A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

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 IC50 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 Q01 site. The bound drug in the Q01 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.

Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.

Theileria annulata subtelomere-encoded variable secreted protein-TA05560 interacts with bovine RNA binding motif protein 39 (RBM39).

Theileria annulata is the only eukaryotic pathogen able to transform bovine leukocytes, including B cells, macrophages and dendritic cells. T. annulata-transformed cells exhibit several cancer-like phenotypes, such as hyperproliferation, immortalization and dissemination. Although several parasite factors involved in bovine cell transformation have been explored, the roles of subtelomere-encoded variable secreted proteins (SVSPs) of the parasite in host-cell interactions are largely unknown. In the present study, the target molecule TA05560, a member of the SVSP multigene family of T. annulata, was identified at the mRNA level during different life cycles through a quantitative real-time PCR assay, and the subcellular distribution of TA05560 was examined via confocal microscopy. The results showed that the parasite molecule TA05560 was transcribed mainly in the schizont stage of T. annulata infection, and the protein was distributed in the nucleus and cytoplasm of the parasitized cells. The potential host cell proteins that interact with TA05560 were screened using the yeast two-hybrid system, and the direct interaction between TA05560 and its prey protein, Bos taurus RNA binding motif protein 39 (RBM39) was further identified in HEK293T cells by using confocal microscopy, coimmunoprecipitation and bimolecular fluorescence complementation assays. Moreover, the interaction between TA05560 and its host protein was observed in T. annulata-infected cells via confocal microscopy. Therefore, our study is the first to show that the T. annulata-secreted TA05560 protein directly binds to both the exogenous and endogenous host cell molecule RBM39, laying the foundation for exploring host-parasite interactions and understanding the transformation mechanisms induced by T. annulata and other transforming parasites.

Characterisation of field tropical Theileriosis and associated risk factors in two bioclimatic areas of Algeria.

Tropical theileriosis (TT) is a tick-borne disease caused by Theileria annulata and commonly infects cattle in tropical and subtropical regions, including Algeria. It is a significant obstacle to cattle breeding programs established to improve production in Algeria. The present investigation aimed to estimate the current molecular prevalence, risk factors, and genetic characterisation of T. annulata in two bioclimatic areas of Algeria. In a cross-sectional study, 679 blood samples (629 from healthy cattle selected on farms and 50 from diseased cattle identified by veterinarians) were collected from the humid (n = 307+50) and semi-arid (n = 322) areas and screened by blood smear examination followed by polymerase chain reaction targeting cytochrome oxidase subunit 3 (cox III) mitochondrial and the 18S ribosomal RNA (18S rRNA) genes for Theileria spp. Seventy-six positive samples (56 clinically healthy and 20 with clinical signs) for Theileria spp. were confirmed to be T. annulata by the merozoïtes surface antigen-1 (Tams1) gene showing a rate of 8.9 % in clinically healthy and 40.0 % in suspected cattle. Among the 307 bloods samples collected from healthy cattle in the humid area, 25 cattle (8.1 %) were positive for T. annulata. Of the 322 healthy cattle from the semi-arid site, 31 (9.6 %) were carriers of T. annulata DNA. In subclinical population, demographic and environmental parameters analysis indicated that T. annulata infection was higher in adult crossbred cattle raised in the intensive and semi-intensive system (P<0.001). The multiple logistic regression analysis showed that age, breed, farming system, and bioclimatic area are potential risk factors for T. annulata infection in cattle (P<0.05). Multiple alignments of cox III sequences of T. annulata showed high heterogeneity with 25 polymorphic sites (nucleotide diversity π = 0.02402), resulting in two haplotypes with a low genetic diversity index (Hd) of 0.533. The 18S rRNA sequence alignment revealed only one T. annulata genotype with 100 % identity to the strains isolated from cattle and ticks in Mediterranean and Asian countries. Our preliminary results will serve as a basis for further studies on the genetic diversity and molecular epidemiology of T. annulata.

