福建省某规模化猪场胸膜肺炎放线杆菌血清1型的分离鉴定

doi:10.16431/j.cnki.1671-7236.2024.12.031

摘要/Abstract

摘要： 【目的】对福建某猪场疑似患有猪接触传染性胸膜肺炎的病死猪肺脏进行胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae，APP)的分离鉴定，并确定APP分离株的血清型及其生物学特性，以期为猪场临床合理用药和制定防疫策略提供科学依据。【方法】对1例疑似APP感染致死的后备母猪的肺脏组织进行病原分离培养、染色镜检和生化鉴定，同时进行16S rRNA和Apx Ⅳ毒素基因PCR扩增及血清型分析，采用药敏试验和动物致病性试验探究其耐药情况及毒力。【结果】从病死猪肺脏中成功分离到1株细菌，菌体形态为表面光滑、边缘整齐、呈灰白色半透明的圆形菌落，革兰染色镜检可见多形性的革兰阴性短杆菌，具有APP的培养特征。生化鉴定结果显示，分离株能利用木糖、葡萄糖、甘露醇，尿素酶、硝酸盐还原和甲基红试验均为阳性，与APP的生化特性一致。16S rRNA与Apx Ⅳ基因测序比对结果显示，扩增序列与GenBank中APP序列的相似性均＞98%，进一步证实分离株为APP，将其命名为APP FJ2022。血清型鉴定结果显示，分离株为生长依赖NAD的血清1型菌株。药敏试验结果显示，分离株对头孢噻肟、头孢呋辛、头孢美唑、阿莫西林等药物敏感，对四环素、氨苄西林、环丙沙星、氟苯尼考等药物耐药。动物致病性试验结果显示，分离株对小鼠具有致病性，其半数致死量(LD₅₀)为7.71×10⁸ CFU。【结论】本研究从福建省某规模化猪场疑似患猪传染性胸膜肺炎病死猪中分离获得1株血清1型APP，其对多种常见抗菌药物表现耐药，致病性较强。试验结果有助于了解福建省APP的血清型，并为猪场防控APP感染提供参考依据。

关键词: 猪胸膜肺炎放线杆菌; 分离; 鉴定; 药敏试验; 致病性

Abstract: 【Objective】 The aim of this experiment was to isolate and identify Actinobacillus pleuropneumoniae (APP) from the lungs of dead pigs suspected to have contracted contagious pleuropneumoniae from a pig farm in Fujian province,and to determine the serotype and biological characteristics of APP strain.In order to provide scientific basis for clinical rational drug use and epidemic prevention strategy in pig farms.【Method】 In this study,the lung tissue of a suspected to have died from APP infection was isolated,cultured,stained and biochemical identified.PCR amplification of 16S rRNA and Apx Ⅳ toxin genes and serotype analysis were performed.Drug susceptibility test and animal pathogenicity test were used to investigate the drug resistance and virulence.【Result】 A strain from diseased pig lung was successfully isolated.The isolate was round colonies with smooth,orderly,greyish-white translucent,Gram staining was negative with short bacilli with polymorphic,which had the culture characteristics of APP.Biochemical identification results showed that the isolate could use xylose,glucose and mannitol,urease,nitrate reduction and methyl red tests were positive,which was consistent with the biochemical characteristics of APP.The sequencing results of 16S rRNA and Apx Ⅳ genes showed that the similarity between the amplified sequence and the APP sequence in GenBank was more than 98%,which further confirmed that the isolate was APP and named it APP FJ2022.Serotype identification showed that the isolate was growth-dependent serotype 1 strain.The results of drug sensitivity test showed that the isolate was sensitive to cefotaxime,cefuroxime and cefmetazole，amoxicillin,and resistant to tetracycline,ampicillin,ciprofloxacin and flufenicol.The results of animal pathogenicity test showed that the isolate was pathogenic to mice,and its median lethal dose (LD₅₀) was 7.71×10⁸ CFU.【Conclusion】 In this study,a serotype 1 APP strain was isolated from dead pig suspected to be infected with pleuropneumonia in a large-scale pig farm in Fujian province.The isolate was resistant to many common antibiotics and showed strong pathogenicity.The experimental results were helpful to understand the serotype of APP in Fujian province,and provide reference for the prevention and control of APP infection in pig farms.

