副猪嗜血杆菌分离株耐药性、血清型及RAPD基因型分析

Antimicrobial susceptibility,serotyping and RAPD genotyping of Haemophilus parasuis isolates

为弄清某规模化养猪场感染和流行的副猪嗜血杆菌(HPS)菌株的耐药性、血清型及基因型,本试验自该猪场发生浆膜炎和关节炎病猪的不同组织样品中分离到6株HPS菌株,通过细菌培养特性、生化试验和PCR扩增细菌16S rRNA基因片段并测序对分离株进行鉴定;进而鉴定分离株对不同类别抗生素的耐药性;最后对分离株进行血清分型和RAPD基因分型并分析两者的相关性。结果显示,自病猪不同组织脏器中分离鉴定了6株HPS菌株,体外培养具有典型"卫星生长"现象;生化试验结果符合HPS反应特性;PCR均可扩增出821 bp的HPS 16S rRNA基因片段,且序列与HPS一致。药敏试验结果显示分离株对头孢菌素类、青霉素类、氨基糖苷类、大环内酯类和四环素类抗生素敏感,而对喹诺酮类、磺胺类、林可霉素类和氯霉素类抗生素产生耐药性。琼脂扩散试验结果显示HPS分离株均为血清5型;RAPD分析显示HPS分离株与15个血清型参考菌株可分为4个基因型。本试验结果为HPS的流行病学调查及防控具有一定的借鉴价值。

To identify and analyze antimicrobial susceptibility,serotype and genotype of Haemophilus parasuis isolates in one large-scale pig farm,samples from pigs with polyserositis and polyarthritis were collected for H. parasuis isolation. Then the isolates were identified and characterized by growth feature,biochemical characteristics,bacterial 16 S rRNA amplification by PCR and sequencing. And the multiple antibiotic resistances of the isolates were analyzed by antimicrobial susceptibility assay. Finally,serotyping and RAPD genotyping of the isolates were investigated to analyze the relationship between them. Totally,6 isolates of H. parasuis were identified and characterized. The growth features showed that the isolates culturing was NAD-dependent,and the typical satellite growth phenomenon was observed upon co-culturing with Staphylococcus epidermidis. The biochemical results conformed to the characters of H. parasuis. The 821 bp fragment of 16 S rRNA was amplified from the genomic DNA of the isolates and sequenced which were consistent with that of H. parasuis. The multiple antibiotic resistance analysis showed that the isolates were susceptible to cephalosporins,penicillins,aminoglycosides,macrolides and tetracyclines,while significant resistance to quinolones,sulfanilamides,lincosamides and chloramphenicols. The isolates were serotyped to serovar 5 of H. parasuis by agar gel precipitation test. All strains containing the isolates and the 15 reference strains were divided into 4 genotypes based on RAPD analysis. The results of this study provides a certain valuable reference in epidemiology and control of H. parasuis.