EXPERIMENTAL PARASITOLOGY 71, l-8 (1990)

Ttypanosoma

cruzi: Partial Prevention of the Natural Infection Guinea Pigs with a Killed Parasite Vaccine

of

MIGUEL ANGEL BASOMBRIO Laboratorio

de Patologia

Experimental, Facultad Calle Buenos Aires

de Ciencias de la Salud, Universidad 177, 4.400 Salta, Argentina

National

de Salta,

BASOMBRIO, M. A. 1990. Trypanosoma cruzir Partial prevention of the natural infection of guinea pigs with a killed parasite vaccine. Experimental Parasitology 71, l-8. Guinea pigs are natural reservoirs of Chagas’ disease. Domestic breeding and local trade of these animals are common practices among andean communities in South America. Infection by Trypanosoma cruzi occurs when the animals live in triatomine-infested houses or yards. The preventive effect of a vaccine consisting of cultured T. cruzi killed by freezing and thawing plus saponin was tested both in mice and in the guinea pig ecosystem. Resistance against T. cruzi challenge in mice was improved by increasing the trypomastigote/epimastigote ratio in live attenuated vaccines but not in killed parasite vaccines. Although the killing of attenuated parasites sharply reduced their immunogenicity for mice, a protective effect against natural T. cruzi infection was detected in guinea pigs. A total of 88 guinea pigs were vaccinated in four intradermal sites on three occasions. Eighty controls received similar inoculations of culture medium plus saponin. All animals were kept in a triatomine-infested yard. Parasitemia was studied with the capillary microhematocrit method. After an exposure time averaging 4 months, natural T. cruzi infection occurred in 55% (44/80) of the controls and in 33% (29/88) of the vaccinated group (P < 0.01). The number of highly parasitemic guinea pigs was also significantly decreased (6180 vs 0188, P < 0.01). Thus, immunizing protocols which are only partially protective against artificial callenge with T. cruzi may nevertheless constrain the exchange of parasites between natural hosts and vectors. 0 IWO Academic Press, Inc.

INDEX DESCRIPTORS:Trypanosoma fection; Reservoir; Vaccination.

cruzi;

Triatoma

infestans;

Guinea pigs; Natural in-

tion with live attenuated T. cruzi (Basombrto et al. 1987a) we have now tested the effect of a killed parasite vaccine in a natural ecosystem of T. cruzi spread.

Domestic

animals are the main reservoir of Trypanosoma cruzi in triatominecolonized houses (Minter 1978). Guinea pigs (Cavia porcelfus) have long been known as important contributors to the spread of the parasite (Torrico 1950). In houses or yards where these animals cohabit with triatomine vectors, a remarkable increase in transmission rates of T. cruzi can occur (Herrer 1955). By using a vaccine consisting of freezethawed T. cruzi epimastigotes plus saponin, MC Hardy (1977) was able to obtain partial, but consistent and reproducible protection against experimental T. cruzi infection of laboratory mice. Such vaccines have never been tested in the field. Based on previous field trials of vaccina-

MATERIALSAND METHODS Our laboratory has a field post in a Chagas’ disease endemic rural area (Figs. 1 and 2). Here guinea pigs are kept in an adobe yard. Local Triatoma in&tans hide in cracks and spaces of the adobe wall and limited proliferation of these insects is allowed between partial insecticide sprayings, so that natural T. cruzi infection occurs regularly in the guinea pigs (Basombrio et al. 1987a). Vaccine. The T. cruzi vaccine was prepared as described by MC Hardy (1977). Culture, cloned strains NIH 0.002 (Crane and Dvorak 1982) and TCC (Basombrlo ef al. 1982a) of T. cruzi were grown in 1000-ml glass bottles containing 100 ml of LIT medium supplemented with 5% fetal bovine serum, 20 kg/ml hemin, 100 U/ml penicillin, and 100 t&ml streptomycin. The parasites, mostly epimastigotes, were harvested in late Field post.

1 0014-4894190 $3.00 Copyright 6 IWO by Academic F’resa. Inc. All rights of reproduction in any form reserved.

FIGS. 1 AND 2. Rural guinea pig (Caviu par ceffus) breeding yard used as field post for experimental purposes. Animals are often exposed to bites from Z’riaroma in&stuns hiding in the adobe cracks and spaces and may be naturally infected by T. cruzi.

2

PROTECTION

AGAINST

exponential phase. At this time, the percentage of trypomastigotes ranged between 7 and 30% in the NIH 0.002 cultures and was under 8.5% in the TCC cultures. After washing the parasites three times in RPM1 medium, the pellet was placed in a plastic tube which was submerged alternatively in liquid nitrogen (- 196°C) and water at 37°C for three cycles. Care was taken not to remove the parasites from these temperatures until they had been completely freezed or thawed for at least 10 min. The absence of viable organisms was confirmed by examination of the parasite material in over 200 high power microscope fields and by culture of 0.2-ml aliquots in LIT medium supplemented as mentioned above. Before injection, the killed parasites were diluted in RPM1 medium so as to inject 7 x 10’ parasites per killogram and 50 pg of Quillaia saponin in 0.1 ml of complete vaccine. Inoculation of guinea pigs. Guinea pigs received simultaneous intradermal injections of the vaccine in four sites of the shaved ventral skin. These injections were repeated on three occasions at U-day intervals and produced infiltrated dermal nodules which disappeared after a month. Preparation and injection of vaccine was always done on the same day or after keeping the parasites overnight in liquid nitrogen. Control guinea pigs received RPMI-saponin inocula not containing parasites. Microhematocrit technique. The occurrence of natural T. cruzi infection in guinea pigs was studied by puncturing the marginal ear’s vein under slight embutal anesthesia and filling three capillary tubes which were processed as described by Freilij et al. (1983). The parasite-rich interphase of each capillary was examined by two observers, each scanning 100 high power microscope fields. Experimental schedule. Guinea pigs born in the yard were brought to the laboratory within 1 week after birth and routinely subjected to xenodiagnosis with 10 Triatoma infestans nymphs, which were examined at 30 and 60 days. A few animals (6.12%) which were found already infected by T. cruzi were discarded. Noninfected guinea pigs were distributed in five experimental series (A, B, C, D, and E) according to birth date, so that age differed less than 90 days within each series. The animals were randomized by age, weight, and sex so as to distribute these equally among vaccinated and control groups. After vaccination, the identified animals were returned to the yard for the rest of the experiment and only brought to the laboratory for short periods (1 week) for parasitological studies. Mouse tests of vaccine batches. In order to compare the effectiveness of attenuated versus killed and of epimastigote versus trypomastigote-enriched vaccines, several preliminary tests were performed in mice. These comparisons were made by vaccinating

T. cruzi INFECTION

3

mice in a single injection site and challenging them with Tulahuen strain blood trypomastigotes (Tables II and III). Except for Experiment A, all vaccine batches used for guinea pigs were also tested in mice. These were vaccinated in the same manner as the guinea pigs regarding time, sites, and saponin dose. Only the number of killed parasites per injection sites was reduced on a dose per killogram basis to adapt to the lighter weight of mice. Twenty days after the last immunizing dose, vaccinated and saponin-injected control mice were challenged with a SC injection of wild-type triatome stool trypomastigotes. Parasitemia after challenge was measured in fresh blood mounts. RESULTS

Effectiveness of the freeze-thawed T. cruzi vaccine in mice. Experiments to compare the effectiveness of epimastigote versus trypomastigote-enriched vaccines were performed in mice (Table I). Neither TCC nor NIH 0.002 live trypomastigotes produced patent infections, thus allowing the measurement of parasitemia after challenge inocula with more infective strains. When live parasites were used as immunogen, a better immunizing effect was seen when trypomastigotes were included in the vaccine. On the other hand, when killed parasites were used, inclusion of trypomastigotes in the vaccine made no remarkable difference, except for one guinea pig experiment (Table IV, series E). Comparison of effectiveness of live versus killed vaccines in mice allways showed a better inhibition of challenge infection in the first case (Table II). The vaccine batches used for guinea pigs rendered in mice the following results: all batches tested produced a partial resistance, so that mice developed after challenge lower parasitemias than controls. Nevertheless, most vaccinated mice presented some degree of parasitemia, as shown by fresh blood mounts (Table III). Effects of vaccination upon natural T. cruzi infection in guinea pigs. The incidence of natural T. cruzi infection in several experimental groups of guinea pigs is given in Table IV. After an average exposure pe-

4

MIGUEL

Compared

Effectiveness

of T. cruzi

ANGEL

Vaccines:

BASOMBRIO

TABLE I Epimastigote

vs Trypomastigote-Enriched

Peak parasitemia Number of vaccine injections

% trypomastigotes in trypomastigoteenriched vaccine

Challenge dose”

A. Live attenuated T. ctuzi Test 1 3 23, 60,22

2 3 4

3 3 3

5 B. Killed 6 I 8 9

95, 20,

100 100 loo0 loo0 loo0

12, 22 30, 37(N)

89(N), 30, 62(N) 2 9400, IWN) T. cruzi plus saponin 2 44, 44 2 44, 44 2 44,44 2 44,44

2000 400 200 40

Vaccines

(? 2 SE)’

A Trypomastigoteenriched vaccine

B Epimastigote vaccine

C ControY+

P A vs B

15 12 1.7 1.7 0.0

45 45 2.6 4.7 0.7

2 + + + +

3 3 1.2 1.4 0.0

83 2 28 64 e 17 78 + 10 4026

P A+B “S c

+ + 2 2 ‘-

6 6 0.7 4.2 0.4

63 63 15.2 16.5 11.7

2 + 2 + +

14 14 6.6 3.4 4.1