Antigen specificity of T-cell response to Mycobacterium avium infection in mice

T cells from Mycobacterium avium-infected C57BL/6 mice reacted to culture filtrate, envelope, and cytosol proteins and to fractions obtained from these proteins. Multiple targets were recognized, such as 29- to 45-kDa and <21-kDa antigens of the culture filtrate, antigens of around 30 kDa in the envelope and cytosol, and 45- to 116-kDa proteins in the envelope.     

Oral infection of ferrets with virulent Mycobacterium bovis or Mycobacterium avium: susceptibility, pathogenesis and immune response

Ferrets are important wildlife reservoirs of tuberculosis in New Zealand, where they acquire infection primarily through scavenging infected carrion. In the present study, groups of laboratory-reared ferrets were infected orally with 5 x 10(6)colony-forming units of Mycobacterium bovis or Mycobacterium avium. Body weight and tuberculin-specific immune reactivity were monitored at intervals (pre-infection, and 4 and 20 weeks post-infection) and animals were killed at 20 weeks post-infection for post-mortem, histopathological and bacteriological examinations. Weight loss was significantly greater in M. bovis -infected than in M. avium -infected ferrets. M. bovis, unlike M. avium, sometimes produced gross necrotic lesions in the mesenteric lymph nodes. M. bovis invariably produced microscopical foci of mycobacterial infection or tissue necrosis typical of tuberculosis, whereas M. avium did so in only one of nine animals. Mycobacteria were recovered from the lymphatic tissues of all M. bovis -infected ferrets but from only five of nine M. avium -infected animals; and the mean bacterial burdens of the lymph nodes of the head and intestinal regions were > 10-fold and > 100-fold greater, respectively, for M. bovis -infected than for M. avium -infected animals. M. bovis, unlike M. avium, evoked tuberculin-specific peripheral blood lymphocyte reactivity and serum antibody responses.     

Pathogenesis of Mycobacterium avium infection

Studying infections with Mycobacterium avium in mouse models has allowed the dissection of the antimycobacterial pathways of the mammalian host. Whereas the paradigm of cell-mediated immunity to intracellular pathogens has been confirmed, namely with regard to the pivotal roles of CD4⁺ T cells, macrophages, and the IL 12-IFNγ cytokine axis, atypical features have been uncovered such as the resistance to NO, the involvement of minor players in the induction of type 1 protective immunity (such as TLR2, CD40, and CD30), and the development of immunopathology during the infection with highly virulent strains such as the development of caseous necrosis of granulomas or the progressive emergence of severe lymphopenia.     

Activation of macrophages and interference with CD4+ T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen-specific CD4+ T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 or CD40 was found. However, the antigen-specific stimulatory capacity of J774 cells for a CD4+ T-cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T-cell hybridoma expressing a T-cell receptor identical to that of the T-cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4+ T-cell line is influenced by the pathogenic bacterium M. ptb.     

Effect of different cell fractions of Mycobacterium avium and vaccination regimens on Mycobacterium avium infection

Because of the availability of uniform genetic stocks and the ability to modulate stress levels, chickens were investigated as a host for the development of an antimycobacterial vaccine. The imposition and the timing of stress significantly influenced the outcome of Mycobacterium avium infection in chickens. Simple, whole cell or lysate vaccines and combinations of vaccine preparations were identified that led to high levels of protection. In addition, short-term stress at the time of vaccination significantly increased the protective efficacy of M. avium vaccine preparations. Post-infection vaccination of M. avium-infected chickens was also shown to significantly reduce the number of lesions and colony counts.     

Inflammatory response following intranasal infection with Mycobacterium avium complex: role of T-cell subsets and gamma interferon.

The role of CD4+ and CD8+ T cells in the response to intranasal infection with a Mycobacterium avium complex isolate (MAC) was investigated. Depletion of CD4+ T cells by injected antibody exacerbated infection in the lung, spleen, and liver. There were decreased numbers of inflammatory cells in the lungs of CD4-depleted mice and a significant decrease in lung cytotoxic activity. The neutrophil response was unaffected, and in CD4-depleted mice, unlike intact infected mice, these cells were found with large numbers of associated MAC. Purified CD4+ splenic T cells produced gamma interferon (IFN-gamma) in vitro in response to MAC antigen. IFN-gamma production by cultured spleen, lung, or mediastinal lymph node cells was markedly reduced in CD4-depleted mice. In contrast, CD8+ T cells did not produce IFN-gamma in vitro, and depletion of CD8+ T cells from infected mice had no effect on bacterial growth or lung cell activation. Depletion of IFN-gamma by injected monoclonal antibody had effects similar to those of CD4 depletion, namely, exacerbation of infection and decreased lung cell cytotoxicity. We conclude that CD4+ T cells are the main T cells involved in the lung response to MAC infection and that this response is at least partially dependent on the production of IFN-gamma.     

