VIRULENCE FOR B A L B / C MICE AND ANTIGENIC DIVERSITY OF EIGHT TOXOPLASMA GONDII STRAINS ISOLATED FROM ANIMALS
AND HUMANS IN BRAZIL
FERREIRA A.M.*, MARTINS M.S.* & VITOR R.W.A.*
S u m m a r y :
With the purpose of establishing alternative parameters to determine the virulence of Toxoplasma gondii strains, the antigenic diversity of eight strains of the parasite isolated in Brazil was evaluated. BALB/c mice were inoculated i.p. with 10°, 101, 1 02 and 1 03 tachyzoites from each strain. The mortality and time to death of the animals showed that T. gondii strains may be divided in three groups: three strains resulted in 100% of mortality, 5-10 days post inoculation (DPI); three strains resulted in 100% of mortality, 7-19 DPI and brain cysts were observed in the mice which were inoculated; two strains resulted in 0% of mortality, 3 0 DPI. The analysis of the antigenic profile of different T. gondii strains through Western blotting, using rabbit antiserum to T.
gondii, revealed that most antigens are similar to all strains. The mAb 4 C 3 H 4 recognized antigens only in the RH, N, AS28 and M E 4 9 strains..
KEY WORDS : Toxoplasma gondii, strain, Brazil, virulence, Western blotting.
I N T R O D U C T I O N
T toxoplasma gondii is an obligate intracellular parasite protozoan that infects a great variety o f vertebrate hosts throughout the world, inclu- ding h u m a n bein gs ( D u b e y & Beattie, 1 9 8 8 ) .
T h e p r e v a l e n c e o f infection b y T. gondii in human b e i n g s is high, with estimates o f c h r o n i c infection in adult individuals varying from 15 to 8 5 % depending o n the g e o g r a p h i c a l region ( D u b e y & Beattie, 1 9 8 8 ) . H o w e v e r , the infections are typically asymptomatic, b e i n g able to cause severe lesions in i m m u n o c o m - p r o m i s e d patients and in congenitally infected fetuses (Luft & Remington 1 9 8 8 ; 1 9 9 2 ) .
* Departamento de Parasitologia, Instituto de Ciencias Biológicas.
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Correspondence: R.W.A. Vitor, Departamento de Parasitologia, Ins- tituto de Ciencias Biológicas, Universidade Federal de Minas Gerais.
Av. Antonio Carlos 6627, C P . 486, Belo Horizonte, MG, CEP 31.270- 901, Brazil. Tel: 0055-31-3499-2875 - Fax: 0055-31-3499-2970.
E-mail: vitorrwa@mono.icb.ufmg.br
Résumé : VIRULENCE POUR LE SOURIS B A L B / c ET DIVERSITÉ ANTIGÉNTQUE DE HUIT SOUCHES DE TOXOPLASMA GONDII ISOLÉES À PARTIR D'ANIMAUX ET D'HOMMES AU BRÉSIL
Dans le but d'établir des paramètres alternatifs pour déterminer la virulence de souches de Toxopasma gondii, la diversité antigénique de huit souches du parasite isolées au Brésil a été évaluée. Les souris BALB/c ont été inoculées i.p. avec 10°, 101,
I02 et I03 tachyzoïtes de chaque souche. La mortalité des animaux a montré que les souches de T. gondii peuvent être réparties en trois groupes : trois souches ont résulté en 100 % de mortalité, 5-10 jours après l'inoculation (DPI) ; trois souches ont entraîné 100 % de mortalité, 7-19 DPI et des kystes ont été observés dans le cerveau des souris qui ont été inoculées; deux souches ont entraîné 0 % de mortalité, 30 DPI. L'analyse du profil antigénique de différentes souches de T. gondii en Western blotting utilisant un antisérum de lapin contre T . gondii a révélé que la plupart des antigènes sont semblables à toutes les souches.
Seules quatre souches (RH, N, AS28 et M E 4 9 ) ont été reconnues par l'anticorps mAb 4C3H4.
MOTS CLÉS : Toxoplasma gondii, souche, Brésil, virulence, Western blotting.
Toxoplasma gondii is r e c o g n i z e d as the only species o f the genus. However, T. gondii strains vary in their virulence. T. gondii has b e e n defined as virulent, avi- rulent or o f intermediate virulence, depending o n its morbidity and mortality in m i c e ( G u o & J o h n s o n , 1995;
H o w e & Sibley, 1 9 9 5 ) . T h e RH strain ( T y p e I) and t h o s e strains w h i c h are g e n e t i c a l l y similar to, are always lethal to mice, irrespective o f d o s e or the strain o f the m o u s e . In contrast, avirulent strains ( T y p e III) s h o w an LD100 greater or equal to 1 03 parasites and c h r o n i c a l infections are easily e s t a b l i s h e d in m i c e ( H o w e et al, 1 9 9 6 ) . T h e strains with intermediate viru- lence (Type II) s e e m to b e strains in transition b e t w e e n the virulent and avirulent p h e n o t y p e s o f the parasite (Literâk et ai, 1 9 9 8 ) .
T. gondii strains have b e e n categorized through stu- dies o f i s o e n z y m e s (Dardé et al., 1 9 9 2 ) , Restriction Fragment Length Polymorphism (Cristina et al., 1995;
H o w e & Sibley 1 9 9 5 ; Literâk et al., 1 9 9 8 ) , RAPD-PCR ( G u o et al, 1 9 9 7 ) , antigenic analysis b y Western blot- ting ( W a r e & Kasper, 1987; W e i s s et al, 1 9 8 8 ; Apple- ford & Smith, 2 0 0 0 ) and reactivity with m o n o c l o n a l antibodies ( G r o s s et al, 1 9 9 1 ; B o h n e et al, 1 9 9 3 ) .
Mémoire - 9 9
Parasite, 2001, 8, 99-105
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2001082099
F E R R E I R A A . M . , M A R T I N S M . S . & V I T O R R . W . A .
With the aim o f characterizing eight T. gondii strains i s o l a t e d in Brazil, w e e v a l u a t e d t h e v i r u l e n c e for B A L B / c m i c e and antigenic diversity o f tachyzoites o f different strains through W e s t e r n blotting with rabbit antiserum to T. gondii and with 4 C 3 H 4 m o n o c l o n a l antibody (Elsaid et al, 1 9 9 9 ) .
M A T E R I A L S A N D M E T H O D S
TOXOPLASMA GONDII STRAINS
E
ight T. gondii strains isolated in Brazil (Sao Paulo state and Minas Gerais state) w e r e e x a m i n e d : AS28, B V , N, E G S , RAR, SAF, C4 and P. T h e RH and ME49 strains w e r e also studied as they represent, respectively, virulent and avirulent strains o f the parasite ( H o w e & Sibley, 1 9 9 5 ) . T h e origin, p l a c e and year o f isolation o f T. gondii strains analyzed in this study are p r e s e n t e d in T a b l e I.
T h e tachyzoites o f T. gondii w e r e o b t a i n e d by inocu
lating from 2 5 0 to 5 0 0 cysts or 1 05 to 1 06 tachyzoites o f different strains b y intraperitoneal (i.p.) injection in Swiss mice. T h e peritoneum o f these mice was w a s h e d two to eight days post inoculation ( D P I ) and the resul
ting material was filtered through p o l y c a r b o n a t e m e m brane o f 3 p m (Millipore). T h e parasites w e r e c o u n t e d and diluted for appropriate concentrations ( 1 03, 1 02, 1 01
and 10° tachyzoites) in PBS pH 7.2.
Strain Origin P l a c e / Y e a r o f isolation References AS28 Mice Sào Paulo/1969 Deane et al., 1971
BV Goat Minas Gerais/1975 Chiari et al, 1985 N Rabbit Sâo Paulo/1952 Nöbrega et al, 1952 EGS Human (CT) Minas Gerais/1998 Castro. 1999 RAR Human (CT) Minas Gerais/1998 Castro, 1999 SAF Human (CT) Minas Gerais/1998 Castro, 1999
C4 Dog Säo Paulo/1972 Jamra & Vieira. 1991 P Dog Sào Paulo/1978 Jamra & Vieira, 1991
RH Human EUA/1939 Sabin, 1941
ME49 Sheep EUA/1965 Darde, 1992
CT = Congenital toxoplasmosis
Table I. - Origin, place and year of isolation of Toxoplasma gondii strains analysed in this study.
