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The widespread chronic enteritis known as Paratuberculosis (PTB) or Johne's disease (JD) is caused by Mycobacterium avium subspecies paratuberculosis (MAP), posing a significant threat to global public health. Given the challenges associated with PTB or JD, the development and application of vaccines are potentially important for disease control. The aim of this study was to design a multi-epitope vaccine against MAP. A total of 198 MAP genomes were analyzed using pan-genome and reverse vaccinology approaches. B-cell and T-cell epitope analysis was performed on the selected promising cross-protective antigens followed by selection of epitopes with high antigenicity, no allergenicity, and no toxicity for the design of the vaccine. The designed vaccine was evaluated through molecular dynamics simulations, molecular docking, and immunological simulations. The results revealed the identification of five promising cross-protective antigens. In total, 10 B-cell epitopes, 10 HTL epitopes, and 9 CTL epitopes were selected for the design of the vaccine. Both the vaccine candidate and the vaccine-TLR4 complex demonstrated considerable stability in molecular dynamics simulations. Molecular docking studies confirmed that the vaccine candidate successfully interacted with TLR4. Immunological simulations showed an increase in both B-cell and T-cell populations after vaccination. Additionally, the vaccine candidate exhibited a codon adaptability index of 1.0 and a GC content of 53.64%, indicating strong potential for successful expression in Escherichia coli . This research developed a multi-epitope vaccine targeting MAP through pan-genomes and reverse vaccinology methods, offering innovative strategies for creating effective vaccines against MAP.

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.

Paratuberculosis (PTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (Map). Vaccination is one of the most cost-effective tools for PTB control, although alternative treatments like the probiotic Dietzia have been explored with promising results. Using a rabbit model, we investigated the association of immunological and microbiota profiles in Gut Associated Lymphoid Tissue (GALT) with the effects in protection induced by the administration of Dietzia spp., the commercial vaccine (Silirum ® ) and the combination of both. The treatment with the probiotic diminished inflammation, but failed to control Map burden, suggesting a detrimental effect. Rabbits treated with the probiotic presented the highest rates of tissue lesion extension, although the immunological profile was not suggestive of an inflammatory state. Map load in both vaccinated groups was similar indicating that both treatments are equally effective in eliminating the infection, suggesting the role of vaccination in eliminating the infection prevails over the immunomodulatory effects of the probiotic. There were slight variations in the presence of some taxonomic groups depending on the treatment, highlighting the complexity of microbial interactions and the need to optimise treatment combinations in the context of each disease and animal species.

We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (Map) strain K-10, the causative agent of Johne's disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon. In silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs suggest overall strong selection for a vast majority of these shared mycobacterial genes, with only 68 ORFs with a synonymous to nonsynonymous substitution ratio of >2. Comparative sequence analysis reveals several noteworthy features of the K-10 genome including: a relative paucity of the PE/PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory during Mtb infection; truncation in the EntE domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map; and Map-specific sequences that are likely to serve as potential targets for sensitive and specific molecular and immunologic diagnostic tests. Taken together, the availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology in Map and enables the development of new generations of diagnostic tests for bovine Johne's disease.

•Antigen presentation to CD4 and CD8 T cells by antigen presenting cells is blocked in the presence of antibody specific for either MHC I or MHC II.•Simultaneous CD4 and CD8 T cell cognate recognition of antigenic epitopes presented by antigen presenting cells is essential for development of CD8 cytotoxic T cells. Studies in cattle show CD8 cytotoxic T cells (CTL), with the ability to kill intracellular bacteria, develop following stimulation of monocyte-depleted peripheral blood mononuclear cells (mdPBMC) with antigen presenting cells (APC, i.e. conventional dendritic cells [cDC] and monocyte-derived DC [MoDC]) pulsed with MMP, a membrane protein from Mycobacterium avium subsp. paratuberculosis (Map) encoded by MAP2121c. CTL activity was diminished if CD4 T cells were depleted from mdPBMC before antigen (Ag) presentation by APC, suggesting simultaneous cognate recognition of MMP epitopes presented by MHC I and MHC II molecules to CD4 and CD8 T cells is essential for development of CTL activity. To explore this possibility, studies were conducted with mdPBMC cultures in the presence of monoclonal antibodies (mAbs) specific for MHC class I and MHC class II molecules. The CTL response of mdPBMC to MMP-pulsed APC was completely blocked in the presence of mAbs to both MHC I and II molecules and also blocked in the presence of mAbs to either MHC I or MHC II alone. The results demonstrate simultaneous cognate recognition of Ag by CD4 and CD8 T cells is essential for delivery of CD4 T cell help to CD8 T cells to elicit development of CTL.

