An Alternative Technique to Reveal Polysaccharides in Toxoplasma gondii Tissue Cysts

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98(7): 915-917, October 2003
915
An Alternative Technique to Reveal Polysaccharides in
Toxoplasma gondii Tissue Cysts
Erick Vaz Guimarães, Laís de Carvalho*, Helene Santos Barbosa/+
Laboratório de Ultra-estrutura Celular, Departamento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz-Fiocruz,
Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil *Laboratório de Cultura de Células, Departamento de Histologia e
Embriologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to
two different methodologies derived from the periodic acid - Schiff’s reagent (PAS) technique. The use of osmium
tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid
resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts,
with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host
cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host
cells.
Key words: Toxoplasma gondii - amylopectin granules - tissue cysts - ultrastructural cytochemistry
Infection with the protozoan parasite Toxoplasma
infected mice were colleted, washed in phosphate buffered
gondii is endemic throughout the world. This obligate
saline (PBS) and homogenized in 30% Dextran (w/v) in
intracellular parasite is able to persist in its hosts by
Hank’s balanced salt solution (HBSS) by 5 passages
differentiating from the replicative tachyzoite stage into
through an 18-23 gauge needle at a concentration of no
cysts forming latent bradyzoites. The reversible stage
more than 1 brain/2.5 ml. This solution was centrifuged at
differentiation in T. gondii makes it an interesting model
3000 g for 10 min and the pellet containing the tissue cysts
system of protozoan differentiation. The energy
was resuspended in HBSS (Freyre 1995, Popiel et al. 1996).
requirements and biosynthetic substrates of coccidian
Transmission electron microscopy - Isolated cysts
parasites are mostly dependent on carbohydrate
were fixed for 1 h at 4°C with 2.5% (v/v) glutaraldehyde in
metabolism. The reserve of polysaccharides in T. gondii
0.1 M Na-cacodylate buffer (pH 7.2). After fixation, the
is found in amylopectin granules, which are scarce or
cysts were washed in PBS and post-fixed for 30 min at
absent in tachyzoites, but abundant in bradyzoites
room temperature with 1% OsO4 in 0.1 M cacodylate buffer
(reviewed in Dubey et al. 1998, Speer et al. 1999).
containing 5 mM CaCl2 and 3.5% sucrose. Cells were then
Bradyzoites enriched with amylopectin granules are
washed in the same buffer, dehydrated in acetone and
colored in red when stained with the periodic acid - Schiff
embedded in PolyBed 812. Unstained thin sections were
reagent (PAS) technique (reviewed in Dubey et al. 1998)
processed for cytochemistry.
besides being less susceptible to the destruction by
Ultrastructural cytochemistry - To characterize the
proteolytic enzymes, as compared with the tachyzoites
polysaccharides expression (amylopectin granules) in
(Jacobs et al. 1960, Dubey 1998). In this report we present
tissue cysts, we have used two methodologies. Thin
the characterization of these amylopectin granules in T.
sections were colleted on gold grids, oxidized for 20 min
gondii tissue cysts by two alternative ultrastructural
with 1% periodic acid, quickly washed twice in distilled
cytochemistry methodologies.
water, and finally incubated for 72 h, in humid chamber at
MATERIALS AND METHODS
room temperature, in 2% thiocarbohydrazide (TCH) diluted
at 20% (v/v) in acetic acid. These grids were washed suc-
Tissue cysts - C57BL/6 mice were infected intra-
cessively in decreasing concentrations of acetic acid
peritoneally with 50-100 cysts of T. gondii ME-49 strain,
(10%, 5%, 3%, and 1%) for 1 min each. The polysaccharide
an avirulent cyst-forming strain. Brains from 4-8 weeks
revelation was made by incubating the grids for 30 min in
an aqueous solution with 1% silver proteinate.
In the second methodology, after the steps described
above, the carbohydrate revelation was accomplished by
incubation of tissue cysts on grids with osmium tetroxide
vapor for 1 min. Using both methodologies after the
Financial support: Conselho Nacional de Desenvolvimento
Científico e Tecnológico, Fundação Carlos Chagas Filho de
revelation, these grids were quickly washed once with
Amparo à Pesquisa do Estado do Rio de Janeiro, Universidade
distilled water, followed by another wash for 10 min.
do Estado do Rio de Janeiro, and Instituto Oswaldo Cruz-
Controls experiments of the reaction were obtained by
Fiocruz
the omission of the oxidation step by the periodic acid, or
+Corresponding author. Fax: +55-21-2260.4434. E-mail:
the treatment of the cysts with TCH to guarantee that the
helene@ioc.fiocruz.br
reaction has been produced by free aldehydes, or by
Received 16 June 2003
reaction of TCH with the biological material, respectively.
Accepted 10 September 2003
Other control was made by omission of the osmium

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916
916
Polysaccharides in T. gondii Tissue Cysts • Erick Vaz Guimarães et al.
Figs 1-2: reaction product for the Thièry technique employing silver proteinate as developing agent of reaction product. Fig. 1: ultrathin
section of a whole cyst containing parasites, the most presenting the electrondense reaction product located in cytoplasmatic organelles,
indicative of the presence of amylopectin granules (AG). Fig. 2: detail of the reaction product in AG inside a bradyzoite (Bz). Figs 3-5:
modification of the Thièry technique using the osmium vapor in Toxoplasma gondii tissue cysts. Fig. 3: detail showing the AG and the
ultrastructural morphology of the parasites (Bz). Some amylopectin granules present the central area lacking the reaction product (arrow);
R: rhoptria; C: conoid. Fig. 4: aspects of the reaction product for the osmium vapor showing intense reactivity in AG and in the cystic wall
and matrix. A contrast reversion was observed with this technique, allowing a detailed visualization of the structures and organelles of the
parasites; CW: cystic wall; MIC: microneme; DG: dense granules; * : matrix. Fig. 5: reaction product observed in AG and in *, appearing
as increase in electrondensity, indicative of the presence of glycoconjugates.

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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98(7), October 2003
917
tetroxide. Finally, the sections were examined unstained
chosen trying to use the osmium technique, and the quality
in a Zeiss EM 10C transmission electron microscope.
of our results was superior than that obtained with the
RESULTS
silver proteinate. The use of osmium allowed to obtain
images of high resolution with great contrast of the
Both methodologies used in the current work revealed
structural cell components, evidentiating besides the
polysaccharides in bradyzoites forms of T. gondii tissue
amylopectin granules, the cellular membranes, nucleus,
cysts located in electrondense oval bodies, the
conoid, rhoptries, dense granules and micronemes. This
amylopectin granules. The granules were observed in large
facilitated the structural visualization of the parasites,
amount mainly in the middle-posterior portion of the
without the presence of precipitates and without affecting
parasites (Figs 1-5). Fig. 1 shows the reaction product
the revelation of the final reaction product.
when silver proteinate was used as a developing agent of
The method used in this study, can be applied to
the aldehyde reactivity. The reaction product was intense,
distinguish between bradyzoite and tachyzoite forms
presenting also a very fine granular deposit in the cyst
inside the tissue cysts, due to the presence of large amount
matrix and in the cyst wall (Figs 1, 2). This reaction was
of amylopectin granules in the bradyzoites, responsible
observed in most intracystic parasites, although some
for great polysaccharides accumulation and as material of
parasites presented no reaction product or small
energy reserve in these parasites. The osmium method
intracellular granules (Fig. 1).
showed to be sensitive to delimitate the matrix and cystic
When the osmium tetroxide vapor was used as a
wall. Our methodology, besides the above-described
developing agent for the reaction, besides increasing the
advantages, can be used for studies on the interaction
electrondensity of the amylopectin granules inside the
between bradyzoites and skeletal muscle cell or other host
cysts, it allowed an excellent visualization of the structures
cells, improving the follow-up of intracellular cycle of T.
of the parasites, such as internal membranes, conoid,
gondii, its reversible differentiation (tachyzoite-
rhoptries and micronemes, which acquired a negative
bradyzoite), and also during the formation of latent
contrast (Figs 3-5). Some granules presented the central
bradyzoites in cysts into the host cells, in vitro.
area without reaction product (Figs 2, 3, 5). The wall, the
granular region, and the matrix of the cysts also showed
ACKNOWLEDGEMENTS
enhanced reactivity, although with lower intensity (Fig.
To Dr Ricardo T Gazzinelli (Universidade Federal de Minas
5). No reaction product was visualized in cells when TCH,
Gerais), for supplying the T. gondii ME-49 strain, to Dr Maurilio
osmium or periodic acid was omitted (data not shown).
José Soares for critically reading this manuscript, and Genésio
L Faria, Ismael CS Gomes, and José L Faria for their excellent
DISCUSSION
technical assistance.
The alternative method for the detection of vic diols
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