Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.

Comparison of gene expression of immune mediators in lung and pulmonary lymph node granulomas from cattle experimentally infected with Mycobacterium bovis.

The cellular infiltrates and macrophage activation pathways may differ in granulomas found in the lungs and pulmonary lymph nodes of cattle infected with Mycobacterium bovis. The aim of this study was to compare the histopathology and gene expression profiles of cytokines and immune mediators for cattle which had these lesions in both sites. Ten Friesian-cross, 15-16 month old cattle were challenged intratracheally with 5 × 10(3)CFU of virulent M. bovis and killed and necropsied at 28 weeks after infection. Seven animals were found to have gross TB granulomas in both their lungs and pulmonary lymph nodes (PLN) and these lesions were fully encapsulated with central necrosis and mineralisation. Neutrophil infiltration was clearly involved in granuloma in lung whereas neutrophils were limited in lesions of PLN. Comparisons were made of immune mediators from these two sites from the same animals as well as those between lesioned PLN tissues and non-lesioned prescapular lymph nodes (PSLN). Gene expressions of the immune mediators were normalised using a housekeeping gene (U1), a monocyte/macrophage marker (CD14) and a common leucocyte marker (CD45). mRNA expression of IFN-γ, IL-17A, IRF5(1) and arginase 1 (Arg1) was significantly up-regulated in lung compared to that for PLN (p<0.05), while mRNA expression of IFN-γ, IL-12p40, TNF-α and iNOs for PLN was significantly higher than that for PSLN (p<0.05). In addition, IL-10 mRNA expression was significantly higher for lung compared to PLN when normalised for CD45 (p<0.05). The results suggested that the stronger proinflammatory immune response in the lesioned lung may be a consequence of enhanced expression of IRF5 promoting IFN-γ and IL-17 production. In contrast, Arg1 expression in the lungs could facilitate the infection through competing with iNOs for l-arginine, preventing generation of nitric oxide for clearance of M. bovis infection.

Diverse cytokine profile from mesenteric lymph node cells of cull cows severely affected with Johne's disease.

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, is able to dampen or distort immune responses at the mucosal sites and coexist with a massive infiltration of immune cells in the gastrointestinal tract. Knowledge of the mechanism by which M. avium subsp. paratuberculosis subverts the immune response at the mucosal level in cattle is important for the development of improved disease control strategies, including new vaccines and diagnostic tests. In this study, 38 cull cows from herds infected with M. avium subsp. paratuberculosis were divided into four groups, based on M. avium subsp. paratuberculosis culture from gut tissues and histopathological lesion scores. Cytokine gene expression and secretion from M. avium subsp. paratuberculosis sonicate-stimulated peripheral blood mononuclear cell (PBMC) and mesenteric lymph node (MLN) cultures of the animals were compared. Antigen stimulation of MLN cells from the severely lesioned group resulted in significant upregulation of the mRNA expression of five cytokines, gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-13, IL-17A, and tumor necrosis factor alpha (TNF-α), which have a diverse range of functions, while there was no significant upregulation of these cytokines by the other groups. There were major differences between the responses of the PBMC and MLN cultures, with higher levels of secreted IFN-γ released from the MLN cultures and, conversely, higher levels of IL-10 released from the PBMC cultures. The upregulation of all five cytokines from cells at the site of infection in the severely lesioned animals suggested a dysregulated immune response, contributing to a failure to clear infection in this group of animals.

Activation of the mucosal immune system in irritable bowel syndrome.

BACKGROUND & AIMS: A role for the mucosal immune system in the pathogenesis of irritable bowel syndrome is suggested by its association with intestinal infections. METHODS: To investigate this, we performed histologic and immunohistologic studies on colonoscopic biopsy specimens from 77 patients with symptoms satisfying the Rome criteria and 28 asymptomatic control patients. RESULTS: Histologic assessment of biopsy specimens from symptomatic patients indicated 3 different groups. The first (38 of 77) had normal conventional histology; however, immunohistology showed increased intraepithelial lymphocytes (median, 1.8-fold; range, 1.74-1.86), lamina propria CD3(+) cells (2-fold; range, 1.55-2.91), and CD25(+) cells (6.5-fold; range, 4.98-8.13) compared with asymptomatic controls. The second group (31 of 77) had nonspecific microscopic inflammation and on immunohistology showed similar increases in lymphocyte populations (not significant vs. the uninflamed group) as well as increased numbers of neutrophil leukocytes and mast cells (P < 0.0001 vs. controls and the uninflamed group). The third group (8 of 77) fulfilled histologic and immunohistologic criteria for classic lymphocytic colitis. CONCLUSIONS: Examination of colonoscopic biopsy specimens from patients meeting the Rome criteria for a clinical diagnosis of irritable bowel syndrome showed subgroups with normal and abnormal conventional histology. All groups showed increased numbers of activated immunocompetent cells in the intestinal mucosa on quantitative immunohistology, implicating the mucosal immune system in pathogenesis.