Project description:Background: Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with features representing more than 23,000 gene transcripts and over 19,000 gene probe sets. Results: Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of genes showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions: This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection Affymetrix GeneChip® Bovine Genome Arrays were used to examine gene expression of peripheral blood leukocytes from cattle infected with Mycobacterium bovis
Project description:Infection of cattle with Mycobacterium bovis causes severe financial hardship in many countries, in addition to presenting a health risk for humans. As an intracellular pathogen, M. bovis, adapted to survive and thrive within the intramacrophage environment. However, little is known about expression patterns in the macrophage, particularly in the bovine host. In this study, DNA microarray analysis was used to detect genes expressed in Holstein bovine macrophages derived from peripheral blood mononuclear cells infected during four hours with two Argentinean strains of M. bovis, a virulent strain, 04-303 and an attenuated strain, 534. Genes encoding antrax toxin receptor, cell division and apoptosis regulator, ankyrin proteins that are found within the membrane associated cytoskeleton, protein of cell differentiation and regulators of endocytic traffic of membrane were more strongly expressed in infected macrophages. Blood from healthy Holstein bovines was taken in sterile conditions and peripheral blood mononuclear cells (PBMC) were separated from heparinized blood. PBMCs were used to prepare ten independent cultures which were incubated at 37C for one week in RPMI 1640 complete medium supplemented with 10% of autologous plasma. Four cultures were infected with viable cells of M. bovis virulent strain 04-303, four with avirulent strain 534 and two were left as uninfected controls. Four hours post infection, the cells were scraped, lysed. RNA was extracted, labeled and hybridized to ten Affymetrix Bovine Genome arrays.
Project description:Infection of cattle with Mycobacterium bovis causes severe financial hardship in many countries, in addition to presenting a health risk for humans. As an intracellular pathogen, M. bovis, adapted to survive and thrive within the intramacrophage environment. However, little is known about expression patterns in the macrophage, particularly in the bovine host. In this study, DNA microarray analysis was used to detect genes expressed in Holstein bovine macrophages derived from peripheral blood mononuclear cells infected during four hours with two Argentinean strains of M. bovis, a virulent strain, 04-303 and an attenuated strain, 534. Genes encoding antrax toxin receptor, cell division and apoptosis regulator, ankyrin proteins that are found within the membrane associated cytoskeleton, protein of cell differentiation and regulators of endocytic traffic of membrane were more strongly expressed in infected macrophages.
Project description:Background: Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with features representing more than 23,000 gene transcripts and over 19,000 gene probe sets. Results: Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of genes showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions: This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection
Project description:Bovine tuberculosis (bTB), caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle and wildlife worldwide. In some regions of Spain, Iberian red deer (Cervus elaphus hispanicus) can serve as reservoir of infection, thus increasing the risk of human and cattle exposure and infection. Mesenteric lymph nodes are naturally infected with M. bovis in Iberian red deer, in which the digestive route of infection is particularly important in Mediterranean Spain. In this study we characterized the differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer naturally infected with M. bovis using a Ruminant Immuno-inflammatory Gene Universal Array (RIGUA) and real-time RT-PCR. Of the 600 genes that were analyzed in the microarray, 157 showed ? 1.2 fold changes in expression in infected or uninfected deer and 17 genes displayed an expression fold change greater than 1.7 with a P-value ? 0.05 and were selected for further analysis. These genes included tight junction proteins (Z02 and occluding), IL-11R, bactenecin, CD62L, CD74, desmoglein, IgA and IgM that constitute new findings and suggest new mechanisms by which M. bovis may modulate host inflammatory and immune responses. Identification of genes differentially expressed in animals and tissues naturally infected with M. bovis contributes to our basic understanding of the mechanisms of pathogenesis and protective immunity to mycobacterial infections and may have important implications for future functional genomic and vaccine studies to aid in the control of bTB in deer and other wildlife reservoir species. Mesenteric lymph node RNA from four different uninfected Iberian red deer stags and two Iberian red deer stags infected with Mycobacterium bovis. Infected animals were naturally infected with M. bovis. All animals were hunter-harvested and the tissues retrieved 2-6 hrs after animal hunting.