一株犬种布鲁氏菌的分离鉴定及其在巨噬细胞内存活能力的研究  

Isolation and identification of a Brucella canis strain and its survival capability in macrophages

作　　者：焉鑫 陈霞 孙世雄 田雪 邵卫星[1] 孙翔翔[1,2] 南文龙 樊晓旭[1] 孙淑芳[1,2] 孙明军[1] YAN Xin;CHEN Xia;SUN Shi-xiong;TIAN Xue;SHAO Wei-xing;SUN Xiang-xiang;NAN Wen-long;FAN Xiao-xu;SUN Shu-fang;SUN Ming-jun(Division of zoonosis diseases surveillance,China Animal Health and Epidemiology Center,Qingdao 266032,China;Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao 266032,China;Chongqing Yubei District Animal Husbandry and Aquatic Products Station,Chongqing 401120,China;Shandong Agricultural University,College of Animal Science and Technology,Taian 271018,China)

机构地区：[1]中国动物卫生与流行病学中心/人兽共患病监测室,山东青岛266032 [2]农业农村部动物生物安全风险预警及防控重点实验室(南方),山东青岛266032 [3]重庆市渝北区畜牧水产站,重庆401120 [4]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国预防兽医学报》2023年第9期889-894,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：中心科技创新基金项目(DW2021002);国家现代农业(奶牛)产业技术体系建设专项(CARS-36)。

摘　　要：为分离鉴定流产犬的致病病原及其生物学特性,本研究从重庆某宠物医院无菌采集流产犬全血2份,将其涂布于布鲁氏菌选择培养基,分别在不含CO_(2)和含7.5%CO_(2)条件下37℃培养72 h,观察菌落形态,并经结晶紫染色,观察细菌类型。按照国家标准鉴定分离菌的生化特性。细菌分离结果显示,在不含CO_(2)和含7.5%CO_(2)的培养箱中2份流产犬全血样品在布鲁氏菌选择培养基上均长出边缘整齐、光滑的圆形菌落,透射光下,菌落呈浅黄色且有光泽;结晶紫染色结果显示分离菌为典型的粗糙型细菌;生化鉴定结果显示,分离株在TSA中的生长不依赖于CO_(2),且不产生H2S,氧化酶和脲酶试验均为强阳性。上述结果表明分离到一株犬种布鲁氏菌,命名为B. canis CQ3。对分离菌进行全基因组测序,利用MAUVE对分离株与GenBank中38株不同种的布鲁氏菌基因组进行比对,并构建分离菌全基因组的进化树。将不同种布鲁氏菌及分离株分别接种于TSB培养基中,通过测定不同时间的OD450nm值绘制各菌株的生长曲线。将各菌株及B. canis CQ3株分别以MOI 100感染小鼠腹腔巨噬细胞RAW264.7,在感染后的不同时间(1 h、24 h、48 h和72 h)收集细胞,经相应处理后涂布TSA平板培养72 h后,经菌落计数分析分离菌株在细胞中的存活能力。全基因组序列比对及进化树结果显示,比对到犬种布鲁氏菌的序列数量最多,且B. canis CQ3株与欧洲分离的犬种布鲁氏菌NR-finland株的亲缘关系最近。生长曲线测定结果显示,在6 h~16 h (对数生长期) B. canis CQ3株的生长速度稍快于羊种布鲁氏菌B. melitensis XJ流行株和牛种布鲁氏菌B. abortus DT流行株;整个生长过程中B. canis CQ3株的生长速度与疫苗株A19和S2基本一致。巨噬细胞感染试验结果显示,感染1 h时,B. canis CQ3株进入巨噬细胞数量多于疫苗株A19和S2、B. melitensis XJ和B. abortus DT株。相比B. melitensis XJ和B. abortuTo explore the pathogenic factors and biological characteristics of aborted dog cases,two whole blood samples of aborted dogs were collected aseptically from a pet hospital in Chongqing,and coated on Brucella selective culture medium.They were cultured at 37℃for 72 hours in incubators without CO_(2) and with 7.5%CO_(2),respectively.Colony morphology was observed,and the bacterial type was observed by crystal violet staining.The biochemical characteristics of isolated bacteria were identified using national standard animal brucellosis diagnostic techniques and WOAH terrestrial animal diagnostic tests and vaccine manual methods.The results of bacterial isolation showed that the isolated bacteria grew neatly edged and smooth circular colonies on the Brucella selective medium in bothe CO_(2) free and 7.5%CO_(2) containing incubators.Under transmitted light,the colonies appeared light yellow and glossy.The results of crystal violet staining showed that the isolates were typical rough bacteria.The biochemical identification results showed that the growth of the isolates in TSA was not dependent on CO_(2) and did not produce H2S,the oxidase and urease tests were strongly positive.The above results indicated that a strain of Brucella canis was isolated and named B.canis CQ3.The whole genome of the isolated strain was sequenced,and compared with genomes of 38 different types of Brucella from GenBank in MAUVE,and the phylogenetic tree of the whole genome of the isolate was constructed.Different types of Brucella and the B.canis CQ3 were inoculated into TSB medium,and the growth curves of each strain were plotted by measuring the OD450nm values at different times.Mouse peritoneal macrophages RAW264.7 were infected with each strain at a MOI of 100.Cells were collected at different times(1 hour,24 hours,48 hours and 72 hours)after infection,and the survival capability of the isolate in the cells was analyzed by colony counting.The results of whole genome sequence alignment and phylogenetic tree showed that the number of seq

关 键 词：犬种布鲁氏菌 分离鉴定 胞内存活 

分 类 号：S852.61[农业科学—基础兽医学]