EXPERIMENTAL PARASITOLOGY 73, 26O-268 (1991)
Trypanosoma cruzi: Early Resistance Induced Culture-Derived Trypomastigotes
by
IRINEU J. B. CAMARGO,PAULO M. F. ARAUJO,JULIA K. SAKURADA, DAGMARR. STACH-MACHADO,AND HUMBERTOA. RANCEL Department0
Microbiologia
e Imunologia, Instituto de Biologia, Sao Paula, Brasil
UNICAMP,
13081 Campinns,
CAMARGO,~. J. B., ARAUJO,P. M. F.,~AKURADA, J. K., STACH-MACHADO, D. R., AND RANGEL, H. A. 1991. Trypanosoma cruzi: Early resistance induced by culture-derived trypomastigotes. Experimental Parasitology 73, 260-268. Previous observations in this labo-
ratory showed that injection of culture-derived trypomastigotes (CT), in CBA/J mice, induced an early increased resistance that was detected 24-72 hr after antigen injection and permitted mice to survive a challenge of 10’ blood trypomastigotes (BT) corresponding to 2000 LD,,%. Present experiments were conducted to determine the optimal conditions for inducing this early resistance and to investigate the early morphological changes which occurred in blood and lymphoid organs of mice infected with either BT or CT. Among nine antigens tested, only living CT showed a protective effect permiting most of mice to survive 30 days after BT challenge, while control mice injected with PBS or other antigens died at 10 k 1 days. A dose-response relationship was seen when different doses of CT were tested, higher doses of CT inducing higher survival and lower parasitemia. Injection of CT by either an im or ip route induced similar degrees of resistance but significantly different results were obtained when mice were challenged by using ip or im routes. Higher parasitemia and lower survival were always obtained when animals were challenged by the ip route. Within 72 hr, mice injected with BT presented a lymphopenia which reached a maximum at 48 hr, a depletion of thymic cortical zone, and splenomegaly with hyperplasia of the white pulp and congestion of the red pulp. No gross alterations were observed in animals infected with CT. Overall data suggest that the early resistance is a specifically induced phenomenon and that BT and CT induce different early reactions in the CBA/J lymphoid organs. The possibility of a correlation between the early resistance and cellular changes induced by CT infection is proposed. 0 1991 Academic Press, Inc. INDEX DESCRIPTORSAND ABBREVIATIONS. Trypanosoma cruzi; CBA/J mice; Early resistance; Culture-derived trypomastigotes (CT); Blood trypomastigotes (BT); Lymphopenia; Thymus; Splenomegaly; intramuscular (im); intraperitoneal (ip).
are highly virulent when tested in Swiss mice. Usually a dose of IO3 blood parasites Infection with Trypanosoma cruzi pre- or less kills mice during the acute phase, sents acute and chronic phases. The acute while doses as high as lo5 of low virulent phase is characterized by a high number of strains (e.g., the Monica strain isolated in bloodstream parasites and a low level of an- our laboratory), although being able to intibodies in the serum, whereas during the fect mice for life, are unable to kill them. The importance of host genetic factors chronic phase a small number of blood parasites and high levels of antibody are de- has been stressed by several authors tected (Krettli and Brener 1976). The out- (Trischmann et al. 1978; Corsini et al. 1980; come of this infection is dependent on ge- Juri et al. 1990). Some strains of mice like netic factors of both parasite and host CBA/J are highly susceptible since they die during the acute phase with a dose of par(Trischmann 1983). Some strains of parasite, like the Y strain asite that is unable to kill mice from resis(Pereira da Silva and Nussenzweig 1953), tant strains like C57BL/6. 260 OO14-4894191 $3.00
T.
