Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis.

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Mycobacterium avium subspecies paratuberculosis is able to manipulate host lipid metabolism and accumulate cholesterol within macrophages.

Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Closely related pathogenic mycobacteria such as M. tuberculosis are capable of altering host lipid metabolism, highlighting the need to explore the role of lipid metabolism contributing to intracellular survival. This study aimed to identify whether MAP is able to manipulate host lipid metabolic pathways and accumulate host cholesterol during early infection. Macrophages were exposed to four different MAP strains and non-pathogenic M. phlei for up to 72 h, with changes to lipid metabolism examined using fluorescent microscopy and gene expression. MAP-infected macrophages displayed strain-dependent differences to intracellular cholesterol levels during early infection, however showed similarly increased intracellular cholesterol at later timepoints. Gene expression revealed that MAP strains similarly activate the host immune response in a conserved manner compared to M. phlei. MAP significantly upregulated host genes associated with lipid efflux and endocytosis. Moreover, lipid biosynthesis genes were differentially regulated in a strain-dependent manner following MAP infection. Collectively, these results demonstrate that MAP manipulates host lipid metabolism during early infection, however the extent of these modulations are strain-dependent. These findings reflect a conserved pathway contributing to intracellular MAP survival.

The humoral immune response is essential for successful vaccine protection against paratuberculosis in sheep.

BACKGROUND: The role played by the humoral immune response in animals vaccinated against a mycobacterial disease such as paratuberculosis, is not well understood. Sheep vaccinated against Mycobacterium avium subsp. paratuberculosis (MAP) can still become infected and in some cases succumb to clinical disease. The strength and location of the humoral immune response following vaccination could contribute to the ability of sheep to clear MAP infection. We examined the peripheral antibody response along with the localised humoral response at the site of paratuberculosis infection, the ileum, to better understand how this contributes to MAP infection of sheep following vaccination and exposure. RESULTS: Through assessing MAP specific serum IgG1 and IgG levels we show that the timing and strength of the humoral immune response directly relates to prevention of infection following vaccination. Vaccinated sheep that subsequently became infected had significantly reduced levels of MAP specific serum IgG1 early after vaccination. In contrast, vaccinated sheep that did not subsequently become infected had significantly elevated MAP specific serum IgG1 following vaccination. Furthermore, at 12 months post MAP exposure, vaccinated and subsequently uninfected sheep had downregulated expression of genes related to the humoral response in contrast to vaccinated infected sheep where expression levels were upregulated. CONCLUSIONS: The timing and strength of the humoral immune response following vaccination against paratuberculosis in sheep directly relates to subsequent infection status. An initial strong IgG1 response following vaccination was crucial to prevent infection. Additionally, vaccinated uninfected sheep were able to modulate that response following apparent MAP clearance, unlike vaccinated infected animals where there was apparent dysregulation of the humoral response, which is associated with progression to clinical disease.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays.

UNLABELLED: Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE: Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.

Role of host- and pathogen-associated lipids in directing the immune response in mycobacterial infections, with emphasis on Mycobacterium avium subsp. paratuberculosis.

Mycobacteria have a complex cell wall with a high lipid content that confers unique advantages for bacterial survival in the hostile host environment, leading to long-term infection. There is a wealth of evidence suggesting the role cell wall-associated lipid antigens play at the host-pathogen interface by contributing to bacterial virulence. One pathway that pathogenic mycobacteria use to subvert host immune pathways to their advantage is host cholesterol/lipid homeostasis. This review focuses on the possible role of pathogen- and host-associated lipids in the survival and persistence of pathogenic mycobacteria with emphasis on Mycobacterium avium subsp. paratuberculosis. We draw upon literature in diverse areas of infectious and metabolic diseases and explain a mechanism by which mycobacterial-induced changes in the host cellular energy state could account for phenomena that are a hallmark of chronic mycobacterial diseases.

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle.

BACKGROUND: Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne's disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne's disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. RESULTS: Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. CONCLUSIONS: This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne's disease progression by warranting further research on the presence of MAP in blood.

Efficient, validated method for detection of mycobacterial growth in liquid culture media by use of bead beating, magnetic-particle-based nucleic acid isolation, and quantitative PCR.

Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10(4)-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n=54) and sheep fecal and tissue (n=90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis.

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611-622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.

IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity.

Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.

Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.

Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection.

Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.

Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 µl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

IP10 is a predictor of successful vaccine protection against paratuberculosis infection in sheep.

