Benchtop nuclear magnetic resonance-based metabolomic approach for the diagnosis of bovine tuberculosis.

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non-tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high-field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low-field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB-vaccinated healthy control (n = 10) and healthy PTB-unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77-1). In summary, plasma fingerprinting using HF and LF-NMR differentiated TB subjects from uninfected animals, and PTB and PTB-vaccinated subjects who may provide a TB-false positive, highlighting the use of LF-NMR-based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods.

Discerning novel drug targets for treating Mycobacterium avium ss. paratuberculosis-associated autoimmune disorders: an in silico approach.

Mycobacterium avium subspecies paratuberculosis (MAP) exhibits 'molecular mimicry' with the human host resulting in several autoimmune diseases such as multiple sclerosis, type 1 diabetes mellitus (T1DM), Hashimoto's thyroiditis, Crohn's disease (CD), etc. The conventional therapy for autoimmune diseases includes immunosuppressants or immunomodulators that treat the symptoms rather than the etiology and/or causative mechanism(s). Eliminating MAP-the etiopathological agent might be a better strategy to treat MAP-associated autoimmune diseases. In this case study, we conducted a systematic in silico analysis to identify the metabolic chokepoints of MAP's mimicry proteins and their interacting partners. The probable inhibitors of chokepoint proteins were identified using DrugBank. DrugBank molecules were stringently screened and molecular interactions were analyzed by molecular docking and 'off-target' binding. Thus, we identified 18 metabolic chokepoints of MAP mimicry proteins and 13 DrugBank molecules that could inhibit three chokepoint proteins viz. katG, rpoB and narH. On the basis of molecular interaction between drug and target proteins finally eight DrugBank molecules, viz. DB00609, DB00951, DB00615, DB01220, DB08638, DB08226, DB08266 and DB07349 were selected and are proposed for treatment of three MAP-associated autoimmune diseases namely, T1DM, CD and multiple sclerosis. Because these molecules are either approved by the Food and Drug Administration or these are experimental drugs that can be easily incorporated in clinical studies or tested in vitro. The proposed strategy may be used to repurpose drugs to treat autoimmune diseases induced by other pathogens.

Regionally Distinct Immune and Metabolic Transcriptional Responses in the Bovine Small Intestine and Draining Lymph Nodes During a Subclinical Mycobacterium avium subsp. paratuberculosis Infection.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative infectious agent of Johne's disease (JD), an incurable granulomatous enteritis affecting domestic livestock and other ruminants around the world. Chronic MAP infections usually begin in calves with MAP uptake by Peyer's patches (PP) located in the jejunum (JE) and ileum (IL). Determining host responses at these intestinal sites can provide a more complete understanding of how MAP manipulates the local microenvironment to support its long-term survival. We selected naturally infected (MAPinf, n=4) and naive (MAPneg, n=3) cows and transcriptionally profiled the JE and IL regions of the small intestine and draining mesenteric lymph nodes (LN). Differentially expressed (DE) genes associated with MAP infection were identified in the IL (585), JE (218), jejunum lymph node (JELN) (205), and ileum lymph node (ILLN) (117). Three DE genes (CD14, LOC616364 and ENSBTAG00000027033) were common to all MAPinf versus MAPneg tissues. Functional enrichment analysis revealed immune/disease related biological processes gene ontology (GO) terms and pathways predominated in IL tissue, indicative of an activated immune response state. Enriched GO terms and pathways in JE revealed a distinct set of host responses from those detected in IL. Regional differences were also identified between the mesenteric LNs draining each intestinal site. More down-regulated genes (52%) and fewer immune/disease pathways (n=5) were found in the ILLN compared to a higher number of up-regulated DE genes (56%) and enriched immune/disease pathways (n=13) in the JELN. Immunohistochemical staining validated myeloid cell transcriptional changes with increased CD172-positive myeloid cells in IL and JE tissues and draining LNs of MAPinf versus MAPneg cows. Several genes, GO terms, and pathways related to metabolism were significantly DE in IL and JE, but to a lesser extent (comparatively fewer enriched metabolic GO terms and pathways) in JELN suggesting distinct regional metabolic changes in IL compared to JE and JELN in response to MAP infection. These unique tissue- and regional-specific differences provides novel insight into the dichotomy in host responses to MAP infection that occur throughout the small intestine and mesenteric LN of chronically MAP infected cows.