CruZi:
TRYPOMASTIGOTES-INDUCED
The phenotypic expression of the genes involved in resistance is not known. Accumulated data indicate that resistance may be associated to specific factors like T lymphocytes (Trischmann 1983; Goncalves da Costa et al. 1984; Brener 1987; Russo et al. 1988) and specific antibodies (Krettli and Brener 1976; Brener 1980; Takehara et al. 1981; Romeiro et al. 1984; Takehara et al. 1990), and to nonspecific factors like protein P amiloid and interferon-y (Scharfstein et al. 1982; James et al. 1982; Plata et al. 1987; Beltz et al. 1989). In a previous paper (Camargo et al. 1989) we reported observations indicating that CBA/J mice were rendered highly resistant some hours after injection of culturederived trypomastigostes, an avirulent stock of the Y strain of T. cruzi. Indeed, CT injection induced an early increased resistance detected 24 to 72 hr after antigen injection by the survival of mice challenged with lo5 bloodstream trypomastigotes, a dose equivalent to 2000-fold the LD,,%. The mechanism of this early resistance is unknown. Present experiments were planned with the aim to determine the optimal conditions for inducing resistance and to investigate the early morphological changes occurring in the lymphoid organs of BT and CT injected animals. This is a first approach to find a clue for understanding the mechanism of the CT-induced early resistance. MATERIALSANDMETHODS Animals and infection. Both male and female, IO- to IZweek-old CBA/J mice were used throughout. The mice were obtained from the specific pathogen free (SPF) stocks bred under barrier sustained conditions at Cemib-Unicamp. Animals were usually infected by the im with 16 bloodstream forms of the Y strain of T. cruzi. In some experiments, indicated below, a newly isolated strain (Monica strain) was used. This strain, which was isolated in our laboratory from the blood of a child accidentally infected by blood transfusion, killed no mice even when a dose of 10’ blood parasites was injected. Both strains were maintained by serial passage either every 7 (Y strain) or 12 (Monica strain)
RESISTANCE
261
days. Experiments used five mice per group with at least three replicates carried out at different occasions. Counring ofparasites. To determine the level of parasitemia, 5 ~1of blood were taken from the tail vein of the mouse and dispersed under coverslip. Parasites were counted on 50 microscopic fields as described by Brener (1980). Statistical evaluation of parasitemia levels were performed using a log,, (count + 1) transformation prior to analysis of variance and comparison by Student’s f test. Parasife crude extracts. Epimastigote forms were obtained from cultures in LIT medium as indicated earlier (Range1 et al. 1981). Blood trypomastigote forms were isolated from infected CBA/J mice as indicated by Camargo et al. (1987). Amastigote and culture-derived trypomastigote forms were harvested from infected Vero cells as indicated previously (Repka er al. 1985). Suspensions of different parasite forms (106/ml) were sonicated for 5 min with a model W-22F sonicator (Ultrasonic, Inc.) and maintained at - 70°C until used (Range1 et al. 1981). Virulence rests. Virulence tests were performed by injecting 16 parasites in CBA/J mice and recording mortality rates over 30 days. Early resistance rests. Unless otherwise stated, early resistance tests were performed by injecting, im, 10’ CT suspended in 0.1 ml PBS in the left limb and challenging, 72 hr later, with 10’ BT, inoculated by the im route in the contralateral limb. Control groups which received an im injection of 0.1 ml of PBS were also challenged 72 hr later with 16 BT. Survival rates were recorded daily over 30 days and parasitemia levels were determined on the seventh day after BT challenge. Hemogrum. Blood from the tail of mice injected with either PBS, BT, or CT was collected in 3% EDTA, 24, 48, and 72 hr after injection. Hematocrit and white blood cells counts were performed by using routine clinical laboratory methods. Differential leukocyte counts were performed on Leishman-stained smear preparations. Hisroparhology. Thymus, spleen, lymph nodes, bone marrow, heart, liver, and pieces of leg muscles taken at the injection site were removed from mice injected 24,48, and 72 hr earlier with either PBS, BT, or CT. Tissues were immediately fixed in Bouin’s fluid, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. RESULTS
In order to verify if early protection could be conferred by different forms of T. cruzi, mice were im injected with varying numbers of either epimastigotes, amastigotes, culture trypomastigotes, or blood trypomastigotes. After varying lengths of
262
CAMARGO ET AL.
time these mice were challenged with lo5 BT and survival rates recorded daily. All mice injected with lo5 CT developed a low parasitemia (4.5 ? 1.5 x lo5 blood parasites/ml) that peaked at the 7th day and survived the 30-day observation period. Mice infected with IO5 BT presented a parasitemia (2.7 ? 0.9 x 106/ml) that also peaked on the 7th day with death occurring at 10 & 1 days. Practically no parasitemia and no deaths were recorded among mice injected with epimastigotes or amastigotes. Data obtained with mice previously injected with one of these parasite forms and challenged later with lo5 BT (Table I) showed that only mice injected with living CT of the Y strain had a significant chance of survival beyond the 12th day. Since no mouse survived the 12th day after challenge in control groups where more than 200 animals were tested, it was concluded that survival beyond this period indicated significant protection. As a rule, survival was higher when mice were challenged 72 hr after CT injection. The observation period was not extended beyond the 30th day after