Wide bovine tick-borne pathogen spectrum: Predominancy of Theileria annulata and the first molecular detection of Ehrlichia minasensis in Turkey.

Vector-borne diseases indulge in severe economic losses in the livestock industry by adversely affecting cattle breeding in tropical and subtropical zone countries, including Turkey, encompassing a wide land area representing diverse climatic conditions. This study aimed to investigate significant bovine tick-borne piroplasm, rickettsia, and some other bacterial agents by genus- or species-specific PCR and nested PCR techniques in Turkey. A total of 210 cattle blood samples were collected from sixteen provinces in different geographical regions of Turkey. PCR analyses were performed targeting the detection of Babesia/Theileria/Hepatozoon sp. 18S rRNA, Babesia/Theileria sp. 18S rRNA (V4), B. bigemina RAP-1a, B. bovis SBP-4, B. ovata AMA-1, B. naoaki AMA-1, T. annulata Tams-1, T. orientalis MPSP, T. mutans 18S rRNA, Anaplasma/Ehrlichia sp. 16S rRNA, A. marginale MSP4, A. bovis 16S rRNA, A. phagocytophilum 16S rRNA, A. capra 16S rRNA, E. ruminantium pSC20, Mycoplasma sp. 16S rRNA, and Coxiella burnetii 16S rRNA genes. Overall, 133 (63.3%) cattle were found to be infected with at least one of the following protozoan or bacterial pathogens; B. bovis, B. bigemina, B. occultans, T. annulata, T. orientalis, A. marginale, A. phagocytophilum, and Mycoplasma sp. The total prevalence of pathogens was determined as follows; 0.5% B. bovis, 0.5% B. bigemina, 1.4% B. occultans, 41.0% T. annulata, 1.4% T. orientalis, 10.5% A. marginale, 13.8% A. phagocytophilum, 0.5% A. bovis, 2.9% Uncultured Anaplasma sp., 0.5% E. minasensis, 0.5% Uncultured Ehrlichia sp., and 23.3% Mycoplasma sp. Moreover, large part of the total infection (n:133) was composed of single infections (63.9%); however, double (24.8%), triple (7.5%), quadruple (2.3%), and quintuple (1.5%) co-infections were also encountered. In addition to some bovine pathogens such as B. occultans, T. orientalis, A. bovis, M. wenyonii, and Candidatus Mycoplasma haemobos, which were rarely reported in Turkey, sequencing and phylogenetic analysis revealed the first detection of Uncultured Ehrlichia sp. (0.5%), and E. minasensis (0.5%) with 100% nucleotide sequence identities. The study also indicates that the spectrum of pathogens harbored by Turkish cattle is quite wide, and these pathogens cause multiple co-infections with various combinations, and T. annulata stands out as the primary bovine pathogen among them.

Population genetic characterization of Theileria annulata based on the cytochrome b gene, with genetic insights into buparvaquone susceptibility in Haryana (India).

The present investigation was aimed at population genetic characterization of Theileria annulata on the basis of the cytochrome b (cyt b) gene along with the evaluation of status of buparvaquone resistance in Haryana (India). The sequences originating from China, Egypt, India, Iran, Iraq, Tunisia, Turkey and Sudan were included in the analysis. The maximum likelihood tree based on the Tamura-Nei (TN93+G) model placed all the sequences of T. annulata into a single clade. The median-joining haplotype network exemplified geographical clustering between T. annulata haplotypes originating from each country. Only five haplotypes (7.81 %) were shared between any two countries, while the remaining 59 haplotypes (92.19 %) were singleton and unique to one country. The values of pairwise genetic distance (FST) between all the populations indicated huge genetic differentiation (> 0.25) between different T. annulata populations, barring the FST value between Iraq and Turkey (0.14454) which suggested a moderate differentiation. Contrary to the FST index, the values of gene flow (Nm) between T. annulata populations were very low. The neutrality indices and mismatch distributions indicated a population expansion in the Indian T. annulata population. Furthermore, the secondary structure and homology modeling of the partial cyt b protein is also reported. The molecular analysis of newly generated sequences for buparvaquone resistance revealed that all the isolates were susceptible to buparvaquone treatment. However, two novel mutations at positions V203I and V219I in between the Q01 and Q02 drug-binding regions of the cyt b gene were observed for the first time.