Crohn's disease: failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen intestine from patients with Crohn's disease.

OBJECTIVES: Although controversial, there is increasing concern that Crohn's disease may be a zoonotic infectious disease consequent to a mycobacterial infection. The most plausible candidate is M. avium subspecies paratuberculosis (MAP) that is unequivocally responsible for Johne's disease in ruminants. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Non-identifiable intestine from patients with documented Crohn's disease was assayed according to the manufacturer's instructions and with suggested modifications. Probes were custom designed for MAP and human ß-actin (as the eukaryotic housekeeping gene) from published genomes. RESULTS: Repetitively, false positive signal was observed in our "No-Probe" negative control. Attempts were made to correct this according to the manufacturer's suggestions (by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters). None prevented false positive signal in the "No-Probe" control. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view® cannot be used to detect MAP in pre-frozen resected intestine of humans with Crohn's disease.

Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis.

The local expression of WC1+ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1+ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1+ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1+ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.

WC1+ and WC1neg γδ T lymphocytes in intestinal mucosa of healthy and Mycobacterium avium subspecies paratuberculosis-infected calves.

Mucosal surfaces such as the gastrointestinal tract, and skin are the front line of host defence and immunity against many pathogens. Gamma delta (γδ) T lymphocytes preferentially localize to the mucosal surfaces in several species including cattle, and are thought to play crucial roles in immunosurveillance and host defence, particularly against mycobacteria. Many γδ T cells are present in young calves, which is the period when calves are thought to be initially exposed to Mycobacterium avium subspecies paratuberculosis (Map). The role of mucosal γδ T cells in cattle, especially during host-pathogen interactions during early pre-clinical phases of infectious disease remains unclear. The purposes of this study were to investigate and characterize WC1+ and WC1neg γδ  T cell subsets in various segments of the gastrointestinal (GI) tract of young calves, and then to examine γδ  T cell subsets in the distal small intestine of calves after experimental intestinal Map infection by direct Peyer's patch inoculation. We show that in healthy calves, the relative proportion of γδ T cells is constant throughout the GI mucosa, though the ileum has significantly more γδ T cells. In the distal intestine, γδ T cells are mainly WC1neg and primarily located within the lamina propria of the jejunum and ileum. In Map-infected intestine, there are higher numbers of γδ T cells in the lamina propria and a greater proportion of WC1+ cells within the epithelial layer compared to control calves. While WC1neg γδ T cells preferentially localize to the distal small intestine of healthy calves, WC1+ γδ T cells are increased in the intestinal mucosa during Map infection, which is suggestive of effector cell function. Further, spectral microscopy and flow cytometry in tandem will lead to improved understanding of the functions of these cells during health and disease.

Core profile of volatile organic compounds related to growth of Mycobacterium avium subspecies paratuberculosis - A comparative extract of three independent studies.

Analysis of volatile organic compounds (VOC) derived from bacterial metabolism during cultivation is considered an innovative approach to accelerate in vitro detection of slowly growing bacteria. This applies also to Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis, a debilitating chronic enteritis of ruminants. Diagnostic application demands robust VOC profiles that are reproducible under variable culture conditions. In this study, the VOC patterns of pure bacterial cultures, derived from three independent in vitro studies performed previously, were comparatively analyzed. Different statistical analyses were linked to extract the VOC core profile of MAP and to prove its robustness, which is a prerequisite for further development towards diagnostic application. Despite methodical variability of bacterial cultivation and sample pre-extraction, a common profile of 28 VOCs indicating cultural growth of MAP was defined. The substances cover six chemical classes. Four of the substances decreased above MAP and 24 increased. Random forest classification was applied to rank the compounds relative to their importance and for classification of MAP versus control samples. Already the top-ranked compound alone achieved high discrimination (AUC 0.85), which was further increased utilizing all compounds of the VOC core profile of MAP (AUC 0.91). The discriminatory power of this tool for the characterization of natural diagnostic samples, in particular its diagnostic specificity for MAP, has to be confirmed in future studies.

