Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis

Abstract

Johne's disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection.

Fig 1. Description of reads mapping to single and multiple locations in the B. taurus genome.

A) Pie chart showing the mean number and percentage of reads that aligned to the reference genome using TopHat and Bowtie. B) Pie chart showing the mean percentage of uniquely mapped reads assigned to coding regions (i.e. known exons), untranslated regions (i.e. 3’ or 5’ untranslated regions), intronic regions (i.e. known introns), intergenic regions (i.e. areas where no known annotated genes are present), and ribosomal RNA based on alignment data from HTSeq and Piccard Tools.

Fig 2. Multidimensional scaling plots of samples from uninfected control, subclinical, and clinical animals based on RNA-seq data.

Distance 1 and distance 2 separate all samples based on the expression values of all 12,133 genes that passed filtering criteria prior to differential gene expression analysis. A) Comparison of all 15 samples. B) Comparison of 14 samples after the removal of uninfected control animal 5 (Cow #8102).

Fig 3. Venn diagram showing the comparison of differentially expressed genes between all group comparisons.

Venn diagram showing the number of DE genes in each group comparison as well as the numbers of genes that are up- (↑) or down-regulated (↓) in each dataset.

Fig 4. Top 10 functional networks that are modulated in group comparisons of differentially expressed genes.

Functional networks that are modulated in the comparison between A) clinical and uninfected control animals, B) subclinical and uninfected control animals, C) clinical and subclinical animals.

Fig 5. Pathway for T-cell receptor signaling for gene regulation comparison between clinical and uninfected control animals.

Genes associated with TCR signaling that are differentially expressed are shaded in grey; genes with a white background represent genes that were not significantly differentially expressed.