409 TRANSACTIONS
OF WE
ROYAL
SOCIEIY
OF TROPICAL.
MEDICINE
AND HYGIENE,
VOL.
73, No.
4, 1979
A comparison of challenge with Trypanosoma cruzi blood-stream trypomastigotes and metacyclic trypomastigotes from Rhodnius prolixus in mice immunized with killed antigens N. MCHARDY AND R. A. NEAL Wellcome Research Laboratories,
Department
Summary
Groups of CD-l mice were immunized with vaccines prepared from freeze-thawed or ultrasonicated epimastigotes, blood trypomastigotes, or “plasma antigen”, of Trypanosoma cruzi strains Y, Ml and Tulahuen. The mice were challenged by the injection of bloodstream trypomastigotes obtained from mice, or of metacyclic trypomastigotes harvested from the rectum of Rhodnius prolixus.
Both challenges induced virulent infections in control mice. Blood-stream trypomastigotes killed mice more rapidly than the same number of bugderived trypomastigotes. Vaccinated mice resisted infection with bug-derived trypomastigotes as well as with blood trypomastigotes, and in some instances better. It is concluded that results obtained with the more convenient, but artificial blood trypomastigote challenge are comparable with the more natural, bug-derived, challenge.
of
Parasitology,
Beckenhant, Kent, UK
Dr. Cerisola, Buenos Aires, in 1962. All were originally derived from human infections. All three strains were maintained as the epimastigote stage by culture in BONE&PARENT’S(1963) liquid medium w>th 5% rabbit serum added. The; were subcultured weeklv. The Y strain had been continuouslv cultured in this way since 1961. For the preseni investigations, fresh isolations of all three strains were made from experimentally-infected mice in 1975, and cultured in Bone and Parent’s medium. The new isolate of strain Y was designated ‘Y2’ (MCHARDY & ELPHICK, 1978). All three strains were also maintained by weekly blood passage in mice. Blood trypomastigotes for challenges were obtained by cardiac puncture, with heparin as anticoagulant. For bug metacyclic trypomastigote challenges, Rhodnius proZixus were fed on mice infected with strain Y, and suspensions of the parasite obtained by maceration of the abdominal region of infected bugs 28 to 30 days after the blood meal (NEAL & MCHARDY, 1977).
Introduction
There is a substantial literature on the effects of immunizing mice with killed and living preparations of Trypan&ome cruzi, e.g. JOHNSONet ai. (1963), GONZALEZ-CAPPAet al. (1968). GOBLE (1970). KIERSZENBAUM 81BIJDZKO(i975);~McHARDY‘(197jj and NEAL & JOHNSON(1977). Trypomastigotes from the blood of an infected mouse were used as the challenge in all these reports, with the exception that NEAL & JOHNSON(1977) challenged in a single experiment with trypomastigotes -of T. crizi derived from the rectum of Rhodius arolixus. MSHELBWALA& ORMEROD(1973) and &AL & MCHARDY (1977) showed that bug-derived trypomastigotes of several strains of T. cruzi are able to establish lethal infections in mice, and that the subcutaneous (s.c.) route of challenge results in a more uniformly lethal infection than the intraperitoneal (i.p.) route. In the present report an investigation was made of the ability of vaccines prepared from various stages of T. cruzi to protect mice against challenge with either blood- or bugderived trypomastigotes. Materials
and Methods
Animals: Randomly bred, male mice, strain CD-l were used. They weighed about 20 g at the time of their first injection. T. cruzi: Three strains of T. cruzi were used for antigen preparation. These were strains Y and Tulahuen (ANDRADE,1974) and Ml, isolated by
Preparation of Antigens: Cultured epimastigotes harvested at the end of the logarithmic growth phase after four days’ incubation at 26”C, were used to prepare freeze-thawed and ultra-sonicated antigens. Virtually all cells were in the epimastigote form. The parasites were thoroughly washed with phosphate-buffered saline (PBS), then disrupted by ultrasonic vibration (MCHARDY, 1978a) or repeated freezing and thawing (MCHARDY, 1977). Blood from very heavily infected mice was used as the source of trypomastigote and plasma antigens. These mice were infected by the S.C.injection of about 10’ blood trypomastigotes. Seven days later they were exsang&nated, &.ing heparin as anticoaeulant. Their blood contained about 1 x 10s trygomastigotes/ml. The blood was centrifuged at 2,000 g for 15 min to sediment erythrocytes and trypomastigotes. The sediment was washed twice with PBS and then frozen and thawed as for the epimastigotes. The plasma supernatant was filtered through a 0.45 pm membrane for use as ‘plasma antigen’. All antigens were stored at -70°C. Immunization and Challenge: Vaccines were prepared for investigation by appropriate dilution with PBS. In Experiments 1, 2 and 4, the disrupted equivalent of 1 x lo* organisms was contained in each vaccine dose. In Experiment 3, the disrupted equivalent of 1 x 10’ epimastigotes or trypomastigotes, was used, and the dose of ‘plasma
410
I~~NIZATION
AGAINST T. cruzi
antigen’ was 0 * 1 ml. This dose was chosen as approximately the volume of plasma from which lo7 trypomastigotes were harvested. In some instances 50 pg Quillaia saponin was added to each vaccine dose, as an immunological adjuvant (JOHNSON er al., 1963). In one experiment (Table II), the saponin-only treatment challenged with metacyclic trypomastigotes, was unusually effective. Since in numerous publications (JOHNSON et al., 1963; MCHARDY, 1977; NEAL
& JOHNSON,
1977;
MCHARDY
& ELPHICK,
