Molecular prevalence, associated risk factors and phylogenetic evaluation of Theileria lestoquardi in the blood samples of small ruminants.

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.

Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi.

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64-90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation.

Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat, India.

BACKGROUND OBJECTIVES: Vector-borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at the identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region in India using microscopy and Polymerase Chain Reaction (PCR). METHODS: A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. RESULTS: The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45 % sensitive and 100 % specific). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. INTERPRETATION CONCLUSION: Despite holding much significance to the animal sector, little work has been undertaken in regional parts of India regarding camel parasites. The present study offers first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

Molecular detection of piroplasms in domestic donkeys in Xinjiang, China.

BACKGROUND: Piroplasmosis is a common and prevalent tick-borne disease that affects equids. OBJECTIVES: To determine the infection and molecular characteristics of the piroplasms in donkeys from Xinjiang, northwestern China, we undertook a cross sectional study by collecting representative samples across several counties within the region. METHODS: A total of 344 blood samples were collected from adult domestic donkeys from 13 counties in Xinjiang. PCR was conducted to test for T. equi and B. caballi in the blood samples based on the equine merozoite antigen-1 (Ema-1) gene and the 48 kDa rhoptry protein (BC48) gene, respectively. RESULTS: Sixteen blood samples tested positive for piroplasms and the overall infection rate was 4.7% (16/344). Seven of the 13 counties were positive for piroplasms. Among the 16 piroplasm-positive samples, 15 were singly infected with T. equi with an infection rate of 4.4% (15/344), and coinfection with T. equi and B. caballi was detected in one sample (0.3%, 1/344) from Wushi. Four T. equi sequence genotypes were identified and grouped into different branches of the evolutionary trees. CONCLUSION: These findings suggest that the infection rate of piroplasms is low in domestic donkeys in southern Xinjiang and that T. equi genotypes have a regional distribution.

Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt.

BACKGROUND: Tick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene. RESULTS: We collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries. CONCLUSION: This study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Expression of IL-10 and TGF-ß1 in horses experimentally infected with T. equi merozoites is associated with antibody production but not modulation of pro-inflammatory responses.

Theileria equi (T. equi) is an apicomplexan parasite that causes severe hemolytic anemia in equids. Presently, there is inadequate knowledge of the immune responses induced by T. equi in equid hosts impeding understanding of the host parasite relationship and development of potent vaccines for control of T. equi infections. The objective of this study was to evaluate the host-parasite dynamics between T. equi merozoites and infected horses by assessing cytokine expression during primary and secondary parasite exposure, and to determine whether the pattern of expression correlated with clinical indicators of disease. Our findings showed that the expression of pro-inflammatory cytokines was very low and inconsistent during both primary and secondary infection. There was also no correlation between the symptoms observed during primary infection and expression of the cytokines. This suggests that the symptoms might have occurred primarily due to hemolysis and likely not the undesirable effects of pro-inflammatory responses. However, IL-10 and TGF-ß1 were highly expressed in both phases of infection, and their expression was linked to antibody production but not moderation of pro-inflammatory cytokine responses.

Molecular detection of tick-borne piroplasmids in camel blood samples collected from Cairo and Giza governorates, Egypt.

Piroplasmosis, a tick-borne disease affecting livestock, including camels, is caused by intracellular apicomplexan parasites belonging to the order Piroplasmida. Despite its importance, there's limited research on piroplasmosis among Egyptian camels. This study aimed to fill this gap by investigating tick-borne piroplasmids in camels from Cairo and Giza Governorates. Out of 181 blood samples collected between October 2021 and March 2022 from apparently healthy one-humped camels (Camelus dromedarius), PCR assays revealed a 41.4 % infection rate with various piroplasmids. Detected species included B. bovis (17.7 %), B. bigemina (12.2 %), B. caballi (8.3 %), B. naoakii (11.6 %), B. microti (1.7 %), T. equi (4.4 %), and Theileria spp. (28.7 %). Phylogenetic analysis revealed the first detection of T. equi genotype E in Egypt and identified a novel B. caballi genotype. Additionally, B. microti isolates were identified as the US-type. These findings shed lights on piroplasmosis among Egyptian camels, and provide valuable information for devising effective control strategies, especially B. microti, a pathogen with potential human health risks.

Unveiling genotypic diversity of Theileria orientalis in lethal outbreaks among bovines in Karnataka, India.

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts.

Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

The interplay of cytokines in bovine tropical theileriosis: a mini review.

Studying cytokine profiling in Theleria annulata infection enhances our understanding of how the immune response unfolds, the intricate interactions between the host and the parasite, the strategies employed by the parasite to evade the immune system, and potential avenues for developing treatments. The generation of pro-inflammatory cytokines plays a pivotal role in the immune response against T. annulata infection. Elevated concentrations of these cytokines potentially contribute to the manifestation of clinical symptoms associated with the disease, such as fever, anemia, exophthalmia, and weight loss. The production of anti-inflammatory cytokines potentially serves as a regulatory mechanism for the immune response, preventing the development of severe disease. Nevertheless, in animals afflicted by T. annulata infection, there is often a notable decrease in the levels of these cytokines, suggesting that they may not be as effective in mitigating the disease as they are in uninfected animals. This knowledge can be harnessed to develop improved diagnostic methods, treatments, and vaccines for tropical theileriosis. The objective of this current mini review is to achieve the same goal by consolidating the available knowledge of cytokine interactions in Bovine Tropical Theileriosis (BTT).

TurboID mapping reveals the exportome of secreted intrinsically disordered proteins in the transforming parasite Theileria annulata.

Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.

Comparative transcriptional analysis identifies genes associated with the attenuation of Theileria parva infected cells after long-term in vitro culture.

