Role of cytokines in the clinical manifestation of exophthalmia in newborn calves with tropical theileriosis.

The present study aimed to evaluate the pathology of the exophthalmia and the host-immune response in naturally Theileria annulata-infected calves. The newborn calves detected positive for theileriosis were grouped into calves with theileriosis and absence of exophthalmia (n = 30), and calves with theileriosis and the presence of exophthalmia (n = 13). Sixteen healthy calves, free from any haemoprotozoal infection, were kept as healthy controls. A significantly (P ≤ .001) higher circulating levels of tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were estimated in diseased calves with and without exophthalmia as compared to healthy controls. Contrarily, significantly (P ≤ .01) lower interferon-γ (IFN-γ) level was estimated in diseased calves. The diseased calves with exophthalmia revealed significantly higher levels of TNF-α (P ≤ .001) and IL-10 (P ≤ .006) as compared to the diseased calves without exophthalmia. The diseased calves were not found to have an elevated intraocular pressure; rather they had significantly (P ≤ .001) lower intraocular pressure compared to the healthy controls. An elevated systemic TNF-α level might be attributed to the exophthalmia in calves with tropical theileriosis. The elevated circulatory IL-10 and reduced IFN-γ levels could be one of the strategies of Theileria annulata to escape the host immunity.

In vitro influence of Theileria annulata on the functions of bovine dendritic cells for stimulation of T lymphocyte proliferation.

The present study was performed on antigen-presenting cells (APCs) of Theileria annulata transformed dendritic cells (TaDCs) and monocyte-derived dendritic cells (MoDCs) to compare differences in antigen presentation and stimulation of T lymphocyte proliferation. Antigen presentation for T lymphocyte proliferation was analysed by flow cytometry. Additionally, the level of mRNA transcription of small GTPases of the Rab family expressed in the TaDC cell line was analysed by quantitative real-time polymerase chain reaction (Q-RT-PCR). The endocytosis rate of TaDCs was significantly (P < 0.01) lower than in MoDCs. In contrast, when T lymphocytes were co-cultured with TaDC-APCs T cell proliferation was similar, while co-culture with MoDC-APC stimulated proliferation of CD4+ cells to a greater degree than CD8+ cells. However, the efficacy of TaDC-APCs to stimulate T lymphocytes dropped as the number of passages of TaDC-APC increased. Likewise, the transcription level of Rab family genes also significantly (P > 0.001) declined with progressive passages (>50) of the TaDC cell line. We conclude that initially the TaDC cell line efficiently presents antigen to stimulate T lymphocyte proliferation to produce a cellular immune response against the presented antigen.

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

BACKGROUND: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. RESULTS: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. CONCLUSIONS: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

Approaches to vaccination against Theileria parva and Theileria annulata.

Despite having different cell tropism, the pathogenesis and immunobiology of the diseases caused by Theileria parva and Theileria annulata are remarkably similar. Live vaccines have been available for both parasites for over 40 years, but although they provide strong protection, practical disadvantages have limited their widespread application. Efforts to develop alternative vaccines using defined parasite antigens have focused on the sporozoite and intracellular schizont stages of the parasites. Experimental vaccination studies using viral vectors expressing T. parva schizont antigens and T. parva and T. annulata sporozoite antigens incorporated in adjuvant have, in each case, demonstrated protection against parasite challenge in a proportion of vaccinated animals. Current work is investigating alternative antigen delivery systems in an attempt to improve the levels of protection. The genome architecture and protein-coding capacity of T. parva and T. annulata are remarkably similar. The major sporozoite surface antigen in both species and most of the schizont antigens are encoded by orthologous genes. The former have been shown to induce species cross-reactive neutralizing antibodies, and comparison of the schizont antigen orthologues has demonstrated that some of them display high levels of sequence conservation. Hence, advances in development of subunit vaccines against one parasite species are likely to be readily applicable to the other.

The level of H2O2 type oxidative stress regulates virulence of Theileria-transformed leukocytes.

Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.

Humoral and cellular immunity in response to an in silico-designed multi-epitope recombinant protein of Theileria annulata.

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.

Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood.

We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata.

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.

Theileria annulata-transformed cell lines are efficient antigen-presenting cells for in vitro analysis of CD8 T cell responses to bovine herpesvirus-1.

Continuously growing cell lines infected with the protozoan parasite Theileria annulata can readily be established by in vitro infection of leukocytes with the sporozoite stage of the parasite. The aim of the current study was to determine whether such transformed cell lines could be used as antigen presenting cells to analyse the antigenic specificity of bovine CD8 T cell responses to viral infections. Bovine herpes virus 1 (BHV-1), which is known to induce CD8 T cell responses, was used as a model. T. annulata- transformed cells were shown to express high levels of CD40 and CD80 and were susceptible to infection with BHV-1, vaccinia and canarypox viruses. The capacity of the cells to generate antigen-specific CD8 T cell lines was initially validated using a recombinant canarypox virus expressing a defined immunodominant T. parva antigen (Tp1). Autologous T. annulata-transformed cells infected with BHV-1 were then used successfully to generate specific CD8 T cell lines and clones from memory T cell populations of BHV-1-immune animals. These lines were BHV-1-specific and class I MHC-restricted. In contrast to previous studies, which reported recognition of the glycoproteins gB and gD, the CD8 T cell lines generated in this study did not recognise these glycoproteins. Given the ease with which T. annulata-transformed cell lines can be established and maintained in vitro and their susceptibility to infection with poxvirus vectors, these cell lines offer a convenient and efficient in vitro system to analyse the fine specificity of virus-specific CD8 T cell responses in cattle.

Ranking control options for tropical theileriosis in at-risk dairy cattle in Tunisia, using benefit-cost analysis.

An economic evaluation of various control programmes against Theileria annulata infection was conducted on a sample of 49 Tunisian dairy farms where clinical cases of tropical theileriosis had been recorded during the summer. Indicators of morbidity and the prevalence of infection, as well as production and demographic indicators (recorded in the present survey ortaken from secondary sources), were used to rank the potential costs and benefits of various control programmes for tropical theileriosis over a time horizon of 15 years. Three options were considered, i.e. vaccination with a local attenuated cell-line vaccine; partial barn upgrading, based on first roughcasting then smoothing all the walls of the animal premises (inner and outer surfaces); and applying acaricides to control the vector tick population on the cattle. The most important loss from this disease, representing between 22% and 38% of the overall losses, is the loss in milk yield from carriers of T. annulata. Upgrading barns produced the highest mean benefit-cost ratio (1.62 to 3.71), while the ratios for vaccination and acaricides ranged from 0.20 to 1.19 and 0.32 to 0.88, respectively. However, the benefit-cost ratio of vaccination increased (from 1.65 to 5.41), when the costs due to carrier state infection, which vaccination does not prevent, were ignored. Upgrading barns is a sustainable eradication policy against tropical theileriosis, based on a single investment, and is environmentally friendly. This control option should be encouraged by national Veterinary Authorities in regions where tropical theileriosis is transmitted by a domestic endophilic tick.

Development and laboratory evaluation of a lateral flow device (LFD) for the serodiagnosis of Theileria annulata infection.

Several DNA-based and serological tests have been established for the detection of Theileria annulata infection, including polymerase chain reaction, reverse line blot and loop-mediated isothermal amplification, indirect enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. In this study, we have applied knowledge from the development and application of a recombinant protein-based indirect ELISA and competitive ELISA to establish a rapid test for point-of-care diagnosis of T. annulata infection in the field to be used by the veterinarian. For the development of a lateral flow test, the recombinantly expressed T. annulata surface protein (TaSP) was applied as the test antigen and anti-TaSP antiserum as the control line. TaSP antigen conjugated to colloidal gold particles was used as the detection system for visualization at the test line for the binding of anti-TaSP antibody present in the serum of infected animals. The developed test specifically detected antibodies in the serum of animals experimentally infected with T. annulata and showed no cross-reactivity with serum from animals infected with other tested bovine pathogens (Trypanosoma brucei, Anaplasma marginale, Babesia bigemina, Babesia bovis, and Theileria parva). Testing of field samples was compared to results obtained by other serological tests, resulting in a sensitivity and specificity of 96.3% and 87.5% compared to indirect fluorescence antibody test, 98.7% and 81.8% compared to indirect ELISA, and 100% and 47.6% compared to competitive ELISA. In conclusion, a rapid test for the detection of T. annulata infection (T. annulata lateral flow device, Ta-LFD) has been developed, which is easy to perform, delivers results to be read by the naked eye within 10 min, and is suitable for the detection of infection in field samples.

Theileria annulata transformation altered cell surface molecules expression and endocytic function of monocyte-derived dendritic cells.

Theileria annulata is a protozoan parasite transmitted by ticks to cattle. The most important processes of T. annulata are the infection and transformation of host monocytes, which promote cell division and generate a neoplastic phenotype. Dendritic cells play an important role in the development of adaptive immune responses against parasites and are traditionally classified into four types. One type of dendritic cell derived from afferent lymph was successfully transformed by T. annulata in vitro in a previous report. However, whether the monocyte-derived dendritic cells could be transformed and how the endocytic function is affected by T. annulata infection were not yet known. Bovine dendritic cells (DCs) derived from blood CD14+ monocytes were cocultured with T. annulata sporozoites in vitro. On day 15 post infection, rounded and continuously proliferating cells were observed. The effect of this transformation on cell phenotype was studied using immunostaining and flow cytometry. After transformation, the cells maintained the expression of the DC-specific marker CD11c, but it was downregulated as were the expression of CD11b, CD14 and CD86. In contrast, CD205, CD45 and MHC class Ⅱ molecules were upregulated in transformed cells. The levels of CD172a, CD21, CD40 and CD80 expression were very low in the transformed cells (<1 %). However, the transformed cells maintained high expression levels of MHC Ⅰ (>99 %). In addition, the normal and transformed DCs were cocultured with OVA-FITC antigen to compare the differences of the endocytic functions between these two types of cells. The results revealed that the endocytic functions of MoDCs were significantly inhibited after transformation by T. annulata.

The protection afforded to cattle immunized with Theileria annulata infected cell line is enhanced by subunit vaccine candidate TaSP.

Tropical theileriosis constraints the development of the dairy industry in the Sudan and vaccination using live attenuated schizont vaccines is considered a promising measure for its control. The present study was carried out to investigate the ability of recombinant T. annulata surface protein (TaSP) to improve the efficacy of the attenuated Atbara cell line in protecting calves against field challenge. To this end, 23 cross-bred (Friesian × Kenana) calves were divided into four groups. Animals in group 1 (n = 5) were left unvaccinated. Group 2 (n = 6) received the Atbara cell line, animals in group 3 (n = 6) were immunized with three doses of TaSP on days 21, 49 and 77, while animals in group 4 (n = 6) received the cell line vaccine on day 0 and three doses of TaSP in Freund's incomplete adjuvant at days 21, 49 and 77. Twenty-eight days after the last TaSP boost, all groups were challenged by exposing them to natural field tick infestation in a region known to be endemic for tropical theileriosis. No thermal reactions, piroplasms or schizonts were observed in the immunized animals following immunization. Upon challenge, all animals showed a range of symptoms of clinical theileriosis with variable degrees of severity. The application of TaSP alone appeared to have no effect in terms of protection. The efficacy of the cell line alone was lower than the 100% level of protection against mortality observed in the group that received the combined cell line vaccine and TaSP, suggesting a synergistic effect of this combination.

Theileria annulata surface protein (TaSP) is a target of cyclin-dependent kinase 1 phosphorylation in Theileria annulata-infected cells.

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T131 PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T131 , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T. annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T. annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.

Field validation of a competitive ELISA for detection of Theileria annulata infection.

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop a recombinant-protein-based indirect enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antibodies in serum of T. annulata-infected animals. To increase the specificity, a competitive ELISA (cELISA) was developed using recombinant TaSP antigen and a monoclonal antibody (1C7) specifically binding to TaSP. Since the cELISA accurately differentiated T. annulata-infected from uninfected animals, a study was performed to analyse the suitability of the cELISA in the field. For this, 230 sera with unknown status from different governorates in the north of Iraq were analysed using both the indirect and competitive ELISA and were compared. There was a significant (p < 0.5 x 10(-19)) correlation (r = 0.556) between the tests, whereby the cELISA detected more sera as negative (44/230) compared to the indirect ELISA (21/270). Accordingly, less sera were determined to be positive in the competitive (186/230) than in the indirect ELISA (209/230). Sensitivity and specificity of the cELISA taking the indirect ELISA as a reference were 84.2% and 52.4%, respectively. Accordingly, the calculated prevalence of T. annulata infection was 90.9%, and the positive predictive value was determined to be 94.6%. Taken together, the cELISA proved its suitability for field application and was found qualified for use in serological surveys to monitor the prevalence of T. annulata infection and to identify carrier animals.

Determination of CD Markers Profile of the Cell Line Infected by S15 Vaccine Strain of Theileria annulata Schizont Using RT-PCR Analysis.

The aim of this study was to identify the cell surface cluster of differentiation (CD) markers of the cell lines infected by Theileria annulata schizont. The CD molecules are very useful for the characterization of cells and different subpopulations of leukocytes. They are usually recognized by specific antibodies using flow cytometry and immunohistochemistry. In the current study, we applied reverse transcriptase-polymerase chain reaction (RT-PCR) to define the profile of cell surface markers in a cell line infected by an attenuated S15 vaccine strain of T. annulata schizont and a new laboratory-established cell line infected by a non-attenuated form. In order to determine the specific markers that can be used for excluding the non-attenuated cell lines, the characterization of the surface proteins profile of the S15 vaccine cell line is important. The RT-PCR was carried out by specifically designed primers using a panel of seven bovine CD markers, as well as beta-actin as an internal control house-keeping gene. We showed that both of the examined cell lines had a consistent expression of CD4, CD5, CD11a, CD14, CD43, and CD45 markers. However, the specific finding in this study was the expression of B-cell markers CD79a and CD5 by the newly-transformed cell line. On the other hand, CD5 as a marker for B-cell subset was expressed by S15 vaccine strain. In conclusion, we consider CD79a surface protein as a new marker for the cell lines infected by non-attenuated T. annulata schizont, while the cell lines infected by the vaccine strain do not express this marker. In addition, the identification of CD marker expression based on the RT-PCR assay might be a suitable and appropriate alternative technique for flow cytometry.

Demonstration of strain-specific CD8 T cell responses to Theileria annulata.

The present study set out to examine the nature and specificity of the bovine CD8 T cell response at the clonal level in a group of eight animals immunized with a cloned population of Theileria annulata. The results demonstrated that immunized animals generated parasite-specific CD8 T cells that produced IFN in response to parasite stimulation but had highly variable levels of cytotoxicity for parasitized cells. The study also demonstrated that these parasite-specific CD8 T cells could be propagated and cloned in vitro from the memory T cell pool of cattle immunized with live T. annulata parasites. Within the small group of animals studied, there was evidence that responses were preferentially directed to antigens presented by an A10+ class I major histocompatibility complex (MHC) haplotype, suggesting that responses restricted by products of this haplotype may be dominant. The A10-restricted responses showed differential recognition of different parasite isolates and clones. By using a cloned population of parasites both for immunization of the animals and for in vitro analyses of the responses, we obtained unambiguous evidence that at least a proportion of CD8 T cells restricted by one MHC haplotype were parasite strain restricted.

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals.

BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane. RESULTS: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum. CONCLUSIONS: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.