1978; MCHARDY, 1978), and in unpublished experiments, saponin was always inactive, it was concluded that antigen had been included in error. The results from this group of mice are not included in Table II. Vaccines were injected S.C.on the abdomen. Two
doses were given, with an interval of 14 days. Challenge doses were injected 14 days after the second immunizing dose, either S.C. between the scapulae, or i.p. Assessment of Results: Experimental groups contained five or 10 mice, individually identified by ear-marking. After challenge the day of death of mice was recorded. At intervals throughout the experiment the degree of parasitaemia in the mice was monitored by microscopical examination of fresh blood smears. 25 fields, each containing about 2,500 erythrocytes, were examined. Parasitaemia was scored on a lo-point scale of doubling parasite numbers. A single parasite in 25 fields was assigned a score of ‘l’, two to three parasites ‘2’, four to six parasites ‘3, and so on up to score ‘lo’, which
Table I-Summary of observations on groups of CD-l mice immunized with freeze-thawed epimastigotes of T. cruzi strain Y, and challenged with homologous blood- or bug-derived trypomastigotes
Immunization
Source of Challenge and Route of Injection
Survival
1. Untreated
Blood s/c
O/5
2. Untreated
Blood i/p
315
3. Epimastigotes
Blood s/c
o/10
4. Epimastigotes
Blood i/p
10/10*
5. Epimastigotes + saponin
Blood s/c
4/10
6. Epimastigotes + saponin
Blood i/p
10/10*
Parasitaemia on Day 11
(E) (I::) (Z) (C)
Maximum Parasitaemia (Survivors only) -
(Z) -
4.8** (l-8) (2)
7. Untreated
Bug
s/c
8. Untreated
Bug
i/p
215
9. Epimastigotes
Bug
s/c
o/10
10. Epimastigotes
Bug
i/p
6/10
11. Epimastigotes + saponin
Bug
s/c
g/10**
12. Epimastigotes + saponin
Bug
i/p
015
(E) (E)
(G) (2;
lo/lo*
* Significant at 0.05 level vs untreated mice receiving similar challenge. ** Significant at 0 *01 level vs untreated mice receiving similar challenge. *** Significant at 0 *001 level vs untreated mice receiving similar challenge.
(E)
2.8*** (o-6) 1*2** (O-4)
411
N. MCHARDYANDR. A. NEAL
Table II-Summary of observations on groups of CD-1 mice immunized with freeze-thawed or ultrasonicated epimastigotes of T. cruzi strain Y2, and challenged with homologous blood- or bugderived trypomastigotes Source of Challenge
Survival
1. Untreated
Blood
O/IO
2. Saponin only
Blood
o/10
3. Freeze-thawed epimastigotes + saponin
Blood
5/10*
4. Ultrasonicated epimastigotes + saponin
Blood
B/10**
5. Untreated
Bug
l/10
6. Freeze-thawed epimastigotes + saponin
Bug
7. Ultrasonicated epimastigotes + saponin
Bug
Immunization
Parasitaemia on Day 11
Maximum Parasitaemia (Survivors only) -
(it 6”, (ii) o-2** (0-l)
* Significant at 0.05 level vs untreated mice receiving similar challenge. ** Significant at 0.001 level vs untreated mice receiving similar challenge. represents at least 16 parasites in each field. All mice which attained a score of 10 died, and some mice developed parasitaemia far greater than 16 parasites per field, A score of ‘1’ represents about 1 x lo5 parasites/ml blood, while score ‘10’ is equivalent to at least 4 x 107/ml. Experiments were terminated 42 days after challenge. Results were analysed statistically using the Mann-Whitney ‘U’-test (MWUT) (MANN & WHITNEY, 1947) and Fisher’s Exact Probability Test (FEPT) (FISHER, 1970), as described by MCHARDY (1977). Results Experiment 1-A comparison of S.C.and i.p. route of challenge, with blood- and bug-derived trypomastigotes
(Table I): Groups of 10 mice were immunized with two doses of strain Y freeze-thawed epimastigote vaccine, injected S.C. and similar groups received vaccine with saponin added as adjuvant. Each vaccine dose contained the equivalent of 1 x 108 epimastigotes. Groups of five mice were kept as unvaccinated controls. The mice were challenged with blood- or bug-derived trypomastigotes, injected S.C.or i.p. Each challenge dose contained 2 x lo* trypomastigotes. The results are shown in Table I. In the unvaccinated mice, challenge with blood trypomastigotes resulted in infections similar to those caused by bug trypomastigotes. In both instances the S.C.route of challenge produced a more rapidly fulminating infection than i.p. All 10 mice challenged S.C.died of the infection (groups 1 and 7), while only five died following i.p. challenge (groups 2 and 8). Similarly among the vaccinated mice,
i.p. challenge was more easily resisted than S.C. Addition of saponin to the vaccine resulted in increased protection against S.C.challenge, but with i.p. challenge the improvement was less evident. Vaccinated mice resisted challenge with bloodor bug-derived trypomastigotes equally well. Experiment 2-A comparison of blood- and bugderived trypomastigote challenge in mice immunized with freeze-thawed or ultrasonicated epimastigote vaccine (Table II):
Groups of 10 mice were immunized with two doses of 1 x 10s freeze-thawed or ultra-sonicated strain Y2 epimastigotes, with saponin, and challenged with 1 x lo4 blood- or bug-derived trypomastigotes, injected S.C. Groups of 10 mice were kept either unvaccinated, or injected with saponin only and given ‘similar challenges. Results are shown in Table II. Blood challenge killed all the control mice. Untreated mice died in a mean of 15 -3 days, and those receiving saponin only, in 18.0 days. Bug challenge killed all 10 unvaccinated mice in 19.7 days. Both vaccines gave significant protection to the mice, but vaccinated mice resisted challenge with bug-derived trypomastigotes more readily than those derived from blood. There was no significant difference between the protection afforded by the two vaccines.
