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Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis that affects cattle worldwide. According to their severity and extension, PTB-associated histological lesions have been classified into the following groups; focal, multifocal, and diffuse. It is unknown whether these lesions represent sequential stages or divergent outcomes. In the current study, the associations between host genetic and pathology were explored by genotyping 813 Spanish Holstein cows with no visible lesions (N = 373) and with focal (N = 371), multifocal (N = 33), and diffuse (N = 33) lesions in gut tissues and regional lymph nodes. DNA from peripheral blood samples of these animals was genotyped with the bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data using the 1000 Bull genomes reference population. A genome-wide association study (GWAS) was performed using the WGS data and the presence or absence of each type of histological lesion in a case-control approach. A total of 192 and 92 single nucleotide polymorphisms (SNPs) defining 13 and 9 distinct quantitative trait loci (QTLs) were highly-associated (P ≤ 5 × 10 -7 ) with the multifocal (heritability = 0.075) and the diffuse (heritability = 0.189) lesions, respectively. No overlap was seen in the SNPs controlling these distinct pathological outcomes. The identified QTLs overlapped with some QTLs previously associated with PTB susceptibility, bovine tuberculosis susceptibility, clinical mastitis, somatic cell score, bovine respiratory disease susceptibility, tick resistance, IgG level, and length of productive life. Pathway analysis with candidate genes overlapping the identified QTLs revealed a significant enrichment of the keratinization pathway and cholesterol metabolism in the animals with multifocal and diffuse lesions, respectively. To test whether the enrichment of SNP variants in candidate genes involved in the cholesterol metabolism was associated with the diffuse lesions; the levels of total cholesterol were measured in plasma samples of cattle with focal, multifocal, or diffuse lesions or with no visible lesions. Our results showed reduced levels of plasma cholesterol in cattle with diffuse lesions. Taken together, our findings suggested that the variation in MAP-associated pathological outcomes might be, in part, genetically determined and indicative of distinct host responses., (© 2021. The Author(s).)

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), responsible for important economic losses in the dairy industry. Current diagnostic methods have low sensitivities for detection of latent forms of MAP infection, defined by focal granulomatous lesions and scarce humoral response or MAP presence. In contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. Our previous RNA-Seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of MAP infected Holstein cows with focal (ABCA13 and MMP8) and diffuse (FAM84A, SPARC and DES) lesions vs. control animals with no detectable PTB-associated lesions in intestine and regional lymph nodes. The aim of the current study was to assess the PTB diagnostic potential of commercial ELISAs designed for the specific detection of these biomarkers. The ability of these ELISAs to identify animals with latent and/or patent forms of MAP infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). ROC analysis revealed that the ABCA13-based ELISA showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (AUC 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (AUC 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular IDEXX ELISA and other conventional diagnostic methods. SPARC and MMP8 showed the highest diagnostic accuracy for the detection of animals with multifocal (AUC 0.852) and diffuse lesions (AUC 0.831), respectively. In conclusion, our results suggest that quantification of ABCA13, SPARC and MMP8 by ELISA has the potential for implementation as a diagnostic tool to reliably identify MAP infection, greatly improving early detection of MAP latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic infectious disease that causes intractable diarrhea in ruminants. To control the occurrence of JD in cattle, a national surveillance is conducted in Japan. Since 2013, real-time quantitative PCR has been used for definite diagnosis. In this study, we compared the amount of fecal MAP DNA with histopathological classification of ileocecal lesions. Multinomial logistic regression models enabled us to predict the probability of finding the histopathological classification from the amount of fecal MAP DNA. These results suggest that shedding level of MAP DNA could act as an indicator of JD progression.

Extracellular microRNAs (miRNAs) are detectable in the peripheral blood and have been touted as potential biomarkers for a range of maladies. The presence and biomarker potential of miRNAs in other biofluids has been less thoroughly explored, particularly in the veterinary realm. Faecal miRNAs are a case in point; while they have been identified largely in rodents and humans, they have not been reported in cattle but may have prognostic or diagnostic value for Johne's Disease (JD) in cattle, a chronic granulomatous inflammation of the ileum caused by Mycobacterium avium subspecies paratuberculosis (MAP). The aim of this study was thus to characterise the bovine faecal miRNome and to determine the utility of these transcripts as biomarkers for JD. Real-time PCR arrays consisting of 752 miRNA targets, optimised for detection of human miRNA, were used to screen RNA purified from faecal samples obtained from confirmed JD clinical cases vs. healthy controls. Two hundred and fifty-eight miRNAs were detected in bovine faeces, three of which are potentially novel orthologs of known human miRNAs. Differential abundance of three miRNA was evident in animals with clinical JD as compared to healthy controls. Our study has therefore identified a variety of miRNAs in bovine faeces and has demonstrated their utility in differentiating healthy animals from those with late-stage JD, providing potential biomarkers for MAP infection and disease progression.

Infection of the host with Mycobacterium avium subsp. paratuberculosis results in chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from an asymptomatic subclinical disease state to advanced clinical disease is not fully understood. In the present study, naturally infected dairy cattle were divided into subclinical and clinical infection groups, along with noninfected control cows of similar parity, to study host immune responses in different stages of infection. Both infection groups had higher levels of secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) than control cows, whereas only clinical cows had increased secretion of IL-10, IL-12, and IL-18 upon stimulation of peripheral blood mononuclear cells (PBMCs) with antigen. Conversely, secretion of IL-17Α was decreased for clinical cows compared to subclinical and control cows. Proinflammatory cytokine genes were upregulated only for subclinical cows, whereas increased IL-10 and IL-17 gene expression levels were observed for both infection groups. Increased CD4 + , CD8 + , and γδ T cell receptor-positive (TCR + ) T cells were observed for subclinical cows compared to clinical cows. Although clinical cows expressed antigen-specific immune responses, the profile for subclinical cows was one of a dominant proinflammatory response to infection. We reason that a complex coordination of immune responses occurs during M. avium subsp. paratuberculosis infection, with these responses shifting as the host transitions through the different stages of infection and disease (subclinical to clinical). A further understanding of the series of events characterized by Th1/Th2/Th17 responses will provide mechanisms for disease progression and may direct insightful intervention strategies., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Three screening tests {(newly developed, six recombinant secretory proteins based 'cocktail ELISA', in-house robust 'indigenous ELISA' based on semi-purified protoplasmic antigens and tissue microscopy were evaluated with 'Gold standard', histo-pathology for the diagnosis of Johne's disease in goats and buffaloes. Serum and tissues {mesenteric lymph nodes and intestines) were driven from farmer's goats (n = 77) and buffaloes (n = 40) slaughtered for harvesting meat and farm goats (n = 77), died and necropsied. Twenty seven (35%) goats and 23 (57.5%) buffaloes were positive in all the four tests. Of 134 tissues screened by histo-pathology, 79.8% MLN and 76.8%, intestines, were positive for MAP infection. In tissue microscopy, 55.2 and 52.3%, goats and buffaloes were positive, respectively. Of 117 sera screened by i_ELISA, 58.4 and 70.0%, goats and buffaloes were positive, respectively. Whereas, c_ELISA detected 55.8 and 62.5%, goats and buffaloes, positives, respectively. Twelve tissues (70.5%) of goats necropsied were positive, both in tissue microscopy and histo-pathology. Most significant gross findings were serous atrophy of the fat and mild to moderate, diffuse thickening of terminal ileum, especially at ileo-caecal junction with or without transverse / longitudinal corrugations. In histo-pathology grade III and IV lesions were significantly low as compared to grade I and II. Of the four tests used for screening 268 samples, histo-pathology was most sensitive (78.3%), followed by i_ELISA (62.3%), c_ELISA (58.9%) and tissue microscopy (58.9%). Between two ELISA tests, c_ELISA using six recombinants secretory proteins, had higher specificity as compared to i_ELISA., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP.

Mycobacterium avium complex (MAC) and Mycobacterium avium paratuberculosis (MAP) cause zoonotic infections transmitted by birds and livestock herds. These pathogens have remained as serious economic and health threats in most areas of the world. As zoonotic diseases, the risk of development of occupational disease and even death outcome necessitate implementation of control strategies to prevent its spread. Zoonotic MAP infections include Crohn's disease, inflammatory bowel disease, ulcerative colitis, sarcoidosis, diabetes mellitus, and immune-related diseases (such as Hashimoto's thyroiditis). Paratuberculosis has classified as type B epidemic zoonotic disease according to world health organization which is transmitted to human through consumption of dairy and meat products. In addition, MAC causes pulmonary manifestations and lymphadenitis in normal hosts and human immunodeficiency virus (HIV) progression (by serotypes 1, 4, and 8). Furthermore, other subspecies have caused respiratory abscesses, neck lymph nodes, and disseminated osteomyelitis in children and ulcers. However, the data over the occupational relatedness of these subspecies is rare. These agents can cause occupational infections in susceptible herd breeders. Several molecular methods have been recognized as proper strategies for tracking the infection. In this study, some zoonotic aspects, worldwide prevalence and control strategies regarding infections due to MAP and MAC and related subspecies has been reviewed., (© 2019 Wiley Periodicals, Inc.)

Macrophages play an important role in the host immune response to Mycobacterium avium subsp. paratuberculosis (MAP) infection, however, MAP is able to disrupt normal macrophage functions to avoid destruction. It is unclear whether the phenotypes of macrophages present in the target tissue play a role in the inability to clear MAP infection. The aim of this study was to identify macrophage phenotypes (host defense or resolution and repair) present within the bovine ileum of naturally infected cattle, as well as to ascertain abundance of each macrophage phenotype present during different stages of MAP infection. Immunofluorescent (IF) labeling was performed on frozen bovine mid-ileal tissue sections collected from 28 Holstein dairy cows. Comprehensive IF staining for cytokines, such as IFN-γ, IL-1Ra, IL-1β, IL-10, TGF-β, TNF-α, and uNOS, along with markers such as CD163, CD206, and TLR4, served to define the macrophage phenotypes. Overall, cows in the clinical stage of disease demonstrated significantly higher numbers of resolution and repair macrophages and lower numbers of host defense macrophages in the ileal tissue. Interestingly, subclinically affected cows with asymptomatic disease had a nearly equal ratio of host defense and resolution and repair macrophage phenotypes, whereas macrophage phenotype was skewed to a host defense macrophage in the tissues of the control noninfected cows. The preponderance of M2-like resolution and repair phenotype for macrophages in the tissues of cows with clinical disease would explain why the host fails to control and/or clear the infection, leading to a higher MAP burden. The results of the current study offer insight into the disparate macrophage phenotypes present in the bovine ileum during different stages of infection., Competing Interests: The authors have declared no competing interests exist.

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., Competing Interests: The author declares that there is no conflict of interest.

Paratuberculosis (PTB) or Johne's disease is a contagious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Ovine PTB is less understood than bovine PTB, especially concerning paucibacillary infection and its evolution into clinical disease. We combined shotgun proteomics, histopathology and immunohistochemistry for the characterization of ileal tissues collected from seven asymptomatic sheep negative to serum ELISA, positive to feces and tissue MAP IS900 and F57 PCR, histologically classified as paucibacillary, actively infected, together with 3 MAP-free controls (K). Following shotgun proteomics with label-free quantitation and differential analysis, 96 proteins were significantly changed in PTB vs K, and were mostly involved in immune defense processes and in the macrophage-MAP interaction. Principal component analysis (PCA) of protein abundances highlighted two PTB sample clusters, PTB1 and PTB2, indicating a dichotomy in their proteomic profiles. This was in line with the PCA of histopathology data and was related to features of type 2 (PTB1) and type 3a (PTB2) lesions, respectively. PTB2 proteomes differed more than PTB1 proteomes from K: 43 proteins changed significantly only in PTB2 and 11 only in PTB1. The differential proteins cathelicidin, haptoglobin, S100A8 and S100A9 were evaluated by immunohistochemistry. K tissues were negative to cathelicidin and haptoglobin and sparsely positive to S100A8 and S100A9. PTB tissues were positive to all four proteins, with significantly more cells in PTB2 than in PTB1. In conclusion, we described several pathways altered in paucibacillary PTB, highlighted some proteomic differences among paucibacillary PTB cases, and identified potential markers for disease understanding, staging, and detection.

Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium that causes Johne's disease in cattle. Although infected cattle can be identified by examining fecal, blood, or milk samples, the gold standard is identification of MAP in tissue samples postmortem. Although tissue samples are commonly frozen, the ability to detect MAP in frozen-thawed tissue samples has apparently not been reported. We therefore determined the ability to detect MAP in tissue samples following freezing. Tissue samples were collected from calves that were either inoculated (IN) 3 mo prior, or contact-exposed (CE) for 3 mo. Following autopsy, tissues were immediately processed for culture, followed by DNA extraction and detection by qPCR. Samples were categorized as positive or negative based on the cycle threshold (Ct) value. The remaining unprocessed tissue samples were frozen at -80°C. After 18 mo, 50 tissue samples designated MAP-positive were thawed and processed for detection of MAP. Four (8%) samples were qPCR-negative, and Ct values of the remaining 46 samples were higher after freezing. Given the small numerical change in Ct values for MAP-positive samples after 18 mo of frozen storage, freezing and thawing may have had some deleterious effects on MAP detection in tissues. Although the decrease in ability to detect MAP-positive samples was minor for IN calves, there may be a greater effect for CE calves that should be considered when freezing tissue samples.

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon translocation from the lumen of the small intestine, mycobacteria have the ability to thwart innate defense mechanisms and persist within the macrophage in the lamina propria. In an effort to understand how the pathology of disease is reflected in current diagnostic tests, immunofluorescent (IFA) labeling was performed to quantitate macrophage and MAP numbers in the ileum of infected cattle and correlate results with common methods for diagnosis of MAP infection; including ELISA, IFN-γ assay, RT-PCR, culture of MAP, and histological classification of tissue sections. Predictive models for clinical and subclinical disease states, histopathology acid-fast (AF), MAP location, granulomatous inflammation and type classifications, as well as macrophage, MAP and macrophages with intracellular MAP IFA labeling were successfully developed. The combination of macrophage number and ELISA were the best predictors of clinical disease state, while macrophage number was the best and only significant predictor of subclinical disease state. Fecal culture and number of MAP were the best predictors of granulomatous inflammation, and of combined AF, MAP location and granuloma type, respectively. Additionally, fecal culture and tissue culture were the best predictors of numbers of macrophages and MAP, respectively, while both ELISA and tissue culture were the best predictors of number of macrophages with intracellular MAP., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paratuberculosis (PTB) or Johne's disease is a chronic intestinal infection of ruminants, caused by Mycobacterium avium subsp. paratuberculosis. The shedding of mycobacteria in the feces starts at the initial stages and increases with disease progression, suggesting that antigens secreted by mycobacteria could be excreted in the feces. Previously, we demonstrated that the alanine and proline-rich antigen (Apa), a secretory antigen of Map, could be detected in the intestine of cows with PTB using a monoclonal antibody. In this study, we verified whether this protein can be found in consistently detectable levels in the feces of cattle with PTB. Feces were obtained from cows with Johne's disease confirmed by laboratory tests, cows with suspected PTB based on seropositivity and from PTB-free control cows. Samples were immunoprecipitated using anti-Apa monoclonal antibody and analyzed by immunoblot. The Apa was detected as a 60/70 kDa doublet band in all samples obtained from animals with laboratory-confirmed disease and in a substantial proportion of seropositive asymptomatic animals, but not in the control samples. Additionally, the antigen was detected in the feces of animals with Johne's disease by ELISA. This study strongly suggests that Apa is a potential fecal biomarker of Johne's disease that could serve for immunodiagnosis.

There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)-an adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptor-has entered human trials based on promising pre-clinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01 vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG, CAF09-primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+ IL-2+). In conclusion, CAF09 supports the development of antibodies along with a high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in cattle and other ruminants., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: J.W, P.A., and C.A. are employed by Statens Serum lnstitut, a non-profit government research facility of which the CAF adjuvants are proprietary products. The authors declare the following patents/patent applications relating to material pertinent to this article: The use of monomycolyl glycerol (MMG) as an adjuvant (WO 2009/003474) and Adjuvant combinations of liposomes mycobacterial lipids for immunization compositions and vaccines (WO 2005/004911). There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Mycobacterium avium subspecies paratuberculosis (MAP) is a cause of contagious and typically fatal enteric disease, primarily affecting ruminant and pseudoruminant species. During a MAP outbreak in a captive collection, six of nine adult Mishmi takin ( Budorcas taxicolor taxicolor) showed marked weight loss over 1-3 mo, followed by an acute deterioration. Fecal culture and microscopy failed to identify MAP shedding. Necropsy findings included grossly normal intestines and marked enlargement of mesenteric lymph nodes. Histological findings included multibacillary granulomatous enteritis, mesenteric lymphadenitis, and periportal hepatitis. MAP was confirmed by culture of intestinal and lymph node tissues from the index case. Results of antemortem serological testing using an indirect ELISA (ID SCREEN® Paratuberculosis Indirect) were corroborated by findings at necropsy or survival of the outbreak. Mishmi takin appear to show high MAP susceptibility and a rapid disease course compared with domestic ruminant species.

Johne's disease (JD) or paratuberculosis is an economically significant, chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Experimental models of JD in cattle are logistically challenging due to the need for long term monitoring, because the clinical disease can take years to manifest. Three trials were undertaken, the largest involving 20 cattle exposed orally to a low dose of C strain MAP and 10 controls studied for 4.75 years. Frequent blood and faecal sampling was used to monitor immunological and infection parameters, and intestinal biopsies were performed at two time points during the subclinical disease phase. Although clinical disease was not seen, there was evidence of infection in 35% of the animals and at necropsy 10% had histopathological lesions consistent with JD, similar to the proportions expected in naturally infected herds. Faecal shedding occurred in two distinct phases: firstly there was intermittent shedding <∼9 months post-exposure that did not correlate with disease outcomes; secondly, in a smaller cohort of animals, this was followed by more consistent shedding of increasing quantities of MAP, associated with intestinal pathology. There was evidence of regression of histopathological lesions in the ileum of one animal, which therefore had apparently recovered from the disease. Both cattle with histopathological lesions of paratuberculosis at necropsy had low MAP-specific interferon-gamma responses at 4 months post-exposure and later had consistently shed viable MAP; they also had the highest loads of MAP DNA in faeces 4.75 year s post-exposure. In a trial using a higher dose of MAP, a higher proportion of cattle developed paratuberculosis. The information derived from these trials provides greater understanding of the changes that occur during the course of paratuberculosis in cattle., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), resulting in inflammation of intestines and persistent diarrhea. The initial host response against MAP infections is mainly regulated by the Th1 response, which is characterized by the production of IFN-γ. With the progression of disease, MAP can survive in the host through the evasion of the host's immune response by manipulating the host immune response. However, the host response during subclinical phases has not been fully understood. Immune regulatory genes, including Th17-derived cytokines, interferon regulatory factors, and calcium signaling-associated genes, are hypothesized to play an important role during subclinical phases of Johne's disease. Therefore, the present study was conducted to analyze the expression profiles of immune regulatory genes during MAP infection in whole blood. Different expression patterns of genes were identified depending on the infection stages. Downregulation of IL-17A, IL-17F, IL-22, IL-26, HMGB1, and IRF4 and upregulation of PIP5K1C indicate suppression of the Th1 response due to MAP infection and loss of granuloma integrity. In addition, increased expression of IRF5 and IRF7 suggest activation of IFN-α/β signaling during subclinical stages, which induced indoleamine 2,3-dioxygenase mediated depletion of tryptophan metabolism. Increased expression of CORO1A indicate modulation of calcium signaling, which enhanced the survival of MAP. Taken together, distinct host gene expression induced by MAP infection indicates enhanced survival of MAP during subclinical stages.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis , is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E 2 (PGE 2 ) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE 2 were performed to investigate the immunosuppressive effect of PGE 2 during Johne's disease. In vitro PGE 2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE 2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE 2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE 2 pathway in cattle. In contrast, downregulation of PGE 2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8 + T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8 + T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8 + T-cell proliferation in vitro , suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease., (Copyright © 2018 American Society for Microbiology.)

