马源马链球菌兽疫亚种新疆株的分离鉴定及遗传特性分析

doi:10.16431/j.cnki.1671-7236.2019.01.027

摘要/Abstract

摘要：

为了解马源马链球菌兽疫亚种（S.zooepidemicus）新疆分离株马链球菌兽疫亚种类M蛋白（SzM）基因的分子进化与变异情况，为该菌引起的感染性疾病的防控提供依据，本试验对新疆地区某马场采集的病马淋巴结样品进行病原菌的分离培养和生化鉴定，并对分离菌株进行药物敏感性试验。根据已发表的马源SzM基因序列设计引物，对其SzM基因进行PCR扩增及序列测定。将获得的SzM序列与GenBank中不同动物源马链球菌兽疫亚种分离株序列进行同源性比对和遗传进化分析。结果显示，分离得到了一株革兰氏阳性链球菌，将其命名为马链球菌兽疫亚种ZMSY15-1。药物敏感性试验结果表明，分离菌株对青霉素、磺胺嘧啶钠耐药，对其他14种药物均敏感。序列分析结果显示，马链球菌兽疫亚种ZMSY15-1与国内外不同动物源分离株SzM基因氨基酸同源性为56.0%~70.0%。遗传进化分析结果显示，这些菌株可分为4个群。马链球菌兽疫亚种ZMSY15-1与猪源分离株SzM蛋白的氨基酸同源性为59.9%，分别属于2个不同的群，其与美国马源分离株NH55426亲缘关系最近。本试验结果可丰富国内马源马链球菌兽疫亚种SzM基因的信息数据，为马链球菌兽疫亚种的致病机制研究和预防控制提供参考依据。

关键词: 马链球菌兽疫亚种; 分离鉴定; SzM基因; 序列分析

Abstract:

To understand the molecular evolution and mutation of SzM gene of Streptococcus equi subsp.zooepidemicus (S.zooepidemicus) from horse in Xinjiang and provide basis for the prevention and treatment of infectious disease,lymph gland samples from sick horse were collected for bacterium culture,biochemical identification,and drug sensitivity test was also tested.The SzM gene was amplified from genomic DNA of S.zooepidemicus isolate by PCR based on the primers designed according to the published sequence in GenBank.Then the amplified SzM gene was cloned,sequenced and analyzed,and phylogenetic tree was constructed.The results showed that the isolate was Gram-positive Streptococcus,which was named as S.zooepidemicus ZMSY15-1.Drug sensitivity test showed that S.zooepidemicus ZMSY15-1 was resistant to penicillin and sulfadiazine sodium salt,and sensitive to other 14 microbials.The amino acid homologies of SzM gene of ZMSY15-1 with different strains at home and abroad were 56.0% to 70.0%.In addition,the phylogenetic analysis indicated that these test strains belonged to 4 different clades,the ZMSY15-1 isolate shared identities of 59.9% with swine isolates and they belonged to two different groups.The ZMSY15-1 isolate was closely related to US horse NH55426.The results could enrich the information data of SzM gene of S.zooepidemicus in domestic horses,and provide experimental data for the pathogenic mechanism and prevention control of S.zooepidemicus.

Key words: S.zooepidemicusi; isolation and identification; SzM gene; sequence analysis

中图分类号:

S852.61

冯凯, 王浩, 周婷婷, 马晓慧, 恩克·博力德, 佘明江, 苏艳. 马源马链球菌兽疫亚种新疆株的分离鉴定及遗传特性分析[J]. 《中国畜牧兽医》, 2019, 46(1): 231-238.

FENG Kai, WANG Hao, ZHOU Tingting, MA Xiaohui, ENKE·Bolide, SHE Mingjiang, SU Yan. Isolation,Identification and Genetic Characterization Analysis of Streptococcus equi subsp. zooepidemicus from Horse in Xinjiang[J]. , 2019, 46(1): 231-238.