Tick-borne pathogens in camels: A systematic review and meta-analysis of the prevalence in dromedaries.

Published data on tick-borne pathogens (TBPs) in camels worldwide have been collected to provide an overview of the global prevalence and species diversity of camelid TBPs. Several TBPs have been detected in dromedary camels, raising concerns regarding their role as natural or maintenance hosts for tick-borne pathogens. Insubstantial evidence exists regarding the natural infection of camels with Babesia spp., Theileria spp., Anaplasma spp., and Ehrlichia spp., particularly because most of the camels were considered healthy at the time of sampling. Based on polymerase chain reaction (PCR) testing, a pooled prevalence of 35.3% (95% CI: 22.6-48.1%) was estimated for Anaplasma, which was the most frequently tested TBP in dromedaries, and DNA of Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma platys, and A. platys-like were isolated, of which ruminants and dogs are reservoirs. Similarly, the estimated pooled prevalence for the two piroplasmid genera; Babesia and Theileria was approximately equal (10-12%) regardless of the detection method (microscopy or PCR testing). Nevertheless, Babesia caballi, Theileria equi, and Theileria annulata DNA have frequently been detected in camels but they have not yet been proven to be natural hosts. Scarce data detected Babesia microti, Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato (s.l.) DNA in blood of dromedaries, although ticks of the genus Ixodes are distributed in limited areas where dromedaries are raised. Interestingly, a pooled seroprevalence of 47.7% (26.3-69.2%) was estimated for Crimean-Congo hemorrhagic fever virus, and viral RNA was detected in dromedary blood; however, their contribution to maintain the viral transmission cycles requires further experimental investigation. The substantially low incidence and scarcity of data on Rickettsia and Ehrlichia species could imply that camels were accidentally infected. In contrast, camels may play a role in the spread of Coxiella burnetii, which is primarily transmitted through the inhalation of aerosols emitted by diseased animals and contaminated environments. Bactrian camels showed no symptoms due to the examined TBPs, meanwhile, clinical disease was seen in alpacas infected with A. phagocytophilum. Similar to dromedaries, accidental tick bites may be the cause of TBP DNA found in the blood of Bactrian camels.

Epidemiology and molecular characterization of Theileria annulata in ticks collected from cattle in the central part of Tamil Nadu, India.

Tick-borne diseases are the most common in cattle in the tropical and subtropical regions of India and lead to substantial economic losses to small and marginal farmers. This study aimed to identify the diverse species of ticks infesting cattle in the central part of Tamil Nadu, India, and to assess the prevalence of Theileria annulata infection in various species of ticks through PCR. Out of 123 cross-bred and 105 native breed cattle examined for tick infestation, 40 (18%) and 29 (12.7%) cattle were infested with Ixodid ticks, respectively. The most prevalent tick species identified was Rhipicephalus microplus (n=589), followed by Hyalomma anatolicum (n=532), Hyalomma marginatum (n=145), Haemaphysalis intermedia (n=79), and Rhipicephalus haemophysaloides (n=1) found in the study area. The prevalence and intensity of the tick infestation were found to be higher in cross-bred (71.04%) than native breed cattle (28.96%), and there was no significant difference between the studied breeds (chi-square value =24; df =20; p value =0.24) was observed. However, a significant difference in the H. anatolicum tick infestation was observed between the Cauvery Delta (14.30%) and the North-Western (20%) zones of Tamil Nadu (p<0.05). DNA fragments of 193 bp derived from 18S rRNA gene sequences of T. annulata were amplified using species-specific primers. Of these, 16 out of 37 (43.2%) and 10 out of 39 (29%) pooled samples of H. anatolicum and 4 out of 18 (22.2%) and 1 out of 5 (20%) pooled samples of H. marginatum were found positive for T. annulata from the Cauvery Delta and North-Western zones, respectively. R. microplus, H. intermedia, and R. haemaphysaloides from these regions were negative. These findings confirm that H. anatolicum (52.17%) is the predominant vector for T.annulata rather than H. marginatum (18.84%), and the PCR is a useful method of determining the infection rates in ticks collected from animals carrying low levels of T. annulata piroplasms.

