Project description:Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite's ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle-cattle and buffalo-cattle transmission. Cattle-cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle-cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

Project description:East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite Theileria parva, transmitted by the three-host tick Rhipicephalus appendiculatus. The African buffalo (Syncerus caffer) is the natural host for T. parva but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between T. parva populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of T. parva populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three T. parva antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8+ and CD4+ T cells in immunized cattle, were used to characterize genetic diversity of T. parva in cattle (n = 126) and buffalo samples (n = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between T. parva populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo T. parva population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of T. parva is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo T. parva population dynamics is needed, as only a comprehensive appreciation of the population genetics of T. parva populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination.

Project description:Corridor disease (CD) is a fatal condition of cattle caused by buffalo-derived Theileria parva. Unlike the related condition, East Coast fever, which results from infection with cattle-derived T. parva, CD has not been extensively studied. We describe in detail the clinical and laboratory findings in cattle naturally infected with buffalo-derived T. parva. Forty-six cattle were exposed to buffalo-derived T. parva under field conditions at the Ol Pejeta Conservancy, Kenya, between 2013 and 2018. The first signs of disease observed in all animals were nasal discharge (mean day of onset was 9 days post-exposure), enlarged lymph nodes (10 days post-exposure), and pyrexia (13.7 days post-exposure). Coughing and labored breathing were observed in more than 50% of animals (14 days post-exposure). Less commonly observed signs, corneal edema (22%) and diarrhea (11%), were observed later in the disease progression (19 days post-exposure). All infections were considered clinically severe, and 42 animals succumbed to infection. The mean time to death across all studies was 18.4 days. The mean time from onset of clinical signs to death was 9 days and from pyrexia to death was 4.8 days, indicating a relatively short duration of clinical illness. There were significant relationships between days to death and the days to first temperature (chi2 = 4.00, p = 0.046), and days to peak temperature (chi2 = 25.81, p = 0.001), animals with earlier onset pyrexia died sooner. These clinical indicators may be useful for assessing the severity of disease in the future. All infections were confirmed by the presence of macroschizonts in lymph node biopsies (mean time to parasitosis was 11 days). Piroplasms were detected in the blood of two animals (4%) and 20 (43%) animals seroconverted. In this study, we demonstrate the successful approach to an experimental field study for CD in cattle. We also describe the clinical progression of CD in naturally infected cattle, including the onset and severity of clinical signs and pathology. Laboratory diagnoses based on examination of blood samples are unreliable, and alternatives may not be available to cattle keepers. The rapid development of CD requires recognition of the clinical signs, which may be useful for early diagnosis of the disease and effective intervention for affected animals.

Project description:The protozoan parasite Theileria parva infects and transforms bovine lymphocytes inducing uncontrolled proliferation. The transforming schizont resides free in the host cell cytoplasm and it is assumed that proteins released from the parasite contribute to host cell transformation and parasite persistence. The identification and characterisation of parasite genes encoding candidate secreted proteins constitutes a first step towards elucidating this complex process. In earlier work, it was shown that the genes encoding subtelomere-encoded variable secreted proteins (SVSPs) are located at the subtelomeres of all four T. parva chromosomes and, with 85 members, form the largest Theileria gene family. The majority of predicted proteins contain signal peptides, suggesting secretion into the host cell cytoplasm. We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Preliminary microarray followed by quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was neither influenced by the cell type transformed by T. parva, nor by the animal background. Instead, the pattern of SVSP mRNA expression was largely defined by the parasite genotype and found to be relatively stable when monitored in vitro over a period of two months. Experiments using antibodies raised against the SVSP TP03_0882 provided first evidence for protein expression. Interestingly, results indicate that SVSP expression in cell lines established from a cloned parasite is limited to only a small percentage of parasites, suggesting SVSP expression by individual parasites is restricted. Expression of epitope-tagged TP03_0882 in mammalian cells revealed nuclear translocation and localisation to different nuclear compartments, including nucleoli, nucleoplasm and other nuclear bodies. Nuclear translocation to the mammalian cell nucleus was shown to involve two different types of nuclear localisation signals present in the conserved C-terminal region of TP03_0882. This first characterisation, opens up possibilities for future studies on the regulation of gene expression and the biological role of these enigmatic proteins. Expression patterns of SVSP genes in different T. parva-infected cloned cell lines Keywords: Gene expression

Project description:BackgroundThe intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm.Methodology/principal findingsWe analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals.ConclusionsOur analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins.

Project description:The protozoan parasite Theileria parva infects and transforms bovine lymphocytes inducing uncontrolled proliferation. The transforming schizont resides free in the host cell cytoplasm and it is assumed that proteins released from the parasite contribute to host cell transformation and parasite persistence. The identification and characterisation of parasite genes encoding candidate secreted proteins constitutes a first step towards elucidating this complex process. In earlier work, it was shown that the genes encoding subtelomere-encoded variable secreted proteins (SVSPs) are located at the subtelomeres of all four T. parva chromosomes and, with 85 members, form the largest Theileria gene family. The majority of predicted proteins contain signal peptides, suggesting secretion into the host cell cytoplasm. We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Preliminary microarray followed by quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was neither influenced by the cell type transformed by T. parva, nor by the animal background. Instead, the pattern of SVSP mRNA expression was largely defined by the parasite genotype and found to be relatively stable when monitored in vitro over a period of two months. Experiments using antibodies raised against the SVSP TP03_0882 provided first evidence for protein expression. Interestingly, results indicate that SVSP expression in cell lines established from a cloned parasite is limited to only a small percentage of parasites, suggesting SVSP expression by individual parasites is restricted. Expression of epitope-tagged TP03_0882 in mammalian cells revealed nuclear translocation and localisation to different nuclear compartments, including nucleoli, nucleoplasm and other nuclear bodies. Nuclear translocation to the mammalian cell nucleus was shown to involve two different types of nuclear localisation signals present in the conserved C-terminal region of TP03_0882. This first characterisation, opens up possibilities for future studies on the regulation of gene expression and the biological role of these enigmatic proteins. Expression patterns of SVSP genes in different T. parva-infected cloned cell lines Keywords: Gene expression We performed a three-condition experiment comparing SVSP gene transcription in three T. parva infected cell lines: (1) 211T-A3 (consisting of CD4+ T cells), (2) 211B-A3 (consisting of B cells), and 951T-F44 (consisting of CD4+ T cells). We used direct two-color experiment design, comparing each cell line with the other, i.e. (1) 211T-A3 vs 211B-A3, (2) 211T-A3 vs 951T-F44 and (3) 211B-A3 vs 951T-F44. There were two biological replicates for each hybridization and dye-flips for each hybridization pair. Microarray consisted of four in-slide replicates peer gene as well as no-DNA spots as negative controls.

Dataset Information

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

Background

Methodology/principal findings

Conclusions/significance

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Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

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