| 多杀性巴氏杆菌与禽白血病病毒亚群混合感染诊断及相关基因分析 |
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| 引用本文： | 罗东还,吴强,温贵兰,胡璇,张小波,文明,欧德渊,龚新勇.多杀性巴氏杆菌与禽白血病病毒亚群混合感染诊断及相关基因分析[J].南方农业学报,2022,53(2):526-537. |
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| 作者姓名： | 罗东还 吴强 温贵兰 胡璇 张小波 文明 欧德渊 龚新勇 |
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| 作者单位： | 1 贵州大学动物科学学院/贵州省动物生物制品工程技术研究中心, 贵阳 550025;2 思南县农业农村局, 贵州铜仁 565100 |
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| 基金项目： | 贵州省科技支撑计划项目;贵州省级农业科技支撑计划项目;课程体系改革项目;贵州省高等学校教学内容 |
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| 摘 要： | 【目的】明确引起贵州某规模化（20万羽）养殖场三黄鸡群发病的病因及科学用药，最大限度减轻经济损失，同时为规模化养殖场防控禽白血病和净化鸡群提供参考依据。【方法】无菌采集送检病鸡的心脏、肝脏和脾脏等组织进行细菌分离培养、生化试验、荚膜分型、毒力基因和耐药基因等分子生物学检测，同时对采集病料进行禽白血病病毒（ALV） PCR检测及gp85基因克隆测序分析。【结果】成功分离获得1株多杀性巴氏杆菌（Pasteurella multocida，Pm），命名为Pm-GZXF2021； Pm-GZXF2021为荚膜A型Pm，具有黏附素（ptfA、hsf-1、hsf-2、pfhA、fimA和tadD）、超氧化物歧化酶（sodA）、外膜蛋白（ompA、ompH、plpB和oma87）和铁摄取（exbB、exbD、tonB、Fur、hgbB和hgbA）相关的毒力基因，同时携带有喹诺酮类的gyrB耐药基因和内酰胺类的tem耐药基因；药敏试验结果表明，Pm-GZXF2021对米诺环素、丁胺卡那、哌拉西林、庆大霉素、多西环素、氧氟沙星、新霉素、头孢拉定、麦迪霉素及头孢哌酮等药物敏感，而对头孢他啶、苯唑西林、羧苄西林及头孢曲松等药物已产生耐药性；健康昆明小鼠在接种Pm-GZXF2021纯培养菌液（1.7×108 CFU/mL）后12 h内全部死亡，剖检可见昆明小鼠小肠均出现胶冻样浸润，肝脏肿大且伴有白色坏死灶。从病鸡的组织样品检测出ALV，命名为ALV-GZXF2021；由基于gp85基因核苷酸序列相似性构建的ALV系统发育进化树可知，ALV-GZXF2021与5株ALV-K参考毒株处于同一分支，与A、B、D、E、J亚群处于不同分支，即ALV-GZXF2021属于K亚群。【结论】贵州某规模化养殖场三黄鸡群发病是由Pm与K亚群外源性ALV混合感染所致。因此，养殖场应采取相应的防控措施，一般包括加强饲养管理，做好生物安全措施及疫苗接种等，尤其是建立无禽白血病的种鸡群。
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| 关 键 词： | 多杀性巴氏杆菌 (Pm) 禽白血病病毒 (ALV) K亚群 混合感染 基因分析 |
| 收稿时间： | 2021-12-06 |
Diagnosis and gene analysis of mixed infection of Pasteurella multocida and avian leukemia virus-K subgroup |
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| LUO Dong-huan,WU Qiang,WEN Gui-lan,HU Xuan,ZHANG Xiao-bo,WEN Ming,OU De-yuan,GONG Xin-yong.Diagnosis and gene analysis of mixed infection of Pasteurella multocida and avian leukemia virus-K subgroup[J].Journal of Southern Agriculture,2022,53(2):526-537. |
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| Authors: | LUO Dong-huan WU Qiang WEN Gui-lan HU Xuan ZHANG Xiao-bo WEN Ming OU De-yuan GONG Xin-yong |
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| Affiliation: | 1 College of Animal Science, Guizhou University/Guizhou Animal Biological Products Engineering Technology Research Center, Guiyang 550025, China;2 Sinan Agriculture and Rural Bureau, Tongren, Guizhou 565100, China |
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| Abstract: | 【Objective】To clarify the etiology of Sanhuang chickens in a large-scale(200000 feather) breeding farm in Guizhou,scientifically used medicine to minimize economic loss,and provide reference for prevention and control of avian leukemia and purification of chickens in large-scale breeding farms.【Method】The heart,liver and spleen tissues of chickens were collected aseptically for bacterial isolation and identification,biochemical test,capsule typing,virulence gene and drug resistance gene detection,avian leukemia virus(ALV) PCR detection,gp85 gene cloning and sequencing analysis,etc.【Result】A Pasteurella multocida(Pm) strain was isolated and named as Pm-GZXF2021. Pm-GZXF2021 was type A capsulatus of Pm with the associated virulence genes of adhesin(ptfA,hsf-1,hsf-2,pfhA,fimA and tadD), superoxide dismutase(sodA),outer membrane protein(ompA,ompH,plpB and oma87) and iron uptake(exbB,exbD, tonB,Fur,hgbB and hgbA). Meanwhile,the isolate strain carried a gyrB resistance gene for quinolones and a tem resistance gene for lactamides. Drug sensitivity test showed that Pm-GZXF2021 was sensitive to minocycline,mikacin,piperacillin,gentamicin,doxycycline,levofloxacin,neomycin,cefradine,medicin and cefoperazone,but resistant to ceftazidime,oxacillin,carbenicillin and ceftriaxone sodium. In addition,all healthy Kunming mice died within 12 h after inoculation with Pm-GZXF2021 pure culture broth(1.7×108 CFU/mL). By necropsy,the small intestine of Kunming mice showed jelly infiltration,and the liver was enlarged with white necrosis. ALV was detected from tissue samples of sick chickens and named ALV-GZXF2021. The ALV phylogenetic tree constructed based on the nucleotide sequence similarity of the gp85 gene showed that ALV-GZXF2021 was in the same branch as the five ALV-K reference strains and was in different branches with subgroups A,B,D,E,and J. In other words,ALV-GZXF2021 belonged to ALV-K.【Conclusion】The incidence of Sanhuang chicken group in a large-scale farm in Guizhou is caused by exogenous ALV infection of Pm and K subgroups. Therefore,the farm should take corresponding prevention and control measures,generally including strengthening feeding management,biosafety measures and vaccination,especially the establishment of breeding chickens without avian leukemia. |
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