1例种鹅鸭瘟病毒与多杀性巴氏杆菌混合感染的诊断及病原鉴定

doi:10.16431/j.cnki.1671-7236.2025.05.029

摘要/Abstract

摘要： 【目的】确定贵州某种鹅场种鹅发病死亡的病因，为种鹅多病原混合感染的防治提供参考。【方法】对发病灰鹅进行临床症状及剖检观察、组织切片观察、细菌分离培养鉴定和6种水禽常见病毒(鸭瘟病毒(DPV)、鸭圆环病毒(DuCV)、禽流感病毒(AIV)、鹅细小病毒(GPV)、鸭病毒性肝炎病毒(DHV)和禽坦布苏病毒(TMUV))PCR检测，进一步对病原菌进行相似性分析、荚膜血清型鉴定、药敏试验和致病性试验及分离病毒的致病性检测。【结果】发病灰鹅头颈肿大，肝脏表面出现灰黄色坏死灶，心脏及肺脏表面有出血点。组织切片结果显示，发病灰鹅肝脏、肺脏和肾脏均出现明显病变。分离菌株菌落呈灰白色、表面光滑的露滴状；革兰染色见红色短杆状阴性菌；瑞氏染色见两极浓染的球杆菌。生化试验结果显示，分离菌株葡萄糖、果糖、甘露醇和麦芽糖等反应为阳性。PCR检测及16S rRNA基因鉴定结果显示，分离菌株扩增出大小为460 bp的多杀性巴氏杆菌特异性基因Kmt1,且与NCBI中巴氏杆菌参考菌株16S rRNA基因序列相似性为98.6%～99.3%，鉴定其为多杀性巴氏杆菌。经血清型鉴定，分离株为A型荚膜血清型多杀性巴氏杆菌。分离菌株对诺氟沙星、庆大霉素和庆大霉素等敏感，对万古霉素中度敏感；将多杀性巴氏杆菌分离株接种小鼠，测得最小致死量(MLD)<5 CFU。病毒PCR检测及测序比对分析显示，发病灰鹅为鸭瘟病毒(DPV)感染，且DPV分离株与NCBI DPV参考株UL6基因序列相似性为98.9%～100%；经鸭胚盲传3代，尿囊液PCR检测出DPV；鸭胚成纤维细胞(DEF)接种DPV，9 h后DEF缩圆，24 h后贴壁的DEF脱落或形成合胞体；经测定，DPV分离株的半数组织培养感染剂量(TCID₅₀)为2.88×10^-9/0.1 mL。【结论】本研究最终诊断发病灰鹅为DPV与多杀性巴氏杆菌混合感染，并分离得到1株毒力较强的A型荚膜血清型多杀性巴氏杆菌与1株毒力较强的DPV，本研究结果可为多杀性巴氏杆菌与DPV感染水禽引起的疾病的防治、病原学调查和多联疫苗等提供科学依据和技术支持。

