Shared antigens between Mycobacterium bovis (BCG) and other bacterial species.

Antigens from Mycobacterium bovis and Listeria monocytogenes bind to serums from normal rabbits as well as from those immunized with unrelated bacteria, especially intracellular parasites. Binding is to the Fab portion of immunoglobulin G and is immunologically specific.

Suppression and immunotherapy of the guinea pig line-10 hepatocarcinoma mediated by nonviable Staphylococcus aureus.

Suppression of growth of the line-10 guinea pig hepatocarcinoma was achieved after the simultaneous injection of line-10 cells and heat-killed Staphylococcus aureus. Growth of tumor was also suppressed when line-10 cells were injected alone, contralaterally, at the same time as the vaccine mixture. Immunity developed to subsequent line-10 cell challenges but not to other syngeneic tumors. Similar results were obtained with the use of protein A-rich or -deficient strains of S. aureus. Multiple intratumor injections of heat-killed S. aureus were therapeutically effective against 6-day tumors. The antitumor effects of nonviable S. aureus were similar in many ways to those of living BCG or bacterial products suspended in oil droplets.

Prevention of tumor growth after intradermal injection of BCG extracts: a comparison of results in strain-2 guinea pigs from the National Institutes of Health and from the National Jewish Hospital and Research Center.

Line 10, a transplantable hepatocellular carcinoma, was obtained originally from an NIH strain-2 male guinea pig fed diethylnitrosamine. The antitumor activity of BCG and BCG extracts was evaluated in strain-2 guinea pigs obtained both from NIH and the National Jewish Hospital and Research Center (NJH). Animals were immunized with these materials and then tested for their capacity to resist the growth of intradermally injected line-10 tumor cells. Tumor growth was not prevented in 18 NIH animals immunized with living BCG. No tumor growth occurred in 1 of 22 NIH animals immunized with a residue that remained after exhaustive methanol extraction of BCG and in 1 of 44 NIH guinea pigs immunized with BCG extracts. In contrast, tumor growth was prevented in 13 of 22 similarly immunized NJH guinea pigs.

Transmission of BCG-associated tumor resistance from maternal to newborn guinea pigs.

Resistance to line-10 hepatocarcinoma was studied in the newborn offspring of inbred strain-2 Sewall Wright guinea pigs that had recovered from this tumor after prior administration of subcellular components of Mycobacterium bovis strain BCG. These mothers gave birth to 15 offspring, 53% of which resisted repeated challenges with this tumor. Offspring of mothers who had been given only BCG components were similarly tested. Complete protection was observed in 23.5% of 17 babies and attenuation of disease in many others. All control offspring from normal, untreated mothers succumbed to this tumor. Resistance was associated with delayed cutaneous hypersensitivity reactions to tumor and mycobacterial antigens.

Shared antigens between bacteria and guinea pig line 10 hepatocarcinoma cells.

This study was undertaken to investigate the possibility that Listeria monocytogenes, Brucella abortus, and Salmonella typhimurium share antigenic components with guinea pig line 10 hepatocarcinoma cells. Rabbits were immunized with sonicates of these bacteria or line 10 tumor cells. Other rabbits were immunized with line 1 cells, a tumor with antigenic characteristics different from those of line 10. The binding of antibodies to radiolabeled antigens prepared from extracts of bacteria and line 10 cells was studied by precipitation of radiolabeled antigen-antibody complexes with anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with these bacteria and line 10 cells bound both the labeled bacteria and line 10 antigens. Antibodies in sera from rabbits immunized with line 1 cells did not bind the bacterial antigens. Inhibition studies involving reactions between radiolabeled Listeria and line 10 antigens and antibodies to Listeria and line 10 cells confirmed that the binding reactions were specific and that line 10 cells shared antigens with Listeria cells. The possibility that B. abortus and S. typhimurium also shared antigens with line 10 cells was suggested. Whether antigens shared by these bacteria and line 10 cells are identical with tumor-specific antigens was not determined.

Immunotherapy of the guinea pig line 10 hepatocarcinoma with a variety of nonviable bacteria.

