Combination of Clofazimine and Atovaquone as a Potent Therapeutic Regimen for the Radical Cure of Babesia microti Infection in Immunocompromised Hosts.

Human babesiosis caused by Babesia microti can be fatal in immunocompromised patients, and the currently used drugs are often ineffective. A recent study found that clofazimine clears B. microti Munich strain in immunocompromised mice. In the present study, we investigated the efficacies of clofazimine and 2-drug combinations involving clofazimine, atovaquone, and azithromycin against B. microti Peabody mjr strain in immunocompromised mice. Treatment with clofazimine alone, clofazimine plus azithromycin, and atovaquone plus azithromycin was ineffective and failed to eliminate the parasites completely, while a 44-day treatment with clofazimine plus atovaquone was highly effective and resulted in a radical cure.

Epidemiology and genetic diversity of Theileria equi and Babesia caballi in Mongolian horses.

Equine piroplasmosis is a tick-borne disease caused by Theileria equi and Babesia caballi in horses. Because of its impact on horse industry, control of this disease is crucial for endemic countries. The control of equine piroplasmosis may be influenced by the genotypic diversity of T. equi and B. caballi. Mongolia, a country with a thriving livestock industry, is endemic for T. equi and B. caballi. However, nationwide epidemiological surveys have not been conducted to determine the current status of infections and genetic diversity of these two parasite species. Therefore, the objective of this research was to investigate the infection rates and genotypes of T. equi and B. caballi in horses across Mongolia. Blood samples were collected from 1353 horses in 15 of Mongolia's 21 provinces, and their DNAs were analyzed with T. equi- and B. caballi-specific PCR assays. Additionally, blood smears were prepared from 251 horses, stained with Giemsa, and examined under a light microscope to identify T. equi and B. caballi. The microscopy revealed that 30 (11.9%) and 4 (1.6%) of the 251 horses were positive for T. equi and B. caballi, respectively. By contrast, PCR assays detected the T. equi and B. caballi in 1058 (78.2%) and 62 (4.6%) horses, respectively. Phylogenetic analysis of 18S rRNA sequences from 42 randomly selected T. equi-positive DNA samples detected the genotypes A and E. On the other hand, the rap-1 sequences from 19 randomly selected B. caballi-positive DNA samples occurred in clades representing the genotypes A and B1, as well as in a distinct clade closely related to the genotype A. Our findings confirm the widespread occurrence of T. equi and B. caballi infections in Mongolian horses, highlighting the need for a comprehensive control approach.

Molecular prevalence and genotypic diversity of Theileria equi and Babesia caballi infecting horses in Kyrgyzstan.

Equine piroplasmosis is caused by Theileria equi and Babesia caballi, which are hemoprotozoan parasites. Understanding the epidemiology and genotypes of T. equi and B. caballi is crucial for developing effective control strategies in endemic countries. However, the endemic status of these two parasite species remains uncertain in Kyrgyzstan due to lack of surveys. Our study, therefore, aimed to detect T. equi and B. caballi infections in Kyrgyzstan and identify their genotypes. Blood samples were collected from 226 horses across all seven provinces of Kyrgyzstan, namely Chuy, Issyk-Kul, Naryn, Talas, Jalal-Abad, Osh, and Batken. These blood samples were subjected to DNA extraction, followed by specific PCR assays targeting T. equi and B. caballi. We found that 56 (24.8%, confidence interval (CI): 19.6-30.8%) and 7 (3.1%, CI: 1.5-6.3%) of the tested horses were positive for T. equi and B. caballi infections, respectively. Theileria equi was detected in all surveyed provinces, whereas B. caballi was found in five provinces, except for Talas and Osh. Subsequent genotype-specific PCR assays showed that T. equi-positive horses harbored all five genotypes: A, B, C (also known as Theileria haneyi), D, and E. On the other hand, phylogenetic analysis of B. caballi rap-1 sequences detected the genotypes A and B1. The prevalence of T. equi and B. caballi suggests a potential risk of clinical equine piroplasmosis among horses in Kyrgyzstan, and the observed genotypic diversity underscores the challenges in managing the disease. Our findings emphasize the need for comprehensive control measures to effectively address equine piroplasmosis in Kyrgyzstan.

