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Piroplasmid parasites such as Theileria luwenshuni protozoa pose a global threat to both animal and human health. However, human theileriosis remains underexplored compared to infections caused by Plasmodium and Babesia species parasites. We investigated potential hemoparasite infections among 1,721 persons with fever, anemia, or both in Yunnan Province, China. Molecular detection identified 13 cases positive for T. luwenshuni protozoa, of which 5 patients were further confirmed by Western blot antibody analysis. We also identified 6 babesiosis cases, 3 infections with B. microti and 3 with novel Babesia spp. Subsequent vector and host investigations in the vicinity of the index cases revealed T. luwenshuni protozoa in 1 tick and 53 livestock animals. Of note, 3.3% combined vector-host samples tested positive for genetically diverse Babesia species. Our findings highlight the endemic circulation of T. luwenshuni and Babesia spp. parasites in southwest China, underscoring their importance as emerging public health concerns.

Raising small ruminants is the main source of income for farmers in Pakistan especially in rural areas of Dera Ghazi Khan in Punjab. Despite having large sheep population, the prevalence of intra-erythrocytic protozoa, Theileria ( T .) lestoquardi , has never been reported from this area. This study was conducted to fill this knowledge gap and 333 blood samples of apparently healthy small ruminants (168 sheep and 165 goats) along with their epidemiological data were collected from Dera Ghazi Khan district during August till November 2022. The polymerase chain reaction (PCR) analysis amplified a 785 base pair amplicon specific for the Merozoite surface antigen ( ms 1–2 ) gene of T . lestoquardi in 2 out of the 168 (3.3%) sheep blood samples, while no goat blood sample out of 165 was found to be infected with T . lestoquardi . DNA sequencing confirmed the presence of Theileria lestoquardi in both samples and phylogenetic analysis revealed that these amplicon resembled the partial ms 1–2 gene sequences detected in small ruminants from Pakistan, India Iran and Egypt. All the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, composition of herd, size of herd and Tick burden on sheep) were not found associated with the prevalence of T . lestoquardi . In conclusion, this study reports a low prevalence of T . lestoquardi infection in the Dera Ghazi Khan District of Punjab, Pakistan. The data generated from this work will help pave the way for the prophylactic detection and control of ovine and caprine theileriosis in the region.

Ticks are significant vectors of bacterial, viral, and protozoan pathogens, impacting both public health and agriculture. In Kyrgyzstan, tick-borne diseases are a growing concern for livestock and human health. While bacterial and viral pathogens are widely studied, and limited previous investigations have focused on specific Babesia and Theileria species in certain host animals, comprehensive data on tick eukaryotic microbiota and potential pathogens across diverse hosts nationwide is scarce. To address this gap, our study provides the comprehensive nationwide assessment of the potential protozoan pathogens in ticks from cattle and sheep, analyzing data of Babesia and Theileria at the genus level. We collected 472 tick samples from cattle and sheep across seven regions of Kyrgyzstan (March-July 2022). Tick species were identified via microscope and Sanger sequencing (mitochondrial COI gene). Eukaryotic microbiota was analyzed using 18S rRNA V9 NGS. Sanger sequencing identified five genera and 11 tick species. NGS analysis revealed Babesia (13.3%) and Theileria (12.7%) as among the most prevalent protozoa detected at the genus level. Babesia was significantly more prevalent in nymph-stage ticks and those collected from sheep, whereas Theileria was detected across a broader range of tick species and host animals, showing less variation across life stages. No significant differences in prevalence were observed based on tick sex or the number of hosts in the tick life cycle. Regionally, Babesia detection was highest in the Osh region, particularly in ticks collected from both cattle and sheep. This is the first comprehensive nationwide analysis of tick eukaryotic metabarcoding study in Kyrgyzstan focusing on pathogenic protozoa detected at the genus level. Findings provide crucial baseline data on Babesia and Theileria geographic and host-specific prevalence. Understanding these information is essential for advancing future research and supporting the development of effective surveillance and control strategies against babesiosis and theileriosis in regional livestock.

Theileria orientalis is a protozoan hemoparasite of cattle vectored by the rapidly emerging invasive Asian longhorned tick ( Haemaphysalis longicornis ). Theileria -associated bovine anemia (TABA) is easily mistaken for bovine anaplasmosis, which can lead to delayed diagnosis in areas where bovine anaplasmosis is endemic and TABA is newly emerging. Our objective was to surveil for infestation of cattle by H. longicornis and infection with T. orientalis on three Missouri cow-calf operations in counties where H. longicornis is known to be established. A total of 147 apparently healthy adult cows from 3 herds were inspected for ticks. Whole blood was collected for T. orientalis and Anaplasma marginale quantitative PCR and was also used for immediate preparation of blood smears and measurement of packed cell volumes. A total of 527 ticks were collected from the cows and taxonomically identified to the species level. Eighteen H. longicornis , including 9 adult females and 9 nymphs, were collected from 16 cows (Farm A, 2 cows; Farm B, 4 cows; Farm C, 10 cows). Intraerythrocytic T. orientalis organisms were presumptively identified on blood smears from 10 cows. Quantitative PCR screening of blood samples with primers designed to amplify all T. orientalis genotypes detected 11 positive samples (Farm A, 7 cows; Farm B, 3 cows; Farm C, 1 cow). Positive samples were re-tested with probes specific for the Ikeda, Chitose, and Buffeli genotypes, which detected the Chitose genotype in 10 samples and the Ikeda genotype in 1 sample. Detection of T. orientalis with concurrent infestation of cows by H. longicornis within these 3 herds, along with collection of H. longicornis from vegetation on the premises, supports local tick-borne transmission of this emerging pathogen.

