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application/pdf, Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T.?equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T.?equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T.?equiperdum. An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum. The luciferase assay would be suitable for the mass screening of chemical libraries against T.?equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains. c 2017 The Authors

Trypanosoma equiperdum primarily parasitizes the genital organs and causes dourine in equidae. We isolated a new T. equiperdum strain, T. equiperdum IVM-t1, from the urogenital tract of a horse definitively diagnosed as having dourine in Mongolia., Trypanosoma equiperdum primarily parasitizes the genital organs and causes dourine in equidae. We isolated a new T. equiperdum strain, T. equiperdum IVM-t1, from the urogenital tract of a horse definitively diagnosed as having dourine in Mongolia. Here, we report the whole-genome sequence, the predicted gene models, and their annotations.

application/pdf@@@application/vnd.ms-powerpoint, Babesia microti is a tick-transmitted zoonotic hemoprotozoan parasite. In the present study, we investigated B. microti infection in questing ticks in Mongolia. A total of 219 questing ticks were collected from three different Mongolian provinces (Bayan-Olgii, Khovsgol, and Selenge). Of these, 63 from Selenge were identified as Ixodes persulcatus, while the remaining 156 (from all three provinces) were identified as Dermacentor nuttalli. When the tick DNA samples were screened using a B. microti-specific nested PCR, 19 (30.2%) of the 63 I. persulcatus ticks were found to be B. microti-positive. The parasite was not detected in D. nuttalli. Subsequently, the 18S rRNA, cox1, and tufA sequences of B. microti were amplified, sequenced, and subjected to phylogenetic analyses. Sequencing analyses showed that the Mongolian 18S rRNA, cox1, and tufA sequences were 99.6-100%, 96.7-97.2%, and 94.7-95.3% homologous, respectively, with B. microti R1 strain US-type sequences from humans. In the phylogenetic analyses, the Mongolian cox1 and tufA sequences were found to be separate lineages, which formed sister-clades to the R1 strain sequences, while all of the Mongolian B. microti 18S rRNA sequences were clustered within US-type clade containing several other sequences of human origin. In conclusion, in addition to reporting the presence of B. microti for the first time in questing ticks in Mongolia, the present study found that Mongolian I. persulcatus ticks were infected with US-type B. microti. These findings warrant large-scale studies to detect and characterize B. microti in ticks, small mammals, and humans. Such studies should provide us with a better understanding of zoonotic Babesia epidemiology in Mongolia. c 2015 Elsevier Ireland Ltd.

Anaplasma marginale is an etiologic agent of bovine anaplasmosis. This study aimed to molecularly detect and characterize A. marginale that is prevalent in Mongolian cattle populations. A highly specific and sensitive nested PCR (nPCR) method based on the Msp5 gene was developed to detect A. marginale (Msp5 nPCR). The method detected A. marginale from the positive DNA samples obtained from different countries, while no amplicons were observed from DNA samples of several other bovine blood pathogens tested. The detection limit of Msp5 nPCR was determined to be 2 copies/μl. The method was tested against field blood DNA samples prepared from 300 Mongolian cattle in 2010. Results indicated a prevalence rate of 8.7% (26 of 300). On the other hand, partial DNA fragments of an Anaplasma sp. closely related to A. ovis (with 95.0% identity) were detected using a different nPCR method based on groEL gene. The phylogenetic analyses based on the Msp5, groEL and 16S rRNA genes demonstrated that A. marginale isolates in Mongolia were not divergent from the isolates distributed in other countries. The present study successfully established a new nPCR assay that can detect A. marginale, and reported the first molecular detection and characterization of A. marginale and an Anaplasma sp. closely related to A. ovis in Mongolian cattle populations.

application/pdf, 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 degrees 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 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.

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. In this paper, we show that human alpha-defensin-5 displays a parasiticidal role against Toxoplasma gondii, the causative agent of toxoplasmosis. Exposure of the tachyzoite form of T. gondii to defensin induced aggregation and significantly reduced parasite viability in a concentration-dependent peptide. Pre-incubation of tachyzoites with human alpha-defensin-5 followed by exposure to a mouse embryonal cell line (NIH/3T3) significantly reduced T. gondii infection in these cells. Thus, human alpha-defensin-5 is an innate immune molecule that causes severe toxocity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that human alpha-defensin-5 causes aggregation, leading to Toxoplasma destruction.

The purpose of this study was to demonstrate the occurrence of equine piroplasmosis in Mongolia, a country in which the disease occurs epidemically in different climatic conditions. Antibodies to Babesia equi and B. caballi were determined in serum samples of 254 pastured horses in different locations of Mongolia using an enzyme-linked immunosorbent assay with recombinant antigens. One hundred and eighty-five (72.8%) and 102 (40.1%) of all serum samples were positive for B. equi and B. caballi infections, respectively. In addition, 78 (30.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread in Mongolia. To our knowledge, this is the first report describing an epidemiological study on equine piroplasmosis in different geographic regions in Mongolia.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4–26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine.

SUMMARYThe dense granule antigen 4 (GRA4) is known as an immundominant antigen ofToxoplasma gondiiand, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-γ) production and survived completely against a subsequent challenge infection with a lethal dose ofT. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronicT. gondiiinfections in mice.

Cats are pivotal in the transmission of Toxoplasma gondii. To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T. gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA). The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T. gondii and sera from normal cats. Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit. Of the 192 samples screened, 42 (21.9%) were positive by ELISA. Among the 42 ELISA-positive samples, 39 were positive by LAT. There was a significant correlation between ELISA and LAT titers. All the 150 ELISA-negative samples were negative by LAT. These results indicate that the ELISA with rSAG2 expressed in E. coli should be a useful method for detection of T. gondii infection in cats.