PCR detection of Theileria equi and Babesia caballi in apparently healthy horses in Paraguay.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses. EP has a global distribution and often leads to a significant socioeconomic impact on the equine industry. Infected animals remain as carriers and become a source of infection for tick vectors, thereby posing an immense challenge in the disease management. Therefore, detection of these carriers is crucial to assess the risk of transmission and to implement appropriate control measures in endemic countries. Paraguay is a tropical country where various tick-borne diseases are common among livestock; however, the status of EP remains unknown in this country. Because the tick vectors capable of transmitting T. equi and B. caballi are endemic in Paraguay, we hypothesised that Paraguayan horses are infected with these parasite species. To test our hypothesis, we prepared blood DNA samples from a total of 545 apparently healthy horses in 16 of the 17 departments of Paraguay and analysed them with specific PCR assays to detect T. equi and B. caballi. The PCR results showed that 178 (32.7%) and 8 (1.5%) of the horses were infected with T. equi and B. caballi, respectively. Among the infected horses, two (0.4%) were infected with both parasite species. Our analyses further indicated that the positive rates of T. equi infection did not differ between horse breeds, males and females, or age groups. We also found that haematological parameters were the same between the non-infected animals and animals with single infections. By contrast, the two horses co-infected with T. equi and B. caballi had haemoglobin and haematocrit values lower than the normal ranges. In conclusion, the present study demonstrated that Paraguayan horses are infected with T. equi and B. caballi and that the rate of T. equi infection is higher than that of B. caballi. Our findings highlight the need to add EP to the list of differential diagnoses when anaemic horses are presented to equine clinics in Paraguay.

THE FIRST SURVEY OF BOVINE BABESIA SPECIES INFECTING YAKS (BOS GRUNNIENS) IN MONGOLIA.

Yak (Bos grunniens) farming is an important part of Mongolia's livestock industry. Yaks survive in harsh mountain environments; provide meat, milk, and wool; and serve as a mode of transportation. In Mongolia, yaks are frequently raised alongside other livestock animals such as cattle, Bactrian camels, sheep, goats, and horses. Recently, we demonstrated that Babesia bovis, Babesia bigemina, and Babesia naoakii-parasites with the potential to cause clinical bovine babesiosis-infect not only cattle but also Bactrian camels in Mongolia. However, yaks have never been surveyed for Babesia infections in this country. In the present study, we surveyed yaks in 8 Mongolian provinces: Bayankhongor, Bayan-Ulgii, Khovd, Khovsgol, Omnogovi, Ovorkhangai, Uvs, and Zavkhan. Blood samples were taken and deoxyribonucleic acid (DNA) was extracted from 375 yaks. Furthermore, Giemsa-stained thin smears were prepared from 315 of the 375 blood samples and then examined for the microscopic detection of Babesia parasites. Microscopy revealed that 34 (10.8%) of 315 blood smears were positive for Babesia parasites. All 375 DNA samples were then tested for B. bovis, B. bigemina, and B. naoakii infection using specific polymerase chain reaction assays. We observed that 238 (63.5%) yaks in all surveyed provinces and 8 (2.1%) yaks in 3 provinces (Bayankhongor, Bayan-Ulgii, and Omnogovi) were positive for B. bovis and B. bigemina, respectively. However, all yaks tested were negative for B. naoakii. This epidemiological survey, the first to report Babesia infection in Mongolian yaks, suggests that disease management strategies for yaks in this country should further address bovine babesiosis.

An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan.

Cattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs.

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes.

BACKGROUND: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. METHODS: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. RESULTS: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. CONCLUSIONS: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.

Phylogenetic analyses of the mitochondrial, plastid, and nuclear genes of Babesia sp. Mymensingh and its naming as Babesia naoakii n. sp.

BACKGROUND: The recently discovered Babesia sp. Mymensingh, which causes clinical bovine babesiosis, has a wide geographical distribution. We investigated the phylogenetic position of Babesia sp. Mymensingh using its mitochondrial, plastid, and nuclear genes. Based on morphological and molecular data, Babesia sp. Mymensingh is a unique species and we named it as Babesia naoakii n. sp. METHODS: A blood DNA sample from a Babesia sp. Mymensingh-infected cow was subjected to genome sequencing to obtain the sequences of mitochondrial, plastid, and nuclear genes. Six phylogenetic trees were then constructed with (1) concatenated amino acid sequences of cytochrome oxidase subunit I, cytochrome oxidase subunit III, and cytochrome b genes of the mitochondrial genome; (2) 16S rRNA of the plastid genome; (3) nucleotide sequences of the elongation factor Tu gene of the plastid genome; (4) ITS1-5.8S rRNA-ITS2; (5) concatenated nucleotide sequences of 89 nuclear genes; and (6) concatenated amino acid sequences translated from the 89 nuclear genes. RESULTS: In all six phylogenetic trees, B. naoakii n. sp. formed a sister clade to the common ancestor of Babesia bigemina and B. ovata. The concatenated nuclear genes of B. naoakii n. sp. and their translated amino acid sequences shared lower identity scores with the sequences from B. bigemina (82.7% and 84.7%, respectively) and B. ovata (83.5% and 85.5%, respectively) compared with the identity scores shared between the B. bigemina and B. ovata sequences (86.3% and 87.9%, respectively). CONCLUSIONS: Our study showed that B. naoakii n. sp. occupies a unique phylogenetic position distinct from existing Babesia species. Our findings, together with morphological differences, identify B. naoakii n. sp. as a distinct parasite species.

