Mycobacterium avium subsp. paratuberculosis infection causes suppression of RANTES, monocyte chemoattractant protein 1, and tumor necrosis factor alpha expression in peripheral blood of experimentally infected cattle

Blood from cattle with subclinical Mycobacterium avium subsp. paratuberculosis infection was stimulated with M. avium subsp. paratuberculosis antigens, and expression of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), RANTES, monocyte chemoattractant protein 1 (MCP-1), and IL-8 was measured. Expression of TNF-alpha, RANTES, and MCP-1 was lower in infected than in uninfected cattle. The reduced response may weaken protective immunity and perpetuate infection.     

Effects of gamma interferon, interleukin-10, and transforming growth factor beta on the survival of Mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle

Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.     

Enhanced expression of interleukin-1alpha and tumor necrosis factor receptor-associated protein 1 in ileal tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis

Infection with Mycobacterium avium subsp. paratuberculosis is associated with high levels of morbidity, decreased production, and early culling in dairy cattle. Clinical symptoms of Johne's disease include persistent diarrhea, inappetence, and resultant weight loss due to chronic inflammation of the small intestine. Although the presence or absence of intestinal lesions cannot be used as a definitive indicator of M. avium subsp. paratuberculosis infection, most infected cattle exhibit significant changes to intestinal mucosa, with the focus of pathology surrounding the ileal cecal junction. Typical pathology of M. avium subsp. paratuberculosis infection includes inflammation, thickening of the lumenal wall, and hyperplasia in draining lymph nodes. To further understand the pathology of Johne's disease, we compared the gene expression profiles of ileal tissues from Johne's disease-positive (n = 6), and Johne's disease-negative (n = 5) Holstein cattle. Gene expression profiles were compared with a bovine total leukocyte (BOTL-3) cDNA microarray. Genes that were expressed at significantly higher levels (>1.5-fold; P < 0.05) in tissues from Johne's disease-infected animals relative to noninfected animals included those encoding tumor necrosis factor receptor-associated protein 1 (TRAF1), interleukin-1alpha (IL-1alpha), MCP-2, N-cadherin, and beta1 integrin (CD29). Dramatic upregulation of IL-1alpha (21.5-fold) and TRAF1 (27.5-fold) gene expression in tissues of Johne's disease-positive cows relative to tissues from control cows was confirmed by quantitative real-time PCR. Western blot analysis confirmed that IL-1alpha and TRAF1 mRNA levels resulted in increased protein expression in tissues of Johne's disease-positive cattle relative to tissues from control cattle. High levels of IL-1alpha can produce symptoms similar to those found in clinical Johne's disease. Taken together, the data presented in this report suggest that many outward symptoms of Johne's disease may be due to IL-1alpha toxicity. In addition, enhanced levels of TRAF1 could result in cells within the lesions of Johne's disease-positive cattle that are highly resistant to TNF-alpha-induced signaling.     

Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-gamma and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-gamma (6 h), and TNF-alpha (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-alpha (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.     

Diverse cytokine profile from mesenteric lymph node cells of cull cows severely affected with Johne's disease

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, is able to dampen or distort immune responses at the mucosal sites and coexist with a massive infiltration of immune cells in the gastrointestinal tract. Knowledge of the mechanism by which M. avium subsp. paratuberculosis subverts the immune response at the mucosal level in cattle is important for the development of improved disease control strategies, including new vaccines and diagnostic tests. In this study, 38 cull cows from herds infected with M. avium subsp. paratuberculosis were divided into four groups, based on M. avium subsp. paratuberculosis culture from gut tissues and histopathological lesion scores. Cytokine gene expression and secretion from M. avium subsp. paratuberculosis sonicate-stimulated peripheral blood mononuclear cell (PBMC) and mesenteric lymph node (MLN) cultures of the animals were compared. Antigen stimulation of MLN cells from the severely lesioned group resulted in significant upregulation of the mRNA expression of five cytokines, gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-13, IL-17A, and tumor necrosis factor alpha (TNF-α), which have a diverse range of functions, while there was no significant upregulation of these cytokines by the other groups. There were major differences between the responses of the PBMC and MLN cultures, with higher levels of secreted IFN-γ released from the MLN cultures and, conversely, higher levels of IL-10 released from the PBMC cultures. The upregulation of all five cytokines from cells at the site of infection in the severely lesioned animals suggested a dysregulated immune response, contributing to a failure to clear infection in this group of animals.     

Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis

Monoclonal antibody neutralization of interleukin-10 (IL-10) increased Johnin purified protein derivative-induced whole-blood gamma interferon (IFN-gamma) secretion 23-fold and also increased IFN-gamma secretion ninefold following in vitro Mycobacterium avium subsp. paratuberculosis infection of peripheral blood mononuclear cells. These results demonstrate the suppressive effect of IL-10 on immune responses to M. avium subsp. paratuberculosis infection in cattle.     

Invasion and persistence of Mycobacterium avium subsp. paratuberculosis during early stages of Johne's disease in calves

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johne's disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johne's disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.     

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.     

