副鸡嗜血杆菌的分离鉴定及16SrRNA序列分析

IDENTIFICATION AND 16SRRNA GENE ANALYSIS OF HAEMOPHILUS PARAGALLINARUM

从云南省和四川省规模化养鸡场鸡传染性鼻炎疑似病例鸡鼻窦分离到2株革兰氏阴性小杆菌,并对其进行生化鉴定、PCR鉴定和16SrRNA序列比对鉴定。16SrRNA分析表明两分离株与GenBank中的其他副鸡嗜血杆菌（Haemophilus paragallinarum,HPg）参考株核苷酸同源性为94.2%~99.2%。两分离菌株鉴定为副鸡嗜血杆菌,分别命名为YNAN20120110和SCPZH20120120株。16SrRNA遗传进化关系表明：分离株与NAD非依赖型副鸡嗜血杆菌血清A型代表株GU951543（HP105strain）的16SrRNA序列位于一个分支上,遗传进化关系最近,它们之间的核苷酸同源性为99.2%;与副鸡嗜血杆菌血清A型参考株AY498867（0083株）核苷酸同源性为96.9%。致病性实验表明分离菌株对成年产蛋鸡有强致病性。抗生素药物敏感试验表明：分离菌株对头孢吡肟、头孢噻吩、氯霉素、卡那霉素和氧氟沙星高敏,对四环素中敏,对磺胺甲唑耐药。

Two strains of Gram negative bacilli were isolated from suspected case of infectious coryza of chickens in a large-scale poultry farm and designated as YNAN20120110 and SCPZH20120120. Both strains were identified as Haemophilus paragallinarum （HPg） by biochemical reactions, PCR and 16SrRNA gene analysis. The two strains shared 94.2%-99.3% of the 16SrRNA sequence homology with other HPg strains in GenBank. Phylogenetic analysis based on 16SrRNA gene showed that both strains were placed on the same branch with the NAD-independent strain GU951543, the serovar A reference strain as their nucleotide homology with GU951543 strain （HP105） was 99.2%. The nucleotide homology between the two strains and serovar A reference strain AY498867 （0083） was 96.9%. The two strains were highly pathogenic to laying hens. Antibiotics sensitivity test indicated that they were highly sensitive to cefepime, cephalothin, chloromycetin, ofloxacin, less sensitive to tetracycline and resistant to sulfarnethoxazole.