Epidemiological situation of bovine tropical theileriosis in an arid region in central Tunisia with a phylogenetic analysis of Theileria annulata.

BACKGROUND: Tropical theileriosis, Theileria annulata infection, is the most prevalent summer disease in Tunisia. It is transmitted by Hyalomma scupense, a two-host tick known to be endophilic. OBJECTIVES: The present study aimed to estimate the infection prevalence of cattle by T. annulata in two districts from central Tunisia. METHODS: Blood samples collected from 270 Holstein cattle from the Sidi Bouzid (140 samples) and Kasserine districts (130 samples) were analysed by Giemsa staining and T. annulata-specific PCR. RESULTS: In both regions, PCR revealed a prevalence of 32.6%. This was significantly higher than the 6.3% prevalence obtained by Giemsa staining blood smears (p < 0.001). Giemsa staining also revealed a low parasitaemia of 0.05%. The PCR-based prevalence was not statistically different between the two districts (31.4 ± 0.04 and 33.8 ± 0.04% in Sidi Bouzid and Kasserine districts, respectively, p = 0.6). On the contrary, the results of blood smear examination (2.85 and 10% in Sidi Bouzid and Kasserine, respectively) differed significantly between the two sampling sites (p = 0.01). There was no evidence of a statistically significant difference between the overall molecular infection prevalence when the samples were segregated based on animals' age or gender (p = 0.1 and 0.2, respectively) and a similar trend was observed for Giemsa staining. Ten PCR amplicons of the Tams1 gene (721 bp) were subsequently sequenced from the two regions. The phylogenetic analyses showed 100% similarity between all sequences. The unique conserved Tams1 sequence was deposited in GenBank under the accession number OP428816 and used to infer its phylogenetic relationships with those available in the GenBank repository. CONCLUSIONS: This is the first report of the presence of T. annulata in this region of central Tunisia which has no history of tropical theileriosis. Priority areas for future studies include understanding the origin of these T. annulata-positive animals in a region where the presence of a known natural vector tick, H. scupense, has not been reported. Given that the disease severely constrains cattle productivity, it would also be worthwhile to investigate if other potential vectors for T. annulata, such as Hyalomma dromedarii, are present in the arid regions.

Comprehensive genetic diversity and molecular evolutionary analysis of Theileria annulata isolates based on TAMS 1 gene.

Molecular epidemiological studies related to the phylogenetic characterization of Theileria annulata are important in delineating the evolutionary history of the parasite. In the current study, the Theileria annulata (T. annulata) merozoite surface antigen 1 (TAMS 1) gene from 14 bovine isolates of T. annulata originating from semi-arid zone of northern India were amplified and sequenced. TAMS 1 gene sequences (n= 337) reported from 16 countries were subsequently analyzed for haplotype network along with genetic diversity. A total of five haplotypes out of the 14 sequenced isolates and 92 haplotypes out of 337 worldwide sequences are documented in this study. Phylogenetic and molecular evolutionary analyses based on TAMS 1 gene sequences showed that T. annulata is dissipated across different countries and numerous strains are closely linked, even though they belong to different geographical locations. The nucleotide homology between 14 isolates from northern India varied between 91.3 and 100%, whereas it was between 31.5 and 100% when sequences across the globe were compared. Haplotype 14 was recognized as most widely distributed haplotype, with 46 isolates circulating in 10 countries. Globally, negligible genetic distance (FST˂0.15) and very high gene flow (Nm˃1) was found in the five populations of the world (South Asia, East Asia, West Asia, Europe and Africa), supporting the absence of clearly defined subgroups in the phylogenetic analysis. Significant negative values of neutrality tests; Tajima's D (D) and Fu and Li's F (F) provided evidence for recent population expansion through positive selection of advantageous variations.