Baseballs, tennis balls, livestock farm manure, the IDH1 mutation, endothelial cell proliferation and hypoxic pseudopalisading (granulomatous) necrosis: Mycobacterium avium subspecies paratuberculosis and the epidemiology, cellular metabolism and histology of diffuse gliomas, including glioblastoma.

An increased rate of diffuse gliomas, including glioblastoma, has been noted in livestock farmers in Western countries. Some researchers have suggested that a zoonotic virus or bacteria present in the livestock animal's feces or manure may be a possible etiologic factor. Mycobacterium avium subspecies paratuberculosis (MAP), the cause of a chronic enteropathy in domestic livestock and a probable zoonosis, is heavily excreted in an infected animal's feces or manure, contaminating soil and ground on the animal's farm. Once excreted in an animal's feces, MAP lasts indefinitely in a dormant but viable form, and easily spreads outside farms to the surrounding environment. MAP's presence throughout the soil in countries where MAP infection of domestic livestock is extensive and long-standing may explain the increased rates of glioblastoma in tennis and baseball players who handle balls coated with MAP-contaminated dirt. MAP infection is consistent with glioblastoma's two defining histopathologic characteristics: endothelial cell proliferation and pseudopalisading necrosis. MAP is a non-tuberculous or atypical mycobacterium, which can cause hypoxic necrotizing granulomas, granulomas that resemble areas of pseudopalisading necrosis. There are known bacterial causes of endothelial cell proliferation. Almost unique amongst intracellular bacteria, MAP's variant isocitrate dehydrogenase 1 (IDH1) enzyme, a type 2-oxoglutarate ferredoxin oxidoreductase, can use a host cell's cytosolic α-ketoglutarate in its own Krebs or tricarboxylic acid cycle. MAP's ability to use a host cell's α-ketoglutarate may explain the survival advantage of the cytosolic IDH1 enzyme mutation for patients with diffuse gliomas including glioblastoma, astrocytoma, and oligdendroglioma, a mutation that results in a reduced supply of cytosolic α-ketoglutarate. MAP may therefore be one possible infectious cause of glioblastoma and the other histologic categories of diffuse glioma.

Nilotinib: A Tyrosine Kinase Inhibitor Mediates Resistance to Intracellular Mycobacterium Via Regulating Autophagy.

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne's disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.

Blocking the mitogen activated protein kinase-p38 pathway is associated with increase expression of nitric oxide synthase and higher production of nitric oxide by bovine macrophages infected with Mycobacterium avium subsp paratuberculosis.

This study evaluated the role of the mitogen-activated protein kinase (MAPK)-p38 pathway in the nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by bovine monocyte-derived macrophages ingesting Mycobacterium avium subsp. paratuberculosis (MAP) organisms in vitro. Bovine monocyte-derived macrophages were incubated with MAP organisms with or without a specific inhibitor of the MAPKp38 pathway and activation of the MAPKp38, interleukin - (IL) IL-10, IL-12, iNOS mRNA expression and NO production were evaluated. Incubation of macrophages with MAP organisms activates the MAPKp38 pathway at early time points post infection. Chemically inhibition of MAPKp38 before incubation of bovine macrophages with MAP resulted in increased expression of IL-12 mRNA at 2, 6 and 24h, decreased expression of IL-10 mRNA at 2, 6 and 24h and increased expression of iNOS mRNA at 2 and 6h. Nitric oxide was evaluated to indirectly determine the effects of MAPKp38 pathway on the anti-microbial activity of bovine macrophages. Incubation of bovine macrophages with MAP resulted in modest increased production of NO at 4 and 6h post infection. Pretreatment of bovine macrophages with the MAPKp38 inhibitor SB203580 before addition of MAP organisms resulted in increased production of NO at 2, 4, 6 and 24h post infection. This study expanded our knowledge of the importance of the MAPKp38 pathway in limiting an appropriate macrophage response to MAP and suggested how activation of MAPKp38 pathway may be a target of this organism to disrupt earlier antimicrobial mechanisms of macrophages. These findings raises the interesting possibility that the cellular manipulation of MAPKp38 may be useful in designing novel vaccines against MAP.

