Judith R. Stabel

Transitions in immune responses to Mycobacterium paratuberculosis

The host immune response to infection with Mycobacterium paratuberculosis is paradoxical, with strong cell-mediated immune responses during the early, subclinical stages of infection and strong humoral responses during the late clinical stages of the disease. Cell-mediated immune responses modulated by various T cell subsets are essential to provide protective immunity and prevent progression of the disease. Secretion of cytokines by T cell populations serves to activate macrophages to kill ingested M. paratuberculosis as well as activate other T cell subsets to contain the infection. This paper reviews the current knowledge of T cell immune responses in M. paratuberculosis infection based upon clinical studies and research using mouse models.

An Improved Method for Cultivation of Mycobacterium Paratuberculosis from Bovine Fecal Samples and Comparison to Three Other Methods

A new method (NADC) for isolation of Mycobacterium paratuberculosis from fecal samples is described and evaluated using fecal samples from a known paratuberculosis-infected herd of cattle. The NADC method includes centrifugation of the total fecal sample supernatant and use of a 2-step decontamination protocol. The growth rate of M. paratuberculosis and contamination rate of cultures when using this method are compared to 3 other published methods: sedimentation, centrifugation, and Cornell. Sensitivity was lowest for the Cornell method precluding detection of some low shedders; however, contamination was not observed for this method. Contamination was the most severe in samples processed by the centrifugation method but was also high for the sedimentation method, resulting in unreadable culture tubes for some fecal samples. The NADC method was 10-fold more sensitive for detection of M. paratuberculosis colonies and contamination was significantly reduced compared to other 3 methods.

Production of γ-Interferon by Peripheral Blood Mononuclear Cells: An Important Diagnostic Tool for Detection of Subclinical Paratuberculosis

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne s disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratuberculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for y-interferon (γ-IFN) production. Cells from subclinical cows produced significantly higher levels of γ-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of γ-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of γ-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that γ-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.

Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis: Evidence for Limited Strain Diversity, Strain Sharing, and Identification of Unique Targets for Diagnosis

ABSTRACT The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohns disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.

Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA

A transformed bovine peritoneal macrophage cell line was developed and characterized. Primary peritoneal macrophages were transformed by calcium-phosphate transfection with SV40 plasmid DNA. The transformed cell line retained the morphology of resident peritoneal macrophages as determined by light microscopy and histochemical analysis showed non-specific esterase activity. In addition, immunohistochemical staining of transformed peritoneal macrophages for lysozyme activity was positive. Transformed cells phagocytized Staphylococcus aureus, lysed chicken red blood cell (RBC) targets with and without opsonization and produced hydrogen peroxide radicals and interleukin-6 upon stimulation with opsonized zymosan and lipopolysaccharide, respectively. Transformed cells were also able to ingest and kill Mycobacterium paratuberculosis, an acid-fast bacillus. These results suggest that this cell line should be useful to study interactions between the bovine and intracellular pathogens.

Biomarker discovery in subclinical mycobacterial infections of cattle

Background Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle. Methodology/Principal Findings We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P≤0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals. Conclusions/Significance The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.

Heat-treated colostrum and reduced morbidity in preweaned dairy calves: Results of a randomized trial and examination of mechanisms of effectiveness

A randomized controlled clinical trial was conducted using 1,071 newborn calves from 6 commercial dairy farms in Minnesota and Wisconsin, with the primary objective being to describe the effects of feeding heat-treated colostrum on serum immunoglobulin G concentration and health in the preweaning period. A secondary objective was to complete a path analysis to identify intermediate factors that may explain how feeding heat-treated colostrum reduced the risk for illness. On each farm, colostrum was collected each day, pooled, and divided into 2 aliquots; then, one aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60 min. Samples of fresh and heat-treated colostrum were collected for standard microbial culture (total plate count and total coliform count, cfu/mL) and for measurement of immunoglobulin G concentrations (mg/mL). Newborn calves were removed from the dam, generally within 30 to 60 min of birth, and systematically assigned to be fed 3.8L of either fresh (FR, n=518) or heat-treated colostrum (HT, n=553) within 2h of birth. Venous blood samples were collected from calves between 1 and 7d of age for measurement of serum IgG concentrations (mg/mL). All treatment and mortality events were recorded by farm staff between birth and weaning. Regression models found that serum IgG concentrations were significantly higher in calves fed HT colostrum (18.0 ± 1.5 mg/mL) compared with calves fed FR colostrum (15.4 ± 1.5 mg/ml). Survival analysis using Cox proportional hazards regression indicated a significant increase in risk for a treatment event (any cause) in calves fed FR colostrum (36.5%, hazard ratio=1.25) compared with calves fed HT colostrum (30.9%). In addition, we observed a significant increase in risk for treatment for scours in calves fed FR colostrum (20.7%, hazard ratio=1.32) compared with calves fed HT colostrum (16.5%). Path analysis suggested that calves fed HT colostrum were at lower risk for illness because the heat-treatment process caused a significant reduction in colostrum total coliform count, which was associated with a reduced risk for illness as a function of improved serum IgG concentrations.

Expression and Immunogenicity of Proteins Encoded by Sequences Specific to Mycobacterium avium subsp. paratuberculosis

ABSTRACT The development of immunoassays specific for the diagnosis of Johnes disease in cattle requires antigens specific to Mycobacterium avium subsp. paratuberculosis. However, because of genetic similarity to other mycobacteria comprising the M. avium complex, no such antigens have been found. Through a comparative genomics approach, 21 potential coding sequences of M. avium subsp. paratuberculosis that are not represented in any other mycobacterial species tested (n = 9) were previously identified (J. P. Bannantine, E. Baechler, Q. Zhang, L. Li, and V. Kapur, J. Clin. Microbiol. 40:1303-1310, 2002). Here we describe the cloning, heterologous expression, and antigenic analysis of these M. avium subsp. paratuberculosis-specific sequences in Escherichia coli. Nucleotide sequences representing each unique predicted coding region were amplified and cloned into two different E. coli expression vectors encoding polyhistidine or maltose binding protein (MBP) affinity purification tags. All 21 of the MBP fusion proteins were successfully purified under denaturing conditions and were evaluated in immunoblotting studies with sera from rabbits and mice immunized with M. avium subsp. paratuberculosis. These studies showed that 5 of the 21 gene products are produced by M. avium subsp. paratuberculosis and are antigenic. Immunoblot analysis with a panel of sera from 9 healthy cattle and 10 cattle with clinical disease shows that the same five M. avium subsp. paratuberculosis proteins are also detected within the context of infection. Collectively, these studies have used a genomic approach to identify novel M. avium subsp. paratuberculosis antigens that are not present in any other mycobacteria. These findings may have a major impact on improved diagnostics for Johnes disease.

Expression Library Immunization Confers Protection against Mycobacterium avium subsp. paratuberculosis Infection

ABSTRACT 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 (∼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.

Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle

BackgroundOur laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.ResultsSera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johnes disease.ConclusionCollectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.