Problems associated with the use of the lecithovitellin (LV) reaction for typing strains of have been defined and and investigated. The haemolysins of the various types of the organism were studied with particular reference to their application to serological neutralisation tests. A haemolysin (HL) neutralisation test has been developed with human red cells as the substrate; the test is recommended for use in parallel with the LV test system in typing procedures. The HL test is more sensitive than the LV test and is of particular value in identifying type-B strains, which frequently produce inadequate amounts of the β-antigen for detection in LV tests with unconcentrated culture products.

Thin-layer chromatographic procedures have been applied and extended to differentiate the various soluble products of that contribute to LV activity in culture concentrates. It was confirmed that strains of types A, B and D consistently decompose lecithin; type-D strains are particularly active in this respect. Potent lipolytic activity was demonstrable in culture products of type-A strains. The lipolytic activity of these strains releases free fatty acids from triglyceride substrates in egg yolk and from the diglyceride products of lecithinase-C (phospholipase-C) activity. Diglycerides accumulate in reaction mixtures of type-B and type-D strains, which do not appear to produce a lipase.

The production, stability and partial purification of the lethal α-toxin were studied. A cytopathic factor (CPF I) in culture products of of types A and B is almost certainly the α-antigen. In concentrated preparations of type-D cultures which contain high concentrations of β-antigen, a separate cytopathic effect was demonstrable. The cytopathogenicity test with CPF I provides a convenient in-vitro assay procedure for the α-antigen.


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