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

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

Development of a dual vaccine against East Coast fever and lumpy skin disease.

East Coast fever is an acute bovine disease caused by the apicomplexan parasite Theileria parva and is regarded as one of the most important tick-vectored diseases in Africa. The current vaccination procedure has many drawbacks, as it involves the use of live T. parva sporozoites. As a novel vaccination strategy, we have constructed the recombinant lumpy skin disease virus (LSDV) named LSDV-SODis-p67HA-BLV-Gag, encoding a modified form of the T. parva p67 surface antigen (p67HA), as well as the bovine leukemia virus (BLV) gag gene for the formation of virus-like particles (VLPs) to potentially enhance p67 immunogenicity. In place of the native sequence, the chimeric p67HA antigen has the human tissue plasminogen activator signal sequence and the influenza hemagglutinin A2 transmembrane domain and cytoplasmic tail. p67HA was detected on the surface of infected cells, and VLPs comprising BLV Gag and p67HA were produced. We also show that higher multiple bands observed in western blot analysis are due to glycosylation of p67. The two vaccines, pMExT-p67HA (DNA) and LSDV-SODis-p67HA-BLV-Gag, were tested for immunogenicity in mice. p67-binding antibodies were produced by vaccinated animals, with higher titers detected in mice vaccinated with the recombinant LSDV. This candidate dual vaccine warrants further testing in cattle.

Theileria annulata SVSP455 interacts with host HSP60.

BACKGROUND: Theileria annulata, a transforming parasite, invades bovine B cells, dendritic cells and macrophages, promoting the uncontrolled proliferation of these cells. This protozoan evolved intricate strategies to subvert host cell signaling pathways related to antiapoptotic signaling to enable survival and proliferation within the host cells. However, the molecular mechanisms of the cell transformation induced by T. annulata remain largely unclear. Although some studies have predicted that the subtelomere-encoded variable secreted protein (SVSP) family plays roles in host-parasite interactions, the evidence for this is limited. METHODS: In the present study, the SVSP455 (TA05545) gene, a member of the SVSP gene family, was used as the target molecule. The expression pattern of SVSP455 in different life-cycle stages of T. annulata infection was explored using a quantitative real-time PCR assay, and the subcellular distribution of SVSP455 was observed using confocal microscopy. The host cell proteins interacting with SVSP455 were screened using the Y2H system, and their interactions were verified in vivo and in vitro using both bimolecular fluorescence complementation and confocal microscopy, and co-immunoprecipitation assays. The role played by SVSP455 in cell transformation was further explored by using overexpression, RNA interference and drug treatment experiments. RESULTS: The highest level of the SVSP455 transcript was detected in the schizont stage of T. annulata, and the protein was located both on the surface of schizonts and in the host cell cytoplasm. In addition, the interaction between SVSP455 and heat shock protein 60 was shown in vitro, and their link may regulate host cell apoptosis in T. annulata-infected cells. CONCLUSION: Our findings are the first to reveal that T. annulata-secreted SVSP455 molecule directly interacts with both exogenous and endogenous bovine HSP60 protein, and that the interaction of SVSP455-HSP60 may manipulate the host cell apoptosis signaling pathway. These results provide insights into cancer-like phenotypes underlying Theilera transformation and therapeutics for protection against other pathogens.

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.

Development and evaluation of a chemiluminescence immunoassay for detecting tropical theileriosis.

Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection.

Immune parameters to p67C antigen adjuvanted with ISA206VG correlate with protection against East Coast fever.

East Coast fever (ECF) is a lymphoproliferative disease caused by the tick-transmitted protozoan parasite Theileria parva. ECF is one of the most serious cattle tick-borne diseases in Sub-Saharan Africa. We have previously demonstrated that three doses of the C-terminal part of the sporozoite protein p67 (p67C) adjuvanted with ISA206VG confers partial protection against ECF at a herd level. We have tested the efficacy of two doses of this experimental vaccine, as reducing the vaccination regimen would facilitate its deployment in the field. We reconfirm that three antigen doses gave a significant level of protection to severe disease (46%, ECF score < 6) when compared with the control group, while two doses did not (23%). Animals receiving three doses of p67C developed higher antibody titers and CD4+ T-cell proliferation indices, than those which received two doses. A new panel of immune parameters were tested in order to identify factors correlating with protection: CD4+ proliferation index, total IgG, IgG1, IgG2 and IgM half maximal titers and neutralization capacity of the sera with and without complement. We show that some of the cellular and humoral immune responses provide preliminary correlates of protection.

Genotyping of Theileria lestoquardi from sheep and goats in Sudan to support control of Malignant Ovine Theileriosis.

Theileriosis, caused by parasitic protozoa of the genus Theileria parasites, are among the major tick-borne diseases of ruminant livestock. The largest economic losses are attributed in particular to those caused by the leukoproliferative species of Theileria: T. parva, T. annulata and T. lestoquardi. Theileria lestoquardi is transmitted by Hyalomma ticks and causes malignant ovine theileriosis (MOT), a disease that is particularly prevalent in Sudan. The disease is considered of a high economic importance in Sudan, where export of sheep is a major component of the national economy. A live vaccine based on a Sudanese isolate of T. lestoquardi (Atbara strain) was previously developed for the control of MOT in Sudan, but not yet deployed in the field. The present study aims to genetically characterize and compare samples of T. lestoquardi circulating in Sudan as well as the live vaccine isolate in order to understand vaccine breakthroughs and failure that may occur. Sheep and goats blood samples were collected from six regions in Sudan that are known to be endemic for T. lestoquardi infection or have experienced outbreaks of MOT. Blood samples infected with T. lestoquardi were identified by PCR or RLB. Genotyping was carried out by (1) sequencing the homologues of two T. parva CD8+ T cell antigen genes, Tp1 and Tp2, and (2) using a panel of seven micro- and mini-satellite markers. A total of 100 T. lestoquardi positive field samples and the T. lestoquardi (Atbara) vaccine were genotyped. The results showed that all samples had mixed genotypes, with several alleles identified at one or more loci. The gene diversity ranged from 0.7840 (TS8) to 0.2133 (TS12) with mean values of 0.5470. PCA revealed three clusters of the parasite in Sudan; interestingly one independent cluster was clearly seen, corresponding to the vaccine isolate. The T. lestoquardi Tp1 homologue showed higher homology with T. annulata than with T. parva sequences included the defined single CD8+ T cell target epitope region. The result indicates that multiple genotypes are a common feature of T. lestoquardi infection in Sudan. Both genotyping and the sequencing results clearly showed that the vaccine isolate is highly distinct from the field samples. This finding raised the question whether vaccination with the prepared lived vaccine will effectively protect animals against challenges by the field isolates of T. lestoquardi. The results of this work will inform on the best approach for controlling MOT in Sudan.

The aetiology, pathogenesis and control of theileriosis in domestic animals.

The Theileria genus includes a large number of species of tick-borne parasites that infect domestic animals and wildlife species, predominantly ruminants. These range from species, such as T. parva and T. annulata, which cause acute lymphoproliferative diseases in cattle resulting in high levels of mortality, to others that are non-pathogenic. In the last decade, several new pathogenic species of Theileria have been identified and pathogenic strains of other previously low-pathogenic species have emerged. Theileria parasites are characterised by developmental stages within leukocytes and erythrocytes. The capacity of the most pathogenic species to undergo extensive multiplication during intra-leukocyte development is central to their ability to cause disease. However, this is not the sole property responsible for disease, as illustrated by T. parva, which grows in a similar mannerin buffalo cells butdoes notcause disease inthisspecies. Because of the highly pathogenic nature of these parasites in livestock and the susceptibility of young animals to disease, control of the diseases is challenging. Control by chemotherapy and prevention of tick infestation has proved expensive and difficult to sustain. Vaccines using live parasites are available for T. parva and T. annulata and have been used with some success in the field. However, their widespread use has been hampered by practical constraints in production and distribution of the vaccines. Studies of the immune responses in immune cattle have helped to elucidate the protective immune responses and identified a number of parasite antigens that are currently being explored for development of alternative vaccines.

Engineering attenuated virulence of a Theileria annulata-infected macrophage.

Live attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata-transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata-infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis.

Attenuation of Theileria lestoquardi infected cells and immunization of sheep against malignant ovine theileriosis.

Malignant ovine theileriosis caused by Theileria lestoquardi is an economically important disease infecting small ruminants in the Sudan. The disease causes massive losses among sheep in many regions of Northern Sudan. The present studies were done to isolate lymphoblastoid cells infected with malignant ovine theileriosis and attenuate them by passage using culture media to develop and produce schizonts candidate vaccine, then test its efficacy and safety by exposing immunized lambs to field challenge in an area endemic with T. lestoquardi. In the present experiments we isolated and established an in vitro culture of T. lestoquardi infected lymphoblast cell line. Long-term culture of T. lestoquardi infected lymphoplastoid cells was shown to result in attenuation of their virulence and lambs inoculated with different doses of such cells at passage 105 exhibited very mild reactions with fever that lasted for 1-5 days and parasitaemia of <0.2%. The experimental lambs immunized with this candidate vaccine were immune and protected when exposed to field challenge in an area endemic of ovine theileriosis, while morbidity and mortality among non-immunized animals reached 76.9% and 46.15%, respectively, and they exhibited the clinical signs of malignant ovine theileriosis that included, high fever, loss of appetite, enlargement of lymph nodes, jaundice, loss of weight and death. The present study demonstrates the efficacy and the safety of this attenuated cell line as a live attenuated candidate vaccine.

Designing bovine T cell vaccines via reverse immunology.

T cell responses contribute to immunity against many intracellular infections. There is, for example, strong evidence that major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) play an essential role in mediating immunity to East Coast fever (ECF), a fatal lymphoproliferative disease of cattle prevalent in sub-Saharan Africa and caused by Theileria parva. To complement the more traditional approaches to CTL antigen identification and vaccine development that we have previously undertaken we propose a use of immunoinformatics to predict CTL peptide epitopes followed by experimental verification of T cell specificity to candidate epitopes using peptide-MHC (pMHC) tetramers. This system, adapted from human and rodent studies, is in the process of being developed for cattle. Briefly, we have used an artificial neural network called NetMHCpan, which has been trained mainly on existing human, mouse, and non-human primate MHC-peptide binding data in an attempt to predict the peptide-binding specificity of bovine MHC class I molecules. Our data indicate that this algorithm needs to be further optimized by incorporation of bovine MHC-peptide binding data. When retrained, NetMHCpan may be used to predict parasite peptide epitopes by scanning the predicted T. parva proteome and known parasite CTL antigens. A range of pMHC tetramers, made "on-demand", will then be used to assay cattle that are immune to ECF or in vaccine trials to determine if CTLs of the predicted epitope specificity are present or not. Thus, pMHC tetramers can be used in one step to identify candidate CTL antigens and to map CTL epitopes. Our current research focuses on 9 different BoLA class I molecules. By expanding this repertoire to include the most common bovine MHCs, these methods could be used as generic assays to predict and measure bovine T cell immune responses to any pathogen.

Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy.

Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses.

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization.

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.

Innate immunity to tropical theileriosis.

The intracellular protozoan parasite Theileria annulata causes a severe, and often fatal, disease of pure and cross-bred cattle in tropical and subtropical countries. The present review refers to the importance of innate immunity as far as it is known to date in this infectious disease. Specifically, macrophages and the mediators produced by these cells are outlined. In addition, the latest findings concerning cattle breed differences in susceptibility to T. annulata infection in relation to macrophage activation are discussed.

Titrating Theileria parva: single stocks against combination of stocks.

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.

Tropical theileriosis: cytotoxic T lymphocyte response to vaccination.

Cattle which survive an infection with Theileria annulata become effectively immune to challenge with the same parasite strain, and are thought to be protected against a heterologous strain of the parasite. T-cells play a crucial role in both induction and maintenance of immunity to T. annulata. The generation of cytotoxic T lymphocytes (CTL) is closely related to the control of the infection - macroschizont-infected cells are killed in an MHC class I restricted manner. Any strain-specificity induced by immunisation is likely to be manifested by CTL. Besides CTLs, CD4+ T-cells also play an important role in protective immunity to T. annulata infection.

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle.

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.

Purification of macroschizonts of a Sudanese isolate of Theileria lestoquardi (T. lestoquardi [Atbara]).

Research on malignant theileriosis is affected by the limited access to biological materials required for studies aiming at controlling the disease through the establishment of diagnostic tools and vaccines. The main aims of this work were to isolate, establish, and characterize a Theileria lestoquardi-infected cell culture (line) as a source of biological material and to generate a schizont cDNA library for further studies aiming at the identification of antigenic proteins. The T. lestoquardi isolate used originated from a sheep showing typical signs of malignant theileriosis in Atbara town in northern Sudan, and was maintained as an infected cell culture. A high-quality representative schizont cDNA library was established by isolating and purifying the schizonts using a nocodazole/aerolysin protocol followed by Percoll gradient ultracentrifugation. As a parameter to assess the quality of the schizont library, a provisional estimation of the percentage of recombinant phage clones originating from T. lestoquardi (Atbara) was undertaken. Ten clones with inserts ranging in size between 600 and 1200 bp were selected randomly, sequenced, and subjected to BLAST similarity searches. As 6 of the 10 sequenced clones showed similarities to T. parva, T. annulata, and other apicomplexan genes, it was concluded that the majority of the library phage clones originated from the parasite and not from host cell transcripts. The cDNA library will be used for screening of antigenic proteins using sera from infected sheep.

Reduction of Theileria annulata infection in ticks fed on calves immunized with purified larval antigens of Hyalomma anatolicum anatolicum.

Larval antigen of Hyalonmma anatolicum anatolicum, the vector of Theileria annulata, was purified by two-step affinity chromatography using anti-tick gut-specific rabbit IgG and IgG from immunized cattle. The purified antigen showed the presence of a single polypeptide of 37 kDa (GHLAgP) on SDS-PAGE. Two groups (I and II) of naive crossbred calves (Bos taurus x B. indicus) were immunized with I mg of GHLAgP in three divided doses. Immunized calves of group I were also infected with a sublethal dose of T annulata along with a group of non-immunized calves (group III). Animals in groups I, II, III as well a control group (group IV) were challenged with live nymphs of H. a. anatolicum on the 10th day of immunization. There was a significant reduction in the number of emerging adults of 56.9% +/- 1.67%, in calves of group I (p < 0.01) and 63.09% +/- 1.26% in calves of group II (p < 0.001) compared to the controls. The calves of groups I and II showed antibody responses to tick antigen up to day 70 post immunization. Infection with T. annulata was determined in the salivary glands of adult ticks that developed from the nymphs used for challenge infection. In ticks taken from group I calves, there was a 75.0% +/- 0.00% infection compared with only 85.0 +/- 2.88% infection in ticks taken from calves of group III. Using PCR, a lower infection (83.33% +/- 3.33%) was detected in ticks that developed from calves of group I compared with calves from group III (90.00% +/- 2.88%). The ground-up tick supernatants (GUTS) of the ticks taken from calves of group III yielded higher infection rate and exhibited higher infectivity titre in in vitro infection assay of bovine mononuclear cells than the GUTS of the ticks taken from calves of group I. The results suggest a partial reduction in growth rate of T. annulata in ticks feeding on calves immunized with GHLAgP.