Mycobacterium Avium Paratuberculosis Infection Research Articles

The role of Mce proteins in Mycobacterium avium paratuberculosis infection

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an infection in the host via the small intestine. This requires the bacterium to adhere to, and be internalised by, cells of the intestinal tract. The effector molecules expressed by MAP for this purpose remain to be fully identified and understood. Mammalian cell entry (mce) proteins have been shown to enable other Mycobacterial species to attach to and invade host epithelial cells. Here, we have expressed Mce1A, Mce1D, Mce3C and Mce4A proteins derived from MAP on the surface of a non-invasive Escherichia coli to characterise their role in the initial interaction between MAP and the host. To this end, expression of mce1A was found to significantly increase the ability of the E. coli to attach and survive intracellularly in human monocyte-like THP-1 cells, whereas expression of mce1D was found to significantly increase attachment and invasion of E. coli to bovine epithelial cell-like MDBK cells, implying cell-type specificity. Furthermore, expression of Mce1A and Mce1D on the surface of a previously non-invasive E. coli enhanced the ability of the bacterium to infect 3D bovine basal-out enteroids. Together, our data contributes to our understanding of the effector molecules utilised by MAP in the initial interaction with the host, and may provide potential targets for therapeutic intervention.

Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Evaluation of ELISA in raw milk for detection of antibodies to Mycobacterium avium subsp. paratuberculosis in dairy herd.

Paratuberculosis (PTBC) is a chronic intestinal disease of animals caused by Mycobacterium avium paratuberculosis (MAP). MAP infection is diagnosed through indirect tests based on the immune response. The aims of this study were to compare the performance of two milk ELISA for the diagnosis of PTBC and to assess the bulk tank milk (BTM) MAP exposure in dairy cattle in Argentina. A total of 357 fecal, serum, and milk samples were collected. The fecal samples were processed by culture for MAP isolation, while both, serum and milk samples were used for the detection of antibodies by two different ELISA tests, "in-house" and commercial kit. MAP was isolated in 3.9% of fecal samples. For milk ELISA, poor concordances were obtained. Optimized cut-off points were calculated. The highest sensitivity and specificity values (64% and 80% respectively) were obtained with the combination of MAP isolation and commercial milk ELISA. The results indicate that the combination of different techniques to identify of dairy cattle infected with MAP increases the efficiency of diagnosis. In addition, BTM samples (n=98) were evaluated to determine herd status using the commercial kit during two seasons, identifying 33.3% of positive samples in autumn and 35.4% in spring.

Folate and Vitamin B12 Deficiency Exacerbate Inflammation during Mycobacterium avium paratuberculosis (MAP) Infection.

Folate and vitamin B12 deficiency is highly prevalent among Crohn's disease (CD) patients. Furthermore, CD pathology can be mediated by Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, the direct effect of folate (B9) and cobalamin (B12) deficiency during MAP infection remains uncharacterized. This study investigates how folate and B12 deficiency impedes macrophage apoptosis and exacerbates the inflammation in macrophages infected with MAP isolated from CD patients. Accordingly, we measured folate and B12 in ex vivo plasma samples collected from CD patients with or without MAP infection (N = 35 per group). We also measured the expression of the pro-inflammatory cytokines IL-1β and TNF-α, cellular apoptosis and viability markers, and bacterial viability in MAP-infected macrophages cultured in folate and B12 deficient media. We determined that MAP-positive CD patients have significantly lower plasma folate and B12 in comparison to MAP-negative CD patients [414.48 ± 94.60 pg/mL vs. 512.86 ± 129.12 pg/mL, respectively]. We further show that pro-inflammatory cytokines IL-1β and TNF-α are significantly upregulated during folate and vitamin B12 deprivation following MAP infection by several folds, while supplementation significantly reduces their expression by several folds. Additionally, depletion of folate, B12, and folate/B12 following MAP infection, led to decreased macrophage apoptosis from 1.83 ± 0.40-fold to 1.04 ± 0.08, 0.64 ± 0.12, and 0.45 ± 0.07 in folate-low, B12-low, and folate/B12-low cells, respectively. By contrast, folate and folate/B12 supplementation resulted in 3.38 ± 0.70 and 2.58 ± 0.14-fold increases in infected macrophages. Interestingly, changes in overall macrophage viability were only observed in folate-high, folate/B12-high, and folate/B12-low media, with 0.80 ± 0.05, 0.82 ± 0.02, and 0.91 ± 0.04-fold changes, respectively. Incubation of Caco-2 intestinal epithelial monolayers with supernatant from infected macrophages revealed that folate/B12 deficiency led to increased LDH release independent of oxidative stress. Overall, our results indicate that folate and B12 are key vitamins affecting cell survival and inflammation during MAP infection.