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

Paratuberculosis (Johne's disease) is an economically significant condition caused by Mycobacterium avium subsp. paratuberculosis. However, difficulties in diagnosis and classification of individual animals with the condition have hampered research and impeded efforts to halt its progressive spread in the global livestock industry. Descriptive terms applied to individual animals and herds such as exposed, infected, diseased, clinical, sub-clinical, infectious and resistant need to be defined so that they can be incorporated consistently into well-understood and reproducible case definitions. These allow for consistent classification of individuals in a population for the purposes of analysis based on accurate counts. The outputs might include the incidence of cases, frequency distributions of the number of cases by age class or more sophisticated analyses involving statistical comparisons of immune responses in vaccine development studies, or gene frequencies or expression data from cases and controls in genomic investigations. It is necessary to have agreed definitions in order to be able to make valid comparisons and meta-analyses of experiments conducted over time by a given researcher, in different laboratories, by different researchers, and in different countries. In this paper, terms are applied systematically in an hierarchical flow chart to enable classification of individual animals. We propose descriptive terms for different stages in the pathogenesis of paratuberculosis to enable their use in different types of studies and to enable an independent assessment of the extent to which accepted definitions for stages of disease have been applied consistently in any given study. This will assist in the general interpretation of data between studies, and will facilitate future meta-analyses.

Johne's disease is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which affects ruminants worldwide and has a significant economic impact. MAP has also been associated with human Crohn's disease, although this connection is not well established. MAP is highly adapted for survival within host macrophages and prevents macrophage activation, blocks phagosome acidification and maturation, and attenuates presentation of antigens to the immune system. The consequence is a very long silent infection before clinical signs are observed. The present work examined the transcriptome of bovine monocyte-derived macrophages (MDM) infected with the L1 strain of MAP at 2h, 6h and 24h post infection using RNA-seq. Pathway over-representation analysis of genes differentially expressed between infected vs. control MDM identified that immune related pathways were affected. Genes belonging to the cytokine-cytokine receptor interaction pathway and members of the JAK-STAT pathway, which is involved in the regulation of immune response, were up-regulated. However, in parallel inhibitors of immune functions were activated, including suppressor of cytokine signaling (SOCS) and cytokine-inducible SH2-containing protein (CISH), which most likely suppresses IFNγ and the JAK/STAT signaling cascade in infected MDM, which may favour MAP survival. After exposure, macrophages phagocytise pathogens, activate the complement cascade and the adaptive immune system through the antigen presentation process. However, data presented here suggest that genes related to phagocytosis and lysosome function are down regulated in MAP infected MDM. Genes of MHC class II and complement pathway were also down-regulated. This study therefore shows that MAP infection is associated with changes in expression of genes related to the host immune response that may affect its ability to survive and multiply inside the host cell., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Exposure to Mycobacterium avium subspecies paratuberculosis (MAP) does not always lead to Johne's disease. Understanding differences in disease susceptibility of individual animals is a key aspect to controlling mycobacterial diseases. This study was designed to examine the susceptibility or resistance of various breeds of sheep to MAP infection. Merino, Suffolk first cross Merino, Border Leicester, and Poll Dorset sheep were orally inoculated with MAP and monitored for 14 months. Clinical disease occurred more frequently in the Merino (42%) and Suffolk first cross Merino (36%) compared to the Border Leicester (12%) and Poll Dorset (11%) breeds. Infection risk, as determined by culture of gut and associated lymphoid tissues, ranged from 75% for the Suffolk first cross Merino to 47% for the Poll Dorset sheep. Significant differences were identified in the site in the intestines of the most severe histopathological lesions and the immune responses to infection between the breeds. However, there was no difference in faecal MAP shedding by clinical cases between breeds. All breeds tested were susceptible to MAP infection, as determined by infection and clinical disease development, although there were differences in the proportions of diseased animals between the breeds. Poll Dorset and Border Leicester sheep were more resilient to MAP infection but there was evidence that more animals could have developed disease if given more time. These findings provide evidence of potential differential disease susceptibility between breeds, further our understanding of disease pathogenesis and risks of disease spread, and may have an influence on control programs for paratuberculosis.

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, -the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This case report shows that Mycobacterium avium subsp. paratuberculosis (MAP) infection can cause clinical disease in domestic dogs, and should be considered as a differential diagnosis for gastrointestinal inflammatory conditions. A male dachshund presented with lethargy and pain. Enlarged mesenteric lymph nodes were found on abdominal ultrasound examination. Cytological examination of lymph node aspirates was consistent with granulomatous inflammation, which was culture-confirmed as MAP. Although we were unable to confirm the source of infection, the dog's history included exposure to sheep in the Western Cape.

