Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: II. Skin Cross-Reactivity in Sensitized Guinea Pigs

A study on skin cross-reactivity between stabilized ¹⁴C-labeled mycobacterial antigens, namely tuberculin purified protein derivative (PPD; from Mycobacterium tuberculosis), PPD-A (M. avium), PPD-Y (M. kansasii), PPD-G (M. scrofulaceum), PPD-B (M. intracellulare), and PPD-F (M. fortuitum), has been carried out in groups of guinea pigs sensitized with one of the following heat-killed mycobacteria: M. tuberculosis, M. avium, M. kansasii, M. scrofulaceum, M. intracellulare, or M. fortuitum. For each type of sensitization, the average response for the corresponding PPD antigen was higher than the average response for any of the other antigens. However, the responses to the heterologous PPD antigens were not necessarily significantly different among themselves, and the significant differences of the heterologous PPD antigens were distributed differently according to the type of sensitization. Therefore, ¹⁴C-PPD antigens skin cross-reacted in guinea pigs essentially in the same manner as reported by others for nonradioactive PPD antigens.     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: I. Preparation of 14C-Labeled Purified Protein Derivative Antigens and Their Adsorption to Glass

Biologically active (14)C-labeled purified protein derivative ((14)C-PPD) has been prepared from the culture filtrates of seven species of mycobacteria, namely Mycobacterium tuberculosis Johnston strain (PPD), M. bovis BCG (PPD-BCG), M. avium (PPD-A), M. kansasii (PPD-Y), M. intracellulare (PPD-B), M. scrofulaceum (PPD-G), and M. fortuitum (PPD-F). These mycobacteria were grown in a culture medium containing a mixture of (14)C-labeled amino acids. The yield and specific radioactivity of the PPD, of the nucleic acid, of the bacterial cells, and of the CO(2) developed during growth have been determined for each of the seven species of mycobacteria. Although the yields of (14)C-PPD antigens differed greatly for the different species of mycobacteria tested, their specific radioactivities were similar. The (14)C-PPD antigens have been used as a means to measure their adsorption to glass. When glass ampoules containing dilute solutions (0.001 mg of PPD per ml) of these PPD antigens (PPD, PPD-BCG, PPD-A, PPD-Y, PPD-G, PPD-B, and PPD-F) were stored for 12 months at 5 C, it was found that they all adsorbed equally well to glass surfaces. In fact, regardless of the origin of the PPD, a loss due to adsorption of about 90% occurred during the first month of storage, and thereafter the PPD content remained practically constant for the rest of the duration of the storage period. The addition of 0.0005% Tween 80 to the PPD solutions effectively reduced the adsorption to glass of most PPD antigens. However, adsorption of PPD-BCG was not quite so effectively prevented, even when the Tween 80 concentration was increased from 0.0005 to 0.0005%.     

Isolation of tuberculin peptides from tuberculin purified protein derivative (PPD).

Tuberculin purified protein derivative (PPD) obtained from the filtrate of Mycobacterium tuberculosis was hydrolysed with proteinase, trypsin, or chymotrypsin. Each hydrolysate consisted of a tuberculin peptides mixture (TPM). From each TPM 16 fractions were obtained by ion-exchange chromatography on Dowex 50W-X8 but only one fraction was isolated from each of the 16 fractions which showed tuberculin activity in guinea pigs sensitized with M. bovis (bcg) or M. tuberculosis. This fraction was designated "purified tuberculin peptide" (PTP). The PTP fraction from the proteinase hydrolysate (PTP-proteinase) was rechromatographed on Dowex 1-X2 and two tuberculin peptide fractions having molecular weights of 3200 and 12,000 were isolated. The potency of these two fractions was assessed in guinea pigs sensitized with M. bovis (BCG) and with M. tuberculosis and they were approximately 4 to 7 times more potent than either the international standaCG and of at least equal potency to either PPD-S or Connaught PPD in guinea pigs sensitized with either M. kansasii, M. scrofulaceum, M. intracellulare, or M. avium whereas very little if any cross-reactivity was elicited by these two fractions. This lack of response indicates that either fraction could be used as an aid to differentiate between sensitization due to M. tuberculosis or M. bovis and sensitization attributed to other mycobacteria.     

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

Tuberculin Activity of Mycobacterial Fractions Obtained by Chromatography

Commercial purified protein derivatives (PPD), old tuberculin (OT), the bacillary extract, and the culture filtrate of Mycobacterium tuberculosis H37Ra were submitted to Sephadex G-25 and diethylaminoethyl (DEAE)-cellulose chromatography. The ability of the fractions obtained to elicit delayed dermal hypersensitivity in M. tuberculosis H37Ra-infected guinea pigs was studied. Skin tests with Sephadex fractions in M. tuberculosis H37Ra-infected guinea pigs showed that the tuberculin activity was localized in the first fraction. All other Sephadex fractions were nonessential and nonspecifically irritating. Fractions from chromatography of Sephadex G-25 fraction 1 on DEAE-cellulose columns showed that all but the first were able to elicit delayed hypersensitivity reactions. There was a variability in the capacity to elicit the tuberculin reaction according to the fraction injected and the stage of tuberculous infection in guinea pigs. Compared to the others, the seven lots of commercial PPD were variable in composition and content. They contained both essential and nonessential materials for the tuberculin reaction. Sephadex fraction 1 would appear to be a better tuberculin as it excludes nonessential nonspecifically irritating elements and contains the complement able to elicit the tuberculin reaction. Its methodological simplicity would be economically advantageous.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Retention of 14C-Labeled Tuberculin Purified Protein Derivative in the Skin of Sensitized and Nonsensitized Animals

Tuberculin purified protein derivative labeled with (14)C ([(14)C]PPD) with a biological potency equivalent to the International Standard for tuberculin PPD was used to study the retention of tuberculin PPD in the skin of sensitized and nonsensitized animals. We found that [(14)C]PPD was almost entirely cleared from the skin test site during the first 18 to 24 h after injection and that when approximately 5% of the initial concentration of [(14)C]PPD was present in the skin test site, the size of the tuberculin skin reaction in sensitized guinea pigs was at its maximum. Furthermore, the addition of 5 or 50 mug of Tween 80 per ml to a solution of PPD did not change either the rate of clearance of PPD from the skin test sites of sensitized guinea pigs or the size of the tuberculin skin reactions. There was no difference in the rate of clearance of [(14)C]PPD from the skin test sites between sensitized and nonsensitized guinea pigs and between guinea pigs of different age. However, there was a significant difference in the rate of clearance of [(14)C]PPD between the guinea pig and the mouse. Finally, the percentage of [(14)C]PPD retained in the site of injection at 24 h was in the neighborhood of 5% of the initial concentration of the solution of PPD injected. The significance of these phenomena is discussed.     

Studies on diagnostic potential of secretory antigens of Mycobacterium bovis

Culture filtrate proteins (CFPs) of M. bovis were produced by culturing the organism for a period of 3 weeks. These CFPs elicited a good delayed hypersensitivity (DTH) reaction in cattle and guinea pig. However, CFPs showed least cross-reactivity when tested in guinea pigs sensitized with M. bovis BCG and M. phlei. CFPs were found to be well recognized by T-cells of single intradermal test (SIDT) positive cattle and were also sero-reactive. Lymphocyte transformation assay revealed the culture filtrate antigens as major T-cell antigens. CFPs induced a good interferon gamma release as compared to conventional antigen, PPD, thereby highlighting their diagnostic capabilities.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Numerical classification of 280 strains of slowly growing mycobacteria. Proposal of Mycobacterium tuberculosis series, Mycobacterium avium series, and Mycobacterium nonchromogenicum series

Numerical classification of 280 strains of slowly growing mycobacteria was carried out by testing each strain for 76 characters. The following fourteen clusters were observed: 1. M. tuberculosis, M. bovis, M. africanum, and M. microti; 2. M. haemophilum; 3. M. ulcerans; 4. M. xenopi; 5. M. kansasii; 6. M. szulgai; 7. M. gordonae; 8. M gastri; 9. M. avium, M. intracellulare, M. scrofulaceum, and M. asiaticum; 10. M. marinum; 11. M. simiae; 12. M. nonchromogenicum, "M. novum," M. terrae, and M. triviale; 13 M. malmoense; 14. M. shimoidei. The clusters composed of M. tuberculosis, M. bovis, M. africanum, and M. microti, of M. avium, M. intracellulare, M. scrofulaceum, and M. asiaticum, and of M. nonchromogenicum, "M. novum," M. terrae, and M. triviale appeared to be reduced to a single species each. The names having priority for each species should be M. tuberculosis, M. avium, and M nonchromogenicum, respectively. However, the clusters may, in practice, be called the M. tuberculosis series (complex), the M. avium series (complex), and the M. nonchromogenicum series (complex). The type species of these series are M. tuberculosis, M. avium, and M. nonchromogenicum, respectively. These series were characterized in this study.     

