我国中东部主要养猪地区死亡育肥猪呼吸道病原菌的流行病学调查及猪多杀性巴氏杆菌的特性鉴定  

Epidemiological Investigation of Respiratory Pathogens in Deceased Fattening Pigs in Major Pig Farming Area of Middle and Eastern China and Characterization of Pasteurella multocida

作　　者：罗素贤 周红[1] 蔺辉星[1] 范红结[1] LUO SuXian;ZHOU Hong;LIN HuiXing;FAN HongJie(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《中国农业科学》2024年第11期2254-2264,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2021YFD1800404);江苏省农业科技自主创新资金项目(CX(23)1029);江苏高校优势学科建设工程专项资金项目(PAPD)。

摘　　要：【目的】从我国中东部主要养猪地区因呼吸道疾病死亡的育肥猪病料中分离主要病原菌,并进行鉴定和分型,为近年流行的猪呼吸道病原菌的防控治疗提供流行病学依据。此外,对分离的猪多杀性巴氏杆菌(Pm)特性进行鉴定,为Pm疫苗研制提供参考。【方法】收集2021-2023年我国中东部主要养猪地区规模化猪场因呼吸道疾病死亡的育肥猪的病猪肺脏;用血琼脂和TSA培养基分离病原菌,通过微生物学和分子生物学方法鉴定分离菌株;合成adk、est、gdh、mdh、pgi、pmi和zwf基因引物,以分离的Pm为模板进行PCR,通过测序7对管家基因对Pm进行MLST分型;通过PCR对Pm进行荚膜分型和脂多糖分型;通过PCR对Pm的毒力因子进行检测;在ICR小鼠对单一分离的A型和部分D、F型Pm进行毒力鉴定;检测JS-65、JS-51和JS-34在ICR小鼠的LD_(50)。【结果】共分离Pm 73株,分离率为15.53%;分离猪链球菌(SS)71株、猪胸膜肺炎放线杆菌(APP)29株和副猪格拉菌(GPS)10株,分离率分别为15.11%、6.17%和2.13%。分型结果表明,Pm主要为A:L3型,占55%;SS主要为9型,占38.03%;APP主要为15型,占51.72%;GPS主要为5/12型,占60%。混合感染包括Pm+SS,Pm+APP,SS+APP和Pm+APP+GPS,混合感染占总猪群的16.67%。共分离到3种Pm荚膜型:A(67%)、D(30%)和F(3%)型;2种脂多糖型:L3型(56%)和L6型(44%);9种ST分型:ST79、ST50、ST7、ST74、ST13、ST27、ST9、ST287和ST370,所占比例分别为33%、26%、16%、10%、4%、4%、3%、3%和1%。毒力基因检测结果表明:ptfA、fimA、hsf-2、exbB、exbD、tonB、fur、nanH、sodA和sodC的阳性率大于95%;hsf-1、pfhA、tadD、hgbA、hgbB、pmHAS、ompA、ompH、oma87和plpB阳性率在40%-90%之间;tbpA和nanB阳性率在10%-30%;未检出toxA。毒力鉴定结果表明A型菌株在剂量小于10^(2)CFU时导致小鼠全部死亡,D型菌株在10^(3)CFU剂量时小鼠死亡率为60%-100%,F型菌株在5×10^(3)CFU剂量时小鼠死亡率为60%。在ICR小鼠检测JS-65、JS-【Objective】This study aims to isolate and identify prominent bacterial respiratory pathogens from samples collected from fattening pigs that have died from respiratory diseases in major domestic pig farming area of middle and eastern China.And identifying these bacterial pathogens will offer valuable evidence for preventing and controlling the significantly prevalent respiratory diseases in recent years.Moreover,the characteristics of Pm were identified,providing reference for the development of Pm vaccines.【Method】The lungs of pigs died from respiratory disease were collected from large-scale farms in major pig farming area of middle and eastern China from 2021 to 2023.Blood agar and TSA were employed for the isolation of pathogens,which were then identified through microbiological and molecular biology methods.Additionally,MLST typing and virulence testing in mice were conducted on Pm isolates.The primers for PCR against seven housekeeping genes of adk,est,gdh,mdh,pgi,pmi and zwf of Pm were designed.Then the productions of amplicons were sequenced and submitted to perform MLST typing.The capsule and lipopolysaccharide typing were detected by PCR.The virulence factor genes were detected by PCR.Single isolates of A type and selected D and F types of Pm were evaluated for virulence in ICR mice.LD_(50)of JS-65,JS-51 and JS-34 were detected in ICR mice.【Result】A total of 73 Pm isolates were obtained,with an isolation rate of 15.53%.Additionally,71 SS isolates,29 APP isolates,and 10 GPS isolates were obtained,with isolation rates of 15.11%,6.17%,and 2.13%respectively.The typing results indicated that the prevailing subtype among Pm isolates was A:L3,accounting for 55%.Among SS isolates,subtype 9 was the prevailing type,accounting for 38.03%.Among APP isolates,subtype 15 was the prevailing type,accounting for 51.72%.Among GPS isolates,subtype 5/12 was the prevailing type,accounting for 60.00%.Co-infection included Pm+SS,Pm+APP,SS+APP and Pm+APP+GPS,accounting for 16.67%of the total pig population.Three cap

关 键 词：流行病学 猪多杀性巴氏杆菌 鉴定 分子分型 

分 类 号：S858.28[农业科学—临床兽医学]