Buparvaquone (BPQ) is used for the treatment of bovine theileriosis, a tickborne disease caused by parasites of the Theileria genus. Studies on T. annulata have linked the mechanism of BPQ resistance predominantly to genetic variations in the parasite cytochrome b ( cytb ) gene. In addition, cryptic mechanisms of resistance involving the parasite peptidyl-prolyl isomerase ( pin1 ) and dihydroorotate dehydrogenase ( dhodh ) genes require assessment. In India, where bovine theileriosis is endemic, and BPQ is widely used for treatment, it is necessary to establish the prevalence of genetic variations linked to BPQ resistance. In this study, multiplexed PCR amplification and nanopore sequencing method was used for genotyping the complete gene loci of the three target genes. Analysis of 454 T. annulata field samples collected from seven different states of India revealed the presence of previously reported BPQ resistance associated variations S129G, A146T and P253S in cytb gene and A53P in pin1 gene. The A146T and I203V variations in cytb were found to be prevalent and mostly co-occurring, and their role in BPQ resistance needs further evaluation. This study has revealed the presence of previously reported BPQ resistance-linked mutations in cytb and pin1 genes in T. annulata infecting dairy cattle in India and establishes an Oxford nanopore sequencing method suitable for large-scale surveillance of genetic variation in Theileria parasites from field samples.

Tick-borne pathogens (TBPs) seriously affect cattle production and can be economically damaging. The epidemiology of these organisms in the Chongqing municipality of China is not well described. This study aimed to investigate the prevalence and risk factors of TBPs including Anaplasma spp., Babesia spp. and Theileria spp. in cattle in Chongqing municipality. The results showed that 43.48% (150/345) of cattle were infected with at least one TBP, of which single infections were detected in 104 (30.14%), double infections in 34 cattle (9.86%) and triple infections in 12 (3.48%) of the cattle. The overall prevalence of Anaplasma spp., Theileria spp. and B. bigemina were 22.32%, 23.19% and 7.24%, respectively. Among these, the prevalence of A. bovis, A. central, A. phagocytophilum, A. platys, A. marginale, T. sinensisi and T. orientalis were 8.41%, 7.83%, 4.93%, 4.35%, 2.61%, 22.32% and 2.60%, respectively. We could not detect B. bovis, T. annulata, T. luwenshuni or T. uilenbergi in cattle. Cattle ≥1-year-old were more likely to be infected with Theileria spp. [adjusted odd ratio (AOR) = 2.70, 95% CI = 1.12-6.56)] compared with younger cattle, while cattle ≥1-year-old had reduced susceptibility to B. bigemina (AOR = 0.14, 95% CI = 0.03-0.60). Cattle living at higher altitude (≥500 m) were more susceptible to B. bigemina (AOR = 6.97, 95% CI = 2.08-23.35) and Theileria spp. infection (AOR = 1.87, 95% CI = 1.06-3.32). The prevalence of Theileria spp. on farms with cats was significantly higher than that without cats (AOR = 2.56, 95% CI = 1.12-5.88). Infection with A. bovis and A. central were significantly associated with A. phagocytophilum infection. Furthermore, there were significant associations between A. bovis and A. central infection, T. sinensisi and A. marginale infection, and B. bigemina and T. orientalis infection. This study provides new data on the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in cattle in Chongqing, and for the first time we reveal a possible relationship between the afore-mentioned pathogens, which will help in formulating appropriate control strategies for these pathogens in this area.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata . The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T . annulata -transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC 50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T . annulata cytochrome B ( Tacytb ). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia . The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q 01 site. The bound drug in the Q 01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T . annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Tropical theileriosis, caused by the protozoan parasite Theileria annulata and transmitted by several species of ixodid ticks of the genus Hyalomma, is an economically important disease of bovines. Concerningly, studies conducted in recent years have shown an increase in the rate of failure when using the primary drug of treatment, buparvaquone (BPQ), particularly in infection caused by T. annulata populations bearing V135A and P253S mutations on the Cytochrome b ( Cyto b ) gene of the parasite mitochondrial genome. The aim of this study was to demonstrate the relationship between BPQ-resistance and V135A and P253S mutations utilising an in vivo experimental set-up and to assess the tick transmissibility of drug-resistant populations. Additionally, the pharmacokinetics of BPQ in healthy and infected calves were compared to evaluate any relationship between plasma drug concentration and treatment failure. The study results demonstrated that, despite four consecutive BPQ treatments, animals infected with the resistant isolates exhibited more severe clinical signs, including longer periods of pyrexia, longer periods of schizont and piroplasm parasitemia, and the death of one animal. In addition, 3-(4,5-dimethylthiazol--yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that all cell lines derived from animals infected with the mutant genotypes exhibited resistance to high BPQ concentrations. Unexpectedly, despite substantial calf-to-calf variation during the experiment, the genetic structure of the parasite population remained largely unchanged and no strong evidence for a major genotypic shift was detected. Plasma BPQ levels were similar in all groups tested. There was no association between plasma concentrations of BPQ and parasitological or clinical response to treatment. Live parasitaemia was observed even at high plasma BPQ levels in animals infected with resistant isolates. Significantly, drug resistant parasite populations harbouring either V135A or P253S mutations was transferred between the host and vector ticks, indicating the potential for resistant parasites to be transmitted from cattle in the field, thereby facilitating their maintenance in natural populations.

