滇南某猪群致死性呼吸道传染病的病原学诊断

Etiological Diagnosis of Lethal Respiratory Disease in a Pig Group in South Yunnan Province

关键词:  猪繁殖与呼吸综合征  猪鼻支原体  类脲气球菌  混合感染  PCR

Key words:  porcine reproductive and respiratory syndrome  Mycoplasma hyorhinis  Aerococus urinae-like organism  mixed infection  PCR

摘要:

近期红河州某规模化养猪场在引种后,发生以发热、腹泻、呼吸困难、濒死前鼻孔出血为特征的仔猪传染病,尝试用多种药物治疗均不能有效控制病情,死亡率很高,其病因值得详细调查。本试验对采集自病死猪只的肺脏、脾脏、肾脏和淋巴结,取病料组织划线于血琼脂平板培养,对形成的溶血性菌落采用高盐培养、革兰染色、生化试验做初步鉴定,同时使用细菌16S rRNA基因通用引物扩增该菌株DNA片段,琼脂糖凝胶电泳回收扩增产物,经TA克隆后测序分析；取病料组织研磨上清,应用聚合酶链式反应(PCR)技术扩增病原核酸,以类似方法分析核酸序列。结果发现,该溶血性菌株为革兰染色阳性球菌,葡萄糖、蔗糖、木糖、山梨醇和6.5% NaCl利用试验呈阳性,麦芽糖、棉籽糖、蕈糖、七叶苷、水杨苷、VP、 PYR、硝酸盐和触酶试验均呈阴性,PCR扩增出1542 bp的特异性条带,其基因序列与脲气球菌同源性最高(94%)；应用PCR技术从病料中分别直接扩增出猪繁殖与呼吸综合征病毒(PRRSV)421 bp和支原体267 bp的特异性条带,前者为高致病性PRRSV毒株特有核酸片段,后者与猪鼻支原体SK76菌株的DNA同源性达99%。本试验结果表明,该猪群发生以高致病性PRRSV感染为主、伴有猪鼻支原体和类脲气球菌混合感染的急性传染病,提示必须高度重视引种携带PRRSV的风险。

Abstract:

There was recently an outbreak of lethal respiratory disease in a large-scale pig farm in Honghe Prefecture shortly after breeding pigs were introduced, which was characteristic of fever, diarrhea, dyspnea, the nose bleeding before dying, leading to a high mortality rate despite of treatment with various drugs, making it worth investigating its etiology in detail. In this experiment, we collected samples including lung, kidneys, spleen and lymph nodes from diseased pigs. The samples were used to inoculate and culture pathogenic bacteria by plate streaking onto the blood agar to form possible hemolytic colonies, followed by high-salt medium culture, Gram staining, biochemical tests for preliminary identification of this isolate. The universal primers for bacterial 16S rRNA genes were then used to amplify its corresponding DNA fragment using polymerase chain reaction (PCR) technique. The resulting PCR product was recovered by agar gel electrophoresis, and then subjected to TA cloning as well as sequence analysis. Supernatant from milled tissue samples was used to amplify nucleic acid fragments of other potential pathogens by PCR, followed by similar procedure. As a result, the isolated strain displayed positive in Gram staining, utilization of glucose, sucrose, xylose, sorbitol and 6.5% NaCl, and, but negative in maltose, raffinose, mushroom sugar, esculin, salicin, VP, pyrrolidone, nitrate and catalase test, and gave a specific band with 1542 bp, which had a highest homology (94%) with Aerococcus urinae; Single porcine reproductive and respiratory syndrome virus (PRRSV) specific 421-bp band and mycoplasma specific 267-bp band were obtained simply by PCR amplification from the tissue samples, respectively, with the former being specific in highly pathogenic PRRSV strains and the latter having a DNA sequence homology up to 99% with Mycoplasma hyorhinis SK76 strain. The above results demonstrated that this outbreak of an acute contagious disease was caused mainly by highly pathogenic PRRSV, with mixed infection caused by M. hyorhinis and A. urinae-like bacterium, indicating the importance of intensive emphasis on potential risk of PRRSV carrying in introduced animals.