•MVA85A boost did not improve the immune response induced by 6611 strain + IFA adjuvant.•Priming with 6611 + ISA201 and boosting with MVA85A elicited the highest immune response.•The protection provided by the 6611 + ISA201 vaccine is enhanced by the MVA85A boost. Vaccination is the best strategy to control Paratuberculosis (PTB), which is a significant disease in cattle and sheep. Previously we showed the humoral and cellular immune response induced by a novel vaccine candidate against PTB based on the Argentinian Mycobacterium avium subspecies paratuberculosis (Map) 6611 strain. To improve 6611 immunogenicity and efficacy, we evaluated this vaccine candidate in mice with two different adjuvants and a heterologous boost with a recombinant modified vaccinia Ankara virus (MVA) expressing the antigen 85A (MVA85A). We observed that boosting with MVA85A did not improve total IgG or specific isotypes in serum induced by one or two doses of 6611 formulated with incomplete Freund’s adjuvant (IFA). However, when 6611 was formulated with ISA201 adjuvant, MVA85A boost enhanced the production of IFNγ, Th1/Th17 cytokines (IL-2, TNF, IL-17A) and IL-6, IL-4 and IL-10. Also, this group showed the highest levels of IgG2b and IgG3 isotypes, both important for better protection against Map infection in the murine model. Finally, the heterologous scheme elicited the highest levels of protection after Map challenge (lowest CFU count and liver lesion score). In conclusion, our results encourage further evaluation of 6611 strain + ISA201 prime and MVA85A boost in bovines.

Mycobacterium avium subsp. paratuberculosis (MAP) primarily invades ruminants' small intestine via the Peyer's patches in the ileum and jejunum. Despite ongoing efforts to develop effective MAP vaccines, the effects of live-attenuated vaccines on mucosal immunity remain poorly understood. Previous studies indicate that the BacA oral vaccine confers localized protection against MAP in the ileum and ileocecal valve of calves, but not in the jejunum. This protection correlates with heightened levels of peripheral blood immune cells exhibiting pro-inflammatory and memory traits. This study aimed to evaluate immune responses induced by oral BacA vaccination in the ileum and jejunum Peyer's patches, comparing protection at both sites through mucosal immune cell profiling and RNA-seq transcriptome analyses. It represents the first exploration of mucosal immune responses in Peyer's patches following oral MAP vaccination. Oral BacA immunization increased CD4 + IFNγ+ and CD4 + TNFα+ cell frequencies, along with the T effector memory to T central memory cell ratio, in the ileum and jejunum of BacA-vaccinated animals challenged with wildtype MAP, compared to the infection control group challenged solely with wildtype MAP. Immune cells isolated from the ileum of vaccinated-challenged animals exhibited significant upregulation in IFNγ, IP-10, TNFα, IL-2, IL-15, and IL-17 expression upon restimulation compared to the uninfected control group, whereas minimal differences were observed in the jejunum under similar conditions. RNA-seq data further indicated a more robust host response in the ileum across all experimental groups. Gene ontology analyses revealed genes associated with increased phagocytic and apoptotic activities in the vaccinated-challenged group. Overall, the BacA oral vaccine's effectiveness appears to vary primarily due to differences in antigen-specific gene expression between the ileum and jejunum, with the ileum showing a more robust host response. Understanding these effects on young calves' mucosal immunity and how live vaccines modulate immune responses is crucial for advancing mucosal vaccine development against MAP.

