No abstract

Pseudomonas aeruginosa (Schroeter) Migula antigen remained in the blood of larvae of the wax moth, Galleria mellonella (L.), during the resistant period of the insect. Bacterial antigen present in the immune blood produces agglutinating titers in rabbits about 10 times as great a s those produced by a n approximately equal volume of standard P. aeruginosa vaccine. Attempts to demonstrate the mechanism that enhances the antigen showed that the active portion was contained in the serum, that the action occurred within several hours in vivo and only reached the same level after 3 days in vitro mixture, and that the action was probably not caused by lysis of the bacterial cells and the consequent liberation of more antigen in the blood. Electrophoretic studies on the blood mixture indicated that the altered or enhanced antigen may be bound to a blood fraction, the exact nature of which was not determined. The larvae were actively or passively immunized against lethal doses of P. aert~gginosa within 20 to 24 hours. Concentration of vaccine had little effect upon the degree of immunity conferred upon the larvae. T h e immunity lasted about three days and was more specific than nonspecific. The larvae were not actively protected against P. aeruginosa by introductioil of albuminous foreign material into the body cavity. True antibodies were not detectable in the immune blood though the bactericidal action of immune blood was a t least twice as great a s that of normal blood. Preliminary investigations on immune responses of other lepidopterous insects to P. aeruginosa antigen and of the wax moth to antigens of some other Gram-negative bacteria indicated similar results. lManuscript received October 17, 1958. Contribution No. 3864,

This paper describes a bacteriological study of strains of Bacillzts cereus Frankland and Frankland producing disease in codling moth larvae and an investigation of their pathogenicity. Strains of the bacterium were pathogenic t o the codling moth by feeding or by injection.

The blood of normal wax moth larvae is not bactericidal for Pseudonzo?tas aerzrginosa. The blood becomes moderately bactericidal when larvae are actively immunized against P. aerzrginosa. This activity was measured b y a modification of Nagington's technique for the estimation of typhoid antibody. Bactericidal activity appears t o be the only measurable type of antibody response against P. aerrrginosa. Cell-free blood was as active as whole blood; 0.02 ml of immune serum kills about 1000 organisms. The blood of normal wax moth larvae is bactericidal for Shigella dysenteriue but the blood of insects immunized against either this organism or P . aerrrginosa shows no increase in activity against S . dysenteriae. A number of non-specific agents, both protein and non-protein, did not stimulate bactericidal activity in serum after their injection into normal larvae. Immune sera prepared against some strains of P. aeruginosa were not active against other strains. Storage a t 37' C or absorption with zymosan both result in blackening of immune blood and loss of bactericidal activity. Bactericidal activity is evident only during the immune state of the insect, i.e. from about 18 hours until the third day after vaccination; it develops a t the same time that inhibition of melanization was observed in the blood from vaccinated larvae.

Pse2ldonzonas acrzlginosa is the chief cause of disease in laboratory cultures of grasshoppers. A large number of strains of the bacterium from different localities fell into five types on the basis of reactions with bacteriophages. The five types and their strains were qualitatively similar to one another in morphology, cultural characteristics, and diagnostic biochemical reactions and to a narned culture of P. aeruginosa from the American Type Culture Collection, and their characteristicsagreed with those listed by most authors in the literature a s diagnostic of the species. The strainsand types showed some quantitative differences in production of pyocyanin, development of iridescence, and degree of haemolysis of blood and of hydrolysis of casein. The symptoms of infection and the loss from tnortality are described. The factors influencing the spread of the infection are discussed and methods of suppression of the disease are recommended. The disease originates from a small number of bacteria intimately associated with a very low percentage of grasshopper eggs collected in the field. Nymphs emerging from these eggs die shortly after emergence and contaminate the food supply. Grasshoppers are normally infected by ingesting food contaminated by the bacteria but the bacteria do not multiply in the gut and most of them are rapidly lcilled or eliminated. The bacteria are not actively invasive, but when a small number gain access to the haemocoel they increase in numbers to about 1 X 10"er insect. Shortly thereafter the host dies frorn the metabolic activities of the parasites, most probably as a result of protein digestion by the strong proteolytic enzymes of the bacterium. There is no evidence that the bacteria produce a specific toxin, and grasshoppers are more resistant to the poisonous action of pyocyanin than are mice.'Manuscript