This study is the first to conduct a sero-surveillance of Bovine Tropical Theileriosis (BTT) caused by the protozoan parasite Theileria annulata (T. annulata) using a recombinant Tams1 protein-based dot-ELISA in cattle, and to compare its efficacy with plate-ELISA, single PCR, nested PCR, and blood microscopy. The goal was to identify the most effective method for the early and accurate detection of theileriosis, which significantly impacts livestock through reduced milk yield and increased mortality. A total of 101 field blood samples were examined using blood smear analysis, single PCR, nested PCR, and dot-ELISA. The recombinant Tams1 protein was successfully cloned and expressed using a pET-30b (+) expression vector in a prokaryotic system. The protein was purified with Ni-NTA chromatography, confirmed for immunoreactivity with T. annulata positive serum via Western blot analysis, and used to optimize both dot-ELISA and plate-ELISA. Both dot-ELISA and plate-ELISA using recombinant Tams1 protein exhibited comparable diagnostic performance, with a kappa value of 0.826 and similar analytical productivity ( P  = 0.6165). Dot-ELISA revealed a BTT seroprevalence of 58.4% in the cattle population, demonstrating good sensitivity (93.33%) and specificity (90%). The diagnostic performance of dot-ELISA was found to be superior to other molecular techniques, including microscopy, single PCR, and nested PCR. Dot-ELISA is also a sustainable solution in comparison to other laboratory diagnostic techniques with benefits of early diagnosis, reduced waste generation, resource efficiency, cost-effective point of care disease surveillance. With its minimal antigen requirement, Tams1 molecule based dot-ELISA is recommended as an effective tool for epidemiological studies and field surveys of BTT.

Theileriosis, babesiosis, anaplasmosis, and ehrlichiosis are the most important constraints to livestock production in Karamoja region, North-eastern Uganda. However, there are no large-scale studies on the prevalence and seasonal variation of tick-borne haemoparasites that are needed to design and implement tick-borne disease control programs. We collected 7080 blood samples from cattle across four districts of north-eastern Uganda during the dry (November 2022 to February 2023) and wet (July to August 2023) seasons. These samples were screened for the most important tick-borne haemoparasites (TBH) by conventional PCR, followed by capillary sequencing of representative PCR amplicons. There was no statistically significant difference [ p  > 0.05] in the overall prevalence of infection with at least one of the screened TBHs during the wet [39.0%; CI 7.3–40.6] and dry seasons [39.2%: CI 37.6–40.9]. Prevalence of the individual TBHs during the dry season were:— Babesia bigemina 11.8% (CI 10.8–12.9), Babesia bovis 11.8% (CI 10.8–12.9), Anaplasma marginale 9.2% (CI 8.2–10.2), Ehrlichia ruminantium 5.1% (CI 4.4–5.8) and Theileria parva 1.3% (CI 1.0–1.8). Prevalence of individual TBHs during the dry season were:— T. parva 22.6% (CI 21.3–24), A. marginale 13.6% (CI 12.5–14.8), B. bigemina 12.7% (CI 11.6–13.8), E. ruminantium 1.4% (CI 1.1–1.9) and B. bovis 0.3% (CI 0.1–0.5). Geospatial location, increasing age, sex, overnight stay in cattle kraals, and cattle breeds were significant predictors of infection with different TBHs during either season. Co-infection with the individual TBHs ranged between 0.14–2.74% and 0–1.64% during the dry and wet seasons respectively. In both seasons, the co-infection rate with all five TBHs was 0.03% (CI 0.0–0.16). Phylogenetic analyses of the representative TBH sequences revealed high level of conservation within the targeted genes of the samples in this study and those within the East Africa region that were retrieved from the GenBank. This study demonstrate high level of infection/co-infection with different TBHs in both dry and wet seasons indicating that ticks and tick-borne diseases are a major impediment to livestock production in Karamoja region. This shows the need of having a ticks and tick-borne disease control program. Moreover, B. bovis was detected for the first time in this region.