1株血清2型鸭疫里默氏杆菌的分离鉴定、致病性研究及灭活疫苗制备

doi:10.16431/j.cnki.1671-7236.2024.12.036

摘要/Abstract

摘要： 【目的】本研究拟对从1例死亡雏鸭体内分离获得的鸭疫里默氏杆菌进行生物学特性和疫苗效果分析，旨在为鸭传染性浆膜炎的防治提供指导。【方法】采集送检雏鸭的脑、心脏和肝脏组织进行细菌分离培养，通过菌落形态观察、革兰染色镜检、16S rDNA PCR扩增测序鉴定菌株并分析其遗传进化关系；采用玻片凝集法鉴定菌株血清型，检测菌株对常见抗菌药物的敏感性及致病性；利用分离株制备灭活疫苗，测定其免疫保护效果。【结果】分离株在血平板上呈半透明、露滴状圆形小菌落，革兰染色可见多个丝状排列的阴性短杆菌，瑞氏染色可见两级浓染的短小杆菌。16S rDNA测序结果显示，分离株与鸭疫里默氏杆菌参考株CZ-RA03处于同一分支，相似性＞99.9％。血清型鉴定结果显示，分离株为鸭疫里默氏杆菌血清2型。药敏试验结果显示，分离株对氧氟沙星、链霉素、卡那霉素等药物耐药，对氟苯尼考、四环素、多西环素等药物敏感。致病性试验结果显示，分离株对7日龄雏鸭的半数致死量(LD₅₀)为1.51×10⁷ CFU，雏鸭感染后肾脏载菌量最高，为2.15×10⁶ CFU/g。利用分离株制备油乳剂灭活疫苗免疫7日龄雏鸭，其免疫攻毒后存活率为86.67%。【结论】本研究从发病雏鸭中分离鉴定出1株具有较高致病性的血清2型鸭疫里默氏杆菌，以该菌制备的油乳剂灭活疫苗具有良好的免疫保护效果。研究结果为鸭传染性浆膜炎的防治提供了理论基础。

关键词: 鸭疫里默氏杆菌; 生物学特性; 免疫原性

Abstract: 【Objective】 The objective of this study was to analyze the biological characteristics and vaccine effectiveness of Riemerella anatipestifer isolated from a dead duckling,in order to provide guidance for the prevention and treatment of infectious serositis of duck.【Method】 The brain,heart and liver tissues of the duckling were collected for bacterial isolation and culture.The strain was identified by colony morphology observation,Gram staining microscopy,16S rDNA PCR amplification and sequencing,and the genetic evolution relationship was analyzed.The serotype of the strain was identified by slide agglutination method,and the susceptibility to common antibiotics and pathogenicity of the strain were detected.The inactivated vaccine was prepared by the isolate and its immunoprotective effect was measured.【Result】 The isolate showed semi-transparent,dewdrop-like round colonies on the blood plate.Gram staining showed multiple filamentous arrangement of negative short bacilli,and Wright’s staining showed two stages of intensely stained short bacilli.16S rDNA sequencing showed that the isolate was in the same branch as the reference strain of Riemerella anatipestifer CZ-RA03，and the similarity was over 99.9%.Serotype identification showed that the isolate was Riemerella anatipestifer serotype 2.The results of drug sensitivity test showed that the isolate was resistant to ofloxacin,streptomycin and kanamycin,and sensitive to flufenicol,tetracycline and doxycycline.The results of pathogenicity test showed that the median lethal dose (LD₅₀)of the isolate to 7-day-old duckling was 1.51×10⁷ CFU,and the kidney bacterial load of infected duckling was the highest (2.15×10⁶ CFU/g).The oil emulsion inactivated vaccine was used to immunize 7-day-old duckling,and the surviral rate was 86.67%.【Conclusion】 In this study,a high pathogenicity strain of Riemerella anatipestifer serotype 2 was isolated and identified from infected ducklings,and the oil emulsion inactivated vaccine prepared by the strain had good immune protection effect.The results provided a theoretical basis for the prevention and treatment of infectious serositis of duck.

