红斑丹毒丝菌分离鉴定及主要保护性抗原基因遗传进化分析

Isolation and Identification of Erysipelothrix rhusiopathiae and Genetic Evolution Analysis of Main Protective Antigen Genes

红斑丹毒丝菌(Erysipelothrix rhusiopathiae)是引起急性、热性猪丹毒的病原菌,是人兽共患病的病原。猪丹毒临床症状和病理变化与非洲猪瘟的症状和病理变化极为相似,容易混淆。从病死猪的肝脏和肺脏中分离到两株革兰氏阳性短杆菌,进行生化试验和药敏试验,并扩增16S rDNA和主要表面保护性抗原(spaA)基因并测序。将纯化后的细菌接种小鼠,测定分离菌的致病性。以国内外已发表的32条spaA基因序列为参考,用DNAstar软件分析分离菌spaA基因的遗传进化关系。登录生物信息学网站在线预测spaA蛋白三级结构。结果表明:两株分离株16S rDNA基因序列与GenBank中丹毒丝菌序列同源性达90%以上,确定为丹毒丝菌,分别命名为LN201801和LN201802。分离菌株均能分解葡萄糖、果糖、乳糖,不分解蔗糖、甘露醇和山梨醇,MR试验和V-P试验均为阴性。LN201801和LN201802对青霉素和头孢噻肟高度敏感,对杆菌肽、恩诺沙星、阿奇霉素耐药。分离菌株spaA基因长1881bp,与已知强毒株Fujisawa序列同源性为99.9%。系统进化树分析表明,LN201801、LN201802与合肥分离株、安徽分离株、湖南分离株、湖北分离株位于同一分支上,提示该病可能存在跨地区传播。蛋白质在线预测结果显示,spaA蛋白分子量为72.294ku,分子式为C3260H5104N854O956S23,为脂溶性蛋白,性质稳定。该蛋白有337个B细胞表位,两个主要结构域(domain)。本研究为猪丹毒的诊断和治疗提供了方法,为该病的防控和开发亚单位疫苗提供候选菌株。

Erysipelothrix rhusiopathiae is a pathogen causing acute and hot swine erysipelas and is a zoonotic pathogen.The clinical symptoms and pathological changes of pig erysipelas are similar to those of African swine fever and other infectious diseases,which are easily confused.In this study,two Gram-positive brevibacterium were isolated from the liver and lung of sick pigs,and biochemical tests and drug susceptibility experiments were carried out.The 16S rDNA and major surface protective antigen(spaA)genes were amplified and sequenced.The pathogenicity of the isolate was determined by inoculating the purified bacteria into mice.Based on the published 32 spaA gene sequences as reference sequences,the genetic evolution relationship of the isolates were analysied by DNASTAR software.The bioinformatics website is online to predict the structure of protective antigenic proteins.The results showed that the 16SrDNA gene sequences of the two isolates were more than 90%homologous to the erysipes in GenBank,and identified as erysipelas,named LN201801 and LN201802,respectively.The biochemical experiments of the two isolates were the same,all of which could decompose glucose,fructose,lactose,and did not decompose sucrose,mannitol and sorbitol.Both the MR test and the V-P test were negative.LN201801 and LN201802 are highly sensitive to penicillin and cefotaxime,and resistant to bacitracin,enrofloxacin and azithromycin.The spaA gene is 1881 bp in length and has 99.9%homology with the known strain Fujisawa.Phylogenetic tree analysis indicated that LN201801 and LN201802 were located on the same branch as Hefei isolate,Anhui isolate,Hunan isolate and Hubei isolate,suggesting that the disease may spread across regions.The results of protein structure prediction showed that the molecular weight of the protein was 72.294 ku,and the molecular formula was C3260H5104N854O956S23,which was a fat-soluble protein with stable properties.The protein has 337 B cell epitopes and two major domains.This study provides a method for the diagnosis and treatment of erysipelas disease,and lays a theoretical foundation for the prevention and control of the disease and the development of subunit vaccines.