1株血清10型副猪嗜血杆菌的分离鉴定与生物学特性分析

doi:10.16431/j.cnki.1671-7236.2024.04.035

摘要/Abstract

摘要： 【目的】阐明引起荆州某猪厂仔猪疑似副猪嗜血杆菌病的病原生理生化特性、毒力与药物敏感性,为今后对该病的防治提供科学依据。【方法】从患病仔猪中分离培养病原菌,通过形态学观察、生理生化鉴定、PCR鉴定、16S rRNA基因测序、血清型鉴定、多序列位点分型(MLST)对分离菌株进行鉴定。通过毒力基因检测、小鼠致病性、药敏试验、中药治疗试验等对分离株进行致病性、耐药性分析。【结果】通过形态学观察、生理生化试验、16S rRNA序列比对、系统进化树分析确定分离菌株为副猪嗜血杆菌。通过血清型与MLST分析确定分离菌株为血清10型、ST型为299。毒力基因检测发现,分离菌株具有CapD、vta1、vta2等15种毒力基因。将分离菌株腹腔注射感染小鼠,可导致小鼠多个脏器发生明显病变,具有较强致病性。耐药基因检测试验发现,分离菌株具有β-内酰胺类耐药基因bla_OXA和氟喹诺酮类耐药基因gyrA。药敏试验结果显示,分离菌株对头孢他啶、新霉素、米诺环素等10种抗菌药物敏感,同时对明雄黄、款冬花、全蝎3种中药敏感。中药治疗试验结果表明,明雄黄和全蝎对分离菌株感染小鼠有较明显的治疗作用。【结论】试验成功分离1株血清10型副猪嗜血杆菌,侵染小鼠可致多个器官出血坏死,该菌株对10种抗菌药物及3种中药敏感,且明雄黄和全蝎对分离菌株感染小鼠治疗效果明显。研究结果可为今后副猪嗜血杆菌的防治和临床诊疗提供参考依据。

关键词: 副猪嗜血杆菌; 血清10型; ST-299型; 毒力基因; 药物敏感性

Abstract: 【Objective】 This study was aimed to elucidate the physiological and biochemical characteristics,virulence and drug sensitivity of the pathogen causing Haemophilus parasuis disease in piglets from a pig factory in Jingzhou,China,so as to provide scientific basis for the prevention and treatment of this disease in the future.【Method】 The pathogenic bacteria were isolated from infected piglets and identified by morphological observation,physiological and biochemical identification,PCR identification,16S rRNA gene sequencing,serotype identification and multi-sequence site typing (MLST).The pathogenicity and drug resistance of the isolate were analyzed by virulence gene detection,pathogenicity test of mouse,drug sensitivity test and traditional Chinese medicine treatment.【Result】 Through morphological observation,physiological and biochemical experiments,16S rRNA sequence alignment and phylogenetic tree analysis,the isolate was identified as Haemophilus parasuis.Serotype and MLST analysis confirmed that the isolate was serotype 10 and ST-299.Virulence gene test showed that the isolate had 15 virulence genes such as CapD,vta1 and vta2.The isolate could cause obvious lesions in many organs of the infected mice by intraperitoneal injection and had strong pathogenicity.The detection of drug resistance genes showed that the isolate had β-lactam resistance gene bla_OXA and fluoroquinolone resistance gene gyrA.The drug sensitivity test showed that the isolate was sensitive to 10 kinds of antibiotics,such as ceftriaxone,neomycin and minocycline,and to 3 kinds of traditional Chinese medicines including realgar,Tussilago farfara and Buthus martensii.The traditional Chinese medicine treatment showed that realgar and Buthus martensii had obvious therapeutic effect on mice infected by the isolate.【Conclusion】 In this study,a strain of Haemophilus parasuis serotype 10 was successfully isolated which could cause bleeding and necrosis of multiple organs in infected mice,and was sensitive to 10 kinds of antibiotics and 3 kinds of traditional Chinese medicines.It was found that realgar and Buthus martensii were effective in treating mice infected by the isolate.The results provided a reference for future prevention and treatment of Haemophilus parasuis.

Key words: Haemophilus parasuis; serotype 10; ST-299 type; virulence gene; drug sensitivity

中图分类号:

S852.61⁺2

戴璐, 张千, 万佳佳, 谢婷婷, 贾一珍, 张锐, 张付贤, 刘峰, 雷连成. 1株血清10型副猪嗜血杆菌的分离鉴定与生物学特性分析[J]. 中国畜牧兽医, 2024, 51(4): 1671-1685.

DAI Lu, ZHANG Qian, WAN Jiajia, XIE Tingting, JIA Yizhen, ZHANG Rui, ZHANG Fuxian, LIU Feng, LEI Liancheng. Isolation,Identification and Biological Characteristics Analysis of a Strain of Haemophilus parasuis Serotype 10[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1671-1685.

