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.

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.

Vaccination of cattle with a high dose of BCG vaccine 3 weeks after experimental infection with Mycobacterium bovis increased the inflammatory response, but not tuberculous pathology.

A study was undertaken to determine whether BCG vaccination of cattle post-challenge could have an effect on a very early Mycobacterium bovis infection. Three groups of calves (n = 12/group) were challenged endobronchially with M. bovis and slaughtered 13 weeks later to examine for tuberculous lesions. One group had been vaccinated prophylactically with BCG Danish vaccine 21 weeks prior to challenge; a second group was vaccinated with a 4-fold higher dose of BCG Danish 3 weeks post-challenge and the third group, remained non-vaccinated. Vaccination prior to challenge induced only minimal protection with just a significant reduction in the lymph node lesion scores. Compared to the non-vaccinated group, BCG vaccination post-challenge produced no reduction in gross pathology and histopathology, but did result in significant increases in mRNA expression of pro-inflammatory mediators (IFN-γ, IL-12p40, IL-17A, IRF-5, CXCL9, CXCL10, iNOs, and TNF-α) in the pulmonary lymph nodes. Although there was no significant differences in the gross pathology and histopathology between the post-challenge BCG and non-vaccinated groups, the enhanced pro-inflammatory immune responses observed in the post-challenge BCG group suggest caution in the use of high doses of BCG where there is a possibility that cattle may be infected with M. bovis prior to vaccination.

Vaccination of Sheep with a Methanogen Protein Provides Insight into Levels of Antibody in Saliva Needed to Target Ruminal Methanogens.

Methane is produced in the rumen of ruminant livestock by methanogens and is a major contributor to agricultural greenhouse gases. Vaccination against ruminal methanogens could reduce methane emissions by inducing antibodies in saliva which enter the rumen and impair ability of methanogens to produce methane. Presently, it is not known if vaccination can induce sufficient amounts of antibody in the saliva to target methanogen populations in the rumen and little is known about how long antibody in the rumen remains active. In the current study, sheep were vaccinated twice at a 3-week interval with a model methanogen antigen, recombinant glycosyl transferase protein (rGT2) formulated with one of four adjuvants: saponin, Montanide ISA61, a chitosan thermogel, or a lipid nanoparticle/cationic liposome adjuvant (n = 6/formulation). A control group of sheep (n = 6) was not vaccinated. The highest antigen-specific IgA and IgG responses in both saliva and serum were observed with Montanide ISA61, which promoted levels of salivary antibodies that were five-fold higher than the second most potent adjuvant, saponin. A rGT2-specific IgG standard was used to determine the level of rGT2-specific IgG in serum and saliva. Vaccination with GT2/Montanide ISA61 produced a peak antibody concentration of 7 × 1016 molecules of antigen-specific IgG per litre of saliva, and it was estimated that in the rumen there would be more than 104 molecules of antigen-specific IgG for each methanogen cell. Both IgG and IgA in saliva were shown to be relatively stable in the rumen. Salivary antibody exposed for 1-2 hours to an in vitro simulated rumen environment retained approximately 50% of antigen-binding activity. Collectively, the results from measuring antibody levels and stablility suggest a vaccination-based mitigation strategy for livestock generated methane is in theory feasible.

Vaccination of cattle with a methanogen protein produces specific antibodies in the saliva which are stable in the rumen.

Methane is produced in the rumen of cattle by a group of archaea (single-celled organisms forming a domain distinct from bacteria and eucarya) called methanogens. Vaccination against methanogens has the potential to reduce methane emissions by inducing antibodies in saliva which are transferred to the rumen and diminish the ability of methanogens to produce methane. Since it is likely that an effective vaccination strategy will need to produce high levels of methanogen-specific antibody in the saliva; the choice of adjuvant, route of vaccination and stability of saliva-derived antibody in the rumen all need to be considered. In this study, stability of IgA and IgG in rumen fluid was determined using an in vitro assay. IgA levels in cattle saliva were reduced by only 40% after 8h exposure to rumen contents while IgG levels were reduced by 80%. These results indicated that antibody is relatively stable in the bovine rumen. A trial was conducted in cattle to investigate induction of immune responses to a methanogen protein, recombinant glycosyl transferase protein (rGT2) from Methanobrevibacter ruminantium M1. Groups of cattle (n=6) were vaccinated subcutaneously with rGT2, formulated with Montanide ISA61 with or without the TLR4 agonist, monophosphoryl lipid A (MPL). A control group (n=6) was not vaccinated. Strong antigen-specific IgG and moderate IgA responses were measured in the serum and saliva of the vaccinated animals and antibody was also detected in the rumen.

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.

