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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

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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