Search

Background: Hemoprotozoan diseases, especially trypanosomosis and theileriosis, adversely affect the productivity, growth, and performance of camels. Regular sampling and investigation of camels are challenging due to several factors. Consequently, there is a lack of knowledge on camel parasite genotyping, cytokine production, and oxidative stress parameters during infection. Methods: The present study investigated two critical blood protozoa infecting camels in Egypt, Trypanosoma evansi and Theileria annulata, using molecular methods, specifically 18S rRNA gene analysis. Following molecular confirmation, experimental infections were induced in Swiss albino mice to assess the expression of immune response genes and oxidative stress parameters. The study further explored the correlation between histopathological alterations and inflammatory reactions in the kidney, spleen, and liver of infected mice, alongside the immunohistochemical expression of caspase-3, proliferating cell nuclear antigen (PCNA), and tumor necrosis factor (TNF). Results: Trypanosoma evansi and T. annulata isolated from naturally infected camels were molecularly identified and deposited in GenBank under accession numbers OR116429 and OR103130, respectively. Infection with T. evansi and T. annulata caused significant adverse effects on the immune condition of infected mice, increasing the pathogenicity of the infection. This was evidenced by a significant increase in oxidative stress parameter levels in both naturally infected camels and experimentally infected mice compared to healthy controls. Furthermore, the expression of immune response genes was significantly elevated in infected mice. Immunohistochemistry analysis showed a pronounced upregulation of caspase-3, PCNA, and TNF in the infected groups relative to the control group. These findings are the first to be reported in Egypt. Conclusions: This study successfully identified and genotyped two economically important blood protozoa, T. evansi and T. annulata, from camels in Egypt. Additionally, the experimental animal model provided valuable insights into the immune response, oxidative stress, and histopathological changes induced by these parasites, demonstrating comparable results to naturally infected camels. These findings highlight the potential of this model to study parasite–host interactions and immune responses, contributing to a better understanding of the pathogenic mechanisms of T. evansi and T. annulata infections. This model may be useful for future studies focused on disease control and therapeutic interventions. [ABSTRACT FROM AUTHOR]

Babesia bigemina and Theileria annulata are tick-borne protozoans that cause piroplasmosis in cattle, resulting in huge damages to the livestock industry. The prevalence of these infections depends on various intrinsic and extrinsic risk factors. In Pakistan, there is no information regarding the molecular characterization of Babesia bigemina and the risk factors associated with piroplasmosis. This study aimed to molecularly characterize Babesia spp. and Theileria spp. infecting various cattle breeds in Khyber Pakhtunkhwa, Pakistan, and to shed light on risk factors associated with these infections. Altogether, 219 blood samples were collected from various symptomatic cattle breeds, including Holstein Friesian (65.3%; 143/219), Jersey (21.5%; 47/219) and Sahiwal (13.2%; 29/219). Isolated genomic DNA from these blood samples was used in PCR for the amplification of the 18S rRNA fragment of apicomplexan parasites. Obtained 18S rDNA sequences from cattle hosts showed 99.5% identity with B. bigemina, or 100% with T. annulata. Having an overall infection rate of 61.6% (135/219), the highest infection rate was recorded for T. annulata (43.8%; 95/219), followed by B. bigemina (18.3%; 40/219). Phylogenetic analysis of 18S rDNA sequences revealed that B. bigemina clustered with corresponding species reported from Bolivia, and South Africa, while T. annulata grouped with same species from Italy, India, and Turkey. Among the different risk factors, the breed, season, and tick infestation were found to have a significant (P < 0.05) association with the piroplasmid infections. The information obtained in this study can be employed for effective surveillance and control of babesiosis and theileriosis in Pakistan. In addition to confirming our previous molecular detection of T. annulata in cattle, this study provides the first molecular surveillance and phylogenetic position of B. bigemina and associated risk factors in the study region. [ABSTRACT FROM AUTHOR]

This study was designed to conduct phylogenetic analysis on the cytochrome b and TaPIN1 genes of Theileria annulata and to detect potential associations between mutations in these two genes and resistance to buparvaquone. Positive PCR products for the cytochrome and TaPIN1 genes of two samples were sequenced and phylogenetically analysed. When cytochrome b gene sequences were aligned to the reference isolate (XM949625), the phylogenetic analysis showed the presence of two new T. annulata cyt b gene isolates. These isolates showed 99.9% homology with each other and exhibited high homology with existing Egyptian and global isolates and were deposited in GenBank under accession numbers (PP920503) and (PP920504) for samples from Alexandria and Beheira governorates, respectively. Also, there is one non-synonymous mutation in our two isolates in codon 146 (Alanine146Threonine), which is in the Qo1 region (one of the buparvaquone binding sites). When TaPIN1 gene sequences were aligned to the reference isolate (XM949502), the phylogenetic analysis showed the presence of two new T. annulata TaPIN1 gene isolates. These isolates showed 98.1% homology with each other and showed high homology with global isolates. These two isolates are the first Egyptian Theileria annulata TaPIN1 gene isolates and were deposited in GenBank under accession numbers (PP920505) and (PP920506) for samples from Alexandria and Beheira governorates, respectively. Moreover, phylogenetically, Also, there are two nonsynonymous mutations in these two isolates in codon 24 (Isoleucine24Valine) and 52 (Methionine52Threonine), which are related to resistance against buparvaquone. Briefly, this is the first Egyptian study that investigated the TaPIN1 gene of Theileria annulata and the first study in Egypt that investigated both the TaPIN1 and cytochrome b genes in the same samples. This study has introduced evidence of point mutations in the cytochrome b and TaPIN1 genes of T. annulata that might be associated with buparvaquone treatment failure in Egypt. [ABSTRACT FROM AUTHOR]