Key words: Actinobacillus pleuropneumoniae; isolation; identification; drug susceptibility test; pathogenicity

中图分类号:

S852.61

杨圆, 于慧, 何乐, 黄文琳, 戴爱玲, 魏春华, 刘建奎. 福建省某规模化猪场胸膜肺炎放线杆菌血清1型的分离鉴定[J]. 中国畜牧兽医, 2024, 51(12): 5449-5458.

YANG Yuan, YU Hui, HE Le, HUANG Wenlin, DAI Ailing, WEI Chunhua, LIU Jiankui. Isolation and Identification of Actinobacillus pleuropneumoniae Serotype 1 from a Swine Farm in Fujian Province[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5449-5458.

参考文献

[1] 焦淼.APP广东分离株生物学特性研究及其ApxⅡ C基因缺失重组载体的构建[D].南京：南京农业大学，2007.JIAO M.The biocharacteristics research of APP Guangdong strain and construction of the ApxⅡ C gene deleted vector[D].Nanjing:Nanjing Agricultural University, 2007.(in Chinese)
[2] 李婷婷.胸膜肺炎放线杆菌relA基因缺失株的构建及其生物学特性的研究[D].哈尔滨：东北林业大学，2017.LI T T.Construction and characterization of relA gene deleted mutant of Actinobacillus pleuropneumoniae serotype 7 strain S8[D].Harbin:Northeast Forestry University, 2017.(in Chinese)
[3] 汪程.猪胸膜肺炎放线杆菌BJ2020株分离鉴定与病原生物学特性分析[D].贵阳：贵州大学，2021.WANG C.Isolation and identification, and pathogenicity of Actinobacillus pleuropneumoniae strain BJ2020[D].Guiyang:Guizhou University, 2021.(in Chinese)
[4] ANTENUCCI F, FOUGEROUX C, BOSSÉ J T, et al.Identification and characterization of serovar-independent immunogens in Actinobacillus pleuropneumoniae[J].Veterinary Research, 2017, 48(1):74.
[5] STRINGER O W, BOSSÉ J T, LACOUTURE S, et al.Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars[J].Veterinary Microbiology, 2021, 55:109021.
[6] 卢碧凯，袁秀芳，徐丽华，等.胸膜肺炎放线杆菌分离株血清型的PCR鉴定和Apx毒素基因检测[J].畜牧兽医学报，2023，54(3)：1341-1346.LU B K, YUAN X F, XU L H, et al.Molecular serotyping and Apx gene profile analysis of Actinobacillus pleuropneumoniae isolates[J].Acta Veterinaria et Zootechnica Sinica, 2023, 54(3):1341-1346.(in Chinese)
[7] 田绍超，王侨，李琦，等.猪胸膜肺炎放线杆菌血清5型的致病性及感染小鼠后的组织分布研究[J].中国预防兽医学报，2018，40(2)：156-159.TIAN S C, WANG Q, LI Q, et al.Study on tissue distribution and pathogenicity of Actinobacillus pleuropneumonise type 5 in mice[J].Chinese Journal of Preventive Veterinary Medicine, 2018, 40(2):156-159.(in Chinese)
[8] KAMP E M, VERMEULEN T M, SMITS M A, et al.Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis[J].Infection and Immunity, 1994, 62(9):4063-4065.
[9] 李凯明，董发明，刘守川，等.洛阳某猪场猪传染性胸膜肺炎放线杆菌的分离鉴定[J].中国畜牧兽医，2020，47(4)：1241-1249.LI K M, DONG F M, LIU S C, et al.Isolation and identification of porcine contagious Actinobacillus pleuropneumoniae in Luoyang[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(4):1241-1249.(in Chinese)
