Pulmonary Tuberculosis Apparently Caused by the Avian Tubercle Bacillus

Avian tubercle bacilli were repeatedly isolated from the sputum of a 65-year-old man who had cavitary disease in the upper lobe of the right lung. The clinical picture resembled that of pulmonary tuberculosis caused by the human type of tubercle bacilli, but the response to antituberculosis chemotherapy was unsatisfactory and the patient''s sputum remained positive. The bacilli were markedly pathogenic to chickens and rabbits but failed to produce progressive disease in guinea pigs. This is in accord with the properties of tubercle bacilli of the avian type.     

Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).     

Granuloma Formation Induced in Mice by Chemically Defined Mycobacterial Fractions

Amounts of trehalose-6,6-dimycolate as small as 1 to 5 mug can, after intravenous injection, induce in the lungs of mice formation of tubercles in which the cellular composition is indistinguishable from that in tubercles formed after an infection with living BCG bacilli. The strongest cellular response in mice was induced by cord factor from Mycobacterium kansasii; the weakest was induced by cord factor from the BCG strain of M. bovis. It was found that three intravenous injections of cord factor induced a more extensive cellular response than did one injection of the same total amount of cord factor. Mice treated intravenously with cord factor were protected against an intravenous challenge with the virulent H37Rv strain of M. tuberculosis. The cellular response in the lungs of mice to intraperitoneal injections of living BCG and cord factor was very weak compared with that after intravenous injections. Intraperitoneal vaccination of mice with cord factor did not protect the mice against a challenge with virulent tubercle bacilli. Mice vaccinated intraperitoneally with BCG were immunized although no granulomas, or very few, were present in the lungs at the time of the challenge. The significance of the cellular response induced by cord factor is discussed.     

Lysosomal enzymes and microbicidal capacity of activated macrophage.

A relation was sought between acid phosphatase contents and the presence of tubercle bacilli inside the peritoneal exudate cells (PEC) of normal guinea pigs and those immunized with BCG. This was done to investigate the role lysosomal enzymes play in the microbicidal capacity of the cell. In both normal and immune animals tubercle bacilli were present only in those PEC that contained acid phosphatase. Cells without acid phosphatase did not contain bacilli. Thus, only activated cells ingested bacilli. Under the conditions of these experiments, macrophage activation, as indicated by the presence of acid phosphatase, was not related to the immune status of the animal. Similarly, stimulation by ingestion of tubercle bacilli was not significant. Also, the number of acid phosphatase grains/cell did not influence the number of bacilli/cell. Thus, the acid phosphatase content of the cell did not correlate with the number of bacilli inside the cell. It was concluded that acid phosphatase may not be one of the factors that contribute to the microbicidal capacity of the cell.     

Immunological memory in tuberculosis. I. Influence of persisting viable organisms

Rats were immunized with 10⁸ BCG and treated with rifampicin plus isoniazid for 9 months; similarly immunized subjects received no chemotherapy. Immediately before the institution of chemotherapy and at predetermined intervals thereafter, the immunological status of these two groups of animals was evaluated with respect to tuberculin hypersensitivity, resistance to reinfection with virulent tubercle bacilli (H37Rv), and the capacity of thoracic duct lymphocytes to consider specific antimicrobial resistance on normal recipients. Treatment with antituberculosis drugs had very little influence upon these parameters of acquired immunity despite the fact that living BCG were eradicated from the animals given chemotherapy. It is concluded that immunological memory in tuberculosis does not depend upon the persistence of viable tubercle bacilli.     

Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or α-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Assessment of the N-PCR Assay in Diagnosis of Pleural Tuberculosis: Detection of M.tuberculosis in Pleural Fluid and Sputum Collected in Tandem

Background

The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized.

Methodology/Principal Findings

Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation.

Conclusions/Significance

To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.     

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.     

Turnover of hyaluronan in the rabbit pleural space.

Hyaluronan influences lung fluid balance. The clearance of lung hyaluronan by way of the pulmonary lymphatics and the pleural space is increased when fluid flux into the interstitium is increased. The purpose of this study was to determine the rate at which hyaluronan is removed from the pleural space. We injected hyaluronan, labeled with tritium in the acetyl group, into the pleural space of six rabbits. The appearance of [3H]H2O in serum was measured over time to calculate the turnover rate of hyaluronan. We found that the half-lives ranged between 8 and 15 h and were positively related to the amount of hyaluronan injected. At the end of the experiment, the contralateral pleural space was irrigated to determine the amount of pleural space hyaluronan, which was 0.3 micrograms/kg body wt.     