Tick and tick-borne pathogens are among the major threats to animal health and production causing huge economic losses in the form of morbidity and mortality throughout the world. Due to the scarcity of epidemiological data and appropriate diagnostic methods, the study was carried out to detect Anaplasma marginale and Theileria annulata from buffaloes of Sheikhupura, Jhang, and Rawalpindi districts by using duplex PCR. A total of 1,152 blood samples of buffaloes were screened for epidemiological investigations. DNA extracted from blood samples was used for the amplification of the msp1ß gene and cytochrome b gene by using single and duplex PCR. The prevalence of these pathogens and associated risk factors were observed through the multiple logistic regression method. The overall prevalence of a single infection of A. marginale was found to be 14.26% and for T. annulata it was 15.28%. The mixed infection, through duplex PCR in the buffalo population, was observed as 12.92%. Different associated risk factors were evaluated to assess their association with the prevalence of these pathogens. Generally, the prevalence was significantly higher in female buffaloes (P=0.0001) during summer season (P=0.0001). Sequencing and phylogenetic analysis of A. marginale showed that Sequence Pak-4* was closely similar to sequences CP006847.1 and appeared in the same clade. Sequence Pak-4* was ascended to Pak-3* isolate and descendant to MK792344.1 Pakistani isolate. On the other hand, phylogenetic analysis of T. annulata showed that the Pak-2* sequence appeared to be descendants of Indian isolate MH78945.1. In the future, this study will be helpful for large-scale analysis of multiple species by using duplex and multiplex PCR and also for improving the control of associated tick-borne diseases in endemic regions through vaccine development. [ABSTRACT FROM AUTHOR]

Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges. [ABSTRACT FROM AUTHOR]

Several miRNA-based studies on Theileria-transformed bovine cells have been conducted; however, the mechanism by which transformed cells exhibit uncontrolled proliferation is not yet fully understood. Therefore, it is necessary to screen more microRNAs that may play a role in the transformation process of host cells infected with Theileria annulata to better understand the transformation mechanisms of Theileria-infected cells. RNA sequencing was used to analyze miRNAs expression in the host bovine lymphocytes infected with T. annulata at different time points after buparvaquone (BW720) treatment and DMSO treatment (control groups). Differential miRNAs related to cell proliferation and apoptosis were identified through comparison with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and a regulatory network of miRNA-mRNA was constructed. In total, 272 differentially expressed miRNAs were found at 36, 60 and 72 h. The miRNAs change of bta-miR-2285t, novel-miR-622, bta-miR-2478, and novel-miR-584 were significant. Analysis of 27 of these co-differential expressed miRNAs revealed that 15 miRNAs were down-regulated and 12 miRNAs were up-regulated. A further analysis of the changes in the expression of each of these 27 miRNAs in the three datasets suggested that bta-miR-2285t, bta-miR-345-5p, bta-miR-34a, bta-miR-150, and the novel-miR-1372 had significantly changed. Predicted target genes for these 27 miRNAs were analyzed by KEGG and the results demonstrated that EZR, RASSF, SOCS1 were mainly enriched in the signaling pathway microRNAs in cancer. MAPKAPK2, RELB, FLT3LG, and GADD45B were mainly enriched in the MAPK signaling pathway, and some genes were enriched in Axon guidance. This study has provided valuable information to further the understanding of the regulatory function of miRNAs in the host microenvironment and host-parasite interaction mechanisms. [ABSTRACT FROM AUTHOR]

Bovine theileriosis is a protozoan disease caused by the intracellular parasite (Theileria spp.) transmitted by ticks and it is considered one of the most significant parasitic diseases, potentially endangering Egyptian cattle herd industry. The present study was conducted for a molecular survey of bovine theileriosis and its associated risk factors (season variations, geographical locations, breeds, age, sex, tick infestation, and acaricide applications) in three Egyptian governorates, Beni-Suef, Al-Faiyum, and Al-Minya for a year extended from December 2021 to November 2022, in addition, genetic diversity of Theileria isolates. A total of 961 cattle were examined for Theileria infection clinically, microscopically, and by Polymerase Chain Reaction (PCR) using 18S rRNA gene for piroplasms DNA detection, Theileria genus-specific primers of the small subunit of rRNA gene, and Theileria annulata specific primers of the Tams-1 gene. The prevalence rate of bovine theileriosis was 9.26%, and 11.86% using Giemsa-stained blood smear and PCR, respectively. All positive samples screened by Theileria genus-specific primers were positive for T. annulata when screened by the specific primers. Based on molecular screening, season, cattle breeds and acaricide applications were considered risk factors for T. annulata infection, while locality, age, sex and tick infestation had insignificant effects with the occurrence of the disease. A potential novel T. annulata haplotype based on the Tam-1 gene was identified with accession numbers OR364144 and OR915851. Therefore, T. annulata was the only Theileria species found and played a significant problem in the cattle population. This study could be the basis for future studies on unexplored regions and different animal species for well-structured prevention and control measures. [ABSTRACT FROM AUTHOR]

Hyalomma anatolicum is a tick of significant one-health importance due to its role as a vector for various pathogens affecting humans, animals and the environment, such as Theileria annulata, which causes tropical theileriosis in cattle, leading to severe economic losses. When infected with pathogens like T. annulata, the salivary glands of H. anatolicum undergo gene expression changes, secrete modified proteins and activate immune responses, all of which facilitate pathogen survival and transmission by modulating the host immune response and optimizing conditions for pathogen development. Understanding these responses is crucial for developing control strategies for tick-borne diseases. To understand the interaction between H. anatolicum and T. annulata, we performed a differential gene expression analysis of H. anatolicum salivary glands. An average of approximately 25 million raw sequencing reads were generated in each replicate using Illumina Sequencing. The sequenced reads were de novo assembled and the assembled transcriptome yielded approximately 50,231 non-redundant transcripts after clustering with CD-HIT using a sequence identity of 95% and alignment coverage of 90%. The assembly quality was evaluated with BUSCO analysis and found to be 86% complete using the Arachnida dataset and then blasted against non-redundant protein sequence database from NCBI followed by counting of reads and differential expression analysis. Overall, around 2400 and 400 genes were found differentially expressed with logFC > 1 and logFC > 2 respectively at FDR < 0.05. Top up-regulated genes included Calpain, Papilin, Neprilysin, and Ankyrin repeat-containing protein. Top down-regulated genes included Scoloptoxin, and Selenoprotein S and other uncharacterized proteins. Many other up-regulated proteins with high significance were uncharacterized suggesting room for further H. anatolicum functional and structural characterization studies. To our best knowledge, this is the first study of H. anatolicum sialotranscriptome which greatly contributes to sialotranscriptome information not only as sequence database but also indicates the potential targets for development of vaccine against ticks and transmission-blocking vaccines against T. annulata. [ABSTRACT FROM AUTHOR]

