Toxoplasma gondii: Electron microscopic study on the dye test reaction

EXPERIMENTAL

PARASITOLOGY

Toxoplasma

40, 170-178

(1976)

gondii:

Electron Dye Test

TAKURO Department Department

ENDO AND

of Parasitology,

of Parasitology,

(Accepted

Microscopic Reaction AKIO

National Instftute

on the

KOBAYASHI

of Health,

The Jikei University Tokyo 105, Japan for publication

Study

9 October

School

Tokyo 141, and of Medicine,

1975)

ENDO, T., AND KOBAYASHI, A. 1976. Toxoplasma gondii: Electron microscopic study on the dye test reaction. Experimental Parasitology 40, 170-178. When intact Toxoplasma trophozoites were stained with isotonic alkaline methylene blue solution, the organelles rich in nucleic acid, i.e., nucleus, free and membrane-bound ribosomes appeared as electron dense areas. When the parasites were incubated with the anti-Toxoplasma antibody and the accessory factor, swelling of the surface membrane occurred first, followed by destruction of the inner structures. In the dye test positive parasites, there were no definite organelles recognizable, as there were in the intact parasites. By the negative staining method, holes (defects) with dark central portions were observed on the surface of the parasites treated with the antibody and the accessory factor, the diameter of the holes measuring about lo-11 nm. These holes, which tended to occur in clusters, were each surrounded by a clear ring. INDEX DESCRIPTORS: Toxoplasma gondii; Parasitic protozoan; Sabin-Feldman dye test; Immune cytolysis; Electron microscopy; Negative staining; Complement-induced hole; Surface membrane; Accessory factor; Complement; Methylene blue; Nucleic acid.

It is well known that the dye test of Sabin and Feldman (1948) is the most reliable serological method for the diagnosis of toxoplasmosis. This test is based on the known fact that the Toxoplasma trophozoite loses its affinity to alkaline methylene blue when the or,ganism is incubated at 37 C for 1 hr together with its antibody and the complement-like accessory factor. The mechanism of the dye test reaction has been an intriguing problem for parasitologists. Kulasiri and Dasgupta (1959), in their histochemical study, observed the disappearance of ribonucleic acid from the cell when the trophozoite was treated with its antibody and the accessory factor. They suggested that the loss of affinity of the 170 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Copyright

parasites for the dye was due to the direct action of the antibody and the accessory factor resulting in the disappearance of or changes in the parasitic ribonucleic acid. Strannegard ( 1967), who studied the action of ferritin-labeled antibody, postulated that damage to the parasitic surface membrane might be the primary step in the immunoinactivation of Toxoplasma gondii and the disintegration of intracellular structures a secondary phenomenon. This study was designed to elucidate (i) the intracellular organelles stainable with alkaline methylene blue and also (ii) possible morphological changes in the parasite affected by the specific immune reaction.

DYE TEST IN TOXOPLASMOSIS MATERIALS

AND METHODS

Toxoplasma Trophoxoites The peritoneal fluid, containing numerous trophozoites of the RH strain (received from Dr. A. B. Sabin in 1952 and maintained by intraperitoneal passage in mice) was drawn from mice infected 3 days earlier. The parasites were washed three times with pH 7.2 phosphate buffered saline before use.

171

tive prevents the decolorization of the dye from the stained parasites. Examination of Changes in the Parasites Affected by the Dye Test

