鸡源豚鼠气单胞菌的分离鉴定及药敏试验

Isolation,identification and drug susceptibilty test of chicken-derived Aeromonas caviae

2021年7月份,河北省昌黎县某蛋鸡场130日龄左右海兰褐蛋鸡发病,主要临床症状为眼睑肿胀且有干酪样渗出物,肛门粘有绿色稀便,剖检可见腿部肌肉组织溶解和腹腔积液。为了查明感染的病原,试验采集病鸡的眼睑干酪样渗出物、气管黏液、腹腔积液及腿部深层肌肉病变组织,进行细菌的分离纯化、形态学观察、革兰氏染色镜检、生化鉴定、16S rRNA基因PCR鉴定,确定感染病原菌种类;对分离纯化的病原菌采用Kirby-Bauer纸片扩散法进行抗生素敏感性试验,采用牛津杯法进行22种中草药敏感性试验,同时对22种中草药提取液进行体外最小抑菌浓度(MIC)和最小杀菌浓度(MBC)的测定;对分离纯化的病原菌进行毒力基因检测,应用Balb/c小鼠进行致病性试验。结果表明:经培养,在绵羊血琼脂培养基上可见边缘整齐、光滑、湿润、呈β溶血的灰色菌落,革兰氏染色可见散在或成对存在的革兰氏阴性短杆菌,与豚鼠气单胞菌形态特征相似,将分离菌株命名为TSQDBJ-1;其生化特性与豚鼠气单胞菌符合率达99%,核苷酸相似性达99.7%;分离菌携带丝氨酸蛋白酶(Ahp)、细胞毒性肠毒素(Act)两种毒力基因;对头孢曲松、乙酰甲喹、头孢噻呋敏感;对苏木、五味子、乌梅、五倍子、诃子、石榴皮提取液的MIC为7.8~15.6 mg/mL,MBC为7.8~31.2 mg/mL;接种该菌菌液的小鼠在第4~6天全部死亡。说明该分离菌为鸡源豚鼠气单胞菌,且具有潜在的致病性。

In July 2021,about 130-day-old Hailan Brown layer hens from a layer hen farm in Changli County,Hebei Province became ill.The main clinical symptoms of the affected chickens were swollen eyelids,cheese-like exudates,and green loose stools sticky to the anus;autopsy revealed muscle dissolution of the leg and effusion in the abdominal cavity.In order to identify the pathogen of infection,the eyelid caseous exudate,tracheal mucus,ascites and the deep tissues of the leg muscle lesions of the sick chickens were tested and the species of infectious pathogens were determined by isolation and purification of bacteria,morphological observation,Gram staining microscopy,biochemical identification,and PCR identification of 16S rRNA gene.Antibiotic susceptibility test was performed by using Kirby-Bauer paper diffusion method for isolated and purified pathogens.The Oxford cup method carried out 22 Chinese herbal susceptibilty tests;at the same time,22 Chinese herbal extracts were determined by minimum bacteriostatic concentration(MIC)and minimum bactericidal concentration(MBC)in vitro.The virulence gene detection was performed on the isolated and purified pathogenic bacteria,and the pathogenicity test was performed in Balb/c mice.The results showed that gray colonies with neat edges,smooth and moist,andβhemolytic were visible on sheep blood agar medium after culture.Gram-negative bacillus could be seen scattered or in pairs,which were similar to the morphological characteristics of Aeromonas caviae,and the isolated strain was named TSQDBJ-1.Its biochemical characteristics were 99%consistent with Aeromonas caviae,and the nucleotide similarity was 99.7%.The isolated strain carried two virulence genes,serine protease(Ahp)and cytotoxic enterotoxin(Act),and was sensitive to ceftriaxone,mequindox and ceftiofur.The MIC and MBC of the extracts of sappanwood,schisandra,dark plum,gallnut,terminalia,and pomegranate peel were 7.8-15.6 mg/mL and 7.8-31.2 mg/mL.All mice inoculated with this bacterial solution died on day 4-6.The results suggested that the isolate was Aeromonas caviae which was potentially pathogenic.