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Outer membrane proteins (OMP) from Neisseria meningitidis cells of groups A, B, C and Y were extracted with CaCl2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of each extract showed a multibanded pattern characterized by the presence of three to four major proteins. Immunization of mice with individual extracts induced homologous bactericidal reactions. In addition, the extracts from groups B and C induced heterologous bactericidal reactions (B-C, C-B). The sera of mice immunized with a mixture of group A, B, and Y extracts showed a high bactericidal titre against the three N. meningitidis groups forming the mixture. Moreover, this bactericidal activity had a large spectrum against different strains from groups A, B, Y and also C. After immunization of mice with the individual extracts, each of the sera was shown by immunoblotting to contain antibodies reacting with some of the corresponding proteins. Mice immunized with the mixture showed a significant reduction of bacteraemia following challenge with either of N. meningitidis groups A, B or C. These results suggest that a mixture of OMP from different N. meningitidis groups may have the potential for a meningococcal polyvalent vaccine.

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The inhibitory substance produced by Staphylococcus haemolyticus strain no. 7 acts on growing as well as resting gonococcal cells, as shown by reductions in viable counts. The optical density of these cell suspensions was only slightly reduced. The inhibitor caused lysis of gonococcal spheroplasts at 24 degrees C and 37 degrees C, but was much less active at 4 degrees C. Acting on intact gonococcal cells, the inhibitor caused a temperature-dependent release of radioactive cytoplasmic material. Electronmicroscopy showed that treated suspensions contained ghost cells with the cell envelope relatively intact. Our results suggest that the inhibitor may act on the cytoplasmic membrane of the gonococcal cell causing cytoplasmic leakage and, eventually, death.

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A strain of Streptococcus faecalis isolated from the urogenital flora was selected for its ability to inhibit the in vitro growth of Neisseria gonorrhoeae. Initially, the inhibitory activity was demonstrated on solid medium only when the inhibitor and the target strains were growing simultaneously, such as in the spot-lawn and flip-flop agar overlay methods. The antigonococcal effect was not due to a shift in the pH or depletion of nutrient in the medium. This activity was not produced in liquid medium nor could it be extracted in a soluble form from either the solid medium or the streptococcal cells. The production of the inhibitory activity could not be enhanced by ultraviolet irradiation or treatment with mitomycin C. The composition of the medium was found to affect the size of the inhibitory zone produced. The inhibitory activity showed a wide antigonococcal spectrum and was susceptible to trypsin and pronase but resistant to alpha- and beta-amylases and catalase. This activity passed through a filtering membrane and was also dialyzable and had an apparent molecular weight of less than 1,000. The addition of bovine serum albumin to solid medium enabled us to show an inhibition even when the producer and the target strains were grown sequentially, thus suggesting that part of the difficulty of studying such inhibitory activity could be due to its instability.

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The purified antigonococcal substance produced by Staphylococcus haemolyticus no. 7 has shown a broad antigonococcal spectrum and a narrow antibacterial spectrum. The inhibitor produced in vitro was also active in the guinea pig subcutaneous chamber. The inhibitor has shown hemolytic activity; the human, horse, and mouse erythrocytes were the most susceptible. Hemolytic and antigonococcal activities were inhibited in the presence of phosphatidylcholine. The amino acid composition of the antigonococcal substance was characterized by the absence of proline, tyrosine, histidine, cysteine, and tryptophan. The molecular weight was found to be 2,565, and the major isoelectric points were 4.8 and 4.9 in the presence of 8 M urea and 4.6 without urea. The inhibitor has some properties similar to those of the delta toxin of Staphylococcus aureus, although the two substances are different based mainly on their chemical characteristics. Also an antiserum directed against the gonococcal inhibitor did not give a precipitation line with the delta toxin, indicating that the two substances are antigenically unrelated.