After incubating the parasites with the antiserum and accessory factor, the trophozoites were collected by gentle centrifugation and fixed in either cold 1% osmium tetroxide in 100 m,M PB with 210 mM sucrose or cold 1.2% glutaraldehyde in 100 mM PB followed by postfixaThe Dye Test tion in osmium tetroxide. Dehydration and Sabin-Feldman dye test was performed, embedding were done as described above. with some modifications, by the method of For a particular study on changes of the Frenkel and Jacobs ( 1958). Fresh human surface membrane of the parasite, the negaplasma was employed as the source of the tive staining method was employed. Folaccessory factor (Kobayashi et al. 1968; Iowing incubation of the parasites with the Wallace 1969). To obtain the plasma, antibody and accessory factor, the trophowhole blood from an accessory factor dozoites were collected and suspended in a nor was mixed with Alsever’s solution at hypotonic PB (20 imOsM ) for 30 min at a ratio of 87:13, and centrifuged at 3000 4 C with agitation. The suspension was rpm for 5 min. then centrifuged at 20,OOOgfor 30 min. As the anti-Toxoplasma antibody, a huSediments thus obtained were washed 3 man serum with a dye test titer of 1:256 times and resuspended in the same hypowas used throughout the present study. tonic solution. Immediately after the treatThe serum was inactivated by heating at ment, the samples were mounted on a grid 56 C for 30 min. The antiserum was apand stained with 2% phosphotungstate plied at 1:64 dilution for the reaction. (pH 7.2) for 30 to 60 sec. As a control, saline was used instead of the antiserum. Examination of Intracellular Organelles An aliquot of the material was examined Stainable with Alkaline Methylene Blue by light microscopy in order to ascertain The intact parasites were stained with that the parasites incubated with the antieither hypotonic or isotonic alkaline methbody and accessory factor were not stained ylene blue solution. The former is rouwith the methylene blue. tinely used in the dye test, whereas the latter was specially prepared by adding RESULTS sucrose to the medium so as to restore the Intracellular Organelles Stainable with isotonicity. The parasites were fixed in cold 1% osmium tetroxide in 100 mM Alkaline Methylene Blue When intact Toxoplasma trophozoites phosphate buffer (PB) with 140 mM suwere treated with ordinary hypotonic alkacrose and 20.3 mM ammonium molybdate. line methylene blue solution, the parasites The organisms were embedded in epon resin after dehydration in a graded series were observed by light microscopy to be of ethyl alcohol solutions. The sections stained a deep blue. An electron microscopic observation rewere examined with a JEM 100-U electron microscope. The control parasites were vealed that the treatment of the parasites treated similarly but without the dye. The with the hypotonic dye solution resulted use of ammonium molybdate in the fixa- in intensive cytoplasmic damage and for-

172

END0

AND

KOBAYASHI

FIG. 1. A section through Toxoplasma gondii trophozoites treated with the hypotonic solution with methylene blue. Island-like highly electron dense areas (arrows) are observed in the parasites: host cell (hc), nucleus (n), parasite (p) (without electron staining). X15,000.

mation of electron dense island-like areas (Fig. 1, arrows). These electron dense areas are considered to be due to the absorption of methylene blue, since the speciments were not treated with lead citrate or uranyl acetate. Hypotonic solution without methylene blue did not produce elec-

tron dense areas in the parasites but caused complete destruction of the inner structure (Fig. 2). Thus, the relation between the localization of the intracellular organelles and the stained areas was obscure when hypotonic dye solution was used. On the other hand, when the intact

FIG. 2. A section through Toxoplasma gondii trophozoites treated tion without methylene blue. The inner structures of the parasites are The electron dense areas are not observed in these cells (with electron

with hypotonic soludestroyed completely. staining). X 13,000.

DYE

TEST

IN

TOXOPLASMOSIS

FIG. 3a and b. Sections through Toxoplasma gondii trophozoites treated with isotonic solution with methylene blue. The electron dense areas (arrows) are observed in relation to the organelles, such as nucleus, free ribosomes and rough endoplasmic reticulum: conoid (c), free ribosomes (fr), dense granule (g), mitochondrion (mt), nucleus (n), rough endoplasmic reticulum (rer), rhoptry (rh) (without electron staining). 3a: X22,500, 3b: ~21,000.

trophozoites were stained with the isotonic methylene blue solution, highly electrondense areas (Figs. 3a and b, arrows) were found in certain organelles rich in nucleic acid, such as nucleus, free ribosomes and rough endoplasmic reticula (membranebound ribosomes), but not in the dense granules, rhoptries or
The control parasites incubated in the isotonic solution without the dye retained their inner structures (Fig. 4). Changes in the Parasites Aflected by the Dye Test Reaction The parasites incubated at 37 C with the antibody and accessory factor showed de-

174

END0

FIG. without nucleus

AND

KOBAYASHI

4. A section through Toxoplasma gondii trophozoites treated with isotonic solution the dye. The inner structures of the parasites remained intact: dense gramrle (g), (n ), rhoptry ( rh) (with electron staining). X 13,000.

veloping morphological changes within the 60 min incubation period. After incubation of the trophozoites for 10 min, swelling of their outer membrane was often observed (Fig. 5, arrows), although the inner structures appeared almost intact. However, when the parasites

Frc. 5. A section accessory factor for granule (g), nucleus

were incubated for more than 30 min, most of the intracellular organelles disappeared, producing different sized vacuoles and swollen mitochondria within the cells (Fig. 6a). Discontinuities of the outer and inner membranes of the parasites (Fig. 6b, arrows) were also observed. In the

through Toxoplasma gondii trophozoites 10 min. Swelling of the outer membrane (n), rhoptry (rh) (with electron staining).

incubated (arrows) X 13,500.

with antibody is observed:

and dense

DYE

TEST

IN

TOXOPLASMOSIS

175

parasites incubated without the antibody for 60 min (Fig. 7), the membrane and inner structures appeared similar to those observed in the nontreated parasites (Fig. 8). The changes in the surface membrane of the affected parasites were studied in detail by the negative staining method. On the surface of the trophozoites treated with the antibody and accessory factor numerous holes with dark central portions, each of which was surrounded by a clear ring, were recognized. The diameter of the holes measured about 10-11 nm. The multiple holes tended to occur in clusters (Fig. 9, arrows) and were never observed on the surface membranes of intact or control parasites (Fig. 10).

