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Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia
BACKGROUND: Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007690/ https://www.ncbi.nlm.nih.gov/pubmed/27580944 http://dx.doi.org/10.1186/s13071-016-1755-3 |
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author | Suganuma, Keisuke Narantsatsral, Sandagdorj Battur, Banzragch Yamasaki, Shino Otgonsuren, Davaajav Musinguzi, Simon Peter Davaasuren, Batdorj Battsetseg, Badgar Inoue, Noboru |
author_facet | Suganuma, Keisuke Narantsatsral, Sandagdorj Battur, Banzragch Yamasaki, Shino Otgonsuren, Davaajav Musinguzi, Simon Peter Davaasuren, Batdorj Battsetseg, Badgar Inoue, Noboru |
author_sort | Suganuma, Keisuke |
collection | PubMed |
description | BACKGROUND: Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection. METHODS: Trypanosomes isolated from the urethral tract of a stallion with suspected dourine, were directly cultivated using soft agarose media at 37 °C in 5 % CO(2). For molecular characterization, 18S ribosomal RNA (rRNA) gene, the internal transcribed spacer (ITS) and 8 maxicircle DNA regions were amplified by a PCR and their sequences were determined. To analyze the ratio of the kinetoplastic/akinetoplastic population, the kinetoplasts and the nuclei of trypanosomes were subjected to Hoechst staining and observed by fluorescence microscopy. RESULTS: In addition to the clinical symptoms and the molecular diagnosis, this stallion was definitively diagnosed with dourine by the detection of trypanosomes in the urethral mucosa. These results strongly suggested that the isolated trypanosome was true T. equiperdum. T. equiperdum isolated from the urethral tract was adapted in vitro using soft agarose media. Based on the results of a phylogenetic analysis of 18S rRNA and ITS, this T. equiperdum isolate was classified into the Trypanozoon clade. In a PCR of the maxicircle DNA region, only NADH-dehydrogenase subunits 4 and 5 was amplified. Clear kinetoplasts were observed in most of the T. equiperdum isolates. In contrast, most culture-adapted T. equiperdum were of the akinetoplastic form. CONCLUSION: We concluded that our isolated trypanosome was the first confirmed case of T. equiperdum in Mongolia and named it “T. equiperdum IVM-t1”. T. equiperdum IVM-t1 was well adapted and propagated in soft agarose media, which indicates that this culture method is useful for isolation of T. equiperdum from horses with dourine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1755-3) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5007690 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-50076902016-09-02 Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia Suganuma, Keisuke Narantsatsral, Sandagdorj Battur, Banzragch Yamasaki, Shino Otgonsuren, Davaajav Musinguzi, Simon Peter Davaasuren, Batdorj Battsetseg, Badgar Inoue, Noboru Parasit Vectors Research BACKGROUND: Trypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection. METHODS: Trypanosomes isolated from the urethral tract of a stallion with suspected dourine, were directly cultivated using soft agarose media at 37 °C in 5 % CO(2). For molecular characterization, 18S ribosomal RNA (rRNA) gene, the internal transcribed spacer (ITS) and 8 maxicircle DNA regions were amplified by a PCR and their sequences were determined. To analyze the ratio of the kinetoplastic/akinetoplastic population, the kinetoplasts and the nuclei of trypanosomes were subjected to Hoechst staining and observed by fluorescence microscopy. RESULTS: In addition to the clinical symptoms and the molecular diagnosis, this stallion was definitively diagnosed with dourine by the detection of trypanosomes in the urethral mucosa. These results strongly suggested that the isolated trypanosome was true T. equiperdum. T. equiperdum isolated from the urethral tract was adapted in vitro using soft agarose media. Based on the results of a phylogenetic analysis of 18S rRNA and ITS, this T. equiperdum isolate was classified into the Trypanozoon clade. In a PCR of the maxicircle DNA region, only NADH-dehydrogenase subunits 4 and 5 was amplified. Clear kinetoplasts were observed in most of the T. equiperdum isolates. In contrast, most culture-adapted T. equiperdum were of the akinetoplastic form. CONCLUSION: We concluded that our isolated trypanosome was the first confirmed case of T. equiperdum in Mongolia and named it “T. equiperdum IVM-t1”. T. equiperdum IVM-t1 was well adapted and propagated in soft agarose media, which indicates that this culture method is useful for isolation of T. equiperdum from horses with dourine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1755-3) contains supplementary material, which is available to authorized users. BioMed Central 2016-08-31 /pmc/articles/PMC5007690/ /pubmed/27580944 http://dx.doi.org/10.1186/s13071-016-1755-3 Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Suganuma, Keisuke Narantsatsral, Sandagdorj Battur, Banzragch Yamasaki, Shino Otgonsuren, Davaajav Musinguzi, Simon Peter Davaasuren, Batdorj Battsetseg, Badgar Inoue, Noboru Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia |
title | Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia |
title_full | Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia |
title_fullStr | Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia |
title_full_unstemmed | Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia |
title_short | Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia |
title_sort | isolation, cultivation and molecular characterization of a new trypanosoma equiperdum strain in mongolia |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007690/ https://www.ncbi.nlm.nih.gov/pubmed/27580944 http://dx.doi.org/10.1186/s13071-016-1755-3 |
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