[10] 张哲玮，袁龙，代小童，等.广西某规模化猪场胸膜肺炎放线杆菌血清5型的分离鉴定[J].畜牧与兽医，2022，54(5)：75-80.ZHANG Z W, YUAN L, DAI X T, et al.Isolation and identification of Actinobacillus pleuropneumoniae serotype 5 from a swine farm in the Guangxi area[J].Animal Husbandry & Veterinary Medicine, 2022, 54(5):75-80.(in Chinese)
[11] TO H, TESHIMA K, KON M, et al.Characterization of nontypeable Actinobacillus pleuropneumoniae isolates[J].Journal of Veterinary Diagnostic Investigation, 2020, 32(4):581-584.
[12] 孙达.猪胸膜肺炎放线杆菌对头孢噻呋耐药判定标准研究[D].武汉：华中农业大学，2019.SUN D.Development of ceftiofur susceptibility breakpoint for Actinobacillus pleuropneumoniae in pigs[D].Wuhan:Huazhong Agricultural University, 2019.(in Chinese)
[13] 郭志英，李郁，吴浩阳，等.猪胸膜肺炎放线杆菌的分离鉴定及其生物学特性的研究[J].中国兽医科学，2016，46(9)：1102-1109.GUO Z Y, LI Y, WU H Y, et al.Isolation and identification of Actinobacillus pleuropneumoniae and studies on characterizations of the APP isolates[J].Chinese Veterinary Science, 2016, 46(9):1102-1109.(in Chinese)
[14] 徐民生，柯海意，施科达，等.广东省猪传染性胸膜肺炎放线杆菌分离鉴定及耐药表型和耐药基因检测与分析[J].中国畜牧兽医，2022，49(8)：3212-3225.XU M S, KE H Y, SHI K D, et al.Isolation and identification of Actinobacillus pleuropneumoniae from Guangdong province and detection and analysis of drug resistance phenotype and gene[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(8):3212-3225.(in Chinese)
[15] 王佳凡，郑博文，杨超，等.四川猪传染性胸膜肺炎放线杆菌耐药基因流行规律与表型相关性研究[J].云南农业大学学报(自然科学)，2021，36(4)：631-639.WANG J F, ZHENG B W, YANG C, et al.Study on the epidemiology regularity and phenotype correlation of drug-resistant genes of infectious Actinobacillus pleuropneumoniae in Sichuan[J].Journal of Yunnan Agricultural University (Natural Science), 2021, 36(4):631-639.(in Chinese)
[16] 车勇良，陈如敬，吴学敏，等.猪胸膜肺炎放线杆菌的分离鉴定及药敏试验[J].养猪，2016，6：102-104.CHE Y L, CHEN R J, WU X M, et al.Isolation and identification of porcine Actinobacillus pleuropneumoniae and drug sensitivity test[J].Swine Production, 2016，6:102-104.(in Chinese)
[17] 贺英，赵萍，储岳峰，等.福建省部分地区猪传染性胸膜肺炎的流行病学调查[J].湖北农业科学，2009，48(6)：1446-1448.HE Y, ZHAO P, CHU Y F, et al.Epidemiological investigation of porcine contagious pleuropneumonia in part area of Fujian province[J].Hubei Agricultural Sciences, 2009, 48(6):1446-1448.(in Chinese)
[18] MATYUS S P, BRAUN P J, WOLAK-DINSMORE J, et al.NMR measurement of LDL particle number using the vantera clinical analyzer[J].Clinical Biochemistry, 2014, 47(16-17):203-210.
[19] COCKERILL F, WIKLER M, BUSH K, et al.Performance standards for antimicrobial susceptibility testing：Twenty-first informational supplement:CLSI document M100-S21[J].Journal of Veterinary Medical Science, 2015, 6(5):30-38.
