巴西龟眼炎病原菌的分离鉴定及组织病理学研究

Isolation and identification of pathogenic bacteria and histopathology in turtle Trachemys scriptaelegans with ophthalmia

为防治龟类细菌性眼炎病,从患眼炎巴西龟Trachemys scriptaelegans (体质量约150. 0 g)眼内分泌液中分离获得1株革兰氏阴性细菌,编号为G0503菌株,通过细菌形态学观察、理化特性、16S r DNA克隆和测序,以及构建系统发育进化树分析等方法,对其进行了鉴定研究。结果表明:该分离菌与甘露糖、半乳糖、肌醇等反应为阳性,与赖氨酸、鸟氨酸脱羧酶等反应为阴性;进一步采用PCR方法扩增其16S r DNA序列,将其亚克隆至p MD18-T载体中测序,获得片段大小为1500 bp,与模式菌株Providencia rettgeri DSM 4542 (AM040492. 1)序列一致性为99. 30%;构建系统发育树分析显示,该菌与Providencia rettgeri(JN644501. 1)自然聚为一支,最终判定G0503菌株为雷氏普罗威登斯菌Providencia rettgeri;药敏试验结果显示,该菌株对头孢哌酮、头孢噻肟、诺氟沙星、左氟沙星、氟苯尼考等药物敏感,对阿莫西林、氨苄西林、磺胺异恶唑、复方新诺明等耐药;用该分离菌人工回归感染巴西龟死亡率达40%,主要呈现眼睛肿大、黏液增多,停止摄食等特征,并且与自然发病龟临床症状一致;组织病理学观察显示,病龟出现眼睑上皮细胞减少、淋巴细胞浸润等病理变化。本研究中首次报道巴西龟眼炎病原的分离鉴定,对该病防治可提供一定的参考依据。

A bacterial strain named as strain G0503 was isolated from the mucus of ophthalmitis turtle Trachemys scriptaelegans(body weight of 150.0 g),and the morphological observation,physiological and biochemical characteristics,and sequencing of 16S rDNA and phylogenetic tree analysis were carried out in this study.Results showed that the G0503 strain was a rod-shaped,and gram-negative bacillus,and was characterized by production of acid from mannose,galactose and inositol,and negative reaction for lysine and ornithine decarboxylase.Furthermore,the 16S rDNA sequences was amplified by PCR method,and subcloned into pMD18-T vector,with a sequencing product of 1500 bp in length.The 16S rDNA sequence had a 99.30%similarity with Providencia rettgeri type strain DSM 4542(AM040492.1),and clustered into one group with Providencia rettgeri(JN644501.1),indicating that the G0503 strain was identified as P.rettgeri.The antibiotic susceptibility test showed that it was sensitive to cefoperazone,cefotaxime,levofloxacin,norfloxacin and florfenicol,and resistant to amoxicillin,ampicillin,sulfamethoxazole,sulfamethoxazole and trimethoprim.The challenge test revealed that the isolate resulted in mortality rate of 40%of the diseased turtle,with clinical signs including asthe swollen,increase in mucus of eye and feeding stop,which were consistent with the natural symptoms of the diseased turtles.Histopathological observation showed that reduction in the epithelium cell number and increase in chromatophores in the eyes.The findings firstly reported the isolation and identification of P.rettgeri from the turtle with ophthalmia,and will provide scientific reference for prevention and treatment in fish.