An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan.

Cattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs.

Molecular investigation of bovine tropical theileriosis outbreak in an organized dairy cattle farm in Madhya Pradesh, India.

The present study aimed to investigate an outbreak of Theileria annulata (T. annulata) infection in an organized dairy cattle farm in Madhya Pradesh, India, using clinical and molecular techniques. Following the deaths of two crossbred cattle in March 2021, 43 blood samples were collected from infected and apparently healthy animals and examined by blood smear and polymerase chain reaction (PCR) techniques. The blood smear examination showed that 23.25% of samples were positive for Theileria organisms, while conventional PCR targeting the 18S ribosomal RNA (18S rRNA) and T. annulata merozoite surface antigen-1 (TAMS-1) genes revealed that 32.55% of samples were positive for T. annulata. PCR targeting cytochrome b (Cytb) gene showed 46.51% of samples were positive for T. annulata. Haematological analysis confirmed clinical signs of infection in affected animals, which were treated with buparvaquone @ 2.5 mg/kg body weight intramuscularly along with supportive medicine. Two 18S rRNA gene amplicons were sequenced and analysed in a phylogenetic tree and haplotype network with 54 Indian and 38 foreign sequences. The phylogenetic tree revealed two groups with a high posterior probability and bootstrap value, while the haplotype network revealed 35 haplotypes, with haplotype 1 (H1) being the most abundant and several single haplotypes clustering around it, indicating fast and widespread expansion. Genetic diversity indices and neutrality tests confirmed that the population was expanding. These studies highlight the significance of prompt and precise diagnosis and management of T. annulata outbreaks and provide insights into its evolutionary history and population dynamics of T. annulata in India, which could aid improving disease preventive and control strategies.

Simultaneous molecular detection of Anaplasma marginale and Theileria annulata in cattle blood samples collected from Pakistan-Afghanistan boarder region.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

Molecular detection of Theileria annulata, Theileria mutans and Theileria velifera but no evidence of Theileria parva infected or vaccinated cattle in Nigeria despite extensive transboundary migrations.

The extensive livestock management system predominant in Nigeria necessitates active disease surveillance for the early detection and prompt control of transboundary animal diseases. Theileriae are obligate intracellular protozoa which infect both wild and domestic bovidae throughout much of the world causing East Coast Fever (Theileria parva), Tropical or Mediterranean theileriosis (Theileria annulata) or benign theileriosis (Theileria mutans; Theileria velifera). This study aimed to detect and characterize Theileria spp. infecting cattle in Nigeria using conventional PCR and sequencing approach. Five hundred and twenty-two DNA samples obtained from different cattle blood samples were subjected to PCR targeting the 18S rRNA gene of piroplasmida and specifically, the p104 kDa and Tp1 genes for the evidence of infection or vaccination respectively, with T. parva. A total of 269 out of 522 (51.5%) of the cattle tested PCR- positive for DNA of piroplasmida. Nucleotide sequence and phylogenetic analyses showed that the cattle were infected with T. annulata, T. mutans and T. velifera. Piroplasmida DNA was associated with sex (ꭓ2 = 7.2; p = 0.007), breed (ꭓ2 = 115; p = 0.000002) of animals and the state where the samples were collected (ꭓ2 = 78.8; p = 0.000002). None of the samples tested positive for T. parva DNA or showed evidence of vaccination (Tp1 gene). This is the first report on the molecular detection and characterization of T. annulata in the blood of cattle from Nigeria. Continuous surveillance of Nigerian cattle for East Coast Fever (ECF) is encouraged considering the recent report of the disease in cattle in the neighboring country, Cameroon, where unregulated transboundary cattle movement into Nigeria has been observed.

