Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The 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 Findings 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. 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. Conclusions Our 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

Development and application of genetic markers linked to A QTL controlling resistance to trypanosomosis on bovine chromosome 7.

Tsetse-transmitted trypanosomosis poses severe constraints to livestock productivity in sub-Saharan Africa, affecting large areas of the continent that hold the greatest potential for agricultural development. Greater exploitation of the genetic resistance to trypanosomosis (trypananotolerance) that is displayed by some breeds of indigenous West African cattle (e.g. N'Dama) is accepted as an important sustainable component for controlling the disease. It is envisaged that within-breed and crossbreeding genetic improvement of indigenous trypanotolerant breeds would be facilitated by the mapping of the chromosomal regions (quantitative trait loci, QTL) controlling trypanotolerance, followed by marker-assisted selection, or introgression of these regions, using their closely linked genetic markers. However, this prospect has been hampered by the absence of practical reliable markers for resistance due to the scarcity of genetic markers on cattle maps. Following a genome-wide scan in an F2 cross between trypanotolerant N'Dama from The Gambia and trypanosusceptible Kenya Boran, a QTL controlling parasitaemia was mapped to a 30 cM region on bovine chromosome 7 (Bta 7). As a means of achieving higher marker density in this QTL region, and to facilitate the identification of the candidate genes mediating trypanotolerance, a combination of chromosome-microdissection, comparative, radiation hybrid (RH) and physical mapping techniques, were exploited to isolate and order new markers in the QTL region. A high-resolution 12,000 rad Bta7 RH map consisting of 55 markers was constructed in this study. The map includes: 41 coding genes, 29 of which are novel previously unmapped genes; 4 sequence tagged sites (STS) from microdissection; and 10 microsatellites from published linkage maps. This marker density substantially exceeds that on all available published bovine maps for this region and therefore represents a significant contribution to the global bovine genome mapping effort. Additionally, 12 BAC clones representing a defined sequence-ready pool of chromosome-specific DNA resource material were isolated using STS from chromosome microdissection. Comparative mapping analysis confirmed that the cattle Bta7 QTL is in a region of conserved synteny relative to two human chromosomes (Hsa19 and Hsa5) and five mouse chromosomes (Mmu8, Mmu9, Mmu11, Mmu17 and Mmu18). The linear order of genes within homology segments is generally conserved (particularly between cattle and humans), with few exceptions where gene order is conserved, but the conserved syntenic segments were inverted in orientation. A previously unknown small region of homology between the Bta7 trypanotolerance QTL and the murine Tir1 trypanotolerance QTL region on chromosome 17 (Mmu17) is also revealed. The size of this region is approximately 342 kb as estimated from the construction of a BAC contig. One of the BAC clones in this region of homology (RP42-102G22) defines a novel evolutionary breakpoint of conserved synteny between the bovine and the mouse genomes. At least nine novel candidate genes, the majority of which are implicated in innate immunity, were identified within the 95% confidence interval of the Bta7 QTL region. These genes have been implicated in: relaying information about infection to transcription factors which up-regulate the transcription of genes involved in innate immune responses (ECSIT and MAP2K7); regulation of gene expression and RNA processing (ELAVL1, EIF3S4 and HNRPM4); B-cell proliferation (particularly memory B-cells) and immunoglobulin secretion (8D6 Antigen/LOC51293); leukocyte migration (SCYA25); haematopoiesis (HSPC240); and free radical mediated destruction of phagocytosed parasites (ACP5). Amongst these genes, HNRPM4, 8D6 Antigen and HSPC240 are located in the region homology between the trypanotolerance QTL on Bta7 and the Tir1 QTL on Mmu17. In addition, a cluster of cytokine genes, plausibly involved in humoral and acquired immune responses was also mapped to the distal portion of the QTL region. The results reported in this thesis have demonstrated the vital role played by comparative genomics in accelerating gene discover

Generation of a bovine BAC pool for chromosome region BTA7q14-22 correlated to the trait trypanotolerance

Trypanosomiasis is one of the most important diseases in African cattle. About 3 million cattle die every year by this disease. However, about 5 percent of the total African cattle have a natural resistance against trypanosomiasis. In a preliminary study markers linked to trypanotolerance have been identified on different chromosomes. The use of these information in breeding programs needs a close linkage of DNA markers to the trait. Therefore, a goal of the research on domestic cattle in Africa is the generation of a high resolution marker map for QTL regions significantly affecting trypanotolerance. An experimental approach for the direct analysis of chromosome regions correlated to economically important traits is microdissection. The chromosome fragment BTA7q14 - q22 which corresponds to the QTL linked to parasitaemia was isolated several times and used for the generation of chromosomal libraries. Primers designed from chromosome fragment specific sequences were used for the isolation of bovine specific BACs (bacterial artificial chromosomes) which now serve as starting material for the identification of informative markers within the QTL region

Establishment of bovine chromosome region specific high resolution maps using microdissection

An alternative approach for the direct analysis of chromosome regions corresponding to economical traits, based on chromosome microdissection, is described. Large fragment clones isolated with primer pairs designed from chromosome fragment-specific DNA sequences were localized by fluorescence in situ hybridization (FISH) to the scraped chromosome region of interest

Fusion of a cell penetrating peptide from HIV-1 TAT to the Theileria parva antigen Tp2 enhances the stimulation of bovine CD8+ T cell responses

Alex N. Tinega, Roger Pellé, Evans L.N. Taracha & Simon P. Graham are ILRI authors.Immunity to the bovine apicomplexan parasite Theileria parva is associated with MHC-I restricted CD8+ T cell responses directed against the intralymphocytic schizont stage of the parasite. A number of schizont-stage antigens that are targets of CD8+ T cell responses from immune animals have been identified but an effective delivery strategy that consistently induces protective CD8+ T cell responses remains to be developed. This study aimed to determine whether fusing Tat, a cell penetrating peptide (CPP) from HIV-1 TAT, to a CD8+ T cell target antigen from T. parva (Tp2) enhances the cytosolic delivery and subsequent stimulation of bovine CD8+ T cell responses in vitro. Using IFN-γ ELISpot and cytotoxicity assays, it was demonstrated that recombinant Tat-Tp2 fusion protein possessed a superior ability to access MHC-I processing and presentation pathway and to stimulate CD8+ T cell responses compared to recombinant Tp2 protein. Exposure of APC to Tat-Tp2 protein for only 30 min was sufficient for protein uptake and stimulation of CD8+ T cells. This work describes for the first time the utility of a CPP to enhance MHC-I presentation in a veterinary species and supports the evaluation of CPP fusion proteins in the induction of CD8+ T cell responses in vivo

Interleukin - 12 p35 encoding gene of cattle and sheep harbours a polymorphic T stretch in intron 4

The paper first describes interleukin-12 P35 encoding gene and discusses its role. It then looks into PCR conditions and sequencing; genotyping; sequencing; polymorphism frequency; mendelian inheritance; chromosomal location in sheep and cattle of different species