Virulent species or strains of hematophagous borne pathogens such as Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp., are lethal to susceptible animals or reduce their productivity on a global scale. Nonetheless, efforts to diagnose the causative agents and assess the genotypic profiles as well as quantify the parasite burden of aforementioned parasites across seasons remain limited. Therefore, the present investigation sought to elucidate the genotypic composition of Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp. The findings revealed heightened infection rates during the summer, manifesting a correlation between Trypanosoma spp. infection and seasonal fluctuations. Among the identified pathogens, Anaplasma marginale emerged as the most dominant species, while the occurrence of Anaplasma platys in Thai cattle was confirmed via the sequencing of the groEL gene. Moreover, the study successfully identified two lineages of Trypanosoma theileri. The findings of this investigation offer valuable insights that can inform the development of preventive strategies for vector-borne diseases, such as considering the appropriate use of insect repellent, mosquito or insect nets, or eliminating breeding places for insects in each season.

Trypanosoma theileri maintains a long-term extracellular infection with a low parasitaemia in bovids. The surface of this parasite is predicted to be decorated with several surface molecules including membrane surface proteases (MSPs), trans-sialidases and T. theileri putative surface proteins (TTPSPs). However, there are no experimental data to verify this hypothesis. Here, we have purified and partially characterized the surface glycoconjugates of T. theileri using biochemical and mass spectrometry-based approaches. The glycoconjugates fall into two classes: glycoproteins and glycolipids. Proteomic analysis of the glycoprotein fraction demonstrated the presence of MSPs and abundant mucin-like TTPSPs, with most predicted to be GPI-anchored. Mass spectrometric characterization of the glycolipid fraction showed that these are mannose- and galactose-containing glycoinositolphospholipids (GIPLs) that are larger and more diverse than those of its phylogenetic relative T. cruzi, containing up to 10 hexose residues and carrying either alkylacyl-phosphatidylinositol or inositol-phospho-ceramide (IPC) lipid components.

Trypanosoma theileri is a worldwide distributed haemoparasite that has been reported throughout the American continent in various species, including bovines, buffaloes and bats. In bovines, high incidence of T. theileri can be harmful when associated with other infections or under stress situations. There is little information on this hemoflagellate in Ecuador, which prompted the study and molecular identification of the trypanosomes collected in two slaughtering centers. Between February and April 2021, a total of 218 samples of bovine blood were collected in abattoirs located in the Andean region of Quito (n = 83) and in the coastal region, in Santo Domingo (n = 135). Quito public Slaughterhouse is the biggest in Ecuador, and for that, they receive animals from all country; on the other hand, Santo Domingo\'s Slaughterhouse is a small one where mainly females from the region are sacrificed and some males. The samples were evaluated using two molecular tests, the PCR cathepsin L-like (CatL) specific for T. theileri and for the positive samples, a Nested PCR that targets the ITS of the 18S gene. The corresponding PCR products were sequenced, analyzed by BLAST/NCBI and the sequences were used to build a concatenated phylogenetic tree, using the MEGA XI software. Overall, 34 out of the 218 samples, (15.6%) were positive to T. theileri by PCR CatL, resulting from 20/83 (24.1%) positives from the Quito abattoir and 14/135 (10.4%) from the Santo Domingo slaughterhouse. These prevalence rates were found to be significantly different (p = 0.006). According to the phylogenetic tree based on the CatL and ITS concatenated sequences (n = 13), the two novel Equatorial T. theileri isolates, ThI (n = 7) and ThII (n = 6) are closely related and associated to the IC, IB and IIB genotypes, present in Brazil, Venezuela and Colombia. Thirty-one out of the thirty-four T. theileri-positive bovines were co-infected with other haemotropic pathogens, Anaplasma marginale Babesia spp and T. vivax. This coinfection could be responsible for additional pathologies and harmful effects on the affected cattle. This study presents the molecular identification and genotypification of T. theileri isolated from cattle in Ecuador through the analysis of CAtL and ITS sequences, and the high frequency of coinfection of this hemoflagellate with other blood haemotropic organisms.

Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein-protein inhibitors.