Experiment 3-A comparison of three antigens challenged with blood- or bug-derived trypomastigotes
(Table III) : Groups of 10 mice were immunized with two doses of 10’ freeze-thawed epimastigotes, 10’
412
IhthWNIZATION
AGAINST T. cruzi
Table III-Summary of observations on groups of CD-l mice immunized with epimastigotes, trypomastigotes or plasma antigen, strain Y, + saponin, and challenged with homologous blood or bug-derived trypomastigotes of T. cruzi
Source of Challenge
Survival
1. Untreated
Blood
o/10
2. Saponin only
Blood
o/10
3. Epimastigotes + saponin
Blood
2/10
4. Trypomastigotes + saponin
Blood
10/10**
2*1** (l-4)
5. Plasma + saponin
Blood
10/10**
1*6** (O-3)
Immunization
6. Untreated 7. Saponin only 8. Epimastigotes + saponin
Bug
o/10
Bug
l/10
Bug
B/10**
Parasitaemia on Day 12
(E) go, (2::;
Maximum Parasitaemia (Survivors only) -
(CO)
(G) (Cl)
9
(:::; 9. Trypomastigotes + saponin 10. Plasma + saponin
Bug
g/10**
2*3** (l-4)
Bug
10/10**
0*9** (O-3)
* Significant at 0 -01 level vs untreated mice receiving similar challenge. ** Significant at 0.001 level vs untreated mice receiving similar challenge. blood-derived trypomastigotes, or 0 * 1 ml “plasma antigen”, all strain Y, and injected S.C.Saponin was included in all vaccines, as adjuvant. As controls, groups of 10 mice were either kept unvaccinated, or given two doses of saponin only. The mice were challenged with 2 x lo4 blood- or bug-derived trypomastigotes injected S.C. Results are given in Table III. The blood-derived challenge killed all 20 control mice in a mean of 14 *8 days, while the bug-derived challenge killed 19 mice in 20.7 days. Trypomastigotes and plasma antigen protected all mice against blood challenge, and all but one against bug challenge. The epimastigote vaccine protected only two mice against blood challenge, but eight against bug challenge. Experiment 4-Comparison of the protection afforded by epimastigote vaccines of four strains against challenge with blood- or bug-derived trypomastigotes of one strain (Table IV):
Groups of 10 mice were immunized by the S.C. injection of two doses of 1 x 10s freeze-thawed epimastigotes of strains Y, Y2, Ml and Tulahuen, with saponin as adjuvant. Groups of 10 unvaccinated
mice were included, as controls. The mice were challenged by the S.C.injection of 5 x lo3 bloodor bug-derived trypomastigotes, of strain Y. The results are shown in Table IV. Blood challenge killed all 10 control mice in a mean of 16.6 days, while bug challenge killed nine mice in 20.0 days. All four strains of epimastigote vaccines gave strong protection against both challenges, perhaps slightly more against bug-derived trypomastigotes (i.e. with bug challenge 37 of 39 mice survived, with blood challenge 33 of 40 survived). Discussion
The results described in this report are in general agreement with those of earlier workers. Trypomastigotes harvested from the rectum of Rhodnius prolixus were found to be slightly less virulent in mice than equivalent numbers of trypomastigotes obtained from the blood of mice. Injection by the i.p. route resulted in less severe infections than following S.C. injection. This was shown for unvaccinated mice by MSHELBWALA & ORMEROD (1973) and NEAL & MCHARDY (1977). MCHARDY (1977) also showed that vaccinated mice resisted
