Johne’s disease (JD) is a life-threatening gastrointestinal disease affecting ruminants, which causes crucial economical losses globally. This ailment is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious intracellular pathogen that belongs to the Mycobacteriaceae family. This acid-fast, hard-to-detect bacterium can resist milk pasteurization and be conveyed to dairy product consumers. Many studies have emphasized the zoonotic nature of MAP, suggesting an association between MAP and some gastroenteric conditions such as Crohn’s disease in humans. This underlines the importance of utilizing efficient pasteurization alongside a state-of-the-art diagnostic system in order to minimize the possible ways this pathogen can be conveyed to humans. Until now, no confirmatory MAP screening technique has been developed that can reveal the stages of JD in infected animals. This is partially due to the lack of an efficient gold-standard reference method that can properly evaluate the performance of diagnostic assays. Therefore, the following research aimed to compare the merits of qPCR and ELISA assessments of milk for the detection of MAP in a total of 201 Sardinian unpasteurized sheep milk samples including 73 bulk tank milk (BTM) and 128 individual samples from a MAP-infected flock (MIF) applying various reference models. Accordingly, milk qPCR and ELISA assessments, together and individually, were used as reference models in the herd-level study, while serum ELISA and fecal PCR were similarly (together and in isolation) considered as the gold standards in the individual-level diagnosis. This study showed that the type of gold-standard test affects the sensitivity and specificity of milk qPCR and ELISA significantly. At the individual level in the MAP-infected flock, serum ELISA in isolation and together with fecal PCR were recognized as the best references; however, the best correlation was seen between milk and serum ELISA (p < 0.0001). Regarding the detection of MAP in BTM, qPCR IS900 was recognized as the most sensitive and specific diagnostic test (p < 0.0001) for monitoring the MAP shedders and animals with clinically developed symptoms within herds, under the condition that both milk qPCR and milk ELISA tests formed a binary reference model. The BTM analyses (qPCR and ELISA) revealed that MAP positivity has a seasonal pattern. This hypothesis was proven through a longitudinal study on 14 sheep herds. Full article

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. Full article

Tuberculosis remains a major public health problem. Conventional tests are inadequate to distinguish between active tuberculosis (ATB) and latent tuberculosis infection (LTBI). We measured antibody responses to Mycobacterium tuberculosis antigens (Mycobacterium tuberculosis chorismate mutase (TBCM), antigen 85B (Ag85B), early secreted antigen-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in ATB, LTBI, and non-infected (NI) individuals. Serum immunoglobulin G (IgG) and immunoglobulin A (IgA) levels were measured and the QuantiFERON-TB Gold In-Tube assay was used to diagnose LTBI. IgG levels against TBCM were significantly higher in LTBI than NI subjects. IgG and IgA levels against Ag85B and IgG levels against CFP-10 were significantly higher in ATB, followed by LTBI, and then NI. When the ATB group was subdivided, IgG levels against Ag85B and CFP-10 were significantly higher in each subgroup compared with those in LTBI and NI groups. Positive correlation trends between interferon-gamma and IgG levels against Ag85B, TBCM, and CFP-10 and IgA levels against Ag85B in LTBI and NI subjects were observed. Age- and sex-adjusted models showed that IgG against TBCM and CFP-10 was independently related to LTBI diagnosis, and IgG against Ag85B was independently related to the diagnosis of ATB and could distinguish between LTBI and ATB. Overall, IgG antibody responses to TBCM, Ag85B, and CFP-10 can discriminate among ATB, LTBI, and NI groups. Full article

Interleukin 2 (IL-2) is considered a key player in exacerbating multiple sclerosis (MS). Therapies targeting its receptor have been developed; however, a resolution of the disease and side effects are still an issue of concern. The involvement of other factors, such as Mycobacterium avium subspecies paratuberculosis (MAP) and envelope protein derived from human endogenous retrovirus type W (HERV-Wenv), in MS pathogenesis has been recently suggested. Here, we investigated the levels of antibodies (Abs) directed against IL-2 and HERV-Wenv in 108 MS patients, 34 patients affected by neuromyelitis optica spectrum disorder (NMOSD), and 137 healthy controls (HCs). Our results show increased levels of Abs specific to IL-2 and HERV-Wenv-su antigens in MS vs. HCs (p < 0.0001 for IL-2, p = 0.0004 for HERV-Wenv) and significantly decreased levels in NMOSD vs. MS. The assessment of different 12-month-long therapies on Abs against IL-2, HERV-Wenv, and MAP lipoarabinomannan (LAM) demonstrated the strongest effect on anti-LAM Abs (p = 0.018), a slight reduction of anti-IL-2 Abs, and small variations for anti-HERV-Wenv Abs. These results highlight the conclusion that the impact of therapy is more correlated with selected epitopes than with the therapeutic agent. Screening for anti-IL-2 and anti-HERV-Wenv Abs has a potential as additional future practice to distinguish between symptomatically similar MS and NMOSD. Full article

Even though anti-TNF therapy significantly improves the rates of remission in inflammatory bowel disease (IBD) patients, there is a noticeable subgroup of patients who do not respond to treatment. Dysbiosis emerges as a key factor in IBD pathogenesis. The aim of the present study is to profile changes in the gut microbiome and transcriptome before and after administration of the anti-TNF agent Infliximab (IFX) and investigate their potential to predict patient response to IFX at baseline. Mucosal biopsy samples from 20 IBD patients and nine healthy controls (HC) were examined for differences in microbiota composition (16S rRNA gene sequencing) and mucosal gene expression (RT-qPCR) at baseline and upon completion of IFX treatment, accordingly, via an in silico pipeline. Significant differences in microbiota composition were found between the IBD and HC groups. Several bacterial genera, which were found only in IBD patients and not HC, had their populations dramatically reduced after anti-TNF treatment regardless of response. Alpha and beta diversity metrics showed significant differences between our study groups. Correlation analysis revealed six microbial genera associated with differential expression of inflammation-associated genes in IFX treatment responders at baseline. This study shows that IFX treatment has a notable impact on both the gut microbial composition and the inflamed tissue transcriptome in IBD patients. Importantly, our results identify enterotypes that correlate with transcriptome changes and help differentiate IFX responders versus non-responders at baseline, suggesting that, in combination, these signatures can be an effective tool to predict anti-TNF response. Full article

Prospective trials of anti-mycobacterial antibiotic therapy (AMAT) have proven efficacious in Crohn’s disease (CD) but use as first-line treatment in CD has not been evaluated. This paper reports the outcomes of patients with CD treated with first-line AMAT. This paper consists of a case series of treatment-naïve CD patients who received AMAT as first-line treatment between 2007 and 2014 at a single center. AMAT treatment consisted of rifabutin, clofazimine and clarithromycin, plus either ciprofloxacin, metronidazole or ethambutol. Symptoms, inflammatory blood markers, colonoscopy and histology results, in addition to, the Crohn’s Disease Activity Index (CDAI) were tabulated from patients’ clinical records, and descriptive statistics were conducted. A Wilcoxon signed-rank test assessed the difference in CDAI scores before and while on AMAT. The statistical significance was set at 5%. Clinical remission (CDAI < 150) with rapid improvement in clinical symptoms and inflammatory markers was seen in all eight patients receiving AMAT as sole therapy by 6 weeks. In all eight patients, the median CDAI score decreased significantly, from 289 prior to treatment to 62 at the 12-month follow-up (p < 0.001). Follow-up colonoscopies showed healing of CD ulcers, no visible mucosal inflammation, restoration of normal vascular patterns and complete mucosal healing on histology samples. AMAT as first-line therapy demonstrated a rapid improvement of Crohn’s disease (not previously seen when used as second-line therapy). Full article

Mycobacterial heat shock protein 65 gene (Hsp65) has been widely used for classification of Mycobacterial species, and detection of Mycobacterial genes by molecular methods and has proven useful in identification of Mycobacterial infection in various clinical conditions. Circulating antibody against Mycobacterial hsp65 has been found in many clinical diseases including autoimmune diseases (Crohn’s disease, lupus erythematosus, multiple sclerosis, diabetes, etc.), atherosclerosis and cancers. The prevalence of anti-Hsp65 antibody in the normal healthy population is unknown. We determined the blood levels of antibody against Mycobacterial hsp65 in the normal population represented by 288 blood donors of the American Red Cross and tested the blood of 109 patients with Crohn’s disease and 28 patients with Sjogren’s syndrome for comparison. The seroprevalence of anti-Hsp65 IgG in the normal population of Red Cross donors was 2.8% (8 of 288 positive). The Hsp65 antibody levels were significantly elevated in patients with Crohn’s disease and Sjogren’s syndrome. The prevalence of Hsp65 antibody in Crohn’s disease patients was 67.9% (74 of 109 patients), and 85.7% for Sjogren’s patients (24 of 28 patients). Our data indicate that anti-Hsp65 antibody is rare in the normal population, but frequent in chronic diseases. The presence of circulating Hsp65 antibody reflects an abnormal immune (adaptive) response to Mycobacterial exposure in patients with chronic diseases, thus differentiating the patients with chronic diseases from those clinical mimics. Full article