Background and Aim: Theileria annulata infection in cattle causes major economic losses in livestock production in many Central Asian countries, including the southern region of Kazakhstan. This study aimed to obtain a recombinant T. annulata surface protein (TaSP) and to investigate its possible use as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of bovine theileriosis. Materials and Methods: Recombinant TaSP was obtained by cloning a polymorphic region of the TaSP gene, expressing it in Escherichia coli strain BL21, and purifying it by metal chelating chromatography. An indirect ELISA using recombinant TaSP as an antigen was developed and evaluated for the detection of T. annulata-specific antibodies in plasma samples from 69 cows polymerase chain reaction (PCR)-positive or PCR-negative for T. annulata and/or Theileria orientalis from southern Kazakhstan. Results: The obtained recombinant protein had a molecular weight of 32 kDa, and mass spectrometry analysis of the purified protein identified it as a fragment of the surface protein of T. annulata. Initial testing of 69 field plasma samples from cattle showed that the results of indirect ELISA using TaSP as an antigen agreed substantially with those of T. annulata PCR (κ: 0.78). The relative sensitivity and specificity of indirect ELISA were 88.7% and 100%, respectively, using PCR as a reference. There was no evidence of cross-reaction with T. orientalis. Conclusion: Initial results using recombinant TaSP as an antigen in indirect ELISA are promising and support the widespread use of this assay for routine diagnosis and T. annulata seroprevalence studies in cattle in Kazakhstan and possibly neighboring countries.

Background and Aim: Theileria annulata infection in cattle causes major economic losses in livestock production in many Central Asian countries, including the southern region of Kazakhstan. This study aimed to obtain a recombinant T. annulata surface protein (TaSP) and to investigate its possible use as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of bovine theileriosis. Materials and Methods: Recombinant TaSP was obtained by cloning a polymorphic region of the TaSP gene, expressing it in Escherichia coli strain BL21, and purifying it by metal chelating chromatography. An indirect ELISA using recombinant TaSP as an antigen was developed and evaluated for the detection of T. annulata-specific antibodies in plasma samples from 69 cows polymerase chain reaction (PCR)-positive or PCR-negative for T. annulata and/or Theileria orientalis from southern Kazakhstan. Results: The obtained recombinant protein had a molecular weight of 32 kDa, and mass spectrometry analysis of the purified protein identified it as a fragment of the surface protein of T. annulata. Initial testing of 69 field plasma samples from cattle showed that the results of indirect ELISA using TaSP as an antigen agreed substantially with those of T. annulata PCR (κ: 0.78). The relative sensitivity and specificity of indirect ELISA were 88.7% and 100%, respectively, using PCR as a reference. There was no evidence of cross-reaction with T. orientalis. Conclusion: Initial results using recombinant TaSP as an antigen in indirect ELISA are promising and support the widespread use of this assay for routine diagnosis and T. annulata seroprevalence studies in cattle in Kazakhstan and possibly neighboring countries. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Theileriosis, a tick borne protozoan disease, drastically affects the productivity of bovine with massive economic repercussion. The current investigation was conducted to determine the molecular prevalence of T. annulata employing cytochrome b gene based PCR assay. Investigation was conducted on 150 blood samples of cattle obtained from three districts, viz. Rajnandgaon (50), Balod (50) and Kabirdham (50) of Chhattisgarh state. Initially microscopic examinations of smears were done followed by PCR analysis of DNA samples. The overall prevalence of tropical theileriosis in plain region of Chhattisgarh was found to be 27.3% (41/150) and 54.0% (81/150) by microscopy and PCR assay, respectively. Among districts, highest prevalence 32% and 60% was recorded in Kabirdham followed by 22% and 54% at Balod and 28% and 50% at Rajnandgaon, by microscopy and PCR assay, respectively. Observations showed that, more number of infected animals were from unorganized farm (62.6%) than organized farm (45%). Present study revealed crossbred cattle more susceptible to T. annulata infection than indigenous animals. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges.

Abstract Theileria annulata is a protozoan parasite with a complex life cycle involving a bovine host and a tick vector. It is transmitted by Hyalomma ticks and is the causative agent of tropical theileriosis, a debilitating and often fatal disease in southern Europe, northern Africa and large parts of Asia. Understanding the biology of different life cycle stages is critical for the control of tropical theileriosis and requires the use of experimental animals which poses an ethical concern. We present for the first time the in vitro infection of red blood cells (RBCs) with T. annulata differentiated schizonts. The Ankara cell line of T. annulata was cultured at 41 °C for nine days to induce merogony and subsequently incubated with purified RBCs for one to three days. Percentage of parasitized erythrocyte (PPE) over the short culture period was estimated by Giemsa staining (0.007–0.01%), Flow cytometry activated sorting (FACS) (0.02–1.1%) and observation of FACS sorted cells by confocal microscopy (0.05–0.4%). There was a significant difference in the PPE between FACS and the two other techniques (one-way ANOVA followed by Tukey test, P = 0.004) but no significant difference was observed between the confocal imaging and Giemsa staining methods (ANOVA one-way followed by Tukey test, P = 0.06). Importantly, all three complementary methods confirmed the invasion of RBCs by T. annulata merozoites in vitro. Although the experimental conditions will require further optimization to increase the PPE, the in vitro infection of RBCs by T. annulata merozoites is pivotal in paving the way for the eventual completion of the T. annulata life cycle in vitro when combined with artificial tick feeding.