The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin.

Murine bone marrow-derived macrophages (Mphis) infected with virulent strains of Mycobacterium avium (TMC 724 and a human clinical isolate) or with an avirulent opaque variant that spontaneously dissociates from the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The efficiency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment period. Growth was assessed by determination of CFU of intracellular bacteria and by a quantitative ultrastructural analysis which allowed us also to determine the extent of bacterial degradation. A similar treatment was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CFU within 7 days of treatment. When applied to Mphis infected with virulent M. avium, clarithromycin immediately arrested bacterial growth but was unable to fully kill and degrade intracellularly growing virulent bacteria. After 21 days of treatment, 25% of intracellular bacteria were still morphologically intact. These bacteria resumed growth upon removal of the antibiotic, with a normal replication rate. These bacteria had not become more resistant to the drug, since the MIC was unchanged as compared to the one determined for the initial stock used to infect Mphis. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation. We propose that the inability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosomes in which clarithromycin accumulates. In accord with our proposal, we show that the avirulent opaque variant, which does not prevent phagosome-lysosome fusions (unpublished data), is finally destroyed by clarithromycin even within the phagosomal environment.     

Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic.

Mycobacterium tuberculosis and Mycobacterium avium multiply in cultured human macrophages (MP) within membrane-enclosed vesicles. These vesicles are generally assumed to be acidic. The evidence most frequently cited for this assumption is that pyrazinamide, which requires an acid pH to be effective, is effective and streptomycin, which loses most of its activity at a low pH, is poorly effective against tubercle bacilli. This assumption was tested by using the two weak bases chloroquine and NH4Cl to raise the pH of acidic vesicles in MP experimentally infected with M. tuberculosis or M. avium. An immunocytochemical locator of acidic regions in the MP was used to monitor the association of intracellular bacilli with acidity. MP were infected with M. tuberculosis or M. avium and incubated with various combinations of the drugs and the weak bases. Replication of the bacteria in the MP was measured by culture counts. Intracellular associations of the mycobacteria with acidity were assessed by electron micrographs and by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, which was detected with colloidal gold-labeled antibodies. It was confirmed by immunocytochemistry that both chloroquine and NH4Cl raise the pH of acidic vesicles in the infected MP. However, neither caused any pH-related change in the antimycobacterial activities of pyrazinamide or streptomycin or of the pH-independent drug isoniazid. Immunochemical analyses showed acidity to be associated with killed but not living mycobacteria in the MP. These findings suggest that living M. tuberculosis and M. avium are located in human MP in vesicles which are not acidic.     

A PE protein expressed by Mycobacterium avium is an effective T-cell immunogen

Infection of mice with Mycobacterium avium or immunization with a novel PE gene expressed by M. avium (MaPE) showed that a dominant T-cell immune response was elicited. Immunization with an MaPE DNA vaccine protected mice against an aerosol challenge with Mycobacterium tuberculosis, suggesting that mycobacteria express PE antigens with cross-protective T-cell epitopes.     

Mycobacterium avium infection in BALB/c and SCID mice.

BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Mycobacterium avium and the numbers of cfu were monitored for 70 days in spleen, liver, lung, kidney, brain and peritoneum. While BALB/c mice formed typical granulomas and controlled bacterial growth in organs, a delay in development of lesions and a modest containment of infection were observed in SCID mice. In the spleen of BALB/c mice, in which bacterial growth was contained, macrophages (Mo) and natural killer (NK) cell numbers increased > or = 4.2 times and T- and B-cell numbers increased > or = 1.8 times after 42 days of infection; conversely, a low recruitment of mononuclear cells was observed in the spleen of SCID mice, where M. avium proliferated efficiently. Unlike visceral organs, a pronounced decrease in the number of cfu was observed in the peritoneum of BALB/c mice, concomitantly with a > or = 31.7-fold increase in Mo and NK cells and a > or = 9.1-fold increase in T and B cells. In the peritoneum of SCID mice only a bacteriostatic effect was observed despite a > or = 56.7-fold increase in Mo and NK cells and a > or = 22.3-fold increase in T and B cells. These results suggest that while an intact immune response can efficiently control M. avium infection in the spleen and peritoneum of BALB/c mice, cells of the innate immune system such as Mo and NK cells play a role in the containment of bacterial growth in the peritoneum, but not spleen, of SCID mice.     

Effect of oestrogen on Mycobacterium avium complex pulmonary infection in mice

The purpose of the present study was to elucidate the role of oestrogen in the pathogenesis of Mycobacterium avium complex (MAC) pulmonary disease, which occurs most frequently in postmenopausal women. The study was carried out in a murine infectious model using ovariectomized DBA/2 female mice. Infection with MAC was established by intratracheal administration of bacilli. In some experiments, ovariectomized mice were treated with exogenous 17 beta-estradiol (E2). The number of bacilli in the lungs of infected mice which received ovariectomy was significantly larger than that in the lungs of sham-operated control mice, and treatment of ovariectomized mice with exogenous E2 restored the burden of bacilli to the same level as that in the sham-operated control mice. We next examined the effect of E2 in vitro using bone marrow-derived macrophages obtained from DBA/2 female mice. The macrophages showed bacteriostatic activity against MAC after treatment with interferon-gamma (IFN-gamma) and this activity was further enhanced by the exogenous addition of E2 to the culture medium. In parallel with these findings, E2 augmented the production of reactive nitrogen intermediates (RNI) by macrophages pretreated with IFN-gamma and stimulated with MAC, as shown by evaluating nitrite production and inducible nitric oxide synthase mRNA expression. These findings taken together suggest that absence of endogenous oestrogen appears to be responsible for the development of MAC pulmonary disease in this mouse model and that the enhancement by E2 of anti-MAC activity of murine macrophages induced through increased RNI production may play some role in resistance to MAC infection.     

Gamma interferon is essential for clearing Mycobacterium genavense infection

Factors determining the in vivo replication of the opportunistic pathogen Mycobacterium genavense are largely unknown. Following intravenous injection of a patient isolate, M. genavense could not be recovered by culture or detected by PCR in the livers or spleens of infected BALB/c mice. In contrast, M. genavense was found to chronically persist and multiply in the livers and spleens of intravenously infected syngeneic gamma-interferon-gene-deficient (GKO) mice as evidenced by acid-fast stains of infected tissues and recovery by both PCR and liquid culture following organ homogenization. In GKO mice, M. genavense elicited a chronic inflammatory response, resulting in marked splenomegaly and extensive lymphadenopathy. Granulomatous lesions in the livers of GKO mice were diffuse, were composed of monocytes, neutrophils, and CD3(+) cells, and were histochemically negative for inducible nitric oxide synthase.     

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.     

Intramacrophage growth of Mycobacterium avium during infection of mice.

Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment.     

Generalized immunological decline during long-term experimental infection with Mycobacterium avium

Terminal loss of immune responsiveness in C57BL/10 mice intranasally infected with Mycobacterium avium was observed in both spleen and lung. It was nonspecific and related to the duration of infection, not the age of the mice. While there was loss of total T cells, the remaining cells were less efficient at gamma interferon production.     

Multigenic control of disease severity after virulent Mycobacterium tuberculosis infection in mice

Following challenge with virulent Mycobacterium tuberculosis, mice of the I/St inbred strain exhibit shorter survival time, more rapid body weight loss, higher mycobacterial loads in organs, and more severe lung histopathology than mice of the A/Sn strain. We previously performed a genome-wide scan for quantitative trait loci (QTLs) that control the severity of M. tuberculosis-triggered disease in [(A/Sn x I/St) F1 x I/St] backcross-1 (BC1) mice and described several QTLs that are significantly or suggestively linked to body weight loss. In the present study we expanded our analysis by including the survival time phenotype and by genotyping 406 (A/Sn x I/St) F2 mice for the previously identified chromosomal regions of interest. The previously identified 12-cM-wide QTL on distal mouse chromosome 3 was designated tbs1 (tuberculosis severity 1); the location of the QTL on proximal chromosome 9 was narrowed to a 9-cM interval, and this QTL was designated tbs2. Allelic variants of the tbs2 locus appeared to be involved in control of both body weight loss and survival time. Also, the data strongly suggested that a QTL located in the vicinity of the H-2 complex on chromosome 17 is involved in control of tuberculosis in mice of both genders, whereas the tbs1 locus seemed to have an effect on postinfection body weight loss in female mice. Interestingly, these loci appeared to interact with each other, which suggests that there might be a basic genetic network for the control of intracellular parasites. Overall, linkage data reported here for F2 mice are in agreement with, and add to, our previous findings concerning the control of M. tuberculosis-triggered disease in the BC1 segregation.     

Multidrug therapy of Mycobacterium avium subsp. avium infection in experimentally inoculated budgerigars (Melopsittacus undulatus)

The aim of the study was to determine whether the four-month experimental therapy of mycobacteriosis in budgerigars may cause a complete recovery. A group of nine budgerigars was infected with a Mycobacterium avium subsp. avium isolate with proven pathogenicity for budgerigars. Five weeks post-inoculation, multidrug therapy was started. Another group comprising six birds received the same treatment but no infection, and the third group also comprising six birds was kept without infection or treatment as a control. The adopted antibiotic regimen included clarithromycin 61?mg/kg b.w., moxifloxacin 25?mg/kg b.w. and ethambutol 60?mg/kg b.w. administered by crop gavage every 12?h for 18 weeks. Despite a significant improvement in the condition of the infected, treated birds, the four-month therapy was not sufficient for the complete recovery of all.