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The comparative anti-tumour activity of cell walls, deproteinized cell walls and cell wall skeleton (CWS) isolated from four BCG substrains (Canadian, Russian, Glaxo and Swedish) was determined against Ehrlich ascitic carcinoma in CF-1 mice and against L1210 lymphoid leukaemia in B6D2F1/J mice. In Ehrlich carcinoma, the protocol of injection was shown to be of critical importance, since high and low levels of protection and even facilitation of tumour growth were observed according to the protocol used. The oil emulsion used as the vehicle for injection of the fractions (control groups) induced facilitation of tumour growth with some protocols. The highest levels of protection were observed when treatment was performed on day -6 for a single i. p. injection or on days -14 and 0 for two injections. Using the former protocol of immunization, the highest level of protection was detected with cell walls and deproteinized cell walls. CWS isolated from the Canadian and Glaxo substrains were found to be inactive, whereas those isolated from the Swedish and Russian substrains induced significant levels of protection. In L1210 leukaemia, very few preparations induced significant protection. CWS preparations did not induce protection. Canadian and Russian deproteinized cell walls and Canadian cell walls (100 and 250 micrograms, respectively) induced some protection. The use of different protocols of injection did not increase the anti-tumour activity of the Canadian cell walls. The most active preparation in both tumour systems was that of Canadian deproteinized cell walls.

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The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity.

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The dynamics of in vivo multiplication of 10 BCG substrains were studied in three different strains of mice: CF-1, B6D2F1/J and C57BL/6. For each substrain, the number of CFU recovered from the spleen of mice previously injected intravenously with BCG was determined weekly for 5 consecutive weeks. On the basis of the statistical analysis of the results, the BCG substrains were classified for each strain of mice in three groups of virulence: high, intermediate and low. The Russian and Japanese substrains were found to be the most virulent, and the Glaxo and Tice 946 BL (I2) the least virulent substrains. No correlation could be established between in vivo multiplication and antitumour activity of each BCG substrain as estimated in preventive treatment against three tumour systems: Ehrlich carcinoma in CF-1 mice, L1210 leukaemia in B6D2F1/J mice, and B16 melanoma in C57BL/6 mice.

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Cross-protection has already been demonstrated in mice after vaccination with a CaCl2 extract from the Neisseria meningitidis group Y Slaterus strain. The immunogenicity of such extracts from group Y cells, cultivated in a fermenter in Neisseria chemically defined medium, against virulent groups A, B, and C meningococci has been evaluated by two different animal models and a microbactericidal procedure. The mouse challenge system has revealed that the active cross-production observed 7 days after a single immunization with the extract was probably nonspecific, since bacillus Calmette-Guérin gave similar results. However, after three vaccinations, active cross-protection was observed, mainly against the strains of groups B and C, for at least 35 days after the last injection. In the mouse bacteremia model, the extract had a protective effect mainly against the homologous group Y strain but in a few experiments a significant protection was also obtained against the strains of groups A and B. The microbactericidal test revealed that even after three injections of mice, guinea pigs, or humans with the extract only the homologous bactericidal activity was induced. Since there was no close correlation between the results obtained with the two animal models and also with the microbactericidal procedure, no definitive conclusion can be drawn on the protective potential of our extract.

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Previous works has indicated that cotton rats treated with Mycobacterium bovis strain BCG are effectively protected against an infection with the metastatically proliferating metacestodes of Echinococcus multilocularis. In an attempt to induce a similar protection in the absence of tubercular granulomatous lesions, cotton rats were treated with BCG cell walls. A single injection of 150 micrograms of cell walls, emulsified in mineral oil-Tween-saline, 2 weeks before the inoculation of the parasite completely protected the animals against infection with E. multilocularis. This protection was correlated with an increase in the numbers of monocytes and, as judged by acid phosphatase activity, an activation of these cells. This study shows that BCG cell walls are as effective in protecting animals against E. multilocularis as the viable organism.