Mycobacterial Polysaccharides II. Comparison of Polysaccharides from Strains of Four Species of Mycobacteria

Evidence from chemical and serological studies indicates that a cellular heteropolysaccharide, also found in lipid extracts and culture filtrate, is present as a group antigen in Mycobacterium tuberculosis H37Ra and in other strains of mycobacteria representing M. kansasii, scotochromogenic and Battey strains. Polysaccharides from the four strains contain the same main sugars, arabinose, and galactose, as revealed by thin-layer chromatography and spectrophotometric studies. In Ouchterlony gel diffusions, bands of identity are produced between the polysaccharides by using rabbit antiserum prepared against any of the four mycobacteria. Immune adsorption studies also confirm the presence of identical antigenic determinant groups. In skin tests with tuberculopolysaccharide I, a skin reaction of about equal size was elicited in guinea pigs sensitized with either M. tuberculosis H37Ra or heterologous mycobacterial antigens in Freund's incomplete adjuvant. In animals sensitized with M. tuberculosis H37Ra, skin tests with both homologous and heterologous polysaccharides elicited similar responses.     

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. I. Production, physiochemical characterization and serological analyses

As a part of a cooperative inter-laboratory WHO supported project raw tuberculins were produced and purified protein derivative (PPD, 18.7 g protein) was prepared. Employing a multistage preparative polyacrylamide gel electrophoresis (PAGE) method the PPD was separated into four fractions corresponding to 15, 7, 4.75 and 3.5% gel concentrations. The PAGE procedure resulted in three lots of material--each representing 11 electrophoretic runs. Immunodiffusion analyses showed that the largest number of precipitinogens was found in the 15% fractions and that some precipitinogens cross-reacted with preparations of Mycobacterium bovis BCG, M. intracellulare, M. kansasii, M. smegmatis and M. vaccae.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Production of a Mycobacterium avium ssp. paratuberculosis purified protein derivative (PPD) and evaluation of potency in guinea pigs.

A Mycobacterium avium ssp. paratuberculosis purified protein derivative (PPD) was produced and the biologic activity evaluated in sensitized guinea pigs. The PPD when adjusted to a protein concentration of 1mg/ml induced a delayed-type hypersensitivity response comparable to USDA Johnin OT 133-8707.     

Passive transfer of cell-mediated immunity in xenogeneic animals

Cell-mediated immunity (delayed hypersensitivity) to 1-chloro-2,4-dinitrobenzene (DNCB) was passively transferred from sensitized guinea pigs to mice. Transfer was accomplished by subcutaneous injection of peritoneal exudate cells of sensitized guinea pigs.     

Antibodies to guinea pig lymphokines. II. Suppression of delayed hypersensitivity reactions by a "second generation" goat antibody against guinea pig lymphokines.

A "second generation" antibody to a highly purified lymphocyte product was raised in a goat against material eluted from a rabbit anti-guinea pig lymphokine immunoadsorbent column. This anti-lymphokine serum, in constrast to anti-lymphocyte serum (ALS) did not appear to contain cytotoxic antibodies directed against membrane antigens on guinea pig lymph node lymphocytes. Furthermore, the anti-lymphokine serum did not inhibit the formation of spontaneous T rosettes nor significantly depress lymphocyte response to mitogens. The anti-lymphokine serum totally suppressed the delayed skin reactivity to PPD and contact sensitivity to DNCB when injected intradermally around the site of antigen challenge. By contrast, intradermally injected ALS did not appear to suppress the PPD response in sensitized guinea pigs. Intravenously and i.p. administered anti-lymphokine serum was somewhat less effective in suppressing the delayed skin response to PPD. The intradermal injection of the antiserum had no effect on nonspecific inflammation evoked by turpentine-olive oil or on the extravasation of circulating Evans blue evoked by intradermally injected histamine. Histologic examination of 24-hr DNCB-induced skin lesions from sensitized guinea pigs treated with intradermally injected anti-lymphokine serum showed marked reduction of mononuclear infiltration of the dermis and of epidermal lesions, as compared with skin sites taken from sensitized animals pretreated with normal goat serum. The anti-lymphokine serum injected i.v. also markedly reduced the perivascular infiltration of the dermis and subcutis in skin reaction sites from sensitized animals challenged with PPD. Intravenous treatment with ALS for 3 consecutive days caused extensive depletion of the paracortical areas of peripheral lymph nodes whereas treatment with normal serum and anti-lymphokine serum caused no such depletion. It is proposed that the anti-lymphokine serum is directed against activated lymphocyte products, one of them being MIF. These products are involved in the mediation of delayed hypersensitivity reactions. This is in marked contrast to ALS, the suppressive action of which appears to be central rather than peripheral.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.