Systematic Determination of TCR-Antigen and Peptide-MHC Binding Kinetics among Field Variants of a Theileria parva Polymorphic CTL Epitope.

CTLs are known to contribute to immunity toward Theileria parva, the causative agent of East Coast fever. The Tp967-75 CTL epitope from the Muguga strain of T. parva is polymorphic in other parasite strains. Identifying the amino acids important for MHC class I binding, as well as TCR recognition of epitopes, can allow the strategic selection of Ags to induce cellular immunity toward T. parva In this study, we characterized the amino acids important for MHC class I binding and TCR recognition in the Tp967-75 epitope using alanine scanning and a series of variant peptide sequences to probe these interactions. In a peptide-MHC class I binding assay, we found that the amino acids at positions 1, 2, and 3 were critical for binding to its restricting MHC class I molecule BoLA-1*023:01. With IFN-γ ELISPOT and peptide-MHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 5-8 in the epitope were required for TCR recognition. Only two of eight naturally occurring polymorphic Tp9 epitopes were recognized by both CTLs. Finally, using a TCR avidity assay, we found that a higher TCR avidity was associated with a stronger functional response toward one of two variants recognized by the CTL. These data add to the growing knowledge on the cross-reactivity of epitope-specific CTLs and specificities that may be required in the selection of Ags in the design of a wide-spectrum vaccine for East Coast fever.

Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells.

There is established evidence that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite Theileria parva However, the mechanism by which the specific CD8+ T cells kill parasitized cells is not understood. Although the predominant pathway used by human and murine CD8+ T cells to kill pathogen-infected cells is granule exocytosis, involving the release of perforin and granzyme B, there is to date a lack of published information on the biological activities of bovine granzyme B. The present study set out to define the functional activities of bovine granzyme B and determine its role in mediating the killing of T. parva-parasitized cells. DNA constructs encoding functional and nonfunctional forms of bovine granzyme B were produced, and the proteins expressed in Cos-7 cells were used to establish an enzymatic assay to detect and quantify the expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parva-specific CD8+ T cell clones were found to be significantly correlated with the levels of granzyme B protein but not the levels of mRNA transcript expression. Experiments using inhibitors specific for perforin and granzyme B confirmed that CD8+ T cell killing of parasitized cells is dependent on granule exocytosis and, specifically, granzyme B. Further studies showed that the granzyme B-mediated death of parasitized cells is independent of caspases and that granzyme B activates the proapoptotic molecule Bid.

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.

Infection and treatment method (ITM) vaccine against East Coast fever: reducing the number of doses per straw for use in smallholder dairy herds by thawing, diluting and refreezing already packaged vaccine.

BACKGROUND: The Infection and Treatment Method (ITM) of vaccination is the only immunization procedure currently available to protect cattle against East Coast fever (ECF), a tick-transmitted disease responsible for losses of several hundreds of millions of dollars per year in sub-Saharan Africa. The vaccine comprises a homogenized preparation of infected ticks packaged in straws and stored in liquid nitrogen. The current manufacturing protocol results in straws containing 30-40 doses (ILRI 0804), which is impractical for immunizing small herds as found in dairy and smallholder farming systems. The ILRI 0804 SD stabilate was prepared as a 1:5 dilution of the parent stabilate, with the aim of producing vaccine stabilate straws containing between four to eight doses and thus suitable for smallholder farming systems. Infectivity of the diluted stabilate was assessed and the protective efficacy of the diluted stabilate was determined by performing experimental and field immunizations. RESULTS: Two groups of six cattle were inoculated with 1 ml of the diluted stabilate at 1:20 (equivalent to the recommended field dose for ILRI 0804, assuming no loss of sporozoite viability during thawing and refreezing) and 1:14 (assuming 30-35% loss of sporozoite viability). Schizonts were detected in all 12 animals, showing viability of sporozoites. Ten animals from the infectivity study and two control animals not previously exposed to T. parva were challenged with the parental ILRI 0804 stabilate. The results show that the two control animals displayed severe ECF reactions and were treated 14 days after challenge. Of the previously infected animals, only one underwent a severe reaction following challenge, a result in accord with the challenge experiments performed previously with the parent stabilate [Ticks Tick-Borne Dis 7:306-314, 2016]. The animal that displayed a severe reaction had no detectable schizonts and did not seroconvert following the initial inoculation with ILRI 0804 SD. In addition, 62 animals immunized under field conditions showed a mean seroconversion rate of 82%. CONCLUSION: The results presented in this article demonstrate that it is possible to prepare straws suitable for use in smallholder herds by thawing, diluting and refreezing already packaged vaccine.

