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 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.

Treatment Efficiency of Combination Therapy With Diminazene Aceturate and Quinapyramine Sulfate in a Horse With Dourine.

Dourine is a lethal protozoan disease of equids, and it is caused by Trypanosoma equiperdum infection via coitus. To date, treatment strategies against the dourine are not recommended because of the frequent relapses; therefore, the World Organisation for Animal Health recommends the stamping-out policy for the control of dourine. Our previous studies have revealed a number of horses with dourine in Mongolia that is the fifth largest horse-breeding country. It is difficult to apply the stamping-out policy for cases of dourine in Mongolia because of an inadequate livestock guarantee system. Therefore, the development of effective treatment measures is an urgent need. In this study, an 8-year-old stallion was definitely diagnosed with dourine based on clinical signs, molecular analysis, and microscopic examination of trypanosomes. Combination therapy with diminazene aceturate and quinapyramine sulfate was applied. Before the treatment, the characteristic clinical signs of dourine were observed, and trypanosomes were detected in the urogenital tract mucosal swab samples by microscopic examination and polymerase chain reaction (PCR). Moreover, positive serological results were obtained. After the treatment, we observed an improvement in the health of the treated horse and no trypanosome infection in its urogenital tract by microscopic examination and PCR. Moreover, serological tests showed seronegative results. The horse has showed no relapse for at least 2.5 years after the treatment, and its reproductive ability has improved. Our result suggests that trypanosomes did not invade cerebrospinal fluid when we started the therapy. In conclusion, the combination therapy has therapeutic potential against dourine at an early phase.

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.

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.

A Seroepidemiological Survey of Theileria equi and Babesia caballi in Horses in Mongolia.

Equine piroplasmosis caused by Theileria equi and Babesia caballi is an economically important disease with a worldwide distribution. The objective of the present study was to investigate the seroepidemiology of T. equi and B. caballi in horses reared in various Mongolian provinces. Serum samples prepared from blood collected from horses in 19 Mongolian provinces were screened for antibodies specific to T. equi and B. caballi using enzyme-linked immunosorbent assays based on recombinant forms of T. equi merozoite antigen-2 and the B. caballi 48-kDa merozoite rhoptry protein, respectively. Of 1,282 horses analyzed, 423 (33%) and 182 (14.2%) were sero-positive for T. equi and B. caballi, respectively. Additionally, 518 (40.4%) were positive for at least 1 parasite species, of which 87 (16.8%) were co-infected with both parasites. Both T. equi and B. caballi were detected in all surveyed provinces, and on a per province basis the positive rates ranged from 19.0 to 74.2% and 4.5 to 39.8%, respectively. Theileria equi- and B. caballi-positive rates were comparable between male horses (31.9 and 14.1%, respectively) and female horses (34.5 and 14.3%, respectively). However, the positive rates were higher in the >3-yr-old age group (37.7 and 15.6%, respectively) compared with the 1-3-yr-old age group (19.4 and 10.0%, respectively). These findings confirmed that T. equi and B. caballi infections are widespread among horses all over Mongolia, and that horse age is a risk factor for infection in this country. Our results will be useful for designing appropriate control measures to minimize T. equi and B. caballi infections among Mongolian horses.

Serosurvey of Babesia bovis and Babesia bigemina in cattle in Mongolia.

Mongolia is an agriculturally rich country with large livestock populations that contribute significantly to its national economy. However, the export market for live animals and livestock products is often constrained for various reasons including infectious diseases. Babesia bovis and B. bigemina, which are bovine hemoprotozoan parasites, cause severe forms of clinical babesiosis, in cattle. However, a country-wide survey to determine the exposure rates in various provinces in Mongolia was not conducted to determine the risk for infections with these parasite species. Therefore, we investigated the frequency of antibodies to B. bovis and B. bigemina in cattle reared throughout Mongolia. B. bovis-and B. bigemina-specific enzyme-linked immunosorbent assays (ELISAs) were used to screen the serum samples sourced from 1946 cattle in 19 of 21 provinces and a provincial municipality (Ulaanbaatar) in Mongolia. We found 351 (18.0%) samples positive for B. bovis and 435 (22.4%) samples positive for B. bigemina infections. The B. bovis- and B. bigemina-positive rates ranged from 0.8 to 61.5% and 4.0 to 50.6%, respectively, among the surveyed provinces. The positive rates of B. bovis and B. bigemina infections were relatively higher in the provinces located in northernmost, northern, eastern, southeastern, and southern Mongolia. Additionally, the B. bovis- and B. bigemina-positive rates were not significantly different between females (18.2 and 22.2%, respectively) and males (17.2 and 18.8%, respectively) or between the 1-3-year-old (16.2 and 19.4%, respectively) and >3-year-old (17.1 and 20.9%, respectively) age groups. The differential seropositivity for B. bovis and B. bigemina infections among the provinces may reflect the variations in the risk of cattle being infected with these parasite species. The findings of the present study highlight the need for country-wide control measures, including tick control programs, to minimize the rates of B. bovis and B. bigemina infections in Mongolian cattle.

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.

The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum.

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.

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.