The cell mediated immune response and ability of immune cells to migrate to the site of infection are both key aspects of protection against many pathogens. Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen and the causative agent of paratuberculosis, a chronic wasting disease of ruminants. Current commercial vaccines for paratuberculosis reduce the occurrence of clinical disease but not all animals are protected from infection. Therefore, there is a need to understand the immune responses triggered by these vaccines at the site of infection, in circulating immune cells and their relationships to vaccine-mediated protection. The magnitude and location of gene expression related to the cell mediated immune response and cellular migration were studied in the ileum of sheep. In addition, longitudinal IP10 (also known as IP10) secretion by circulating immune cells was examined in the same sheep. Animals were grouped based on vaccination status (vaccinated vs non-vaccinated) and MAP exposure (experimentally exposed vs unexposed). Vaccination of unexposed sheep increased the expression of IP10, CCL5 and COR1c. Sheep that were successfully protected by vaccination (uninfected following experimental exposure) had significantly reduced expression of IP10 in the ileum at 12 months post exposure compared to vaccine non-responders (those that became infected) and non-vaccinated infected sheep. Successfully protected sheep also had significantly increased secretion of IP10 in in vitro stimulated immune cells from whole blood compared to vaccine non responders at 4 months post exposure. Therefore, the IP10 recall response has the potential to be used as marker for infection status in vaccinated sheep and could be a biomarker for a DIVA test in sheep.

Association between major histocompatibility complex haplotypes and susceptibility of unvaccinated and vaccinated cattle to paratuberculosis.

Bovine Johne's disease (BJD) or paratuberculosis is caused by Mycobacterium avium spp. paratuberculosis (MAP) and is a worldwide problem among domestic and wild ruminants. While vaccines are available, natural differences in background immunity between breeds within species and between individuals within herds suggest that genetic differences may be able to be exploited in marker-assisted selection as an aid to disease control. The major histocompatibility complex (MHC) is an important component in immune recognition with considerable genetic variability. In this study, associations between the MHC and resistance to BJD were explored in dairy cattle across two herds in which some of the cattle had been vaccinated with Silirum® (n = 540 cows). A BJD susceptible animal was exposed to MAP and became infected, while a resistant animal was exposed but did not become infected. There are different ways to define both exposure and infection, with different levels of stringency, therefore many classifications of the same set of animals are possible and were included in the analysis. The polymorphic regions of major histocompatibility complex class I (MHC I) and class II (MHC II) genes were amplified from the genomic DNA by PCR and sequenced, targeting exons 2 and 3 of the classical and non-classical MHC I genes and exon 2 from the DRB3, DQA1, DQA2 + 3 and DQB MHC II genes. The frequencies of MHC I and MHC II haplotypes and alleles were determined in susceptible and resistant populations. In unvaccinated animals, seven MHC I haplotypes and seven MHC II haplotypes were associated with susceptibility while two MHC I and six MHC II haplotypes were associated with resistance (P < 0.05). In vaccinated animals, two MHC I and three MHC II haplotypes were associated with susceptibility, while one MHC I and two MHC II haplotypes were associated with resistance (P < 0.05). The alleles in significant haplotypes were also identified. Case definitions with higher stringency resulted in fewer animals being included in the analyses, but the power to detect an association was not reduced and there was an increase in strength and consistency of associations. Consistent use of stringent case definitions is likely to improve agreement in future association studies.

Global Phylogeny of Mycobacterium avium and Identification of Mutation Hotspots During Niche Adaptation.

Mycobacterium avium is separated into four subspecies: M. avium subspecies avium (MAA), M. avium subspecies silvaticum (MAS), M. avium subspecies hominissuis (MAH), and M. avium subspecies paratuberculosis (MAP). Understanding the mechanisms of host and tissue adaptation leading to their clinical significance is vital to reduce the economic, welfare, and public health concerns associated with diseases they may cause in humans and animals. Despite substantial phenotypic diversity, the subspecies nomenclature is controversial due to high genetic similarity. Consequently, a set of 1,230 M. avium genomes was used to generate a phylogeny, investigate SNP hotspots, and identify subspecies-specific genes. Phylogeny reiterated the findings from previous work and established that Mycobacterium avium is a species made up of one highly diverse subspecies, known as MAH, and at least two clonal pathogens, named MAA and MAP. Pan-genomes identified coding sequences unique to each subspecies, and in conjunction with a mapping approach, mutation hotspot regions were revealed compared to the reference genomes for MAA, MAH, and MAP. These subspecies-specific genes may serve as valuable biomarkers, providing a deeper understanding of genetic differences between M. avium subspecies and the virulence mechanisms of mycobacteria. Furthermore, SNP analysis demonstrated common regions between subspecies that have undergone extensive mutations during niche adaptation. The findings provide insights into host and tissue specificity of this genetically conserved but phenotypically diverse species, with the potential to provide new diagnostic targets and epidemiological and therapeutic advances.

Mycobacterium avium subsp. paratuberculosis exploits miRNA expression to modulate lipid metabolism and macrophage polarisation pathways during infection.