Multiplex qPCR for differentiation of Mycobacterium avium subspecies paratuberculosis in active and passive infection of goats.

Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 1011 to 4.14 × 104 for IS900 (FAM), 1.27 × 1011 to 1.27 × 104 for IS900 mRNA (Cy5), and 3.68 × 1010 to 3.68 × 104 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 104, 1.27 × 104, and 3.68 × 104 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: • Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. • ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. • The LOD was computed using copy numbers with 4.14 × 104 and 3.68 × 104 copies for IS900 and ModD, respectively.

MiR-146a rs2910164 G\u2009>\u2009C polymorphism modulates Notch-1/IL-6 signaling during infection: a possible risk factor for Crohn\u2019s disease

BackgroundMiR-146a, an effector mediator, targets Notch-1 and regulates the innate and adaptive immune systems response. Recently, we reported that Notch-1 signaling plays a key role in macrophage polarization and response during infection. We employed Mycobacterium avium paratuberculosis (MAP) infection in Crohn’s disease (CD) as a model to demonstrate the role of Notch-1/IL-6 signaling on MCL-1 based apoptosis and intracellular MAP infection and persistence. This study was designed to investigate the impact of polymorphisms in miR146a on the immune response and infection in our MAP-CD model.MethodsWe determined the incidence of miR-146a rs2910164 G > C in 42 blood samples from clinical CD patients and controls. We also measured the effect of rs2910164 on expression of Notch-1 and IL-6, and plasma IL-6 protein levels in our study group. Finally, we analyzed the blood samples for MAP DNA and studied any correlation with miR-146a polymorphism. Samples were analyzed for statistical significance using unpaired tow-tailed t-test, unpaired two-tailed z-score and odds ratio. P < 0.05 considered significant.ResultsMiR-146a rs2910164 GC was detected at a higher incidence in CD (52.6%) compared to healthy controls (21.7%) rs2910164 GC Heterozygous polymorphism upregulated Notch-1 and IL-6, by 0.9 and 1.7-fold, respectively. As expected, MAP infection was detected more in CD samples (63%) compared to healthy controls (9%). Surprisingly, MAP infection was detected at a higher rate in samples with rs2910164 GC (67%) compared to samples with normal genotype (33%).ConclusionsThe data clearly associates miR-146a rs2910164 GC with an overactive immune response and increases the risk to acquire infection. The study is even more relevant now in our efforts to understand susceptibility to SARS-CoV-2 infection and the development of COVID-19. This study suggests that genetic variations among COVID-19 patients may predict who is at a higher risk of acquiring infection, developing exacerbating symptoms, and possibly death. A high scale study with more clinical samples from different disease groups is planned.

Notch-1 Signaling Modulates Macrophage Polarization and Immune Defense against Mycobacterium avium paratuberculosis Infection in Inflammatory Diseases.

Despite the extensive research on Notch signaling involvement in inflammation, its specific role in macrophage response in autoimmune disease and defense mechanisms against bacterial infection, such as Mycobacterium avium paratuberculosis (MAP), remains unknown. In this study, we investigated the molecular role of Notch-1 signaling in the macrophage response during MAP infection. In particular, we measured the in vitro effect of MAP on Notch-1 signaling and downstream influence on interleukin (IL)-6 and myeloid cell leukemia sequence-1 (MCL-1) and consequent cellular apoptosis, MAP viability, and macrophage polarization. Overall, the data show significant upregulation in Notch-1, IL-6, and MCL-1 in MAP-infected macrophages, parallel with a decrease in apoptosis and elevated pro-inflammatory response in these infected cells. On the contrary, blocking Notch signaling with γ-secretase inhibitor (DAPT) decreased MAP survival and burden, increased apoptosis, and diminished the pro-inflammatory response. In particular, the treatment of infected macrophages with DAPT shifted macrophage polarization toward M2 anti-inflammatory phenotypic response. The outcome of this study clearly demonstrates the critical role of Notch signaling in macrophage response during infection. We conclude that MAP infection in macrophages activates Notch-1 signaling and downstream influence on IL-6 which hijack MCL-1 dependent inhibition of apoptosis leading to its chronic persistence, and further inflammation. This study supports Notch-1 signaling as a therapeutic target to combat infection in autoimmune diseases such as Crohn’s disease and Rheumatoid Arthritis.

Notch‐1 signaling through MCL‐1 modulates macrophage polarization and immune defense against Mycobacterium avium paratuberculosis infection in autoimmune disease

We previously reported that IL‐6 plays a major role in the survival of Mycobacterium avium paratuberculosis (MAP) in infected macrophages. MAP has been associated with Crohn’s disease (CD) and Rheumatoid Arthritis (RA) pathogenesis. The role of Notch‐1 signaling in macrophages during intracellular infection such as MAP is not clearly understood. We hypothesis that IL‐6 expression in macrophages is induced by Notch‐1 signaling which inhibits apoptosis through Myeloid cell leukemia sequence‐1 (MCL‐1), which ultimately induce persistence of chronic infection. In this study, we measured the in vitro effect of MAP infection on Notch‐1 signaling and downstream influence on IL‐6 and MCL‐1. Specifically, we evaluated the role of Notch‐1 signaling in apoptosis and consequent MAP viability in infected macrophages and its effect on macrophage polarization and inflammatory response. Overall, the data showed that MAP infection upregulated Notch‐1, IL‐6, and MCL‐1 by (1.2, 1.8 and 4.2 fold expression, respectively) in activated THP‐1 compared to uninfected macrophages. Interestingly, treatment of MAP‐infected macrophages with IL‐6 showed upregulation of Notch‐1 and MCL‐1, which suggest a role for Notch‐1 and IL‐6 in the upregulation of MCL‐1 and possibly regulation of apoptosis in MAP infected macrophages. Targeting Notch signaling by a gamma‐secretase inhibitor resulted in decreased expression of IL‐6 and MCL‐1 (1.38 ± 0.32 and 0.63 ± 0.034, respectively) in infected macrophages compared to untreated macrophages (2.7 ± 0.25 and 2.0 ± 0.21, respectively). Moreover, gamma‐secretase inhibitor decreased MAP viability by 1.3 log CFU/ml. On the other hand, MAP modulates the macrophage polarization toward M1 macrophage polarization (M1/M2=2.20) compared uninfected macrophages (M1/M2=1.27), whereas the Notch inhibitor promotes M2 and diminish M1 macrophage polarization (M1/M2=0.76). Collectively, targeting Notch‐1 signaling will regulates the inflammatory response and enhances bacterial clearance in MAP infected macrophages. The study provides significant data that support the mechanistic role of Notch‐1 signaling in chronic infection in autoimmune disease.Support or Funding InformationThis work was funded, in part, by the Florida Legislative Grant.

Polymorphisms in TNF Receptor Superfamily 1B (TNFRSF1B:rs3397) are Linked to Mycobacterium avium paratuberculosis Infection and Osteoporosis in Rheumatoid Arthritis.