Whole-blood gene-expression profiles of cows infected with Mycobacterium avium subsp. paratuberculosis reveal changes in immune response and lipid metabolism.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic debilitating disease affecting ruminants worldwide. In the present study, we aimed to determine the major gene networks and pathways underlying the immune response to MAP infection using whole-blood cells, as well as provide the potential transcriptional markers for identifying the status of MAP infection. We analyzed the transcriptional profiles of whole-blood cells of cattle identified and grouped according to the presence of MAP-specific antibodies and the MAP shed by them. The grouping was based on the results obtained by ELISA and PCR analyses as follows: i) Test1 group: MAP-negative results obtained by ELISA and positive results obtained by PCR; ii) Test2 group: MAP-positive results obtained by ELISA and negative results obtained by PCR; iii) Test3 group: MAP-positive results obtained by ELISA and positive results obtained by PCR; iv) uninfected control: MAP-negative results obtained both by ELISA and PCR analysis. The results showed down-regulated production and metabolism of reactive oxygen species in the Test1 group, activation of pathways related to the host-defense response against MAP (LXR/RXR activation and complement system) in the Test2 and Test3 groups, and anti-inflammatory response (activation of IL-10 signaling pathway) only in the Test3 group. Our data indicate a balanced response that serves the immune-limiting mechanism while the host-defense responses are progressing.

Metabolomic profiling in cattle experimentally infected with Mycobacterium avium subsp. paratuberculosis.

The sensitivity of current diagnostics for Johne's disease, a slow, progressing enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), is too low to reliably detect all infected animals in the subclinical stage. The objective was to identify individual metabolites or metabolite profiles that could be used as biomarkers of early MAP infection in ruminants. In a monthly follow-up for 17 months, calves infected at 2 weeks of age were compared with aged-matched controls. Sera from all animals were analyzed by 1H nuclear magnetic resonance spectrometry. Spectra were acquired, processed, and quantified for analysis. The concentration of many metabolites changed over time in all calves, but some metabolites only changed over time in either infected or non-infected groups and the change in others was impacted by the infection. Hierarchical multivariate statistical analysis achieved best separation between groups between 300 and 400 days after infection. Therefore, a cross-sectional comparison between 1-year-old calves experimentally infected at various ages with either a high- or a low-dose and age-matched non-infected controls was performed. Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS DA) yielded distinct separation of non-infected from infected cattle, regardless of dose and time (3, 6, 9 or 12 months) after infection. Receiver Operating Curves demonstrated that constructed models were high quality. Increased isobutyrate in the infected cattle was the most important agreement between the longitudinal and cross-sectional analysis. In general, high- and low-dose cattle responded similarly to infection. Differences in acetone, citrate, glycerol and iso-butyrate concentrations indicated energy shortages and increased fat metabolism in infected cattle, whereas changes in urea and several amino acids (AA), including the branched chain AA, indicated increased protein turnover. In conclusion, metabolomics was a sensitive method for detecting MAP infection much sooner than with current diagnostic methods, with individual metabolites significantly distinguishing infected from non-infected cattle.

Dose-dependent interferon-gamma release in dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis.

The interferon-gamma (IFN-γ) release assay is considered useful for diagnosis of subclinical paratuberculosis. However, interpretation can be subjective and complex; therefore, additional information regarding the course of the cellular immune response and effects of age and dose at infection would be helpful. Thirty-three calves were randomly allocated to 10 challenge groups and a negative control group. Calves were inoculated orally at 2 weeks or at 3, 6, 9, or 12 months of age. Within each age group, calves received either a high or low dose of Mycobacterium avium subspecies paratuberculosis (MAP). Monthly blood samples were collected, stimulated with Purified Protein Derivative (PPD) Johnin in vitro, and the subsequent release of IFN-γ measured. Calves inoculated with a high dose had earlier and stronger IFN-γ responses than low-dose calves. Furthermore, calves inoculated at 2 weeks of age produced less IFN-γ compared to those inoculated later in life. The IFN-γ response peaked (on average) 4 months after exposure; therefore, this would be an optimal interval to test cattle for MAP-infection (although the timing of field-based infections is unknown and clearance of infection a possibility). To conclude, the IFN-γ release assay could be a valuable diagnostic test on herd-level to indicate exposure to MAP.