Trypanosoma theileri is a cosmopolitan opportunistic haemoparasite described in wild and domestic ruminants, and also in arthropod vectors. The presence of this parasite has been reported in several South American countries, including Amazonian regions. Despite the importance of livestock production, Ecuador possesses scarce studies about trypanosomosis and no T. theileri reports in its territory. Here, we showed molecular evidences of the presence of T. theileri in cattle from a province located in the Ecuadorian Amazon. Bovine blood samples were collected from 2014 to 2019, during campaigns to detect haemoparasites in the Ecuadorian provinces of Orellana and Sucumbíos. DNA was extracted from the buffy coat and used in PCR assays with three different molecular markers, ITS1, 18S and Cathepsin L-like. T. theileri was detected only in the Sucumbíos province, with a specific molecular prevalence of 8.6% (3/35) using the three primers and an additional animal detected as positive (11.4% prevalence) only by the ITS1 marker. DNA sequences derived from the generated amplicons were subjected to phylogenetics maximum parsimony and maximum likelihood analysis, which indicate the presence of TthI and TthII genotypes circulating in the evaluated animals. Molecular surveillance should be continually implemented in Ecuador in order to deepen the epidemiological and evolutionary knowledge about T. theileri as well other haemoparasites in the amazon parts of the country.

OBJECTIVE: The present study reports a very rare case of Trypanosoma theileri like infection in the peritoneal fluid of a 6-year-old female Holstein Friesian (HF) cattle brought to Veterinary Clinical Complex, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar. The aim of the clinical study was to report an unusual case of T. theileri-like infection associated with peritonitis and its therapeutic management.
METHODS: For confirmatory diagnosis, peritoneal fluid and blood sample was collected from animal for haemato-biochemical examination, buffy coat examination and detection of Trypanosoma antigen by monoclonal antibody-based latex agglutination test.
RESULTS: Animal had history of anorexia, high fever, oedematous swelling on the abdominal area and decrease in milk yield since 1 week. Clinical examination revealed high fever, congested conjunctival mucus membrane and oedematous inflammatory swelling on the abdominal region. Haematological examination on Day 1 revealed relative neutrophilia and eosinophilia, whereas other parameters measured were on the lower side of normal range. Thin blood smear examination did not reveal any parasite on first attempt. However, after confirmation of Trypanosoma species in peritoneal fluid collected by FNAC, buffy coat examination of blood sample was also found positive for Trypanosoma species. On Day 2, haematological examination revealed neutrophilic leucocytosis, eosinophilia and anaemia. Blood sample was also found serologically positive for Trypanosoma antigen by monoclonal antibody based latex agglutination test. Biochemical examination revealed increased blood urea nitrogen (BUN), gamma glutamyl transferase (GGT) levels, hyperglycemia, hyperprotienemia and decreased levels of sodium and chloride ions. Animal was primarily treated with isometamidium chloride (Nyzom®) @ 0.5 mg/kg body weight (IM, single dose) along with supportive therapy.
CONCLUSIONS: Animal showed clinical recovery after 10 days of treatment. Post-recovery, blood sample of the animal was re-examined for parasite and other blood parameters and it was found negative for presence of Trypanosoma infection along with restoration of haematological values. Hence, the present study concludes the confirmatory diagnosis and therapeutic use of isometamidium hydrochloride along with anti-inflammatory drugs to Trypanosoma infection.

Trypanosomes belonging to Trypanosoma theileri group are mammalian blood parasites with keds and horse fly vectors. Our aim is to study to vector specificity of T. theileri trypanosomes. During our bloodsucking Diptera survey, we found a surprisingly high prevalence of T. theileri trypanosomes in mosquitoes (154/4051). Using PCR and gut dissections, we detected trypanosomes of T. theileri group mainly in Aedes mosquitoes, with the highest prevalence in Ae. excrucians (22%), Ae. punctor (21%), and Ae. cantans/annulipes (10%). Moreover, T. theileri group were found in keds and blackflies, which were reported as potential vectors for the first time. The vectorial capacity was confirmed by experimental infections of Ae. aegypti using our isolates from mosquitoes; sand fly Phlebotomus perniciosus supported the development of trypanosomes as well. Infection rates were high in both vectors (47-91% in mosquitoes, 65% in sandflies). Furthermore, metacyclic stages of T. theileri trypanosomes were observed in the gut of infected vectors; these putative infectious forms were found in the urine of Ae. aegypti after a second bloodmeal. On the contrary, Culex pipiens quinquefasciatus was refractory to experimental infections. According to a phylogenetic analysis of the 18S rRNA gene, our trypanosomes belong into three lineages, TthI, ThII, and a lineage referred to as here a putative lineage TthIII. The TthI lineage is transmitted by Brachycera, while TthII and ThIII include trypanosomes from Nematocera. In conclusion, we show that T. theileri trypanosomes have a wide range of potential dipteran vectors, and mosquitoes and, possibly, sandflies serve as important vectors.