We previously discovered that single nucleotide polymorphisms (SNPs) in PTPN2/22 (T-cell negative-regulators) occur in 78% of rheumatoid arthritis (RA), along with Mycobacterium avium paratuberculosis (MAP) infection in 33% of patients. In Crohn’s disease, we reported that SNPs in TNFα and receptors (TNFRSF1A/TNFRSF1B) benefited intracellular MAP-survival, increased infection, and elevated inflammatory response mimicking the poor response to anti-TNFα treatment in some patients. Here, we studied the frequency and effects of SNPs in TNFα/TNFRSF1A/TNFRSF1B in RA including gene expression, MAP infection, and osteoporosis marker levels in blood (54 RA and 48 healthy controls). TNFα:rs1800629 (GA) was detected in 19/48 (40%) RA and 8/54 (15%) controls (p-value < 0.05, odds ratio (OR) = 3.6, 95% CI: 1.37–9.54). TNFRS1B:rs3397 (CT) was detected in 21/48 (44%) RA and 10/54 (19%) controls (p-value < 0.05, OR = 4.43, 95% CI: 1.73–11.33). In RA, rs3397 downregulated TNFRSF1B expression (CC > CT (0.34 ± 0.14) and CC > TT (0.27 ± 0.12)), compared to wildtype CC (0.51 ± 0.17), p-value < 0.05. MAP DNA was detected significantly in 17/48 (35.4%) RA compared to 11/54 (20.4%) controls (p-value < 0.05, OR = 2.14, 95% CI: 1.12–5.20). The average osteocalcin level was significantly lower (p-value < 0.05) in RA (2.70 ± 0.87 ng/mL), RA + MAP (0.60 ± 0.31 ng/mL), RA + TNFRSF1B:rs3397 (TT) (0.67 ± 0.35 ng/mL), compared to the healthy control (5.31 ± 1.39 ng/mL), and MAP-free RA (3.85 ± 1.31 ng/mL). Overall, rs3397 appears to downregulate TNFRSF1B, increase MAP infection, worsen inflammation, and cause osteocalcin deficiency and possibly osteoporosis in RA.

643 RHB-104, a Fixed-Dose, Oral Antibiotic Combination Against Mycobacterium Avium Paratuberculosis (MAP) Infection, Is Effective in Moderately to Severely Active Crohn's Disease

INTRODUCTION: MAP US was a global multicenter randomized trial comparing the efficacy and safety of RHB-104, a fixed-dose oral combination of clarithromycin, rifabutin and clofazimine against MAP infection, in moderately to severely active Crohn's disease (CD). We report evaluations of efficacy, symptom improvement and exposure-response by individual RHB-104 components. METHODS: Patients with active CD (CDAI ≥220 and ≤450) and failure with conventional therapies were randomized 1:1 to RHB-104 or placebo for up to 52 Wks. Concomitant corticosteroids, immunosuppressives (IS) and anti-TNF agents were permitted. The primary endpoint was clinical remission (CDAI &lt;150) at Wk 26. Secondary endpoints included clinical response at Wk 26 (CDAI decrease ≥100 points), early remission at Wk 16 and remission at Wk 52. A population pharmacokinetic (PK) and exposure-response analysis was performed based on PK assessments and remission at Wk 26; a subset of patients underwent colonoscopy at baseline and Wk 26. RESULTS: 331 subjects were randomized at 92 sites. The proportion achieving remission at Wk 26 (36.7% vs 23%, P = .007), remission at Wk 16 (42.2% vs 29.1%, P = .015) and response at Wk 26 (44% vs 30.9%, P = .017) were significantly greater with RHB-104 vs placebo. The superiority of RHB-104 was more pronounced in patients receiving RHB-104 with concomitant anti-TNF or IS treatment. Despite the small numbers (n = 35), a greater proportion of RHB-104 patients achieved endoscopic response by SES-CD 50 (28.6% vs 4.8%, P = .11). Differences in fecal calprotectin (FCP) increased over time (Figure 1); improvements in PRO-2 symptoms appeared by Wk 4 and reached significance by Wk 16 (Figure 2). A significantly greater proportion of patients receiving RHB-104 achieved clinical remission with at least a 50% reduction from baseline in either FCP or CRP concentration at Wk 16 (25.9% vs 9.7%, P = .0002), Wk 26 (21.1% vs 9.1%, P = .0003) and Wk 52 (16.9% vs 7.9%, P = .02). Exposure-response modeling demonstrated the combination of the individual components of RHB-104 contributed to the higher rate of remission compared to placebo and that the probability of achieving remission was most sensitive to clofazimine. CONCLUSION: RHB-104 demonstrated meaningful improvement in efficacy and biomarkers of active inflammation, with exposure-response for each drug component, early onset of response and benefit in patients with and without concomitant anti-TNF or IS therapy.

