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目的应用形态学和PCR方法对浙江省发现的两例疑似感染锥虫的鼠血样进行鉴定。方法采集两例疑似感染锥虫的鼠血样,制作血涂片进行显微镜检查,测量锥虫的相关形态学指标。提取两例鼠血样DNA,应用PCR法扩增锥虫属18S r RNA基因特异性片段。扩增产物测序后,应用NCBI中Blast分析程序对测序序列进行比对分析,并将测序序列提交至Gen Bank。结果两份疑似感染锥虫的鼠血样分别来源于野外黄毛鼠和室内黄胸鼠。制作血涂片进行显微镜检查,测量锥虫的相关形态学指标。镜下观察发现,血涂片中的锥虫均具备核、鞭毛和动基体等锥虫特征性结构。分离自黄毛鼠和黄胸鼠的锥虫总体长分别为27.50μm和23.80μm,游离鞭毛的长度分别为9.60μm和9.20μm,核指数分别为0.74和1.05,动基体指数分别为1.40和1.57,属于啮齿动物内寄生的匍锥虫亚属(Herpetosoma)锥虫。应用PCR法扩增锥虫属18S r RNA基因的特异性片段,片段大小约为700 bp,对扩增产物测序后将序列提交至Gen Bank,登录号分别为KP098535和KP098536。分离自黄毛鼠锥虫的18S r RNA基因序列(KP098535)与Trypanosoma rabinowitschae(AY491765.1)、T.blanchardi(AY491764.1)和T.musculi(AJ223568.1)等序列的18S r RNA基因序列的同源性均为100%,可以确定其为匍锥虫亚属,但尚不能确定具体种。分离自黄胸鼠锥虫的序列(KP098536)与Gen Bank中路易斯锥虫(T.lewisi)的18S r RNA基因序列(AB242273.1和AJ009156.1)的同源性为100%,可判定其为路易斯锥虫。结论分离自黄胸鼠的锥虫为路易斯锥虫,分离自黄毛鼠的锥虫隶属于匍锥虫亚属,可判为类路易斯锥虫。
Objective To identify the murine blood samples of two suspected trypanosomates in Zhejiang Province by morphological and PCR methods. Methods Two murine blood samples suspected of being infected with trypanosomes were collected, and blood smears were collected for microscopic examination and related morphological indicators of trypanosomes were measured. Two murine samples of blood were extracted and PCR amplified specific 18S rRNA gene fragment of Trypanosoma. After the amplified product was sequenced, the Blast analysis program in NCBI was used to analyze the sequencing sequence and the sequencing sequence was submitted to Gen Bank. Results Two mouse blood samples suspected of being infected with trypanosomes were derived from wild rodents and domestic rodents. Blood smears were prepared for microscopic examination and trypanosome-associated morphological parameters were measured. Microscope observation found that trypanosomes in blood smears have nuclei, flagella and dynamic substrate such as trypanosomes characteristic structure. The total length of Trypanosomes isolated from Rattus norvegicus and Rattus flavipectus were 27.50μm and 23.80μm, respectively. The length of free flagella were 9.60μm and 9.20μm, the nuclear indices were 0.74 and 1.05, respectively, and the dynamic basal indices were 1.40 and 1.57 Belongs to the species of T. infestans (Herpetosoma) parasitized in rodents. The specific fragment of 18S rRNA gene of Trypanosoma was amplified by PCR. The fragment was about 700 bp in length. The amplified product was sequenced and submitted to Gen Bank with accession numbers KP098535 and KP098536 respectively. The 18S rRNA gene sequence (KP098535) isolated from T. trypanosoma and the 18S rRNA gene sequence of Trypanosoma rabinowitschae (AY491765.1), T. blanchardi (AY491764.1) and T. musculi (AJ223568.1) The homology was 100%, it can be determined that it is a subgenus of Trypanosoma, but no specific species can be identified yet. The sequence (KP098536) isolated from T. rhamnosus was 100% identical to the 18S rRNA gene sequence of T. lewisi (AB242273.1 and AJ009156.1) in GenBank, For Trypanosoma brucei Conclusion Trypanosomes isolated from Rattus flavipectus are Trypanosoma brucei, and Trypanosomes isolated from Rattus norvegicus are subgenus of Trypanosoma, which may be classified as Trypanosoma brucei.