牛多杀性巴氏杆菌和溶血性曼氏杆菌的分离鉴定及致病力评估

Isolation,identification,and pathogenicity evaluation of Pasteurella multocida and Mannheimia haemolytica

牛呼吸道疾病(bovine respiratory disease,BRD)是牛场中重要的疾病之一,给我国养牛业造成了严重的经济损失。多杀性巴氏杆菌(Pasteurella multocida,Pm)和溶血性曼氏杆菌(Mannheimia haemolytica,Mh)是与BRD相关的主要细菌性病原。为分离纯化到目前Pm和Mh流行的优势毒株,建立合适的小动物攻毒模型,给BRD疫苗研制提供有效的疫苗候选株。本研究首先从患BRD牛的病料中进行Pm和Mh的检测、分离鉴定,确定基因型且评估分离株对小鼠的致病性,筛选合适的疫苗候选株。结果显示,本研究在2022年11-12月从我国3个省份的6个牛场采集到48份患有BRD的牛鼻拭子,PCR检测结果显示Pm的检出率为47.9%(95%CI:33.3~62.8),Mh的检出率为37.5%(95%CI:24.0~52.6),Pm/Mh共感染的检出率为18.8%(95%CI:8.9~32.6)。在阳性样本中分离纯化得到12株Pm,经血清型鉴定均为A型多杀性巴氏杆菌(PmA);分离到11株Mh,其中7株为A1型,4株为A6型,且在同一头牛的鼻拭子中同时分离到A1型和A6型的Mh。随后建立PmA和Mh的小鼠感染模型,筛选到PmA和Mh的强毒株分别是PmA GDP001株和Mh GDM019株,并且测定PmA GDP001株对小鼠的LD_(50)为10^(1.1) CFU,Mh GDM019株对小鼠的LD_(50)为10^(8.2) CFU。本研究揭示了我国部分地区牛场中BRD主要细菌性病原的流行病学,分离到了重要的病原(PmA和Mh),并且鉴定了分离株的血清型,且通过小鼠感染致病模型筛选到毒力强的PmA和Mh菌株,为BRD多联多价疫苗的研发提供了良好的疫苗候选株。

Bovine respiratory disease(BRD) is one of the important diseases in cattle farms,causing serious economic losses to cattle industry in China.The major bacterial species involved in BRD are Pasteurella multocida(Pm) and Mannheimia haemolytica(Mh).The purpose of this study is to isolate the dominant strains of Pm and Mh,establish suitable small animal models for virus attack,and provide effective vaccine candidates for BRD vaccine development.In this study,Pm and Mh were detected,isolated,and identified from diseased samples of cattle with BRD.The genotype of the strain was determined by PCR and the pathogenicity of the isolated strain to mice was evaluated to screen suitable vaccine candidates.In this study,48 nasal swabs collected from cattle with clinical BRD from 6 cattle farms in 3 provinces of China during November to December 2022.The results showed that the detection rate of Pm was 47.9%(95% CI:33.3-62.8),the detection rate of Mh was 37.5%(95% CI:24.0-52.6),and the detection rate of Pm/Mh coinfection was 18.8% (95% CI:8.9-32.6).Twelve strains of Pm were isolated and purified from the nasal swabs,and their serotypes were identified as Pasteurella multocidatype A(PmA).Eleven strains of Mh were isolated,of which 7strains were identified as A1type and 4strains as A6type.Both A1type and A6type Mh strains were isolated from the same nasal swab.In this study,the mouse infection model infected with PmA and Mh were established,and the highly virulent strains of PmA and Mh were selected as PmA GDP001strain and Mh GDM019strain,respectively.The LD_(50) of PmA GDP001strain and Mh GDM019strain to mice was determined to be 10^(1.1) CFU and 10^(8.2) CFU,respectively.In summary,this study revealed the epidemiology of the main bacterial pathogens of BRD in some regions of China,isolated important pathogens(PmA and Mh),and identified the serotypes of the isolated strains.Highly virulent strains of PmA and Mh were screened,providing the good vaccine candidate strains for the development of BRD multivalent vaccines.