Theileria parva antigens recognized by CD8+ T cells show varying degrees of diversity in buffalo-derived infected cell lines.

The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.

Immunization with one Theileria parva strain results in similar level of CTL strain-specificity and protection compared to immunization with the three-component Muguga cocktail in MHC-matched animals.

BACKGROUND: The tick-borne protozoan parasite Theileria parva causes a usually fatal cattle disease known as East Coast fever in sub-Saharan Africa, with devastating consequences for poor small-holder farmers. Immunity to T. parva, believed to be mediated by a cytotoxic T lymphocyte (CTL) response, is induced following natural infection and after vaccination with a live vaccine, known as the Infection and Treatment Method (ITM). The most commonly used version of ITM is a combination of parasites derived from three isolates (Muguga, Kiambu 5 and Serengeti-transformed), known as the "Muguga cocktail". The use of a vaccine comprising several strains is believed to be required to induce a broad immune response effective against field challenge. In this study we investigated whether immunization with the Muguga cocktail induces a broader CTL response than immunization with a single strain (Muguga). RESULTS: Four MHC haplotype-matched pairs of cattle were immunized with either the trivalent Muguga cocktail or the single Muguga strain. CTL specificity was assessed on a panel of five different strains, and clonal responses to these strains were also assessed in one of the MHC-matched pairs. We did not find evidence for a broader CTL response in animals immunized with the Muguga cocktail compared to those immunized with the Muguga strain alone, in either the bulk or clonal CTL analyses. This was supported by an in vivo trial in which all vaccinated animals survived challenge with a lethal dose of the Muguga cocktail vaccine stabilate. CONCLUSION: We did not observe any substantial differences in the immunity generated from animals immunized with either Muguga alone or the Muguga cocktail in the animals tested here, corroborating earlier results showing limited antigenic diversity in the Muguga cocktail. These results may warrant further field studies using single T. parva strains as future vaccine candidates.

CD8 T-cell responses against the immunodominant Theileria parva peptide Tp249-59 are composed of two distinct populations specific for overlapping 11-mer and 10-mer epitopes.

Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide-MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106 ), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249-59 -reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross-reactivity. The Tp249-59 and Tp250-59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide-MHC I complexes adopt structurally different conformations and Tcell receptor ß sequence analysis showed that Tp249-59 and Tp250-59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant.

Sequence diversity between class I MHC loci of African native and introduced Bos taurus cattle in Theileria parva endemic regions: in silico peptide binding prediction identifies distinct functional clusters.

There is strong evidence that the immunity induced by live vaccination for control of the protozoan parasite Theileria parva is mediated by class I MHC-restricted CD8(+) T cells directed against the schizont stage of the parasite that infects bovine lymphocytes. The functional competency of class I MHC genes is dependent on the presence of codons specifying certain critical amino acid residues that line the peptide binding groove. Compared with European Bos taurus in which class I MHC allelic polymorphisms have been examined extensively, published data on class I MHC transcripts in African taurines in T. parva endemic areas is very limited. We utilized the multiplexing capabilities of 454 pyrosequencing to make an initial assessment of class I MHC allelic diversity in a population of Ankole cattle. We also typed a population of exotic Holstein cattle from an African ranch for class I MHC and investigated the extent, if any, that their peptide-binding motifs overlapped with those of Ankole cattle. We report the identification of 18 novel allelic sequences in Ankole cattle and provide evidence of positive selection for sequence diversity, including in residues that predominantly interact with peptides. In silico functional analysis resulted in peptide binding specificities that were largely distinct between the two breeds. We also demonstrate that CD8(+) T cells derived from Ankole cattle that are seropositive for T. parva do not recognize vaccine candidate antigens originally identified in Holstein and Boran (Bos indicus) cattle breeds.

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 genomes of three stocks comprising the most widely utilized live sporozoite Theileria parva vaccine exhibit very different degrees and patterns of sequence divergence.

