大鳄龟感染蛙病毒的PCR检测及组织病理分析

PCR Detection and Pathohistological Analysis of Ranavirus Infection in Alligator Snapping Turtle

2013年3月成都市某海洋馆送检2只发病大鳄龟（Macrochelys temminckii）,临床特征表现为：精神状态萎靡,爬行无力,对外界刺激反应迟钝;颈部和四肢局部红肿,腹甲溃烂,严重部位甚至穿孔,最后死亡。为明确患病大鳄龟的病因,进行了细菌学、组织病理学和PCR检查。细菌学检查阴性;病理组织学观察发现,大鳄龟多组织、器官均发生严重病变,尤其是肾、肝、肺和心的损伤最为严重,表现为明显的变性、坏死和炎症细胞浸润,并在一些病变组织细胞浆内见嗜酸性或嗜碱性包涵体。针对蛙病毒（Ranavirus）病毒的特异性PCR检测扩增出蛙病毒主要衣壳蛋白（MCP）基因500 bp目的片段,测序后的DNA序列与Gen Bank中已知核酸序列进行Blast比对,发现其与Gen Bank中的蛙病毒主要衣壳蛋白基因同源性达95%~99%。根据组织病理特点及PCR检测结果推测大鳄龟的死亡是感染蛙病毒所致。

In March 2013, two diseased alligator snapping turtles （Macrochelys temmincMi） were collected from an aquarium in Chengdu （Sichuan Province, China） and they were transferred to the Key Laboratory of Animal Disease and Human Health of Sichuan Province. Symptoms included energetic dispirited, crawled in weakness, slowed response to external stimulation; local redness and swelling in the neck and limbs, as well as plastron ulcerated and perforated （Fig. 1）. Bacteriologic test, pathological observation and PCR detectionwere performed to identify the etiological factors of these two alligator snapping turtles. Bacteriologic test was negative; histopathologically, the lesions appeared in multi-organs and tissues, especially in the kidney, liver, lung and heart. The main pathologic lesions were degeneration, necrosis and infiltration of the inflammation cells, with acidophilic or basophilic cytoplasm of inclusions. Intracytoplasmic inclusions were found in various cell types （Fig. 2）. In addition, The PCR results of two samples were positive for Ranavirus. A 500 bp fragment （Fig. 3） of the major capsid protein gene was amplified and sequenced. GenBank BLAST searches on the sequences revealed an identitiy of 95% to 99% with that of Ranavirus. From the PCR results and pathological observation, we speculated that Ranavirus infection had caused the death of the alligator snapping tm＇tles.