Revaccination of cattle with bacille Calmette-Guérin two years after first vaccination when immunity has waned, boosted protection against challenge with Mycobacterium bovis.

In both humans and animals, controversy exists concerning the duration of protection induced by BCG vaccine against tuberculosis (TB) and whether revaccination enhances protection. A long-term study was undertaken to determine whether BCG-vaccinated calves would be protected against challenge with Mycobacterium bovis 2½ years after vaccination and to determine the effect of revaccination after 2 years. Seventy-nine calves were divided into five groups (n = 15-17 calves/group) with four of the groups vaccinated subcutaneously with 105 CFU of BCG Danish at 2-4 weeks of age and the fifth group serving as non-vaccinated controls. Three of the four BCG-vaccinated groups were revaccinated 2 years after the initial vaccination. One BCG-vaccinated group was revaccinated with BCG. A second group was vaccinated subcutaneously with a TB protein vaccine consisting of biopolyester particles (Biobeads) displaying two mycobacterial proteins, ESAT-6 and Antigen 85A, mixed with an adjuvant. A third group was vaccinated with TB proteins from M. bovis culture filtrate, mixed with an adjuvant. Twenty-three weeks after the BCG revaccination, all animals were challenged endotracheally with virulent M. bovis and a further 13 weeks later, animals were killed and necropsied to determine protection against TB. The BCG-vaccinated animals produced positive tuberculin caudal fold intradermal (15 of 62 animals) and IFN-γ TB test responses (six of 62 animals) at 6 months after vaccination, but not at subsequent time-points compared to the non-vaccinated animals. Calves receiving a single vaccination with BCG vaccine 2½ years prior to challenge were not protected against TB, while those revaccinated with BCG 2 years after the initial vaccination displayed significant reductions in lung and pulmonary lymph node lesion scores compared to the non-vaccinated animals. In contrast, no reduction in lesion scores was observed in the animals revaccinated with the TB protein vaccines with their immune responses biased towards induction of antibody.

Altered patterns of toll-like receptor gene expression in cull cows infected with Mycobacterium avium subsp. paratuberculosis.

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease of cattle. The mechanism how MAP can co-exist in the gastro-intestinal tract despite a massive infiltration of immune cells is not known. Toll-like receptors (TLRs) are known to play an important role in both innate and acquired immune responses but it is unclear what role different TLRs play in response to MAP. In this study, 38 cull cows from herds infected with MAP were classified into four groups, based on MAP culture from gut tissues and histopathological lesion scores. The expression of TLR1, 2 and 4 mRNA from MAP antigen-stimulated mesenteric lymph node (MLN) cultures and peripheral blood mononuclear cells (PBMCs) and in the MLN and ileum tissues of these animals was determined. MAP antigen-specific expression of TLR1 in MLN and PBMC was significantly lower in the MAP-infected groups than the non-infected control group, suggesting that in MAP-infected animals there is impairment in the up-regulation of TLR1 in response to MAP antigen. TLR4 expression in MLN tissues was significantly higher in the severely infected group than the control group suggesting up-regulation of endogenous TLR4 expression at a site of MAP infection in animals severely affected with Johne's disease. A preliminary screening of TLR1, 2 and 4 in the cull cows revealed the presence of polymorphisms in TLR1 and TLR2. In summary, one mechanism how MAP may subvert the immune system is that there is an apparent lack of recognition of MAP antigens as foreign by TLR1 in MAP-infected cows.

Immune responses associated with progression and control of infection in calves experimentally challenged with Mycobacterium avium subsp. paratuberculosis.

This study examined the immune responses related to the infection, progression and control of Mycobacterium avium subsp. paratuberculosis (MAP) infection in calves. Twenty calves were challenged orally with MAP and 11 non-challenged calves served as controls. Approximately half the calves from each group were sacrificed at either 7 or 15 months post-challenge (PC). The majority of the challenged calves (19/20) shed MAP in feces 2-4 months PC, but thereafter fecal shedding reduced markedly. The severity of infection was reduced at 15 months PC compared to that at 7 months PC as seen from a significantly lower isolation of MAP from tissues and lower lesion scores (P<0.05). In addition, there was a reduction in the upregulation of gene expression of gamma interferon, interleukin-10 (IL-10) and inducible nitric oxide synthase from the antigen-stimulated mesenteric lymph node (MLN) cultures of the challenged calves. No evidence of infection was detected in the control calves. The severity of the infection in individual calves at 15 months PC as indicated from the number of tissue culture positive sites, was negatively related to IL-10 released from antigen-stimulated peripheral blood mononuclear cells (P<0.05). Collectively the data indicated that the severity of the MAP infection was reduced in the calves at 15 months PC and in a specific time period during infection, IL-10 may play a role in reducing the severity of this disease.

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.