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.

CD4 T Cell Responses to Theileria parva in Immune Cattle Recognize a Diverse Set of Parasite Antigens Presented on the Surface of Infected Lymphoblasts.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

Integral Use of Immunopeptidomics and Immunoinformatics for the Characterization of Antigen Presentation and Rational Identification of BoLA-DR-Presented Peptides and Epitopes.

MHC peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein Ags to elicit functional T cell responses. Liquid chromatography-mass spectrometry analysis of MHC-eluted ligand data has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of Ag presentation have reached a high level of accuracy for both MHC class I and class II. In this study, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte Ag class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by eluted ligand data derived from bovine cell lines expressing a range of DRB3 alleles prevalent in Holstein-Friesian populations. The model generated (NetBoLAIIpan, available as a Web server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR-restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced mass spectrometry peptidomics with immunoinformatics for characterization of the BoLA-DR Ag presentation system and provide a prediction tool that can be used to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

Interaction between transforming Theileria parasites and their host bovine leukocytes.

Theileria are tick-transmitted parasites that cause often fatal leuko-proliferative diseases in cattle called tropical theileriosis (T. annulata) and East Coast fever (T. parva). However, upon treatment with anti-theilerial drug-transformed leukocytes die of apoptosis indicating that Theileria-induced transformation is reversible making infected leukocytes a powerful example of how intracellular parasites interact with their hosts. Theileria-transformed leukocytes disseminate throughout infected cattle causing a cancer-like disease and here, we discuss how cytokines, noncoding RNAs and oncometabolites can contribute to the transformed phenotype and disease pathology.

Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle.

East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

Pathogenesis of liver lesions in Theileria orientalis-inoculated cattle and severe combined immunodeficiency mice with bovine erythrocyte transfusion.

Theileria orientalis (T. orientalis) is a bovine protozoal disease similar to malaria in humans. Although the common outcome of malaria in humans and T. orientalis infection in cattle is hepatic disorder, the mechanisms of its development remain unknown. In this study, we investigated hepatocyte injury characterized by accumulation of macrophages with ingested erythrocytes in sinusoid and extramedullary hematopoiesis in cattle and mice experimentally infected with T. orientalis (T. orientalis-infected cattle and T. orientalis-infected mice). Vacuolization of hepatic cells was frequently observed in the vicinity of the aggregated macrophages in the liver sinusoids of T. orientalis-infected mice. A significant percentage of the macrophages accumulated in the liver sinusoids of the severely infected cattle and mice (14.6% and 24.2 to 53.2%, respectively) reacted positively with interleukin-1, interleukin-6 and TNF-α antibodies. Increase in the production of these cytokines was confirmed in T. orientalis-infected cattle and mice by real-time RT-PCR. These findings strongly suggest that increased cytokine production by the macrophages that have phagocytosed T. orientalis-infected erythrocytes causes hepatic disorder in T. orientalis-infected animals.

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.

Evaluation of cytokines and sialic acids contents in horses naturally infected with Theileria equi.

This study was undertaken to assess the effects of T. equi infection on serum concentrations of some important cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-1 beta (IL-1ß), IL-1α, IL-4, IL-6, IL-8, IL-10, IL-12α, IL-12ß, IL-18, as well as total, protein and lipid binding sialic acids (TSA, PBSA and LBSA). Furthermore, any probable relation among the parasitemia, cytokines and sialic acids (SAs) were calculated using Pearson correlation and simple linear regression. Almost 300 draft horses (Kurdish-breed) with age of 3-4 years old from north-west of Iran were examined and an infected group comprised of 28 mares, naturally infected with T. equi, was identified and divided into 3 subgroups according to their parasitemia rates (low <1 %, moderate 1-3 % and high 3-5 %). Twenty healthy horses were considered as a control. Characterization and differentiation of piroplasmosis were conducted using routine hematological procedures and specific PCR assay. The results revealed a significant increase (P < 0.05) in all of the cytokines and SAs in a parasitic burden-dependent fashion. Additionally, a strong and positive relation was detected among the parasitemia, cytokines and SAs. Conclusively, T. equi infection is associated with induction of severe inflammatory processes in horses and SA plays a pivotal role in pathophysiology of the disease as it is tightly correlated with the parasitemia rate.

Changes in the Molecular and Functional Phenotype of Bovine Monocytes during Theileria parva Infection.

