我国猪群中多杀性巴氏杆菌的基因型分析
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摘要
本研究旨在了解我国猪群中流行的多杀性巴氏杆菌(Pm)的主要基因型。利用荚膜基因分型、脂多糖(LPS)基因分型、多位点序列分型(MLST)对2013年12月—2017年12月4年间来源于我国各地区规模化猪场患有疑似呼吸系统疾病的病死猪肺、鼻拭子、气管、肝等样品中分离鉴定的Pm进行基因型分析,并对23种主要毒力基因进行检测。结果表明,当前在我国猪群中流行的Pm的荚膜基因型为A(48.85%)、D(42.75%)、F(2.67%),优势基因型为A和D;LPS基因型为L3(25.00%)和L6(75.00%)型,优势基因型为L6;MLST基因型为ST3(21.25%)、ST10(27.50%)、ST11(42.50%)、ST12(2.50%)、ST16(2.50%)、ST74(1.25%)及ST75(2.50%),优势基因型为ST3、ST10和ST11。如果将荚膜基因型、LPS基因型和MLST基因型组合起来看,当前在我国猪群中流行的Pm的主要基因型为荚膜:脂多糖:MLST基因型A:L3:ST3(20.00%)、A:L6:ST10(26.25%)及D:L6:ST11(42.50%)。毒力基因检测的结果发现部分毒力基因的分布表现出一定的"基因型偏好性"。结果提示,D:L6:ST11是我国猪群中流行的多杀性巴氏杆菌的主要基因型,可能与我国猪群中由多杀性巴氏杆菌导致的呼吸道疾病密切相关。
The aim of this study was to understand the genotypical characteristics of swine P. mul-tocida currently circulating in China. Capsular genotyping, LPS genotyping, MLST genotyping were performed on P. multocida isolates from the clinical samples including lungs, nasal swabs, tracheas, livers, and other tissues of pigs with respiratory disorders in different farms in different regions of mainland China; virulence genotyping based on the detection of different virulence gene profiles was also set for the isolates recovered herein. The results showed that the capsular genotypes of the swine P. multocida currently circulating in China were A(48.85%), D(42.75%), and F(2.67%); the LPS genotypes were L3(25.00%) and L6(75.00%); the MLST genotypes were ST3(21.25%), ST10(27.50%), ST11(42.50%), ST12(2.50%), ST16(2.50%), ST74(1.25%), and ST75(2.50%). When combining the capsular genotypes with the LPS genotypes and the MLST genotypes, it was found that the predominant capsule: LPS: MLST genotypes prevalent in pigs in China were A: L3: ST3(20.00%), A: L6: ST10(26.25%), and D: L6: ST11(42.50%). During the virulence genotyping, some virulence-associated genes were found to show a certain level of "genotype-preference", which means the detection rate of those genes in some genotypes is significantly higher than their detection in other genotypes. These results indicate that a capsule: LPS: MLST genotype D: L6: ST11 of P. multocida is likely to be strongly associated with swine respiratory disease in China.
The aim of this study was to understand the genotypical characteristics of swine P. mul-tocida currently circulating in China. Capsular genotyping, LPS genotyping, MLST genotyping were performed on P. multocida isolates from the clinical samples including lungs, nasal swabs, tracheas, livers, and other tissues of pigs with respiratory disorders in different farms in different regions of mainland China; virulence genotyping based on the detection of different virulence gene profiles was also set for the isolates recovered herein. The results showed that the capsular genotypes of the swine P. multocida currently circulating in China were A(48.85%), D(42.75%), and F(2.67%); the LPS genotypes were L3(25.00%) and L6(75.00%); the MLST genotypes were ST3(21.25%), ST10(27.50%), ST11(42.50%), ST12(2.50%), ST16(2.50%), ST74(1.25%), and ST75(2.50%). When combining the capsular genotypes with the LPS genotypes and the MLST genotypes, it was found that the predominant capsule: LPS: MLST genotypes prevalent in pigs in China were A: L3: ST3(20.00%), A: L6: ST10(26.25%), and D: L6: ST11(42.50%). During the virulence genotyping, some virulence-associated genes were found to show a certain level of "genotype-preference", which means the detection rate of those genes in some genotypes is significantly higher than their detection in other genotypes. These results indicate that a capsule: LPS: MLST genotype D: L6: ST11 of P. multocida is likely to be strongly associated with swine respiratory disease in China.
引文
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[13] LIU H S,ZHAO Z Q,XI X J,et al.Occurrence of Pasteurella multocida among pigs with respiratory disease in China between 2011 and 2015[J].Irish Vet J,2017,70:2.
[14] PENG Z,WANG H N,LIANG W,et al.A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China[J].Arch Microbiol,2018,200(1):107-118.
[15] 林星宇,胡凌,王印,等.猪源多杀性巴氏杆菌的分离鉴定及荚膜血清型分型[J].浙江农业学报,2016,28(4):558-562.LIN X Y,HU L,WANG Y,et al.Isolation,identification and capsule serotyping of Pasteurella multocida originated from swine[J].Acta Agriculturae Zhejiangensis,2016,28(4):558-562.(in Chinese)
[16] 席晓剑,赵战勤,薛云,等.猪源多杀性巴氏杆菌的病原流行病学及其毒力特性[J].中国兽医学报,2015,35(8):1205-1210.XI X J,ZHAO Z Q,XUE Y,et al.Prevalence and virulence of Pasteurella multocida in pig farms in central China[J].Chinese Journal of Veterinary Science,2015,35(8):1205-1210.(in Chinese)
[17] 彭忠,王庚,吴广敬,等.猪源多杀性巴氏杆菌的分离与鉴定[J].中国兽医科学,2015,45(9):943-951.PENG Z,WANG G,WU G J,et al.Isolation and identification of Pasteurella multocida from swine[J].Chinese Veterinary Science,2015,45(9):943-951.(in Chinese)
[18] 郭大和,郑明,潘松年.我国多杀性巴氏杆菌荚膜抗原的血清学鉴定[J].畜牧兽医学报,1979,10(2):67-78.GUO D H,ZHENG M,PAN S N.Serological identification of capsular antigens of Pasteurella multocida in China[J].Acta Veterinaria et Zootechnica Sinica,1979,10(2):67-78.(in Chinese)
[19] TANG X B,ZHAO Z Q,HU J Y,et al.Isolation,antimicrobial resistance,and virulence genes of Pasteurella multocida strains from swine in China[J].J Clin Microbiol,2009,47(4):951-958.
[20] 方超,李润成,魏榕,等.猪源荚膜血清F型多杀性巴氏杆菌的分离鉴定、毒力基因检测与致病性研究[J].中国预防兽医学报,2017,39(3):210-214.FANG C,LI R C,WEI R,et al.Identification,virulent factors detection and pathogenesis investigation of capsule serotype F Pasteurella multocida isolated from pigs[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(3):210-214.(in Chinese)
[21] 林星宇,王印,杨泽晓,等.猪源荚膜血清F型多杀性巴氏杆菌的分离鉴定[J].中国兽医科学,2015,45(6):567-571.LIN X Y,WANG Y,YANG Z X,et al.Isolation and identification of a Pasteurella multocida strain belonged to capsular type F from swine[J].Chinese Veterinary Science,2015,45(6):567-571.(in Chinese)
[22] 王羽,董文龙,王巍,等.猪源荚膜血清F型多杀性巴氏杆菌分离鉴定[J].中国兽医杂志,2017,53(6):31-33,36.WANG Y,DONG W L,WANG W,et al.Isolation and identification of a Pasteurella multocida strain belonged to capsular type F from swine[J].Chinese Journal of Veterinary Medicine,2017,53(6):31-33,36.(in Chinese)
[23] 王林柏,孙久鹤,郭东春,等.国内部分地区多杀性巴氏杆菌荚膜血清型和基因型的研究[J].中国预防兽医学报,2016,38(2):116-119.WANG L B,SUN J H,GUO D C,et al.Identification of capsule serotype and genotype of Pasteurella multocida in some areas of China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(2):116-119.(in Chinese)
[24] HARPER M,ST MICHAEL F,JOHN M,et al.Pasteurella multocida Heddleston serovar 3 and 4 strains share a common lipopolysaccharide biosynthesis locus but display both inter-and intrastrain lipopolysaccharide heterogeneity[J].J Bacteriol,2013,195(21):4854-4864.
[25] HARPER M,ST MICHAEL F,JOHN M,et al.Structural analysis of lipopolysaccharide produced by Heddleston serovars 10,11,12 and 15 and the identification of a new Pasteurella multocida lipopolysaccharide outer core biosynthesis locus,L6[J].Glycobiology,2014,24(7):649-659.
[26] 王豪男.我国部分省区猪源多杀性巴氏杆菌的分子流行病学调查[D].武汉:华中农业大学,2017.WANG H N.Molecular epidemiology of Pasteurella multocida from swine in some provinces of China[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[27] GARCíA-ALVAREZ A,VELA A I,SAN MARTíN E,et al.Characterization of Pasteurella multocida associated with ovine pneumonia using multi-locus sequence typing (MLST) and virulence-associated gene profile analysis and comparison with porcine isolates[J].Vet Microbiol,2017,204:180-187.
