Host responses to Mycobacterium avium subsp. paratuberculosis: a complex arsenal

The immune system is not always successful in recognizing and destroying pathogens it may encounter. Host immunity to mycobacteria is characterized by a very complex series of events, designed to clear the infection. The first line of defense is uptake and processing of the pathogen by macrophages, followed by the initiation of cell-mediated immunity. The secretion of pro-inflammatory cytokines such as IFN-gamma is credited with containment of mycobacterial infections. Yet it is clear that activated T-cells may contain but fail to clear the infection in some hosts. Further, it is recognized that if infection progresses to a more clinical state, the production of pro-inflammatory cytokines is suppressed and expression of anti-inflammatory cytokines is increased. It is unclear what defines a host that can successfully contain the infection versus one that succumbs to severe immunopathologic disease. This review will address some of the key elements in host immunity to mycobacterial pathogens, with an emphasis on Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), in an attempt to understand the dialogue between immune cells and their mediators during infection and what causes this discourse to go awry.     

Divergent cytokine responses of macrophages to Mycobacterium avium subsp. paratuberculosis strains of Types II and III in a standardized in vitro model

Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1?, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1? and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1β mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.     

Variation in the immuno-pathological responses of lambs after experimental infection with different strains of Mycobacterium avium subsp. paratuberculosis

Ruminant infection by Mycobacterium avium subsp. paratuberculosis (MAP) causes a granulomatous inflammatory response in the intestine and associated lymph nodes. Differences either in the affected organs or in the inflammatory infiltrate were observed between species and individuals. Such differences are usually attributed to variations in host immune responses or to inconsistent effects among different MAP strains. To evaluate if different MAP strains induce different immuno-pathological responses in lambs, 28 one-month-old individuals were divided into six groups and inoculated with different MAP strains. Groups 1 and 2 were inoculated with two bovine strains isolated in Argentina that showed different genetic patterns after BstEII-IS900-RFLP (hereafter strains E and A respectively). Group 3 was inoculated with a bovine strain isolated in Spain obtained after a previous step of culture (patterns C1). Group 4 was inoculated with a homogenate of intestinal mucosa of a clinical case affected by the same bovine strain as that of group 3. Group 5 was inoculated with an ovine strain that was directly purified from the intestinal mucosa of a clinical case, and group 6 was kept as control (i.e. no inoculation). Peripheral immune responses were assessed until 150 days post-infection (dpi), when lambs were humanely killed. Pathological studies were performed in tissues from the intestine and lymph nodes. Lesion types and inflammatory infiltrates were examined as indicators of pathogenicity. All the lambs infected with bovine MAP strains showed a common lesion pattern regardless of the strain type. Such pattern was characterized by focal lesions mainly in the mesenteric lymph nodes, the presence of fibrous tissue, and, occasionally, necrosis in the granulomas as well as the presence of numerous giant cells. Differences in lesion severity were observed among groups: lambs from groups 1 and 2 had the highest number of granulomas and the largest lymph node area affected. Lesions in animals from group 5 (infected with an ovine strain) were more severe and occurred mostly in the intestinal lymphoid tissue; necrosis, fibrosis or giant cells were never detected in this group. These results indicate that the MAP strain type induces different pathological responses in lambs.     

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Measurement of secreted interferon (IFN)-gamma has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-gamma assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-gamma were observed. Intradermal injection resulted in significant increases in secreted IFN-gamma for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-gamma was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-gamma was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-gamma, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.     

Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) to identify individual proteins within the complexes. Identity of individual proteins within complexes was further confirmed by MS upon excision of spots from 2D SDS-PAGE gels. Among the seven putative membrane complexes observed, major membrane protein (MAP2121c), a key MAP antigen involved in invasion of epithelial cells, was found to form a complex with cysteine desulfurase (MAP2120c). Other complexes found included those involved in energy metabolism (succinate dehydrogenase complex) as well as a complex formed by Cfp29, a characterized T cell antigen of Mycobacterium tuberculosis. To determine antigenicity of proteins, Western blot was performed on replicate 2D SDS-PAGE gels with sera from noninfected control cows (n = 9) and naturally infected cows in the subclinical (n = 10) and clinical (n = 13) stages of infection. Clinical animals recognized MAP2121c in greater proportion than subclinical and control cows, whereas cysteine desulfurase recognition was not differentiated by infection status. To further characterize antigenicity, recombinant proteins were expressed for 10 of the proteins identified and evaluated in an interferon-gamma (IFN-γ) release assay as well as immunoblots. This study reveals the presence of protein complexes in the cell envelope of MAP, suggesting protein interactions in the envelope of this pathogen. Furthermore the identification of antigenic proteins with potential as diagnostic targets was characterized.     