Automated identification of tubercle bacilli in sputum. A preliminary investigation

OBJECTIVE: To use an automated method to detect tubercle bacilli in sputum specimens. STUDY DESIGN: Using fluorescence microscopy, tubercle bacilli were identified on auramine-stained sputum specimens. Images were then captured with a digital camera and enhanced through imaging processing techniques. The bacilli were recognized using neural network classifiers. RESULTS: This preliminary investigation demonstrated a sensitivity of 94.1% for the identification of individual bacilli. As there are usually fairly numerous tubercle bacilli in the sputum of patients with active pulmonary tuberculosis, the overall diagnostic accuracy of sputum smear-positive patients can be expected to be very high. CONCLUSION: Potential benefits of automated screening for TB bacilli are: rapid, accurate, inexpensive diagnosis; the ability to screen larger numbers of people; increased resources to monitor patients; and reduction in health risks to staff.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Effect of a single exposure to ultraviolet radiation on Mycobacterium bovis bacillus Calmette-Guerin infection in mice

BALB/c and C3H mice were exposed on the dorsal skin to 45 kJ/m2 of UVB radiation from FS-40 sunlamps 3 days before infection with 1 x 10(6) live units of Mycobacterium bovis bacillus Calmette-Guérin (BCG) (Tice strain) in the footpad. At regular intervals, groups of mice were tested for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli, and the course of infection was monitored by measuring the size of the infected footpad, enlargement of the draining lymph node, and the number of bacteria in the spleen and lymph node. In both strains the DTH response to PPD was significantly delayed in UV-treated mice compared to unirradiated mice, when tested 21 and 42 days after BCG infection. By day 50, no significant difference was detected in the DTH response between irradiated and unirradiated mice. UV treatment reduced the size of the lymph node draining the site of BCG infection in both strains of mice and the size of the infected footpad in C3H mice but not in BALB/c mice. In both strains of mice the total number of bacteria in the spleen and the draining lymph node increased after UV irradiation. When irradiated 3, 5, 18, or 21 days after BCG infection, BALB/c mice also showed a significant decrease in their DTH response to PPD, indicating that the UV-induced suppression of BCG occurs both at the induction and the elicitation stages of the immune response. Thus, mice exposed to a single dose of UV radiation either before or after BCG infection showed an impaired DTH response to mycobacteria, which was accompanied by an increase in the multiplication of bacteria in the tissues, even though the organisms were introduced at an unirradiated site. These studies demonstrate that a systemic effect of UV irradiation can interfere with the development and expression of immunity to pathogenic bacteria in mice.     

Liquid and protein dynamics using a new minimally invasive pleural catheter in rabbits.

To obtain continuous access to the pleural space without causing injury, we tested a new transdiaphragmatic pleural catheter for its ability 1) to drain the pleural space without injury and 2) to drain liquid at a rate equal to normal pleural liquid production. In 13 anesthetized rabbits, we opened the abdomen and dissected through the diaphragm to insert a flared-tip catheter into the ventral pleural space on one side and then turned the rabbit prone. In 10 of the rabbits (8 for 6 h, 2 for 24 h), we continuously collected draining pleural liquid, and in 3 rabbits (6 h), we did not open the catheter. We injected radiolabeled albumin intravenously as a protein marker. Terminally, we collected pleural liquid from both pleural spaces and lavaged for total radioactivity. In 14 awake control rabbits without catheters, we measured normal pleural liquid production by the rate of equilibration of radiolabeled albumin from plasma to pleural liquid. We found that, although the percentage of neutrophils was increased on the side with the catheter (54 vs. 1% in control rabbits), the pleural liquid volume, protein concentration, specific activity of albumin, and total radioactivity in the pleural space were the same on the side with the catheter as on the opposite side and in the control rabbits. The liquid flow rate through the catheter over 6 h was 53 +/- 23 microliters/h [0.017 +/- 0.008 (SD) ml.kg-1.h-1], which was not significantly different from the computed rate of normal pleural liquid production in the control rabbits, 49 +/- 14 microliters/h (0.016 +/- 0.004 ml.kg-1.h-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the MER tubercle bacillus fraction on the production of antibodies in vitro: I. Effect on the primary response

The effect of the methanol extraction residue (MER) fraction of BCG tubercle bacilli on the primary antibody response in vitro to sheep red blood cells (SRBC), TNP conjugates, and the monovalent hapten DNP-glycine was studied. Addition of MER to whole splenocyte cultures simultaneously with antigen presentation potentiated the antibody response to SRBC and TNP-SRBC, and facilitated reactivity to DNP-glycine; there was no effect on the response to the T-independent entity TNP-LPS (lipopolysaccharide from E. coli 055-B5). Immunopotentiating activity of MER for SRBC and DNP-glycine was also evident in macrophage-depleted cultures. Peritoneal exudate cells (PEC) taken from MER-treated donors were more efficient than PEC from untreated donors in reconstituting antibody formation to SRBC by macrophage-depleted spleen cell populations. The results obtained indicate that activation of both macrophages and of certain lymphocyte population(s) by MER may play a role in the potentiation of antibody responsiveness in vitro by this agent.     

Pleural space thickness in situ by light microscopy in five mammalian species

The thickness of the pleural space was measured by a focusing method using a light microscope (X157, 2.5-micron depth of focus). In anesthetized animals, thin transparent parietal pleural windows were made by dissection of intercostal muscle. Multiple postmortem measurements were made of the combined thickness of the pleural space and the window by focusing in sequence on the lung surface and on 1- to 2-micron tantulum particles sprayed on the window. The window thickness was measured after creating a pneumothorax and retracting the lungs. In supine rabbits the pleural space measured at various heights on the costal surface was of uniform thickness (16 micron) except for a thicker region (62 micron) located within 3 mm of the most dependent part of the lung. The thicker region reverted to the uniform thickness after it was placed in a nondependent position by inverting the animal from the supine to prone position, indicating fluid drainage by gravity. In the prone position near midchest, pleural space thickness (t) averaged 6.9 micron in the mouse, 10.2 in the rat, 17.2 in the rabbit, 18.3 in the cat, and 23.6 in the dog. Animals of larger body mass (M, kg) had a wider pleural space: t = 13.1 X M0.20. There was no contact between the two pleurae, indicating that fluid lubrication facilitates sliding between the lung and chest wall. Based on the t vs. M relationship and estimates of the viscous flow of pleural liquid, pleural fluid exchange rate would be proportional to body mass and the work of sliding as a fraction of the work of breathing would be smaller in larger animals.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.     

Alteration of the immune response to Mycobacterium bovis BCG in mice exposed chronically to low doses of UV radiation

BALB/c mice were exposed on shaved dorsal skin to 1 minimal erythemal dose (MED) of UVB radiation (2.25 kJ/m2) from a bank of six FS-40 sunlamps three times per week. The total number of irradiations ranged from 1 to 27. At regular intervals, groups of mice were injected in the left hind foot pad with 1 x 10(6) live mycobacteria (Mycobacterium bovis BCG) 3 days after the last UVB exposure. The mice were tested 21 and 42 days after infection for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli by injecting PPD into the right hind foot pad and measuring the foot pad swelling 24 hr later. The course of infection was followed by assessing the number of bacterial colony forming units in the lymph node draining the site of BCG infection and the spleen. Mice exposed from 1 to 15 times to 1 MED of UV radiation showed a significant suppression in their DTH response to PPD compared with the unirradiated mice. At the same time, the number of bacterial colony-forming units in the lymph node and spleen of the UV-irradiated mice was greater than in control mice. With continued exposure to UVB, however, the DTH response recovered to a normal level, and there was no longer an increase in the number of viable bacteria in the lymphoid organs. These results indicate that early in the course of chronic UV irradiation, mice were impaired in their ability to mount a DTH response to BCG and to clear these bacteria from their lymphoid organs; later the mice recovered from these effects of UV, with continued treatment. A dose-response study using single doses of UV radiation indicated that a dose of 2.7 kJ/m2 suppressed the DTH response by 50%. Thus, exposure of mice to a single or multiple low doses of UV radiation prior to infection can interfere with systemic immunity to mycobacteria.