A variety of heat-killed bacteria were tested for their capacity to induce regressions of established line 10 hepatocarcinomas in syngeneic guinea pigs. Multiple intralesional injections of heat-killed Escherichia coli, Streptococcus mutans, Listeria monocytogenes, and Propionibacterium acnes resulted in complete regression of the tumor in a majority of guinea pigs. Repeated injections of heat-killed Mycobacterium bovis strain Bacillus Calmette-Guérin caused no regressions. Surviving animals were immune to subsequent challenge with line 10 cells but not L2C cells, another syngeneic tumor.

Isolation of mycobacteria from clinical specimens by use of selective 7H11 medium.

7H11 agar containing carbenicillin, amphotericin B, polymyxin B and trimethoprim lactate (selective 7H11 or S7H11) was used for the selective isolation of mycobacteria from clinical specimens. This medium as previously described contained 100 mug. carbenicillin per ml. It was found that reducing the concentration of carbenicillin to 50 mug. per ml. made S7H11 less inhibitory to certain strains of mycobacteria. However, some strains of M. kansaii, M. intracellulare, and M. gordonae still do not grow well on S7H11. Of 3,134 clinical specimens (mostly sputum) received, processed, and plated on 7H11 agar and the S7H11 medium, 508 positive specimens were isolated. Of these, 402 were positive on both types of media, 30 were positive on 7H11 only, 19 were positive on S7H11 only, 52 were contaminated on 7H11 but positive on S7H11, while only 5 were contaminated on S7H11 and positive on the plain medium. Thus, the total positive specimens on 7H11 was 437 and total positive specimens on the selective medium was 473. Used in conjunction with nonselective media, S7H11 agar appears to be a valuable culture medium for used in diagnostic mycobacteriology laboratories.

Rapid method of microbial susceptibility testing.

A rapid method of microbial susceptibility testing is described. Bacterial growth is measured by determining the incorporation of (14)C-tryptophan into cellular protein in the presence of antimicrobial drugs.

Mechanism of Action of Isoniazid on Mycobacterium bovis Strain BCG.

The mechanism of action of isoniazid (INH) on Mycobacterium bovis strain BCG was studied. The rates of synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein after the addition of INH to growing cultures were followed by measuring the incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-l-valine, respectively. After the addition of INH, the rate of DNA synthesis began to decrease and was abolished within 4 hr. RNA synthesis ceased after 6 hr, and protein synthesis was inhibited after 7 hr. Thus, it appears that inhibition of the synthesis of DNA is one of the earliest events after INH addition. The inhibition of the synthesis of DNA was further found to correspond to losses in viability of treated cultures. Degradation of preexisting DNA in INH-treated strain BCG was not detected.

The seroagglutination test in the study of nontuberculous mycobacteria.

The Schaefer seroagglutination test is useful in epidemiologic studies of nontuberculous mycobacteria, especially the Mycobacterium avium-intracellulare-scrofulaceum complex. Serovars 8, 16, 4, 19, 9, 42, and 1 were isolated most frequently from patients in the United States in the period 1976 to mid-1978. Several so-called double serovars were found serologically. It appears that this is an artifact due to cross-reacting antigens, because only one antigen is seen using chromatographic analyses. Further development of this system and its use in conjunction with other methods offer a valuable method for the study of nontuberculous mycobacteria.

Heterogeneity of the stability of messenger ribonucleic acid in Salmonella typhimurium.

Cells of Salmonella typhimurium strain SL 282, deflagellated by mechanical shear, regenerated their flagella in the absence of tryptophan, an amino acid required for growth but not found in flagellin. Ribonucleic acid (RNA) synthesis was severely inhibited by tryptophan starvation. These findings suggested that the messenger RNA (mRNA) for flagellin might be stable. Actinomycin D was used to inhibit RNA synthesis in ethylenediaminetetraacetate-treated bacteria. The introduction of an F(lac) episome into strain SL 282 permitted the simultaneous study of the synthesis of flagellin, beta-galactosidase, and total protein. In the actinomycin-treated bacteria protein and beta-galactosidase syntheses were inhibited by 90%, whereas flagellin synthesis was unaffected. We conclude that the mRNA for flagellin synthesis is stable and that species of mRNA vary with respect to metabolic stability in S. typhimurium.