Theileria parva is the causative agent of East Coast fever (ECF), a tick-borne disease that kills over a million cattle each year in sub-Saharan Africa. Immune protection against T. parva involves a CD8+ cytotoxic T cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role played by innate immune cells in ECF pathogenesis and T. parva control. Here, we demonstrate an increase in intermediate monocytes (CD14++ CD16+) with a concomitant decrease in the classical (CD14++ CD16-) and nonclassical (CD14+ CD16+) subsets at 12 days postinfection (dpi) during lethal infection but not during nonlethal T. parva infection. Ex vivo analyses of monocytes demonstrated upregulation of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) mRNA and increased nitric oxide production during T. parva lethal infection compared to nonlethal infection at 10 dpi. Interestingly, no significant differences in peripheral blood parasite loads were observed between lethally and nonlethally infected animals at 12 dpi. In vitro stimulation with T. parva schizont-infected cells or Escherichia coli lipopolysaccharide (LPS) resulted in significant upregulation of IL-1ß production by monocytes from lethally infected cattle compared to those from nonlethally infected animals. Strikingly, monocytes from lethally infected animals produced significant amounts of IL-10 mRNA after stimulation with T. parva schizont-infected cells. In conclusion, we demonstrate that T. parva infection leads to alterations in the molecular and functional phenotypes of bovine monocytes. Importantly, since these changes primarily occur in lethal infection, they can serve as biomarkers for ECF progression and severity, thereby aiding in the standardization of protection assessment for T. parva candidate vaccines.

The haematological, proinflammatory cytokines and IgG changes during an ovine experimental theileriosis.

Malignant ovine theileriosis is caused by Theileria lestoquardi, which is highly pathogenic in sheep. Theileriosis involves different organs in ruminants. Little is known about the role of proinflammatory cytokines in the pathogenesis of T. lestoquardi infection. The aim of this study was to measure concentration changes of proinflammatory cytokines and immunoglobulin G (IgG) during an ovine experimental theileriosis and correlate it with clinical and haematological parameters. During an experimental study, seven healthy Baluchi sheep (four females and three males) about 6-8 months old were infected with T. lestoquardi by feeding of infected unfed ticks on the sheep's ears. The infected sheep were clinically examined during the study and blood samples were collected on days 0, 2, 5, 7, 10, 12, 14, 17 and 21. The haematological parameters were analysed by an automatic veterinary haematology cell counter and the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IgG were measured by enzyme-linked immunosorbent assay. All infected sheep had temperatures above 40 °C on days 3-4 post infection (PI). The maximum temperature was noted on day 7, and it remained high until day 21. The parasitaemia of T. lestoquardi infection increased from 0.01% (day 7 PI) to 3.3% (day 21 PI). The mean white blood cell (WBC), red blood cell (RBC), lymphocyte, neutrophil and platelet values slightly increased on day 2 PI and decreased by day 17 and day 21 PI. The percentage parasitaemia and fever had a negative correlation with the numbers of WBCs, RBCs, lymphocytes, neutrophils and platelets. The serum concentration of IL-6, TNF-α and IFN-γ cytokines increased and peaked on day 12 and thereafter decreased to levels lower than 0. Out of all tested cytokines, the concentration of IL-6 was significantly higher, as early as day 2 PI. No significant changes were observed for the IgG levels during the course of disease. A significant and strong correlation was observed between IL-6, TNF-α and IFN-γ values and a moderate correlation between IL-6 and the numbers of lymphocytes in the present study. A strong correlation was determined between the percentage parasitaemia and haematological parameters in T. lestoquardi-infected sheep. In addition, preliminary results indicate that the measurement of the serum concentrations of IL-6 in combination with haematological parameters could be considered a good marker to estimate the pathogenicity of T. lestoquardi strain.

Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells.

As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles.

East Coast Fever Caused by Theileria parva Is Characterized by Macrophage Activation Associated with Vasculitis and Respiratory Failure.

Respiratory failure and death in East Coast Fever (ECF), a clinical syndrome of African cattle caused by the apicomplexan parasite Theileria parva, has historically been attributed to pulmonary infiltration by infected lymphocytes. However, immunohistochemical staining of tissue from T. parva infected cattle revealed large numbers of CD3- and CD20-negative intralesional mononuclear cells. Due to this finding, we hypothesized that macrophages play an important role in Theileria parva disease pathogenesis. Data presented here demonstrates that terminal ECF in both Holstein and Boran cattle is largely due to multisystemic histiocytic responses and resultant tissue damage. Furthermore, the combination of these histologic changes with the clinical findings, including lymphadenopathy, prolonged pyrexia, multi-lineage leukopenia, and thrombocytopenia is consistent with macrophage activation syndrome. All animals that succumbed to infection exhibited lymphohistiocytic vasculitis of small to medium caliber blood and lymphatic vessels. In pulmonary, lymphoid, splenic and hepatic tissues from Holstein cattle, the majority of intralesional macrophages were positive for CD163, and often expressed large amounts of IL-17. These data define a terminal ECF pathogenesis in which parasite-driven lymphoproliferation leads to secondary systemic macrophage activation syndrome, mononuclear vasculitis, pulmonary edema, respiratory failure and death. The accompanying macrophage phenotype defined by CD163 and IL-17 is presented in the context of this pathogenesis.