challenge since it was noticed that the number of deaths recorded after that period was negligible. Test were also performed to see if the following antigens could induce early protection: CT crude extract (tested at l/l, l/3, and l/10 dilutions), BT crude extract (tested at varying concentrations from l/l to l/100), LPS (25 and 50 pg), living and avirulent Listeria monocytogenes ( lo6 and 107), and Bio-Gel (Bio-Rad Laboratories) used as indicated by Fauve et al. (1987). In these experiments not one mouse, challenged between 24 and 72 hr after antigen injection, survived beyond the 12th day. Virulence tests of different CT suspensions showed that parasites became avirulent after 2 or 3 passages in Vero cell cultures. However early resistance tests showed that only CT with more than 10 passages presented unequivocal positive results. To study the relationship between doseage of CT and degree of protection mice were injected im with varying numbers of CT (12th passage) and challenged 72 hr
TABLE I Survival of CBA/J Mice Injected with Different Antigens and Challenged with lo5 BT
Antigens lo5 Living CT Y strain 10’ Living Epimastigotes Y strain 10’ Living Amastigotes Y strain ld Living BT Y strain ld Living BT Monica strain PBS
Percentage survival at day
Time between antigen and challenge (hr)
12
15
20
25
30
24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72
80 80 loo 0 0 0 0 40 20 0 0 0 20 0 20 0 0 0
40 60 100
20 40 100
20 20 100
0 0 80
0 0
0 0
T. cruzi:
TRYPOMASTIGOTES-INDUCED
later with lo5 BT. Data obtained showed that increasing doses of CT induced increasing degrees of survival (Fig. 1A) and decreasing levels of parasitemia (Fig. 1B). To determine whether the route of injection had some influence on the degree of protection, mice were injected either im or ip with lo5 CT and challenged, 72 hr later, by the ip or im routes with lo5 BT. Results obtained showed no significant difference in the mortality rates of control groups im or ip injected with lo5 BT (Fig. 2A). However the level of parasitemia of the control groups im injected was significantly lower than that of controls groups ip injected (Fig. 2B). Among groups of mice previously injected with CT and challenged later with BT, higher degrees of survival and lower levels of parasitemia were always observed when mice were im challenged (Figs. 2A and 2B), the highest degree of protection being observed when both CT and BT were im injected. However, no significant differ-
RESISTANCE
ence was observed in the level of parasitemia of mice ip or im injected with CT and im challenged with BT (Fig. 2A). No alteration was detected in the hematocrit values of mice injected with either BT or CT. The number of leukocytes in mice injected with BT was reduced during the early phase. This number, which 24 hr after injection was significantly lower than normal values, reached a minimum level at 48 hr and began to recover at 72 hr. The leukocyte counts remained within normal levels in mice injected with CT (Fig. 3). Differential cell counting showed that leukopenia of BT injected mice was due to a reduction of the number of lymphocytes. No significant alteration was observed in the number of polymorphonuclear cells (Fig. 4). Histopathological observation detected no alteration in the sections of heart, liver, bone marrow, and lymph nodes of mice infected with BT or CT up to 72 hr previously. Thymuses of BT infected mice
DAYS
AFTER
CHALLENGE
263
NUMBER
Cf
CT
INJECTED
FIG. 1. (A) Survival and (B) level of parasitemia of CBA/J mice injected with IO5(O), lo4 (X), lo3
(O), and lo2 (A) CT or PBS (Cl) and challenged, 72 hr later, with lo5 BT.
CAMARGO
ET AL.
6
DAYS
AFTER
CTi.p + BTtp
CHALLENGE
CTtp. + BTlm
CTi + BTap
BTtm
FIG. 2. (A) Survival rate and (B) level of parasitemia of CBA/J mice injected with lo5 CT by either
im (0) or ip (W) routes and challenged 72 hr later with lo5 BT, injected by im (0) or ip (0) routes.
showed depletion of the cortical areas as soon as 24 hr after infection. This depletion was very marked 3 days after infection (Fig. 5A). No histopathological alteration was observed in the thymuses of PBS and CT injected mice during this period (Fig. -
5B). Also no alteration was seen in the thymuses of those CT injected mice that were challenged with BT. Splenomegaly with hyperplasia of the white pulp and congestion of the red pulp were seen in BT injected animals. Only minor alterations were observed in the spleens of CT injected mice. The spleens of those CT injected mice that were challenged with BT also presented only minor alterations. DISCUSSION
Lr 3T HOURS
PB
POST
IB-r
h
3scTKr ?2
INFECTION
FIG. 3. Leucocyte counts in blood of mice for vary-
ing times after im injection with PBS, lo5 CT or lo5 BT.
Previous observations (Camargo et al. 1989) showing that the carbon clearance activity of BT injected animals was decreased while that of CT injected animals was normal or slightly increased suggested that CTinduced early resistance could be associated with nonspecific factors. The data presented in this report show that only living CT was able to induce an early resistance, here defined as the increase in resistance detected within 24 to 72 hr after antigen injection, permitting animals to overcome a
T. cruzi:
TRYPOMASTIGOTES-INDUCED
.E61
0
1..
0
(2
.