Key words: Riemerella anatipestifer; biological characteristics; immunogenicity

中图分类号:

S852.61

戴婷婷, 刘荣昌, 陈海玉, 庄丽云, 韦玉帆, 谭慧敏, 刘洋, 唐福川, 黄瑜, 程艳青, 包银莉, 郑新添. 1株血清2型鸭疫里默氏杆菌的分离鉴定、致病性研究及灭活疫苗制备[J]. 中国畜牧兽医, 2024, 51(12): 5499-5506.

DAI Tingting, LIU Rongchang, CHEN Haiyu, ZHUANG Liyun, WEI Yufan, TAN Huimin, LIU Yang, TANG Fuchuan, HUANG Yu, CHENG Yanqing, BAO Yinli, ZHENG Xintian. Isolation, Identification, Pathogenicity Analysis and Inactivated Vaccine Preparation of a Strain of Riemerella anatipestifer Serotype 2[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5499-5506.

参考文献

[1] HU Q, QI J, BO H, et al.Complete genome sequence of Riemerella anatipestifer serotype 10 strain HXb2[J].Microbiology Resource Announcements, 2017, 5(18):1110-1128.
[2] 吕泽昊, 韩姗姗, 秦立廷, 等.3株鸭疫里默氏杆菌的分离鉴定和耐药性分析[J].中国兽医杂志, 2023, 59(6):87-92.LYU Z H, HAN S S, QIN L T, et al.Isolation, identification and drug resistance analysis of 3 strains of Riemerella anatipestifer[J].Chinese Journal of Veterinary Medicine, 2023, 59(6):87-92.(in Chinese)
[3] OREN A, GARRITY G M.List of new names and new combinations previously effectively, but not validly, published[J].International Journal of Systematic and Evolutionary Microbiology, 2020, 70(1):1-5.
[4] GARCIA-LOPEZ M, MEIER-KOLTHOFF J P, TINDALL B J, et al.Analysis of 1000 type-strain genomes improves taxonomic classification of bacteroidetes[J].Frontiers in Microbiology, 2019, 23(10):2083.
[5] ZHU X H, LI Y L, WANG Z W, et al.Antibiotic resistance of Riemerella anatipestifer and comparative analysis of antibiotic-resistance gene detection methods[J].Journal of Poultry Science, 2023, 102(3):102405.
[6] 吴植, 董洪燕, 郭长明, 等.鸭疫里默氏杆菌的分离鉴定及生物学特性分析[J].中国家禽, 2023, 45(10):122-126.WU Z, DONG H Y, GUO C M, et al.Isolation, identification and biological characteristics analysis of Riemerella anatipestifer[J].China Poultry, 2023, 45(10):122-126.(in Chinese)
[7] 程龙飞, 傅光华, 施少华, 等.鸭传染性浆膜炎二价灭活疫苗的研制[J].福建农业学报, 2014, 29(11):1058-1061.CHENG L F, FU G H, SHI S H, et al.Development of bivalent inactivated vaccine against infectious serositis in ducks[J].Fujian Journal of Agricultural Sciences, 2014, 29(11):1058-1061.(in Chinese)
[8] YANG S, DONG W, LI G, et al.A recombinant vaccine of Riemerella anatipestifer OmpA fused with duck IgY Fc and Schisandra chinensis polysaccharide adjuvant enhance protective immune response[J].Microbial Pathogenesis, 2019, 136(9):103707.
[9] CHANG F F, CHEN C C, WANG S H, et al.Epidemiology and antibiogram of Riemerella anatipestifer isolated from waterfowl slaughterhouses in Taiwan[J].Journal of Veterinary Research, 2019, 63(1):79-86.
[10] LYU Z, HAN S, LI J, et al.Epidemiological investigation and drug resistance characteristics of Riemerella anatipestifer strains from large-scale duck farms in Shandong province, China from March 2020 to March 2022[J].The Journal of Poultry Science, 2023, 102(7):102759.
[11] HAN W, CHEN Z, NIU P, et al.Development of a colloidal gold immunochromatographic strip for rapid detection of Riemerella anatipestifer in ducks[J].The Journal of Poultry Science, 2020, 99(10):4741-4749.