Characterization of novel coding sequences specific to Mycobacterium avium subsp. paratuberculosis: implications for diagnosis of Johne's Disease

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's Disease, an economically important intestinal ailment of ruminants. Due to the considerable genetic and serologic cross-reactivity with closely related and ubiquitous members of the M. avium complex, a species-specific method for the serological diagnosis of Johne's disease is unavailable. Computational and PCR-based analysis of the complete genome sequence of M. avium subsp. paratuberculosis led to the identification of 13 open reading frames with no identifiable homologs. One of these sequences is a putative insertion element present in six copies on the M. avium subsp. paratuberculosis genome. These novel M. avium subsp. paratuberculosis genes were cloned into Escherichia coli expression vectors, and nine were successfully expressed as recombinant fusion proteins. Five of these proteins were purified in sufficient amounts to allow immunoblot analyses of their reactivity with sera from naturally infected cattle as well as mice and rabbits exposed to M. avium subsp. paratuberculosis. Fusion proteins representing MAP0862, MAP3732c, and MAP2963c were recognized by nearly all of the sera tested, including those from cattle in the clinical stages of disease. Notably, further analysis of the protein encoded by MAP0862 showed that it reacted with sera from additional infected cattle but not with sera from uninfected control animals. The fusion product of MAP0860c did not react with any of the sera tested, while the remaining four proteins were variably recognized by sera from M. avium subsp. paratuberculosis-infected cattle. Collectively, the results of this study demonstrate the utility of genomic data to identify potential diagnostic sequences.     

Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp. paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle

The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-gamma) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-gamma responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in M. bovis and M. avium subsp. paratuberculosis) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from M. avium subsp. paratuberculosis. These results indicate that the M. avium subsp. paratuberculosis Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.     

Mycobacterium avium subsp. paratuberculosis Inhibits Gamma Interferon-Induced Signaling in Bovine Monocytes: Insights into the Cellular Mechanisms of Johne's Disease

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection by M. avium subsp. paratuberculosis depends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by which M. avium subsp. paratuberculosis subverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected and M. avium subsp. paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation of M. avium subsp. paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates that M. avium subsp. paratuberculosis blocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed in M. avium subsp. paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this critical M. avium subsp. paratuberculosis behavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.     

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.     

Comparison of histopathology, cultivation of tissues and rectal contents, and interferon-gamma and serum antibody responses for the diagnosis of bovine paratuberculosis

The diagnosis of infection with Mycobacterium avium subsp. paratuberculosis was studied in 12 cattle from infected dairy herds and four from non-infected herds. A comparison was made of (1) histopathological examination and bacterial culture of tissues, (2) culture of serial samples of rectal contents, and (3) examination of repeated blood samples for interferon-gamma (IFN-gamma) and antibody responses. Tissue samples were taken from the small and large intestine and corresponding mesenteric lymph nodes, and from the pharyngeal tonsil and other lymphoid nodes (retropharyngeal, mediastinal, hepatic and supramammary). Histopathological examination and bacterial culture of tissues each revealed that six of the 16 cattle were infected, whereas repeated culture of rectal contents revealed only five infected animals. Except for the hepatic lymph node of a single animal, culture-positive tissues were confined to the intestinal tract and related lymph nodes. Bacterial culture of tissues from the ileum, caecum and lymph nodes draining the jejunum revealed the highest number of infected animals. Moreover, slightly greater numbers of positive tissues were revealed by culture than by histopathological examination. For both the IFN-gamma response and the antibody response, the means of the values for the final three samples before slaughter were significantly higher in infected than in non-infected cattle. However, these immunological responses were too variable to provide a reliable indication of infection.     

Correlation of cytokine gene expression with pathology in white-tailed deer (Odocoileus virginianus) infected with Mycobacterium bovis

Mycobacterium bovis-infected white-tailed deer (WTD) in northeast Michigan are a reservoir of mycobacteria that pose a threat to both domestic animals and humans. Relatively little work has been done to characterize the immune response of WTD to M. bovis infection; however, an understanding of the immune response to infection and pathogenesis may be critical to the development of an effective vaccine. Immunological responses to infection were characterized by monitoring cytokine gene expression in M. bovis-infected and uninfected deer. Peripheral blood leukocytes (PBL) from infected WTD expressed more gamma interferon (IFN-gamma), interleukin-12p40 (IL-12p40), granulocyte-monocyte colony-stimulating factor, and IL-4 mRNA than did PBL from uninfected deer; however, differences were not detected in expression of IL-10 and transforming growth factor-beta mRNA. Infected animals could be divided into two groups based on pathology. Lesions were confined primarily to the lymph nodes of the head in animals with less severe pathology. Animals with more severe pathology had lesions in the lung and associated lymph nodes as well as the lymph nodes of the head. More robust IFN-gamma mRNA expression correlated with pathology early in infection. These findings indicate that IFN-gamma expression likely plays a role in both protection and pathogenesis.     

Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp. paratuberculosis infection in the red deer (Cervus elaphus)

Johne's disease (JD) infection, caused by Mycobacterium avium subsp. paratuberculosis, represents a major disease problem in farmed ruminants. Although JD has been well characterized in cattle and sheep, little is known of the infection dynamics or immunological response in deer. In this study, typing of M. avium subsp. paratuberculosis isolates from intestinal lymphatic tissues from 74 JD-infected animals showed that clinical isolates of M. avium subsp. paratuberculosis from New Zealand farmed red deer were exclusively of the bovine strain genotype. The susceptibility of deer to M. avium subsp. paratuberculosis was further investigated by experimental oral-route infection studies using defined isolates of virulent bovine and ovine M. avium subsp. paratuberculosis strains. Oral inoculation with high (10(9) CFU/animal) or medium (10(7) CFU/animal) doses of the bovine strain of M. avium subsp. paratuberculosis established 100% infection rates, compared to 69% infection following inoculation with a medium dose of the ovine strain. The high susceptibility of deer to the bovine strain of M. avium subsp. paratuberculosis was confirmed by a 50% infection rate following experimental inoculation with a low dose of bacteria (10(3) CFU/animal). This study is the first to report experimental M. avium subsp. paratuberculosis infection in red deer, and it outlines the strong infectivity of bovine-strain M. avium subsp. paratuberculosis isolates for cervines.     

Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection

Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization.