犬隐球菌病病原分离与ITS区序列分析鉴定

Isolation of Pathogenic Agent of Canine Cryptococcosis and Identification by ITS Region Sequence Analysis

为确定引起格力犬皮肤病发生的病原菌种属并提供科学的诊治方案,从病犬发病部位真皮层刮取病料,采用沙氏培养基对病料进行分离、培养,根据菌落形态特征与镜检结果对分离到的病原菌进行初步鉴定。采用真菌鉴定通用引物ITS1及ITS4对分离菌的ITS区序列进行PCR扩增、测序,将测序结果与GenBank中的新生隐球菌进行比对分析并构建系统发育树。结果表明,分离到的病原菌经形态学鉴定初步判定为隐球菌属真菌,其ITS区序列与GenBank中的新生隐球菌(JN939462.1和JN939461.1)的ITS序列相似性为99%,且在系统发育树上属同一分支,提示引起该犬隐球菌病的病原菌为隐球菌属新生隐球菌。

In order to identify the pathogenic agent causing skin disease of a greyhound dog and provide scientific diagnosis and treatment protocols, the samples were collected from the dermis of the pathological site of the diseased dog. The samples were cultured with Sabouraud＇s medium to isolate the pathogenic agent. Preliminary identification of suspected isolates was made according to the morphological characteristics of the colony and microscopic examination result. The fungi identification universal primers of ITSland ITS4 were used for amplification and sequencing of the ITS region sequence of the isolates. The phylogenetie tree based on the ITS region sequences of the isolates and published Cryptococcus neoformans strains in GenBank was established. The isolates were phenotypically identified as Cryptococcus strains, and the similarity of the ITS region sequence of the isolates and the published Cryptococcus neoformans strains （JN939462.1and JN939461.1 ） was 99%.The strains isolated in this study and published in GenBank belonged to the same branch on the phylogenetic tree. It was indicated that the pathogenic agent causing skin disease of the greyhound dog was Cryptococcus neoformans.