First detection of Theileria equi in free-roaming donkeys (Equus africanus asinus) in Sri Lanka.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses, donkeys, zebras, and mules. It is globally endemic with significant economic impact on the equine industry. Infected animals may serve as carriers, and they may be a source of infection for ticks, thereby posing a great challenge for disease management. Sri Lanka is a tropical country, where infections by various tick-borne parasites are common among livestock animals. However, infections by T. equi and B. caballi remain unstudied in Sri Lanka. Therefore, in the present study, we conducted an epidemiological survey to investigate the presence of T. equi and B. caballi in apparently healthy free-roaming donkeys. Blood samples were randomly taken from 111 donkeys in Mannar (n = 100) and Kilinochchi (n = 11) districts in Sri Lanka. Thin blood smears were prepared from the blood samples and subjected to microscopic examination. Additionally, blood DNA samples were prepared and screened for T. equi and B. caballi infections using species-specific PCR assays. Our results showed that 64 (57.7%) and 95 (85.6%) of the donkeys were positive for T. equi by microscopy and PCR, respectively. However, all samples were negative for B. caballi. Phylogenetic analysis of the T. equi 18S rRNA sequences detected two distinct genotypes, namely C and D. To our knowledge, this is the first report of T. equi in Sri Lanka and of genotype C in donkeys. The present study highlights the importance of monitoring the shrinking donkey population in Sri Lanka owing to EP caused by T. equi.

Molecular survey of bovine Babesia species in Bactrian camels (Camelus bactrianus) in Mongolia.

Bovine babesiosis, which is caused by species of genus Babesia, is a leading cause of considerable economic losses to the cattle industry each year. Bovine Babesia species have frequently been detected in non-cattle hosts, such as water buffalo (Bubalus bubalis), from which the parasites can be transmitted by ticks to cattle. Therefore, Babesia infections should be minimized not only in cattle but also in non-cattle carriers. In the present study, we surveyed the Bactrian camels (Camelus bactrianus) in Mongolia for three clinically significant bovine Babesia species, including Babesia bovis, B. bigemina, and Babesia sp. Mymensingh, which had been detected previously in Mongolian cattle. We screened blood DNA samples from 305 Bactrian camels in six Mongolian provinces for these species, using parasite-specific PCR assays. Our findings showed that the Bactrian camels in Mongolia were infected with all three Babesia species surveyed. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 32.1%, 21.6%, and 24.3%, respectively, whereas 52.5% of the surveyed animals were infected with at least one parasite species. We also found that the female Bactrian camels and the Mongolian native camel breed had significantly higher Babesia positive rates than the male Bactrian camels and the Hos Zogdort breed. In Mongolia, cattle and Bactrian camels usually share common pasture lands for grazing; furthermore, tick species infesting cattle also infest Bactrian camels. Our findings, together with these observations, suggest that the tick transmission of bovine Babesia species might be possible between cattle and Bactrian camels. Therefore, strategies for the control of bovine babesiosis in Mongolia should include methods to minimize bovine Babesia species infections in Bactrian camels.

Molecular survey of Babesia, Theileria, Trypanosoma, and Anaplasma infections in camels (Camelus dromedaries) in Egypt.

The one-humped camel (Camelus dromedarius) or dromedary is an economically important domestic animal. However, infectious diseases, including those caused by vector-borne hemopathogens, frequently compromise the health and production of camels. In this study, we examined infections caused by Babesia, Theileria, Trypanosoma, and Anaplasma species in camels in Egypt. We analyzed blood DNA samples from 148 camels reared in six Egyptian governorates (Giza, Asyut, Sohag, Qena, Luxor, and the Red Sea) using pathogen-specific Polymerase Chain Reaction (PCR) assays. Our results indicated that 29 (19.6%), 22 (14.9%), 1 (0.7%), 2 (1.4%), 1 (0.7%), 2 (1.4%), and 28 (18.9%) of the surveyed animals were infected with Babesia bovis, B. bigemina, Babesia sp. Mymensingh, Theileria sp. Yokoyama, Theileria equi, Trypanosoma evansi, and Anaplasma marginale, respectively. We found that a total of 68 (45.9%) animals were infected with at least one of the detected hemopathogens. Sequencing analyses of PCR amplicons confirmed our diagnostic results. This study is the first to report Theileria sp. Yokoyama and Babesia sp. Mymensingh in Egypt. This is also the first report of infection with these two species in one-humped camel. In conclusion, this study found that camels in Egypt are infected with several vector-borne hemopathogens, including novel parasite species.

Molecular epidemiological survey of Babesia bovis, Babesia bigemina, and Babesia sp. Mymensingh infections in Mongolian cattle.

Bovine babesiosis caused by Babesia species is an economically significant disease of cattle. Severe clinical babesiosis in cattle is caused by Babesia bovis, B. bigemina, and the recently discovered Babesia sp. Mymensingh. Mongolia is an agricultural country with a large cattle inventory. Although previous studies have detected active infections of B. bovis and B. bigemina in Mongolian cattle, only a few provinces were surveyed. Additionally, the endemicity of Babesia sp. Mymensingh in Mongolia remains unknown. We screened blood DNA samples from 725 cattle reared in 16 of the 21 Mongolian provinces using B. bovis-, B. bigemina-, and Babesia. sp. Mymensingh-specific PCR assays. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 27.9% (n = 202), 23.6% (n = 171), and 5.4% (n = 39), respectively. B. bovis and B. bigemina were detected in cattle in all surveyed provinces; whereas Babesia sp. Mymensingh was detected in 11 of the 16 surveyed provinces. On a per province basis, the B. bovis- B. bigemina-, and Babesia sp. Mymensingh-positive rates were 5.9-52.0%, 9.1-76.3%, and 0-35.7%, respectively. In conclusion, this is the first report of Babesia sp. Mymensingh in Mongolia. In addition, we found that species of Babesia that are capable of causing bovine clinical babesiosis, including B. bovis, B. bigemina, and Babesia sp. Mymensingh, are widespread throughout the country.

Nationwide serological surveillance of non-tsetse-transmitted horse trypanosomoses in Mongolia.

In Mongolia, horses play important roles, not only in livestock production, but also in terms of culture, tradition, and Mongolian beliefs. Although the presence of non-tsetse-transmitted horse trypanosomoses, which are caused by infections with Trypanosoma evansi (surra) and T. equiperdum (dourine), has been reported in the country, whether there is a nationwide epidemic of these infectious diseases is unknown. In the present study, a nationwide surveillance of horse trypanosomoses was performed. The sample sizes for each province, the whole country, and male and female horses were, respectively, 96, 2,400, and 316 and 306. In total, 3,641 samples of horse sera were collected by simple random sampling. The rTeGM6-4r-based ELISA, which was applied for surra against cattle and water buffalo and dourine against horse, revealed that the overall sero-prevalence of the diseases in Mongolia was 4.8%. Among them, high sero-prevalences were observed in the central provinces (5.2-11.0%, p < 0.05) of the country. The sero-prevalence was significantly higher in females than in males (6.0% and 4.0%, p < 0.05, respectively) and in non-castrated males (8.4%, p < 0.01) compared with castrated males (3.0%). These results suggested that currently, horse trypanosomoses are a nationwide endemic problem in Mongolia. Knowledge of the nationwide endemic status of non-tsetse-transmitted horse trypanosomoses in Mongolia will be useful to prevent these diseases.

Draft Genome Sequence of Trypanosoma equiperdum Strain IVM-t1.

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.

Molecular detection of Anaplasma ovis in small ruminants and ixodid ticks from Mongolia.

Anaplasma ovis is a tick-borne obligate intracellular rickettsial bacterium that causes anaplasmosis in domestic and wild small ruminants. Sheep and goats, whose combined population is approximately 48.5-million in Mongolia, play a vital role in the country's economy. In this study, we conducted an epidemiological survey of A. ovis in sheep and goats from 19 of 21 provinces in Mongolia. Additionally, DNA samples extracted from unfed ticks collected in 11 Mongolian provinces were also screened for A. ovis. Of 1179 and 871 blood DNA samples from sheep and goats, 813 (69.0%) and 621 (71.3%), respectively, were positive for A. ovis when screened by a PCR assay based on major surface protein 4 gene (msp4). On a per province basis, A. ovis infection rates ranged from 7.4%-93.3% and 13.3%-100% in sheep and goats, respectively. Subsequently, DNA samples prepared from 721 unfed ticks, including Dermacentor nuttalli (n = 378), Ixodes persulcatus (n = 95), Haemaphysalis pospelovashtromae (n = 120), and Hyalomma asiaticum (n = 128), were screened for A. ovis using the same PCR assay. Although nine D. nuttalli were A. ovis-positive, all other tick DNA samples were negative. In addition to reporting A. ovis in sheep and goats from all over Mongolia, this study identified D. nuttalli as a potential transmission vector of A. ovis in Mongolia. The present data highlight the importance of monitoring Mongolian sheep and goats for possible episodes of clinical anaplasmosis and controlling D. nuttalli throughout the country.

The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis.

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.

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 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 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.