Resistance to infection and activation of the monocyto-macrophage system caused by Bacillus firmus and its fractions.

Crude lipids isolated from Bacillus firmus, but not from other bacilli, were previously found to induce significant resistance against Listeria monocytogenes infection in mice. In this study, formaldehyde- and heat-killed bacterins of eight Bacillus species and some cellular fractions of B. firmus were prepared and tested for further immunomodulatory activities. Crude lipids, their aqueous extract, LTA, Protodyne and Pex-residue preparations exhibited a strong anti-infection activity, whereas Pextract, P40 and all bacterins tested had no effect. Formaldehyde-killed bacterins, live bacteria and the P40 preparation of both B. firmus strains, as well as bacterins of both B. subtilis strains, induced pronounced splenomegaly in mice. Peptidoglycan and Pex-residue induced significant depression of cytochrome P-450 in mouse liver microsomes after application of 0.1 mg per mouse. Optimal conditions for obtaining a bacterial suspension exhibiting these immunomodulatory properties were elaborated.

Effect of crude bacterial lipids on the course of Listeria infection in mice.

Crude lipids from 37 strains belonging to 32 bacterial species were isolated. By injecting mice with lipids 5 d prior to challenge with a virulent strain of Listeria monocytogenes, immunostimulatory activity in 19 preparations was found. In general, lipids of Gram-negative bacteria appeared to be more effective. As to bacilli, an extraordinary activity was found in the lipids of Bacillus firmus. Lipids of various species of the genus Listeria were found to be active in approximately one-half of cases. Among other Gram-positive bacteria, significant activity of lipids was found in Corynebacterium xerosis, Propionibacterium acnes and BCG. The composition of fatty acids in the lipids did not differ significantly from that reported in the literature and their mutual differences could not explain the different biological activity. In selected strains of Gram-negative bacteria lipids were repeatedly purified with anhydrous chloroform; these preparations were found to be inactive as compared with original chloroform-methanol lipids.

Cellular fatty acids and fatty aldehydes of Listeria and Erysipelothrix.

The cellular fatty acid composition determined by gas chromatography-mass spectrometry, was found not to differ among Listeria monocytogenes, L. innocua and L. ivanovii. Slight quantitative differences found in the fatty acid pattern of L. welshimeri were significantly pronounced in L. denitrificans. L. murrayi and L. grayi displayed characteristic closely related patterns. Considerable amounts of fatty aldehydes and their dimethyl acetals were observed in hydrolysates and methanolysates of L. seeligeri. The fatty acid composition of Erysipelothrix rhusiopathiae was found to strongly differ from that of Listeria.

The immunological properties of listerial complex Ei.

Factor Ei, besides exerting toxic reactions, is amphipathic, antigenic, chemotaxinogenic, causes blastic transformation, adjuvant effect, hypersensitivity (MIF, skin test), activates the RES (splenomegaly), increases the macrophage production, prevents listerial infection in mice, mycobacterial infection in guinea pigs and enhances the effect of BCG experimental tuberculosis and neoplasia in mice (Sa 180).

Phospholipase C in Listeria.

The cooperative and antagonistic effect of extracellular bacterial proteins of Streptococcus agalactiae, Rhodococcus equi, Corynebacterium ovis, and Corynebacterium haemolyticum with proteins of listerial strains of various sources, species and serovars of the membrane of sheep erythrocytes was investigated. Listeria monocytogenes and Listeria ivanovii produce, beside listeriolysin, further proteins. Their specific effect on the sheep erythrocyte membrane becomes apparent after the appearance of substances produced by the organisms under study. Ten strains of L. ivanovii produced phospholipase C responsible for the zone of incomplete haemolysis. It was inhibited by the non-haemolytic sphingomyelinase D of C. ovis and C. haemolyticum. Chromatographic analysis revealed that phospholipase C splits sphingomyelin in the membrane of sheep erythrocytes. The inhibition of double haemolysis of L. ivanovii by sphyngomyelinase D of C. ovis on agar plates with washed (!) sheep erythrocytes can be utilized as a specific and rapid identification test of L. ivanovii.

Biological and immunological properties of avirulent strain of Listeria innocua.

The strain isolated by Dr. J. H. Welshimer from plants has antigenic formula V (VI) IX; XV; XI; AB, C--serovar 6a, is non-haemolytic, produces lipase, and toxic factor Ei, is avirulent for adult mice, but causes encephalitis in sucklings. In organs of intravenously injected mice the strain persists and multiplies for 1-3 weeks. The protective effect against listerial infections in mice of this strain administered 2-14 days before challenge is dose depending. After 3 weeks induces resistance of guinea pigs to infection with Mycobacterium tuberculosis H37Rv measured by spleen weight and Feldman index. The hypersensitivity induced in animals is detectable by factor Ei and PPD or OT tuberculins using MIF method. A suspension of living cells of this strain injected intraperitoneally causes resistance to Mycobacterium kansasii in mice, measured by inhibition of loss of weight and decrease of the number of bacillus in their lungs.

Experimental listeriosis in immunized sheep.

Sheep immunized with live or inactivated vaccine were challenged with a virulent strain of Listeria monocytogenes. Clinical manifestations, penetration of listeriae into the mucous membranes and persistence in organs were noted. Bacteriaemia caused by intravenous injection of high doses of virulent strain disappears in 3 days. Readily after inoculation, listeriae penetrate into the gastrointestinal tract where they persist for 8 days. Listeriae appear from the 3rd day in the conjunctiva and nasal mucous membranes with clinical symptoms of conjunctivitis and rhinitis whence they are eliminated in 8 to 14 days. The brain, liver and spleen of animals that died in 5 to 7 days were loaded with listeriae. Organs from immune animals sacrificed 14 days after challenge were negative by cultivation even when listeriomas were in the liver and spleen. Rapid penetration and persistence of listeriae are an expression of their increased affinity to epithelia of systems which are not only the portal of their entry but even the site of their secondary propagation and thus a source of infection.