BACKGROUND: There are no commercially available vaccines against human protozoan parasitic diseases, despite the success of vaccination-induced long-term protection against infectious diseases. East Coast fever, caused by the protist Theileria parva, kills one million cattle each year in sub-Saharan Africa, and contributes significantly to hunger and poverty in the region. A highly effective, live, multi-isolate vaccine against T. parva exists, but its component isolates have not been characterized. Here we sequence and compare the three component T. parva stocks within this vaccine, the Muguga Cocktail, namely Muguga, Kiambu5 and Serengeti-transformed, aiming to identify genomic features that contribute to vaccine efficacy. RESULTS: We find that Serengeti-transformed, originally isolated from the wildlife carrier, the African Cape buffalo, is remarkably and unexpectedly similar to the Muguga isolate. The 420 detectable non-synonymous SNPs were distributed among only 53 genes, primarily subtelomeric antigens and antigenic families. The Kiambu5 isolate is considerably more divergent, with close to 40,000 SNPs relative to Muguga, including >8,500 non-synonymous mutations distributed among >1,700 (42.5 %) of the predicted genes. These genetic markers of the component stocks can be used to characterize the composition of new batches of the Muguga Cocktail. CONCLUSIONS: Differences among these three isolates, while extensive, represent only a small proportion of the genetic variation in the entire species. Given the efficacy of the Muguga Cocktail in inducing long-lasting protection against infections in the field, our results suggest that whole-organism vaccines against parasitic diseases can be highly efficacious despite considerable genome-wide differences relative to the isolates against which they protect.

Genetic and antigenic diversity of Theileria parva in cattle in Eastern and Southern zones of Tanzania. A study to support control of East Coast fever.

This study investigated the genetic and antigenic diversity of Theileria parva in cattle from the Eastern and Southern zones of Tanzania. Thirty-nine (62%) positive samples were genotyped using 14 mini- and microsatellite markers with coverage of all four T. parva chromosomes. Wright's F index (F(ST) = 0 × 094) indicated a high level of panmixis. Linkage equilibrium was observed in the two zones studied, suggesting existence of a panmyctic population. In addition, sequence analysis of CD8+ T-cell target antigen genes Tp1 revealed a single protein sequence in all samples analysed, which is also present in the T. parva Muguga strain, which is a component of the FAO1 vaccine. All Tp2 epitope sequences were identical to those in the T. parva Muguga strain, except for one variant of a Tp2 epitope, which is found in T. parva Kiambu 5 strain, also a component the FAO1 vaccine. Neighbour joining tree of the nucleotide sequences of Tp2 showed clustering according to geographical origin. Our results show low genetic and antigenic diversity of T. parva within the populations analysed. This has very important implications for the development of sustainable control measures for T. parva in Eastern and Southern zones of Tanzania, where East Coast fever is endemic.

Use of "one-pot, mix-and-read" peptide-MHC class I tetramers and predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle.

Peptide-major histocompatibility complex (p-MHC) class I tetramer complexes have facilitated the early detection and functional characterisation of epitope specific CD8+ cytotoxic T lymphocytes (CTL). Here, we report on the generation of seven recombinant bovine leukocyte antigens (BoLA) and recombinant bovine ß2-microglobulin from which p-MHC class I tetramers can be derived in ~48 h. We validated a set of p-MHC class I tetramers against a panel of CTL lines specific to seven epitopes on five different antigens of Theileria parva, a protozoan pathogen causing the lethal bovine disease East Coast fever. One of the p-MHC class I tetramers was tested in ex vivo assays and we detected T. parva specific CTL in peripheral blood of cattle at day 15-17 post-immunization with a live parasite vaccine. The algorithm NetMHCpan predicted alternative epitope sequences for some of the T. parva CTL epitopes. Using an ELISA assay to measure peptide-BoLA monomer formation and p-MHC class I tetramers of new specificity, we demonstrate that a predicted alternative epitope Tp229-37 rather than the previously reported Tp227-37 epitope is the correct Tp2 epitope presented by BoLA-6*04101. We also verified the prediction by NetMHCpan that the Tp587-95 epitope reported as BoLA-T5 restricted can also be presented by BoLA-1*02301, a molecule similar in sequence to BoLA-T5. In addition, Tp587-95 specific bovine CTL were simultaneously stained by Tp5-BoLA-1*02301 and Tp5-BoLA-T5 tetramers suggesting that one T cell receptor can bind to two different BoLA MHC class I molecules presenting the Tp587-95 epitope and that these BoLA molecules fall into a single functional supertype.

A longitudinal assessment of the serological response to Theileria parva and other tick-borne parasites from birth to one year in a cohort of indigenous calves in western Kenya.

