牦牛多杀性巴氏杆菌烈性噬菌体的生物学特性与全基因组序列分析

Biological Characteristics and Complete Genome Sequence Analysis of Lytic Bacteriophage vB_PmuP_Pa7 of Pasteurella multocida from Yak

为了鉴定1株防治牦牛出血性败血症的噬菌体,并评价其开发为新型环保生物制剂的潜力,本试验用双层平板法从牦牛的粪便污水中分离获得1株新型的B型牦牛多杀性巴氏杆菌噬菌体vB_PmuP_Pa7(简称Pa7),并使用透射电镜观察其形态,同时对噬菌体进行热稳定性、pH、最佳感染复数(MOI)、一步生长曲线和裂解谱等生物学特性分析,采用Illumina测序技术进行全基因组测序。结果显示:Pa7有1个正廿多面体头部(直径约55 nm)和1个短尾(直径约20 nm),属于短尾噬菌体科;Pa7能耐受一定的高温,60℃以上不稳定;Pa7在pH 5.0~9.0活性较为稳定;Pa7的最佳感染复数(MOI)为0.01;一步生长曲线试验结果显示,Pa7潜伏期大约持续20 min,平均裂解量为162 PFU/cell;Pa7裂解谱很窄,仅能裂解B型多杀性巴氏杆菌;基因测序结果显示,Pa7基因组为双链DNA,基因组大小长度为37402 bp,G+C含量40.9%,拥有41个开放阅读框(ORFs),基因组无耐药性因子,未检测到tRNA基因。结果表明,噬菌体Pa7为1株多杀性巴氏杆菌烈性噬菌体,具有开发为抗多杀性巴氏杆菌制剂的潜力。

In order to identify a lytic phage with potential as a new environmental protection biological agent for prevention and treatment of hemorrhagic septicemia in yak,this study utilized double-layer plate method to isolate a lytic bacteriophage of Pasteurella multocida,named vB_PmuP_Pa7(Pa7),from yak feces,transmission electron microscopy(TEM)to visualize Pa7,other biological techniques to assess the thermal stability and pH,the multiplicity of infection(MOI),one-step growth curve and lytic ability of Pa7,and Illumina sequencing technology to determine the complete genome sequence of Pa7.The results showed that under electron microscopy,Pa7 had an icosahedral head(about 55 nm in diameter)and a short tail(about 20 nm in diameter),characteristic of the Caudovirales family;Pa7 could endure high temperature,but appeared unstable above 60℃;Pa7 was stable over a broad pH range(pH 5.0-9.0);Pa7 displayed MOI of 0.01,and had a latent period of 20 min and burst size of 162 PFU/cell in one-step growth curve analysis;Pa7 had monovalent lytic ability,and only lysed serotype B of P.multocida.Genome sequencing confirmed a linear dsDNA genome of 37402 bp in length,with a G+C content of 40.9%and 41 putative open reading frames(ORFs),without any drug resistant and tRNA genes.Thus,Pa7 is a lytic bacteriophage specific for P.multocida,and has potential to be developed as a novel alternative for the control of P.multocida-associated infections.