Paratuberculosis or Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteritis affecting both domestic and wild ruminants. The present work is part of a wider set of studies designed to assess the prevalence of paratuberculosis in free ranging red deer (Cervus elaphus). With that purpose, 877 free-ranging red deer legally hunted in the Centre-eastern Portugal were submitted to necropsy and sampled for molecular methods, microbiology and histopathology. Thirty-seven (4.2%) kidneys revealed acid-fast bacilli when screened with the Ziehl-Neelsen technique. Map was detected by IS900 polymerase chain reaction (PCR) in thirty (81.1%) of the Ziehl-Neelsen positive kidneys. Subsequent PCR and/or culture from the different organs of the 37 examined animals allowed us to detect 86.4% (32 animals) infected red deer. Our results suggest that renal involvement in Map infected deer may be underdiagnosed and thus the routine examination of this organ and its inclusion in PCR techniques designed for Map detection could substantially improve the diagnostic of paratuberculosis in red deer.

Available diagnostic assays for Mycobacterium avium subsp. paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN-γ, ELISA-antibody and fecal shedding experimental sensitivity scores for MAP infection detection and disease progression. We used both statistical methods and dynamic mathematical models to (i) evaluate the empirical assays (ii) infer and explain biological mechanisms that affect the time evolution of the biomarkers, and (iii) predict disease stages of 57 animals that were naturally infected with MAP. This analysis confirms that the fecal test is the best marker for disease progression and illustrates that Th1/Th2 (IFN-γ/ELISA antibodies) assays are important for infection detection, but cannot reliably predict persistent infections. Our results show that the theoretical simulated macrophage-based assay is a potential good diagnostic marker for MAP persistent infections and predictor of disease specific stages. We therefore recommend specifically designed experiments to test the use of a based assay in the diagnosis of MAP infections.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4 + T cell infiltration into the colonic lamina propria . Functional analysis identified a critical role of CD4 + T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis affecting ruminants worldwide. Depending on the MAP-Type (MAP-C or MAP-S, cattle or sheep type), strains differ in virulence and host preference. There is not yet any strong evidence indicating that individual field strains of the same MAP-subgroup exhibit differences in virulence. The aim of this study was to evaluate a potential association between the genotype of individual field strains belonging to the MAP-C group and the presence of macroscopic intestinal lesions characteristic of paratuberculosis in the infected animals. 88 MAP-C isolates were sampled from clinically healthy cows at slaughter. Cows were grouped as A (n=46) with, and B (n=42) without macroscopic intestinal lesions. Sampled cows from both the A and B groups came from different farms and had a similar age distribution. MAP isolates were characterized by MIRU-VNTR and IS900-RFLP analysis. Resulting genotypes were examined for an association with the presence of macroscopic intestinal lesions characteristic of paratuberculosis. MAP isolates from groups A and B exhibited similar strain diversity: 20 and 18 combined genotypes, altogether 32 genotypes. Six of these genotypes were detected in both groups. Although no association was found between individual combined genotypes and presence of macroscopic intestinal lesions, IS900-RFLP-(BstEII)-Type-C1 (the most common type worldwide) was found more often in group A (p<0.01). The data give only weak indication for the existence of differences in virulence among MAP-cattle type isolates. Differences in the development and severity of lesions may rather depend on unknown host factors or inoculation dose. Virulence properties of IS900-RFLP-(BstEII)-Type-C1 isolates should be examined in more detail., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the etiological agent of Johne's disease in ruminants. Johne's disease is an important enteric infection causing large economic losses associated with infected herds. In an attempt to fight this infection, we created two novel live-attenuated vaccine candidates with mutations in sigH and lipN (pgsH and pgsN, respectively). Earlier reports in mice suggested these vaccines are promising candidates to fight Johne's disease in ruminants. In this study, we tested the performances of the two constructs as vaccine candidates using the goat model of Johne's disease. Both vaccines appeared to provide significant immunity to goats against challenge from wild-type M. paratuberculosis The pgsH and pgsN constructs showed a significant reduction in histopathological lesions and tissue colonization compared to nonvaccinated goats and those vaccinated with an inactivated vaccine. Unlike the inactivated vaccine, the pgsN construct was able to eliminate fecal shedding from challenged animals, a feature that is highly desirable to control Johne's disease in infected herds. Furthermore, strong initial cell-mediated immune responses were elicited in goats vaccinated with pgsN that were not demonstrated in other vaccine groups. Overall, the results indicate the potential use of live-attenuated vaccines to control intracellular pathogens, including M. paratuberculosis, and warrant further testing in cattle, the main target for Johne's disease control programs., (Copyright © 2017 American Society for Microbiology.)

Animals infected with Mycobacterium avium subspecies paratuberculosis show a variety of granulomatous lesions that range from focal forms, seen in the subclinical stages, to diffuse lesions associated with clinical signs. The aim of this study was to phenotypically characterize the macrophages present in the different lesion types using immunohistochemical methods. Lesions from a total of 23 animals with bovine paratuberculosis, natural and experimental, were examined by immunohistochemistry. Antibodies against inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), CD163, interleukin 10 (IL-10), transforming growth factor β (TGF-β), major histocompatibility complex (MHC) class II, natural resistance-associated macrophage protein 1 (Nramp-1), calprotectin, Ki-67, CD68, lysozyme, and ionized calcium-binding adaptor molecule 1 (Iba-1) molecules were employed. Samples were scored semiquantitatively using a complete histological score (H-score), reflecting the staining intensity and the percentage of immunolabeled macrophages. Differences in the H-score were seen depending on the lesion type. In focal lesions, with none or few acid-fast bacilli (AFB), macrophages were polarized toward M1 phenotype, with high H-scores for iNOS and TNF-α. Diffuse multibacillary lesions showed M2 differentiation, with high expression of CD163, IL-10, and TGF-β as well as Nramp-1 and MHC class II antigens. Macrophages in diffuse paucibacillary forms showed high H-scores for iNOS but low ones for TNF-α. Diffuse lesions, either multibacillary or paucibacillary, showed high calprotectin and low Ki-67 expression, suggesting a progressive character, while focal forms, with low H-scores for these antigens, would be consistent with latency. Lysozyme and CD68 expression were related to the amount of AFB. H-score for Iba-1 antibody was similar among all types. The findings of this study provide insights into the polarization status of macrophages and lesion development in bovine paratuberculosis.

