中国寄生虫学与寄生虫病杂志

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应用形态学与PCR技术鉴定黄胸鼠和黄毛鼠体内锥虫

张玲玲1,倪庆翔2,姚立农1,张孝和2,陈华良1,丰燕1,张轩1,余可根1,阮卫1 *   

  1. 1 浙江省疾病预防控制中心,杭州 310051;2 温州市疾病预防控制中心,温州 325001
  • 出版日期:2015-08-30 发布日期:2015-09-10

Identification of Trypanosoma Species in Rattus flavipectus and R. losea using Microscopy and PCR Method

ZHANG Ling-ling1,NI Qing-xiang2,YAO Li-nong1,ZHANG Xiao-he2,CHEN Hua-liang1,FENG Yan1,ZHANG Xuan1,YU Ke-gen1,RUAN Wei1 *   

  1. 1 Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou 310051,China;2 Wenzhou Center for Disease Control and Prevention,Wenzhou 325001,China
  • Online:2015-08-30 Published:2015-09-10

摘要:

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 rRNA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GenBank。 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27.50 μm和23.80 μm,游离鞭毛的长度分别为9.60 μm和9.20 μm,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 rRNA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GenBank,登录号分别为KP098535和KP098536。分离自黄毛鼠锥虫的18S rRNA基因序列(KP098535)与Trypanosoma rabinowitschae(AY491765.1)、T. blanchardi(AY491764.1)和T. musculi(AJ223568.1)等序列的18S rRNA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GenBank中路易斯锥虫(T. lewisi)的18S rRNA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。

关键词: 锥虫, 路易斯锥虫, 黄胸鼠, 黄毛鼠, PCR

Abstract:

Objective  To identify two suspected Trypanosoma species in Rattus found in Zhejiang Province using microscopy and PCR method.  Methods  Blood samples were collected from Rattus losea and R. flavipectus. Blood smears were prepared, and observed under microscope. The morphological indices of trypanosomes were measured and calculated. The genomic DNA was extracted from the trypanosomes, and the specific fragment of Trypanosoma 18S rRNA gene was amplified using PCR. The products were further sequenced and submitted to GenBank. Blast analysis was performed on line in NCBI.  Results  Blood samples from Rattus flavipectus and R. losea were collected from Lucheng District and Wencheng County of Wenzhou, respectively. The parasites from R. losea and R. flavipectus were found to possess the characteristic features of Trypanosoma species, such as nucleus, free flagellum, and kinetoplast, etc. The body length was 27.50 μm and 23.80 μm, and the free flagellum length was 9.60 μm and 9.20 μm, respectively. The nucleus index was 0.74 and 1.05, the kinetoplast index was 1.40 and 1.57, respectively. Based on the morphological characteristics and host specificity, the parasites from R. losea and R. flavipectus were identified as Herpetosoma species, mainly found in rodents. The amplified products were about 700 bp by 18S rRNA gene PCR with the DNA isolated from the trypanosomes. The products were further sequenced, and the resulting sequences were submitted to GenBank with assession numbers of KP098535(from R. losea) and KP098536(from R. flavipectus). Blast analysis showed that KP098535 was completely homologous with the sequences from Herpetosoma subgenus (AY491765.1, AY491764.1, and AJ223568.1), and KP098536 was completely homologous with Trypanosoma lewisi (AB242273.1, AJ009156.1).  Conclusion  The Trypanosoma species found from Rattus flavipectus is Trypanosoma lewisi, and the other one belongs to Herpetosoma subgenus, which may be named as Trypanosoma lewisi-like trypanosome.

Key words: Trypanosome, Trypanosoma lewisi, Rattus flavipectus, Rattus losea, PCR