This study showed that total percentage of infection with Theileria species in 100 blood samples of cattle in Mosul City was 38% with no significant differences between male and female of cattle. Cattle older than 1 year old had a high percentage of Thieleria infection 40% compared to younger cattle 34.28%, with no significant variations in Thieleria infection and age groups of cattle. Theileria measurement in red blood cells ranged from 0.5 to 2 microns, while parasitemia levels ranged from 0.2 to 15%, with an average of 6.572%. The results of molecular diagnosis confirmed the detection Theileria genus, Theileria annulata, and Theileria parva in blood samples of examined cattle and PCR revealed that the amplification products were 237bp, 690bp, and 700bp, respectively. In this study, we recorded new strains of Theileria in cattle of the Nineveh governorate by using the BLAST program consisting of three strains of T. annulata, one strain of Theileria ovis and two strains of Theileria parva which differs from the normal isolates in many nucleotides in different worlds. The phylogenetic tree showed the relative relation among the Theileria spp, the results matching species between 8 and 11, 1 and 4, 6 and 10, 3 and 9, and 2 and 12; the species 7 were more closely linked to 11 and 8, the species five similar to 6 and 10.

Theileriosis is a protozoan parasite that is transmitted by ticks and infects a wide range of animals worldwide. This study aims to assess the molecular prevalence and related risk factors of theileriosis in Balochistan, Pakistan. Standard microscopy methods, polymerase chain reaction (PCR), 18S small subunit ribosomal RNA gene sequencing, and phylogenetic analysis were used. For this purpose, a total of 408 blood samples were collected from tick-infested cattle in Zhob, Loralai, and Quetta districts of Balochistan, Pakistan. Microscopy and subsequent PCR analysis confirmed the highest prevalence of Theileria annulata in Loralai district (11.76% and 12.75%), followed by Zhob district (11.27% and 12.25%), and Quetta district (8.34% and 9.56%), respectively. Moreover, the prevalence of T. annulata was higher in young cattle (85.82%), followed by female cattle (58.87%), and exotic crossbred cattle (33.33%) in the study area. However, various variable such as sex, area, and breeds of the cattle were not significantly correlated (P>0.05) with the presence of T. annulata, except for the age of animals (P

Theileriosis is a protozoan parasite that is transmitted by ticks and infects a wide range of animals worldwide. This study aims to assess the molecular prevalence and related risk factors of theileriosis in Balochistan, Pakistan. Standard microscopy methods, polymerase chain reaction (PCR), 18S small subunit ribosomal RNA gene sequencing, and phylogenetic analysis were used. For this purpose, a total of 408 blood samples were collected from tick-infested cattle in Zhob, Loralai, and Quetta districts of Balochistan, Pakistan. Microscopy and subsequent PCR analysis confirmed the highest prevalence of Theileria annulata in Loralai district (11.76% and 12.75%), followed by Zhob district (11.27% and 12.25%), and Quetta district (8.34% and 9.56%), respectively. Moreover, the prevalence of T. annulata was higher in young cattle (85.82%), followed by female cattle (58.87%), and exotic crossbred cattle (33.33%) in the study area. However, various variable such as sex, area, and breeds of the cattle were not significantly correlated (P>0.05) with the presence of T. annulata, except for the age of animals (P<0.05). In addition, sequencing and phylogenetic analyses revealed that the isolated T. annulata was closely related to the isolates from Türkiye, Italy, Egypt, Iran, and Pakistan. Hence, these findings will contribute to the development of more effective control strategies for theileriosis in the cattle population of Balochistan, as well as in Pakistan on a broader scale. [ABSTRACT FROM AUTHOR]

Antitheilerial resistance and treatment failure are growing international concerns. Against this backdrop, the first study aimed to compare the efficacy of Juglone (a naturally occurring naphthoquinone) and buparvaquone against bovine tropical theileriosis during natural infection. A total of 60 adult cattle (2-4 years) were selected and divided into five groups, comprising 15 animals each. The animals of Group-I were administered two doses of buparvaquone (BPQ) at a dose rate of 2.5mg/kg body weight intramuscular, 48 hours apart. The cattle of Group-II were administered with BPQ and oxytetracycline (OXY) at the dose rate of 2.5 mg/kg (two doses at 48-hour intervals) and 20 mg/kg intramuscular 3 doses at 48-hour intervals, respectively. While infected animals of Group-III were treated with Juglone (JUG) at a dose rate of 0.48 mg/kg IV on alternate days for 7 days. Whereas, Group-IV acted as a healthy control. The hemogram revealed normocytic normochromic anemia in infected animals. A significant decrease of neutrophils along with decrease of total leukocytes and lymphocytes was noticed in recovered animals from all treated groups (Group I to III). After 21 days, the results of the therapeutic trial showed the highest efficacy of Group II (BPQ+OXY) (93.3%; 14/15), followed by Group III (JUG) (73.3%) and Group I (BPQ) (66.7%; 10/15) groups, three weeks after treatment. It can be concluded that BPQ+OXY had the highest efficacy; whereas the efficacy of Juglone is also comparable, this can be used as an alternative treatment against tropical theileriosis. [ABSTRACT FROM AUTHOR]

Background: The production of bovine theileriosis vaccine involves in vitro cultivation of Theileria annulata schizont-infected cell lines. Fetal bovine serum (FBS) is commonly used in animal cell culture, including the Theileria cell line. However, we aimed to reduce the amount of serum needed for cell culture by modifying the Stoker culture medium with supplements such as excretion factor and serum substitutes. Methods: To evaluate the effectiveness of these modifications, techniques such as cell counting, cell viability assays, and genomic analysis were employed in the Parasitic Vaccines Production Department of Razi institute of Iran, from 2020 to 2022. Statistical analysis was used to compare the results of different experimental conditions. Results: The three experimental media were as effective as the commonly used 10% Stoker medium in supporting the growth and viability of cells. Conclusion: The significant reduction in the required amount of serum and the remarkable cell growth achieved by using defined serum replacements for the production of cell culture media is a significant step towards the preparation of a proper cell culture medium for the production of bovine Theileriosis vaccine. [ABSTRACT FROM AUTHOR]

