一株血清5型副猪嗜血杆菌的分离鉴定及致病性分析

Isolation,identification and pathogenicity of a serotype 5 Haemophilus parasuis

为了对一例疑似副猪嗜血杆菌感染发病猪进行确诊,试验对采集的病料组织进行细菌分离,对分离菌株采用形态学观察、培养特性、生化特性、PCR方法进行鉴定,同时对分离菌株进行琼脂扩散试验、基因分子分型鉴定、测序分析,以及仔猪致病力试验。结果表明:所分离菌株为革兰氏阴性菌,呈多形性,菌落形态为圆形、光滑、灰白色、半透明、边缘整齐;具有烟酰胺腺嘌呤二核苷酸(NAD)依懒性;呈现典型"卫星生长"现象,不产生溶血现象;生化试验结果符合副猪嗜血杆菌相关生化特性;PCR鉴定扩增出1090 bp大小的条带,经琼脂凝胶扩散试验确定其为血清5型。分子分型鉴定分离株仅扩增出468 bp大小片段,经测序比对再次证实其为血清5型。在仔猪致病力试验中,空白对照猪未出现异常,攻毒猪表现出由副猪嗜血杆菌感染引起的发热、关节炎、胸腹腔多脏器表面纤维素性渗出为主的典型症状。说明发病猪感染了副猪嗜血杆菌,且分离得到血清5型副猪嗜血杆菌。

In order to confirm a suspected case of Haemophilus Parasuis infection in a pig,in this experiment,bacteria were isolated form the collected infected tissue,and the isolated strains were identified by morphology observation,culture characterization,biochemical characterization and PCR methods.Meanwhile,agar diffusion test,genotyping,sequencing analysis and pathogenicity test were carried out on the isolated strain.The results showed that the isolated strains were gram-negative bacteria with pleomorphism,and the colony morphology was round,smooth,gray-white,semi-transparent with neat edge.It showed NAD-dependent,typical satellite growth phenomenon,and no hemolysis phenomenon.The biochemical test results were consistent with the biochemical characteristics of Haemophilus Parasuis.A 1090 bp band was amplified by PCR,and was identified as serovar 5 by agar gel diffusion test.The isolate was identified by genotyping and only 468 bp fragment was amplified,which was confirmed to be serovar 5 by sequencing comparison.In the pathogenicity test of pig,there was no abnormality in the blank control pigs,and the infected pigs showed typical clinical symptoms of fever,arthritis and fibrinous polyserositis on the surface of multiple organs of chest and abdomen caused by Haemophilus parasuis.The results suggested that the infected pig was infected with Haemophilus parasuis,and the serovar 5 Haemophilus parasuis strain was isolated in this research.