Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

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.

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.

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.

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.

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.

Utilization of crude and recombinant ELISAs for serodiagnosis of camel trypanosomosis in Sudan.

This study was carried out to evaluate the application of CATT/T. evansi, crude and recombinant (TeGM6-4r) antigen ELISAs in the diagnosis of camel trypanosomosis caused by two trypanosome species, T. evansi and T. vivax, in Sudan. Concurrently, the current situation of camel trypanosomosis was investigated based on the results of a serological analysis. The recombinant tandem repeat antigen TeGM6-4r is conserved among salivarian trypanosome species and was highly sensitive in the detection Trypanozoon, and T. vivax. It has been validated in the diagnosis of surra in cattle and water buffalo but not in camels. A comparative evaluation of a crude antigen ELISA and a recombinant antigen GM6 (rTeGM6-4r) ELISA was performed using 189 blood samples, which included 148 samples obtained from different camel herds in Eastern Sudan and 41 samples from camels that had been brought from Western Sudan to local markets. The results showed that the rTeGM6-4r ELISA detected the greatest number of positive samples (n = 118, 62%), while CATT/T. evansi and the crude antigen ELISA detected the lowest number of positive samples (n = 73, 39%). The kappa value of rTeGM6-4r as compared to TeCA ELISA was 0.5515, which indicated moderate agreement. We concluded that the rTeGM6-4r ELISA is the test of choice for use in screening camel for trypanosomosis caused by T. evansi and T. vivax in Sudan.

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 utility of an rTeGM6-4r-based immunochromatographic test for the serological diagnosis of non-tsetse-transmitted equine trypanosomosis in rural areas of Mongolia.

Our previous studies report epidemics of non-tsetse-transmitted equine trypanosomosis in Mongolia. However, the current status of non-tsetse-transmitted equine trypanosomosis endemicity remains to be clarified in some parts of Mongolia. We previously reported the potential application of rTeGM6-4r-based diagnostic tools, an rTeGM6-4r-based immunochromatographic test (ICT) and an enzyme-linked immunosorbent assay (ELISA), in the serological surveillance of equine trypanosomosis in Mongolia. In the present study, the utility of the rTeGM6-4r-based ICT was validated. The rTeGM6-4r-based ICT accurately diagnosed positive reference sera that had been prepared from dourine horses in Mongolia, similarly to the rTeGM6-4r-based ELISA. The diagnostic performance of the rTeGM6-4r-based ICT was maintained when the strips were preserved for at least 2 months under dry conditions. The ICT detected 42 positive serum samples from a total of 1701 equine sera that had been collected from all 21 provinces of Mongolia. The κ-value, sensitivity and specificity of rTeGM6-4r-based ICT were 0.58, 50.0% (95% CI, 37.7-62.3%) and 99.3% (95% CI, 98.7-99.6%), respectively, in comparison to the rTeGM6-4r-based ELISA. Our field-friendly rTeGM6-4r-based ICT was found to be useful for the serological diagnosis of non-tsetse-transmitted equine trypanosomosis in rural areas of Mongolia.

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.

Clofazimine Inhibits the Growth of Babesia and Theileria Parasites In Vitro and In Vivo.

The present study evaluated the growth-inhibitory effects of clofazimine, currently used for treating leprosy, against Babesia bovis, B. bigemina, B. caballi, and Theileria equi in in vitro culture and against Babesia microti in mice. The 50% inhibitory concentrations (IC50s) of clofazimine against the in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi were 4.5, 3, 4.3, and 0.29 µM, respectively. In mice infected with B. microti, treatment with 20 mg/kg of body weight of clofazimine administered orally resulted in a significantly lower peak parasitemia (5.3%) than that in the control group (45.9%), which was comparable to the subcutaneous administration of 25 mg/kg diminazene aceturate, the most widely used treatment for animal piroplasmosis. Although slight anemia was observed in both clofazimine- and diminazene aceturate-treated infected mice, the level and duration of anemia were lower and shorter, respectively, than those in untreated infected mice. Using blood transfusions and PCR, we also examined whether clofazimine completely killed B. microti On day 40 postinfection, when blood analysis was performed, parasites were not found in blood smears; however, the DNA of B. microti was detected in the blood of clofazimine-treated animals and in several tissues of clofazimine- and diminazene aceturate-treated mice by PCR. The growth of parasites was observed in mice after blood transfusions from clofazimine-treated mice. In conclusion, clofazimine showed excellent inhibitory effects against Babesia and Theileria in vitro and in vivo, and further study on clofazimine is required for the future development of a novel chemotherapy with high efficacy and safety against animal piroplasmosis and, possibly, human babesiosis.

Inhibitory effects of pepstatin A and mefloquine on the growth of Babesia parasites.

We evaluated the inhibitory effects of pepstatin A and mefloquine on the in vitro and in vivo growths of Babesia parasites. The in vitro growth of Babesia bovis, B. bigemina, B. caballi, and B. equi was significantly inhibited (P < 0.05) by micromolar concentrations of pepstatin A (50% inhibitory concentrations = 38.5, 36.5, 17.6, and 18.1 µM, respectively) and mefloquine (50% inhibitory concentrations = 59.7, 56.7, 20.7, and 4 µM, respectively). Furthermore, both reagents either alone at a concentration of 5 mg/kg or in combinations (2.5/2.5 and 5/5 mg/kg) for 10 days significantly inhibited the in vivo growth of B. microti in mice. Mefloquine treatment was highly effective and the combination treatments were less effective than other treatments. Therefore, mefloquine may antagonize the actions of pepstatin A against babesiosis and aspartic proteases may play an important role in the asexual growth cycle of Babesia parasites.