[目的]通过探讨青海省西宁市某规模化奶牛场病死牛源多杀性巴氏杆菌的荚膜血清型、耐药性和毒力基因分布情况，为多杀性巴氏杆菌引起的牛呼吸道疾病的治疗和预防提供参考。[方法]对病死牛肺脏样本进行细菌分离，通过菌落观察和染色镜检、生化鉴定、特异性引物扩增、16S rRNA测序分析及系统发育树构建、荚膜血清及脂多糖分型、毒力基因检测、致病性试验和药敏试验对分离菌进行鉴定和病原特性评价。[结果]肺脏样本中分离得到的菌落呈现灰白色中间微凹陷的露珠样，未出现溶血环;革兰染色可见粉红色球杆菌;瑞氏染色可见两端浓染的蓝色球杆菌。生化鉴定结果显示，分离菌株D-葡萄糖、D-甘露醇、D-甘露糖、酪氨酸芳胺酶、磷酸酶、COURMARATE和ELLMAN反应结果为阳性;16S rRNA测序结果显示，分离菌株与多种多杀性巴氏杆菌相似性＞95％，鉴定分离得到的菌株为多杀性巴氏杆菌，命名为XN222。荚膜血清分型及脂多糖分型结果显示，分离菌株XN222的荚膜血清分型为D型，其脂多糖分型为L3和L5型。毒力基因扩增结果显示，分离菌株XN222携带ptfA、fimA、hsf-2、sodA、tbpA、sodC等共计19种毒力基因。小鼠致病性试验结果显示，注射0。2 mL 1。5 × 108 CFU/mL菌液，可导致100％(10/10)小鼠死亡，说明分离菌株XN222具有较强的致病能力。药敏试验结果显示，分离菌株XN222对头孢噻呋、左氧氟沙星、恩诺沙星、马波杀星、四环素和青霉素等15种抗菌药均敏感，对泰乐菌素中度敏感。[结论]本研究分离获得1株致病力较强的D型多杀性巴氏杆菌，该菌株的发现丰富了国内牛源多杀性巴氏杆菌亚型，可为多杀性巴氏杆菌引起的牛呼吸道疾病的防治、病原学调查和巴氏杆菌多价苗研制等提供生物素材。
Isolation,Identification and Pathogen Characteristics Evaluation of a Strain of Pasteurella multocida D∶L3∶L5 from Cattle
[Objective]The experiment was aimed to explore the distribution of capsule serotypes,drug resistance and virulence genes of P asteurella multocida from a large-scale dairy farm in Xining city,Qinghai province,in order to provide reference for the treatment and prevention of respiratory diseases caused by Pasteurella multocida in cattle.[Method]Bacteria were isolated from the lung sample of a dead cattle.And then,through the methods of colony observation,dyeing microscopy,biochemical identification,specific primer amplification,16S rRNA clone sequencing analysis,phylogenetic tree construction,capsular serum and lipopolysaccharide type,virulence gene detection,pathogenicity test and drug sensitivity test,the isolated Pasteurellamultocida was identified and its pathogen characteristics were evaluated.[Result]The colonies isolated from the lung samples were dewdrop-like with a slight depression in the gray middle,and no hemolysis ring appeared.Gram staining showed coccidia pink,Wright's staining showed blue coccidia with dense staining at both ends.Biochemical identification showed that the results of D-glucose,D-mannitol,D-mannose,tyrosine arylaminase,phosphatase,COURMARATE and ELLMAN reaction were positive.16S rRNA sequencing results showed that the strain was more than 95％similar to a variety of Pasteurella multocida,and the isolated strain was identified as Pasteurella multocida and named XN222.The results of capsule serum typing and lipopolysaccharide type showed that XN222 was classified as type D,and its lipopolysaccharide types were L3 and L5.The results of virulence gene amplification showed that XN222 carried 19 virulence genes,including ptfA,fimA,hsf-2,sodA,tbpA,sodC,and so on.The results of pathogenicity test in mice showed that injection of 0.2 mL 1.5 ×108 CFU/mL bacterial solution resulted in 100％(10/10)death of mice,indicating that the isolated strain XN222 had strong pathogenicity.The results of drug sensitivity test showed that XN222 was sensitive to 15 kinds of antimicrobial agents,such as ceftiofur,levofloxacin,enrofloxacin,marbofloxacin,tetracycline and penicillin,and was moderately sensitive to tylosin.[Conclusion]In this study,a highly pathogenic Pasteurella multocida type D strain was isolated.The discovery of this strain enriched the domestic bovine subtype of Pasteurella multocida,and provided biological materials for the prevention and control of bovine respiratory diseases caused by Pasteurella multocida,etiological investigation and development of Pasteurella multocida multivalent vaccine.
Pasteurella multocida type Disolation and identificationpathogen characteristics
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