参考文献

[1] BROCKMEIER S L,REGISTER K B,KUEHN J S,et al.Virulence and draft genome sequence overview of multiple strains of the swine pathogen Haemophilus parasuis[J].PLoS One,2014,9(8):e103787.
[2] GU W,CHEN S W,CHEN G P,et al.Enhancement of Haemophilus parasuis serovar 5 yields by medium optimization[J].Letters in Applied Microbiology,2015,61(1):44-49.
[3] RAFIEE M,BLACKALL P J.Establishment,validation and use of the Kielstein-Rapp-Gabrielson serotyping scheme for Haemophilus parasuis[J].Australian Veterinary Journal,2000,78(3):172-174.
[4] 林尖兵,高清清,易新健,等.江苏地区副猪嗜血杆菌病流行现状调查[J].畜牧与兽医,2020,52(2):107-110. LIN J B,GAO Q Q,YI X J,et al.Epidemiological survey of Haemophilus parasuis infection in Jiangsu area[J]. Animal Husbandry & Veterinary Medicine, 2020,52(2):107-110.(in Chinese)
[5] MULLINS M A,REGISTER K B,BRUNELLE B W,et al.A curated public database for multilocus sequence typing (MLST) and analysis of Haemophilus parasuis based on an optimized typing scheme[J].Veterinary Microbiology,2013,162(2/4):899-906.
[6] 梁娟.猪呼吸系统病原菌五重PCR检测方法的建立与应用[D].重庆:西南大学,2020. LIANG J.Establishment and application of 5-plex PCR method for detection of porcine respiratory pathogenic bacteria[D].Chongqing:Southwest University,2020.(in Chinese)
[7] BOONE D R,CASTENHOLZ R W,GARRITY G M.Bergey’s Manual^® of Systematic Bacteriology[M].8th ed.New York:Springer,1984.
[8] 王玲彩,王青,王晓斐,等.14型副猪嗜血杆菌的分离鉴定及其致病性检测[J].中国预防兽医学报,2020,42(8):766-771. WANG L C,WANG Q,WANG X F,et al.Isolation and identification of Haemophilus parasuis type 14 and assessment of its pathogenicity[J].Chinese Journal of Preventive Veterinary Medicine,2020,42(8):766-771.(in Chinese)
[9] GREISEN K,LOEFFELHOLZ M,PUROHIT A,et al.PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria,including bacteria found in cerebrospinal fluid[J].Journal of Clinical Microbiology,1994,32(2):335-351.
[10] 王同兆,张付贤,雷连成.1株副猪嗜血杆菌血清4型的分离鉴定及耐药性分析[J].中国畜牧兽医,2022,49(3):1096-1105. WANG T Z,ZHANG F X,LEI L C.Isolation,identification and drug resistance analysis of a strain of Haemophilus parasuis serotype 4[J]. China Animal Husbandry & Veterinary Medicine,2022,49(3):1096-1105.(in Chinese)
[11] HOWELL K J,PETERS S E,WANG J,et al.Development of a multiplex PCR assay for rapid molecular serotyping of Haemophilus parasuis[J].Journal of Clinical Microbiology,2015,53(12):3812-3821.
[12] MLYNARCIK P,RODEROVA M,KOLAR M.Primer evaluation for PCR and its application for detection of carbapenemases in Enterobacteriaceae[J].Journal of Antimicrobial Chemotherapy,2016,9(1):e29314.
[13] NADEZHDA K,FURSOVA I,ASTASHKIN,et al.The spread of bla_OXA-48 and bla_OXA-244 carbapenemase genes among Klebsiella pneumoniae, Proteus mirabilis and Enterobacter spp.isolated in Moscow,Russia[J].Annals of Clinical Microbiology & Antimicrobials,2015,14:46.
[14] 王治方,徐引弟,朱文豪,等.副猪嗜血杆菌血清7型菌株毒力及耐药性分析[J].中国畜牧兽医,2022,49(8):3245-3255. WANG Z F,XU Y D,ZHU W H,et al.Analysis of virulence and drug resistance of Haemophilus parasuis serotype 7 strains[J]. China Animal Husbandry & Veterinary Medicine,2022,49(8):3245-3255.(in Chinese)
[15] 李新果,刘占,马宁宁,等.副猪嗜血杆菌重组抗原蛋白对小鼠的免疫效果评价[J].中国预防兽医学报,2021,43(5):534-540. LI X G,LIU Z,MA N N,et al.Evaluation of immune efficacy of recombinant antigen protein of Haemophilus parasuis on mice[J]. Chinese Journal of Preventive Veterinary Medicine,2021,43(5):534-540.(in Chinese)
[16] ZHANG J,XU C,GUO L,et al.Prevalence and characterization of genotypic diversity of Haemophilus parasuis isolates from Southern China[J].Canadian Journal of Veterinary Research,2012,76(3):224-229.
[17] WU C,LABRIE J,TREMBLAY Y D,et al.Zinc as an agent for the prevention of biofilm formation by pathogenic bacteria[J].Journal of Applied Microbiology,2013,115(1):30-40.
