1株血清10型副猪嗜血杆菌的分离鉴定与生物学特性分析

Isolation,Identification and Biological Characteristics Analysis of a Strain of Haemophilus parasuis Serotype 10

【目的】阐明引起荆州某猪厂仔猪疑似副猪嗜血杆菌病的病原生理生化特性、毒力与药物敏感性,为今后对该病的防治提供科学依据。【方法】从患病仔猪中分离培养病原菌,通过形态学观察、生理生化鉴定、PCR鉴定、16S rRNA基因测序、血清型鉴定、多序列位点分型(MLST)对分离菌株进行鉴定。通过毒力基因检测、小鼠致病性、药敏试验、中药治疗试验等对分离株进行致病性、耐药性分析。【结果】通过形态学观察、生理生化试验、16S rRNA序列比对、系统进化树分析确定分离菌株为副猪嗜血杆菌。通过血清型与MLST分析确定分离菌株为血清10型、ST型为299。毒力基因检测发现,分离菌株具有CapD、vta 1、vta 2等15种毒力基因。将分离菌株腹腔注射感染小鼠,可导致小鼠多个脏器发生明显病变,具有较强致病性。耐药基因检测试验发现,分离菌株具有β-内酰胺类耐药基因bla OXA和氟喹诺酮类耐药基因gyrA。药敏试验结果显示,分离菌株对头孢他啶、新霉素、米诺环素等10种抗菌药物敏感,同时对明雄黄、款冬花、全蝎3种中药敏感。中药治疗试验结果表明,明雄黄和全蝎对分离菌株感染小鼠有较明显的治疗作用。【结论】试验成功分离1株血清10型副猪嗜血杆菌,侵染小鼠可致多个器官出血坏死,该菌株对10种抗菌药物及3种中药敏感,且明雄黄和全蝎对分离菌株感染小鼠治疗效果明显。研究结果可为今后副猪嗜血杆菌的防治和临床诊疗提供参考依据。

【Objective】This study was aimed to elucidate the physiological and biochemical characteristics,virulence and drug sensitivity of the pathogen causing Haemophilus parasuis disease in piglets from a pig factory in Jingzhou,China,so as to provide scientific basis for the prevention and treatment of this disease in the future.【Method】The pathogenic bacteria were isolated from infected piglets and identified by morphological observation,physiological and biochemical identification,PCR identification,16S rRNA gene sequencing,serotype identification and multi-sequence site typing(MLST).The pathogenicity and drug resistance of the isolate were analyzed by virulence gene detection,pathogenicity test of mouse,drug sensitivity test and traditional Chinese medicine treatment.【Result】Through morphological observation,physiological and biochemical experiments,16S rRNA sequence alignment and phylogenetic tree analysis,the isolate was identified as Haemophilus parasuis.Serotype and MLST analysis confirmed that the isolate was serotype 10 and ST-299.Virulence gene test showed that the isolate had 15 virulence genes such as CapD,vta 1 and vta 2.The isolate could cause obvious lesions in many organs of the infected mice by intraperitoneal injection and had strong pathogenicity.The detection of drug resistance genes showed that the isolate hadβ-lactam resistance gene bla OXA and fluoroquinolone resistance gene gyrA.The drug sensitivity test showed that the isolate was sensitive to 10 kinds of antibiotics,such as ceftriaxone,neomycin and minocycline,and to 3 kinds of traditional Chinese medicines including realgar,Tussilago farfara and Buthus martensii.The traditional Chinese medicine treatment showed that realgar and Buthus martensii had obvious therapeutic effect on mice infected by the isolate.【Conclusion】In this study,a strain of Haemophilus parasuis serotype 10 was successfully isolated which could cause bleeding and necrosis of multiple organs in infected mice,and was sensitive to 10 kinds of antibiotics and 3 kinds of traditional Chinese medicines.It was found that realgar and Buthus martensii were effective in treating mice infected by the isolate.The results provided a reference for future prevention and treatment of Haemophilus parasuis.