A long-term study was undertaken to monitor immune responses, faecal cultures and clinical disease in sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map) strain Telford. New Zealand Merino lambs (N=56) were challenged with three oral doses of Map suspension. The lambs were weighed and faecal and blood samples obtained at different time-points. At 63 weeks post-challenge, surviving sheep were euthanised and samples of liver, ileo-caecal valve and mesenteric lymph node were collected for histopathology and Map culture. High IFN-γ and antibody responses were evident as early as 8 weeks post-C1 which persisted until the end of the trial. Approximately 92% of the sheep shed Map in faeces at 36 weeks post-challenge, with the prevalence decreasing to around 40% at the end of the trial. Thirteen sheep progressively lost weight and were euthanised between weeks 32 and 58 post-challenge. Nearly 58% of surviving sheep exhibited histo-pathological lesions in at least one of the three tissues sampled, while 42% showed acid-fast bacilli in at least one tissue. A positive Map culture in at least one tissue was obtained from approximately 85% of sheep. These results indicate that the three doses of Map challenge were highly effective in establishing Johne's disease in NZ Merino lambs., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Johne's disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease of ruminants. Hallmark symptoms of clinical Johne's disease include diarrhea, progressive weight loss, and premature death; symptoms due largely to chronic inflammation in the small intestine. MAP colonizes resident macrophages within the ileum of the small intestine, subsequently establishing a persistent infection in the host. It has been proposed that regulatory T cells may play a role in the progression of Johne's disease, either through promotion of tolerance to MAP or via a loss in homeostasis that subsequently allows widespread inflammation. In this report, we evaluated the presence of Tregs, as well as other immune parameters, in the ileum and draining lymph nodes of MAP associated lesions. A lesion classification scheme was developed to categorize severity of MAP-induced lesions within infected tissues and subsequently regulatory T cell presence and overall immune activity were assessed corresponding to lesions of varying severity, in comparison to tissues from healthy control animals. Our results revealed a relationship between animal health and overall lesion severity within the infected tissues, as well as a relationship between bacterial burden and severity of pathology. Regulatory T cell abundance was shown to decrease with increasing lesion severity. Within the ileum, the expression of many Th1, Th2, and Treg-associated genes increased in mild lesions and decreased in severe lesions, whereas in the lymph nodes the expression of these genes tended to increase with increasing lesion severity. Based on our results, we conclude that a local loss of T cell (including Treg) activity occurs within severe ileal lesions associated with MAP, resulting in a loss of homeostasis that ultimately leads to the progression of clinical Johne's disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Several farms in the Northeast of Brazil were investigated for Mycobacterium avium subsp. paratuberculosis infection in order to identify the occurrence of paratuberculosis in buffaloes. Samples were obtained from 17 farms, two slaughter houses, and a quarantine area in the Northeast. About 15,000 buffaloes of the Murrah, Mediterranean, and Jafarabadi breed as well as their crossbreeds were evaluated for meat, dairy, and mixed farms with semi-intensive or extensive breeding practices. For diagnostic purposes, postmortem and histopathological examination, including Ziehl-Neelsen test of fecal smears and scraped intestinal mucosa were performed. PCR was applied for fecal samples, mesenteric lymph nodes, and intestines. Six Johne's disease-positive farms, which together with those previously identified, indicate that the disease is spread through the Brazilian Northeast, similar to what occurs in cattle herds in other regions of the country. The increase in prevalence of paratuberculosis is a consequence of introduction of animals from other regions without adequate veterinary assistance and due to the little official attention paid to this initially silent and chronic disease.

Milk yield, milk ingredients, health and other, production-related parameters of subclinically infected, Mycobacterium avium ssp. paratuberculosis (MAP-) shedding (positive faecal PCR, n = 20) and non-shedding (negative faecal PCR, n = 10) dairy cows were compared in the period from 10 days prepartum to 120 days postpartum. Body condition, rumen fill and faeces scores were lower in the MAP-shedding cows. There was no significant difference in plasma or urine metabolic parameters between the groups. Milk yield and lactose content tended to be lower (P = 0.074 and 0.077, respectively), somatic cell count tended to be higher (P = 0.097), while milk fat content was significantly higher (P = 0.006) in MAP-shedding cows than in the controls. Milk protein content did not differ between the groups. All other health and production parameters [number of reproductive tract treatments, number of udder treatments, number of artificial inseminations (AIs), calving interval, and service period] were significantly better in the control group. It is concluded that MAP infection, even in a subclinical form, has a significant impact on some production and health parameters of dairy cows.

Two different forms of clinical paratuberculosis in sheep are recognised, related to the level of bacterial colonization. Paucibacillary lesions are largely composed of lymphocytes with few bacteria, and multibacillary pathology is characterized by heavily-infected macrophages. Analysis of cytokine transcripts has shown that inflammatory Th1/Th17 T cells are associated with development of paucibacillary pathology and Th2 cytokines are correlated with multibacillary disease. The master regulator T cell transcription factors TBX21, GATA3, RORC2 and RORA are critical for the development of these T cell subsets. Sequence variations of the transcription factors have also been implicated in the distinct disease forms of human mycobacterial and gastrointestinal inflammatory diseases. Relative RT-qPCR was used to compare expression levels of each transcript variant of the master regulators in the ileo-caecal lymph nodes of uninfected controls and sheep with defined paucibacillary and multibacillary pathology. Low levels of GATA3 in multibacillary sheep failed to confirm that multibacillary paratuberculosis is caused simply by a Th2 immune response. However, high levels of TBX21, RORC2 and RORC2v1 highlights the role of Th1 and Th17 activation in paucibacillary disease. Increased RORAv1 levels in paucibacillary tissue suggests a role for RORα in Th17 development in sheep; while elevated levels of RORAv4 hints that this variant might inhibit RORα function and depress Th17 development in multibacillary sheep.

Several studies have demonstrated that wildlife reservoirs of mycobacteria are responsible for the maintenance and spreading of the infection to livestock and wildlife counterparts. Recent data report the role of wild boar (Sus scrofa) as a reservoir for Mycobacterium bovis. This study was conducted to evaluate the chronic inflammatory response in the mesenteric lymph nodes (MLN) of wild boar with granulomatous lymphadenitis (n=30). Morphological parameters of the lesions were recorded. The expression of CD3 and CD79α molecules was evaluated by immunohistochemistry. Molecular genotyping and culture to identify mycobacteria were performed. The lesions consisted mainly of stage III and stage IV granulomas. CD3 and CD79α positive cells were observed in 15 (50%) and in 11 (36.6%) MLN, respectively. In these lesions, higher percentages of T lymphocytes were found and a limited number of animals exhibited a tendency for an increased percentage of B lymphocytes. Our results suggest that there are similar percentages and distribution patterns of CD3 and CD79α in the lesions, regardless of the presence of Mycobacterium avium subsp. paratuberculosis (Map), M. bovis or Map-M. bovis co-infection, and confirm that wild boar is both susceptible and could be an important Map and M. bovis wild reservoir in the study area., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was "proliferation of endothelial cells", indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as "proliferation of endothelial cells" (bta-miR-196 b), "bacteria recognition" (bta-miR-146 b), and "regulation of the inflammatory response" (bta-miR-146 b).