The control of Tropical Theileriosis, a tick-borne disease with a strong impact on cattle breeding, can be facilitated using marker-assisted selection in breeding programs. Genome-wide association studies (GWAS) using high-density arrays are extremely important for the ongoing process of identifying genomic variants associated with resistance to Theileria annulata infection. In this work, single-nucleotide polymorphisms (SNPs) were analyzed in the Portuguese autochthonous cattle breeds Alentejana and Mertolenga. In total, 24 SNPs suggestive of significance (p ≤ 10−4) were identified for Alentejana cattle and 20 SNPs were identified for Mertolenga cattle. The genomic regions around these SNPs were further investigated for annotated genes and quantitative trait loci (QTLs) previously described by other authors. Regarding the Alentejana breed, the MAP3K1, CMTM7, SSFA2, and ATG13 genes are located near suggestive SNPs and appear as candidate genes for resistance to Tropical Theileriosis, considering its action in the immune response and resistance to other diseases. On the other hand, in the Mertolenga breed, the UOX gene is also a candidate gene due to its apparent link to the pathogenesis of the disease. These results may represent a first step toward the possibility of including genetic markers for resistance to Tropical Theileriosis in current breed selection programs. [ABSTRACT FROM AUTHOR]

Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man® probe for detection and quantification of Theileria orientalis and Theileria annulata simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically Theileria–suspected cows of three Theileria–endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed T. annulata and T. orientalis in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the Theileria spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of T. annulata and T. orientalis simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of Thelieria spp. [ABSTRACT FROM AUTHOR]

Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tamsl 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes. [ABSTRACT FROM AUTHOR]

Tick-borne diseases have a major adverse effect on livestock worldwide, causing enormous economic losses in meat and milk production as well threatening animal and public health. In this study, we aimed to detect and characterize piroplasms isolated from cattle and buffalo in southern Egypt, using molecular techniques. Three hundred blood samples were collected from cattle and buffalo in two governorates in southern Egypt. All 300 samples (100%) were confirmed to contain DNA, as they exhibited bands of bovine β-actin gene at the expected 227 bp for cattle and buffalo. The samples were analyzed by PCR for the presence of piroplasms, specifically Babesia bovis, Babesia bigemina, and Theileria annulata. Samples positive for the piroplasma 18S ribosomal RNA gene were further examined for two additional genes, spherical body protein 4 gene, to provide an enhanced degree of specificity for the identification of B. bovis and B. bigemina, and the major merozoite surface antigen gene for T. annulata. The infection rate for piroplasma spp. was 60/300 (20%). The positivity rates were 10.7% (32/300) for T. annulata, 5.3% (16/300) for B. bovis, and 4% (12/300) for B. bigemina. By host species, 42/150 (28%) cattle and 18/150 (12%) buffalo were positive for piroplasms. None of the isolates sequenced for the B. bovis isolates from buffalo in this study showed 100% identity with any sequence deposited in GenBank for the small subunit ribosomal RNA gene (maximum identity value = 99.74%). Similarly, no T. annulata small subunit ribosomal RNA gene sequence identified in this study exhibited 100% identity with any sequence deposited in GenBank (maximum identity value = 99.89%). The current study provides a partial sequence of the T. annulata merozoite-piroplasm surface antigen gene, as well as the B. bovis and B. bigemina spherical body protein 4 genes, in cattle and buffalo in southern Egypt, and is the first report on these piroplasma genes in cattle and buffalo in southern Egypt.

Background: The production of bovine theileriosis vaccine involves in vitro cultivation of Theileria annulata schizont-infected cell lines. Fetal bovine serum (FBS) is commonly used in animal cell culture, including the Theileria cell line. However, we aimed to reduce the amount of serum needed for cell culture by modifying the Stoker culture medium with supplements such as excretion factor and serum substitutes. Methods: To evaluate the effectiveness of these modifications, techniques such as cell counting, cell viability assays, and genomic analysis were employed in the Parasitic Vaccines Production Department of Razi institute of Iran, from 2020 to 2022. Statistical analysis was used to compare the results of different experimental conditions. Results: The three experimental media were as effective as the commonly used 10% Stoker medium in supporting the growth and viability of cells. Conclusion: The significant reduction in the required amount of serum and the remarkable cell growth achieved by using defined serum replacements for the production of cell culture media is a significant step towards the preparation of a proper cell culture medium for the production of bovine Theileriosis vaccine.

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

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen's kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata. [ABSTRACT FROM AUTHOR]

Tropical theileriosis, babesiosis, and anaplasmosis are the most dominant tick-borne infections in North Africa where they cause significant economic losses in ruminants' industry. The aim of the present work was to study infections and co-infection patterns in 66 cattle with clinical signs of piroplasmosis and/or anaplasmosis in two localities, Beni Hamidene and Grarem Gouga, districts of Constantine and Mila (Northeast of Algeria), respectively. This study was conducted between early May and late September during four years 2017, 2018, 2020, and 2021. PCR showed that the most frequent pathogen in cattle with clinical signs of piroplasmosis and/or anaplasmosis was Theileria annulata (66/66; 100%) followed by Babesia bovis (21/66; 31.8%), Anaplasma marginale (15/66; 22.7%), and Babesia bigemina (3/66; 4.5%) (p < 0.001). Giemsa-stained blood smears examinations revealed that 66.7% (44/66); 10.6% (7/66); and 9.1% (6/66) of cattle were infected by T. annulata, Babesia spp., and A. marginale, respectively (p < 0.001). PCR revealed seven co-infection patterns: T. annulata/A. marginale (15/66; 22.7%), T. annulata/B. bovis (21/66; 31.8%), T. annulata/B. bigemina (3/66; 4.5%), T. annulata/A. marginale/B. bovis (7/66; 10.6%), T. annulata/B. bovis/B. bigemina (2/66; 3%), T. annulata/A. marginale/B. bigemina (1/66; 1.5%), and T. annulata/A. marginale/B. bigemina/B. bovis (1/66; 1.5%). Phylogenetic analyses showed that T. annulata Tams1 and B. bigemina gp45 sequences were identical to isolates from Mauritania and South Africa, respectively. The three A. marginale amplicons obtained herein had 99.63 to 99.88% similarity between them. This study provides data that can be used to improve control programs targeting these cattle hemopathogens. [ABSTRACT FROM AUTHOR]