Artemisinin derivatives induce oxidative stress leading to DNA damage and caspase-mediated apoptosis in Theileria annulata-transformed cells.

BACKGROUND: Bovine theileriosis caused by the eukaryotic parasite Theileria annulata is an economically important tick-borne disease. If it is not treated promptly, this lymphoproliferative disease has a significant fatality rate. Buparvaquone (BPQ) is the only chemotherapy-based treatment available right now. However, with the emergence of BPQ resistance on the rise and no backup therapy available, it is critical to identify imperative drugs and new targets against Theileria parasites. METHODS: Artemisinin and its derivatives artesunate (ARS), artemether (ARM), or dihydroartemisinin (DHART) are the primary defence line against malaria parasites. This study has analysed artemisinin and its derivatives for their anti-Theilerial activity and mechanism of action. RESULTS: ARS and DHART showed potent activity against the Theileria-infected cells. BPQ in combination with ARS or DHART showed a synergistic effect. The compounds act specifically on the parasitised cells and have minimal cytotoxicity against the uninfected host cells. Treatment with ARS or DHART induces ROS-mediated oxidative DNA damage leading to cell death. Further blocking intracellular ROS by its scavengers antagonised the anti-parasitic activity of the compounds. Increased ROS production induces oxidative stress and DNA damage causing p53 activation followed by caspase-dependent apoptosis in the Theileria-infected cells. CONCLUSIONS: Our findings give unique insights into the previously unknown molecular pathways underpinning the anti-Theilerial action of artemisinin derivatives, which may aid in formulating new therapies against this deadly parasite. Video abstract.

Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle.

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

miR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes.

MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.

Identification of a Novel Interaction between Theileria Prohibitin (TaPHB-1) and Bovine RuvB-Like AAA ATPase 1.

Theileriosis is a tick-borne disease caused by Theileria annulata, an intracellular parasite that belongs to the phylum Apicomplexa. The infective forms of the parasite to cattle are sporozoites that are introduced into the host when the infected ticks take a blood meal. The sporozoites selectively invade bovine B cells, macrophages, or monocytes, leading to their cellular transformation. The parasite factors involved in the host cell transformation are not well explored. In pursuit of this, we revisited the probable secretome of the parasite and, following a stringent downscaling criterion, have identified Theileria prohibitin (TaPHB-1) as one of factors secreted into the host cells. Interestingly, in infected cells, TaPHB-1 localized both on the parasite surface and in the host cytoplasm, and independent approaches such as coimmunoprecipitation, yeast two-hybrid screening (Y2H), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed RuvB-like AAA ATPase 1 (RUVBL-1) as one of its interacting partners. Further, the T. annulata infection does not affect the localization of bovine prohibitin. Mitigating the expression of bovine RUVBL-1 precluded the translocation of TaPHB-1 in the host cell cytoplasm without affecting the host cell viability. Taken together, we report for the first time a unique interaction of TaPHB-1 with bovine RUVBL-1 that is likely needed to cause cancer-like hallmarks during theileriosis. IMPORTANCE Theileria annulata is an apicomplexan parasite that causes tropical theileriosis in cattle. It is the only eukaryotic pathogen able to cause cellular transformation of host cells yielding a cancer-like phenotype. The parasite factors responsible for the transformation of the host cell are largely unknown. This study demonstrates for the first time the partial role of Theileria prohibitin (TaPHB-1) in maintaining the transformed state of the host cell and its interaction with RuvB-like AAA ATPase 1 (RUVBL-1) in a T. annulata-infected bovine cell line. Interestingly, the knockdown of bovine RUVBL-1 rendered the parasites metabolically inactive, implying that the identified interaction is critical for parasite survival. This study contributes to our understanding the Theileria-host interactions and offers scope for novel therapeutic interventions to control theileriosis.

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone.

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydin region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal.