SARCOCYSTIS, TOXOPLASMA AND RELATED PROTOZOA 



335 



blood of parasites. Humoral antibodies 

 are not effective against intracellular 

 parasites, however. 



At least 2 types of humoral antibodies, 

 complement fixing and cytoplasm modify- 

 ing, are produced against Toxoplasma. 

 The latter are revealed by the Sabin-Feld- 

 man dye test. They appear earlier in the 

 course of the disease than complement 

 fixing antibodies and persist much longer. 



The dye test was introduced by Sabin 

 and Feldman (1948) and has been des- 

 cribed in detail by Sabin et al. (1952). It 

 is based on the fact that both the cyto- 

 plasm and nucleus of Toxoplasma tropho- 

 zoites stain deeply with alkaline methylene 

 blue after incubation with normal serum, 

 but that after incubation with antibody- 

 containing serum only the nuclear endo- 

 some will stain. According to Lelong and 

 Desmonts (1952), the dye test antibodies 

 act by producing partial lysis of the or- 

 ganisms thru a modified Pfeiffer phenom- 

 enon in which the parasites lose those 

 cytoplasmic constituents which are ordi- 

 narily stained by methylene blue. Kulasiri 

 and Dasgupta (1959) found that ribonucleic 

 acid disappears during incubation in a pos- 

 itive reaction, and suggested that this is 

 the reason the organisms no longer stain. 



The antibody itself is heat stable, but 

 a fairly large amount of a heat-labile, 

 complement-like "accessory" factor is 

 also necessary. This is apparently a 

 mixture of the C2, C3 and C4 factors of 

 complement plus properdin (Gronroos, 

 1956). 



In carrying out the dye test, a series 

 of serum dilutions is used, and a titer of 

 1:16 is considered diagnostic. The dye 

 test titer usually reaches a high level by 

 the end of the second week after infection; 

 in active disease, titers above 1:1000 are 

 found in a month or more. These anti- 

 bodies usually persist for a number of 

 years, probably for more than a decade, 

 altho their titer declines slowly. 



The trophozoites used in the dye test 

 can be obtained from peritoneal exudate 

 or tissue culture. These fluids sometimes 



contain a soluble antigen in sufficiently 

 high titer to block the test partly or com- 

 pletely (Jacobs and Cook, 1954). Antibody 

 in mouse peritoneal fluid may give rise to 

 false positive tests (Frenkel, 1956). In 

 addition, a prozone phenomenon may often 

 occur, so that a full range of dilutions up 

 to 1:1024 at least must be tested. 



The dye test is not necessarily spe- 

 cific for Toxoplas))ia. Muhlpfordt (1951) 

 and Awad and Lainson (1954) reported 

 cross reactions with Sarcocystis tenella, 

 and Awad (1954) even developed a modified 

 dye test for Toxoplasma, using S. tenella 

 trophozoites. 



On the other hand, Cathie and Cecil 

 (1957) were unable to confirm this latter 

 test. Moscovici (1954) found no dye test 

 cross reaction between T. gondii and S. 

 tenella. Jacobs (1956) found no dye test 

 cross reaction between Toxoplasma and 

 Trypanosoma criizi, Plasmodium berghei, 

 P. galUnaceum, Eimeria tenella, Hepato- 

 zoon sp. in squirrels, or Sarcocystis in 

 rhesus monkeys, but did observe cross 

 reactions at titers up to 1:4 between Toxo- 

 plasma and Enceplialitozoon in rats and 

 up to 1:16 between Toxoplasma and Bes- 

 noitia jellisoni in rabbits. Cathie (1957) 

 found the dye test to be specific for Toxo- 

 plasma, for human sera at least; the test 

 sera should be inactivated. 



The complement fixation test was de- 

 veloped by Warren and Sabin (1943) and 

 Sabin (1949). Complement fixing anti- 

 bodies rarely appear earlier than 1 month 

 after infection, and decrease relatively 

 rapidly with time. In 60 children with 

 congenital toxoplasmosis studied by Eich- 

 enwald (1956), complement fixing anti- 

 bodies had disappeared from 44 at 5 years 

 of age and from 8 more at 7 years, altho 

 all but 3 still had dye test antibodies. In 

 15 cases of active toxoplasmosis studied 

 by Makstenieks and Verlinde (1957), the 

 complement fixation reaction became neg- 

 ative in 6 to 9 months while the dye test 

 was still positive at the end of 4 years. 



A positive complement fixation titer 

 of 1:32 or above is considered to indicate 

 relatively recent infection. 