413
N. MCHARDYANDR. A. NRAL.
Table IV-Summary of observations on groups of CD-l mice immunized with freeze-thawed epimastigotes of 4 strains of T. cruzi + saponin, and challenged with blood- or bug-derived trypomastigotes strain Y
Immunization
Source of Challenge
Survival
1. Untreated
Blood
o/10
2. Y epimastigotes
Blood
g/10***
Parasitaemia on Day 12
Maximum Parasitaemia (Survivors only)
2.g*** (l-6)
3. Y2 epimastigotes
Blood
lo/lo***
4. Ml epimastigotes
Blood
7/10**
5. Tulahuen epimastigotes
Blood
g/IO***
5. Untreated
Bug
l/l0
7. Y epimastigotes
Bug
g/10***
8. Y2 epimastigotes
Bug
g/10***
9. Ml epimastigotes
Bug
lo/lo***
Bug
g/g ***
10. Tulahuen epimastigotes
1.1*** (O-5) 2.3*** (O-6) 3-o** (O-7) (E)
0*8** (O-2) (-,.(j”** (O-2)
* Significant at 0 *05 level vs untreated mice receiving similar challenge. ** Significant at 0.01 level vs untreated mice receiving similar challenge. **:+ Significant at 0.001 level vs untreated mice receiving similar challenge.
challenge with blood trypomastigotes injected i.p. significantly better than when they were given S.C. All these workers associated the greater virulence of S.C. challenge with its similarity to the natural percutaneous route of infection. The finding that vaccinated mice resist bugderived challenge at least as well as the bloodderived challenge is encouraging. In some instances, such as epimastigote vaccine plus saponin in Experiment 3, protection against the more natural challenge was significantly greater than against blood challenge. The finding that epimastigote vaccines prepared from four strains of T. cruzi (Experiment 4) are all able to protect against bugderived trypomastigote challenge is also encouraging and is in agreement with the findings of MCHARDY & ELPHICK (1978), who found strong crossprotection between five strains, using bloodderived challenge. These results with “natural”, bug-derived challenge clearly validate earlier results with “artificial”, blood-derived challenge. The greater convenience of blood-trypomastigote challenge
makes it likely that it will continue to be used routinely, and it seems probable that results obtained with this method will be as relevant to the natural situation. Successful immunization prevented death of mice from acute infections of T. cruzi and by the end of the observation period the tail blood of most mice was microscopically negative. Although the animals in the present experiments were not studied further, it is now known that immunization by these methods does not eliminate the parasite although mice survive for prolonged periods. Sub-patent parasitaemias in similar experiments were revealed by blood subinoculation into clean mice (McHAR~Y, 1977) and by xenodiagnosis and haemoculture (NEAL, unpublished observations, Mcl%~n~,in preparation) by methods described by NEAL & MILES (1977). Acknowledgements We are grateful to Jane P. Elphick, Gillian Hamilton, Susan Featherstone and Lesley Holmes for their expert technical assistance with this work.
414
IhiMUNIZATION
References
Andrade, S. C. (1974). CaracterizacBo de cepas do Trypanosoma cruzi isoladas no reconcavo baiano. Revista Patologia Tropical, 3, 65-121. Bone, G. J. & Parent, G. (1963). Stearic acid, an essential growth factor for Trypanosoma cruzi. Journal of General Microbiology,
31,261-266.
Fisher, R. A. (1970). Statistical Methods for Research Workers. Edinburgh: Oliver and Boyd. Goble, F. C. (1970). South AmericanTrypanosomes. In: Immunity to Parasitic Animals, Vol. 2, Jackson, G. J., Herman, R. & Singer, I. (Editors). New York: Appleton-Century-Crofts, pp. 597889. Gonzalez-Cappa, S. M., Schmufiis, G. A., Traversa, 0. C., Yanovsky, J. F. & Parodi, A. S. (1968). Complement fixation tests, skin tests, and artificial immunization with antigens of Trypanosoma cruzi prepared under pressure. American Journal of Tropical 709-715.
Medicine
and Hygiene,
17,
Johnson, P., Neal, R. A. & Gall, D. (1963). Protective effect of killed trypanosome vaccines with incorporated adjuvants. Nature, 200, 83. Kierszenbaum, F. & Budzko, D. B. (1975). Immunization against experimental Chagas’ disease by using culture forms of Trypanosoma cruzi killed with a solution of sodium perchlorate. Infection
and Immunity,
12, 461-467.
McHardy, N. (1977). Immunization of mice against Trvaanosoma cruzi: The effect of size of dose. and rouie of injection, of immunizing and challenge inocula. Tropenmedizin und Parasitologic, 28, 11-16. McHardy, N. (1978a). Immunization of mice against Trypanosoma cruzi : the effect of chemical treatment or immune serum on an epimastigote
AGAINST
T.
cruzi
~5cini2
Tropenmedizin und Parasitologic,
29,
.
McHardy,N. (1978b). Effect of sexof miceinrelation to their response to immunization with vaccines prepared from Trypanosoma cruzi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 201-202. McHardy, N. & Elphick, J. P. (1978). Immunization of mice against infection with Trypanosoma cruzi. Cross-immunization between five strains of the parasite using freeze-thawed vaccines containing epimastigotes of up to five strains. International Journal for Parasitology, 8, 25-31. Mann, H. B. & Whitney, D. R. (1947). On a test of whether one of two random variables is stochastically larger than the other. Annals of Mathematical Statistics,
18. 52-54.
Mshelbwala, A. S. & Ormerod, W. E. (1973). Measurements of the infectivity of Trypanosoma cruzi in faecesof Rhodnius by comparison of doseresponse curves. Journal of General Microbiolony, -75;339-350.
-
-
Neal, R. A. & Johnson, P. (1977). Immunization against Trypanosoma cruzi using killed antigens, and with saponin as adjuvant. Acta Tropica, 34, 87-96.
Neal. R. A. & McHardv. N. (1977). Comparison of infectivity of Trypanbsoma cruzi blood stream trypomastigotes and metacyclic trypomastigotes from Rhodnius brolixus. Acta Trotica, 34. 79-85. Neal, R. A. & ales, R. A. (1977):Thk sensitivity of culture methods to detect experimental infections of Trypanosoma cruzi and comparison with xenodiagnosis. Revista do Instituto de Medicina
Tropical de Sgo Paulo, 19, 170-176.
Accepted for publication
28th November, 1978.
OF WE
ROYAL
SOCIEIY
OF TROPICAL.
MEDICINE
AND HYGIENE,
VOL.
73, No.
4, 1979
A comparison of challenge with Trypanosoma cruzi blood-stream trypomastigotes and metacyclic trypomastigotes from Rhodnius prolixus in mice immunized with killed antigens N. MCHARDY AND R. A. NEAL Wellcome Research Laboratories,
Department
Summary
Groups of CD-l mice were immunized with vaccines prepared from freeze-thawed or ultrasonicated epimastigotes, blood trypomastigotes, or “plasma antigen”, of Trypanosoma cruzi strains Y, Ml and Tulahuen. The mice were challenged by the injection of bloodstream trypomastigotes obtained from mice, or of metacyclic trypomastigotes harvested from the rectum of Rhodnius prolixus.
Both challenges induced virulent infections in control mice. Blood-stream trypomastigotes killed mice more rapidly than the same number of bugderived trypomastigotes. Vaccinated mice resisted infection with bug-derived trypomastigotes as well as with blood trypomastigotes, and in some instances better. It is concluded that results obtained with the more convenient, but artificial blood trypomastigote challenge are comparable with the more natural, bug-derived, challenge.
of
Parasitology,
Beckenhant, Kent, UK
Dr. Cerisola, Buenos Aires, in 1962. All were originally derived from human infections. All three strains were maintained as the epimastigote stage by culture in BONE&PARENT’S(1963) liquid medium w>th 5% rabbit serum added. The; were subcultured weeklv. The Y strain had been continuouslv cultured in this way since 1961. For the preseni investigations, fresh isolations of all three strains were made from experimentally-infected mice in 1975, and cultured in Bone and Parent’s medium. The new isolate of strain Y was designated ‘Y2’ (MCHARDY & ELPHICK, 1978). All three strains were also maintained by weekly blood passage in mice. Blood trypomastigotes for challenges were obtained by cardiac puncture, with heparin as anticoagulant. For bug metacyclic trypomastigote challenges, Rhodnius proZixus were fed on mice infected with strain Y, and suspensions of the parasite obtained by maceration of the abdominal region of infected bugs 28 to 30 days after the blood meal (NEAL & MCHARDY, 1977).
Introduction
There is a substantial literature on the effects of immunizing mice with killed and living preparations of Trypan&ome cruzi, e.g. JOHNSONet ai. (1963), GONZALEZ-CAPPAet al. (1968). GOBLE (1970). KIERSZENBAUM 81BIJDZKO(i975);~McHARDY‘(197jj and NEAL & JOHNSON(1977). Trypomastigotes from the blood of an infected mouse were used as the challenge in all these reports, with the exception that NEAL & JOHNSON(1977) challenged in a single experiment with trypomastigotes -of T. crizi derived from the rectum of Rhodius arolixus. MSHELBWALA& ORMEROD(1973) and &AL & MCHARDY (1977) showed that bug-derived trypomastigotes of several strains of T. cruzi are able to establish lethal infections in mice, and that the subcutaneous (s.c.) route of challenge results in a more uniformly lethal infection than the intraperitoneal (i.p.) route. In the present report an investigation was made of the ability of vaccines prepared from various stages of T. cruzi to protect mice against challenge with either blood- or bugderived trypomastigotes. Materials
and Methods
Animals: Randomly bred, male mice, strain CD-l were used. They weighed about 20 g at the time of their first injection. T. cruzi: Three strains of T. cruzi were used for antigen preparation. These were strains Y and Tulahuen (ANDRADE,1974) and Ml, isolated by
Preparation of Antigens: Cultured epimastigotes harvested at the end of the logarithmic growth phase after four days’ incubation at 26”C, were used to prepare freeze-thawed and ultra-sonicated antigens. Virtually all cells were in the epimastigote form. The parasites were thoroughly washed with phosphate-buffered saline (PBS), then disrupted by ultrasonic vibration (MCHARDY, 1978a) or repeated freezing and thawing (MCHARDY, 1977). Blood from very heavily infected mice was used as the source of trypomastigote and plasma antigens. These mice were infected by the S.C.injection of about 10’ blood trypomastigotes. Seven days later they were exsang&nated, &.ing heparin as anticoaeulant. Their blood contained about 1 x 10s trygomastigotes/ml. The blood was centrifuged at 2,000 g for 15 min to sediment erythrocytes and trypomastigotes. The sediment was washed twice with PBS and then frozen and thawed as for the epimastigotes. The plasma supernatant was filtered through a 0.45 pm membrane for use as ‘plasma antigen’. All antigens were stored at -70°C. Immunization and Challenge: Vaccines were prepared for investigation by appropriate dilution with PBS. In Experiments 1, 2 and 4, the disrupted equivalent of 1 x lo* organisms was contained in each vaccine dose. In Experiment 3, the disrupted equivalent of 1 x 10’ epimastigotes or trypomastigotes, was used, and the dose of ‘plasma
410
I~~NIZATION
AGAINST T. cruzi
antigen’ was 0 * 1 ml. This dose was chosen as approximately the volume of plasma from which lo7 trypomastigotes were harvested. In some instances 50 pg Quillaia saponin was added to each vaccine dose, as an immunological adjuvant (JOHNSON er al., 1963). In one experiment (Table II), the saponin-only treatment challenged with metacyclic trypomastigotes, was unusually effective. Since in numerous publications (JOHNSON et al., 1963; MCHARDY, 1977; NEAL
& JOHNSON,
1977;
MCHARDY
& ELPHICK,
1978; MCHARDY, 1978), and in unpublished experiments, saponin was always inactive, it was concluded that antigen had been included in error. The results from this group of mice are not included in Table II. Vaccines were injected S.C.on the abdomen. Two
doses were given, with an interval of 14 days. Challenge doses were injected 14 days after the second immunizing dose, either S.C. between the scapulae, or i.p. Assessment of Results: Experimental groups contained five or 10 mice, individually identified by ear-marking. After challenge the day of death of mice was recorded. At intervals throughout the experiment the degree of parasitaemia in the mice was monitored by microscopical examination of fresh blood smears. 25 fields, each containing about 2,500 erythrocytes, were examined. Parasitaemia was scored on a lo-point scale of doubling parasite numbers. A single parasite in 25 fields was assigned a score of ‘l’, two to three parasites ‘2’, four to six parasites ‘3, and so on up to score ‘lo’, which
Table I-Summary of observations on groups of CD-l mice immunized with freeze-thawed epimastigotes of T. cruzi strain Y, and challenged with homologous blood- or bug-derived trypomastigotes
Immunization
Source of Challenge and Route of Injection
Survival
1. Untreated
Blood s/c
O/5
2. Untreated
Blood i/p
315
3. Epimastigotes
Blood s/c
o/10
4. Epimastigotes
Blood i/p
10/10*
5. Epimastigotes + saponin
Blood s/c
4/10
6. Epimastigotes + saponin
Blood i/p
10/10*
Parasitaemia on Day 11
(E) (I::) (Z) (C)
Maximum Parasitaemia (Survivors only) -
(Z) -
4.8** (l-8) (2)
7. Untreated
Bug
s/c
8. Untreated
Bug
i/p
215
9. Epimastigotes
Bug
s/c
o/10
10. Epimastigotes
Bug
i/p
6/10
11. Epimastigotes + saponin
Bug
s/c
g/10**
12. Epimastigotes + saponin
Bug
i/p
015
(E) (E)
(G) (2;
lo/lo*
* Significant at 0.05 level vs untreated mice receiving similar challenge. ** Significant at 0 *01 level vs untreated mice receiving similar challenge. *** Significant at 0 *001 level vs untreated mice receiving similar challenge.
(E)
2.8*** (o-6) 1*2** (O-4)
411
N. MCHARDYANDR. A. NEAL
Table II-Summary of observations on groups of CD-1 mice immunized with freeze-thawed or ultrasonicated epimastigotes of T. cruzi strain Y2, and challenged with homologous blood- or bugderived trypomastigotes Source of Challenge
Survival
1. Untreated
Blood
O/IO
2. Saponin only
Blood
o/10
3. Freeze-thawed epimastigotes + saponin
Blood
5/10*
4. Ultrasonicated epimastigotes + saponin
Blood
B/10**
5. Untreated
Bug
l/10
6. Freeze-thawed epimastigotes + saponin
Bug
7. Ultrasonicated epimastigotes + saponin
Bug
Immunization
Parasitaemia on Day 11
Maximum Parasitaemia (Survivors only) -
(it 6”, (ii) o-2** (0-l)
* Significant at 0.05 level vs untreated mice receiving similar challenge. ** Significant at 0.001 level vs untreated mice receiving similar challenge. represents at least 16 parasites in each field. All mice which attained a score of 10 died, and some mice developed parasitaemia far greater than 16 parasites per field, A score of ‘1’ represents about 1 x lo5 parasites/ml blood, while score ‘10’ is equivalent to at least 4 x 107/ml. Experiments were terminated 42 days after challenge. Results were analysed statistically using the Mann-Whitney ‘U’-test (MWUT) (MANN & WHITNEY, 1947) and Fisher’s Exact Probability Test (FEPT) (FISHER, 1970), as described by MCHARDY (1977). Results Experiment 1-A comparison of S.C.and i.p. route of challenge, with blood- and bug-derived trypomastigotes
(Table I): Groups of 10 mice were immunized with two doses of strain Y freeze-thawed epimastigote vaccine, injected S.C. and similar groups received vaccine with saponin added as adjuvant. Each vaccine dose contained the equivalent of 1 x 108 epimastigotes. Groups of five mice were kept as unvaccinated controls. The mice were challenged with blood- or bug-derived trypomastigotes, injected S.C.or i.p. Each challenge dose contained 2 x lo* trypomastigotes. The results are shown in Table I. In the unvaccinated mice, challenge with blood trypomastigotes resulted in infections similar to those caused by bug trypomastigotes. In both instances the S.C.route of challenge produced a more rapidly fulminating infection than i.p. All 10 mice challenged S.C.died of the infection (groups 1 and 7), while only five died following i.p. challenge (groups 2 and 8). Similarly among the vaccinated mice,
i.p. challenge was more easily resisted than S.C. Addition of saponin to the vaccine resulted in increased protection against S.C.challenge, but with i.p. challenge the improvement was less evident. Vaccinated mice resisted challenge with bloodor bug-derived trypomastigotes equally well. Experiment 2-A comparison of blood- and bugderived trypomastigote challenge in mice immunized with freeze-thawed or ultrasonicated epimastigote vaccine (Table II):
Groups of 10 mice were immunized with two doses of 1 x 10s freeze-thawed or ultra-sonicated strain Y2 epimastigotes, with saponin, and challenged with 1 x lo4 blood- or bug-derived trypomastigotes, injected S.C. Groups of 10 mice were kept either unvaccinated, or injected with saponin only and given ‘similar challenges. Results are shown in Table II. Blood challenge killed all the control mice. Untreated mice died in a mean of 15 -3 days, and those receiving saponin only, in 18.0 days. Bug challenge killed all 10 unvaccinated mice in 19.7 days. Both vaccines gave significant protection to the mice, but vaccinated mice resisted challenge with bug-derived trypomastigotes more readily than those derived from blood. There was no significant difference between the protection afforded by the two vaccines.
Experiment 3-A comparison of three antigens challenged with blood- or bug-derived trypomastigotes
(Table III) : Groups of 10 mice were immunized with two doses of 10’ freeze-thawed epimastigotes, 10’
412
IhthWNIZATION
AGAINST T. cruzi
Table III-Summary of observations on groups of CD-l mice immunized with epimastigotes, trypomastigotes or plasma antigen, strain Y, + saponin, and challenged with homologous blood or bug-derived trypomastigotes of T. cruzi
Source of Challenge
Survival
1. Untreated
Blood
o/10
2. Saponin only
Blood
o/10
3. Epimastigotes + saponin
Blood
2/10
4. Trypomastigotes + saponin
Blood
10/10**
2*1** (l-4)
5. Plasma + saponin
Blood
10/10**
1*6** (O-3)
Immunization
6. Untreated 7. Saponin only 8. Epimastigotes + saponin
Bug
o/10
Bug
l/10
Bug
B/10**
Parasitaemia on Day 12
(E) go, (2::;
Maximum Parasitaemia (Survivors only) -
(CO)
(G) (Cl)
9
(:::; 9. Trypomastigotes + saponin 10. Plasma + saponin
Bug
g/10**
2*3** (l-4)
Bug
10/10**
0*9** (O-3)
* Significant at 0 -01 level vs untreated mice receiving similar challenge. ** Significant at 0.001 level vs untreated mice receiving similar challenge. blood-derived trypomastigotes, or 0 * 1 ml “plasma antigen”, all strain Y, and injected S.C.Saponin was included in all vaccines, as adjuvant. As controls, groups of 10 mice were either kept unvaccinated, or given two doses of saponin only. The mice were challenged with 2 x lo4 blood- or bug-derived trypomastigotes injected S.C. Results are given in Table III. The blood-derived challenge killed all 20 control mice in a mean of 14 *8 days, while the bug-derived challenge killed 19 mice in 20.7 days. Trypomastigotes and plasma antigen protected all mice against blood challenge, and all but one against bug challenge. The epimastigote vaccine protected only two mice against blood challenge, but eight against bug challenge. Experiment 4-Comparison of the protection afforded by epimastigote vaccines of four strains against challenge with blood- or bug-derived trypomastigotes of one strain (Table IV):
Groups of 10 mice were immunized by the S.C. injection of two doses of 1 x 10s freeze-thawed epimastigotes of strains Y, Y2, Ml and Tulahuen, with saponin as adjuvant. Groups of 10 unvaccinated
mice were included, as controls. The mice were challenged by the S.C.injection of 5 x lo3 bloodor bug-derived trypomastigotes, of strain Y. The results are shown in Table IV. Blood challenge killed all 10 control mice in a mean of 16.6 days, while bug challenge killed nine mice in 20.0 days. All four strains of epimastigote vaccines gave strong protection against both challenges, perhaps slightly more against bug-derived trypomastigotes (i.e. with bug challenge 37 of 39 mice survived, with blood challenge 33 of 40 survived). Discussion
The results described in this report are in general agreement with those of earlier workers. Trypomastigotes harvested from the rectum of Rhodnius prolixus were found to be slightly less virulent in mice than equivalent numbers of trypomastigotes obtained from the blood of mice. Injection by the i.p. route resulted in less severe infections than following S.C. injection. This was shown for unvaccinated mice by MSHELBWALA & ORMEROD (1973) and NEAL & MCHARDY (1977). MCHARDY (1977) also showed that vaccinated mice resisted
413
N. MCHARDYANDR. A. NRAL.
Table IV-Summary of observations on groups of CD-l mice immunized with freeze-thawed epimastigotes of 4 strains of T. cruzi + saponin, and challenged with blood- or bug-derived trypomastigotes strain Y
Immunization
Source of Challenge
Survival
1. Untreated
Blood
o/10
2. Y epimastigotes
Blood
g/10***
Parasitaemia on Day 12
Maximum Parasitaemia (Survivors only)
2.g*** (l-6)
3. Y2 epimastigotes
Blood
lo/lo***
4. Ml epimastigotes
Blood
7/10**
5. Tulahuen epimastigotes
Blood
g/IO***
5. Untreated
Bug
l/l0
7. Y epimastigotes
Bug
g/10***
8. Y2 epimastigotes
Bug
g/10***
9. Ml epimastigotes
Bug
lo/lo***
Bug
g/g ***
10. Tulahuen epimastigotes
1.1*** (O-5) 2.3*** (O-6) 3-o** (O-7) (E)
0*8** (O-2) (-,.(j”** (O-2)
* Significant at 0 *05 level vs untreated mice receiving similar challenge. ** Significant at 0.01 level vs untreated mice receiving similar challenge. **:+ Significant at 0.001 level vs untreated mice receiving similar challenge.
challenge with blood trypomastigotes injected i.p. significantly better than when they were given S.C. All these workers associated the greater virulence of S.C. challenge with its similarity to the natural percutaneous route of infection. The finding that vaccinated mice resist bugderived challenge at least as well as the bloodderived challenge is encouraging. In some instances, such as epimastigote vaccine plus saponin in Experiment 3, protection against the more natural challenge was significantly greater than against blood challenge. The finding that epimastigote vaccines prepared from four strains of T. cruzi (Experiment 4) are all able to protect against bugderived trypomastigote challenge is also encouraging and is in agreement with the findings of MCHARDY & ELPHICK (1978), who found strong crossprotection between five strains, using bloodderived challenge. These results with “natural”, bug-derived challenge clearly validate earlier results with “artificial”, blood-derived challenge. The greater convenience of blood-trypomastigote challenge
makes it likely that it will continue to be used routinely, and it seems probable that results obtained with this method will be as relevant to the natural situation. Successful immunization prevented death of mice from acute infections of T. cruzi and by the end of the observation period the tail blood of most mice was microscopically negative. Although the animals in the present experiments were not studied further, it is now known that immunization by these methods does not eliminate the parasite although mice survive for prolonged periods. Sub-patent parasitaemias in similar experiments were revealed by blood subinoculation into clean mice (McHAR~Y, 1977) and by xenodiagnosis and haemoculture (NEAL, unpublished observations, Mcl%~n~,in preparation) by methods described by NEAL & MILES (1977). Acknowledgements We are grateful to Jane P. Elphick, Gillian Hamilton, Susan Featherstone and Lesley Holmes for their expert technical assistance with this work.
414
IhiMUNIZATION
References
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Johnson, P., Neal, R. A. & Gall, D. (1963). Protective effect of killed trypanosome vaccines with incorporated adjuvants. Nature, 200, 83. Kierszenbaum, F. & Budzko, D. B. (1975). Immunization against experimental Chagas’ disease by using culture forms of Trypanosoma cruzi killed with a solution of sodium perchlorate. Infection
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McHardy,N. (1978b). Effect of sexof miceinrelation to their response to immunization with vaccines prepared from Trypanosoma cruzi. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 201-202. McHardy, N. & Elphick, J. P. (1978). Immunization of mice against infection with Trypanosoma cruzi. Cross-immunization between five strains of the parasite using freeze-thawed vaccines containing epimastigotes of up to five strains. International Journal for Parasitology, 8, 25-31. Mann, H. B. & Whitney, D. R. (1947). On a test of whether one of two random variables is stochastically larger than the other. Annals of Mathematical Statistics,
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Neal, R. A. & Johnson, P. (1977). Immunization against Trypanosoma cruzi using killed antigens, and with saponin as adjuvant. Acta Tropica, 34, 87-96.
Neal. R. A. & McHardv. N. (1977). Comparison of infectivity of Trypanbsoma cruzi blood stream trypomastigotes and metacyclic trypomastigotes from Rhodnius brolixus. Acta Trotica, 34. 79-85. Neal, R. A. & ales, R. A. (1977):Thk sensitivity of culture methods to detect experimental infections of Trypanosoma cruzi and comparison with xenodiagnosis. Revista do Instituto de Medicina
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Accepted for publication
28th November, 1978.
