Fluorescent Antibody Reactions in Trypanosoma rhodesiense and T. gambiense in Experimental Animals

Abstract

A fluorescent antibody technique for the serodiagnosis of African trypanosomiasis in experimental animals is described. The indirect fluorescent antibody test employing the trypanosome form from rats as antigen appears to be highly sensitive in rabbits infected with T. rhodesiense and T. gambiense. Results of 29 sera from rabbits infected with T. rhodesiense organisms yielded 29 positive reactions, whereas 21 positive reactions and one weak reaction were obtained from 22 rabbits infected with T. gambiense organisms. The reproducibility of this test employing different lots of trypanosomes and labeled antiglobulin was good. Animals were inoculated with varying doses from 4 X to 5 X 106 trypanosomes and were bled at weekly intervals. Antibodies were detected between 1 and 2 weeks after inoculation with no apparent correlation between the early appearance of antibodies and the size of the inoculum as previously reported with helminthic infections. The similarity of observable reactions between the African trypanosomes and T. lewisi as antigen is of great interest. Since T. lewisi is nonpathogenic for humans and can be maintained readily in most laboratories, it is of great importance in areas where sufficient precautions cannot be taken to prevent human infections. Several parasitic protozoans have been stained with fluorescent antibody techniques (Goldman, 1953, 1957; Ristic et al., 1957; McEntegard et al., 1958). However, only rarely (Fife and Muschel, 1959; Tobie et al., 1961) has this procedure been successfully utilized for the immunodiagnosis of human protozoan infections. Recently a reliable and practical fluorescent antibody technique has been described for the serodiagnosis of schistosomiasis (Sadun et al., 1960). Adaptations of this test to permit the utilization of minute amounts of dried blood on filter paper and of preserved organisms have provided simple, rapid, and reliable serological techniques for the laboratory diagnosis of schistosomiasis (Sadun et al., 1961; Anderson et al., 1961a, 1961b) and trichinosis (Sadun et al., 1962b). The current report presents the results of studies in which the indirect fluorescent antibody technique was used to stain the various stages in the life cycle of T. rhodesiense and T. gambiense. Furthermore, attempts were made to determine the reproducibility of speReceived for publication 21 December 1962. cific immunological staining, to determine the time of initial appearance of antibodies in experimentally infected animals, and to determine whether organisms which are not pathogenic for man, such as T. lewisi, could be used as a source of antigen for the serological diagnosis of African trypanosomiasis. MATERIALS AND METHODS Trypanosoma rhodesiense (Wellcome strains) and T. gambiense (Wellcome and Cheick strains) maintained in albino rats and in cultures were used as the source of antigen. The rats were inoculated intraperitoneally with approximately 100,000 trypanosomes in rat blood diluted with 0.85% saline and subinoculated into healthy rats twice weekly. Trypanosome cultures were maintained on a diphasic blood agar medium (Tobie et al., 1950) and subinoculated into fresh cultures every 10 to 12 days. Antisera were obtained by bleeding rabbits from the medial artery of the ear at various intervals following a single intraperitoneal inoculation of varying numbers of trypanosomes. Estimates of the number of organisms in the inoculum were based on counts of trypanosomes in Hayem's red blood cell diluting fluid in a Neubauer cell-counting chamber. Thin smears were made on clean slides from rat tail blood or from the supernate of trypanosome cultures. Usually, 48 to 72 hr were required for