[28] GARCíA-ALVAREZ A,CHAVES F,FERNáND-EZ A,et al.An ST11 clone of Pasteurella multocida,widely spread among farmed rabbits in the Iberian Peninsula,demonstrates respiratory niche association[J].Infect Genet Evol,2015,34:81-87.
[29] EWERS C,LüBKE-BECKER A,BETHE A,et al.Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[J].Vet Microbiol,2006,114(3-4):304-317.
[30] GARCíA N,FERNáNDEZ-GARAYZáBAL J F,GOYACHE J,et al.Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain[J].Vet Rec,2011,169(14):362.
[31] FURIAN T Q,BORGES K A,LAVINIKI V,et al.Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine[J].Braz J Microbiol,2016,47(1):210-216.
[32] SARANGI L N,THOMAS P,GUPTA S K,et al.Virulence gene profiling and antibiotic resistance pattern of Indian isolates of Pasteurella multocida of small ruminant origin[J].Comp Immunol Microbiol Infect Dis,2015,38:33-39.
[2] WILKIE I W,HARPER M,BOYCE J D,et al.Pasteurella multocida:diseases and pathogenesis[M]//AKTORIES K,ORTH J H C,ADLER B.Pasteurella multocida.Berlin,Heidelberg:Springer,2012,361:1-22.
[3] TOWNSEND K M,BOYCE J D,CHUNG J Y,et al.Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system[J].J Clin Microbiol,2001,39(3):924-929.
[4] HARPER M,JOHN M,TURNI C,et al.Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus[J].J Clin Microbiol,2015,53(2):477-485.
[5] HARPER M,BOYCE J D,ADLER B.Pasteurella multocida pathogenesis:125 years after pasteur[J].FEMS Microbiol Lett,2006,265(1):1-10.
[6] PENG Z,LIANG W,LIU W J,et al.Genomic characterization of Pasteurella multocida HB01,a serotype A bovine isolate from China[J].Gene,2016,581(1):85-93.
[7] PENG Z,LIANG W,LIU W J,et al.Genome characterization of Pasteurella multocida subspecies septica and comparison with Pasteurella multocida subspecies multocida and gallicida[J].Arch Microbiol,2017,199(4):635-640.
[8] LIANG W L,PENG Z,LIU W J,et al.Complete genome sequence of P.multocida serotype A strain HB03,isolated from swine[C]//Proceedings of the 23rd International Pig Veterinary Society Congress.Cancun,Mexico:IPVS,2014,2:30.
[9] PENG Z,LIANG W,WANG F,et al.Genetic and phylogenetic characteristics of Pasteurella multocida isolates from different host species[J].Front Microbiol,2018,9:1408.
[10] LIU W J,YANG M J,XU Z F,et al.Complete genome sequence of Pasteurella multocida HN06,a toxigenic strain of serogroup D[J].J Bacteriol,2012,194(12):3292-3293.
[11] PENG Z,LIANG W,WANG Y G,et al.Experimental pathogenicity and complete genome characterization of a pig origin Pasteurella multocida serogroup F isolate HN07[J].Vet Microbiol,2017,198:23-33.
[12] KHAMESIPOUR F,MOMTAZ H,MAMOREH M A.Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran[J].Front Microbiol,2014,5:536.
[13] LIU H S,ZHAO Z Q,XI X J,et al.Occurrence of Pasteurella multocida among pigs with respiratory disease in China between 2011 and 2015[J].Irish Vet J,2017,70:2.
[14] PENG Z,WANG H N,LIANG W,et al.A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China[J].Arch Microbiol,2018,200(1):107-118.
[15] 林星宇,胡凌,王印,等.猪源多杀性巴氏杆菌的分离鉴定及荚膜血清型分型[J].浙江农业学报,2016,28(4):558-562.LIN X Y,HU L,WANG Y,et al.Isolation,identification and capsule serotyping of Pasteurella multocida originated from swine[J].Acta Agriculturae Zhejiangensis,2016,28(4):558-562.(in Chinese)
[16] 席晓剑,赵战勤,薛云,等.猪源多杀性巴氏杆菌的病原流行病学及其毒力特性[J].中国兽医学报,2015,35(8):1205-1210.XI X J,ZHAO Z Q,XUE Y,et al.Prevalence and virulence of Pasteurella multocida in pig farms in central China[J].Chinese Journal of Veterinary Science,2015,35(8):1205-1210.(in Chinese)
[17] 彭忠,王庚,吴广敬,等.猪源多杀性巴氏杆菌的分离与鉴定[J].中国兽医科学,2015,45(9):943-951.PENG Z,WANG G,WU G J,et al.Isolation and identification of Pasteurella multocida from swine[J].Chinese Veterinary Science,2015,45(9):943-951.(in Chinese)
[18] 郭大和,郑明,潘松年.我国多杀性巴氏杆菌荚膜抗原的血清学鉴定[J].畜牧兽医学报,1979,10(2):67-78.GUO D H,ZHENG M,PAN S N.Serological identification of capsular antigens of Pasteurella multocida in China[J].Acta Veterinaria et Zootechnica Sinica,1979,10(2):67-78.(in Chinese)
[19] TANG X B,ZHAO Z Q,HU J Y,et al.Isolation,antimicrobial resistance,and virulence genes of Pasteurella multocida strains from swine in China[J].J Clin Microbiol,2009,47(4):951-958.