[20] 张飞，曹三杰，文心田，等.猪传染性胸膜肺炎疫苗的研究进展[J].中国兽医学报，2015，35(10)：111-113.ZHANG F, CAO S J, WEN X T, et al.Advance in vaccines against porcine contagious pleuropneumonia[J].Chinese Journal of Veterinary Science, 2015, 35(10):111-113.(in Chinese)
[21] 张东超，杨宁宁，林静，等.猪传染性胸膜肺炎放线杆菌的分离鉴定及药敏试验[J].中国畜牧兽医，2016，43(6)：1604-1609.ZHANG D C, YANG N N, LIN J, et al.Isolation, identification and drug sensitive tests of porcine contagious Actinobacillus pleuropneumoniae[J].China Animal Husbandry & Veterinary Medicine, 2016, 43(6):1604-1609.(in Chinese)
[22] 仇保丰，宋鸿雁，刘文斌，等.猪胸膜肺炎放线杆菌、副猪嗜血杆菌和多杀性巴氏杆菌的生化及分子鉴定[J].动物医学进展，2014，35(4)：72-76.QIU B F, SONG H Y, LIU W B, et al.Biochemical and molecular identification of Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida[J].Progress in Veterinary Medicine, 2014, 35(4):72-76.(in Chinese)
[23] 袁龙.规模化猪场PRDC细菌病原感染情况调查及APP临床强毒株的筛选[D].武汉：华中农业大学，2022.YUAN L.Investigation of PRDC bacterial pathogens infection in large-scale pig farms and screening of APP virulent clinical strains[D].Wuhan:Huazhong Agricultural University, 2022.(in Chinese)
[24] 陈旺.新疆部分地区猪常见呼吸道细菌性疾病病原的检测[D].乌鲁木齐：新疆农业大学，2023.CHEN W.Detection of pathogens of common respiratory tract bacterial diseases in pigs in some areas of Xinjiang[D].Urumqi:Xinjiang Agricultural University, 2023.(in Chinese)
[25] KIM B, HUR J, LFF J Y, et al.Molecular scrotyping and antimicrobial resistance profiles of Actinobacillus pleuropoeumooiue isolated from pigs in South Korea[J]. Veterinary Quarterly, 2016, 36(3):137-144.
[26] SWEENEY M T, LINDEMAN C, JOHANSEN L, et al.Antimicrobial susceptibility of Actinobacillus pleuropneumoniae, Pasteurella multocida, Streptococcus suis, and Bordetella bronchiseptica isolated from pigs in the United States and Canada, 2011 to 2015[J].Journal of Swine Health and Production, 2017, 25(3):106-120.
[27] ZUTIC J, PAVLOVIC L, ROHANSEN L, et al.Antimicrobial susceptibility of Actinobacillus pleuropneumoniae strains isolated from pig’s clinical samples in Serbia[J].Medycyna Weteryn Aryjna-Veterinary Medicine-Science and Practice, 2016, 72(10):637-640.
[28] 朱秀高，李艳青.猪胸膜肺炎放线杆菌感染和血清型分布[J].动物医学进展，2017，38(10)：115-117.ZHU X G, LI Y Q.Review of prevalence and serotype distribution for Actinobacillus pleuropeumoniae[J].Progress in Veterinary Medicine, 2017, 38(10):115-117.(in Chinese)
[29] 夏新萌，连伟民，李成蒙.我国部分地区猪传染性胸膜肺炎放线杆菌的血清型流行病学调查[J].中国猪业，2015，10(12)：50-53.XIA X M, LIAN W M, LI C M, et al.Serotype epidemiological investigation of Actinobacillus pleuropneumoniae in pigs in some areas of China[J].China Swine Industry, 2015, 10(12):50-53.(in Chinese)