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African trypanosomiasis and toxoplasmosis
CORRESPONDENCE
723
MARCIAL-ROJAS, R. A. (1971). Pathology of Protozoal and Helminthic Diseases with Clinical Correlation. p. 727. Baltimore: Williams & Wilkins Company. REDEWlLL, F. H. (1949). Urol. Cutan. Rev., 53, 609. WHITEHALL, R. & MILLER, M. H. (1944). Bull. Johns Hopk. Hosp., 75, 1969. YAMAGUCHI (1925). Quoted by Faust, E. C. & Russell, P. F., Clinical Parasitology, 7th ed., 1964, p. 363. Philadelphia: Lea & Febiger.
AFRICAN TRYPANOSOMIASIS AND TOXOPLASMOSIS
SIR,--The question of whether human African trypanosomiasis sera can give false positives in the indirect fluorescent antibody test for toxoplasmosis has been raised by SEAH and GABRIELIAN (1972). There is some evidence that this is not the case as cross reaction has not been shown in the complement fixation, direct agglutination, indirect fluorescent antibody or dye tests (BEVERLEY, 1957; CATHIE, 1957; FULTON and VOLLER, 1964). I am, etc., MILES B. M.ARKUS, Department of Zoology and Applied Entomology, Imperial College, 16 July, 1973 University of London, S.W.7. REFERENCES
BEVERLEY, J. K. A. (1957). Trans. R. Soc. trop. Med. Hyg., 51, 118. CATHIE, I. A. B. (1957). Ibid., 51, 104. FULTON, J. D . & VOLLER, A. (1964). Br. reed. J., 2, 1173. SEAH, S. K. K. & GABRIELIAN, S. (1972). Trans. R. Soc. trop. Med. Hyg., 66, 807.
ISOLATION OF TRYPANOSOMES
SIR,--The technique of LANHAM and GODFREY (1970) has proved invaluable for the isolation and concentration of the blood forms of the African trypanosomes but is not so efficacious for those of T. cruzi. T h e observations by GALLIARD(1929, 1930 and 1952) on T. cruzi trypanomastigotes in the peritoneum of mice suggested to us the possibility of using peritoneal washings from infected mice as a source of parasites and then separating the trypanosomes from white cells by means of a cellulose column. We initially used the Y strain of T. cruzi, described by SILVAand NUSSENZWEIG(1953), on the 7th day after blood passage when a maximum number of parasites occur in the blood. 2 ml. of sterile saline or citrate-saline were injected intraperitoneally into anaesthetized mice and aspirated after 2-5 rain. Large numbers of trypanosomes were encountered in the fluid. A column of Cellex-SE cation exchange resin (Bio-Rad Laboratories, Richmond, California), 0.9 cm. diameter and 4 cm. high was prepared in a glass syringe plugged with a small wad of cotton wool. Immediately before addition of the peritoneal fluid 1 ml. of sterile saline was added and when almost completely absorbed the parasite preparation was added. When most of this fluid had entered the cellulose a further 1 ml. of saline was added and very gentle pressure was applied by means of the syringe piston. T h e first 1 ml. of eluate contained very few trypanosomes and was discarded. Active trypanosomes were recovered from the subsequent eluate. U p to 50% recoveries of trypanosomes have been obtained whereas virtually all host leucocytes are removed by the column. Killing the mice immediately after i.p. injection and allowing some minutes for blood clotting to occur prior to the aspiration of the peritoneal fluid reduced to negligible levels the contamination of the preparation by erythrocytes. Trypanomastigotes were not found in preliminary observations on peritoneal fluids from mice infected with the C L (BRE~mR, 1965) and Brazil (original source unknown) strains of T. cruzi at times when blood parasitaemias were present. These latter strains differ from the Y strain in possessing predominantly "stout" blood trypanomastigotes (BRENER and CHIARI, 1963). T h e M R strain (BRENER and CHIARI, 1963) also possesses predominantly "stout" blood trypanomastigotes however, but these forms were found in the peritoneum of mice examined on the 10th day after blood passage. T h e "stout" forms could also be separated from peritoneal cells by the cellulose column.
723
MARCIAL-ROJAS, R. A. (1971). Pathology of Protozoal and Helminthic Diseases with Clinical Correlation. p. 727. Baltimore: Williams & Wilkins Company. REDEWlLL, F. H. (1949). Urol. Cutan. Rev., 53, 609. WHITEHALL, R. & MILLER, M. H. (1944). Bull. Johns Hopk. Hosp., 75, 1969. YAMAGUCHI (1925). Quoted by Faust, E. C. & Russell, P. F., Clinical Parasitology, 7th ed., 1964, p. 363. Philadelphia: Lea & Febiger.
AFRICAN TRYPANOSOMIASIS AND TOXOPLASMOSIS
SIR,--The question of whether human African trypanosomiasis sera can give false positives in the indirect fluorescent antibody test for toxoplasmosis has been raised by SEAH and GABRIELIAN (1972). There is some evidence that this is not the case as cross reaction has not been shown in the complement fixation, direct agglutination, indirect fluorescent antibody or dye tests (BEVERLEY, 1957; CATHIE, 1957; FULTON and VOLLER, 1964). I am, etc., MILES B. M.ARKUS, Department of Zoology and Applied Entomology, Imperial College, 16 July, 1973 University of London, S.W.7. REFERENCES
BEVERLEY, J. K. A. (1957). Trans. R. Soc. trop. Med. Hyg., 51, 118. CATHIE, I. A. B. (1957). Ibid., 51, 104. FULTON, J. D . & VOLLER, A. (1964). Br. reed. J., 2, 1173. SEAH, S. K. K. & GABRIELIAN, S. (1972). Trans. R. Soc. trop. Med. Hyg., 66, 807.
ISOLATION OF TRYPANOSOMES
SIR,--The technique of LANHAM and GODFREY (1970) has proved invaluable for the isolation and concentration of the blood forms of the African trypanosomes but is not so efficacious for those of T. cruzi. T h e observations by GALLIARD(1929, 1930 and 1952) on T. cruzi trypanomastigotes in the peritoneum of mice suggested to us the possibility of using peritoneal washings from infected mice as a source of parasites and then separating the trypanosomes from white cells by means of a cellulose column. We initially used the Y strain of T. cruzi, described by SILVAand NUSSENZWEIG(1953), on the 7th day after blood passage when a maximum number of parasites occur in the blood. 2 ml. of sterile saline or citrate-saline were injected intraperitoneally into anaesthetized mice and aspirated after 2-5 rain. Large numbers of trypanosomes were encountered in the fluid. A column of Cellex-SE cation exchange resin (Bio-Rad Laboratories, Richmond, California), 0.9 cm. diameter and 4 cm. high was prepared in a glass syringe plugged with a small wad of cotton wool. Immediately before addition of the peritoneal fluid 1 ml. of sterile saline was added and when almost completely absorbed the parasite preparation was added. When most of this fluid had entered the cellulose a further 1 ml. of saline was added and very gentle pressure was applied by means of the syringe piston. T h e first 1 ml. of eluate contained very few trypanosomes and was discarded. Active trypanosomes were recovered from the subsequent eluate. U p to 50% recoveries of trypanosomes have been obtained whereas virtually all host leucocytes are removed by the column. Killing the mice immediately after i.p. injection and allowing some minutes for blood clotting to occur prior to the aspiration of the peritoneal fluid reduced to negligible levels the contamination of the preparation by erythrocytes. Trypanomastigotes were not found in preliminary observations on peritoneal fluids from mice infected with the C L (BRE~mR, 1965) and Brazil (original source unknown) strains of T. cruzi at times when blood parasitaemias were present. These latter strains differ from the Y strain in possessing predominantly "stout" blood trypanomastigotes (BRENER and CHIARI, 1963). T h e M R strain (BRENER and CHIARI, 1963) also possesses predominantly "stout" blood trypanomastigotes however, but these forms were found in the peritoneum of mice examined on the 10th day after blood passage. T h e "stout" forms could also be separated from peritoneal cells by the cellulose column.