The Hypervariable Tpr Multigene Family of Theileria Parasites, Defined by a Conserved, Membrane-Associated, C-Terminal Domain, Includes Several Copies with Defined Orthology Between Species.

Multigene families often play an important role in host-parasite interactions. One of the largest multigene families in Theileria parva, the causative agent of East Coast fever, is the T. parva repeat (Tpr) gene family. The function of the putative Tpr proteins remains unknown. The initial publication of the T. parva reference genome identified 39 Tpr family open reading frames (ORFs) sharing a conserved C-terminal domain. Twenty-eight of these are clustered in a central region of chromosome 3, termed the "Tpr locus", while others are dispersed throughout all four nuclear chromosomes. The Tpr locus contains three of the four assembly gaps remaining in the genome, suggesting the presence of additional, as yet uncharacterized, Tpr gene copies. Here, we describe the use of long-read sequencing to attempt to close the gaps in the reference assembly of T. parva (located among multigene families clusters), characterize the full complement of Tpr family ORFs in the T. parva reference genome, and evaluate their evolutionary relationship with Tpr homologs in other Theileria species. We identify three new Tpr family genes in the T. parva reference genome and show that sequence similarity among paralogs in the Tpr locus is significantly higher than between genes outside the Tpr locus. We also identify sequences homologous to the conserved C-terminal domain in five additional Theileria species. Using these sequences, we show that the evolution of this gene family involves conservation of a few orthologs across species, combined with gene gains/losses, and species-specific expansions.

The antigen recognition portion of African buffalo class I MHC is highly polymorphic, consistent with a complex pathogen challenge environment, and the 3' region suggests distinct haplotype configurations.

African buffalo (Syncerus caffer) have been distinct from the Auroch lineage leading to domestic cattle for 5 million years, and are reservoirs of multiple pathogens, that affect introduced domestic cattle. To date, there has been no analysis of the class I MHC locus in African buffalo. We present the first data on African buffalo class I MHC, which demonstrates that gene and predicted protein coding sequences are approximately 86-87% similar to that of African domestic cattle in the peptide binding region. The study also shows concordance in the distribution of codons with elevated posterior probabilities of positive selection in the buffalo class I MHC and known antigen binding sites in cattle. Overall, the diversity in buffalo class I sequences appears greater than that in cattle, perhaps related to a more complex pathogen challenge environment in Africa. However, application of NetMHCpan suggested broad clustering of peptide binding specificities between buffalo and cattle. Furthermore, in the case of at least 20 alleles, critical peptide-binding residues appear to be conserved with those of cattle, including at secondary anchor residues. Alleles with six different length transmembrane regions were detected. This preliminary analysis suggests that like cattle, but unlike most other mammals, African buffalo appears to exhibit configuration (haplotype) variation in which the loci are expressed in distinct combinations.

Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite.

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.

Genotyping of Theileria parva populations in vaccinated and non-vaccinated cattle in Malawi.

Theileria parva is an apicomplexan protozoan parasite that causes bovine theileriosis (East Coast Fever; ECF) in central, eastern and southern Africa. In Malawi, ECF is endemic in the northern and central regions where it has negatively affected the development of dairy industry. Despite its endemic status the genetic population structure of T. parva in Malawi is currently unknown. To obtain an understanding of T. parva in Malawi, we performed population genetics analysis of T. parva populations in cattle vaccinated with the Muguga cocktail live vaccine and non-vaccinated cattle using mini- and microsatellite markers covering all the four T. parva chromosomes. The T. parva Muguga strain was included in this study as a reference strain. Linkage disequilibrium was observed when all samples were treated as a single population. There was sub-structuring among the samples as shown by the principal coordinate analysis. Majority of the samples clustered with the T. parva Muguga reference strain suggesting that the isolates in Malawi are closely related to the vaccine component, which support the current use of Muguga cocktail vaccine to control ECF. The clustering of samples from non-endemic southern region with those from endemic central region suggests expansion of the distribution of T. parva in Malawi.

Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva.

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

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.

Characterization of tabanid flies (Diptera: Tabanidae) in South Africa and Zambia and detection of protozoan parasites they are harbouring.

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

Prevalence and risk factors associated with Theileria parva infection in cattle in three regions of Tanzania.

Ticks and tickborne diseases (TBDs) are serious constraints to cattle production in Tanzania and other tropical and subtropical countries. Among the TBDs, East Coast fever (ECF) is the most important as it causes significant economic losses to the cattle industry in Tanzania. However, control of ECF in Tanzania has continued to be a challenge due to inadequate epidemiological information. The main objective of this study was to determine the epidemiological situation of Theileria parva infections in cattle kept under pastoral and agro-pastoral farming systems in Mara, Singida, and Mbeya regions of Tanzania. Blood samples were collected from 648 cattle in the three regions. Genomic DNA was extracted and amplified in a polymerase chain reaction (PCR) using T. parva-specific primers targeting the 104-kD antigen (P104) gene. In addition, information was collected on the possible risk factors of T. parva infection (animal age, region, animal sex, tick burden, tick control method, and frequency of acaricide application). The prevalence of T. parva across the three regions was 14.2%. There was variation in prevalence among the three regions with Mara (21.8%) having a significantly higher (p = 0.001) prevalence than the other regions. Moreover, Mbeya exhibited relatively lower prevalence (7.4%) compared to the other regions. Factors found to be significantly associated with an animal being PCR positive for T. parva were region (p = 0.001) and tick burden (p = 0.003). Other factors were not found to be significant predictors of being PCR positive for T. parva. The present study showed high variation in tick burden and T. parva prevalence across the regions. Therefore, different strategic planning and cost-effective control measures for ticks and T. parva infection should be implemented region by region in order to reduce losses caused by ticks and ECF in the study area.

A review on prevalence, control measure, and tolerance of Tanzania Shorthorn Zebu cattle to East Coast fever in Tanzania.

In Tanzania, control of East Coast fever (ECF) has predominantly relied on tick control using acaricides and chemotherapy, little on ECF vaccination, and very little on dissemination regarding animal immunization. In this paper, the prevalence, control measure, and tolerance of Tanzania Shorthorn Zebu (TSHZ) cattle to ECF are reviewed. In addition, the opportunities available for reducing the use of acaricides for the benefit of the farmers in terms of reduction of costs of purchasing acaricides and environmental pollution are described. The tick distribution and epidemiological factors for ECF such as the agro-ecological zones (AEZ), livestock production systems (LPS), strain, and age of the animals are also described. These factors influence the epidemiology of ECF and the distribution of TSHZ strains in different geographic locations of Tanzania. We have further showed that there is a tendency of farmers to select among the strains of TSHZ for animals which can tolerate ticks and ECF and crossbreed them with their local strains with the aim of benefiting from the inherent characteristics of the most tolerant strains. Generally, many strains of TSHZ cattle are tolerant to tick infestation and ECF infection and can be bred to respond to the needs of the people. In this review paper, we recommend that in future, ECF epidemiological studies should account for factors such as livestock production system, agro-climate, breed of animal, tick control strategy, and the dynamic interactions between them. In conclusion, we have demonstrated that an integrated control method involving use of acaricides, immunization, and ECF-tolerant/-resistant animals is required.

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.

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.

Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium.

East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy.

Seroprevalence and risk factors associated with Theileria parva infection among calves in Narok County, Kenya.