Tick-borne diseases are a major impediment to improved productivity of livestock in sub-Saharan Africa. Improved control of these diseases would be assisted by detailed epidemiological data. Here we used longitudinal, serological data to determine the patterns of exposure to Theileria parva, Theileria mutans, Babesia bigemina and Anaplasma marginale from 548 indigenous calves in western Kenya. The percentage of calves seropositive for the first three parasites declined from initial high levels due to maternal antibody until week 16, after which the percentage increased until the end of the study. In contrast, the percentage of calves seropositive for T. mutans increased from week 6 and reached a maximal level at week 16. Overall 423 (77%) calves seroconverted to T. parva, 451 (82%) to T. mutans, 195 (36%) to B. bigemina and 275 (50%) to A. marginale. Theileria parva antibody levels were sustained following infection, in contrast to those of the other three haemoparasites. Three times as many calves seroconverted to T. mutans before seroconverting to T. parva. No T. parva antibody response was detected in 25 calves that died of T. parva infection, suggesting that most deaths due to T. parva are the result of acute disease from primary exposure.

p67 gene alleles sequence analysis reveals Theileria parva parasites associated with East Coast fever and Corridor disease in buffalo from Zambia.

Theileriosis caused by Theileria parva infections is responsible for high cattle mortalities in Zambia. Although infected buffalo are a risk to cattle, the characterization of T. parva parasites occurring in this host in Zambia has not been reported. Furthermore, considering the advances in the development of a p67 subunit vaccine, the knowledge of p67 genetic and antigenic diversity in both cattle and buffalo associated T. parva is crucial. Therefore, blood samples from buffalo (n=43) from Central, Eastern and Southern provinces, and cattle (n=834) from Central, Copperbelt, Eastern, Lusaka, and Southern provinces, were tested for T. parva infection and the parasites characterized by sequencing the gene encoding the p67 antigen. About 76.7 % of buffalo and 19.3 % of cattle samples were PCR positive for T. parva. Three of the four known p67 allele types (1, 2 and 3) were identified in parasites from buffalo, of which two (allele types 2 and 3) are associated with T. parva parasites responsible for Corridor disease. Only allele type 1, associated with East Coast fever, was identified from cattle samples, consistent with previous reports from Zambia. Phylogenetic analysis revealed segregation between allele type 1 sequences from cattle and buffalo samples as they grouped separately within the same sub-clade. The high occurrence of T. parva infection in buffalo samples investigated demonstrates the risk of Corridor disease infection, or even outbreaks, should naïve cattle co-graze with infected buffalo in the presence of the tick vector. In view of a subunit vaccine, the antigenic diversity in buffalo associated T. parva should be considered to ensure broad protection. The current disease control measures in Zambia may require re-evaluation to ensure that cattle are protected against buffalo-derived T. parva infections. Parasite stocks used in 'infection and treatment' immunization in Zambia, have not been evaluated for protection against buffalo-derived T. parva parasites currently circulating in the buffalo population.

MDM2 regulates a novel form of incomplete neoplastic transformation of Theileria parva infected lymphocytes.

Our efforts are concerned with identifying features of incomplete malignant transformation caused by non viral pathogens. Theileria parva (T. parva) is a tick-transmitted protozoan parasite that can cause a fatal lymphoproliferative disease in cattle. The T. parva-infected lymphocytes display a transformed phenotype and proliferate in culture media like the other tumor cells, however those cells will return to normal after antiprotozoal treatment reflecting the incomplete nature of transformation. To identify signaling pathways involved in this form of transformation of T. parva-infected cells, we screened a library of anticancer compounds. Among these, TIBC, a specific inhibitor of MDM2, markedly inhibited proliferation of T. parva-infected lymphocytes and promoted apoptosis. Therefore we analyzed MDM2 function in T. parva-infected cells. Several T. parva-infected cell lines showed increased expression level of MDM2 with alternatively spliced isoforms compared to the lymphoma cells or ConA blasts. In addition, buparvaquone affected MDM2 expression in T. parva transformed cells. Moreover, p53 protein accumulation and function were impaired in T. parva-infected cells after cisplatin induced DNA damage despite the increased p53 transcription level. Finally, the treatment of T. parva-infected cells with boronic-chalcone derivatives TIBC restored p53 protein accumulation and induced Bax expression. These results suggest that the overexpression of MDM2 is closely linked to the inhibition of p53-dependent apoptosis of T. parva-infected lymphocytes. Aberrant expression of host lymphocyte MDM2 induced by cytoplasmic existence of T. parva, directly and/or indirectly, is associated with aspects of this type of transformation of T. parva-infected lymphocytes. This form of transformation shares features of oncogene induced malignant phenotype acquisition.

Escape from CD8+ T cell response by natural variants of an immunodominant epitope from Theileria parva is predominantly due to loss of TCR recognition.

