Sheep nitric oxide synthase 2, ind

Objective / Background:

Paratuberculosis (Johne’s disease) is a chronic infectious granulomatous enteritis that mainly affects ruminants and is caused by Mycobacterium avium ssp. paratuberculosis (MAP). The disease is highly prevalent throughout the world with significant economic losses. MAP has also been linked to human Crohn’s disease. There is a strong correlation between the immune response and the development of various types of pathologies in ruminants.

The polarization of the immune response, which is critical to the clinical outcome of paratuberculosis infection, is controlled by the differential expression of certain cytokines and inducible nitric oxide synthase (iNOS) in Johne’s disease. In previous studies, the role of different cytokines (Th1 and Th2) in ovine paratuberculosis has been occasionally studied. In the present study, we studied the differential expression of the genes interferon (IFN) -γ, interleukin (IL) -1α, IL-10, transforming growth factor (TGF) -β, iNOS, and TRAF1 in sheep infected with MAP and she established a relationship with different pathologies.

Methods:

Tissue sections (small intestine, ileocecal junction, and mesenteric lymph nodes) were collected from sheep suspected of Johne’s disease and appropriately preserved for RNA extraction, polymerase chain reaction (PCR) analysis, and histopathology. The pathological classification was made on the basis of the nature and extent of cellular infiltration, granuloma formation, and the abundance of acid-fast bacilli. Six sheep each with pauci (PB) and multibacillary (MB) lesions and six healthy control sheep were selected for cytokine studies. MAP in sheep genomic DNA extracted from tissue was quantified by a quantitative PCR assay.

The RNA extracted from tissue was reverse transcribed to prepare cDNA from which a quantitative reverse transcription PCR (qRT-PCR) was performed to amplify IFN-γ, IL-1β, IL-10, TGF-β, β- actin, TRAF1, and iNOS with Quantitect SYBR Green Master Mix. The qRT-PCR data were analyzed using the 2-ΔΔCT method using the β-actin gene as a control. All qRT-PCR results were compared by one-way analysis of variance (minimum difference of significance and Duncan’s tests) for the p-value using SPSS (version 7.5) for the expression of each gene in tissues of infected and control sheep.

Results:

In the small intestine, PB sheep showed significant improvements in the expression of IL-10, TGF-β, iNOS, and IFN-γ compared to similar tissues from uninfected control sheep. IL-1α expression was significantly reduced (p <0.01). IL-10 expression in mesenteric lymph node (MLN) tissue of PB sheep was significantly increased (p <0.01) compared to control sheep.

MB sheep revealed significantly improved TGF-β mRNA expression and a reduction in IL-1α expression compared to control sheep. In MLN from MB sheep, IL-10 and TGF-β expressions were significantly increased (p <0.01), and IFN-γ was significantly reduced compared to uninfected control sheep. When cytokine expression was compared between two clearly infected groups, MB sheep showed a very significant decrease (p <0.01) in the expression of iNOS and IFN-γ in the small intestine and IFN-γ in MLN tissues.

Conclusion:

The present study indicated that IFN-γ and iNOS were found to play an important role in inducing Th1-type immune response in PB sheep. MB sheep had a significant reduction in IFN-γ and iNOS expression and elevation of IL-10 and TGF-β, which was typical of the Th2 cytokine pattern. The elevated expression of IL-10 and TGF-β in PB cases possibly suggests the role of regulatory T cells and may follow an independent mechanism that is not typical of the Th1 pattern. In view of the significantly reduced expression in both forms of the disease, IL-1α may not be an important cytokine in ovine paratuberculosis.

Keywords:

Cytokines, Inducible nitric oxide synthase, Multibacillary, Mycobacterium avium ssp. Paratuberculosis, Paucibacillary, Sheep

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