参考文献

[1] STEWARD K F,ROBINSON C,HOLDEN M T G,et al.Diversity of Streptococcus equi subsp.zooepidemicus strains isolated from the Spanish sheep and goat population and the identification,function and prevalence of a novel arbutin utilisation system[J].Veterinary Microbiology,2017,207(7):231-238.
[2] VELINENI S,DENEGRI R,ARTIUSHIN S C,et al.Comparison of specificities of serum antibody responses of horses to clinical infections caused by Streptococcus equi or zooepidemicus[J].Veterinary Microbiology, 2015,180(3-4):253-259.
[3] GRCSZYNSKI K,YOCNG A,LEVINE S J,et al.Streptococcus equi subsp.zooepidemicus infections associated with guinea pigs[J].Emerging Infectious Diseases, 2015,21(1):156-158.
[4] 曲立春,徐涛,尹春花.猪链球菌病研究进展[J].动物医学进展,2008,29(10):79-81. QU L C,XU T,YIN C H.Advance in swine streptococcosis[J].Progress in Veterinary Medicine,2008,29(10):79-81.(in Chinese).
[5] SALASIA S I,WIBAWAN I W,PASARIBU F H,et al.Persistent occurrence of a single Streptococcus equi subsp.zooepidemicus clone in the pig and monkey population in Indonesia[J].Journal of Veterinary Science,2004,5(3):263-265.
[6] 杜润慈,刘建华,史茜,等.TLRS介导的信号通路在马链球菌感染TAW264.7细胞中的作用[J].中国畜牧兽医,2016,43(5):1270-1276. DU R C,LIU J H,SHI Q,et al.Effect of TLRs mediated signal pathway on Streptococcus equi infection in RAW264.7 cells[J].China Animal Husbandry & Veterinary Medicine, 2009,43(5):1270-1276.(in Chinese)
[7] URAL O,TUNCER I,DIKICI N,et al.Streptococcus zooepidemicus meningitis and bacteraemia[J].Scandinavian Journal of Infectious Diseases,2003,35(3):206-207.
[8] HOLDEN M T G,HEATHER Z,PAILLOT R,et al.Genomic evidence for the evolution of Streptococcus equi:Host restriction,increased virulence,and genetic exchange with human pathogens[J].PLoS Pathogens,2009,5(3):e1000346.
[9] TIMONEY J F,ARLIUSHIN S C,BOSCHWITZ J S.Comparison of the sequences and functions of Streptococcus equi M-like protein Sem and SzPse[J].Infection & Immunity,1997,65(9):3600-3605.
[10] BOSCHWITZ J S,TIMONEY J F.Inhibition of C3 desposition on Streptococcus equi subsp. equi by M protein:A mechanism for survival in equine blood[J].Infection & Immunity,1994,62(8):3515-3520.
[11] WALKER J A,TIMONEY J F.Molecular basis of variation in protective SzP proteins of Streptococcus zooepidemicus[J].American Journal of Veterinary Research,1998,59(9):1129-1133.
[12] LIBARDONI F,VIELMO A,FARIAS L,et al.Diversity of seM in Streptococcus equi subsp. equi isolated from strangles outbreaks[J].Veterinary Microbiology,2013,162(2-4):663-669.
[13] LINDAHL S,SÖDERLUND R,FROSTH S,et al.Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the SeM gene and pulsed-field gel electrophoresis[J].Veterinary Microbiology,2011,153(1):144-149.
[14] 李书光,李天芝,李峰,等.快速鉴定猪链球菌和马链球菌兽疫亚种双重PCR方法的建立[J].中国畜牧兽医,2013,40(7):43-45. LI S G,LI T Z,LI F,et al.Establishment of double PCR for rapid identification of Streptococcus suis and Streptococcus equi subspecies[J].China Animal Husbandry & Veterinary Medicine, 2013,40(7):43-45.(in Chinese)
