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.

BackgroundHaemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient.MethodsAn insertional mutation in tonB was constructed and the impact of the mutation on virulence and fitness in a chinchilla model of otitis media was determined. The tonB insertion mutant strain was significantly impacted in both virulence and fitness as compared to the wildtype strain in this model.ConclusionsThe tonB gene of H. influenzae is required for the establishment and maintenance of middle ear infection in this chinchilla model of bacterial disease.

BackgroundThe RNA binding protein Hfq of Haemophilus influenzae is highly homologous to Hfq from other bacterial species. In many of these other bacteria, Hfq affects the expression of a broad range of genes and enhances the ability to respond to stressful environments. However, the role of Hfq in H. influenzae is unknown.ResultsDeletion mutants of hfq were generated in the nontypeable H. influenzae strains R2866 and 86-028NP to assess the role of Hfq in these well characterized but genotypically and phenotypically divergent clinical isolates. A deletion mutation of hfq had no effect on growth of H. influenzae in nutrient rich media and had no effect on survival in several stressful conditions in vitro. However, the mutation resulted in a reduced ability to utilize heme from hemoglobin. The mutant and wild type strains were assessed for virulence and competitive fitness in models of invasive disease and otitis media. In the chinchilla model of otitis media, the hfq mutant of 86-028NP exhibited impaired competitive fitness when compared to its wild type progenitor but exhibited no apparent defect in virulence. In the infant rat model, deletion of hfq in R2866 resulted in reduced bacterial titers in blood and a shorter duration of infection when compared to the wild type strain in the competitive fitness study.ConclusionWe conclude that Hfq is involved in the utilization of essential nutrients and facilitates infection by H. influenzae.

Serum samples were obtained from Holstein dairy control cows and cows naturally infected with Mycobacterium avium ssp. paratuberculosis (MAP) to evaluate the effects of disease status on serum 25-hydroxyvitamin D 3 (25OHD 3 ) levels. Disease status was stratified for infected cows into asymptomatic, subclinical infection (n = 25), and cows demonstrating clinical signs (n = 20), along with noninfected control (n = 12) cows for comparison. In addition, portions of the ileocecal valve were taken from a subsample of cows (n = 5 per treatment group) at necropsy and processed for RNA sequencing gene transcription studies. Genes associated with vitamin D metabolism were queried to determine any association between infection and gene expression. Serum 25OHD 3 levels were significantly lower in cows in the clinical stage of disease compared with either cows in the subclinical stage and noninfected control cows. Differential expression for genes associated with the vitamin D pathway such as CYP27A1, CYP27B1, vitamin D-binding protein (DBP), and IFNG was dependent upon infection status. An upregulation of CYP27A1 was noted for cows in subclinical status, whereas CYP27B1 expression was enhanced for clinical cows. Increased expression of vitamin D-binding protein was observed for infected cattle, regardless of infection status. In summary, decreases in circulating 25OHD 3 for animals with clinical disease may suggest that these cows have reduced innate immune responses, thereby influencing the ability of animals to fight infection.