[18] 项明源.副猪嗜血杆菌生物被膜的生物学特征及转录组分析[D].成都:四川农业大学,2020. XIANG M Y.Biological characteristics and transcriptome analysis of Haemophilus parasuis biofilm[J].Chengdu:Sichuan Agricultural University,2020.(in Chinese)
[19] JIN H,ZHOU R,KANG M,et al.Biofilm formation by field isolates and reference strains of Haemophilus parasuis[J]. Veterinary Microbiology,2006,118(1):117-123.
[20] VAN C N,THANH T,ZOU G,et al.Characterization of serotypes and virulence genes of Haemophilus parasuis isolates from Central Vietnam[J].Veterinary Microbiology,2019,230:117-122.
[21] 张艳禾,李婷,谢芳,等.副猪嗜血杆菌相关毒力因子的研究进展[J].中国预防兽医学报,2016,38(10):835-838. ZHANG Y H,LI T,XIE F,et al.Research progress on the virulence of Haemophilus parasuis[J].Chinese Journal of Preventive Veterinary Medicine,2016,30(10):835-838.(in Chinese)
[22] CAO Q,WEI W,WANG H,et al.Cleavage of E-cadherin by porcine respiratory bacterial pathogens facilitates airway epithelial barrier disruption and bacterial paracellular transmigration[J].Virulence,2021,12(1):2296-2313.
[23] PINA S,OLVERA A,BARCELO A,et al.Trimeric autotransporters of Haemophilus parasuis:Generation of an extensive passenger domain repertoire specific for pathogenic strains[J].Journal of Bacteriology,2009,191(2):576-587.
[24] CORREA-FIZ F,GALOFRE-MILA N,COSTA-HURTADO M,et al.Identification of a surface epitope specific of virulent strains of Haemophilus parasuis[J].Veterinary Microbiology,2017,198:116-120.
[25] LI G,NIU H,ZHANG Y,et al. Haemophilus parasuis cytolethal distending toxin induces cell cycle arrest and p53-dependent apoptosis[J].PLoS One,2017,12(5):e177199.
[26] JINADASA R N,BLOOM S E,WEISS R S,et al.Cytolethal distending toxin:A conserved bacterial genotoxin that blocks cell cycle progression,leading to apoptosis of a broad range of mammalian cell lineages[J].Microbiology (Reading),2011,157(Pt7):1851-1875.
[27] ALVAREZ-ESTRADA A,MARTINEZ-MARTINEZ S,MARTIN C G,et al.Immunogenic characterization of vaccines based on Haemophilus parasuis Nagasaki strain,OmpP2,OmpP5 and OmpD15,in colostrum-deprived pigs experimentally challenged with the same strain[J].Research in Veterinary Science,2018,119:292-301.
[28] ZHAO Y,GUO L,LI J,et al.Characterization of antimicrobial resistance genes in Haemophilus parasuis isolated from pigs in China[J].PeerJ,2018,6:e4613.
[29] 杜娟,刘亚辉,苏玉贤.河南省安阳市副猪嗜血杆菌流行菌株调查与耐药性分析[J].中国动物检疫,2023,40(8):20-27. DU J,LIU Y H,SU Y X.Investigation on the prevalent strains of Haemophilus parasuis in Anyang city,Henan province and analysis on its drug resistance[J].China Animal Health Inspection,2023,40(8):20-27.(in Chinese)
[30] 董婉玉,王馨雨,张聚民,等.2020年~2021年我国副猪嗜血杆菌和链球菌的血清型、耐药基因型流行情况分析[J].中国预防兽医学报,2022,44(10):1045-1051. DONG W Y,WANG X Y,ZHANG J M,et al.Epidemiological analysis of serotypes and drug-resistant genotypes of Haemophilus parasuis and Streptococcus in China in 2020-2021[J]. Chinese Journal of Preventive Veterinary Medicine,2022,44(10):1045-1051.(in Chinese)
[31] 卢昱希,余波,周思璇,等.中药消除细菌耐药性研究进展[J].黑龙江畜牧兽医,2020,9:50-53. LU Y X,YU B,ZHOU S X,et al.Research progress on eliminating bacterial drug-resistance by traditional Chinese medicine[J]. Heilongjiang Animal Science and Veterinary Medicine,2020,9:50-53.(in Chinese)
[32] 刘华清.节肢动物地龙和全蝎抗菌化学成分研究[D].洛阳:河南科技大学,2018. LIU H Q.Research on the antibacterial chemical composition from Pheretima asperfillm E.Perrier and Buthus martensi Karsch[D].Luoyang:Henan University of Science and Technology,2018.(in Chinese)
[33] 刘嵘,濮德敏.雄黄的研究进展[J].时珍国医国药,2007,4:982-984. LIU R,PU D M.Research progress on realgar[J].Lishizhen Medicine and Materia Medica Research,2007,4:982-984.(in Chinese)