Effect of Different Immunization Procedures on Agglutination and Precipitation Reactions of Trichomonas gallinae

Abstract

Immune sera were prepared in rabbits by intravenous or subcutaneous inoculations of an avirulent T. gallinae substrain JB(VI)C, derived from the highly pathogenic Jones' Barn strain by prolonged in vitro cultivation. Living or disrupted antigen was used for intravenous injections, while only disrupted organisms were administered via the subcutaneous route. In some rabbits inoculations of the antigen were given in combination with the complete Freund's adjuvant. The highest agglutination and hemagglutination titers, but the fewest and weakest precipitin lines on gel diffusion plates, were observed with sera from rabbits inoculated intravenously with living flagellates. In contrast, the lowest agglutination and hemagglutination titers and the most numerous and strongest precipitin lines were recorded with sera from rabbits which received disrupted trichomonads via the subcutaneous route. Sera prepared by several other variations of the immunization method fell between the above two in their agglutinating, hemagglutinating, and precipitating capacities. The employment of the complete Freund's adjuvant appeared to enhance somewhat the strength of all immune sera, its effects being most clearly evident in the results of gel diffusion experiments. A survey of the literature reveals that various forms of agglutination have been the methods used most extensively in immunologic studies of trichomonads (Honigberg, 1970). In many investigations the entire immunization series involved intravenous inoculations of living parasites into rabbits; in at least one study (Samuels and Chun-Hoon, 1964) only part of the immunizing series was administered by this method. The superiority of living flagellates injected intravenously into rabbits in stimulating production of agglutinating antibodies to Trichomonas vaginalis was suggested by the comparative studies of Trussell (1946) and Teras (1961, 1962). Much lower titers were obtained with sera of animals which received intravenous injections of killed organisms, and the results of subcutaneous or intramuscular inoculations were still less satisfactory. No extensive comparative investigations have been reported with respect to any other trichomonad species, but according to Robertson (1941, 1960), living Received for publication 22 June 1970. * This investigation was supported by Research Grants AI00742-13, 14, from NIAID, U. S. Public Health Service. t Dr. Stepkowski, Associate Professor of Veterinary Science and Chief of the Laboratory of Poultry Diseases, College of Agriculture, Lublin, Poland, was a Guest Scientist in the Department of Zoology, University of Massachusetts at Amherst, from January 1968 to March 1969. Tritrichomonas foetus were more effective than dried flagellates in the production of agglutinating antibodies. Nonetheless, satisfactory agglutination, hemagglutination, precipitation, and/or fluorescent antibody reactions with trichomonads were obtained by several workers who used killed trichomonads, prepared in various ways, for immunization of rabbits and other animals (Morisita, 1939; Kerr and Robertson, 1943, 1954; MacDonald and Tatum, 1948; Osaki and Oka, 1962; Hoffmann and Gorczynski, 1964; Stepkowski, 1966; Goldman and Honigberg, 1968; Honigberg and Goldman, 1968). Subcutaneous inoculations of disrupted Trichomonas gallinae in the presence of adjuvant gave very strong rabbit sera for precipitation and fluorescent antibody tests (Goldman and Honigberg, 1968; Honigberg and Goldman, 1968), and the advantages of using adjuvant in immunization were brought out also by agglutination experiments with Tritrichomonas augusta (Samuels and Chun-Hoon, 1964). The present investigation was undertaken to ascertain which immunization method would give the most satisfactory rabbit sera for agglutination, hemagglutination, and precipitation (gel diffusion) reactions involving T. gallinae. MATERIALS AND METHODS