Increasing evidence links a worldwide bacterial infection of cattle and other animal species by Mycobacterium avium ssp. paratuberculosis (MAP) to Crohn’s disease (CD). A large, FDA phase 2/3 controlled clinical trial of combination antimycobacterial antibiotic therapy for CD has been completed, and the report describing the trial is pending publication. The identification of MAP infection in CD patients will become increasingly important. Thus, it is desirable to develop MAP-based tests that accurately predict which CD patients have a MAP infection. A prospective, case-control laboratory test study of 199 subjects (61 CD patients and 138 non-CD controls) was performed using a panel of MAP antigens, including Hsp65, PknG, PtpA, CL1, and MAP IDEXX, which were measured under blind conditions in the plasma of the 199 subjects. Results showed that compared to any individual MAP antigen, combinations of antigens showed improved CD classification performance. For the Hsp65 antigen, the sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), correct classification (CC), and area under the curve (AUC) were 59.02%, 58.70%, 38.71%, 76.42%, 59.3% and 0.606, respectively. For the best combination of MAP antibodies (Hsp65 and PknG), the SEN, SPE, PPV, NPV, CC, and AUC were 59.02%, 60.87%, 40.00%, 77.06%, 60.30%, and 0.631, respectively. Further improvement of the CD classification performance was achieved by combining IFN-γ, IL-8, and IL-17 cytokines with antibodies against MAP antigens, yielding SEN, SPE, PPV, NPV, CC, and AUC of 62.3%, 62.32%, 42.22%, 78.9%, 62.31% and 0.708, respectively. Thus, combinations of antibodies against MAP antigens and cytokine levels yield better CD diagnostic predictive performance than any individual antibodies against MAP antigens.

Vaccination against paratuberculosis, before or after infection with Mycobacterium avium subsp. paratuberculosis (Map), could affect the progression of paratuberculosis, the development of lesions, the peripheral and local immune response, or the colonization of Map in tissues and its elimination through feces. An experimental study was conducted with thirty-five 1.5-month-old kids, which were separated into 6 experimental groups that include different intervention combinations (vaccinated, non-vaccinated, challenged and non-challenged) at different points and slaughtered at 120 and 330 days post-infection. The use of an inactivated vaccine against paratuberculosis could avoid clinical disease manifestation but does not prevent the tissue colonization, even when applied before Map exposure, achieving a reduction in the presence of viable bacteria in tissues and limiting the progression toward diffuse lesions. The therapeutic effect in vaccinated animals could not be confirmed. In this sense, vaccination not only modulates the immune response in terms of the production of IFN-γ and antibodies in peripheral blood and reduces tissue damage but also contributes to limiting the spread of infection through reduced bacterial shedding especially in goats vaccinated before Map infection.

Background Mycobacterium avium subspecies Paratuberculosis (MAP) is a bacterium known to cause Johne’s disease in ruminants and has been implicated in several autoimmune diseases. This study aimed to investigate the potential association between MAP infection and Rheumatoid Arthritis (RA). Methods A total of 119 patients with RA and 120 healthy controls (HCs) were enrolled in the study. The participants were outpatient attendees at a rheumatology specialist’s clinic, selected according to the 2010 ACR/EULAR Classification Criteria for RA. Their serum samples were analyzed for antibodies against two peptides, MAP_4027 18–32 and IRF5 424-434 , using an indirect enzyme-linked immunosorbent assay (ELISA). Results A significant difference was found in the levels of anti-MAP antibodies between RA patients and HCs. RA patients were more likely to have anti-MAP_4027 18–32 antibodies (44.5%) vs. 10.8% in HCs. Among RA patients, treatment group patients had more antibodies (51.6%) against MAP_4027 18–32 than no-treatment group patients (36.4%), but this difference was not statistically significant. The antigen IRF5 424-434 showed the highest antibody seroreactivity, being present in a higher percentage of RA patients (60.5%) compared to HCs (8.3%). This difference was statistically significant. There was a moderate correlation between IRF5 424-434 and its MAP_4027 18-32 homolog. Conclusions The study findings suggest that anti-MAP antibodies are more prevalent in RA patients compared to healthy controls, potentially implicating MAP in the development of RA. The strong immunological response to the antigen IRF5 424-434 warrants further investigation. Although the difference in antibody levels between previously diagnosed and newly diagnosed RA patients was not statistically significant, the overall higher prevalence of these antibodies in the RA cohort supports the hypothesis of MAP’s involvement.