鸭源阿培依姆沙门菌的生物学特性及致病性分析

The analysis of biological properties and pathogenicity of duck Salmonella apeyeme

为探究鸭源阿培依姆沙门菌的生物学特性及对雏鸭的致病性,本实验将本研究室前期分离到的1株阿培依姆沙门菌BJ1999分别接种至不同培养基测定菌落生长特征,接种至LB肉汤培养基测定细菌生长特性,通过生化反应试验测定其生化反应特征,结果显示,分离菌BJ1999株在Rambach培养基、XLD培养基、亮绿琼脂培养基和麦康凯琼脂培养基中均生长,且呈典型沙门菌菌落颜色和形态;在LB肉汤中生长良好;生化反应结果也符合沙门菌属细菌的特征。通过结晶紫染色法测定该分离菌生物被膜(BF)形成能力,结果显示,分离菌在LB肉汤中培养48 h和96 h的BF形成能力均极显著弱于鼠伤寒沙门菌ATCC14028株(P<0.001)。采用PCR方法检测该分离菌的毒力基因,结果显示扩增到胞内存活(msg A、spiA等)、Ⅲ型分泌系统结构和功能(invA、hilA等)、菌毛结构和功能(lpfA、lpfC)、铁代谢(sitC)等相关的毒力基因共18个。进一步选用健康雏鸭,以10^(9)cfu/只、10^(8)cfu/只皮下注射和10^(9)cfu/只口服感染该菌,评估分离菌对雏鸭的致病性。结果显示,10^(9)cfu/只和10^(8)cfu/只皮下注射雏鸭表现为精神沉郁、食欲下降,分别导致60%和30%死亡率;剖检可见病死雏鸭肝脏明显出血和白色坏死点。以10^(9)cfu/只口服感染后1 d雏鸭肝脏细菌检出率40%(2/5)、脾脏检出率100%(5/5),感染后3 d肝脏和脾脏平均载菌量分别为640 cfu/g和2140 cfu/g,感染后7 d肝脏内细菌基本被清除(1/5),脾脏平均载菌量下降至560 cfu/g。结果表明,阿培依姆沙门菌BJ1999株具有典型的沙门菌属细菌的生物学特性,并且对雏鸭具有一定致病性。国内鲜见对阿培依姆沙门菌的系统研究,尤其未见对禽源阿培依姆沙门菌的研究,本研究首次对鸭源阿培依姆沙门菌的生物学特性及致病性进行了分析,为该菌的系统性研究奠定了基础,同时丰富了沙门菌属细菌的流行病学资料。

To investigate the biological properties of Salmonella apeyeme and its pathogenicity to ducklings,the BJ1999 strain previously isolated in our laboratory was inoculated into various medium to analyze colony growth properties,and inoculated into LB broth to determine the growth characteristic,and the biological properties were characterized by biochemical reaction assay.The result suggested that typical colony color and morphology of Salmonella was observed on Rambach medium,XLD medium,brilliant green medium and MacConkey agar.The isolate could grow favorably in LB broth.Biomedical reaction was accordance with other Salmonella serovars.The biofilm formation ability of the isolated S.apeyeme BJ1999 strain was determined by crystal violet staining,and it showed that the isolate presented lower biofilm production ability than S.typhimurium ATCC14028 after broth culture for 48 hours and 96 hours(P<0.001).Virulence-associated genes were identified by conventional PCR,and it showed that 18 virulence genes related to TTSS(invA,hilA),structure and function of fimbriae(lpfA,lpfC),intracellular survival(msgA,spiA)and iron metabolism(sitC)were identified.The healthy ducklings were infected orally with 10^(9)cfu/bird and subcutaneously with 10^(9) cfu and 10^(8)cfu/bird,respectively,to evaluate the pathogenicity of the isolate to ducklings,it showed that 60%and 30%mortality rate of ducklings infecting subcutaneously with 10^(9)cfu and 10^(8)cfu were formatted.The ducklings showed depression and decreased appetite.Hemorrhage and white necrosis point in liver of dead ducklings were observed.The rate of bacterial recovery was 40%(2/5)in liver and 100%(5/5)in spleen at 1 day post oral infection with 10^(9)cfu/bird.The average bacterial load of liver and spleen was 640cfu/g and 2,140cfu/g at 3 days post infection(3dpi).The bacteria in liver basically were basically cleared(1/5)and the bacterial load of spleen dropped to 560cfu/g at 7dpi.The results showed that S.apeiem BJ1999 strain had typical biological characteristics of Salmonella and had certain pathogenicity to ducklings.There are few systematic studies on S.apeiem in China,especially on S.apeiem from poultry.In this study,the biological characteristics and pathogenicity of S.apeiem from ducks were investigated for the first time,which laid a foundation for the study of S.apeiem and further enriched the epidemi-ological data of Salmonella.