Background and Aim: East Coast fever (ECF), caused by Theileria parva, is a devastating disease that causes significant economic losses to cattle production in sub-Saharan Africa. Prevention and control of ECF are challenging in pastoral settings due to inadequate epidemiological information. This study aimed to estimate the seroprevalence and risk factors associated with T. parva infection among calves in different production systems to help design appropriate control interventions. Materials and Methods: Blood samples were collected from 318 calves and tested using an indirect enzyme-linked immunosorbent assay targeting antibodies against polymorphic immunodominant molecules found on the surface of T. parva. Information on calf characteristics and management practices was also collected during sampling. Descriptive statistics and logistic regression were used to analyze potential risk factors, such as age and acaricide application, where p < 0.05 was considered significant. Results: Of the 318 calves sampled, 41 (12.89%) were positive for T. parva, with a higher proportion in pastoral systems (36.58%) than in mixed farming systems (34.10%) and agropastoral systems (29.27%). From univariate analysis, calf age (p = 0.002), body weight (p = 0.001), suckling status (p = 0.026), rectal temperature (p = 0.06), calves on pasture (p = 0.022), other feeds (p = 0.004), feed grown within the farm (p = 0.004), acaricide application (p = 0.001), and acaricide application frequency (p = 0.001) were significantly associated with seropositivity. However, calf age (odds ratio [OR], 0.96; 95% confidence interval [CI], 0.91-0.99; p = 0.04), other feeds (OR, 8.82; 95% CI, 1.74-44.63; p = 0.009), and suckling status (OR, 0.38; 95% CI, 0.15-0.99; p = 0.05) were significantly associated with T. parva infection in the multivariable mixed logistic model. Conclusion: T. parva is circulating in young calves in the study area (and possibly in cattle populations due to maternal transfer of antibodies to the calves). There is a need for molecular surveillance to determine the presence and burden of T. parva infection.

Molecular and serological prevalence of corridor disease (buffalo associated Theileria parva infection) in cattle populations at the livestock/game interface of KwaZulu-Natal province, South Africa.

Theileria parva are intracellular protozoal parasites responsible for three disease syndromes in cattle, namely East Coast fever (ECF), Corridor disease (CD) and Zimbabwean theileriosis. The increase in reports of CD outbreaks in recent years has raised questions about the probability of adaptation of buffalo-derived T. parva strains in cattle herds adjacent to game reserves. A cross-sectional study was conducted from March 2016 to December 2018 to investigate the extent of occurrence of T. parva infections in cattle in the CD-controlled area of KwaZulu-Natal Province. Blood samples were collected from 1137 cattle from 14 herds and analysed by quantitative real-time PCR (qPCR) and indirect fluorescent antibody test (IFAT) to determine the prevalence of T. parva. A total of 484 samples from 4 of the 14 herds were further tested on qPCR for the presence of T. taurotragi infections. The data were analysed using descriptive statistics and a chi-square test was used to assess association between variables. The overall prevalence of T. parva was 1.3% (95%CI:1-2%) and 19.9% (95%CI:17-22%) on qPCR and IFAT, respectively. The qPCR positive samples were detected in March and May while IFAT positive samples were detected in all seasons sampled, with higher numbers during summer months. The Pearson Chi-squared test showed that T. parva prevalence rates based on both qPCR and IFAT were positively associated with herds with previous history of CD outbreaks (χ2 = 8.594, p = 0.003; χ2 = 69.513, p < 0.001, respectively). The overall prevalence of T. taurotragi was 39.4% (95% CI: 35-44%) with the herd-level prevalence ranging between 35.0% and 43.4%. Possible cross-reaction of T. parva IFAT to T. taurotragi was detected on few samples, however, there was no significant association between T. taurotragi infections and IFAT positivity (χ2 = 0.829, p = 0.363). Results from this study demonstrated the extent of occurrence of subclinical carriers and the level of exposure to T. parva infections in cattle populations at a livestock/game interface area of KwaZulu-Natal Province. The molecular and seroprevalence rates were low when compared with other areas where cattle-adapted T. parva infections are endemic. The adaptation of buffalo-derived T. parva in cattle population resulting in cattle-cattle transmissions seem to be unlikely under the current epidemiological state.