Identification of loci associated with pathological outcomes in Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis using whole-genome sequence data.

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.

Detection of latent forms of Mycobacterium avium subsp. paratuberculosis infection using host biomarker-based ELISAs greatly improves paratuberculosis diagnostic sensitivity.

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.

Identification of microRNAs in bovine faeces and their potential as biomarkers of Johne's Disease.

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.

The association between detection of Mycobacterium avium subsp. paratuberculosis DNA in feces and histopathological classification.

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.

Divergent Antigen-Specific Cellular Immune Responses during Asymptomatic Subclinical and Clinical States of Disease in Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis.

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.

Evaluation of newly developed 'six recombinant secretary proteins based 'cocktail ELISA' and 'whole cell lysate' based 'indigenous ELISA' and tissue microscopy' with 'Gold standard' histo-pathology for the diagnosis of Johne's disease in slaughtered goats and buffaloes.

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.

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.

Phenotypes of macrophages present in the intestine are impacted by stage of disease in cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

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-1ß, IL-10, TGF-ß, 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.

Quantification of Macrophages and Mycobacterium avium Subsp. paratuberculosis in Bovine Intestinal Tissue During Different Stages of Johne's Disease.

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 paratuberculosis and Mycobacterium avium complex and related subspecies as causative agents of zoonotic and occupational diseases.

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.

Characterization of paucibacillary ileal lesions in sheep with subclinical active infection by Mycobacterium avium subsp. paratuberculosis.

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.

Relationship between the pathology of bovine intestinal tissue and current diagnostic tests for Johne's disease.

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.

Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves.

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.

Identification of the Apa protein secreted by Mycobacterium avium subsp. paratuberculosis as a novel fecal biomarker for Johne's disease in cattle.

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.

Effects of freezing on ability to detect Mycobacterium avium subsp. paratuberculosis from bovine tissues following culture.

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.

CLINICAL SIGNS, ANTEMORTEM DIAGNOSTICS, AND PATHOLOGICAL FINDINGS ASSOCIATED WITH MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS INFECTION IN MISHMI TAKIN ( BUDORCAS TAXICOLOR TAXICOLOR).

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.

Immunopathological changes and apparent recovery from infection revealed in cattle in an experimental model of Johne's disease using a lyophilised culture of Mycobacterium avium subspecies paratuberculosis.

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.

Gene expression profiles of immune-regulatory genes in whole blood of cattle with a subclinical infection of Mycobacterium avium subsp. paratuberculosis.

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/ß 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.

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis.

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.

Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease.

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 E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 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. PGE2 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, PGE2 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 PGE2 pathway in cattle. In contrast, downregulation of PGE2 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.