一株猪丹毒杆菌的分离鉴定及其对小鼠的致病性研究

Isolation,identification and pathogenicity in mouse of a strain of Erysipelothrix rhusiopathiae

为确定湖南省某猪场猪只发病死亡的病原,从送检猪组织中分离纯化获得1株革兰阳性菌,通过生化鉴定及16S rDNA测序对分离菌进行了鉴定,药敏试验验证分离菌的耐药性,并进一步测序分析了其毒力因子基因的遗传变异;最后通过动物试验研究了分离菌对小鼠的致病性。结果显示,分离菌呈针尖大小、半透明,生化鉴定为猪丹毒杆菌。药敏试验结果显示该分离株对青霉素、苯唑西林、氨苄西林等15种抗生素敏感。分离株16S rDNA序列与猪丹毒疫苗株G4T10的同源性为100%;与G4T10株相比,spaA基因高突变区存在4个突变位点,分别是A509C、C510A、A924C和C925T,相对应的氨基酸有3个突变位点,包括P170H、D308E和F309L,而其他毒力因子基因包括神经氨酸酶基因、荚膜多糖A、B和C基因序列则无变化。小鼠攻毒试验显示,当剂量为1.466×10^(3)CFU或更高时,小鼠呈全身败血症病理变化,死亡率100%,说明该菌株毒力较强;从死亡小鼠各脏器组织中可分离到与分离株形态特征一致的菌株。本研究成功分离鉴定了1株毒力较强的猪丹毒杆菌,为湖南省猪丹毒的流行病学及致病性提供了参考。

To determine the cause of pig death in a pig farm in Hunan Province,a strain of Gram-positive bacterium was isolated and purified in the collected tissues.The strain was identified through biochemical characteristics and 16S rDNA sequencing,antimicrobial susceptibility test was used to verify drug resistance of the isolated bacteria.Further sequencing analysis was conducted to determine the genetic variation of virulence factor genes of the isolated strain.In addition,the pathogenicity of the strain in mice was studied.The results showed that the isolated bacterium was characterized by needlepoint-sized,translucent colonies,and identified as Erysipelothrix rhusiopathiae.The isolated strain was sensitive to 15 antibiotics such as penicillin,oxacillin,ampicillin and etc.The 16S rDNA sequence analysis revealed that the isolated strain was 100%homology to the vaccine strain G4T10.The sequence of the hypervariable region in spaA gene showed four nucleotide mutations in the sites of A509C,C510A,A924C and C925T compared with the G4T10 strain,with three mutations in its amino acids including P170H,D308E and F309L.However,sequences of other virulence factor genes,including sialidase gene,capsular polysaccharide A,B,and C gene,remained unchanged.The animal experiment showed that all mice exhibited systemic sepsis with 100%of mortality when the mice were challenged with 1.466×10^(3) CFU or higher titers of the isolate,indicating that the isolated strain is highly virulent.Bacteria were isolated from the tissues of the dead mice which were consistent with the morphological characteristics of the strain.In this study,a high virulence strain of E.rhusiopathiae was successfully isolated and identified,the data could provide a reference for the epidemiology and pathogenicity of swine erysipelas in Hunan Province.