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When given by direct s.c. injection into the Ehrlich solid carcinoma 1 week after s.c. tumor transfer, viable crude spores of Clostridium perfringens type A (attenuated mutant strain LNG11 ATCC 29348) inhibited tumor growth and significantly prolonged the life span of male outbred Swiss mice. Under these conditions a concentrated sterile supernatant of a C. perfringens culture proved to be slightly more effective than were viable crude spores. In contrast viable crude spores were ineffective in the treatment of female Swiss mice, but the sterile supernatant retained significant activity. When given at the time and site of s.c. grafting of Ehrlich tumor cells, a concentrated sterile supernatant of a C. perfringens culture prevented tumor growth in 80% of male outbred Swiss mice. Under these conditions viable crude spores prevented tumor growth in 70% of mice and significantly prolonged the life span in the other 30%. When given by i.p. injection and before i.p. grafting of tumor cells, viable crude spores of C. perfringens prevented Ehrlich ascites tumor in 5 of 12 Swiss mice and prolonged life span in the other 7. In contrast concentrated sterile supernatant and viable purified spores were ineffective in the prevention or delay of the growth of Ehrlich ascites tumor. These data indicate that C. perfringens can be a potent antitumor agent without producing the harmful anaerobic infection of solid tumors (clostridial oncolysis.

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Crude ribosomal fractions isolated from the BCG strain of Mycobacterium bovis are capable of inducing antituberculous immunity in CF-1 mice. These crude preparations contain varying amounts of polysaccharides in addition to ribosomal ribonucleic acid and proteins. Whereas these latter two constituents of the crude fractions were found inactive, high levels of antituberculous immunity were induced by a ribosomal sub-fraction significantly enriched with polysaccharides. These results strongly suggest that polysaccharides play an important role in the induction of antituberculous immunity in CF-1 mice immunized with crude ribosomal fractions isolated from the BCG strain and that if ribosomal proteins and/or RNA are of a certain importance, their role would be secondary (complexes providing physical support to polysaccharides, adjuvant, etc.).

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Levels of antituberculous immunity similar to those induced by live BCG vaccine were detected in CF1 mice immunized with ribosomal fractions isolated from Mycobacterium tuberculosis var. bovis, strain BCG, and challenged 3 weeks later with the virulent H37Rv strain of Mycobacterium tuberculosis var. hominis. The activity of the crude ribosomal preparations was found to be a function of the immunizing doses and the immunity induced by 1.0-mg doses remained at the same high level after 4 weeks of storage at 4 degrees C but decreased markedly thereafter. Dialysis and lyophilization had no detrimental effects on the activity of the crude preparations whereas purification by column chromatography on Sephadex G-200 annihilated their biological activity. Crude low-polysaccharide-containing preparations were found inactive even at the 1.0-mg dose level and results of experiments performed with crude ribosomal fractions of varying polysaccharide contents strongly suggest that polysaccharides, or RNA-polysaccharide complexes, may play an important role in the induction of immunity with crude ribosomal fractions isolated from the BCG strain of Mycobacterium tuberculosis var. bovis.

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Suspensions in dilute Sauton's medium of 14 mycobacterial strains grown under identical conditions were prepared with bacilli harvested at their midlog phase of growth,and were frozen and stored at -55 degrees C. Survivals, estimated by CFU determinations after periodic intervals of storage, showed no adverse effect of freezing and thawing on any of the strains. Effectiveness of preservation, however, varied from strain to strain and no correlation existed between effectiveness and species of mycobacteria. No noticeable changes in the virulence of the H37Rv strain and in the immunizing activity of BCG were detected after prolonged storeage. Results of experimentsperformed with two strains suspended in seven diluents suggest that some are more suitable for long-term preservation, particularly so for the more sensitive one.

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Two menaquinone-deficient and one aromatic-deficient mutants of Staphylococcus aureus were unable to reduce nitrate to nitrite. Reinitiation of menaquinone synthesis in the aromatic-deficient mutant by growing it with shikimic acid restored its nitrate respiratory activity. The results clearly demonstrate a role for menaquinone in nitrate respiration in Staphylococcus aureus.

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