[15] 闫若潜,吴志明,张志凌,等.猪链球菌1、7、9型多重PCR诊断方法的建立及初步应用[J].中国畜牧兽医,2009,36(7):154-157. YAN R Q,WU Z M,ZHANG Z L,et al.Establishment and application of multiplex PCR assay for detection of Streptococcus suis serotypes 1,7 and 9[J]. China Animal Husbandry & Veterinary Medicine,2009,36(7):154-157.(in Chinese)
[16] LIANG H,TANG B,ZHAO P,et al.Identification and characterization of a novel protective antigen,Sec_205 of Streptococcus equi ssp. zooepidemicus[J].Vaccine,2018,36(6):788-793.
[17] WATSON J R,LEBER A,VELINENI S,et al.Recurrent Streptococcus equi subsp.zooepidemicus bacteremia in an infant[J]. Journal of Clinical Microbiology,2015,53(9):3096-3099.
[18] LIU P H,SHUN S F,WANG Y K,et al.Identification of swine Streptococcus isolates in Shanghai[J].Chinese Journal of Veterinary Medicine,2001,21(1):42-46.
[19] ACKE E,MIDWINTER A C,LAWRENCE K,et al.Prevalence of Streptococcus dysgalactiae subsp.equisimilis and S.equi subsp.zooepidemicus in a sample of healthy dogs,cats and horses[J].New Zealand Veterinary Journal,2015,63(5):265-271.
[20] SOWMAN H R,CAVE N J,DUNOWSKA M.A survey of canine respiratory pathogens in New Zealand dogs[J].New Zealand Veterinary Journal, 2018,66(5):236-242.
[21] BRITTON A P,BLUM S E,LEGGE C,et al.Multi-locus sequence typing of Streptococcus equi subspecies zooepidemicus strains isolated from cats[J].The Journal of Veterinary Diagnostic Investigation,2018,30(1):126-129.
[22] PELKONEN S,LINDAHL S B,SUOMALA P,et al.Transmission of Streptococcus equi subspecies zooepidemicus infection from horses to humans[J].Emerging Infectious Diseases,2013,19(7):1041-1048.
[23] BJÖRNSDÓTTIR S,HARRIS S R,SVANSSON V,et al.Genomic dissection of an Icelandic epidemic of respiratory disease in horses and associated zoonotic cases[J]. American Society for Microbiology, 2017,8(4):e00826-17.
[24] MOORE B O,BRYANS J T.Antigenic classification of group C animal strepcocci[J].Journal of American Veterinary Medicine Association,1969,155(2):416-420.
[25] TIMONEY J F,WALKER J,ZHOU M,et al.Cloning and sequence analysis of a protective M-like protein genefrom Streptococcus equi subsp. zooepidemicus[J].Infection & Immunity,1995,63(4):1440-1445.
[26] 范红结,陆承平,唐佳琪.猪源马链球菌兽疫亚种中国分离株类M基因特性分析[J].中国农业科学,2006,39(1):210-214. FAN H J,LU C P,TANG J Q.Analysis of the M-like gene of Streptococcus zooepidemicus Chinese isolates from pig[J]. Scientia Agricultura Sinica,2006,39(1):210-214.(in Chinese)
[27] IVENS P A,MATTHEW S D,WEBB K,et al.Molecular characterization ‘Strangles’ outbreaks in the UK:The use of M-protein typing of S.equi subsp.equi[J].Equine Verterinary Journal,2011,43(3):359-364.
[28] 王淑杰,姜成钢,武嘉男,等.致病性马兽疫链球菌的分离鉴定、生物学特性及其M-like基因的序列分析[J].中国预防兽医学报,2012,34(11):878-881. WANG S J,JIANG C G,WU J N,et al.Isolation and identification of a Streptococcus equi subsp.zooepidemicus and the M-like gene analysis[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(11):878-881.(in Chinese)
[29] JAVED R,TAKU A K,GANGIL R,et al.Molecular characterization of virulence genes of Streptococcus equi subsp.equi and Streptococcus equi subsp.zooepidemicus in equines[J]. Veterinary World,2016,9(8):875-881.