Thirty Holstein calves were obtained from 2 dairy farms in central Iowa at birth and randomly assigned to 1 of 6 treatment groups: (1) colostrum deprived (CD), no vitamins; (2) colostrum replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; and (6) CR, vitamins A, D3, E, with 5 calves per treatment in a 14-d study. Calves were fed pasteurized whole milk (CD) or fractionated colostrum replacer (CR) at birth (d 0) and injected with vitamins according to treatment group. From d 1 through d 14 of the study, all calves were fed pasteurized whole milk (PWM) supplemented with vitamins as assigned. All calves were inoculated with Mycobacterium avium ssp. paratuberculosis on d 1 and 3 of age. Calves fed CR acquired IgG1 and haptoglobin in serum within 24 h of birth, whereas CD calves did not. The CR-fed calves were 2.5 times less likely to develop scours, and CR calves supplemented with vitamins D3 and E also demonstrated a decreased incidence of scours. Serum vitamin levels of A, D, and E increased within treatment group by d 7 and 14 of the study. Interestingly, synergistic effects of supplemental vitamins A, D3, and E on serum 25-(OH)-vitamin D were observed at d 7, resulting in higher levels than in calves administered vitamin D only. Further, vitamin D3 deficiency was observed in CD and CR calves fed a basal diet of pasteurized whole milk and no supplemental vitamins. Colonization of tissues with Mycobacterium avium ssp. paratuberculosis was negligible and was not affected by colostrum feeding or vitamin supplementation. Results demonstrated passive transfer of haptoglobin to neonatal calves, and potential health benefits of supplemental vitamins D3 and E to calves fed pasteurized whole milk., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) causes Johne's disease, a chronic enteric infection in ruminants with severe economic impact on the dairy industry in the USA and worldwide. Currently, available vaccines have limited protective efficacy against disease progression and does not prevent spread of the infection among animals. Because of their ability to elicit wide-spectrum immune responses, we adopted a live-attenuated vaccine approach based on a sigH knock-out strain of M. paratuberculosis (ΔsigH). Earlier analysis of the ΔsigH mutant in mice indicated their inadequate ability to colonize host tissues, unlike the isogenic wild-type strain, validating the role of this sigma factor in M. paratuberculosis virulence. In the present study, we evaluated the performance of the ΔsigH mutant compared to inactivated vaccine constructs in a vaccine/challenge model of murine paratuberculosis. The presented analysis indicated that ΔsigH mutant with or without QuilA adjuvant is capable of eliciting strong immune responses (such as interferon gamma-γ, IFN-γ) suggesting their immunogenicity and ability to potentially initiate effective vaccine-induced immunity. Following a challenge with virulent strains of M. paratuberculosis, ΔsigH conferred protective immunity as indicated by the reduced bacterial burden accompanied with reduced lesions in main body organs (liver, spleen and intestine) usually infected with M. paratuberculosis. More importantly, our data indicated better ability of the ΔsigH vaccine to confer protection compared to the inactivated vaccine constructs even with the presence of oil-adjuvant. Overall, our approach provides a rational basis for using live-attenuated mutant strains to develop improved vaccines that elicit robust immunity against this chronic infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Five calves were inoculated orally at 2 weeks of age with a dose of 5 × 10(9) colony-forming units of Mycobacterium avium subspecies paratuberculosis (MAP) on 2 consecutive days. Two calves developed clinical Johne's disease at 12 and 16 months of age after being consistently positive for MAP on fecal culture and antibody enzyme-linked immunosorbent assay (ELISA), starting 2 to 3 weeks and 4 to 5 months after inoculation, respectively.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in cattle, has significant impacts on the livestock industry and has been implicated in the etiology of Crohn's disease. Macrophages play a key role in JD pathogenesis, which is driven by the manipulation of host immune mechanisms by MAP. A change in the macrophage microenvironment due to pathogenic or host-derived stimuli can lead to classical (M1) or alternative (M2) polarization of macrophages. In addition, prior exposure to antigenic stimuli has been reported to alter the response of macrophages to subsequent stimuli. However, macrophage polarization in response to MAP exposure and its possible implications have not been previously addressed. In this study, we have comprehensively examined monocyte/macrophage polarization and responsiveness to antigens from MAP-exposed and unexposed animals. At 3 years post-exposure, there was a heterogeneous macrophage activation pattern characterized by both classical and alternate phenotypes. Moreover, subsequent exposure of macrophages from MAP-exposed cattle to antigens from MAP and other mycobacterial species led to significant variation in the production of nitric oxide, interleukin-10 and tumour necrosis factor α. These results indicate the previously unreported possibility of changes in the activation state and responsiveness of circulating monocytes/macrophages from MAP-exposed cattle., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rabbits are susceptible to infection by Mycobacterium avium subspecies paratuberculosis (MAP) in both wildlife and experimental conditions. Under the hypotheses that nutrient balance might influence the establishment of infection, we designed an experiment where MAP intestinal colonization was assessed under three dietary regimens: high fiber, high protein, and regular diet in New Zealand white rabbits submitted to oral challenge with MAP. Lowest weight gain (F=5.17, p=0.024), higher tissue culture positivity rates (X(2)=7.43, p=0.024) and especially extended MAP-compatible lesions (F=5.78, p=0.017) were detected in the regular diet. Taken altogether, results indicate that paratuberculosis infection was achieved affecting mostly regular diet animals and showing that dietary changes may modulate the course of the infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cell-mediated immunity has been exploited historically in the diagnosis of mycobacterial diseases through elicitation of a delayed-type hypersensitivity (DTH) reaction following intradermal injection of an antigen. Here we describe the histopathological features of the cutaneous DTH reaction and its association with intestinal pathology and systemic immune responses in sheep with Mycobacterium avium subspecies paratuberculosis (MAP) infection. A mixed mononuclear cellular infiltrate dominated the DTH reaction and was present in perivascular and periadnexal patterns. Multiple multinucleate giant cells were present in the cellular infiltrate in one sheep while plasma cells were an obvious feature in six others. Sheep with paucibacillary intestinal lesions had the greatest degrees of cutaneous induration, more severe cellular infiltration in DTH lesions and high systemic interferon (IFN)-γ production. In contrast, sheep with multibacillary intestinal lesions, and particularly those with dissemination of MAP to extra-intestinal tissues, had minimal cutaneous induration, nil to mild cellular infiltration in DTH lesions and high serum anti-MAP antibody levels. Systemic IFN-γ production generally was augmented following skin sensitization. In general, the gross and histopathological features of the cutaneous DTH response matched the stage of paratuberculosis reflected by intestinal pathology and systemic measures of humoral and cellular immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFβ. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.

