LKR/SDH Plays Important Roles throughout the Tick Life Cycle Including a Long Starvation Period

BACKGROUND:Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. CONCLUSIONS/SIGNIFICANCE:Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods

Next-generation sequencing metabarcoding assays reveal diverse bacterial vector-borne pathogens of Mongolian dogs

Bacterial vector-borne pathogens (BVBPs) negatively impact canine health worldwide, with several also being zoonotic, posing an additional disease risk to humans. To date, BVBPs have been reported in humans and various sylvatic and domestic animal hosts across multiple Mongolian aimags (provinces); however, there has been no published data on these pathogens within Mongolia’s canine populations. Collection of such data is important given Mongolia’s size, diverse number of climatic regions, and large population of dogs, most of which closely share their environment with humans and livestock. Therefore, a bacteria-targeting next-generation sequencing metabarcoding (mNGS) assay was used to test the feasibility of mNGS as a proof-of-concept study to ascertain the detection of BVBP in 100 Mongolian dogs. The majority of dogs (n = 74) were infected with at least one of six BVBPs identified; including three species of haemoplasmas (also known as haemotropic mycoplasmas, n = 71), Bartonella rochalimae (n = 3), Ehrlichia spp. (n = 2) and Anaplasma platys (n = 1). Univariable analysis found sex, housing, and role of the dog to be associated with BVBP infection. Male dogs had 4.33 (95% CI: 1.61–11.62, P = 0.003) times the odds of infection with BVBPs compared to females. The majority of dogs included in this study were kept outdoors and had regular direct contact with both livestock and humans, indicating that dogs may contribute to the transmission and dissemination of BVBPs in Mongolia and could act as epidemiological sentinels. This study underscores the importance of pathogen surveillance studies in under-researched regions, reinforces the efficacy of mNGS as an explorative diagnostic tool, and emphasises the need for further larger-scale seroprevalence studies of Mongolian dogs

The First Survey of Bovine Babesia Species Infecting Yaks (Bos grunniens) in Mongolia

application/pdfYak (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. © American Society of Parasitologists 2023

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. Keywords: Colorimetric assay, Drug screening system, Liquid culture, Luciferase assay, Trypanosoma equiperdu