of Haemophilus parasuis.At the same time, a trivalent inactivated vaccine of Haemophilus parasuis serotype 4, serotype 5 and type 12 was developed in our laboratory.Evaluation of immune protection of BALB/c mice. 1 isolation and identification of Haemophilus suis in Yangzhou University Animal Hospital and a slaughterhouse of Yangzhou were collected and cultured on chocolate medium.The 16s rRNA of Haemophilus parasuis was detected by PCR.PCR positive strains were purified and preserved.After PCR identification, 8 Haemophilus parasuis strains were isolated from dead pigs and 26 Haemophilus parasuis strains were isolated from healthy pigs, the isolation rate was 7.12%.Three strains of Haemophilus parasuis were sequenced. The results showed that the homology of 16 SrRNA gene sequence was over 99%.All isolates were classified by glassy plate agglutination.Three strains of serotype 5, two strains of serotype 12, one strain of serotype 14 and two strains of untyped serotype were isolated from dead pigs.Preliminary development of a trivalent inactivated vaccine against Haemophilus parasuis serotype 4, serotype 4, serotype 15. 38, serotype 5 and serotype 3. 85, serotype 11, type 11, serotype 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, type 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11, strain 11Type 12 and type 12 isolates were used to attack guinea pigs intraperitoneally, respectively.Results all guinea pigs of serotype 5 and 12 died, and 1 guinea pig of serotype 4 died.The peritoneal effusion, pleural effusion and a small amount of cellulose exudation were found in the dead guinea pigs, which were similar to the symptoms of Haemophilus parahaemophilus in the diseased pigs.Three different doses of Haemophilus parahaemophilus serotype 4 serotype 5 and 12 strains were used to attack BALB/c mice intraperitoneally.Results when the amount of the three serotypes was 5.0 脳 109CFU, all the BALB / c mice died.At the dose of 1.0 脳 109CFU, one strain of serotype 4 and one strain of serotype 5 died (2 of them), while the strain of serotype 12 did not.BALB/c mice were immunized subcutaneously with trivalent inactivated vaccine 1 (containing 3 serotype strains 1.0 脳 10 9 CFUU each), trivalent inactivated vaccine 2 (containing 3 serotypes of 2. 0 脳 10 9 CFU), trivalent inactivated vaccine 3 (containing 4 0 脳 10 9 CFU each of 3 serotypes) and commercial inactivated vaccine.After the second immunization for 14 days, the mice were given intraperitoneal attack.Results the resistance of trivalent inactivated vaccine groups 1 and 2 to 4 and 5 strains was similar to that of commercial vaccine groups, and the protective rate was about 40%.However, the protective rates of trivalent inactivated vaccine groups 1 and 2 reached 60%, while that of commercial vaccines was only 20%.The protective rate of trivalent inactivated vaccine against 4 and 5 strains was 20% higher than that of commercial vaccine group, and the protective rate was 60%.The protective rate of trivalent inactivated vaccine group 3 was 60% higher than that of commercial vaccine group on resistance to strain 12.To sum up, the immune protective effect of the trivalent inactivated vaccine of Haemophilus parais was better than that of the commercial inactivated vaccine, and it had a better immune protection ability to Haemophilus parais serotype 4, serotype 5 and serotype 12.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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