关键词: 鹅; A型多杀性巴氏杆菌; 鸭瘟病毒; 分离鉴定; 病原特性

Abstract: 【Objective】 This experiment was conducted to determine the cause of morbidity and mortality of breeding geese in a goose farm in Guizhou,and provide reference for the prevention and treatment of mixed infection of multiple pathogens in breeding geese.【Method】 Clinical symptoms,necropsy observation,tissue section observation,bacterial isolation,culture and identification,and PCR detection of 6 common waterfowl viruses (Duck plague virus (DPV),Duck circovirus (DuCV),Avian influenza virus(AIV),Goose parvovirus (GPV),Duck viral hepatitis virus (DHV) and Avian Tambusu virus (TMUV)) were performed on the affected gray geese.Furthermore,the similarity analysis,capsular serotype identification,drug sensitivity test and pathogenicity test of the pathogenic bacteria were carried out,and the pathogenicity of the isolated virus was detected.【Result】 The head and neck of the affected gray goose were enlarged,and there were gray-yellow necrotic foci on the surface of the liver,and hemorrhagic spots on the surface of the heart and lung.The results of tissue sections showed that the affected gray goose had obvious lesions in liver,lung and kidney.The isolated bacterial colonies were gray-white,like dew drops with smooth surface.Gram staining showed short red rod-shaped negative bacteria.Wright’s staining showed highly stained coccobacillus at both poles.The results of biochemical tests showed that the isolated strain was positive for glucose,fructose,mannitol and maltose.PCR detection and 16S rRNA gene identification results showed that the isolated strain amplified a 460 bp Pasteurella multocida specific gene Kmt1.The similarity of 16S rRNA gene sequence between the isolate and NCBI reference Pm strains was 98.6%-99.3%,which indicated that Pm isolate was identified as Pasteurella multocidal (Pm).The Pm isolate was identified as A capsular strain by serotype analysis.The Pm isolate was susceptible to norfloxacin,gentamicin and gentamycin,and moderately susceptible to vancomycin.The minimum lethal dose (MLD) of Pm strain to mouse was less than 5 CFU.The PCR and sequencing analysis of the virus showed that the affected gray geese were infected by Duck plague virus (DPV),the UL6 gene sequence of the DPV isolate shared 98.9%-100% similarity with the reference DPV strains of NCBI.After three passages of blind transmission from duck embryos,DPV was detected by PCR in allantois fluid.Duck embryo fibroblasts (DEF) were inoculated with DPV.After 9 h,DEF shranked,and attached DEF fell off or formed syncytia after 24 h.The median tissue culture infective dose (TCID₅₀) of DPV isolate was 2.88×10^-9/0.1 mL.【Conclusion】 In this study,the infected grey goose was finally diagnosed as mixed infection of DPV and Pm,and one virulent Pm strain of capsular serotype A and one virulent DPV were isolated.The results of this study could provide scientific basis and technical support for the prevention and control of diseases caused by Pm and DPV infection of waterfowl,etiological investigation and multi-combination vaccine.

Key words: goose; Pasteurella multocida type A; Duck plague virus; isolation and identification; characteristics of pathogens

中图分类号:

吴鹏, 袁阳, 杨芸芸, 冯轶, 王艳, 杨颖, 陈江凤, 姜海波, 文明. 1例种鹅鸭瘟病毒与多杀性巴氏杆菌混合感染的诊断及病原鉴定[J]. 中国畜牧兽医, 2025, 52(5): 2253-2265.

WU Peng, YUAN Yang, YANG Yunyun, FENG Yi, WANG Yan, YANG Ying, CHEN Jiangfeng, JIANG Haibo, WEN Ming. Diagnosis and Pathogen Identification of a Case of Mixed Infection of Duck Plague Virus and Pasteurella multocida in Breeding Goose[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2253-2265.

