内蒙古包头市1例绵羊肺腺瘤病的诊断与病毒env基因分析

Diagnosis and JSRV env Gene Analysis of a Case of Ovine Pulmonary Adenomatosis in Baotou,Inner Mongolia

绵羊肺腺瘤(Ovine pulmonary adenomatosis,OPA)是由绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)引起的慢性、进行性、接触性绵羊肺肿瘤疾病。本研究通过尸体剖检、病理组织学、PCR、免疫组织化学和蛋白免疫印迹等方法确诊了1例绵羊肺腺瘤病,用病毒env基因测序结果进行基因结构和遗传进化分析。病理诊断结果显示,尸体剖检见肺肿大且质地坚实,肺表面有大量白色结节,肺叶上有数个有光泽的实变区,肺切面可见大面积实变区;光镜下可见肺泡Ⅱ型上皮细胞增生明显,呈立方状或柱状生长形成典型的“菜花样”腺瘤灶,肺泡腔内可见巨噬细胞。在病原(JSRV)的env和U3区分别设计两对引物的PCR检测结果显示,所扩增的目的条带大小与预期扩增片段大小一致,同时序列分析结果显示JSRV env与本实验室上传至GenBank中的参考序列JQ837489同源性为97.7%,JSRV env与JQ837489和新疆株KP691837聚为一支,亲缘关系较近;采用本实验室制备的JSRV的囊膜蛋白(Env)的多克隆抗体为一抗进行免疫组织化学检测,结果显示在增生的肺泡Ⅱ型上皮细胞中出现棕黄色深染的阳性信号;蛋白免疫印迹实验检测肺组织中JSRV Env相对表达量的结果显示,在80 kDa处表达特异性条带。研究结果表明,内蒙古包头地区已存在JSRV感染病例,这为今后OPA的诊断提供参考依据。

Ovine pulmonary adenomatosis(OPA)is a chronic,progressive and contact sheep pulmonary tumor caused by Jaagsiekte sheep retrovirus(JSRV).In this study,a case of ovine pulmonary adenomatosis was diagnosed through necropsy,histopathology,PCR,immunohistochemistry,and Western blotting.The gene structure and genetic evolution were analyzed by using the JSRV env gene sequencing results.A large number of gray nodules were observed on the lung surface,several lustrous consolidation areas were observed on the lung lobe,and a large area of consolidation was observed on the section of the lung.Under the light microscope,type II pneumocytes showed obvious hyperplasia,which showed cuboidal or columnar growth and formed typical adenoma foci.Macrophages were observed in the alveolar cavity.Two pairs of primers were designed in JSRV env and U3 region,respectively.The PCR results showed that the size of the target bands was consistent with that of the expected amplified fragment.Meanwhile,the sequence analysis showed that the JSRV env had 97.7%homology with the reference sequence JQ837489 uploaded to GenBank in this laboratory.The JSRV env was grouped with JQ837489 and Xinjiang strain KP691837,which was closely related to each other.The polyclonal antibody of JSRV envelope protein(Env)prepared in this laboratory was used as the primary antibody for immunohistochemical detection.The results showed that positive signals of brownish yellow staining appeared in the hyperplasia of type II pneumocytes.The relative expression of JSRV Env in lung tissue was detected by the western blot,and specific bands were found at 80KDa.In summary,this case was diagnosed as JSRV infection.This study fully indicated that a case of JSRV infection has already existed in Baotou,Inner Mongolia,and timely prevention and control measures should be taken to control the spread of the disease.This paper provided a reference for diagnosing the JSRV infection in the future.