Immunization to Vi and O Antigens of Salmonella Typhi Using a Stable Antigen in Oil

Abstract

It has been shown by a number of workers that saline suspensions of Salmonella typhi lose their Vi activity rapidly, while dried organisms retain the ability to stimulate antibody production and to react with Vi antibody. Chu & Hoyt (1954) found that the Vi haptene, although very soluble in water, retains its antigenic specificity even when autoclaved for 30 min. at 20 lb. pressure. A vaccine prepared by harvesting Vi-positive organisms in alcohol, which were then dried and resuspended in peanut oil, was found to produce high antibody titres against both 0 and Vi antigens when a single injection was given to rabbits. These observations have formed the basis for the preparation of a vaccine, whose properties are described below. METHODSS. typhi, strain Ty2, was grown in Blake bottles on nutrient agar. The organisms were shown to have 0, H and Vi antigens. After 18 hr. incubation, the growth was harvested by washing the surface of the agar with 20 ml. of absolute ethyl alcohol. The alcohol suspension was transferred to a 250 ml. centrifuge bottle and diluted with 200 ml. absolute ethyl alcohol. The bottle was stoppered, shaken vigorously to wash the bacteria and centrifuged. The supernatant alcohol was discarded and the washing was repeated once. The bacterial sediment was placed in the 370 C. incubator to dry overnight. The dried sediment was ground to a fine powder by means of a glass stirring rod attached to a small laboratory stirrer, under aseptic conditions. A loopful of the dry powder was cultured to test for the persistence of living organisms. When the cultures indicated that the organisms were dead, the powder was weighed on an analytical balance; 20 mg. of powdered bacteria were added to 40 ml. peanut oil of a pharmaceutical grade. The dried bacteria were suspended in the oil by grinding in a Pyrex test-tube using a glass stirring rod. When the organisms were evenly suspended the oil emulsion was transferred to vaccine bottles and autoclaved at 15 lb. for 30 min. The oil vaccine was tested for sterility and used for inoculation of animals.A saline vaccine was prepared by suspending 20 mg. dried organisms in 40 ml. sterile saline. The vaccine was prepared at the time of administration and was not heated before use. IMMUNIZATION OF ANIMALSRabbits weighing 2-2-5 kg. were subjected to a preliminary bleeding before immunization. Those which showed no antibodies against Vi antigen of Ballerup (XXIX, Vi) and 0 antigen of S. typhi, strain 0-901, were used; the vaccine was