DISCUSSION Electron microscopic observations on the Sabin-Feldman dye test have been reported by Bringmann and Holz ( 1953), BraunSteiner et al. (1957), Ludvic et al. (1964), Matsubayashi and Akao (1966) and Str%nnegard ( 1967). Most of these workers have observed vacuoles occurring in the dye test positive parasites. The positive reaction is probably the result primarily of damage to the parasite membrane, followed by the Donnan effect. The destruction of the inner structures accompanying vacuole formation should be regarded as a secondary phenomenon. Strannegard ( 1967) also observed similar phenomena in his antibody-activator system. The dye test reaction can be considered as an immune lysis of the parasites, in which the accessory factor behaves like complement in many respects. However, in comparison with the immune hemolysis, the dye test requires much more of the complement-like accessory factor. It has been suggested that the accessory factor consists of complement and properdin (Feldman 1956; StrHnnegard and Lycke 1966). Guinea pig serum in fairly large amount was also found to serve as a source

FIG. 6. Sections through Toxoplasma gondii trophozoites incubated with antibody and accessory factor for more than 30 min. a: Some large and small vacuoles and swollen mitochondria are observed in the parasite: mitochondrion (mt), vacuole (v) (with electron staining). X15,000. b: Discontinuities of the inner and outer membranes (arrows) are observed (with electron staining). X50,000.

of the accessory factor (Kobayashi et al. 1969). Suzuki and Tsunematsu ( 1973) presented evidence that, in the dye test reaction, the trypsinized Toxoplasma trophozoites underwent cytolysis in the presence of guinea pig serum at concentrations as low as l&1.5%, suggesting that the properdin system might not be involved in the reaction. Suzuki ( 1974) speculated that some subfactor in the accessory factor might exert trypsin-like activity in the surface of the parasite. In this respect, however, little convincing evidence has been presented. Holes, with dark central portions and surrounded by a clear ring, were observed on the surface of the parasites affected by the reaction. The diameter of these holes

176

END0

AND

KOBAYASHI

FIG. 7. A section through Toxoplasma gondii trophozoites incubated with accessory factor alone for 60 min as the negative control of the dye test. The membrane and inner structure of the parasite seem intact: conoid (c), dense granule (g), nucleus (n), rhoptry (rh), free ribosomes (arrows ) (with electron staining). X 16,000.

FIG.

8. A section

through

Toxoplasma gondii trophozoites: granule (g), rhoptry (rh), tron staining).

mitochondrion free ribosomes X 16,000.

the nontreated control conoid (c), dense (mt), nucleus (n), (arrows) (with elec-

measured 16-11 nm. The occurrence of such surface membrane defects resulting from an immunolytic reaction with protozoan parasites has not, to our knowledge, been previously observed. There have been similar findings, however, with human erythrocytes. According to Rosse et al. ( 1966), the diameter appeared to depend upon the source of the complement used and not the source of the antibody or the type of the cell; the size of the holes induced by human complement ranged from 9.3-11.2 nm. The holes observed on the affected parasites correspond in size to those reported by Rosse and his colleagues with human erythrocytes. The production of multiple clustered holes on the parasite membrane may be due to excessive amounts of the complement in proportion to antibody as was suggested by Humphrey and Dourmashkin (1969) for the immune hemolytic system when the number of holes exceeded what had been predicted from the one-hit et theory. According to Miiller-Eberhard al. ( 1966), the degree of hemolysis was

DYE

TEST

IN

177

TOXOPLASMOSIS

FIG. 9. The surface of Toxoplasma gondii trophozoite membrane treated with antibody and accessory factor. The holes with dark central portions, each surrounded by a clear ring, tend to occur in clusters (arrows). The diameter of the hole is about lo-11 nm (negative staining). X 100,000.

proportional to the number of the third component of complement molecules. A high level of binding of C3 molecules per cell was found to be a prerequisite for the production of immune hemolysis. The hole clusters may result in intensive focal dam-

FIG. 10. The surface of Toxoplasma go&ii factor alone as a negative control. The holes parasites (negative staining). X 100,000.

age of the parasite membrane. As compared with ordinary immune hemolysis, it may be that in the Toxoplasma dye test a greater number of holes is necessary and therefore a proportionally larger amount of the accessory factor would be required.

trophozoite are never

membrane treated with observed on the surface

accessory of control

178

END0

AND

ACKNOWLEDGMENTS We wish to thank Dr. Tatsushi Ishizaki and Keizo Inoue, National Institute of Health of suggestions and enJaw, for their valluable couragements throughout this study. We are also appreciative of Dr. Akio Suzuki, the Jikei University School of Medicine, for his useful advice and technical help in providing us with the facilities to use the JEM 100-U electron microscope. This study was partly supported by a research grant from the Japan Ministry of Education and Welfare. Dr.

REFERENCES

BRAUNSTEINER, H., PAKESCH, F., AND THALHAMMER, 0. 1957. Elektronenmikroskopische Untersuchungen uber die Morphologie des Toxoplasma gondii und das Wesen des Farbtestes nach Sabin und Feldman. Wiener Zeitschrift fiir innere Me&in und ihre Grenzgebiet 38, 16-27. ERINGMANN, G., AND HOLZ, J. 1953. Lichtund elektronenmikroskopische Untersuchungen zum Sero-Farbtest auf Toxoplasmose nach Sabin und Feldman. Zeitschrift fiir Hygiene 138, 151-154. FELDMAN, H. A. 1956. The relationship of Toxoplasma antibody activator to the serum-properdin system. Annals of the New York Academy of Science 66, 263-267. FRENKEL, J. K., AND JACOBS, L. 1958. Ocular toxoplasmosis; pathogenesis, diagnosis, and treatment. A.M.A. Archives of Ophthalmology 59, 260-279. HUMPHREY, J. H., AND DOURMASHKIN, R. R. 1969. The lesions in cell membranes caused by complement. Advances in Immunology 11, 75115. KOBAYASHI, A., KUMADA, M., SUZUKI, M., AND TSUNEMATSU, Y. 1969. Studies on the accessory factor for Toxoplasma dye test. Guinea pig serum as a source of the accessory factor. Japanese Journal of Medical Science and Biology 22, 327-336. KOBAYASHI, A., KUMADA, M., AND TSUNEMATSU, Y. 1968. Effects of anticoagulants on the dye test for Toxoplasmosis. Japanese Journal of Medical Science and Biology 21, 71-89. KULASIRI, C., AND DASGUPTA, B. 1959. A cyto-

KOBAYASHI

chemical investigation of the Sabin-Feldman phenomenon in Toxoplasma gondii and an explanation of its mechanism on this basis. Parasitology 49, 586593. LUDVIK, J., SIIM, J, Cnn., AND BIRCH-ANDERSEN, A. 1964. Studies on the ultrastructure of Toxoplasma gondii after treatment with specific antibodies. In “3rd European Regional Conference on Electron Microscopy.” Praha, NCSAV 2, 193-194. MATSUBAYASIII, H., AND AKAO, S. 1966. Immunoelectron microscopic studies on Toxoplasma gondii. American Journal of the Tropical Medicine and Hygiene 15, 486-491. MULLER-EBERHARD, H. J., DALMASSO, A. P., AND CALCOTT, M. A. 1966. The reaction mechanism of &-globulin (C3) in immune hemolysis. The Journal of Experimental Medicine 123, 33-54. ROSSE, W. F., DouRhxAsHIuN, R. R., AND HUMPHREY, J. H. 1966. Immune lysis of normal human and paroxysmal nocturnal hemoglobinuria (PNH) red blood cells. The JouTnaZ of Experimental Medicine 123, 969-984. SABIN, A. B., AND FELDMAN, H. A. 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (Toxoplasma). Science 108, 660-663. STRANNEGARD, 0. 1967. An electron microscopic study on the immuno-inactivation of Toxoplasma gondii. Acta Pathologica et Microbiologica Scandinavica 71, 463-470. STRKNNEGARD, O., AND LYCKE, E. 1966. Properdin and the antibody-effect on Toxoplasma gondii. Acta Pathologica et Microbiologica Scandinavica 66, 227-238. SUZUKI, M. 1974. Mechanism of the Sabin-Feldman’s dye test. Japanese Journal of Parasitology 23, ( Suppl. ) 20-21 (in Japanese). SUZUKI, M., AND TSUNEMATSU, Y. 1973. Toxoplasma gondii: Trypsin treatment of trophozoites for enhanced immune cytolysis in the dye test. Experimental Parasitology 33, 433436. WALLACE, G. D. 1969. Sabin-Feldman dye test for toxoplasmosis. The use of sodium citrate in accessory factor, and a method for collecting and storing blood on paper discs. American Journal of Tropical Medicine and Hygiene 18, 395-398.