Browsing by Author "Suganuma Keisuke"

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    The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis
    (Elsevier, 2017) Davaasuren Batdorj; Amgalanbaatar Tovuu; Musinguzi Simon Peter; Suganuma Keisuke; Otgonsuren Davaajav; Mossaad Ehab; Narantsatsral Sandagdorj; Battur Banzragch; Battsetseg Badgar; Xuan Xuenan; Inoue Noboru
    Trypanosoma equiperdum, which is the etiological agent of dourine, spreads through sexual intercourse in equines. Dourine (T. equiperdum) has been reported in Mongolia, where it is considered an economically important disease of horses. T. evansi has also been reported in Mongolian domestic animals. The objective of this study was to evaluate the potential application of recombinant T. evansi GM6 (rTeGM6-4r)-based diagnostic methods on a farm with an outbreak of non-tsetse transmitted horse trypanosomosis. Ninety-seven percent homology was found between the amino acid sequences of T. equiperdum GM6 and the GM6 of another Trypanozoon, which also shared the same cellular localization. This finding suggests the utility of rTeGM6-4r-based serodiagnostic methods for epidemiological studies and the diagnosis of both surra and dourine in Equidae. Fifty blood samples were examined from a herd of horses. The diagnostic value of an rTeGM6-4r-based ELISA and an rTeGM6-4r-based immunochromatographic test (ICT) were measured in comparison to a T. evansi crude antigen-based ELISA, which is a diagnostic method recommended by the OIE. However, this is not a perfect diagnostic method for trypanosomosis. Positive serum samples were detected in 46%, 42% and 28% of the tested horses using an rTeGM6-4r-based ELISA, crude antigen-based ELISA and rTeGM6-4r-based ICT, respectively. The sensitivity of rTeGM6-based ELISA was 81%, the specificity was 79%, and the agreement was moderate. We conclude that rTeGM6-4r-based ELISA and ICT represent alternative options for baseline epidemiological studies and the on-site diagnosis of horse trypanosomoses in the field, respectively.
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    Identification And Characterization of Guanosine 5′-Monophosphate Reductase of Trypanosoma Congolense as a Drug Target
    (2017) Musinguzi Peter; Sarwono Albertus Eka Yudistira; Suganuma Keisuke; Mitsuhashi Shinya; Okada Tadashi; Shigetomi Kengo; Inoue Noboru; Ubukata Makoto
    Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000_5_1940, as a guanosine 5′-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.
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    Identification and characterization of guanosine 5′-monophosphate reductase of Trypanosoma Congolese as a drug target.
    (Parasitology international, 2017) Musinguzi Simon Peter; Eka Albertus; Sarwono Yudistira; Suganuma Keisuke; Mitsuhashi Shinya; Okada Tadashi; Shigetomi Kengo; Inoue Noboru; Ubukata Makoto
    Trypanosoma Congolese is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. Congolese is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. Congolese, TcIL3000_5_1940, as a guanosine 5′-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.
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    The incrimination of three trypanosome species in clinically affected German shepherd dogs in Sudan
    (Springer, 2014) Mossaad Ehab; Satti A. Rawan; Fadul Abdeen; Suganuma Keisuke; Salim Bashir; Elamin E. A.; Musinguzi Simon Peter; Xuan Xuenan; Inoue Noboru
    Canine trypanosomosisis (CT) is a common disease caused by tsetse- and non-tsetse-transmitted trypanosomes worldwide. The severity of the disease varies from acute, sub-acute to chronic with non-specific clinical signs. Here, we attempt in a cross-sectional study to assess the current situation of CT and the role of dogs in transmitting trypanosomes to other domesticated animals. The study was carried out during July 2016 on 50 caged German shepherd dogs in Khartoum State to investigate the prevalence of dog trypanosomosis using both serological (CATT/Trypanosoma evansi) and molecular (KIN-PCR, RoTat1.2 VSG-PCR and TviCatL-PCR) tests to detect possible trypanosome infections. CATT/T. evansi detected antibodies against T. evansi in 15 (30%) dogs, while parasite DNA was detected in 17 (34%) dogs by RoTat1.2 PCR. In contrast, a KIN-PCR detected the subgenus Trypanozoon, Trypanosoma congolense savannah, T. congolense Kenya and T. vivax in 36 (72%), 3 (6%), 1 (2%), and 2 (4%) dogs, respectively. However, a species-specific PCR for Trypanosoma vivax was detected 7 (14%) positive cases. We concluded that CT was caused by at least three species of trypanosomes, namely T. evansi, T. vivax and T. congolense. Trypanozoon other than T. evansi could not be ruled out since other tsetse-transmitted trypanosomes have also been detected and species-specific PCRs were not used. This study illustrates that dogs play an important role in the transmission dynamic and the epidemiology of the abovementioned trypanosome species.
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    Isolation, cultivation and molecular characterization of a new Trypanosoma equiperdum strain in Mongolia
    (2016) Musinguzi Simon Peter; Suganuma Keisuke; Sandagdorj Narantsatsral; Banzragch Battur; Shino Yamasaki; Davaajav Otgonsuren; Batdorj Davaasuren; Badgar Battsetseg; Inoue Noboru
    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 % CO2. 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 cultureadapted 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. Keywords: Dourine, In vitro culture, Maxicircle DNA, Mongolia, Soft agarose media, Trypanosoma equiperdum Abbreviations: 18S rRNA, 18S ribosomal RNA; ALP, Alkaline phosphatase; ALT, Alanine transaminase; AST, Aspartate aminotransferase; BUN, Blood urea nitrogen; CFT, Complement fixation test; CSF, Cerebrospinal fluid; ELISA, Enzymelinked immunosorbent assay; HCT, Hematocrit; HGB, Hemoglobin; HMI-9, Hirumi’s modified Isocove’s medium-9; ICT, Immunochromatographic test; ITS, Internal transcribed spacer; kDNA, Kinetoplast DNA; MCH, Mean corpuscular hematocrit; MCHC, Mean corpuscular hemoglobin concentration; MCV, Mean corpuscular volume; ND4-ND5, NADHdehydrogenase subunits 4 and 5; PLT, Platelet; RBC, Red blood cell; WBC, White blood cell