Theileria annulata (T. annulata) is an intracellular protozoan, transmitted by ixodid ticks of the genus Hyalomma and affects camels. There are few epidemiological data on T. annulata infection and its associated risk factors in Egyptian camels. Thus, the present study aimed to determine the prevalence of T. annulata in camels using PCR and assess the associated risk factors for infection. A total of 380 blood samples were collected from camels raising in three Egyptian governorates and examined by PCR assay targeting 30-kDa gene to detect the presence of T. annulata infection, beside statistical analysis of associated factors. The results revealed presence of T. annulata with overall prevalence of 21.1%. In addition, the univariate analysis revealed significant (P<0.05) association between prevalence of T. annulata in camels and locality, age, sex, tick infestation, and application of acaricides. Whereas the prevalence of T. annulata was higher in camels of age group >6 to 10 years (38%), females (25.7%) and in infested camels with ticks (29%) and in case of absence of acaricides application (25%). In contrast, the body condition of camels had not significant effect on prevalence of theileriosis in camels. The current study concluded that T. annulata is prevalent in Egyptian camels and that a tick control program is required to reduce the risk of infection. [ABSTRACT FROM AUTHOR]

In this study, we aimed to investigate haematological, pro‐inflammatory, inflammatory, anti‐inflammatory and immunological responses in naturally Theileria annulata‐infected cattle. The study material consisted of 25 Simmental cattle, 2–4 years of age, one of which was a control group consisting of healthy animals (Control group, n = 10), and the other was a Theileria group that include animals positive for Theileria annulata (Theileria group, n = 15). Haematological analysis (red blood cell [RBC], haemoglobin [HGB], haematocrit [HCT]), pro‐inflammatory (tumour necrosis factor‐α [TNF‐α], nuclear factor kappa B [NF‐ĸB] and interleukin‐1 beta, [IL‐1β]), inflammatory (neutrophil‐lymphocyte ratio [NLR]), anti‐inflammatory (interleukin‐10 [IL‐10]) and antimicrobial peptide (CAMP) analyses were performed by using ELISA kit from blood samples. It was found that the rectal temperature of the Theileria group was found to be significantly higher (p <.001) than that of the control group. Haematological and biochemical analysis revealed that the RBC and HGB count and HCT percentage decreased (p <.001), while NF‐ĸB (p <.001), TNF‐α (p =.002), IL‐1β (p <.001), IL‐10 (p =.012), NLR (p <.001) and CAMP (p =.037) levels increased in Theileria group compared to the control group. There was a strong correlation between NF‐ĸB and TNF‐α, NF‐ĸB and IL‐10, NLR and IL‐1β, NF‐ĸB and CAMP, TNF‐α and CAMP and IL‐10 and CAMP. As a result of this study, it was revealed that a pro‐inflammatory and immunological response also occurs along with the anti‐inflammatory response in the inflammatory process. [ABSTRACT FROM AUTHOR]

Tropical theileriosis is a tick-borne disease caused by the protozoan Theileria annulata and transmitted by numerous species of Ixodid ticks of the genus Hyalomma. The main clinical signs are fever, lymphadenopathy, and anemia responsible for heavy economic losses, including mortality, morbidity, vaccination failure, and treatment cost. Development of poor cell-mediated immunity (CMI) has been observed in the case of many bovine pathogens (bacteria, viruses, and parasites). Quantification of CMI is a prerequisite for evaluating vaccine efficacy against theileriosis caused by T. annulata. The current study evaluated the CMI in calves administered with two types of T. annulata vaccine (live attenuated and killed). We prepared a live attenuated T. annulata vaccine by attenuation in a rabbit model and also prepared killed vaccine from non-attenuated T. annulata. For the evaluation of immune response in experimental groups including control, 20 calves were divided into four different groups (A, B, C, and D). They were either inoculated subcutaneously with live rabbit-propagated-Theileria-infected RBCs (5 × 106) (group A) or with killed T. annulata vaccine (2 × 109 schizonts) with Freund's adjuvant (group B), along with an infected group (group C) and a healthy control group (group D). The protection of vaccinated calves was estimated with challenge infection. Our results showed that with a single shot of live-attenuated and killed vaccine with a booster dose elicited cell-mediated immune responses in immunized calves. We observed a significant elevation in CD4 + and CD8 + T cells in immunized calves. A significant difference in the CD8 + T cell response between the post-challenge stage of killed and live vaccine (p < 0.0001) was observed, whereas no other difference was found at both pre- and post-immunization stages. A similar finding was recorded for the CD4 + T cells at a post-challenge stage, where a significant difference was seen between killed and live vaccine (p < 0.0001). Another significant difference was observed between the CD8 + T cells and CD4 + T cells at the post-challenge stage in the live vaccine group, where there was a significantly higher induction of CD4 + T cell response (p < 0.0001). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The current work was conducted to unveil the current situation for the infection by Theileria annulata in cattle in Al-Hilla City, Iraq. A total of 225 blood samples (200 from suspected infected animals and 25 from clinically healthy animals as a control group) were collected. These samples were subjected to a direct slide-smearing for detection using a microscope and DNA sequencing, targeting the cytochrome b (Cyt b) gene of 10 polymerase chain reaction (PCR) products. The thin smear findings of the 200 suspected cases revealed that 63 (31.5%) were infected with Theileria spp., while 115 (57.5%) cases had no Theileria but other blood parasites; however, only 22 (11%) suspected cases showed no presence of any parasites. Unsurprisingly, the 25 blood samples from the control group demonstrated no presence of any blood parasite. Moreover, the DNA sequencing demonstrated that the Theileria spp. belonged to T. annulata species, and these sequences were nucleotide-based similar to Gene-Bank isolates from Tunisia (ON035604, ON035605, ON035606, ON035607, ON035608, ON035609, ON035610, ON035611, ON035612, and ON035613). The present study outcomes indicate that theileriosis is the dominant parasitic infection in cattle in Al-Hilla City and is highly caused by Theileria annulata.