[20] 方超,李润成,魏榕,等.猪源荚膜血清F型多杀性巴氏杆菌的分离鉴定、毒力基因检测与致病性研究[J].中国预防兽医学报,2017,39(3):210-214.FANG C,LI R C,WEI R,et al.Identification,virulent factors detection and pathogenesis investigation of capsule serotype F Pasteurella multocida isolated from pigs[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(3):210-214.(in Chinese)
[21] 林星宇,王印,杨泽晓,等.猪源荚膜血清F型多杀性巴氏杆菌的分离鉴定[J].中国兽医科学,2015,45(6):567-571.LIN X Y,WANG Y,YANG Z X,et al.Isolation and identification of a Pasteurella multocida strain belonged to capsular type F from swine[J].Chinese Veterinary Science,2015,45(6):567-571.(in Chinese)
[22] 王羽,董文龙,王巍,等.猪源荚膜血清F型多杀性巴氏杆菌分离鉴定[J].中国兽医杂志,2017,53(6):31-33,36.WANG Y,DONG W L,WANG W,et al.Isolation and identification of a Pasteurella multocida strain belonged to capsular type F from swine[J].Chinese Journal of Veterinary Medicine,2017,53(6):31-33,36.(in Chinese)
[23] 王林柏,孙久鹤,郭东春,等.国内部分地区多杀性巴氏杆菌荚膜血清型和基因型的研究[J].中国预防兽医学报,2016,38(2):116-119.WANG L B,SUN J H,GUO D C,et al.Identification of capsule serotype and genotype of Pasteurella multocida in some areas of China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(2):116-119.(in Chinese)
[24] HARPER M,ST MICHAEL F,JOHN M,et al.Pasteurella multocida Heddleston serovar 3 and 4 strains share a common lipopolysaccharide biosynthesis locus but display both inter-and intrastrain lipopolysaccharide heterogeneity[J].J Bacteriol,2013,195(21):4854-4864.
[25] HARPER M,ST MICHAEL F,JOHN M,et al.Structural analysis of lipopolysaccharide produced by Heddleston serovars 10,11,12 and 15 and the identification of a new Pasteurella multocida lipopolysaccharide outer core biosynthesis locus,L6[J].Glycobiology,2014,24(7):649-659.
[26] 王豪男.我国部分省区猪源多杀性巴氏杆菌的分子流行病学调查[D].武汉:华中农业大学,2017.WANG H N.Molecular epidemiology of Pasteurella multocida from swine in some provinces of China[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[27] GARCíA-ALVAREZ A,VELA A I,SAN MARTíN E,et al.Characterization of Pasteurella multocida associated with ovine pneumonia using multi-locus sequence typing (MLST) and virulence-associated gene profile analysis and comparison with porcine isolates[J].Vet Microbiol,2017,204:180-187.
[28] GARCíA-ALVAREZ A,CHAVES F,FERNáND-EZ A,et al.An ST11 clone of Pasteurella multocida,widely spread among farmed rabbits in the Iberian Peninsula,demonstrates respiratory niche association[J].Infect Genet Evol,2015,34:81-87.
[29] EWERS C,LüBKE-BECKER A,BETHE A,et al.Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[J].Vet Microbiol,2006,114(3-4):304-317.
[30] GARCíA N,FERNáNDEZ-GARAYZáBAL J F,GOYACHE J,et al.Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain[J].Vet Rec,2011,169(14):362.
[31] FURIAN T Q,BORGES K A,LAVINIKI V,et al.Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine[J].Braz J Microbiol,2016,47(1):210-216.
[32] SARANGI L N,THOMAS P,GUPTA S K,et al.Virulence gene profiling and antibiotic resistance pattern of Indian isolates of Pasteurella multocida of small ruminant origin[J].Comp Immunol Microbiol Infect Dis,2015,38:33-39.