参考文献

[1] 田琴,张明华,周碧君,等.鸭瘟病毒感染鸭十二指肠黏膜炎症相关基因及调控通路的筛选[J].中国畜牧兽医,2020,47(10):3323-3333.TIAN Q,ZHANG M H,ZHOU B J,et al.Screening of duodenal mucosal inflammation related genes and regulatory pathways in ducks with Duck plague virus infection[J]. China Animal Husbandry & Veterinary Medicine,2020,47(10):3323-3333.(in Chinese)
[2] 杨伏春,刘芮,李晓涵,等.鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析[J].畜牧兽医学报,2022,53(8):2689-2696.YANG F C,LIU R,LI X H,et al.Construction of UL41 gene deletion strain of Duck enteritis virus and analysis of its replication ability in vitro[J].Acta Veterinaria et Zootechnica Sinica,2021,53(8):2689-2696.(in Chinese)
[3] 包涛涛,彭彩丽,杨先富,等.1起鸭感染鸭瘟病毒的诊治[J].养禽与禽病防治,2023,1:21-24.BAO T T,PENG C L,YANG X F,et al.Diagnosis and treatment of a Duck plague virus infection[J].Poultry Husbandry and Disease Control,2023,1:21-24.(in Chinese)
[4] 杨树宇.鸭瘟病毒间接ELISA与胶体金免疫层析试纸检测方法的建立[D].新乡：河南科技学院,2023.YANG S Y.Establishment of indirect ELISA and colloidal gold immunochromatography test strip detection method for Duck blast virus[D].Xinxiang: Henan Institute of Science and Technology,2023.(in Chinese)
[5] 冼永杰.一例人工饲养丹顶鹤感染禽霍乱的诊断与体会[J].福建畜牧兽医,2024,46(3):66-67.XIAN Y J.Diagnosis and experience of a case of red-horned crane infected with avian cholera in artificial breeding[J].Fujian Journal of Animal Husbandry and Veterinary Medicine,2024,46(3):66-67.(in Chinese)
[6] 张蕾,陈亮,金振华,等.1株羊源多杀性巴氏杆菌的分离鉴定及病原特性研究[J].中国畜牧兽医,2024,51(1):347-356.ZHANG L,CHEN L,JIN Z H,et al.Isolation,identification and pathogenic characteristics of one Pasteurella multocida isolate from sheep[J].China Animal Husbandry & Veterinary Medicine,2024,51(1):347-356.(in Chinese)
[7] 郭伟娜,王紫苇,马佰贺,等.鸭源多杀性巴氏杆菌的分离鉴定及药敏试验[J].安徽科技学院学报,2024,38(1):1-6.GUO W N,WANG Z W,MA B H,et al.Isolation,identification and antimicrobial susceptibility test of Pasteurella multocida from duck[J].Journal of Anhui Science and Technology University,2024,38(1):1-6.(in Chinese)
[8] 胡璇,蔡平,李红婷,等.一株猪源A型多杀性巴氏杆菌的分离鉴定[J].中国兽医科学,2021,51(12):1568-1578.HU X,CAI P,LI H T,et al.Isolation and identification of a strain of Pasteurella multocida type A from swine[J].Chinese Veterinary Science,2021,51(12):1568-1578.(in Chinese)
[9] CLINICAL AND LABORATORY STANDARDS INSTITUTE.Performance standards for antimicrobial susceptibility testing[S].28th ed.CLSI supplement M100.Wayne,PA:Clinical and Laboratory Standards Institute,2018.
[10] 刘跃清,翁习琴.家禽常见疾病的临床症状与治疗[J].畜禽业,2021,32(8):101-102.LIU Y Q,WENG X Q.Clinical symptoms and treatment of common poultry diseases[J].Livestock and Poultry Industry,2021,32(8):101-102.(in Chinese)
[11] 吕明哲.四种禽病流行状况的简要分析[J].现代畜牧科技,2021,11:118.LYU M Z.A brief analysis of the epidemic status of four avian diseases[J].Modern Animal Husbandry Science & Technology,2021,11:118.(in Chinese)
[12] MUBAMBA C,RAMSAY G,ABOLNIK C,et al.Is syndromic data from rural poultry farmers a viable poultry disease reporting tool and means of identifying likely farmer responses to poultry disease incursion?[J].Preventive Veterinary Medicine,2018,153:84-93.
[13] LI N,HONG T,LI R,et al.Pathogenicity of duck plague and innate immune responses of the Cherry Valley ducks to Duck plague virus[J].Scientific Repots,2016,6:32183.