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.     

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.     

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.     

Use of long-term vaccination with a killed vaccine to prevent fecal shedding of Mycobacterium avium subsp paratuberculosis in dairy herds

OBJECTIVES: To determine whether vaccination with a killed vaccine prevents fecal shedding of Mycobacterium avium subsp paratuberculosis, to compare effectiveness of a culture and cull program in vaccinated and nonvaccinated herds, and to compare paratuberculosis-related preventive management in vaccinated and nonvaccinated herds. SAMPLE POPULATION: 58 commercial Dutch dairy herds. DESIGN: Cross-sectional study (study A) in vaccinated (n = 25) and nonvaccinated (29) herds of dairy cows. Longitudinal study (study B) in vaccinated (n = 2) and nonvaccinated (2) herds of dairy cows. PROCEDURE: In study A, fecal samples were obtained from adult cows in herds with and without a history of vaccination with a killed vaccine. Management measures were evaluated. In study B, fecal samples were obtained 4 times at 6-month intervals from cows older than 6 months. Cows that had positive test results were removed from the herd directly after the outcome of the culture. RESULTS: In study A, differences were not detected among the 25 herds that were vaccinated; culture results were positive for M avium subsp paratuberculosis in 4.4% of herds. In 29 herds that had not been vaccinated, culture results were positive in 6.7%. In study B, the percentage of positive results on culture decreased from 10.9% and 5.7% to 3.5% and 0%, respectively in the 2 vaccinated herds. In the 2 nonvaccinated herds, percentages decreased from 6.1% and 16.5% to 0% and 2.3%, respectively. Management practices were different between herds that were vaccinated and herds that were not; owners of herds that were not vaccinated followed more preventive management procedures and practiced less feeding of raw milk to calves. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination of calves with a killed vaccine does not prevent transmission of M avium subsp paratuberculosis; therefore, hygienic practices remain essential in herd management.     

Review of Mycobacterium avium subsp. paratuberculosis antigen candidates with diagnostic potential

Mycobacterium avium subsp. paratuberculosis (MAP) is a slow growing bacterium that can infect ruminants and remain latent for years without development of any clinical signs or disease. Diagnosis is often based on detection of MAP antibodies in milk or serum samples or culture of bacteria from faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen candidates described to date with special emphasis on antigen candidates tested for CMI responses. Relevant information on 115 different MAP antigens was systematically extracted from literature and summarized in 6 tables of CMI antigens, secreted antigens, cell wall and membrane antigens, lipoprotein antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified for this application. Most of the novel diagnostic candidates were evaluated in few animals and it is recommended that an appropriate sample size is included for evaluation of antigen candidates in future studies.     

Lewis rats are not susceptible to oral infection with Mycobacterium avium subsp. paratuberculosis

Pathogenesis studies of Mycobacterium avium subsp. paratuberculosis infection in ruminants are hampered by the long incubation time of the disease. A laboratory animal model with a shorter incubation time would facilitate research in this field. Although small rodents are usually considered to be resistant to M.a. paratuberculosis infection, several susceptible murine strains have been found. To our knowledge, there are no detailed reports with regard to susceptibility in rats. The Lewis rat is a valuable model for inflammatory bowel disease studies as well as autoimmune diseases involving mycobacteria as inducing agents. In this study Lewis rats were used to investigate their potential as a small laboratory animal model for paratuberculosis.In total 28 female Lewis rats were orally inoculated with M.a. paratuberculosis. The rats were first inoculated at 3 weeks of age, and 12 more inoculations followed in increasing intervals during the 3 months to follow. Eight control rats received a sham inoculation. Over 9 months, two rats from each group were sacrificed at regular intervals and immunological and histopathological examinations were performed on the gastrointestinal tract, the liver and the spleen.None of the rats developed lesions which were indicative of mycobacterial infection as determined by histology with HE and Ziehl-Neelsen staining. The bacteria could not be recultured from samples taken from the gut, the liver or the spleen. The immunological tests however, showed that bacteria had entered via the intestinal tract. From this study it appears that Lewis rats are resistant to oral inoculation with M. a. paratuberculosis, and not suitable as a model to study the immunopathogenesis of paratuberculosis as it occurs in ruminants.     