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus.

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.

Biological properties of alpha-toxin mutants of Staphylococcus aureus.

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Biological properties of alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:575-579. 1966.-Twenty nonhemolytic mutants of Staphylococcus aureus were isolated after treatment of a hemolytic strain with ultraviolet light or nitrous acid. Thirteen strains isolated were completely lacking in the synthesis of alpha toxin or immunologically related proteins, presumably the result of a single mutational event. Although the strains were nonhemolytic on rabbit blood-agar plates, six of them retained the dermonecrotic and lethal activities usually associated with staphylococcal alpha toxin, as well as slight hemolytic activity for rabbit erythrocyte suspensions. The active mutants and one inactive mutant produced a protein that reacted immunologically with antibody to alpha toxin. Mutations which alter the alpha toxin molecule can effect the lethal, dermonecrotic, and hemolytic activities separately or in varying ratios.

Niacin, nitrate and pyrazinamide studies using Middlebrook 7H10 Broth.

An innovative and rapid method for testing mycobacteria was developed using Middlebrook 7H10 Tween Broth in place of conventional media. Niacin production, nitrate reduction and the breakdown of pyrazinamidase were determined in 198 mycobacteria isolates. Less than nine days were required to obtain positive test results, and the correlation of tween broth with conventional test methods exceeded 98%.

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.

Shared antigens between heterologous bacterial species.

Sera from normal rabbits were shown to have antibodies that bound radiolabeled test antigens derived from many taxonomically unrelated bacteria. Sera from rabbits that had been immunized with sonically treated material of 12 different bacteria, including M. bovis strain BCG, had antibodies that bound not only radiolabeled homologous test antigens but also radiolabeled antigens from many unrelated bacteria. Binding by normal and immunized sera to radiolabeled test antigens was inhibited by homologous unlabeled test antigens but not by substances such as bovine serum albumin, polyvinylpyrrolidone, sheep erythrocytes, and endotoxin. The broad range of shared or cross-reactivity among antigens in bacteria may explain the presence of antibodies to many bacteria in sera from normal humans and previously unimmunized experimental animals. The presence of these antibodies raises the question whether resistance to many bacterial infections may be partly due to immune mechanisms, whether cellular or humoral, that have been stimulated by unrelated bacteria.

Induction of Resistance by Listeria monocytogenes Cell Wall Fraction.

A crude cell wall fraction of Listeria monocytogenes was prepared by sonic disruption and differential centrifugation of viable, washed cultures. When injected into mice, this sterile, crude cell wall fraction protected mice against an intraperitoneal challenge with 18 to 85 50% mean lethal dose of L. monocytogenes. Resistance was greatly enhanced when bacterial endotoxin (lipopolysaccharide) was injected along with the cell wall fraction. Resistance was measured both by enumerating the bacteria in the livers and spleens of vaccinated and control mice and by survival studies. Two major lines of evidence suggest that the resistance induced by cell wall fraction is at least in part specific. Unlike non-specific resistance, the cell wall fraction-induced resistance was relatively long-lived, (i.e., it was demonstrable 6 weeks after the last injection of cell wall fraction and lipopolysaccharide). In addition, cell wall fraction protected against challenge with L. monocytogenes, but not against challenge with S. typhimurium.

Urease testing of mycobacteria with BACTEC radiometric instrumentation.

A total of 140 mycobacterial isolates from patients treated at Fitzsimons Army Medical Center or the National Jewish Hospital and Research Center and from animal specimens submitted to the National Veterinary Services Laboratory were tested by using a urease procedure modified for use with a BACTEC model 301. Mycobacterial suspensions were prepared by using Middlebrook 7H10 Tween broth. Of the 98 mycobacteria isolates which were urease positive utilizing standard methodology, all were positive using the radiometric procedures. Similarly, all 42 urease-negative isolates were also negative employing the new methodology. Although maximum radiometric readings were observed at 48 h, all positive strains were readily identified 24 h after inoculation without sacrificing either test sensitivity or specificity. Thus, urease testing of mycobacteria, using the modified BACTEC radiometric methodology, was as sensitive, as specific, and more rapid than conventional methods.