Divergent Antigen-Specific Cellular Immune Responses during Asymptomatic Subclinical and Clinical States of Disease in Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

Infection of the host with Mycobacterium avium subsp. paratuberculosis results in chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from an asymptomatic subclinical disease state to advanced clinical disease is not fully understood. In the present study, naturally infected dairy cattle were divided into subclinical and clinical infection groups, along with noninfected control cows of similar parity, to study host immune responses in different stages of infection. Both infection groups had higher levels of secretion of gamma interferon (IFN-), tumor necrosis factor-alpha (TNF-), and interleukin-2 (IL-2) than control cows, whereas only clinical cows had increased secretion of IL-10, IL-12, and IL-18 upon stimulation of peripheral blood mononuclear cells (PBMCs) with antigen. Conversely, secretion of IL-17 was decreased for clinical cows compared to subclinical and control cows. Proinflammatory cytokine genes were upregulated only for subclinical cows, whereas increased IL-10 and IL-17 gene expression levels were observed for both infection groups. Increased CD4, CD8, and T cell receptor-positive (TCR) T cells were observed for subclinical cows compared to clinical cows. Although clinical cows expressed antigen-specific immune responses, the profile for subclinical cows was one of a dominant proinflammatory response to infection. We reason that a complex coordination of immune responses occurs during M. avium subsp. paratuberculosis infection, with these responses shifting as the host transitions through the different stages of infection and disease (subclinical to clinical). A further understanding of the series of events characterized by Th1/Th2/Th17 responses will provide mechanisms for disease progression and may direct insightful intervention strategies

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host. RESULTS: Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a steady decline in viability over the next six days. Antibodies against M. paratuberculosis were affinity purified and used in conjunction with transmission electron microscopy to track the development of intracellular bacilli. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. Conversely, when macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed. CONCLUSIONS: We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and viability counts. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years

Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein

BACKGROUND: The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed. RESULTS: MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows. CONCLUSIONS: Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay

Disparate Host Immunity to Mycobacterium avium subsp. paratuberculosis Antigens in Calves Inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii, and M. bovis

The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratu- berculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN- responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta () T, and B cells for the calves that were inoculated with either M. avium subsp. para- tuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO CD25 T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although re- combinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differ- ences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis

Comparison of Sheep, Goats, and Calves as Infection Models for Mycobacterium avium subsp. paratuberculosis

Animal infection models to study Mycobacterium avium subsp. paratuberculosis (MAP) infection are useful for evaluating the efficacy of vaccines and other therapeutics for the prevention or treatment of infection. The goal of the present study was to compare smaller ruminants, sheep and goats, with calves as infection models. Neonatal sheep, goats, and calves (n = 4) received 109 cfu of a cattle isolate of MAP in milk replacer on days 0, 3 and 6 in a 12-month study and sampled monthly thereafter. Results demonstrated a robust antigen-specific IFN-γ response at 90 days post-inoculation for sheep and goats, with lower responses noted for calves. By 360 days, IFN-γ responses were 50 and 82% higher for calves than for goats and sheep, respectively. Although MAP- specific antibody responses were first observed in sheep at 90 days, calves had higher antibody responses throughout the remainder of the study. Following pass-through shedding on day 7, fecal shedding was fairly negligible across treatments but remained higher for calves throughout the study. Colonization of tissues was variable within treatment group and was higher for calves and sheep for the majority of tissues. Upon antigen stimulation of PBMCs, higher populations of CD4 + T cells cells and lower populations of γδ TCR + and NK cells were observed for goats and calves compared to sheep. Relative gene expression of IL-4, IL-12, and IL-17 in PBMCs was higher in goats, corresponding to lower tissue colonization with MAP. These data suggest that ruminant species are fairly comparable as infection models for MAP, but discrete differences in host responses to MAP infection exist between species

Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis

Johne’s disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and pro-vides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up-regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection

Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

Antigens extracted using ethanol (EtOH) and incorporated in the EtOH vortex ELISA (EVELISA) test have previously shown high specificity and sensitivity for detecting Mycobacterium avium subspecies paratuberculosis (Map) and M. bovis infections in cattle. The objective of this study is to define the components present in the EtOH extract. We show that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of Map which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a Map genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne’s disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in M. bovis EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in Map EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle