绵羊大肠杆菌和艾美尔球虫混合感染病原分离与病理组织学观察

Isolation and pathological histology of mixed infection of Escherichia coli and Eimeria in sheep

【目的】对宁夏某规模化绵羊场大肠杆菌和艾美耳球虫混合感染进行分离鉴定并对靶器官的组织病理学变化进行分析。【方法】采用固体培养基进行细菌病原分离,饱和盐水漂浮法进行寄生虫检测;采用大肠杆菌16S rRNA引物进行基因序列扩增和测序,使用DNA Star软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比较;采用MEGA 7.0软件中的邻接法,依据16S rRNA序列构建分离株系统发育树并进行遗传进化分析;对靶器官组织进行病理组织学观察。【结果】在伊红美蓝培养基中观察到金属光泽的黑色单一菌落,在血平板中有溶血环的灰色圆形单一菌落,染色为革兰氏阴性杆菌,共分离得到3株菌株;麦克马斯特法检测显示,粪便中有卵圆形、表面光滑的孢子化艾美尔球虫卵囊,其平均OPG为2200,属于轻度感染;对3株菌株16S rRNA扩增片段与阳性对照一致,16S rRNA基因序列构建的系统发育树与大肠杆菌在同一分支;病理组织学观察显示,肠系膜淋巴结淋巴细胞数量减少,有中性粒细胞浸润;肠组织黏膜层绒毛溶解坏死、黏膜上皮结构丢失;回肠固有层内有球虫虫体,呈嗜酸性圆形;空肠间质有中性粒细胞浸润,腺腔有镰刀状球虫裂殖体;结肠局部黏膜有溃疡灶,并有大量弱嗜碱性短杆状物质沉积。【结论】结合病原形态学和分子生物学结果分析表明,该例绵羊群腹泻是由大肠杆菌和艾美尔球虫共同感染所致。

【Objective】The present paper aimed to isolate and identify mixed infections of Escherichia coli and Eimeria in a large-scale sheep farm in Ningxia and analyze the histopathological changes of target organs.【Method】Solid culture medium was used to isolate bacterial pathogens, and saturated saline floating method was used to detect parasites. The gene sequence was amplified and sequenced using 16S rRNA primers from E. coli, and the DNA Star software was used to compare the sequencing results of the isolated strains with the sequence of the standard strains in GenBank. The adjacency method in MEGA 7.0 software was used for genetic evolution analysis. The phylogenetic tree of isolates was constructed based on 16S rRNA using the adjacency method in MEGA 7.0 software. The target organ tissues were subjected to histopathological observation.【Result】A metallic black single colony was observed in eosin methylene blue medium, and a grey circular single colony with a hemolytic ring was observed in the blood plate. It was stained as Gram negative bacilli.A total of 3 strains were isolated.McMaster test showed that there were ovoid, smooth surface sporulated Eimeria oocysts in the feces, with an average OPG of 2200, indicating a mild infection. The amplified fragment of 16S rRNA from three strains was consistent with the positive control, and the phylogenetic tree constructed based on 16S rRNA gene sequence was in the same branch as E. coli. Histopathological observation showed that the number of lymphocytes in mesenteric lymph nodes decreased and neutrophils infiltrated. There was dissolution and necrosis of villi in the mucosal layer of intestinal tissue, and loss of mucosal epithelial structure. There were coccidia bodies in the lamina propria of the ileum, which were eosinophilic and round in shape. There were neutrophils infiltrating the interstitium of the jejunum, and there were schizoites of coccidia falciformis in the glandular cavity. There were ulcerative foci in the local mucosa of the colon, with a large amount of weakly basophilic short rod-shaped substances deposited.【Conclusion】The comprehensive analysis of pathogenic morphology and molecular biology results showed that the diarrhea in this sheep group was caused by the co-infection of E. coli and Eimeria.