(2
24
46
24
46
HOURS
72
c
72
POST INFECTION
FIG. 4. Lymphocyte and polymorphonuclear cells (PMN) counts in the blood of mice previously injected with PBS, CT, or BT.
challenge of 10’ BT. No early resistance was detected when living epimastigotes, blood trypomastigotes, amastigotes, and parasite extracts, including those obtained from Y strain CT forms, were tested. Also no early resistance could be observed when testing L. monocytogenes and Bio-Gel, known inducers of inflammatory reactions (Fauve et al. 1987), thus suggesting that CT-induced resistance is not associated with nonspecific factors but is rather specifically induced by CT epitopes that may be masked, absent, or in very low concentration in other parasite forms and their extracts. The expression of these putative resistance epitopes in CT seems to depend on one or more virulence factors of the para-
RESISTANCE
265
site since early resistance was detected only when virulence was lost or diminished by serial passage in cell cultures. Indeed, CT shortly regain virulence once in the host environment (Camargo et al. 1989), which stimulated by CT epitopes is now able to cope with the virulence factor(s). Comparison of CT and BT permitted detection of some differences in regard to infectivity for L cells and interaction with macrophages (Nogueira et al. 1980; Meireles et al. 1982; Kloetzel et al. 1984). However, comparison of BT and CT antigenic patterns, by using Western blotting analysis, showed an extensive homology, with only minor differences in the intensity of two antigens being detected (Silva et al. 1988). Thus CT seems to differ from BT only by very few epitopes. Experiments performed to establish the dose-response relationship showed that, within the limits tested, the higher the injected dose of CT, the higher the increase in resistance measured as higher survival rates and lower levels of parasitemia. Best results were obtained by using IO5 CT, when most of mice survived for 30 days while 100% of control died within 10 4 1 days. No difference in survival rates were observed in control groups injected with BT by the ip or im routes, probably due to the fact that they received a very large number of virulent parasites. However, mice im injected presented significantly different levels of parasitemia. Although injection of CT by either im or ip routes induced similar degrees of resistance, significantly higher degrees of survival and lower levels of parasitemia were seen when mice were challenged by the im route. Best results were always obtained when both CT and BT were administered by the im route. These data confirm observations from other laboratories (Kierzenbaum and Howard, 1976; Liew et al. 1985; Murtin et al. 1989) stressing the importance of the route of antigen injection for the features of host response.
266
CAMARGO
ET AL.
FIG. 5. Microscopic aspect of thymuses of mice, injected 72 hr previously, with: (A) phosphatebuffered saline (PBS); (B) culture trypomastigotes (CT); (C and D) blood trypomastigotes (BT). Magnification of 80X for (A), (B) and (C) and of 300X for (D). Note the peripheric thin cortical zone of BT infected mice.
Hematological changes during the acute et al. 1989); however, no information presphase were described in a man accidentally ently exists permitting one to determine if infected (HoMin et al. 1987), in children lymphopenia and thymic cortical depletion (Rassi 1979), and in mice experimentally in- result from cell lysis or from rapid removal fected (Cardoso and Brenner 1980; Repka from the blood and thymic cortical areas et al. 1985). However, to our knowledge, with sequestration in other organs. The no register was made of the first 3 days after possibility that these findings are secondary infection. Present observation indicates to a stress-derived increase in corticostethat during this early period BT exerts a roides (Savino et al. 1989) cannot be exmarked action on lymphoid organs while no cluded. The fact that no alteration was detected significant morphological alterations occurs in CT injected animals, suggesting that BT in the lymphoid organs of mice injected virulence is exerted through an early action with CT and challenged with BT permits on lymphoid cells. Early lymphopenia was the hypothesis that early increased resisobserved in T. musculi infections (Duffey et tance may result from stimulation of these al. 1985) and thymic cortical depletion in T. lymphoid cells involved in the control of cruzi has been recently described (Savino virulent parasites. This view is somewhat
T. cruzi: TRYPOMASTIGOTES-INDUCED
RESISTANCE
267
supported by evidence indicating that T FAUVE, R. M., FONTAN, E., HEVIN, M. B., SAKLANI, H., AND PARKER,F. 1987. Remote effects of inflamcells play an important role in resistance mation on non-specific immunity. Immunology Letagainst T. cruzi (Tarleton and Scott 1987; ters 16, 199-204. Russo et al. 1988; Tarleton 1990) and that, GONFALVES DA COSTA, S. C., LAGRANGE, P. H., during infection with virulent parasites, seHURTHREL, B., KERR, I., AND ALENCAR, A. 1984. Role of T lymphocytes in the resistance and immuvere T cell abnormalities result (Minomio nopathology of experimental Chagas’ disease. Anet al. 1986; Hontebrye-Joskowicz et al. nales d’ Immunologic (Znstitut Pasteur) 135C, 3171987; Curotto de Lafaille et al. 1990).
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ACKNOWLEDGMENTS We thank Professor M. A. Cruz Hofling for help in histopathological studies and Dime Lima Gabriel for technical help. This study was partially supported by FAPESP and CNPq grants.
