Abstract
Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection.
Highlights
Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP) is a chronic granulomatous enteritis of ruminants, both domestic and wild, including cattle, sheep, deer, and other mammalian species [1]
Our analysis revealed a marked reduction in the number of differentially expressed (DE) genes at the 24 h time point compared to the two earlier infection time points; these results indicated that majority of transcriptional changes induced by infection occur within the first 6 h of infection, with differential gene expression having largely abated 24 h post-infection
We examined the correlation between the log2 expression values generated using the RNA sequencing (RNA-seq) and microarray platforms for all genes that passed the filtering criteria and for which a definite gene length could be determined (RPKM values cannot be computed for genes with splicing events)
Summary
Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP) is a chronic granulomatous enteritis of ruminants, both domestic and wild, including cattle, sheep, deer, and other mammalian species [1]. While prevalence figures of Johne’s disease in cattle are difficult to determine – due, in part, to limited sensitivity and specificity of MAP diagnostic tests – current estimates in European countries vary from 31 to 71% [5,6,7,8]. In the United States, Johne’s disease is estimated to cost the economy between $200 million and $1.5 billion annually, with that figure rising concurrently with herd-level MAP prevalence [9, 10]. The bacilli traverse the M cells by transcytosis and migrate to the basolateral side of the cell where they are recognized and phagocytosed by intestinal macrophages. Macrophage recognition of MAP bacilli is mediated by host pathogen recognition receptors (PRRs), including www.frontiersin.org
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