Mycobacterium avium ssp. paratuberculosis (MAP) is an infection of the ruminant intestine. In cows, a long subclinical phase with no or low intermittent shedding precedes the clinical phase with high shedding. It is generally considered that an adaptive cell-mediated immune response controls the infection during the subclinical phase, followed by unprotective antibodies later in life. Based on recent observations, we challenge the importance of adaptive immunity and instead suggest a role of the structural organization of infected macrophages in localized granulomatous lesions. We investigated this hypothesis by mathematical modelling. Our first model describes infection in a villus, assuming a constant lesion volume. This model shows the existence of two threshold parameters, the MAP reproduction ratio R MAP determining if a lesion can develop, and the macrophage replacement ratio R MF determining if recruitment of macrophages is sufficient for unlimited growth. We show that changes in R MF during a cow's life - i.e. changes in the innate immune response - can cause intermittent shedding. Our second model describes infection in a granuloma, assuming a growing lesion volume. This model confirms the results of the villus model, and can explain early slow granuloma development: small granulomas grow slower because bacteria leave the granuloma quickly through the relatively large surface area. In conclusion, our models show that the long subclinical period of MAP infection can result from the structural organization of the infection in granulomatous lesions with an important role for innate rather than adaptive immunity. It thus provides a reasonable hypothesis that needs further investigation.

Johne's disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), occurs in domestic and wild animals worldwide, causing a significant economic loss to livestock industries. After a prolonged incubation time, infected cattle shed MAP bacilli into feces and spread the disease to an uninfected animal population. It is largely unknown how (or whether) the interplay between the pathogen and the host immunity determines timing of shedding after the long incubation time. Such information would provide an understanding of pathogenesis in individual animals and the epidemiology of MAP infection in animal populations. In this review, we summarize current knowledge of bovine Johne's disease pathology, pathogenesis, immunology and genetics. We discuss knowledge gaps that direly need to be addressed to provide a science-based approach to diagnostics and (immuno)prophylaxis. These knowledge gaps are related to anatomical/clinical manifestation of MAP invasion, interaction of bacteria with phagocytes, granuloma formation, shedding, establishment and kinetics of adaptive immune responses in the pathogenesis of the disease. These topics are discussed at the molecular, cellular and tissue levels with special attention to the within host dynamics including the temporal and the spatial context relevant for the various host-pathogen interactions.

Following an outbreak of Johne's disease on an elk farm in northern Alberta, Canada, fecal culture, fecal polymerase chain reaction (PCR), and serum enzyme-linked immunosorbent assay (ELISA) tests were performed on individual animals. The magnitude of the outbreak is described and the challenges associated with poor test agreement, as well as herd management options, are discussed.

Background: Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) is difficult to control due to a long phase of clinically non-apparent (latent) infection for which sensitive diagnostics are lacking. A defined animal model for this phase of the infection can help to investigate host-MAP interactions in apparently healthy animals and identify surrogate markers for disease progress and might also serve as challenge model for vaccines. To establish such a model in goats, different age at inoculation and doses of oral inoculum of MAP were compared. Clinical signs, faecal shedding as well as MAP-specific antibody, IFN-γ and IL-10 responses were used for in vivo monitoring. At necropsy, about one year after inoculation (pi), pathomorphological findings and bacterial organ burden (BOB) were scored., Results: MAP infection manifested in 26/27 inoculated animals irrespective of age at inoculation and dose. Clinical signs developed in three goats. Faecal shedding, IFN-γ and antibody responses emerged 6, 10-14 and 14 wpi, respectively, and continued with large inter-individual variation. One year pi, lesions were detected in 26 and MAP was cultured from tissues of 23 goats. Positive animals subdivided in those with high and low overall BOB. Intestinal findings resembled paucibacillary lesions in 23 and multibacillary in 4 goats. Caseous and calcified granulomas predominated in intestinal LNN. BOB and lesion score corresponded well in intestinal mucosa and oGALT but not in intestinal LNN., Conclusions: A defined experimental infection model for the clinically non-apparent phase of paratuberculosis was established in goats as suitable basis for future studies.

The development of lesions after infection with Mycobacterium avium subsp paratuberculosis (MAP) was examined in an experimental infection model. Goat kids were orally inoculated 10 times with 10 mg bacterial wet mass of MAP (total dose 2.6 × 10(8) colony-forming units). Six to 7 inoculated goats and 3 controls were autopsied 3, 6, 9, and 12 months postinoculation (mpi), lesions were documented, and samples were collected for histology, immunohistochemistry (IHC), and bacterial culture. Twenty-five of the 26 inoculated goats did not develop clinical signs. Macroscopic lesions were detected in 3 of the 7 inoculated goats as soon as 3 mpi. Jejunal Peyer's patches (JPPs) were thickened and had ulcerated surfaces and circumscribed serositis. Characteristic granulomatous infiltrates were seen in all goats in gut-associated lymphoid tissues (GALTs), especially JPPs and lymphoid tissue at the ileocecal valve and in intestinal lymph nodes. Granulomatous intestinal infiltrates not associated with GALT were seen beginning at 6 mpi and with increasing frequency thereafter. Interindividual differences in lesions were most pronounced at 12 mpi, varying from mild focal paucibacillary to severe diffuse multibacillary patterns. Bacterial culture of MAP confirmed the IHC findings but was more sensitive and revealed widespread dissemination at 3 and 12 mpi. Granulomatous arteritis was found in intestinal submucosa of several goats. This may contribute to the spreading of MAP to the intestinal wall and possibly systemically. The different lesions observed during the clinically inapparent period of paratuberculosis are most likely indicators for the later progression of infection and development of clinical disease., (© The Author(s) 2014.)