奶牛腐蹄病坏死杆菌分离鉴定及白细胞毒素基因lktA1序列分析

doi:10.16431/j.cnki.1671-7236.2015.09.014

《中国畜牧兽医》---唯一指定的官方网站 ›› 2015, Vol. 42 ›› Issue (9): 2308-2312.doi: 10.16431/j.cnki.1671-7236.2015.09.014

奶牛腐蹄病坏死杆菌分离鉴定及白细胞毒素基因lktA1序列分析

高晶, 李春秋, 姚爽, 张立春, 王欣宇, 王志慧, 赵喜文, 魏姗, 王恩雨, 孙东波, 武瑞, 郭东华

黑龙江八一农垦大学动物科技学院, 大庆 163319

收稿日期:2015-04-14 出版日期:2015-09-20 发布日期:2015-09-25
通讯作者: 郭东华 E-mail:dh_guo@126.com
作者简介:高晶(1990-),女,黑龙江齐齐哈尔人,硕士生,主要从事牛坏死杆菌研究,E-mail:arisia@126.com
基金资助:
黑龙江省高校青年学术骨干计划(1253G003)

Isolation,Identification and Sequence Analysis of Leukotoxin Gene lktA1 of Fusobacterium necrophorum of Cow Footrot

GAO Jing, LI Chun-qiu, YAO Shuang, ZHANG Li-chun, WANG Xin-yu, WANG Zhi-hui, ZHAO Xi-wen, WEI Shan, WANG En-yu, SUN Dong-bo, WU Rui, GUO Dong-hua

College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Received:2015-04-14 Online:2015-09-20 Published:2015-09-25

摘要/Abstract

摘要： 为分离纯化奶牛腐蹄病坏死杆菌,分析其与其他菌株的亲缘关系,本研究利用坏死杆菌白细胞毒素特异性引物,对奶牛腐蹄病病牛蹄部拭子样品进行了PCR检测,利用厌氧培养基对PCR检测阳性样品进行了坏死杆菌的分离培养,以分离的坏死杆菌基因组DNA为模板,对白细胞毒素基因进行了克隆和序列分析。结果显示,9份奶牛腐蹄病病牛蹄部拭子样品PCR检测结果均为阳性,对其中一份样品中的坏死杆菌进行分离培养,获得了纯培养物,命名为bFR13-1。坏死杆菌bFR13-1菌株白细胞毒素基因测序结果显示,与GenBank已发表的H05、A25和B35菌株的白细胞毒素基因在核苷酸水平的同源性分别为98.40%、98.35%和90.79%,推导氨基酸的同源性分别为97.7%、97.6%和89.0%。进化树分析结果显示,坏死杆菌bFR13-1菌株白细胞毒素与H05菌株的同源性最高,bFR13-1菌株与H05菌株和A25菌株呈较近亲缘关系。结果表明,不同坏死杆菌分离株的白细胞毒素呈现一定的变异性,这种变化是否与坏死杆菌致病性相关,值得深入研究。

关键词: 奶牛腐蹄病; 坏死杆菌; 白细胞毒素

Abstract: For separation and purification of Fusobacterium necrophorum of cow footrot, and analysis of genetic relationship with other strains, the hoof ministry swab samples were detected by PCR based on specific primers of leukotoxin gene, and genomic DNA were isolated from PCR positive samples of Fusobacterium necrophorum culturing in anaerobic medium.The genes of leukotoxin were cloned and sequenced.The results showed that nine of hoof ministry swab samples were all PCR positive samples, and we obtained Fusobacterium necrophorum pure culture from one of the samples which named bFR13-1.The gene sequencing results indicated that the homologies of leukotoxin gene nucleotide sequence of bFR13-1 strain compared with H05, A25 and B35 strains from GenBank were 98.40%, 98.35% and 90.79%, respectively, and the homologies of deduced amino acid sequence were 97.7%, 97.6% and 89.0%, respectively.Phylogenetic tree analysis results showed that leukotoxin gene of Fusobacterium necrophorum bFR13-1 and H05 had high homology and bFR13-1, H05 and A25 showed a close genetic relationship.The result indicated that leukotoxin showed variability between different Fusobacterium necrophorum isolated strains, and it was worth to study whether this change and pathogenicity of Fusobacterium necrophorum were related.

