Immunopathological changes and apparent recovery from infection revealed in cattle in an experimental model of Johne's disease using a lyophilised culture of Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) or paratuberculosis is an economically significant, chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Experimental models of JD in cattle are logistically challenging due to the need for long term monitoring, because the clinical disease can take years to manifest. Three trials were undertaken, the largest involving 20 cattle exposed orally to a low dose of C strain MAP and 10 controls studied for 4.75 years. Frequent blood and faecal sampling was used to monitor immunological and infection parameters, and intestinal biopsies were performed at two time points during the subclinical disease phase. Although clinical disease was not seen, there was evidence of infection in 35% of the animals and at necropsy 10% had histopathological lesions consistent with JD, similar to the proportions expected in naturally infected herds. Faecal shedding occurred in two distinct phases: firstly there was intermittent shedding <∼9 months post-exposure that did not correlate with disease outcomes; secondly, in a smaller cohort of animals, this was followed by more consistent shedding of increasing quantities of MAP, associated with intestinal pathology. There was evidence of regression of histopathological lesions in the ileum of one animal, which therefore had apparently recovered from the disease. Both cattle with histopathological lesions of paratuberculosis at necropsy had low MAP-specific interferon-gamma responses at 4 months post-exposure and later had consistently shed viable MAP; they also had the highest loads of MAP DNA in faeces 4.75 year s post-exposure. In a trial using a higher dose of MAP, a higher proportion of cattle developed paratuberculosis. The information derived from these trials provides greater understanding of the changes that occur during the course of paratuberculosis in cattle.

Integrated vaccine screening system: using cellular functional capacity in vitro to assess genuine vaccine protectiveness in ruminants.

Experimental trials in the natural host are essential for development and screening of effective vaccines. For chronic diseases of livestock such as paratuberculosis, these can be lengthy and costly in nature. An alternative is to screen vaccines in vitro; however, previous studies have found that vaccine success in vitro in existing screening assays does not translate to in vivo efficacy. To overcome these issues, we have developed a system that combines both in vivo and in vitro aspects. We hypothesise that the effectiveness of vaccine-induced immune responses mounted in vivo could be gauged by assessing the ability of immune cells to 'control' an in vitro infection. Monocytes from Merino wethers (n = 45) were infected with Mycobacterium avium subspecies paratuberculosis (MAP) in vitro, cultured with autologous lymphocytes and remaining viable intracellular MAP was quantified. Cells from MAP exposed sheep had a higher capacity to kill intracellular MAP compared to non-exposed controls (P = 0.002). Importantly, cells from MAP exposed uninfected sheep had a greater capacity to kill intracellular MAP compared to vaccinated animals that were infected (ineffective vaccination), indicating that this in vitro assay has the potential to gauge actual protectiveness, or lack thereof, of a vaccine.

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis.

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

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.

Macrophage polarization in cattle experimentally exposed to Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in cattle, has significant impacts on the livestock industry and has been implicated in the etiology of Crohn's disease. Macrophages play a key role in JD pathogenesis, which is driven by the manipulation of host immune mechanisms by MAP. A change in the macrophage microenvironment due to pathogenic or host-derived stimuli can lead to classical (M1) or alternative (M2) polarization of macrophages. In addition, prior exposure to antigenic stimuli has been reported to alter the response of macrophages to subsequent stimuli. However, macrophage polarization in response to MAP exposure and its possible implications have not been previously addressed. In this study, we have comprehensively examined monocyte/macrophage polarization and responsiveness to antigens from MAP-exposed and unexposed animals. At 3 years post-exposure, there was a heterogeneous macrophage activation pattern characterized by both classical and alternate phenotypes. Moreover, subsequent exposure of macrophages from MAP-exposed cattle to antigens from MAP and other mycobacterial species led to significant variation in the production of nitric oxide, interleukin-10 and tumour necrosis factor α. These results indicate the previously unreported possibility of changes in the activation state and responsiveness of circulating monocytes/macrophages from MAP-exposed cattle.

Lymphoproliferative and gamma interferon responses to stress-regulated Mycobacterium avium subsp. paratuberculosis recombinant proteins.

Johne's disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinant M. avium subsp. paratuberculosis proteins known to be upregulated under in vitro stress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with live Mycobacterium avium subsp. paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis.

Enzyme-linked immunospot: an alternative method for the detection of interferon gamma in Johne's disease.

To date, the sensitivity of the interferon gamma (IFN-gamma) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN-gamma detection in the early stages of infection, an alternate assay needs to be developed. The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive technique for the detection of cytokines and has the potential to improve the diagnosis of JD. Of the variables examined, choice of capture antibody and the method by which the peripheral blood mononuclear cells were isolated significantly affected the ability to enumerate IFN-gamma-secreting cells. The ELISPOT assay was as sensitive as or better than the IFN-gamma ELISA at detecting ovine JD and could also detect disease at early time points postinoculation. The IFN-gamma ELISPOT could distinguish infected from unexposed animals; however, neither the IFN-gamma ELISA nor the ELISPOT assay could distinguish between sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis and those exposed to the bacterium but diagnosed as uninfected at necropsy.