Auriol C. Purdie

Does a Th1 over Th2 dominancy really exist in the early stages of Mycobacterium avium subspecies paratuberculosis infections

The immune response of ruminants to Johnes disease has been long associated with a cell mediated immune (CMI) response in the early stages of infection with a switch to an antibody response later as the disease manifests. This study examines the immune response in sheep to Mycobacterium avium subspecies paratuberculosis (Map) infections, specifically the antigen-specific interferon gamma (IFN-γ) and antibody responses as surrogates of T helper-1 (Th1) and Th2 immunity. The difference in IFN-γ production between paucibacillary and multibacillary diseased animals was also examined. The results show that sheep are more likely to have a combined antibody and IFN-γ response (seen in 50% of the animals) rather than a switch from an IFN-γ to antibody response (39%). Multibacillary diseased animals were found to have a decrease in functional ability to produce IFN-γ from cells stimulated with MAP-specific antigens and non-specific mitogens. This indicates that the immune responses to Map infections are more complex than thought, where both antibody and cellular immunity may play key roles in the early stages of disease manifestation or resistance. The loss of the cellular response in multibacillary animals may be an indication that the entire immune response is dysfunctional, with the cell mediated responses becoming affected first.

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: A review

Paratuberculosis (Johnes disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohns disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohns disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.

High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

ABSTRACT Johnes 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 McNemars 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.

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

ABSTRACT Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johnes 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.

Can early host responses to mycobacterial infection predict eventual disease outcomes

Diagnostic tests used for Johnes disease in sheep either have poor sensitivity and specificity or only detect disease in later stages of infection. Predicting which of the infected sheep are likely to become infectious later in life is currently not feasible and continues to be a major hindrance in disease control. We conducted this longitudinal study to investigate if a suite of diagnostic tests conducted in Mycobacterium avium subspecies paratuberculosis (MAP) exposed lambs at 4 months post infection can accurately predict their clinical status at 12 months post infection. We tracked cellular and humoral responses and quantity of MAP shedding for up to 12 months post challenge in 20 controls and 37 exposed sheep. Infection was defined at necropsy by tissue culture and disease spectrum by lesion type. Data were analysed using univariable and multivariable logistic regression models and a subset of variables from the earliest period post inoculation (4 months) was selected for predicting disease outcomes later on (12 months). Sensitivity and specificity of tests and their combinations in series and parallel were determined. Early elevation in faecal MAP DNA quantity and a lower interferon gamma (IFNγ) response were significantly associated with sheep becoming infectious as well as progressing to severe disease. Conversely, early low faecal MAP DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. Combination of early elevated faecal MAP DNA or lower IFNγ response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would become infectious. Collectively, these results highlight the potential for combined test interpretation to aid in the early prediction of sheep susceptibility to MAP infection.

Expression of genes associated with the antigen presentation and processing pathway are consistently regulated in early Mycobacterium avium subsp. paratuberculosis infection

The primary objective of this study was to evaluate early gene expression profiles associated with paratuberculosis in cattle exposed to known infectious doses of Mycobacterium avium sub-species paratuberculosis (MAP). A Johnes disease experimental infection field trial was conducted on a mixed population of Holstein and Holstein Red cattle. Blood samples from four MAP exposed and four unexposed cattle, selected based on IFNγ expressions were taken at 9, 13 and 21 weeks and RNA processed to Affymetrix GeneChip™ Bovine Genome arrays. Ontological analysis revealed consistent differences in gene expression between MAP exposed and control animals. A stark variation was observed in expression of a number of genes along antigen presentation pathways, suggesting that MAP exposure potentially results in the host immune response switching to a CD8(+) biased antigen presentation profile. This requires further in-depth analysis since it exposes a hitherto unconfirmed association between MAP exposure and in vivo MHC gene modulation.

Indoleamine 2,3-Dioxygenase, Tryptophan Catabolism, and Mycobacterium avium subsp. paratuberculosis: a Model for Chronic Mycobacterial Infections†

ABSTRACT 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 Johnes 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.

Toll-like receptor (TLR)6 and TLR1 differentiation in gene expression studies of Johne's disease

Johnes disease (JD) is a mycobacterial infection of the gut affecting ruminants and other species caused by Mycobacterium avium subspecies paratuberculosis (MAP). The role of toll-like receptors (TLR) in the pathogenesis of JD has been previously identified at the level of gene expression. Gene expression studies using reverse transcriptase (RT)-PCR are widely used and powerful, but the results obtained from such studies are dependent on the specificity of the assay. Here we describe an assay designed to detect ovine TLR6 in blood and tissues from sheep. Discrimination between TLR1 and TLR6 at the level of gene expression was challenging due to extensive tracts of homology and identity within the two sequences. Both TLR1 and 6 can form heterodimers with TLR2 in order to bind the ligands of microbial pathogens. The expression of TLR6 was increased in the ileum and jejunum of sheep infected with MAP, with a trend towards TLR6 upregulation in peripheral blood cells in response to exposure to MAP. A likely role for TLR6/TLR2 heterodimers in the pathogenesis of JD was identified. TLR6 may be a potential marker of exposure and could aid in the development of a gene signature for sheep resistant to MAP infection.

In silico identification of epitopes in Mycobacterium avium subsp. paratuberculosis proteins that were upregulated under stress conditions.

ABSTRACT Johnes disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts.

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

ABSTRACT 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 Johnes 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 104-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.