Crohn's disease-specific anti-CUZD1 pancreatic antibodies are absent in ruminants with paratuberculosis

Pancreatic autoantibodies (PABs) specifically recognizing GP2 and/or CUZD1 are present in more than 35% of patients with Crohn's disease (CrD). We have recently provided evidence of the presence of GP2-specific PABs in ruminants with paratuberculosis (ptb), a Mycobacterium avium paratuberculosis (MAP)-induced disease resembling CrD. To assess whether anti-CUZD1 antibodies are also present in ruminants with ptb. A total of 110 samples (73 cattle/37 sheep) were studied including 40 with ptb (24 cattle/16 sheep; 20 anti-GP2 antibody pos) and 70 without ptb (49 cattle/21 sheep; 10 anti-GP2 antibody pos). The samples were pre-characterized for anti-MAP and anti-GP2 antibodies by ELISA. Evidence of MAP was confirmed by PCR. Anti-CUZD1 antibody testing was performed by indirect immunofluorescence (IIF) based on transfected HEK293 cells expressing CUZD1. Anti-sheep or anti-cattle specific antisera were used as revealing antibodies. None of the ruminant sera had anti-CUZD1 antibodies by IIF testing at dilutions varying from 1/10 to 1/160. Methodological flaws were prevented by a series of tests. Control sera from anti-CUZD1 positive CrD samples have shown anti-CUZD1 antibody reactivity at various concentrations. Antibody reactivity to GP2-expressing HEK293 cells has confirmed the reactivity to GP2 in ruminant sera found positive for anti-GP2 antibodies by ELISA. The present study has found no evidence of anti-CUZD1 PABs in MAP-induced ptb. Our findings indicate that the induction of CUZD1-specific PABs is unrelated to MAP infection and that the mechanisms responsible for the loss of tolerance to GP2 and CUZD1 are probably quite distinct.

Paratuberculosis in European wild rabbits from the Iberian Peninsula

Of the non-ruminant wildlife species known to harbor Mycobacterium avium paratuberculosis (MAP), the rabbit ( Oryctolagus cuniculus) is thought to pose the greatest risk of transmission to cattle. We analyzed 80 hunter-harvested wild rabbits from a core study area in southern Spain, and sera from 157 wild rabbits sampled opportunistically on seven additional sites. Gross lesions compatible with paratuberculosis were observed in two of 80 necropsied rabbits. Histopathology revealed focal to diffuse multibacillary MAP-compatible lesions in 8 of 10 rabbits examined. Presence of MAP was confirmed in one rabbit with gross lesions by positive amplification curves for both IS900 and ISMAP02. However, no isolate was obtained from 47 samples by culture. We adapted an indirect ELISA for the detection of MAP antibodies. At the established cut-off of 0.5, 6 of 237 wild rabbit sera (2.5%) yielded a positive ELISA result. Antibodies were detected in rabbits from 3 of 8 sampling sites. Considering the increasing relevance of MAP infection for animal health, these results open a challenging field for future research.

Case-control Study

Mycobacterium avium paratuberculosis (MAP), the cause of Johne's disease (JD), is common in US dairy herds with over 68% of herds containing infected cattle. As there is no approved or effective treatment for JD, control depends on minimizing transmission of MAP to uninfected cattle. Early identification and removal of infected cattle would help minimize JD transmission. Unfortunately, because of the slow progression of JD, early identification of infected cattle is problematic due to poor sensitivity and specificity of currently available diagnostic tests in young, subclinically infected heifers. Moreover, it is unclear whether testing young heifers is cost-effective, given the constraints and costs of current tests, as well as the indefinite effects of subclinical MAP infection on individual cow productivity reported in the literature. Repeated exposure to high levels of MAP accelerates JD progression such that clinical signs are exhibited in relatively young cattle. Accordingly, these cattle should test positive for MAP at a younger age. Multiple studies have documented fecal shedding of MAP in naturally infected calves less than 24 months of age, but none have followed these calves into adulthood to see how they perform as mature cows. The objective of this study was to compare herd longevity, production performance, and adult MAP test status of cows testing positive for MAP &lt;24 months of age to age-matched herdmates.