Trypanosoma (Megatrypanum) theileri is a non-pathogenic or weakly pathogenic parasite of domestic cattle that is cyclically transmitted by blood-sucking insects, mainly tabanid flies. It has been reported in several countries like Brazil, Venezuela, Japan, Taiwan, Thailand, Vietnam, and the Philippines. Although the ruminant industry is actively expanded in Malaysia, T. theileri and T. theileri-like trypanosomes have never been reported from Malaysia. The low pathogenicity of this species might be the main reason for overlooking T. theileri in this country. This paper describes an unforeseen finding of T. theileri from the outbreak of T. evansi in the state of Kelantan, Malaysia. This is the first time T. theileri reported in Malaysia, and also the first time T. theileri is reported in equid. Clinical signs compatible with infection by blood protozoa were observed; however, it was uncertain whether they were due to T. theileri infection. The detection of T. theileri from the blood sample and Tabanus sp. were confirmed through molecular analysis with PCR and DNA sequencing. In the present study, T. theileri from one horse and one Tabanus sp. were clustered with sequences of the previously described phylogenetic lineages from Japan, Chad and Brazil cattle. Even though this species is claimed to be host-specific with ruminant host restriction, the finding from this study suggested that T. theileri can infect equine whilst other isolates are known to infect ruminant species only. It is suspected there were two genotypes of T. theileri circulating in at least two districts of Kelantan. Thus, further study on multiple DNA regions should be conducted to determine the strains of detected T. theileri in Malaysia. Its impact on the horse and cattle industry should also be revised.

Trypanosoma theileri is considered a non- or low-pathogenic trypanosome that generally causes latent infection in apparently healthy cattle; however, T. theileri propagates in the bloodstream and may cause clinical disease in pregnant animals or co-infection with bovine leukemia virus or Theileria orientalis. In the current study, a monthly survey of T. theileri infection over one year was carried out in a research dairy farm in Hokkaido, Japan to determine the 1) seasonal variations in the prevalence, 2) genetic characterization of T. theileri, and 3) associations of milk and blood parameters in dairy cattle with T. theileri infection, including data of metabolic profile tests and dairy herd performance tests, using linear mixed models. We found that 1) the prevalence of T. theileri infection was significantly higher in summer and winter than in other seasons; 2) T. theileri possibly showed genetic diversity in Eastern Hokkaido; and 3) T. theileri infection was associated with significantly lower levels of blood urea nitrogen, milk protein, and solids-not-fat, which are caused by a low rumen fermentation level. This is the first study to report the negative impact of T. theileri infection in dairy cattle, and our study indicates that control of T. theileri infection can improve the productivity of dairy cattle.

The taxonomy of ruminant Trypanosoma theileri and its relatives (Kinetoplastida: Trypanosomatidae) is controversial, with recent phylogenetic studies segregating T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers. In the present study, T. theileri-like trypanosomes isolated from Honshu sika deer (Cervus nippon) in the western Japan (YMG isolate) were genetically characterized using a number of genetic markers. Sika deer trypanosomes of the YMG isolate were genetically different from the Trypanosoma sp. TSD1 isolate previously recorded from Hokkaido sika deer in northern Japan, with the former trypanosome isolate being genetically closer to European cervid trypanosomes and the bovine T. theileri TthII lineage. In contrast, the latter isolate exhibited greater relatedness to North American cervid trypanosomes and the bovine T. theileri TthI lineage, although a clear genetic distinction between these was apparent. Furthermore, trypanosomes in Honshu sika deer from the central part of Japan harboured additional genetic diversity and were closer to either TSD1 or YMG isolates, while distinct from known T. theileri-related genotypes. Importantly, cervids and wild ruminants worldwide might harbour divergent descendants of a T. theileri ancestor, which exhibit rigid host specificity to either bovines or cervid species.