DETERMINATION OF VIRULENCE OF T. GONDII STRAINS IN MICE
Female B A L B / c mice, from six to eight w e e k s o f age, w e r e used for experimental inoculations. For e a c h strain, five animals w e r e inoculated i.p. with e a c h o n e o f the concentrations o f tachyzoites and the mortality and time to death w e r e o b s e r v e d for a period o f 3 0 days. Five normal animals inoculated i.p. with PBS pH 7.2 w e r e maintained as negative control. T h e m i c e w e r e kept under conventional conditions and fed with pelleted food.
O n the 3 0t h day after inoculation, the surviving m i c e w e r e bled b y the retro-orbital plexus and the sera w e r e tested b y indirect fluorescent a n t i b o d y test (IFAT), performed in a c c o r d a n c e with the t e c h n i q u e described b y Camargo ( 1 9 6 4 ) . T h e m i c e w h i c h did not s e r o c o n - vert w e r e excluded from the experiment. All o f the sur
viving m i c e w e r e sacrificed b y cervical dislocation for searching tissue cysts in the brain.
PREPARATION OF T. GONDII ANTIGEN FOR WESTERN BLOTTING
T. gondii tachyzoites w e r e withdrawn from Swiss m i c e infected as d e s c r i b e d a b o v e . T h e tachyzoites w e r e w a s h e d three times with P B S pH 7.2 b y centrifugation at 1,500 g, for 15 minutes and filtered in p o l y c a r b o n a t e m e m b r a n e o f 3 p m ( M i l l i p o r e ) . A l i q u o t s o f 1 04 t a c h y z o i t e s w e r e s t o c k e d at - 2 0 ° C until t h e m o m e n t o f use. Cells o b t a i n e d from peritoneal cavi
ties o f Swiss m i c e not infected with T. gondii w e r e purified b y using the s a m e t e c h n i q u e described a b o v e . T h e RAR strain was not studied b y W e s t e r n blotting.
SDS-PAGE AND WESTERN BLOTTING
Sodium dodecyl sulfate - polyacrylamide gel e l e c t r o phoresis (SDS-PAGE) and electrotransference o f pro
teins to the nitrocellulose m e m b r a n e o f 0.45 p m p o r o sity w e r e performed in a c c o r d a n c e with what was previously described (Vitor et al, 1 9 9 9 ) . After transfe
rence, the m e m b r a n e s w e r e b l o c k e d with s k i m m e d milk at 10 % in P B S - T w e e n 2 0 0.05 % for two hours.
Afterwards, the m e m b r a n e s w e r e incubated with rabbit antiserum to T. gondii, immunized with d e a d tachy
zoites o f N strain, diluted 1:50 in P B S c o n t a i n i n g b o v i n e serum albumin ( B S A ) at 3 %, for o n e hour under agitation at room temperature or with the m o n o clonal antibody, 4C3H4, anti-P32, d e v e l o p e d against tachyzoites o f the N strain (Elsaid et al., 1 9 9 9 ) . After three washings in P B S - T w e e n 2 0 0.05 %, the m e m b r a n e s w e r e incubated with anti-immunoglobulin G ( I g G ) o f rabbit conjugated with p e r o x i d a s e o r anti-IgG o f m i c e c o n j u g a t e d w i t h p e r o x i d a s e ( S I G M A ) in
1:5000 dilution in PBS pH 7.2 for 1h, at r o o m t e m p e rature. Proteins w e r e shown by developing m e m b r a n e s b y using 4 - c h l o r o - l - n a p h t h o l as substrate.
STATISTICAL ANALYSIS
T h e differences o b s e r v e d b e t w e e n the m e a n day o f mice death inoculated with different strains o f T. gondii and a m o n g the m e a n s o f the n u m b e r o f brain cysts in surviving mice after 3 0 days o f infection w e r e analyzed by Student's t test, using the significance level o f 9 5 % (Armitage & Berry, 1 9 9 4 ) . T h e correlation b e t w e e n the n u m b e r o f T. gondii tachyzoites inoculated and the n u m b e r o f brain cysts in the m i c e w a s analyzed b y the Linear Regression (Armitage & Berry, 1 9 9 4 ) .
R E S U L T S
VIRULENCE OF TOXOPLASMA GONDII STRAINS IN BALB/c MICE
T
a b l e II presents the virulence c o m p a r i s o n for B A L B / c m i c e infected i.p. with different inocula o f tachyzoites o f T. gondii strains analyzed in this study.Infections with RH, AS28, B V , a n d N strains resulted in 100 % o f mortality o f m i c e , with the death o f ani
mals occurring 5-10 DPI, with the e x c e p t i o n o f the ino
culum o f 1 01 tachyzoites o f the RH strain, in which o n e m o u s e ( 2 0 % ) survived 3 0 D P I . Nevertheless, this m o u s e presented negative serology by IFAT and nega
tive brain examination, suggesting that the animal w a s not infected. Brain cysts w e r e not o b s e r v e d in any o f the m i c e inoculated with t h e s e strains.
T h e infections with the E G S , RAR, and SAF strains resulted in 100 % o f mortality from s e v e n to 19 DPI.
For the i n o c u l u m o f 10° tachyzoites o f the RAR strain, only o n e m o u s e ( 2 0 % ) died 18 DPI. Meanwhile, the s u r v i v i n g m i c e p r e s e n t e d n e g a t i v e s e r o l o g y f o r
T. gondii b y IFAT, without brain cysts.
Brain cysts w e r e observed in mice inoculated with EGS, RAR and SAF strains. T i m e to death w a s significantly l o n g e r than that o b s e r v e d for m i c e infected with dif
ferent inocula o f tachyzoites o f the RH strain (P < 0.05), e x c e p t for the inoculum o f 1 03 tachyzoites.
T h e ME49, C4 and P strains w e r e not virulent for m i c e in the inocula effectuated. All animals survived after the 30-day-period o f observation, with the e x c e p t i o n o f a m o u s e inoculated with 1 02 tachyzoites o f the P strain, w h i c h died 19 DPI. T h e brain o f this animal w a s positive for tissue cysts.
VIRULENCE OF TOXOPLASMA CONDII STRAINS
BRAIN CYSTS IN BALB/C MICE INOCULATED WITH ME49, C4 AND P STRAINS OF TOXOPLASMA GONDII
Brain cysts w e r e found in all m i c e inoculated with C4 and P strains, surviving 3 0 DPI and w h i c h presented positive s e r o l o g y for T. gondii. Brain cysts w e r e found in 2, 4, 1 and 0 m i c e inoculated, respectively, with 1 03, 1 02, 1 01 and 10° tachyzoites o f the ME49 strain. All o f t h e m w e r e positive by IFAT.
As p r e s e n t e d in T a b l e III, the n u m b e r o f brain cysts in m i c e infected with C4 and P strain w a s significantly different than that found in m i c e infected with the M E 4 9 strain (P < 0 . 0 5 ) . T h e C4 strain formed a greater n u m b e r o f cysts than the P strain, in all o f the tested Strain Number
o f tachyzoites inoculated i.p.
Number o f surviving*/
inoculated mice
Mean n u m b e r o f brain cysts
± SD
ME49 10° 4 5 -
101 5 5 20 ± 44.7
102 S 5 80 ± 44.7
10' S 5 40 ± 54.8
P 10° 2 5 350 ± 70.71
101 5/5 380 ± 258.8
1 02 4/5 600 ± 141.4
1 03 5 5 1,000 ± 254.9
C4 10° 4/5 500 ± 230.9
101 5/5 1,960 ± 1,608.7
ln- 5/5 2,040 ± 1,556.6
1 03 5/5 2,000 ± 902.8
* mice with positive IFAT
Table III - Number of brain cysts in BALB/c mice 30 days post ino
culation with tachyzoites of ME49, P and C4 strains of Toxoplasma gondii.
Number o f tachyzoites inoculated i.p.
(five m i c e e a c h dilution)
10° 10' 102 103
Strain D/S* d* D/S d D/S d D/S d
RH 5/0 10,0 4/0 9,5 5/0 8,0 5/0 6,8
AS28 5 0 H.I s
o
- . ( ) 5 (1 6,0 S o 5,0BV 5/0 8,8 5/0 8,0 5 11 7,2 5/0 6,0
N 5/0 8,8 5/0 8,0 S 1) 7,2 5 0 6,8
EGS 5 0 19,2 5 0 13,2 5 0 9,4 5/0 7,0
RAR 1/0 18,0 5/0 16,2 5/0 13,0 5/0 10,2
SAF 5/0 18,8 5/0 16,4 5/0 10,8 5/0 8,4
ME49 0/4
-
0/5-
0/5 - 0/5-
C4 0/4
-
0/5-
0/5-
0/5-
P 0/2
-
0/5-
1 -) 19,0 0/5-
* D/S = dead/surviving mice (with positive IFAT) + d = mean day of death
Table II. - Virulence comparison for BALB/c mice inoculated by intraperitoneal injection with tachyzoites of different Toxoplasma gondii strains.
Parasite, 2001, 8, 99-105
Mémoire -101
FERREIRA A.M., MARTINS M.S. & VITOR R.W.A.
inocula. However, the n u m b e r o f cysts was significantly different only in m i c e inoculated with 1 03 tachyzoites (P < 0 . 0 5 ) . In addition, there w a s a greater n u m b e r o f cysts in m i c e infected with greater inocula o f tachy
zoites. H o w e v e r , the correlation w a s significant only in animals infected with the P strain (r = 0 . 9 6 ) . ANTIGENIC CHARACTERIZATION OF T. GONDII STRAINS ISOLATED IN BRAZIL
N u m e r o u s antigens o f similar m o l e c u l a r weight w e r e identified b y the rabbit antiserum to T. gondii in all strains: 8 0 , 7 2 - 7 4 , 67, 6 4 , 56, 4 9 , 4 4 , 3 9 , 3 6 , 32 and 21 k D a (Fig. 1 ) . T h e r e w a s n o reactivity o f antibodies with antigens o f peritoneal cells o f m i c e .
T h e mAb 4 C 3 H 4 reacted with antigens o f 32, 21 and 15 k D a o f the highly virulent N and RH strains, and with antigen o f 2 6 k D a o f the M E 4 9 strain, not pre
senting reactivity with any o f the other strains (Fig. 2 ) . In order to verify if this mAb could recognize antigens o f low expression in other strains, a greater n u m b e r o f tachyzoites ( 1 05 per lane) o f RH, EGS, AS28 and N strains was used. As it can b e observed in Fig. 3, the mAb reacted with antigens with molecular weight o f 32, 21 and 15 kDa in RH, AS28 and N strains. There was n o reactivity with tachyzoites o f the EGS strain, confirming that the mAb 4 C 3 H 4 recognizes, preferentially antigens o f T. gondii strains highly virulent for mice.
Fig. 1 - Western blotting analysis of antigens of P, C4, SAF, EGS, AS28, BV, N, ME49 and RH strains of Toxoplasma gondii reacted with rabbit antiserum to T. gondii. Equal numbers ( 1 04) of purified tachyzoites from each strain were subjected to 12,5 % polyacryla- mide gel and electrophoretically transferred to nitrocellulose mem
brane. Molecular weight markers (in kilodaltons) are indicated on the left. Cell - cells obtained from peritoneal cavities of Swiss mice not infected with T. gondii.
D I S C U S S I O N
I
n t h e s e e x p e r i m e n t s o f m i c e inoculated with 10° to 1 03 tachyzoites o f the AS28, B V , N, EGS, RAR, SAF, C4 and P strains o f T. gondii isolated in Brazil, a n d o f RH (virulent) and M E 4 9 (avirulent) strains, reported in this study, it w a s o b s e r v e d that the T. gondii strainsFig. 2 - Western blotting analysis of antigens of P, C4, SAF, EGS, AS28, BV, N, ME49 and RH strains of Toxoplasma gondii reacted with mAb 4C3H4. Equal numbers ( 1 04) of purified tachyzoites from each strain were subjected to 12,5 % polyacrylamide gel and elec
trophoretically transferred to nitrocellulose membrane. Molecular weight markers (in kilodaltons) are indicated on the left. Cell - cells obtained from peritoneal cavities of Swiss mice not infected with
T. gondii.
Fig. 3 - Western blotting analysis of antigens of RH. EGS, AS28 and N strains of Toxoplasma gondii reacted with mAb 4C3H4. Equal num
bers ( 1 05) of purified tachyzoites from each strain were subjected to 12,5 % polyacrylamide gel and electrophoretically transferred to nitrocellulose membrane. Molecular weight markers (in kilodaltons) are indicated on the left. Cell - cells obtained from peritoneal cavi
ties of Swiss mice not infected with T. gondii.
analyzed m a y b e divided in three groups, according to the virulence for B A L B / c m i c e .
T h e AS28, B V and N strains p r e s e n t e d high virulence, similar to that observed for the RH strain, with the mor
tality o f all m i c e infected with the different n u m b e r o f tachyzoites. T h e s e strains h a v e not p r o d u c e d brain cysts. H o w e v e r , as the animals died prematurely, the research o f cysts may h a v e had a false-negative result.
In m i c e , tissue cysts are formed within three and four
VIRULENCE OF TOXOPLASMA GONDII STRAINS
days after p a r e n t e r a l i n o c u l a t i o n with t a c h y z o i t e s ( D u b e y & Beattie, 1 9 8 8 ) . Nevertheless, in the begin
ning o f the infection, the visualization is m a d e diffi
cult b y its small size. H o w e & Sibley ( 1 9 9 5 ) o b s e r v e d that highly virulent strains, as the RH strain, lost the ability to form tissue cysts or they form a very reduced n u m b e r o f cysts.
T h e E G S , RAR, and SAF strains, isolated from c a s e s o f h u m a n congenital toxoplasmosis formed the s e c o n d group o f strains o b s e r v e d in this study. Despite being virulent for mice, killing 100 % o f animals, the presence o f brain cysts w a s o b s e r v e d in m i c e w h i c h died after the infection and time to death was longer than that o b s e r v e d for the m i c e inoculated with the strains o f the first group.
T h e C4 and P strains present an avirulent behavior, as the M E 4 9 strain. T h e inoculation o f tachyzoites o f these strains led to the d e v e l o p m e n t o f brain cysts without killing the mice, e v e n with the inoculum o f 1 03 tachyzoites. A m o u s e inoculated with 1 02 tachy
zoites o f the P strain died 19 DPI, exemplifying indi
vidual characteristics o f r e s p o n s e to the infections, e v e n in populations o f isogenic animals.
Within the strains analyzed in this study, the AS28 strain calls attention by the fact o f having b e e n primarily des
cribed causing an infection in m i c e tending to b e chronic, developing a great n u m b e r o f brain cysts in the animals ( D e a n e et al., 1 9 7 1 ) . In our study, this strain s h o w e d to b e highly virulent for mice, killing 100 % o f the animals in less than 10 DPI, and the pre
s e n c e o f brain cysts was not observed.
J a c o b s & Melton ( 1 9 5 4 ) s h o w e d that successive passage o f tachyzoites from o n e strain in mice modifies the viru
l e n c e o f T. gondii. Nevertheless, w e agree with D u b e y
& Frenkel ( 1 9 7 3 ) w h o stated that the adaptation o f a c e r t a i n h o s t differs a m o n g t h e s e v e r a l strains o f T. gondii. For instance, the RH strain killed four o f five mice infected in the first passage from 17 to 21 days s o o n after its isolation in 1939, but after only three intra
peritoneal passages it started to kill all the mice ino
culated from three to five days (Sabin, 1 9 4 1 ) . O n the other hand, with M-7741 strain, e v e n after 62 passages, the m i c e infected with 1 05 tachyzoites survived for m o r e than nine days ( D u b e y & Frenkel, 1973).
Similarly, the C4 and P strains analyzed in this study maintain the s a m e avirulent behavior since its isolation, while the B V and N strains, isolated from animals, and the E G S , RAR and SAF strains, isolated from h u m a n cases o f congenital toxoplasmosis, killed 100 % o f mice since the first passage after its isolation.
T h e n u m b e r o f brain cysts differed a m o n g the m i c e infected with the ME49, C4 and P strains o f T. gondii.
T h e n u m b e r o f brain cysts in m i c e infected with C4 and P strains was greater than the o n e found in m i c e infected with the ME49 strain. T h e C4 strain formed
more brain cysts than the P strain in mice infected with 1 03 tachyzoites. Suzuki et al. ( 1 9 8 9 ) also o b s e r v e d dif
ferences in the n u m b e r o f cysts formed in the brain o f CBA/Ca mice, infected with ME49 and DAG strains o f T. gondii. T h e authors declared that the pathoge
nesis o f encephalitis by T. gondii should b e considered in the context o f the strain o f the parasite involved and its potential for a continuous activity during the acute infection in mice.
T h e analysis by Western blotting o f different strains o f T. gondii s h o w e d that antigens o f similar molecular weight, varying from 21 to 8 0 kDa w e r e identified in all strains. T h e s e data differ from the results described in the literature as, through the technique o f Western blotting, it was verified that there is an antigenic diver
sity among different strains o f T. gondii (Ware & Kasper, 1987; Weiss et al., 1988; Appleford & Smith, 2 0 0 0 ) . However, C a z a b o n n e et al. ( 1 9 9 4 ) performed a study o f kinetics and characterization o f e x c r e t e d / s e c r e t e d antigens from three strains o f T. gondii o f different viru
l e n c e and o b s e r v e d that the antigens w e r e similar for all the strains. T h e s e strains p r o d u c e d the s a m e immu
nological r e s p o n s e in mice. In our study, most anti
gens w e r e similar to all strains analyzed, w h i c h pro
bably occurred due to the fact that the polyclonal serum used is capable o f recognizing antigens with the same molecular weight in the different T. gondii strains analyzed.
T h e reactivity comparison o f T. gondii strains with mAb 4C3H4 (Elsaid et al., 1 9 9 9 ) revealed three antigens reco
gnized only in the RH and N strains w h i c h are highly virulent for mice, and o n e antigen o f 2 6 kDa, reco
gnized only in the M E 4 9 strain. T h e reactivity with m o r e than o n e antigen suggests that the mAb 4C3H4 recognizes epitopes w h i c h are c o m m o n in proteins o f different molecular weight.
Using a greater n u m b e r o f parasites per lane, the mAb 4 C 3 H 4 also reacted with antigens o f the AS28 strain, suggesting a low expression in this strain. Neverthe
less, there w a s n o reaction o f the mAb with antigens o f the EGS strain. T h e s e results suggest that the spe
cific recognition o f virulent strains b y mAb 4C3H4 is related to the lost o f the ability to m a k e cyst. Studies o f molecular biology may help to explain the function and r e l e v a n c e o f these antigens, particularly referring to its role in the virulence o f strains.
While analyzing the reactivity o f mAb 5 B 1 0 , developed against the RH strain, Gross et al. ( 1 9 9 D o b s e r v e d that this mAb detected an antigen o f 2 3 k D a e x p r e s s e d b y the virulent strains, but not by the strains with low viru
l e n c e , isolated from clinical c a s e s o f toxoplasmosis in Europe. B o h n e et al. ( 1 9 9 3 ) differed virulent strains from avirulent o n e s o f T. gondii by using the mAb T B 6 G 5 , d e v e l o p e d a g a i n s t t h e NTE strain w h i c h reacted with eight avirulent strains for mice, but not
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F E R R E I R A A.M.,. M A R T I N S M . S . & V I T O R R . W . A .
with virulent strains. T o g e t h e r with the m o n o c l o n a l antibodies d e v e l o p e d b y these authors, the analysis o f reactivity o f T. gondii strains with mAb 4C3H4, u s e d in this study, c o u l d b e an additional parameter for the serological classification o f T. gondii in virulent and avi- rulent strains for mice.
The results o f the present study s h o w e d the occurrence o f T. gondii strains o f varied virulence in Brazil. Fur
ther studies using g e n e t i c a p p r o a c h e s that are n o w available for T. gondii (Sibley et al, 1 9 9 9 ) will b e o f great value in establishing g e n e t i c relationship a m o n g these Brazilian T. gondii strains.
ACKNOWLEDGEMENTS
We w o u l d like to thank Rosalida E. N. Lopes for technical assistance. T h e RH and ME49 strains w e r e o b t a i n e d from Ricardo T. Gaz- zinelli ( D e p a r t a m e n t o de B i o q u í m i c a e Imunologia, ICB, U F M G , Brazil). This w o r k w a s s u p p o r t e d b y FAPEMIG and CNPq.
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Recu le 8 novembre 2000 Accepte le 26 mars 2001
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Mémoire 105