Expression of genes associated with cholesterol and lipid metabolism identified as a novel pathway in the early pathogenesis of Mycobacterium avium subspecies paratuberculosis-infection in cattle.

Johne's disease (JD) is a chronic disease affecting ruminants and other species caused by the pathogenic mycobacterium, Mycobacterium avium subsp. paratuberculosis (MAP). MAP has developed a multitude of mechanisms to persist within the host, and these in turn are counteracted by the host through various immune pathways. Identifying and characterising the different strategies employed by MAP to alter the host immune system in its favour, and thereby persist intracellularly, could hold the key to developing strategies to fight this disease. In this study we analysed a subset of bovine microarray data derived from early time points after experimental infection with MAP. A specifically developed integrated approach was used to identify and validate host genes involved in cholesterol homeostasis (24DHCR, LDLR, SCD-1), calcium homeostasis and anti-bacterial defence mechanisms, (CD38, GIMAP6) which were downregulated in response to MAP exposure. A trend for upregulation of granulysin gene expression in MAP-exposed cattle in comparison to unexposed cattle was also observed. From these analyses, a model of potential pathogen-host interactions involving these novel pathways was developed which indicates an important role for host lipids in mycobacterial survival and persistence.

Host responses to the pathogen Mycobacterium avium subsp. paratuberculosis and beneficial microbes exhibit host sex specificity.

Differences between microbial pathogenesis in male and female hosts are well characterized in disease conditions connected to sexual transmission. However, limited biological insight is available on variances attributed to sex specificity in host-microbe interactions, and it is most often a minimized variable outside these transmission events. In this work, we studied two gut microbes-a pathogen, Mycobacterium avium subsp. paratuberculosis, and a probiotic, Lactobacillus animalis NP-51-and the interaction between each agent and the male and female gastrointestinal systems. This trial was conducted in BALB/c mice (n=5 per experimental group and per sex at a given time point), with analysis at four time points over 180 days. Host responses to M.avium subsp. paratuberculosis and L. animalis were sensitive to sex. Cytokines that were significantly different (P ≤ 0.05) betweenthe sexes included interleukin-1α/ß (IL-1α/ß), IL-17, IL-6, IL-10, IL-12, and gamma interferon (IFN-) and were dependent on experimental conditions. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-13/23 showed no sex specificity. A metabolomics study indicated a 0.5- to 2.0-fold (log2 scale) increase in short-chain fatty acids (butyrate and acetate) in males and greater increases in o-phosphocholine or histidine from female colon tissues; variances distinct to each sex were observed with age or long-term probiotic consumption. Two genera, Staphylococcus and Roseburia, were consistently overrepresented in females compared to males; other species were specific to one sex but fluctuated depending on experimental conditions. The differences observed suggest that male and female gut tissues and microbiota respond to newly introduced microorganisms differently and that gut-associated microorganisms with host immune system responses and metabolic activity are supported by biology distinct to the host sex.

Gene expression profiling and putative biomarkers of calves 3 months after infection with Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (JD), a chronic granulomatous intestinal inflammation of ruminants. Current diagnostic tools lack sensitivity to detect JD early in infection; therefore, alternatives are desired. The objective was to identify potential biomarkers in whole blood of high- and low-dose (LD) MAP-challenged Holstein-Friesian calves 3 months after inoculation. Infected calves were designated MAP-positive using the IFN-γ release assay. Differential expression of transcripts in whole blood was compared between non-infected controls and HD, as well as LD calves, using the Affymetrix(®) GeneChip(®) Bovine Genome Array. Microarray data were analyzed using RMA and PLIER algorithms; 296 transcripts were differentially expressed (17 had ≥ 1.5 fold change). The HD and LD calves had differential gene expression profiles for up to 80% of differentially expressed genes. Pathway analyses using Ingenuity Pathway Analysis (IPA(®)) indicated inhibition of several defence mechanisms, including apoptosis, leukocyte and lymphocyte trafficking, overall repression of gene expression and potentially hydrogen peroxide production in macrophages. Further validation using qPCR verified increased expression of CD46, ICOS, and CEP350, but decreased expression of CTLA4, YARS, and PARVB in infected calves. Additionally, a comparison of seropositive and seronegative infected calves identified transcripts predictive of seroconversion. We concluded that IL6ST/gp130 and CD22 may have important roles in the induction of antibodies against MAP. Putative biomarkers of early MAP infection with roles in immune responses were identified; in addition, the importance of infective dose on biomarkers was determined.