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Trypanosoma cruzi: Early Resistance Induced Culture-Derived Trypomastigotes
by
IRINEU J. B. CAMARGO,PAULO M. F. ARAUJO,JULIA K. SAKURADA, DAGMARR. STACH-MACHADO,AND HUMBERTOA. RANCEL Department0
Microbiologia
e Imunologia, Instituto de Biologia, Sao Paula, Brasil
UNICAMP,
13081 Campinns,
CAMARGO,~. J. B., ARAUJO,P. M. F.,~AKURADA, J. K., STACH-MACHADO, D. R., AND RANGEL, H. A. 1991. Trypanosoma cruzi: Early resistance induced by culture-derived trypomastigotes. Experimental Parasitology 73, 260-268. Previous observations in this labo-
ratory showed that injection of culture-derived trypomastigotes (CT), in CBA/J mice, induced an early increased resistance that was detected 24-72 hr after antigen injection and permitted mice to survive a challenge of 10’ blood trypomastigotes (BT) corresponding to 2000 LD,,%. Present experiments were conducted to determine the optimal conditions for inducing this early resistance and to investigate the early morphological changes which occurred in blood and lymphoid organs of mice infected with either BT or CT. Among nine antigens tested, only living CT showed a protective effect permiting most of mice to survive 30 days after BT challenge, while control mice injected with PBS or other antigens died at 10 k 1 days. A dose-response relationship was seen when different doses of CT were tested, higher doses of CT inducing higher survival and lower parasitemia. Injection of CT by either an im or ip route induced similar degrees of resistance but significantly different results were obtained when mice were challenged by using ip or im routes. Higher parasitemia and lower survival were always obtained when animals were challenged by the ip route. Within 72 hr, mice injected with BT presented a lymphopenia which reached a maximum at 48 hr, a depletion of thymic cortical zone, and splenomegaly with hyperplasia of the white pulp and congestion of the red pulp. No gross alterations were observed in animals infected with CT. Overall data suggest that the early resistance is a specifically induced phenomenon and that BT and CT induce different early reactions in the CBA/J lymphoid organs. The possibility of a correlation between the early resistance and cellular changes induced by CT infection is proposed. 0 1991 Academic Press, Inc. INDEX DESCRIPTORSAND ABBREVIATIONS. Trypanosoma cruzi; CBA/J mice; Early resistance; Culture-derived trypomastigotes (CT); Blood trypomastigotes (BT); Lymphopenia; Thymus; Splenomegaly; intramuscular (im); intraperitoneal (ip).
are highly virulent when tested in Swiss mice. Usually a dose of IO3 blood parasites Infection with Trypanosoma cruzi pre- or less kills mice during the acute phase, sents acute and chronic phases. The acute while doses as high as lo5 of low virulent phase is characterized by a high number of strains (e.g., the Monica strain isolated in bloodstream parasites and a low level of an- our laboratory), although being able to intibodies in the serum, whereas during the fect mice for life, are unable to kill them. The importance of host genetic factors chronic phase a small number of blood parasites and high levels of antibody are de- has been stressed by several authors tected (Krettli and Brener 1976). The out- (Trischmann et al. 1978; Corsini et al. 1980; come of this infection is dependent on ge- Juri et al. 1990). Some strains of mice like netic factors of both parasite and host CBA/J are highly susceptible since they die during the acute phase with a dose of par(Trischmann 1983). Some strains of parasite, like the Y strain asite that is unable to kill mice from resis(Pereira da Silva and Nussenzweig 1953), tant strains like C57BL/6. 260 OO14-4894191 $3.00
T.
CruZi:
TRYPOMASTIGOTES-INDUCED
The phenotypic expression of the genes involved in resistance is not known. Accumulated data indicate that resistance may be associated to specific factors like T lymphocytes (Trischmann 1983; Goncalves da Costa et al. 1984; Brener 1987; Russo et al. 1988) and specific antibodies (Krettli and Brener 1976; Brener 1980; Takehara et al. 1981; Romeiro et al. 1984; Takehara et al. 1990), and to nonspecific factors like protein P amiloid and interferon-y (Scharfstein et al. 1982; James et al. 1982; Plata et al. 1987; Beltz et al. 1989). In a previous paper (Camargo et al. 1989) we reported observations indicating that CBA/J mice were rendered highly resistant some hours after injection of culturederived trypomastigostes, an avirulent stock of the Y strain of T. cruzi. Indeed, CT injection induced an early increased resistance detected 24 to 72 hr after antigen injection by the survival of mice challenged with lo5 bloodstream trypomastigotes, a dose equivalent to 2000-fold the LD,,%. The mechanism of this early resistance is unknown. Present experiments were planned with the aim to determine the optimal conditions for inducing resistance and to investigate the early morphological changes occurring in the lymphoid organs of BT and CT injected animals. This is a first approach to find a clue for understanding the mechanism of the CT-induced early resistance. MATERIALSANDMETHODS Animals and infection. Both male and female, IO- to IZweek-old CBA/J mice were used throughout. The mice were obtained from the specific pathogen free (SPF) stocks bred under barrier sustained conditions at Cemib-Unicamp. Animals were usually infected by the im with 16 bloodstream forms of the Y strain of T. cruzi. In some experiments, indicated below, a newly isolated strain (Monica strain) was used. This strain, which was isolated in our laboratory from the blood of a child accidentally infected by blood transfusion, killed no mice even when a dose of 10’ blood parasites was injected. Both strains were maintained by serial passage either every 7 (Y strain) or 12 (Monica strain)
RESISTANCE
261
days. Experiments used five mice per group with at least three replicates carried out at different occasions. Counring ofparasites. To determine the level of parasitemia, 5 ~1of blood were taken from the tail vein of the mouse and dispersed under coverslip. Parasites were counted on 50 microscopic fields as described by Brener (1980). Statistical evaluation of parasitemia levels were performed using a log,, (count + 1) transformation prior to analysis of variance and comparison by Student’s f test. Parasife crude extracts. Epimastigote forms were obtained from cultures in LIT medium as indicated earlier (Range1 et al. 1981). Blood trypomastigote forms were isolated from infected CBA/J mice as indicated by Camargo et al. (1987). Amastigote and culture-derived trypomastigote forms were harvested from infected Vero cells as indicated previously (Repka er al. 1985). Suspensions of different parasite forms (106/ml) were sonicated for 5 min with a model W-22F sonicator (Ultrasonic, Inc.) and maintained at - 70°C until used (Range1 et al. 1981). Virulence rests. Virulence tests were performed by injecting 16 parasites in CBA/J mice and recording mortality rates over 30 days. Early resistance rests. Unless otherwise stated, early resistance tests were performed by injecting, im, 10’ CT suspended in 0.1 ml PBS in the left limb and challenging, 72 hr later, with 10’ BT, inoculated by the im route in the contralateral limb. Control groups which received an im injection of 0.1 ml of PBS were also challenged 72 hr later with 16 BT. Survival rates were recorded daily over 30 days and parasitemia levels were determined on the seventh day after BT challenge. Hemogrum. Blood from the tail of mice injected with either PBS, BT, or CT was collected in 3% EDTA, 24, 48, and 72 hr after injection. Hematocrit and white blood cells counts were performed by using routine clinical laboratory methods. Differential leukocyte counts were performed on Leishman-stained smear preparations. Hisroparhology. Thymus, spleen, lymph nodes, bone marrow, heart, liver, and pieces of leg muscles taken at the injection site were removed from mice injected 24,48, and 72 hr earlier with either PBS, BT, or CT. Tissues were immediately fixed in Bouin’s fluid, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. RESULTS
In order to verify if early protection could be conferred by different forms of T. cruzi, mice were im injected with varying numbers of either epimastigotes, amastigotes, culture trypomastigotes, or blood trypomastigotes. After varying lengths of
262
CAMARGO ET AL.
time these mice were challenged with lo5 BT and survival rates recorded daily. All mice injected with lo5 CT developed a low parasitemia (4.5 ? 1.5 x lo5 blood parasites/ml) that peaked at the 7th day and survived the 30-day observation period. Mice infected with IO5 BT presented a parasitemia (2.7 ? 0.9 x 106/ml) that also peaked on the 7th day with death occurring at 10 & 1 days. Practically no parasitemia and no deaths were recorded among mice injected with epimastigotes or amastigotes. Data obtained with mice previously injected with one of these parasite forms and challenged later with lo5 BT (Table I) showed that only mice injected with living CT of the Y strain had a significant chance of survival beyond the 12th day. Since no mouse survived the 12th day after challenge in control groups where more than 200 animals were tested, it was concluded that survival beyond this period indicated significant protection. As a rule, survival was higher when mice were challenged 72 hr after CT injection. The observation period was not extended beyond the 30th day after
challenge since it was noticed that the number of deaths recorded after that period was negligible. Test were also performed to see if the following antigens could induce early protection: CT crude extract (tested at l/l, l/3, and l/10 dilutions), BT crude extract (tested at varying concentrations from l/l to l/100), LPS (25 and 50 pg), living and avirulent Listeria monocytogenes ( lo6 and 107), and Bio-Gel (Bio-Rad Laboratories) used as indicated by Fauve et al. (1987). In these experiments not one mouse, challenged between 24 and 72 hr after antigen injection, survived beyond the 12th day. Virulence tests of different CT suspensions showed that parasites became avirulent after 2 or 3 passages in Vero cell cultures. However early resistance tests showed that only CT with more than 10 passages presented unequivocal positive results. To study the relationship between doseage of CT and degree of protection mice were injected im with varying numbers of CT (12th passage) and challenged 72 hr
TABLE I Survival of CBA/J Mice Injected with Different Antigens and Challenged with lo5 BT
Antigens lo5 Living CT Y strain 10’ Living Epimastigotes Y strain 10’ Living Amastigotes Y strain ld Living BT Y strain ld Living BT Monica strain PBS
Percentage survival at day
Time between antigen and challenge (hr)
12
15
20
25
30
24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72
80 80 loo 0 0 0 0 40 20 0 0 0 20 0 20 0 0 0
40 60 100
20 40 100
20 20 100
0 0 80
0 0
0 0
T. cruzi:
TRYPOMASTIGOTES-INDUCED
later with lo5 BT. Data obtained showed that increasing doses of CT induced increasing degrees of survival (Fig. 1A) and decreasing levels of parasitemia (Fig. 1B). To determine whether the route of injection had some influence on the degree of protection, mice were injected either im or ip with lo5 CT and challenged, 72 hr later, by the ip or im routes with lo5 BT. Results obtained showed no significant difference in the mortality rates of control groups im or ip injected with lo5 BT (Fig. 2A). However the level of parasitemia of the control groups im injected was significantly lower than that of controls groups ip injected (Fig. 2B). Among groups of mice previously injected with CT and challenged later with BT, higher degrees of survival and lower levels of parasitemia were always observed when mice were im challenged (Figs. 2A and 2B), the highest degree of protection being observed when both CT and BT were im injected. However, no significant differ-
RESISTANCE
ence was observed in the level of parasitemia of mice ip or im injected with CT and im challenged with BT (Fig. 2A). No alteration was detected in the hematocrit values of mice injected with either BT or CT. The number of leukocytes in mice injected with BT was reduced during the early phase. This number, which 24 hr after injection was significantly lower than normal values, reached a minimum level at 48 hr and began to recover at 72 hr. The leukocyte counts remained within normal levels in mice injected with CT (Fig. 3). Differential cell counting showed that leukopenia of BT injected mice was due to a reduction of the number of lymphocytes. No significant alteration was observed in the number of polymorphonuclear cells (Fig. 4). Histopathological observation detected no alteration in the sections of heart, liver, bone marrow, and lymph nodes of mice infected with BT or CT up to 72 hr previously. Thymuses of BT infected mice
DAYS
AFTER
CHALLENGE
263
NUMBER
Cf
CT
INJECTED
FIG. 1. (A) Survival and (B) level of parasitemia of CBA/J mice injected with IO5(O), lo4 (X), lo3
(O), and lo2 (A) CT or PBS (Cl) and challenged, 72 hr later, with lo5 BT.
CAMARGO
ET AL.
6
DAYS
AFTER
CTi.p + BTtp
CHALLENGE
CTtp. + BTlm
CTi + BTap
BTtm
FIG. 2. (A) Survival rate and (B) level of parasitemia of CBA/J mice injected with lo5 CT by either
im (0) or ip (W) routes and challenged 72 hr later with lo5 BT, injected by im (0) or ip (0) routes.
showed depletion of the cortical areas as soon as 24 hr after infection. This depletion was very marked 3 days after infection (Fig. 5A). No histopathological alteration was observed in the thymuses of PBS and CT injected mice during this period (Fig. -
5B). Also no alteration was seen in the thymuses of those CT injected mice that were challenged with BT. Splenomegaly with hyperplasia of the white pulp and congestion of the red pulp were seen in BT injected animals. Only minor alterations were observed in the spleens of CT injected mice. The spleens of those CT injected mice that were challenged with BT also presented only minor alterations. DISCUSSION
Lr 3T HOURS
PB
POST
IB-r
h
3scTKr ?2
INFECTION
FIG. 3. Leucocyte counts in blood of mice for vary-
ing times after im injection with PBS, lo5 CT or lo5 BT.
Previous observations (Camargo et al. 1989) showing that the carbon clearance activity of BT injected animals was decreased while that of CT injected animals was normal or slightly increased suggested that CTinduced early resistance could be associated with nonspecific factors. The data presented in this report show that only living CT was able to induce an early resistance, here defined as the increase in resistance detected within 24 to 72 hr after antigen injection, permitting animals to overcome a
T. cruzi:
TRYPOMASTIGOTES-INDUCED
.E61
0
1..
0
(2
.
(2
24
46
24
46
HOURS
72
c
72
POST INFECTION
FIG. 4. Lymphocyte and polymorphonuclear cells (PMN) counts in the blood of mice previously injected with PBS, CT, or BT.
challenge of 10’ BT. No early resistance was detected when living epimastigotes, blood trypomastigotes, amastigotes, and parasite extracts, including those obtained from Y strain CT forms, were tested. Also no early resistance could be observed when testing L. monocytogenes and Bio-Gel, known inducers of inflammatory reactions (Fauve et al. 1987), thus suggesting that CT-induced resistance is not associated with nonspecific factors but is rather specifically induced by CT epitopes that may be masked, absent, or in very low concentration in other parasite forms and their extracts. The expression of these putative resistance epitopes in CT seems to depend on one or more virulence factors of the para-
RESISTANCE
265
site since early resistance was detected only when virulence was lost or diminished by serial passage in cell cultures. Indeed, CT shortly regain virulence once in the host environment (Camargo et al. 1989), which stimulated by CT epitopes is now able to cope with the virulence factor(s). Comparison of CT and BT permitted detection of some differences in regard to infectivity for L cells and interaction with macrophages (Nogueira et al. 1980; Meireles et al. 1982; Kloetzel et al. 1984). However, comparison of BT and CT antigenic patterns, by using Western blotting analysis, showed an extensive homology, with only minor differences in the intensity of two antigens being detected (Silva et al. 1988). Thus CT seems to differ from BT only by very few epitopes. Experiments performed to establish the dose-response relationship showed that, within the limits tested, the higher the injected dose of CT, the higher the increase in resistance measured as higher survival rates and lower levels of parasitemia. Best results were obtained by using IO5 CT, when most of mice survived for 30 days while 100% of control died within 10 4 1 days. No difference in survival rates were observed in control groups injected with BT by the ip or im routes, probably due to the fact that they received a very large number of virulent parasites. However, mice im injected presented significantly different levels of parasitemia. Although injection of CT by either im or ip routes induced similar degrees of resistance, significantly higher degrees of survival and lower levels of parasitemia were seen when mice were challenged by the im route. Best results were always obtained when both CT and BT were administered by the im route. These data confirm observations from other laboratories (Kierzenbaum and Howard, 1976; Liew et al. 1985; Murtin et al. 1989) stressing the importance of the route of antigen injection for the features of host response.
266
CAMARGO
ET AL.
FIG. 5. Microscopic aspect of thymuses of mice, injected 72 hr previously, with: (A) phosphatebuffered saline (PBS); (B) culture trypomastigotes (CT); (C and D) blood trypomastigotes (BT). Magnification of 80X for (A), (B) and (C) and of 300X for (D). Note the peripheric thin cortical zone of BT infected mice.
Hematological changes during the acute et al. 1989); however, no information presphase were described in a man accidentally ently exists permitting one to determine if infected (HoMin et al. 1987), in children lymphopenia and thymic cortical depletion (Rassi 1979), and in mice experimentally in- result from cell lysis or from rapid removal fected (Cardoso and Brenner 1980; Repka from the blood and thymic cortical areas et al. 1985). However, to our knowledge, with sequestration in other organs. The no register was made of the first 3 days after possibility that these findings are secondary infection. Present observation indicates to a stress-derived increase in corticostethat during this early period BT exerts a roides (Savino et al. 1989) cannot be exmarked action on lymphoid organs while no cluded. The fact that no alteration was detected significant morphological alterations occurs in CT injected animals, suggesting that BT in the lymphoid organs of mice injected virulence is exerted through an early action with CT and challenged with BT permits on lymphoid cells. Early lymphopenia was the hypothesis that early increased resisobserved in T. musculi infections (Duffey et tance may result from stimulation of these al. 1985) and thymic cortical depletion in T. lymphoid cells involved in the control of cruzi has been recently described (Savino virulent parasites. This view is somewhat
T. cruzi: TRYPOMASTIGOTES-INDUCED
RESISTANCE
267
supported by evidence indicating that T FAUVE, R. M., FONTAN, E., HEVIN, M. B., SAKLANI, H., AND PARKER,F. 1987. Remote effects of inflamcells play an important role in resistance mation on non-specific immunity. Immunology Letagainst T. cruzi (Tarleton and Scott 1987; ters 16, 199-204. Russo et al. 1988; Tarleton 1990) and that, GONFALVES DA COSTA, S. C., LAGRANGE, P. H., during infection with virulent parasites, seHURTHREL, B., KERR, I., AND ALENCAR, A. 1984. Role of T lymphocytes in the resistance and immuvere T cell abnormalities result (Minomio nopathology of experimental Chagas’ disease. Anet al. 1986; Hontebrye-Joskowicz et al. nales d’ Immunologic (Znstitut Pasteur) 135C, 3171987; Curotto de Lafaille et al. 1990).
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ACKNOWLEDGMENTS We thank Professor M. A. Cruz Hofling for help in histopathological studies and Dime Lima Gabriel for technical help. This study was partially supported by FAPESP and CNPq grants.
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