Polymorphism of immunodominant CD8(+) T cell epitopes can facilitate escape from immune recognition of pathogens, leading to strain-specific immunity. In this study, we examined the TCR ß-chain (TRB) diversity of the CD8(+) T cell responses of cattle against two immunodominant epitopes from Theileria parva (Tp1(214-224) and Tp2(49-59)) and investigated the role of TCR recognition and MHC binding in determining differential recognition of a series of natural variants of the highly polymorphic Tp2(49-59) epitope by CD8(+) T cell clones of defined TRB genotype. Our results show that both Tp1(214-224) and Tp2(49-59) elicited CD8(+) T cell responses using diverse TRB repertoires that showed a high level of stability following repeated pathogenic challenge over a 3-y period. Analysis of single-alanine substituted versions of the Tp2(49-59) peptide demonstrated that Tp2(49-59)-specific clonotypes had a broad range of fine specificities for the epitope. Despite this diversity, all natural variants exhibited partial or total escape from immune recognition, which was predominantly due to abrogation of TCR recognition, with mutation resulting in loss of the lysine residue at P8, playing a particularly dominant role in escape. The levels of heterozygosity in individual Tp2(49-59) residues correlated closely with loss of immune recognition, suggesting that immune selection has contributed to epitope polymorphism.

Identification of hard ticks (Acari: Ixodidae) and seroprevalence to Theileria parva in cattle raised in North Kivu Province, Democratic Republic of Congo.

This study aimed to identify tick species and to determine their relationship with the Theileria parva seroprevalence in cattle raised under an extensive farming system in North Kivu Province, Democratic Republic of Congo in two agro-ecological zones namely medium (1,000-1,850 m) and high (>1,850 m) altitude. Among the 3,215 ticks collected on 482 animals, from February to April 2009, Rhipicephalus appendiculatus (64.26 %), the main vector of T. parva, was the most abundant species followed by Rhipicephalus decoloratus (35.49 %) and Amblyomma variegatum (0.25 %). The mean burden of R. appendiculatus tick per infested animal appeared significantly higher at medium (6.5 ± 0.22 ticks) than at high (0.07 ± 0.3 ticks) altitude (P < 0.05). However, an indirect fluorescent antibody test carried out on 450 blood samples revealed a global T. parva seroprevalence of 43 % (95 % CI: 38-47) which was not significantly (P > 0.05) different between medium (48.4 %; 95 % CI: 38-49) and high (41.9 %; 95 % CI: 35-49) altitude. These relatively low seroprevalences suggest that there is a state of endemicity to T. parva infection in the study area. The presence of the tick vector on animals was associated with an increased risk of being seropositive to T. parva infection (odds ratio = 2.04; 95 % CI: 1.8-2.3; P < 0.001). The results suggest the need for a longitudinal study to investigate the seasonal dynamics of tick species and T. parva infection. The rate of tick infection should also be evaluated in order to determine the intensity of T. parva transmission to cattle.

A Theileria parva isolate of low virulence infects a subpopulation of lymphocytes.

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1(+) γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4(+), CD8(+), and WC1(+) T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8(+) T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+), or WC1(+) T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8(+) lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.

MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors.

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.

Spatial variation of tick abundance and seroconversion rates of indigenous cattle to Anaplasma marginale, Babesia bigemina and Theileria parva infections in Uganda.

Tick abundance and seroconversion rates of 640 indigenous cattle in a mixed crop-livestock system in Uganda were investigated in a 14 months longitudinal study. Up to 100% of the cattle in Buyimini, Kubo, Nanjeho, Ojilai and Sitengo villages (high tick challenge zone) were consistently infested with Rhipicephalus appendiculatus, whereas on average 50% of the cattle in Bunghaji, Hitunga and Magoje villages (low tick challenge zone) were inconsistently infested. Likewise, up to 50% of the cattle in Buyimini, Kubo, Nanjeho, Ojilai and Sitengo villages were consistently infested with R. (Boophilus) decoloratus ticks, while on average 30% of the cattle in Bunghaji, Hitunga and Magoje were inconsistently infested. Seroconversion rates of cattle to Anaplasma marginale infection under low tick challenge were higher than those under high tick challenge, but the reverse was true for Babesia bigemina infection. For Theileria parva infection, seroconversion rates of cattle older than 6 months under low tick challenge were significantly higher than those under high tick challenge (P < 0.05). However, the likelihood of occurrence of theileriosis cases among calves (0-6 m) under high tick challenge was 6 times (Odds ratio = 5.82 [1.30-36.37]) higher than under low tick challenge. The high density of anti-tick plants Lantana camara and Ocimum suave that were widespread in villages with low tick challenge, among other factors, was probably the cause for unfavourable tick survival.