Key words: cow footrot; Fusobacterium necrophorum; leukotoxin

中图分类号:

S852.61

高晶, 李春秋, 姚爽, 张立春, 王欣宇, 王志慧, 赵喜文, 魏姗, 王恩雨, 孙东波, 武瑞, 郭东华. 奶牛腐蹄病坏死杆菌分离鉴定及白细胞毒素基因lktA1序列分析[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(9): 2308-2312.

GAO Jing, LI Chun-qiu, YAO Shuang, ZHANG Li-chun, WANG Xin-yu, WANG Zhi-hui, ZHAO Xi-wen, WEI Shan, WANG En-yu, SUN Dong-bo, WU Rui, GUO Dong-hua. Isolation,Identification and Sequence Analysis of Leukotoxin Gene lktA1 of Fusobacterium necrophorum of Cow Footrot[J]. , 2015, 42(9): 2308-2312.

参考文献

[1] Sun D,Zhang H,Guo D,et al.Shotgun proteomic analysis of plasma from dairy cattle suffering from footrot:Characterization of potential disease-associated factors[J].PLoS One,2013,8(2):e55973.

[2] Langworth B F.Fusobacterium necrophorum:Its characteristics and role as an animal pathogen[J].Bacteriol Rev,1977,41:373-390.

[3] Batty A,Wren M W,Gal M.Fusobacterium necrophorum as the cause of recurrent sore throat:Comparison of isolates from persistent sore throat syndrome and Lemierre's disease[J].J Infect,2005,51(4):299-306.

[4] 胡柏冬,谷城,吕思文,等.运动场环境对奶牛肢蹄健康的影响[J].黑龙江畜牧兽医,2014,12:42-43.

[5] Nagaraja T G,Narayanan S K,Stewart G C,et al.Fusobacterium necrophorum infections in animals:Pathogenesis and pathogenic mechanisms[J].Anaerobe,2005,11:239-246.

[6] 孙东波,吕思文,高晶,等.牛坏死杆菌溶血素基因克隆、表达和抗原性分析[J].黑龙江畜牧兽医,2015,1:140-142.

[7] Narayanan S K,Nagaraja T G,Chengappa M M,et al.Leukotoxins of gram-negative bacteria[J].Vet Microbiol,2002,84(4):337-356.

[8] 郭东华,孙东波,武瑞,等.坏死杆菌白细胞毒素作为腐蹄病亚单位疫苗候选抗原的研究前景[J].中国畜牧兽医,2009,36(3):137-139.

[9] Guo D H,Sun D B,Wu R,et al.An indirect ELISA for serodiagnosis of cattle footrot caused by Fusobacterium necrophorum[J].Anaerobe,2010,16(4):317-320.

[10] Nagaraja T G,Lechtenberg K F.Liver abscesses in feedlot cattle[J].Vet Clin North Am Food Anim Pract,2007,23:351-369.

[11] 郭顺元,王淑华,郑星道,等.奶牛坏死杆菌性子宫内膜炎的研究[J].黑龙江畜牧兽医,1996,7:31-33.

[12] 郭东华,王君伟,孙玉国,等.牛腐蹄病坏死梭杆菌白细胞毒素重组亚单位疫苗诱导小鼠免疫保护效果的观察[J].中国预防兽医学报,2008,30(5):398-402.

奶牛腐蹄病坏死杆菌分离鉴定及白细胞毒素基因lktA1序列分析

Isolation,Identification and Sequence Analysis of Leukotoxin Gene lktA1 of Fusobacterium necrophorum of Cow Footrot

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