Project description:Streptococcus equi subspecies equi, strain 1691 grown on COBA streptococcal selective agar shows classical mucoid colony morphology in addition to a reduced capsule phenotype. This project aimed to identify changes in the transcriptional profile between the two morphologies.
Project description:The objectives of this study were 2-fold: 1) to compare the expression profiles at specific ages of blood leukocytes from foals stimulated with virulent R. equi with those of unstimulated leukocytes; and, 2) to characterize the age-related changes in the gene expression profile associated with blood leukocytes in response to stimulation with virulent R. equi. Peripheral blood leukocytes were obtained from 6 foals within 24 hours (h) of birth (day 1) and 2, 4, and 8 weeks after birth. The samples were split, such that half were stimulated with live virulent R. equi, and the other half served as unstimulated control. For one hyb design: we hybridized 6 samples stimulated and 6 unstimulated at each time point (30 mL of blood was treated with live virulent R. equi (Strain ATCC 33701) and the remaining 30 mL of blood with PBS ). For time course: 6 samples from day1 stimulated hybridized to week2 stimulated; day1 stimulated hyb to week 4 stimulated and day1 stimulated hyb to week8 stimulated. RNA was extracted and the generated cDNA was labeled with fluorescent dyes for microarray hybridizations using an equine microarray.
Project description:Understanding the pathogenic mechanisms and host responses to Johne's disease, caused by Mycboacterium avium subspecies paratuberculosis (MAP), is complicated by a number of challenges of the disease progression and lack of ex vivo models to study the disease. We describe novel cell culture passage model which mimics the course of infection in vivo. Using this model we determined that cell culture passaged MAP develops an inflammatory phenotype initiating higher levels of pro-inflammatory cytokines and chemokines compared to levels initiated by non-passaged MAP. To verify that the MAP phenotypes were changing, we conducted transcriptome analysis of each population during the passage model and identified a change in gene expression, specifically and upregulation of genes involved in the biosynthesis of lipid components within the cell. Lipidomic analysis showed dramatically different lipid profiles between the 2 populations. These transcriptome changes were validated by the identification of high levels of upregulated transcripts from MAP-infected cattle ileal tissue. In total, this study described a novel model used to show that MAP changes phenotype during intracellular passage, thus changing its lipid profile and triggering an inflammatory response in the host leading to the severe clinical phage of Johne's disease. In total 6 slides were analyzed. Of the 2 phenotypes under investigation, the P0 samples (non-inflammatory) were analyzed in triplate and the P2 samples (inflammatory) were analyzed in triplicate. Each slide fluorescence intensity was measured at PMT400, 600 and 750. Each oligo on each slide was spotted in triplicate and each slide had its own internal controls.
Project description:Rhodococcus equi is an intracellular bacterium that affects young foals and immuno-compromised individuals causing severe pneumonia. Currently, the genetic mechanisms which confer susceptibility and or resistance to R. equi are not fully understood. Previously, using a SNP-based genome-wide association study, we identified a region on equine chromosome 26 associated with clinical pneumonia. To better characterize this region and understand the relationship of the SNPs associated with disease, we performed RNA-Seq on 12 horses representing the 3 allelic states of the SNP identified. Differential expression analyses identified differentially expressed genes in the innate immune response pathway when comparing homozygous A allele horses with the AB and BB horses. Isoform analyses of the RNA-Seq data predicted multiple transcripts with evidence of differential expression to exist at the TRPM2 locus. This finding is consistent with previously demonstrated work in human cell lines in which isoform specific expression of TRPM2 was critical to cell viability. This work demonstrates that SNPs in TRPM2 are associated with differences in gene expression, suggesting that modulation of expression of this innate immune gene contributes to susceptibility to R. equi pneumonia. Overall design: Analysis of RNA expression derived from 3 genotypes
Project description:CsrRS is a two component regulator which is known to regulate 10-18 % of the genome of the closely related Streptococcus pyogenes. We aimed to look for transcriptional differences between pairs of naturally occurring Streptococcus equi strains where one possessed a SNP in csrRS whereas the other did not. Such SNPs have not been identified in isolates from acute disease but have been identified in persistent isolates.