Pathogenic mycobacteria including Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, manipulate host macrophages to persist and cause disease. In mycobacterial infection, highly plastic macrophages, shift between inflammatory M1 and permissive M2 phenotypes which alter the disease outcome and allow bacteria to survive intracellularly. Here we examine the impact of MAP infection on polarised macrophages and how increased lipid availability alters macrophage phenotype and bacterial persistence. Further, we assess if host microRNA (miRNA) are sensitive to macrophage polarisation state and how MAP can drive their expression to overcome innate responses. Using in vitro MAP infection, we find that increasing lipid availability through supplementing culture media with exogenous lipid increases cellular nitric oxide production. Lipid-associated miRs -19a, -129, -24, and -24-3p are differentially expressed following macrophage polarisation and lipid supplementation and are further regulated during MAP infection. Collectively, our results highlight the importance of host lipid metabolism in MAP infection and demonstrate control of miRNA expression by MAP to favour intracellular persistence.

Correlates of vaccine protection against Mycobacterium avium sub-species paratuberculosis infection revealed in a transcriptomic study of responses in Gudair® vaccinated sheep.

A critical hindrance in the development of effective vaccine strategies to combat infectious disease is lack of knowledge about correlates of protection and of the host responses necessary for successful adaptive immunity. Often vaccine formulations are developed by stepwise experimentation, with incomplete investigation of the fundamental mechanisms of protection. Gudair® is a commercially available vaccine registered for use in sheep and goats for controlling spread of Mycobacterium avium sub-species paratuberculosis (MAP) infections and reduces mortality by up to 90%. Here, using an experimental infection model in sheep, we have utilized a transcriptomics approach to identify white blood cell gene expression changes in vaccinated, MAP-exposed Merino sheep with a protective response in comparison to those vaccinated animals that failed to develop immunity to MAP infection. This methodology facilitated an overview of gene-associated functional pathway adaptations using an in-silico analysis approach. We identified a group of genes that were activated in the vaccine-protected animals and confirmed stability of expression in samples obtained from naturally exposed commercially maintained sheep. We propose these genes as correlates of vaccine induced protection.

Sheep vaccinated against paratuberculosis have increased levels of B cells infiltrating the intestinal tissue.

Systemic immunisation delivered subcutaneously is currently used to control paratuberculosis, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). These vaccines do not provide complete protection and a small cohort of animals still succumb to clinical disease. The aim of this study was to assess mycobacterial infection site-specific variations in immune cells in vaccinated sheep that did or did not develop the disease following controlled exposure to MAP. Immunohistochemical staining of terminal ileum demonstrated that vaccination increased infiltration of CD4 + T cells and B cells. Infiltration of large numbers of CD4 + T and B cells was also seen in sheep that successfully cleared infection. Vaccination promoted the polarisation of macrophages to an M1 activation state. The presence of certain cells at the site of infection, especially CD4 + T cells, is likely to contribute to vaccine success by increasing the speed and potency of the local immune response. Systemic immunisation against MAP can alter the composition of innate and adaptive immune cell populations at the predilection site for MAP infection in the ileum one year after vaccination. This informs understanding of the impact of vaccination at the site of infection and also the duration of vaccine-elicited changes. This information may assist vaccine development and allow targeting of protective immune responses in the gut of ruminants.

Indoleamine 2,3-dioxygenase, tryptophan catabolism, and Mycobacterium avium subsp. paratuberculosis: a model for chronic mycobacterial infections.

Virulent mycobacterial infections progress slowly, with a latent period that leads to clinical disease in a proportion of cases. Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen that causes paratuberculosis or Johne's disease (JD), a chronic intestinal disease of ruminants. Indoleamine 2,3-dioxygenase (IDO), an enzyme that regulates tryptophan metabolism, was originally reported to have a role in intracellular pathogen killing and has since been shown to have an important immunoregulatory role in chronic immune diseases. Here we demonstrate an association between increased IDO levels and progression to clinical mycobacterial disease in a natural host, characterizing gene expression, protein localization, and functional effects. IDO mRNA levels were significantly increased in M. avium subsp. paratuberculosis-infected monocytic cells. Levels of both IDO gene and protein expression were significantly upregulated within the affected tissues of sheep with JD, particularly at the site of primary infection, the ileum, of animals with severe multibacillary disease. Lesion severity was correlated with the level of IDO gene expression. IDO gene expression was also increased in the peripheral blood cells of M. avium subsp. paratuberculosis-exposed sheep and cattle. IDO breaks down tryptophan, and systemic increases were functional, as shown by decreased plasma tryptophan levels, which correlated with the onset of clinical signs, a stage well known to be associated with Th1 immunosuppression. IDO may be involved in downregulating immune responses to M. avium subsp. paratuberculosis and other virulent mycobacteria, which may be an example of the pathogen harnessing host immunoregulatory pathways to aid survival. These findings raise new questions about the host-mycobacterium interactions in the progression from latent to clinical disease.