Autologous administration of attenuated Theileria parva-infected cells induces immunity to T. parva in cattle. The mechanism of attenuation, however, is largely unknown. Here, we used RNA sequencing of pathogenic and attenuated T. parva-infected T-cells to elucidate the transcriptional changes underpinning attenuation. We observed differential expression of several host genes, including TRAIL, PD-1, TGF-ß and granzymes that are known to regulate inflammation and proliferation of infected cells. Importantly, many genes linked with the attenuation of the related T. annulata-infected cells were not dysregulated in this study. Furthermore, known T. parva antigens were not dysregulated in attenuated relative to pathogenic cells, indicating that attenuation is not due to enhanced immunogenicity. Overall this study suggests that attenuation is driven by a decrease in proliferation and restoration of the inflammatory profile of T. parva-infected cells. Additionally, it provides a foundation for future mechanistic studies of the attenuation phenotype in Theileria-infected cells.

Evaluation of haematological parameters in haemolytic anaemia caused by tick-borne pathogens in grazing cattle.

BACKGROUND: No tick-borne pathogens (TBPs) causing haemolytic anaemia in cattle have been reported, except Theileria orientalis and complete blood count (CBC) profile is the only haematological parameter to determine the severity of regenerative haemolytic anaemia. OBJECTIVES: To identify the causative agents of TBP-induced haemolytic anaemia and determine haematological parameters that indicate haemolytic anaemia in grazing cattle. METHODS: Eighty-two Korean indigenous cattle (Hanwoo) were divided into two groups: grazing (n = 67) and indoor (n = 15) groups. CBC and serum biochemistry were performed. PCR was conducted using whole blood-extracted DNA to investigate the prevalence of TBPs. RESULTS: TBP-induced haemolytic anaemia was observed in the grazing group. In grazing cattle, co-infection (43.3%, 29/67) was most frequently detected, followed by T. orientalis (37.6%, 25/67) and Anaplasma phagocytophilum infections (1.5%, 1/67). In indoor cattle, only co-infection (20%, 3/15) was identified. Grazing cattle exhibited regenerative haemolytic anaemia with marked monocytosis, mild neutropenia, and thrombocytopenia. According to grazing frequency, the 1st-time grazing group had more severe anaemia than the 2nd-time grazing group. Elevations in indirect bilirubin and L-lactate due to haemolytic anaemia were identified, and correlations with the respective markers were determined in co-infected grazing cattle. CONCLUSIONS: Quantitative evaluation of haematocrit, mean corpuscular volume, and reticulocytes (markers of regenerative haemolytic anaemia in cattle) was performed for the first time. Our results show that, in addition to T. orientalis, A. phagocytophilum is strongly associated with anaemia. The correlation between haemolytic anaemia severity and haematological parameters (indirect bilirubin, reticulocytes, and L-lactate) was confirmed.

Molecular detection and characterization of prevailing Theileria equi genotype in equine from northern India.

Equine piroplasmosis caused by Theileria equi is a febrile, tick-borne disease of equids. However, there is limited literature about the genotyping of T. equi in India. Blood samples were collected from 202 horses and subjected to microscopy and PCR to detect T. equi. Initially, a universal screening primer pair targeting 18S ribosomal RNA genes common for Babesia caballi and T. equi was employed to amplify the DNA of both parasites. Thereafter additional primers were employed for species-specific detection resulting in amplification of approximately 435 bp specific for T. equi. T.equi was detected in 9.9% and 20.79% of horses screened by microscopy and PCR, respectively. The representative samples confirmed positive by PCR were sequenced, submitted to NCBI (OR651254, OR687254, OR685656, OR650830, OR650834), and used for genotype characterization and phylogenetic analysis. Employing Genetool and MEGA X software, the T. equi Indian isolates and across the globe were compared, and the results demonstrated 99.05-100% and 95.86-100% homologies, respectively. All the T. equi Indian isolates belonged to genotype A. Phylogeny based on the EMA-1 gene of five isolates (OR731831, OR731833, OR731829, OR731830, OR731832) were also characterized by sequencing and support the previous findings. Genotypes C and D, as well as genotypes B and E, exhibited lower levels of evolutionary divergence compared to other genotypes. The EMA-1 gene exhibited limited diversity and might not be the most suitable target for assessing variability within T. equi populations. The findings also reveal a significant association (p < 0.01) between T. equi infection and the presence of ticks.

Establishment and application of TaqMan real-time PCR method for detection of Theileria annulata resistant to buparvaquone.

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application.

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.

Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Theileria parasites sequester host eIF5A to escape elimination by host-mediated autophagy.

Intracellular pathogens develop elaborate mechanisms to survive within the hostile environments of host cells. Theileria parasites infect bovine leukocytes and cause devastating diseases in cattle in developing countries. Theileria spp. have evolved sophisticated strategies to hijack host leukocytes, inducing proliferative and invasive phenotypes characteristic of cell transformation. Intracellular Theileria parasites secrete proteins into the host cell and recruit host proteins to induce oncogenic signaling for parasite survival. It is unknown how Theileria parasites evade host cell defense mechanisms, such as autophagy, to survive within host cells. Here, we show that Theileria annulata parasites sequester the host eIF5A protein to their surface to escape elimination by autophagic processes. We identified a small-molecule compound that reduces parasite load by inducing autophagic flux in host leukocytes, thereby uncoupling Theileria parasite survival from host cell survival. We took a chemical genetics approach to show that this compound induced host autophagy mechanisms and the formation of autophagic structures via AMPK activation and the release of the host protein eIF5A which is sequestered at the parasite surface. The sequestration of host eIF5A to the parasite surface offers a strategy to escape elimination by autophagic mechanisms. These results show how intracellular pathogens can avoid host defense mechanisms and identify a new anti-Theileria drug that induces autophagy to target parasite removal.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.