No change in mRNA expression of immune-related genes in peripheral blood mononuclear cells challenged with Theileria annulata in Murrah buffalo (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) act as carrier to Theileria annulata and show less clinical sign of tropical theileriosis as compared to indigenous and exotic cattle. Differential expression of immune-related genes such as major histocompatibility complex, class II, DQ alpha 1 (MHC-DQα), signal-regulatory protein alpha (SIRPA), prion protein (PRNP), Toll-like receptor 10 (TLR10), c-musculoaponeurotic fibrosarcoma oncogene homolog (cMAF) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) genes influence host resistance to this disease in exotic, crossbred and indigenous cattle. In the present study we examined the differential mRNA expression of the abovesaid immune-related genes in response to T. annulata infection in buffaloes. Peripheral blood mononuclear cells (PBMCs) harvested from blood samples of buffaloes were challenged with ground-up tick supernatant carrying T. annulata sporozoites in vitro. After 48h of in vitro challenge qPCR was employed to measure the relative mRNA expression of MHC-DQα, SIRPA, PRNP, TLR10, cMAF and MAFB genes in infected and control PBMCs. In the current study, the selected genes showed no change in mRNA expression after T.annulata infection which indicates that they have little role in providing host resistance to theileriosis in buffaloes.

The mRNA expression of immune-related genes in crossbred and Tharparkar cattle in response to in vitro infection with Theileria annulata.

Tropical theileriosis is a major protozoan disease of cattle and is associated with high rates of morbidity and mortality. Indigenous cattle (Bos indicus) are less affected by this disease than exotic and crossbred cattle. Genetic basis of resistance to tropical theileriosis in indigenous cattle is not well studied. Recent reports suggest that number of immune response genes expressed differentially in exotic and indigenous breeds play an important role in breed specific resistance to tropical theileriosis. Such studies comparing expression of these genes in crossbred cattle and indigenous cattle are lacking. The present study compares the mRNA expression of immune-related genes in response to Theileria annulata infection in indigenous and crossbred cattle. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of indigenous (Tharparkar) and crossbred (HF/BS/Jersey × Hariana) cattle and challenged with prepared ground-up tick supernatant carrying Theileria annulata sporozoites in vitro. qPCR was employed to measure relative mRNA expression of toll-like receptor 10 (TLR10), signal-regulatory protein alpha (SIRPA), MHC class II DQα (BoLA-DQA), musculoaponeurotic fibrosarcoma (MAF) and prion protein (PRNP) genes in infected and control PBMCs from crossbred and indigenous cattle. On the basis of comparative fold change analysis, significant up-regulation in SIRPA, PRNP and MHC DQα genes and significant down-regulation in TLR10, cMAF and MAFB genes in crossbreds as compared to indigenous cattle was observed. Results of the present study suggest that breed specific differential expression of the genes under study may contribute to the breed specific resistance to Theileria annulata infection in indigenous cattle compared to crossbred cattle.

Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis.

Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs.

NKp46+ CD3+ cells: a novel nonconventional T cell subset in cattle exhibiting both NK cell and T cell features.

The NKp46 receptor demonstrates a high degree of lineage specificity, being expressed almost exclusively in NK cells. Previous studies have demonstrated NKp46 expression by T cells, but NKp46+ CD3+ cells are rare and almost universally associated with NKp46 acquisition by T cells following stimulation. In this study we demonstrate the existence of a population of NKp46+ CD3+ cells resident in normal bovine PBMCs that includes cells of both the αß TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+ CD3+ cells express transcripts for a broad repertoire of both NKRs and TCRs and also the CD3ζ, DAP10, and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+ CD3+ cells confirm that NKp46, CD16, and CD3 signaling pathways are all functionally competent and capable of mediating/redirecting cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+ CD3+ cells exhibit cytotoxic activity against autologous Theileria parva-infected cells in vitro, and during in vivo challenge with this parasite an expansion of NKp46+ CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results in this study identify and describe a novel nonconventional NKp46+ CD3+ T cell subset that is phenotypically and functionally distinct from conventional NK and T cells. The ability to exploit both NKRs and TCRs suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Comparative in vitro toll-like receptor ligand induced cytokine profiles of Toda and Murrah buffaloes-Identification of tumour necrosis factor alpha promoter polymorphism.

The objective of this study was to assess cytokine production upon activation of pattern recognition receptors responsible for sensing bacterial and viral pathogen associated molecular patterns in two genetically diverse buffalo breeds, Toda and Murrah. A very limited molecular-epidemiological analysis showed a higher prevalence of Anaplasma and Theileria in Murrah than Toda buffaloes. Toda buffalo peripheral blood mononuclear cells (PBMC) produced significantly higher levels of IFN γ and/or TNF α mRNAs in response to peptidoglycan, poly I:C, lipopolysaccharide, imiquimod and CpG. Flagellin stimulation did not result in any significant differences in the expression levels of the cytokines tested between these breeds. The levels of ligand induced IFN γ and TNF α mRNA and proteins also correlated except when induced with CpG. The proximal promoter region of TNF α across these two breeds were also sequenced to detect SNPs and promoter assay performed to determine their role in altering the transcriptional activity. Two polymorphisms were identified at -737 (T/A) and -1092 (G/T) positions in Toda buffalo TNF α promoter and promoter assay revealed higher transcription activity in Toda buffalos than in Murrah. This suggests that disease tolerance of these buffalo breeds could be due to the differences in their cytokine transcription levels in response to the respective PAMPs that may be at least in part determined by polymorphisms in the cytokine promoter regions.

Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva.

Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo.

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.