[14] KONG J,WU X,LIAO L,et al.Duck plague virus infection alter the microbiota composition and intestinal functional activity in Muscovy ducks[J].Poultry Science,2023,102(3):102365.
[15] 赵楠.鹅禽霍乱的流行病学、临床症状、实验室诊断及防控[J].现代畜牧科技,2021,3:114-115.ZHAO N.Epidemiology,clinical symptoms,laboratory diagnosis and control of goose and poultry cholera[J].Modern Animal Husbandry Science & Technology,2021,3:114-115.(in Chinese)
[16] 朱海行,陈淑芳.一例浙东白鹅禽霍乱病例报告[J].科学种养,2020,5:49.ZHU H X,CHEN S F.A case report of White goose and poultry cholera in Eastern Zhejiang province[J].Kexue Zhongyang,2020,5:49.(in Chinese)
[17] 侯庆同.1例猪圆环病毒1型和巴氏杆菌混合感染的诊治[J].养殖与饲料,2023,22(3):71-73.HOU Q T.Diagnosis and treatment of a case of mixed infection of Porcine circovirus type 1 and Pasteurella[J].Animals Breeding and Feed,2023,22(3):71-73.(in Chinese)
[18] 李玉峰,于可响,胡峰,等.山东省水禽主要疫病流行现状[J].中国禽业导刊,2024,41(6):49-56.LI Y F,YU K X,HU F,et al.The epidemic status of major diseases in waterfowl of Shandong province[J].Guide to Chinese Poultry,2024,41(6):49-56.(in Chinese)
[19] 李文军.探析规模化养鸭常见疾病的诊断及防治[J].畜牧兽医科技信息,2022,9:236-237.LI W J.Analysis on the diagnosis and prevention of common diseases in large-scale duck breeding[J]. Chinese Journal of Animal Husbandry and Veterinary Medicine,2022,9:236-237.(in Chinese)
[20] 李玉琪.基于LORF5基因5'UTR区鉴别鸭瘟强、弱毒的PCR方法建立及应用[D].雅安：四川农业大学,2022.LI Y Q.Establishment and application of PCR method based on LORF5 gene 5'UTR region to identify virulent and attenuated strains of DPV[D].Ya’an:Sichuan Agricultural University,2022.(in Chinese)
[21] 陈非玥.miR-27b和miR-223在白术多糖缓解LPS诱导的雏鹅肝脏炎性损伤中的作用[D].广州：仲恺农业工程学院,2022.CHEN F Y.The role of miR-27b and miR-223 in Atractylodes polysaccharide alleviating LPS-induced inflammatory liver injury in gosling[D].Guangzhou：Zhongkai University of Agriculture and Engineering,2022.(in Chinese)
[22] RICHARDSON N I,RAVENSCROFT N,KUTTEL M M.Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides[J].Glycobiology,2023,33(9):745-754.
[23] HAMADA K,KAWASHIMA S,HOSHINOO K,et al.Draft genome sequence of Pasteurella multocidastrain BD1769 with untypable capsular serotype isolated from a layer chicken[J].Microbiology Resource Announcements,2023,12(11):e0059423.
[24] 刘洪港,赵杰,石艳红,等.一株鸭源多杀性巴氏杆菌的分离鉴定及耐药性分析[J].家禽科学,2024,46(7):23-30.LIU H G,ZHAO J,SHI Y H,et al.Isolation,identification and drug resistance analysis of a duck-origin Pasteurella multocida strain[J]. Poultry Science,2021,46(7):23-30.(in Chinese)
[25] 贾惠言,周潇,戴久丽,等.浙东白鹅种鹅多杀性巴氏杆菌分离鉴定及药敏试验[J].中国畜禽种业,2023,19(11):139-144.JIA H Y,ZHOU X,DAI J L,et al.Isolation,identification and antimicrobial susceptibility test of Pasteurella multocida from White goose of Eastern Zhejiang province[J].The Chinese Livestock and Poultry Breeding,2023,19(11):139-144.(in Chinese)
[26] 杨泽晓,刘常钰,王印,等.一株鹅源多杀性巴氏杆菌强毒株的分离鉴定及其血清型鉴定相关基因的克隆[J].浙江农业学报,2016,28(6):935-943.YANG Z X,LIU C Y,WANG Y,et al.Isolation,identification of a virulent strain goose Pasteurella multocida and gene cloning of its serotype associated genes[J].Acta Agriculturae Zhejiangensis,2016,28(6):935-943.(in Chinese)