Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling of disease progression in bovine tuberculosis and the identification of potential diagnostic biomarkers

ABSTRACT: The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bovine tuberculosis in cattle. We have used the advantages of the murine model to identify promising candidates in the host transcriptome post-infection. RNA from BALB/c splenocytes and lung cells after aerogenic Mycobacterium bovis infection were with high-density microarrays to defining biomarkers of disease progression. In antigen-stimulated splenocytes we found statically significant modulation of 1109 genes early after infection and 1134 at later time-point post-infection. 618 of these genes were modulated at both time points. In the lung 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were granzyme A in spleen, and cxcl9, granzyme B, interleukin-22, interluekin-17A receptor, and ccr6 in lungs. The expression of 20 of the most up-regulated genes identified in the murine studies were evaluated using PBMC from uninfected and naturally infected cattle. We could show that the expression of cxcl9, granzyme A and interleukin-22 following in vitro stimulation with PPD was significantly increased in infected cows compared to naïve animals. Thus, we have demonstrated that murine transcriptome analysis can be used to predict responses in cattle. In addition, we have prioritised CXCL9, GnzA and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis. Two groups of 5 BALB/c mice each were infecetd with approx 600 CFU M. bovis via the intranasal route. At days 3 and 14 post challenge five mice per group were euthanized and spleens and lungs harvested. One group of 5 mice were used as a control. Their splenocytes and lung cells were stimulated in vitro for 3 days with M7 protein pool. Antigen cell culture stimulations in mice were performed using an equal pool of seven secreted, immunogenic recombinant mycobacterial proteins (Rv1886c, Rv3019c, Rv3763, Rv3804c, Rv0251, Rv0287 and Rv0288) common to M. bovis and BCG, referred here as M7 protein cocktail. Each protein was used at final concentration of 2μg/ml in 3-day culture.

ORGANISM(S): Mus musculus

SUBMITTER: ELIHU Aranday-Cortes

PROVIDER: E-GEOD-33058 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cells types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. Results: Comparison of M. bovis-challenged MDM gene expression profiles with the non-challenged MDM controls at each time point identified 3,529 differentially expressed genes after 2 hours post-challenge, with 5,211 and 6,150 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤ 0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly larger than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors (PRRs)-receptors; and (3) apoptosis. Conclusions: The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support important roles for MYD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis, which to our knowledge have not been reported previously. Affymetrix GeneChip® Bovine Genome Arrays were used to examine gene expression of bovine monocyte-derived macrophages (MDM) after in vitro challenge with Mycobacterium bovis across a time series of 2 hr, 6 hr and 24 hr post-challenge. A 0 hr control treatment was also generated and seven different age-matched female Holstein-Friesian cattle were used for each time-point/treatment combination for a total of 49 microarrays.

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

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Transcriptional profiling of disease progression in bovine tuberculosis and the identification of potential diagnostic biomarkers

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