Immunoreactivity of protein tyrosine phosphatase A (PtpA) in sera from sheep infected with Mycobacterium avium subspecies paratuberculosis.

Evasion of host defense mechanisms and survival inside infected host macrophages are features of pathogenic mycobacteria including Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne's disease in ruminants. Protein tyrosine phosphatase A (PtpA) has been identified as a secreted protein critical for survival of mycobacteria within infected macrophages. The host may mount an immune response to such secreted proteins. In this study, the humoral immune response to purified recombinant M. avium subsp. paratuberculosis PtpA was investigated using sera from a cohort of sheep infected with M. avium subsp. paratuberculosis and compared with uninfected healthy controls. A significantly higher level of reactivity to PtpA was observed in sera collected from M. avium subspecies paratuberculosis infected sheep when compared to those from uninfected healthy controls. PtpA could be a potential candidate antigen for detection of humoral immune responses in sheep infected with M. avium subspecies paratuberculosis.

Divergent immune responses to Mycobacterium avium subsp. paratuberculosis infection correlate with kinome responses at the site of intestinal infection.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/ß-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection.

Global gene expression profiling of monocyte-derived macrophages from red deer (Cervus elaphus) genotypically resistant or susceptible to Mycobacterium avium subspecies paratuberculosis infection.

Mycobacterium avium subspecies paratuberculosis (MAP) can cause a chronic inflammatory bowel disease, Johne's disease (JD), in ruminant animals. This study has explored the molecular basis of resistance and susceptibility to this disease in red deer breeds previously confirmed to express polarised phenotypes by experimental infection trials and following natural infection. Monocyte-derived macrophage cultures were obtained from uninfected red deer selected for either a resistant or susceptible phenotype. Cells were infected with MAP in vitro and gene expression analysed by RNA-Seq. Transcriptome analysis revealed a more disrupted gene expression profile in macrophages from susceptible animals compared with cells from resistant animals in terms of the number of genes up- or downregulated. Highly upregulated genes were related to chemotaxis (CXCL10, CSF3, and CCL8) and type 1 interferon signalling (RSAD2, IFIT1, IFIT2, ISG12, ISG15, USP18, and HERC6). Upregulation of these genes was observed to be greater in macrophages from susceptible animals compared to cells from resistant animals in response to in vitro MAP infection. These data support the use of transcriptomic approaches to enable the identification of markers associated particularly with susceptibility to MAP infection.

Intestinal strictures, fibrous adhesions and high local interleukin-10 levels in goats infected naturally with Mycobacterium avium subsp. paratuberculosis.

This study describes pathological findings and their association with the production of interferon (IFN)-γ and interleukin (IL)-10 in goats infected naturally with Mycobacterium avium subsp. paratuberculosis (MAP). Twenty-seven goats were subjected to pathological examination. More than half of the animals had severe, diffuse, transmural granulomatous enteritis, often with abundant acid-fast bacilli (AFB), which was most evident in the proximal jejunum. Jejunal strictures and fibrous, peritoneal adhesions were findings that are not often reported in animals with paratuberculosis. Immunohistochemical labelling of IL-10 was seen within diffuse, granulomatous lesions and this may have prevented optimal local IFN-γ production and exacerbated the disease. However, since IFN-γ production was detected in cells from blood, jejunum and jejunal lymph nodes of goats with severe lesions by enzyme-linked immunosorbent assay, intracellular labelling and in-situ hybridization, the up-regulation of IL-10 might have been a consequence rather than a cause of the severe disease. The IL-10 labelling was co-localized with major histocompatibility complex (MHC) class II(+) cells, but rarely with CD4(+) cells. Comparable numbers of CD4(+) and CD8(+) T cells were recruited to both severe, diffuse lesions and small to moderate granulomatous lesions, while few T cells expressing the γδ form of the T-cell receptor were associated with both types of lesions.