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Lysogenic conversion has been suggested as a mechanism of control of group A streptococcal pyrogenic exotoxin type A production. Digestion of DNA from two converting bacteriophages, 3GL16 and T12, with a variety of restriction endonucleases yielded identical DNA fragments upon electrophoresis in agarose gels. Several known A toxin-positive strains that did not appear to produce converting phage upon induction were analyzed for toxin and phage DNA. Strains, including NY5, 594, and C203S, were shown by hybridization studies to carry the A toxin gene (speA) adjacent to chromosomally inserted phage fragments, homologous to phage T12 DNA, which may represent defective converting phages. The phage T12 att site mapped adjacent to speA. These data suggest that phage T12 acquired the A toxin gene from the bacterial genome. All streptococcal strains tested that were A toxin negative by Ouchterlony immunodiffusion failed to show any hybridization to speA-specific probes.

A defined medium was developed in which easily measured quantities of exotoxin A (PE) were produced by Pseudomonas aeruginosa PA-103. The medium contained three L-amino acids (arginine, aspartic acid, and alanine), basal and trace salts including 14 mM K2HPO4, 14 mM glucose, and 140 mM glycerol. The concentrations of amino acids which yielded most satisfactory results were 6 mM alanine, 13 mM aspartic acid, and 16 mM arginine. The identity of PE in the culture supernatant fluid was demonstrated by adenosine diphosphate-ribosyl transferase activity and by immunodiffusion with sheep antitoxin elicited with purified PE and with PE produced in Trypticase soy broth dialysate and pure PE as controls. PE production was also demonstrated by mouse lethality and passive hemagglutination. As compared to Trypticase soy broth dialysate, P. aeruginosa produced 25 to 50% PE in the defined medium. Different strains of P. aeruginosa produced PE in the defined medium in proportions relative to those in Trypticase soy broth dialysate.

Pasteurella haemolytica serotype 1 was transferred daily for 128 serial passages on both unsupplemented brain heart infusion agar and the same basal medium supplemented with bovine blood, horse serum, and yeast extract. Repeatedly transferred cultures were shown to retain the ability to produce both capsular material and leukotoxin. Furthermore, intact organisms were found to be as toxic in vitro for bovine leukocytes and as virulent for mice as unpassaged cultures. These results indicate that the precaution of using only freshly isolated cultures in the study of this organism may not be necessary.

An epoxy derivative of linoleate, 9,10-epoxy-12-octadecenoate, was demonstrated to be biosynthesized by neutrophils from various sources such as canine and human blood, and guinea-pig peritonea. It was nominated as leukotoxin from its 'toxic' activity onto mitochondrial respiration. From the reaction mixture of leukocytes with linoleate, an isomer of leukotoxin, 12,13-epoxy-9-octadecenoate, and a 'non-toxic' hydroxy derivative of linoleate, 9-hydroxy-12-octadecenoate, were detected. Such a cascade reaction of linoleate by leukocytes was discussed. Biosynthesis of leukotoxin by neutrophils was substantially enhanced by the presence of calcium ion and calcium-ionophore, A23187. Neutrophils contained leukotoxin, ca. 7 f moles/cell, which was extractable by 60% ethanol, but little of the isomer.