[12] 顾玉美, 侯军, 钟万金, 等.鸭疫里默氏杆菌流行菌株的血清型分析与最小感染量测试[J].中国禽业导刊, 2023, 40(3):60-63.GU Y M, HOU J, ZHONG W J, et al.Serotype analysis and minimum infection dose test of epidemic strains of Riemerella anatipestifer[J].Guide to Chinese Poultry, 2023, 40(3):60-63.(in Chinese)
[13] 林志敏, 林彬彬, 谢碧林, 等.鸭疫里默氏杆菌PT-RA1的分离鉴定、遗传进化分析及致病性试验[J].微生物学通报, 2024, 51(1):1-15.LIN Z M, LIN B B, XIE B L, et al.Isolation, identification, genetic evolution and pathogenicity test of PT-RA1 from Riemerella anatipestifer[J].Microbiology China, 2024, 51(1):1-15.(in Chinese)
[14] HASAN A, BOSE P, AKTAR M T, et al.gro EL gene-based molecular detection and antibiogram profile of Riemerella anatipestifer from duck in Bangladesh[J].Journal of Advanced Veterinary and Animal Research, 2022, 9(4):684-693.
[15] HUANG M, LIU M, LIU J, et al.Evaluation of the immunoprotection efficacy of Riemerella anatipestifer fur-deficient mutant as an attenuated vaccine[J].The Journal of Poultry Science, 2023, 102(3):102450.
[16] LI J, ZHANG Y, WANG Y, et al.Immunogenicity of live phoP gene deletion strain of Riemerella anatipestifer serotype 1[J].The Journal of Poultry Science, 2023, 102(1):102294.
[17] KE T, YANG D, YAN Z, et al.Identification and pathogenicity analysis of the pathogen causing spotted spleen in Muscovy duck[J].Frontiers in Veterinary Science, 2022, 23(9):846298.
[18] 张燕.鸭疫里默氏杆菌安徽分离株耐药性分析与致病性研究[D].扬州:扬州大学, 2023.ZHANG Y.Analysis of drug resistance and pathogenicity of Anhui isolates of Riemerella anatipestifer[D].Yangzhou:Yangzhou University, 2023.(in Chinese)
[19] WU H C, CHANG W C, WU M C, et al.Assessment of immunization regimens of duck Riemerella anatipestifer vaccines[J].Journal of Applied Microbiology, 2020, 129(5):1185-1192.
[20] LIU H, WANG X, DING C, et al.Development and evaluation of a trivalent Riemerella anatipestifer-inactivated vaccine[J].Clinical and Vaccine Immunology, 2013, 20(5):691-697.
[21] 嵇辛勤, 傅心亮, 阮涌, 等.贵州省鸭疫里默氏杆菌血清2型灭活疫苗制备及免疫研究[J].中国预防兽医学报, 2016, 38(2):151-154.JI X Q, FU X L, RUAN Y, et al.Preparation and immunity of serotype 2 inactivated vaccine against Riemerella anatipestifer in Guizhou province[J].Chinese Journal of Preventive Veterinary Medicine, 2016, 38(2):151-154.(in Chinese)
[22] 田宇杰, 李婷, 代国滔, 等.A∶L1型鸭源多杀性巴氏杆菌灭活疫苗制备及免疫效力研究[J].中国畜牧兽医, 2022, 49(7):2716-2724.TIAN Y J, LI T, DAI G T, et al.Preparation and immunological efficacy of A∶L1 inactivated Pasteurella multocida vaccine from ducks[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(7):2716-2724.(in Chinese)
[23] 徐景峨, 李婷, 陈汝佳, 等.鸭疫里氏杆菌二价灭活疫苗制备及免疫效力研究[J].中国畜牧兽医, 2023, 50(1):290-299.XU J E, LI T, CHEN R J, et al.Study on preparation and immune efficacy of bivalent inactivated vaccine against Riemerella anatipestifer[J].China Animal Husbandry & Veterinary Medicine, 2023, 50(1):290-299.(in Chinese)
[24] 马光强, 刘丽娟, 陈国权, 等.鸭疫里默氏杆菌贵州流行株蜂胶灭活疫苗制备[J].中国畜牧兽医, 2022, 49(8):3163-3170.MA G Q, LIU L J, CHEN G Q, et al.Preparation of propolis inactivated vaccine against Riemerella anatipestifer endemic strain in Guizhou[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(8):3163-3170.(in Chinese)