Tropical theileriosis is a protozoan infection caused by Theileria annulata, which significantly affects cattle worldwide. This study was aimed to analyze the TaSPAG1 protein and design a novel multi-epitope vaccine candidate. Online tools were employed for the prediction of Physico-chemical properties, antigenicity, allergenicity, solubility, transmembrane domains and signal peptide, posttranslational modification (PTM) sites, secondary and tertiary structures as well as intrinsically disordered regions, followed by identification and screening of potential linear and conformational B-cell epitopes and those peptides having affinity to bind bovine major histocompatibility complex class I (MHC-I) molecules. Next, a multi-epitope vaccine construct was designed and analyzed. This 907-residue protein was hydrophilic (GRAVY: -0.399) and acidic (pI: 5.04) in nature, with high thermotolerance (aliphatic: 71.27). Also, 5 linear and 12 conformational B-cell epitopes along with 8 CTL epitopes were predicted for TaSPAG1. The 355-residue vaccine candidate had a MW of about 35 kDa and it was antigenic, non-allergenic, soluble and stable, which was successfully interacted with cattle MHC-I molecule and finally cloned into the pET28a(+) vector. Further wet studies are required to assess the vaccine efficacy in cattle. [ABSTRACT FROM AUTHOR]

Background: Tropical theileriosis is the most prevalent hemoprotozoan disease in Pakistan. Aims: The study aimed to investigate the epidemiology and evolutionary relationship of Theileria annulata in bovines in diverse agro-climatic regions of Punjab, Pakistan. Methods: 800 blood specimens were collected from asymptomatic cattle (n=480) and buffaloes (n=320) using a multistage sampling method from Sargodha (n=400) and Multan (n=400) districts. The samples were assessed for blood smear microscopy and cytochrome b gene based PCR. Twenty samples were collected from each union council of each district. Results: The overall prevalence of T. annulata infection in bovines was 9% and 17.13% as determined by blood smear analysis and PCR, respectively. The disease positivity in cattle and buffaloes was respectively 10.21% and 20.42% by blood smear screening and 7.19%, 12.19% by PCR. The overall PCR based prevalence in the Sargodha and Multan districts was 19% and 15.25%, respectively. Absence of rural poultry, tick infestation, and a history of tick-borne diseases had significant effect in cattle. Tick infestation and age were the main statistically significant disease determinants in buffaloes. The evolutionary analysis of the cytochrome b gene showed that the Pakistani isolate infecting buffalo was related to those from Iran, India, Egypt, and Sudan. The isolate from cattle was genetically close to those from Pakistan, India, Iran, Iraq, and Turkey. Conclusion: It can be concluded that biotic and abiotic factors contribute to disease occurrence. The current study will help to devise control strategies to prevent substantial economic losses. [ABSTRACT FROM AUTHOR]

Tick-borne maladies of canines are increasing in sub-tropical and tropical areas around the globe. Theileriosis is an emerging issue in canines, especially dogs but the data regarding the disease prevalence in Asian countries is scarce. Therefore, the current study was designed to check the molecular prevalence of Theileria spp. in dogs of Lahore, Pakistan. A total of 102 dog blood samples were initially screened by microscopy and later on by PCR. The results revealed that 10.78% (11/102) and 6.86% (7/102) of pet dogs were found positive by PCR and microscopy, respectively. The phylogenetic analysis showed the sequences resemblance up to 98-99% with cytb gene fragments of Theileria annulata isolates from Iran, India, Turkey, Sudan, China, and Tunisia. Assumed risk factors revealed a significant (P

The tick-borne intra-cellular parasite, Theileria annulata, causes mortality in bovines. Schizont of the parasite in lymphocytes divides continuously stimulating the clonal expansion of lymphoid cells in lymph nodes leading to the neoplasticlike lymphoid cell proliferation and sometimes may also cause lymphoma, especially in chronic conditions. Two cattle, with a history of fever and respiratory distress, were presented to the Large Animal Unit, Teaching Veterinary Clinical Complex, GADVASU. On clinical examination, the animals had a high fever (106-107ºF), anemia, icteric mucus membranes, and prescapular lymph node enlargement. Whole blood and fine needle aspirate from the lymph node were collected. A blood smear examination revealed anemia, leucopenia with relative lymphocytosis, and thrombocytopenia. Further, there was the presence of schizonts (Koch blue bodies) in the lymphocytes and piroplasms of T. annulata in the red blood cells. Lymph node aspirate examination revealed the feature that mimics lymphoma by the presence of a monomorphic population of lymphoblasts and with all stages of mitotic figures along with lymphoglandular bodies. Numerous schizonts were also recorded in the lymphoblasts during the examination of lymph node aspirate.

Abstract Background There had been isolated reports of the presence of novel Theileria annulata genotypes based on the 18S rRNA gene sequence data from India, Pakistan and Saudi Arabia; but, these studies were restricted to limited field samples. Additionally, no comparative study has been conducted on all the isolates of this parasite from different countries whose sequences are available in the nucleotide databases. Therefore, we aimed to study the genetic diversity of T. annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. Out of a total of 312 gene sequences of T. annulata available in the NCBI database, only 70 nearly complete sequences (> 1527 bp) were used for multiple sequence alignment. Results The maximum likelihood tree obtained using TN93 + G + I model manifested two major clades. All the valid host-cell transforming Theileria species clustered in one clade. The T. annulata designated sequences occupying this clade clustered together, excluding two isolates (DQ287944 and EU083799), and represented the true T. annulata sequences (n = 54). DQ287944 and EU083799 exhibited close association with Theileria lestoquardi. In addition, 14 Indian sequences formed a large monophyletic group with published Theileria orientalis sequences. The broad range of sequence identity (95.8–100%) of T. annulata designated sequences indicated the presence of different Theileria spp. A closer analysis revealed the presence of three Theileria spp., namely, T. annulata, T. orientalis, and two isolates (DQ287944 and EU083799) closely related to T. lestoquardi. The true T. annulata sequences manifested 98.8–100% nucleotide identity within them. EU083799 and 14 misidentified Indian T. annulata sequences exhibited the highest similarity with T. lestoquardi (98.6–98.8%) and T. orientalis (98.0-99.9%) in comparison with the other Theileria spp. of domestic and wild ruminants. Conclusion In the course of analyzing the genetic diversity of T. annulata, we identified the nearly complete 18S rRNA gene sequences of other Theileria spp. that have not only been misidentified as T. annulata in the GenBank™, but are also published as T. annulata. Moreover, a high level of sequence conservation was noticed in the 18S rRNA gene of true T. annulata and T. orientalis sequences.

