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.

Metal nanoparticles restrict the growth of protozoan parasites.

The Trypanosoma and Toxoplasma spp, are etiological agents of diseases capable of causing significant morbidity, mortality and economic burden, predominantly in developing countries. Currently, there are no effective vaccines for the diseases caused by these parasites; therefore, therapy relies heavily on antiprotozoal drugs. However, the treatment options for these parasitic diseases are limited, thus underscoring the need for new anti-protozoal agents. Here, we investigated the anti-parasite action of nanoparticles. We found that the nanoparticles have strong and selective in vitro activity against T. b. brucei but moderate in vitro activity against T. congolense and T. evansi. An estimation of the in vitro anti-Trypanosoma efficacy showed that the nanoparticles had ≥200-fold selective activity against the parasite versus mammalian cells. Moreover, the nanoparticle alloys moderately suppressed the in vitro growth of T. gondii by ≥60%. In our in vivo study, the nanoparticles appeared to exhibit a trypanostatic effect, but did not totally suppress the rat parasite burden, thereby failing to appreciably extend the survival time of infected animals compared with the untreated control. In conclusion, this is the first study to demonstrate the selective in vitro anti-Trypanosoma action of nanoparticles and thus supports the potential of nanoparticles as alternative anti-parasitic agents.

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.

Oral administration of azithromycin ameliorates trypanosomosis in Trypanosoma congolense-infected mice.

Animal trypanosomosis is a devastating parasitic disease that is of economic importance to livestock production. The infection includes animal African trypanosomosis, surra, and dourine. The treatment is based solely on few compounds that were discovered decades ago and which are associated with severe toxicity. Furthermore, it is likely that the parasite has developed resistance towards them. Thus, there is an urgent need for new, accessible, and less toxic drugs. Azithromycin is an antibiotic with documented efficacy against Toxoplasma, Babesia, and Plasmodium. The current study investigated its effects against animal trypanosomes. An in vitro system was used to determine the trypanocidal effects of azithromycin against Trypanosoma congolense, Trypanosoma brucei brucei, and Trypanosoma evansi, and cytotoxicity in Madin-Darby bovine kidney (MDBK) and NIH 3T3 cells. Furthermore, the trypanocidal effects of azithromycin were investigated in T. congolense-infected mice. In vitro, azithromycin had an IC50 of 0.19 ± 0.17; 3.69 ± 2.26; 1.81 ± 1.82 µg/mL against T. congolense, T. b. brucei, and T. evansi, respectively. No cytotoxic effects were observed in MDBK and NIH 3T3 cells. The efficacy of orally administered azithromycin was investigated in short-term and long-term treatment protocols. Although the short-term treatment protocol showed no curative effects, the survival rate of the mice was significantly prolonged (p < 0.001) in comparison to the control group. The long-term treatment yielded satisfying curative effects with doses of 300 and 400 mg/kg achieving 80 and 100% survival, respectively. In conclusion, long-term oral azithromycin treatment has trypanocidal effects against T. congolense.

An ATP-Based Luciferase Viability Assay for Animal African Trypanosomes Using a 96-Well Plate.

Cell viability assays using multi-well cell culture plates are frequently used for in vitro drug screening. We herein describe an ATP-based luciferase viability assay for animal African trypanosomes using a 96-well plate. This assay could be further applied to the screening of novel compounds for the treatment of animal African trypanosomiasis.

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.