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. Full article

Environmental factors such as bacterial infections may play an important role in the development of autoimmune diseases. Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate pathogen of ruminants able to use the host’s cholesterol for survival into macrophages and has been associated with multiple sclerosis (MS), type 1 diabetes (T1DM) and rheumatoid arthritis (RA) through a molecular mimicry mechanism. Here, we aimed at investigating the correlation between humoral reactivity against MAP and serum lipoprotein levels in subjects at T1DM risk (rT1DM) grouped by geographical background and in patients affected by MS or RA. Our results showed significant differences in HDL, LDL/VLDL and Total Cholesterol (TC) levels between patients and healthy controls (p < 0.0001). Patients positive to anti-MAP Abs (MAP+) had lower HDL levels in comparison with Abs negative (MAP-) subjects, while opposite trends were found for LDL/VLDL concentrations (p < 0.05). TC levels varied between MAP+ and MAP- patients in all three assessed diseases. These findings suggest the implication of anti-MAP Abs in fluctuations of lipoprotein levels highlighting a possible link with cardiovascular disease. Further studies will be needed to confirm these results in larger groups. Full article

Bacille Calmette–Guerin (BCG) vaccination is widely practiced around the world to protect against the mycobacterial infection tuberculosis. BCG is also effective against the pathogenic mycobacteria that cause leprosy and Buruli’s ulcer. BCG is part of the standard of care for bladder cancer where, when given as an intravesicular irrigant, BCG acts as an immunomodulating agent and lessens the risk of recurrence. Mycobacterium avium ss. paratuberculosis (MAP) causes a fatal enteritis of ruminant animals and is the putative cause of Crohn’s disease of humans. MAP has been associated with an increasingly long list of inflammatory/autoimmune diseases: Crohn's, sarcoidosis, Blau syndrome, Hashimoto’s thyroiditis, autoimmune diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis, lupus and Parkinson’s disease. Epidemiologic evidence points to BCG providing a “heterologous” protective effect on assorted autoimmune diseases; studies using BCG vaccination for T1D and MS have shown benefit in these diseases. This article proposes that the positive response to BCG in T1D and MS is due to a mitigating action of BCG upon MAP. Other autoimmune diseases, having a concomitant genetic risk for mycobacterial infection as well as cross-reacting antibodies against mycobacterial heat shock protein 65 (HSP65), could reasonably be considered to respond to BCG vaccination. The rare autoimmune disease, relapsing polychondritis, is one such disease and is offered as an example. Recent studies suggesting a protective role for BCG in Alzheimer’s disease are also explored. BCG-induced energy shift from oxidative phosphorylation to aerobic glycolysis provides the immunomodulating boost to the immune response and also mitigates mycobacterial infection—this cellular mechanism unifies the impact of BCG on the disparate diseases of this article. Full article

Increasingly, Johne’s disease of ruminants and human Crohn’s disease are regarded as the same infectious disease: paratuberculosis. Mycobacterium avium ss. paratuberculosis (MAP) is the cause of Johne’s and is the most commonly linked infectious cause of Crohn’s disease. Humans are broadly exposed to MAP in dairy products and in the environment. MAP has been found within granulomas such as Crohn’s disease and can stimulate autoantibodies in diseases such as type 1 diabetes (T1D) and Hashimoto’s thyroiditis. Moreover, beyond Crohn’s and T1D, MAP is increasingly associated with a host of autoimmune diseases. This article suggests near equivalency between paucibacillary Johne’s disease of ruminant animals and human Crohn’s disease and implicates MAP zoonosis beyond Crohn’s disease to include T1D. Full article