摘要
【目的】为确诊安阳动物园一起蓝孔雀急性死亡病例,本研究开展了临床诊断与实验室检测。【方法】观察病死孔雀临床症状和病理变化,通过鉴别培养基、革兰染色、生化鉴定、16S rRNA基因PCR扩增及构建系统发育树、动物试验等方法分离鉴定病原菌,利用结晶紫染色法对分离菌进行生物被膜形成能力检测,采用K-B纸片法进行药敏试验,通过PCR检测耐药基因。【结果】死亡孔雀体表无明显症状,对孔雀进行剖检观察发现,其心包积液增多、浑浊,肝脏肿大,肾脏有尿酸盐沉积,肠道出血。无菌采集死亡孔雀肝脏组织,划线接种于麦康凯培养基,分离出粉红色菌落,镜检观察到病原菌呈红色短杆状,单个或成对散在分布,疑似肠杆菌科细菌。生化鉴定结果显示,分离菌吲哚、MR、戊糖、山梨醇等为阳性,西蒙氏枸橼酸盐、V-P、尿素、硫化氢等为阴性;16S rRNA基因PCR扩增片段经测序得到大小约为1500 bp的序列(GenBase登录号:C_AA110994.1)。系统发育树显示,分离菌株与大肠杆菌相似性最高,为100%,确定该分离菌为大肠杆菌;分离菌比标准菌株被膜形成能力强,且能导致BALB/c小鼠肝脏和脾脏肿大、肺脏淤血、肠壁变薄、肠道出血、肠绒毛脱落等。分离菌对左氧氟沙星、诺氟沙星等药物敏感,对四环素、链霉素等耐药。耐药基因PCR扩增结果显示,分离菌检测到tetA、ampC、blaTEM、Sul1、Sul2和strA-strB 6个耐药基因。【结论】本研究分离到1株蓝孔雀源致病性大肠杆菌。药敏试验结果显示,该大肠杆菌菌株对左氧氟沙星、诺氟沙星等药物敏感,对四环素、链霉素等耐药,并携带6种耐药基因。本研究结果可为蓝孔雀大肠杆菌病的诊断和临床用药提供借鉴。
【Objective】To confirm an acute death case of Pavo cristatus at Anyang zoo,this study carried out clinical diagnosis and laboratory tests.【Method】The clinical symptoms and pathological changes of dead peacocks were observed.The pathogenic bacteria were isolated and identified by methods such as identification medium,Gram staining,biochemical identification,16S rRNA gene PCR amplification,phylogenetic tree construction and animal experiments.The biofilm-forming ability of the isolated bacteria was detected by crystal violet staining method,and the drug sensitivity test was conducted by K-B disk method.Drug resistance genes were detected by PCR.【Result】There was no obvious symptoms on the body surface of dead peacocks.The results of the peafowl necropsy showed that there was an increase and turbidity in pericardial effusion,and the liver was enlarged.There was urate deposition in the kidneys,and bleeding in the intestines.The liver tissues of dead peacocks were aseptically collected and streaked and inoculated on MacConkey medium,and red colonies were isolated.Microscopic examination revealed that the pathogenic bacteria were pink short rods,scattered singly or in pairs,and suspected to be bacteria of the Enterobacteriaceae family.The results of biochemical identification indicated positive reactions for indole,MR,pentose,sorbitol,and so on,and negative reactions for Simonsons citrate,V-P,urea,hydrogen sulfide,and so on.16S rRNA gene PCR amplified fragment was sequenced to obtain a sequence of about 1500 bp in length(GenBase accession No.:C_AA110994.1).The phylogenetic tree indicated that the isolated strain had the highest similarity(100%)to Escherichia coli(E.coli),confirming that the isolated bacterium was E.coli.The isolated bacteria had stronger biofilm-forming ability than standard strain and could cause liver and spleen enlargement,lung congestion,intestinal wall thinning,intestinal bleeding,and villus shedding in BALB/c mice,eventually leading to death.The isolated bacteria were sensitive to levofloxacin,norfloxacin and so on,but resistant to tetracycline,streptomycin and other drugs,and carried six resistance genes tetA,ampC,blaTEM,Sul1,Sul2 and strA-strB.【Conclusion】In this study,the pathogenic E.coli srtain was isolated and identified form Pavo cristatus.The results of drug sensitivity test showed that this E.coli strain was sensitive to levofloxacin,norfloxacin,and so on,but resistant to tetracycline,streptomycin,and so on.This study provided a valuable reference for the diagnosis and clinical medication of E.coli-related diseases in Pavo cristatus.
作者
连凯琪
王宇航
孟祥凤
张明亮
周玲玲
LIAN Kaiqi;WANG Yuhang;MENG Xiangfeng;ZHANG Mingliang;ZHOU Lingling(School of Biotechnology and Food Science,Anyang Institute of Technology,Anyang 455000,China;Henan Joint International Research Laboratory of Veterinary Biologics Research and Application,Anyang 455000,China)
出处
《中国畜牧兽医》
北大核心
2026年第1期448-458,共11页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金青年项目(31802170)
河南省科技攻关计划项目(242102110073)
安阳市重点研发与推广专项(2023C01NY013)
安阳工学院博士启动基金项目(BSJ2024005)。
关键词
蓝孔雀
大肠杆菌
分离鉴定
耐药性
Pavo cristatus
Escherichia coli
isolation and identification
drug resistance