Abstract Background When Theileria annulata infects host cells, it undertakes unlimited proliferation as tumor cells. Although the transformed cells will recover their limited reproductive characteristics and enter the apoptosis process after treatment with buparvaquone (BW720c), the metabolites and metabolic pathways involved are not clear. Methods The transformed cells of T. annulata were used as experimental materials, and the buparvaquone treatment group and DMSO control group were used. Qualitative and quantitative analysis was undertaken of 36 cell samples based on the LC–QTOF platform in positive and negative ion modes. The metabolites of the cell samples after 72 h of drug treatment were analyzed, as were the different metabolites and metabolic pathways involved in the BW720c treatment. Finally, the differential metabolites and metabolic pathways in the transformed cells were found. Results A total of 1425 metabolites were detected in the negative ion mode and 1298 metabolites were detected in the positive ion mode. After drug treatment for 24 h, 48 h, and 72 h, there were 56, 162, and 243 differential metabolites in negative ion mode, and 35, 121, and 177 differential metabolites in positive ion mode, respectively. These differential metabolites are mainly concentrated on various essential amino acids. Conclusion BW720c treatment induces metabolic disturbances in T. annulata-infected cells by regulating the metabolism of leucine, arginine, and l-carnitine, and induces host cell apoptosis. Graphical abstract

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. [ABSTRACT FROM AUTHOR]

Schizont-infected nucleated cells and anemia-induced hypoxia may cause acute kidney injury (AKI) in cattle with tropical theileriosis. In most cases, serum creatinine (sCr) fails to detect early kidney injury. Recently, urine biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL), Cystatin C (CysC), and Clusterin (Clu) have outperformed sCr in identifying AKI in various animal species. However, these markers have not been tested in cattle for AKI detection. This study investigated AKI in cattle with tropical theileriosis using serum and urinary NGAL, CysC, and Clu and compared them to standard renal function markers such as sCr, urine protein to creatinine (uPC) ratio and urine-specific gravity (USG). Seventeen cattle were infected with Theileria annulata (Th) and ten were controls (CTRL) in this investigation. The urinary NGAL (uNGAL) in the Th group was considerably (P<0.001) greater than that in the CTRL group. uCysC increased significantly in the Th group compared to CTRL group (P<0.001). The Th group had higher sClu than the CTRL group (P<0.001). There was a significant difference (p=0.005) in sCr concentrations between Th and CTRL groups, but the values were within the reference range in both groups. Th group had a higher uPC ratio than CTRL (p= 0.002), whereas USG was lower in Th group than in CTRL group (P<0.001). These results suggested that cattle with tropical Theileriosis have acute tubular injury. [ABSTRACT FROM AUTHOR]

The present study was carried out to investigate the epidemiological and clinical status of bovine Theileriosis in Aswan governorate. During a 2-year study, 265 cattle were clinically suspected upon careful clinical examination as Theileria annulata (T. annulata) infected animals. Conventional diagnosis based on blood and lymph smears examinations showed that, the prevalence of Tropical Theileriosis in cattle in Aswan governorate was 56 (21.13%). Stained blood smears showed the presence of macro and/or micro-schizonts inside lymphocyte (Koch's blue bodies. Intraerythrocytic stages of Theileria annulata piroplasms inside RBCs. Polymerase chain reactions of T. annulata merozoite-piroplasms surface antigen Targeting gene: (Tams-1), revealed positive 29 (58%) animals confirmed by visualization of specific bands at 768 bp. Positive results could be detected in suspected cattle that showed positive or negative blood smear results that confirmed the high sensitivity of the PCR technique compared with the conventional method for diagnosis of bovine tropical Theileriosis. PCR proved to be a highly sensitive and accurate method for diagnosis of bovine tropical theileriosis especially in the detection of samples that was negative on blood and lymph smears. [ABSTRACT FROM AUTHOR]

Bovine tropical theileriosis caused by Theileria annulata, is a serious constraint to Indian dairy industry with more fatal infections in exotic cattle and substantial losses to cross-bred and indigenous zebu cattle. The present communication is to place on record the first report of molecular based confirmed case of cerebral theileriosis caused by T. annulata coupled with its morphological detection, clinical manifestations, haematological alterations and therapeutic management in a cross bred cattle calf from India. After preparation of peripheral thin blood smear from cross bred cattle calf at the site of collection and fixation with methanol, blood sample brought to Department of Veterinary Parasitology, College of Veterinary Science and A.H, Jabalpur and stained by standard protocol for Giemsa staining. Genomic DNA was isolated from the collected blood sample using QIAamp® DNA blood mini kit following the manufacturer's recommendations and PCR was performed. The cross bred cow calf revealed high rise in temperature (105.5°F), increased heart rate, labored breathing with seromucous nasal discharge, enlargement of prescapular lymph node and animal exhibited tonic clonic convulsions in response to any sudden noise. Giemsa stained thin blood smear revealed intraerythrocytic piroplasm and Koch'sblue bodies of T. annulata within the cytoplasm of lymphocytes. The species of Theileria was confirmed by molecular amplification of genomic DNA as T. annulata. [ABSTRACT FROM AUTHOR]