Modulation of gammadelta T cells and CD1 in Mycobacterium avium subsp. paratuberculosis infection

M.a. paratuberculosis is the causal agent of paratuberculosis (Johne's disease). Recent work has suggested that gammadelta T cells may play an important role in the early immunological response to mycobacterial diseases, and that CD1 may act as a non-classical MHC molecule in antigen presentation to these gammadelta T cells. Experimental infection of neonatal lambs with M.a. paratuberculosis was used to investigate the changes in gammadelta T cells and CD1 molecules in the gut associated lymphoid tissue 4 weeks after inoculation. Immunohistochemistry was used to label the gammadelta lymphocytes and CD1 molecules. An increase in the number of gammadelta T cells was noted in both the jejunal and ileal Peyer's patches in the gut of infected lambs, but no statistically significant change was found in the mesenteric lymph nodes. There were no obvious changes in the CD1 molecules in any tissue. This work suggests that gammadelta T cells may play a role in the initial immunological events of paratuberculosis infection.     

In utero infection of cattle with Mycobacterium avium subsp. paratuberculosis: a critical review and meta-analysis

Mycobacterium avium subsp. paratuberculosis (Mptb) causes Johne's disease in ruminants. Disease control programmes aim to break the faecal-oral cow-calf transmission cycle through hygienic calf rearing and removal of affected cows from the herd, but these programmes do not take account of the potential for congenital infection. The aims of this study were to critically review research on in utero infection, determine the prevalence of fetal infection in cattle through meta-analysis and estimate the incidence of calves infected via the in utero route. About 9% (95% confidence limits 6-14%) of fetuses from subclinically infected cows and 39% (20-60%) from clinically affected cows were infected with Mptb (P<0.001). These are underestimates for methodological reasons. The estimated incidence of calf infection derived via the in utero route depends on within-herd prevalence and the ratio of sub-clinical to clinical cases among infected cows. Assuming 80:20 for the latter, estimates of incidence were in the range 0.44-1.2 infected calves per 100 cows per annum in herds with within-herd prevalence of 5%, and 3.5-9.3 calves in herds with 40% prevalence. These estimates were not markedly sensitive to the value chosen for the proportion of clinical cases. In utero transmission of Mptb could retard the success of disease control programmes if the opportunities for post natal transmission via colostrum/milk and environmental contamination were able to be controlled. The consequences of fetal infection for the calves so infected are discussed in the context of diagnosis and vaccination together with recommendations for future research.     

Novel antigens for detection of cell mediated immune responses to Mycobacterium avium subsp. paratuberculosis infection in cattle

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring specific cell mediated immune responses, using the whole blood interferon-γ (IFN-γ) assay. Available IFN-γ assays use purified protein derivative of MAP (PPDj) which are complex antigen mixtures with low specificity. The objectives of this study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The study included blood samples from 26 heifers from a MAP infected herd, collected three times with four to five-week intervals, and blood samples from 60 heifers of a non-infected herd collected once. Heifers of the non-infected herd were used to establish cut-off values for each antigen. The case definition was an animal with ≥ 2 positive tests for ≥ 4 antigens, resulting in 13 cases and 13 non-cases. IFN-γ levels of cases were higher compared to IFN-γ levels of non-cases (P<0.05). The results of the IFN-γ assay using PPDj did not correlate well with the results using the novel antigens. PPDj produced elevated IFN-γ responses of samples from both the non-infected and the MAP infected herd, indicating unspecific IFN-γ responses and showed low consistency. Three latency proteins, LATP-1, LATP-2 and LATP-3 gave positive IFN-γ tests that correlated very well with the case definition suggesting high immunogenicity. Three tested antigens, LATP-2, MAP-1 and MAP-2 have no homologue in the M. avium subsp. avium or M. bovis genome and could be promising diagnostic antigens, especially LATP-2 correlated highly with the case definition.     

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.     

Comparison of DNA probe test and cultivation methods for detection of Mycobacterium avium subsp paratuberculosis in caprine and ovine faeces

OBJECTIVE: To compare a DNA probe test with two cultivation methods for the detection of Mycobacterium avium subsp paratuberculosis in goat and sheep faeces. DESIGN: Comparison of the results of the three methods with histological examination as the reference standard. PROCEDURE: Faecal specimens were obtained from goats and sheep originating from flocks known to be affected with paratuberculosis and tested for Mycobacterium avium subsp paratuberculosis with a DNA probe test and two cultivation methods (old conventional culture and new double incubation method in Herrold's and Lowenstein-Jensen medium). RESULTS: In goats, the sensitivity of the various tests were for the DNA probe test 17.2%, for the double incubation culture method 25.4% and for the old conventional culture method 22.8% using the histopathological results as reference. In sheep the sensitivity of the various tests were for the DNA probe test 13.2%, for the double incubation culture method 8.8% and for the old conventional culture method 5.9% using the histopathological results as reference. The specificity of the above tests was 100% in goats and sheep and the specificity of the double incubation culture method in goats was 91%. CONCLUSIONS: The DNA probe test is a rapid and specific test that could be used in a control program if the sensitivity of the test were improved and possibly in combination with another test.