534 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 72, No. 5,1978

Immunization

* Departamento

and enhancement properties of the circulating Trypanosoma cruzi

antigens

FAUSTO G. ARAUJO*, EVALDO NASCIMENTO* AND MARIA J. F. MORATO** de Parasitoiogia, Instituto de Ciencias Biologicas, Universidade Federal de Minas 30000 Belo Horizonte, MG, Brasil ** CP ‘Rene Rachou’, FIOCRUZ, Belo Horizonte, Bras2

Circulating antigens or exoantigens of trypanosomal origin were first demonstrated in the serum of rats infected with Trypanosoma brucei by WEITZ (1960). Subsequently the presence of these types of antigens has been demonstrated in hosts infected with T. vivax by GRAY (1961), with T. Iewisi by D’ALESSANDRO (1972), and with T. cruzi by SIQUEIRA et al. (1966), DZBENSKI (1974) and ARAUJO (1976). The nature and the role of these antigens in the course of the infection are not yet clear. WEITZ (1960) reported that rats can be immunized by repeated injection of exoantigens of T. brucei, and that exoantigens of this trypanosome are able to enhance an infection when added to the trypanosome inoculum just before inoculation into rats (WEITZ, 1963). In this paper we present results of experimental investigations of the immunizing and enhancing activities of exoantigens of T. cruzi. Material and Methods Mice Albino females, 18-20 g, were from the mouse colony of the ICB/UFMG. Exoantigens Obtained from plasma of mice acutely infected with the strain Y of T . cruzi (see SILVA & NUSSENZWEIG, 1953) according to a iechnique previously described (ARAUTO. 19761. Brieflv. acutelv infected mice were ‘bled L&i&g heparin as &ticoag;lant. The red cells were sedimented at 600 g for 10 min and the plasma centrifuged at 6000 g for 30 min to sediment the trypanosomes. The final supernatant was filtered through millipore (pore size 0.45 mc) and kept at - 20°C. Collection of the blood was in an ice bath and all centrifugations were at 5°C. The presence of exoantigens in the plasma was demonstrated through agar-gel diffusion and counterimmuno-electrophoresis (ARAUJO, 1976). Results Time of appearance of exoantigens Seven mice were inoculated intraperitoneally with 5 X lo4 washed blood trypomastigotes of the Y strain of T. cruzi, bled through the retro-orbital sinus at two-day intervals and their blood and plasma tested for the presence of trypanosomes and exoantigens. T. cru.zi was first detected on day three

of

Gerais,

after inoculation but exoantigens were only demonstrated on day seven. By this time, the range of the parasitaemia in all mice was between 1 X 10” and 3 x lOa trypomastigotes per 0.1 ml of blood. The mice started dying on day eight and by day ten only one mouse was still alive. Its parasitaemia was 1 x lOa trypomastigotes/O. 1 ml blood and its plasma was still positive for exoantigens. As measured through the methods of MANCINI et al. (1965) the amount of exoantigens in the plasma of these mice correlated roughly with the level of parasitaemia. Enhancement of the infection by exoantigens Seven mice were injected intravenously with 0 a25 ml of plasma rich in exoantigens 30 min before being inoculated intraperitoneally with 1 x IO” washed blood T. cruzi. Controls received plasma of normal mice. Parasitaemia was determined on days three, five and seven after inoculation and the mortality recorded daily. There were no significant differences between the test and control mice regarding either the level of parasitaemia or the rate of mortality. A similar experiment performed by mixing T, cruzi with plasma containing exoantigens and injecting the mixture intraperitoneally into mice yielded the same results. In both experiments mice started dying on day eight after inoculation and by day ten all had died. Immunization of mice with exoantigens This experiment was to determine whether the repeated injection of plasma containing exoantigens would stimulate the immune system in order to protect mice against infection with T. cruzi. Seven mice were injected intraperitoneally with 0.25 ml of plasma containing exoantigens at weekly intervals for three weeks. Controls received plasma of normal mice. One week after the last injection the mice were challenged with 1 X lo3 washed blood trypanosomes intraperitoneally. Parasitaemia was determined on different days and mortality recorded daily. The results are shown in Table 1. Two immunized mice had very low parasitaemias as compared with the controls. All control mice had died by day 17 after challenge, but three immunized All mice mice survived longer than 90 days. injected with exoantigens formed antibodies against them as demonstrated by gel diffusion.

F. G. ARAUJOet al. Table I-Parasitaemia same parasite

in mice

immunized

4

Immunized

exoantigens

Parasitaemia*

Mouse

Control

with

7

11

13

15

2820-b

2538 1128

423 564

-

3 4

0 0

423 1692

423 564

282 -

282

2 7

0 i

1269 423 564

987 564 423

423 282

-

:: 3 4 5 6 7

1410

4089 1269 0 141 2397 1974 564

423 846 0 141 987 282 564

423 282

-0

: 0

of T. cruzi and challenged

at day

::

:

535 with

the

Death on day 17

28

32 13 12

-

17 12 :.: 14

14:

0

0 0

141 282

141 141

141

141 0

0 0

0

0

* 5mm3 blood in slide, coverslip 22 X 22mm, count 50, 40X fields X microscope blood. + not detected - dead

15 16 >90 >90 13 17 >90

index (141) = approxi-

mate number trypanosomes/5mm3

Discussion These experiments show that exoantigens of T. cruzi are present in the blood of mice seven days after the infection when the parasitaemia is at a peak for the strain employed. The amount of exoantigens correlated roughly with the level of parasitaemia. Contrary to the findings reported for T. brucei by WEITZ (1963), the exoantigens of T. cruzi, in the doses employed, did not enhance an infection by this same parasite, either when injected separately or when mixed with the inoculum of trypanosomes. A protective effect was demonstrated in mice immunized with repeated injections of plasma containing exoantigens. The protection afforded was not absolute since all immunized mice showed patent parasitaemia which was, however, very low in at least two mice. This and the lower total mortality noted in the immunized mice suggest that the immunization procedure was able to stimulate a defence mechanism which, to some extent, controlled the proliferation of the parasite. JOHNSON et al. (1963) showed that plasma of mice acutely infected with T. cruzi when injected with adjuvant was more effective in protecting mice than were dead trypanosomes. The protection is unlikely to be due to passive transfer of antibody since large amounts of antibody are required to produce an effect (KAGAN & NORMAN, 1961; KIERSZENBAUM & HOWARD, 1976), and the antibodies are at a low level and of a low affinity, IgM class (VATTUONE et al., 1974). The fact that mice injected with plasma formed antibodies against exoantigens suggests that the protection was due solely to the immunizing activity of the exoantigens. The functions of the exoantigens in the course of the infection and their origin remain obscure. Work is in progress to determine the usefulness of the exoantigens in evaluating the activity of the infection.

Acknowledgement This work was supported by grant SIP 081029 from Conselho National de Pesquisas (CNPq) of Brasil. References Araujo, F. G: (1976). Immunology of Chagas’ disease. I. Circulating antigens in mice experimentally infected with Trypanosoma cruzi. Revista do Instituto de Medicina Tropical de Sao Paula, 18; 433-439. D’Alessandro, P. (1972) Trypanosoma lewisi: Production of exoantigens during infection in the rat. Experimental Parasitology, 32, 149-164. DZBENSKI, T. H. (1974). Exoantigen of Trypanosoma cruzi in vivo. Tro~enmedizin- und Parasitologic, 25.485-491. Gray, A. R. (1961). Soluble antigens of Trypanosoma vivax and of other trypanosomes. Immunology, 4, 253-261. Johnson, I’., Neal, R. A. & Gall, D. (1963). Protective effect of killed trypanosome vaccines with incorporated adjuvants. Nature, 198, 83. Kagan, I. G. & Norman, L. (1961). Immunologic studies on Trypanosoma cruzi. III. Duration of acquired immunity in mice initially infected with a North American strain of T. cruzi. Journal of Infectious Diseases, 108, 213-217. Kierszenbaum, F. & Howard, J. G. (1976). Mechanisms of resistance against experimental infection with T. cruzi: The importance of antibodies and antibody forming capacity in the Biozzi high and low responder mice. Journal of Immunology, 116, 1208-1211. Mancini, G., Carbonara, A. 0. & Heremans, J. F. (1965). Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235-254.

536

PROPERTIES OF CIRCULATING ANTIGENS OF T. cruzi

Silva, L. H. I?. & Nussenzweig, V. (1953). Sobre tuna cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Fohu Clinica Biologica (SHo Paulo), 20, 191-207. Siqueira, A. F.j Ferriolli, F., & Carvalheiro, J. R. (1966). Urn antigen0 soluvel presente no soro de ratos infectados corn Trypunosomu cruzi. Rev&u do Instituto de Medicine tropical de Sio Puulo, 8, 148. Vattuone, N. H., Gonzalez-Cappa, S. M., Menes, S. & Schmunis, G. A. (1974). Cell mediated and humoral immune response of mice infected with

Trypunosomu cruzi. Tropenmedizin und Purasitologie, 2.5, 267-272. Weitz, B. (1960). A soluble protective antigen of Trypunosomu brucei. Nature, 185, 788-789. Weitz, B. (1963). The antigenicity of some African trypanosomes. In: Immunity to Protozoa. Garnham, I’. C. C. (Editor) Oxford: Blackwell Scientific Publications.

Accepted for publication

3rd February,

1978.

Book Review Donald W. Roberts & Mary Ann Strand. Pathogens of medically important arthropods. Bulletin of the World Health Organization, Vol. 55, Supplement No. 1, 418 pp. Geneva: WHO. 1977 [ISBN 92 4 068551 01. This useful compendium is built upon the labours of 25 individual contributors, admirably collated by the two editors, with the active support of the Division of Vector Biology and Control (VBC) of the World Health Organization, Geneva. Editors, contributors and VBC alike are to be congratulated on bringing together between one set of covers, and in an admirably summarized form, the large but diffuse literature (circa 1,100 citations) on the pathogens of medically important arthropods. The new publication very effectively supplements the earlier “Pathogens, narasites and predators of medically important arthropods”, published as a suonlement to Vol. 30 of Bull. Wld Hlth Ow. (Jenkins, D. W., 1964), although the new supplement does not cover the field of predators. The pathogens dealt with are currently the subject of renewed interest as potential alternatives, or supplements, to chemical methods of pest and vector control. The appearance of this valuable publication is therefore timely and, with the editors and contributors, one hopes that it will stimulate further active research in a rather neglected area; it certainly provides a very useful entry to the field of biological control by means of pathogenic A companion publication covering organisms. predators and other biological agents would be a welcome addition in the future. The Editors express the intention to keep the material continually up to date for future editions or addenda; to this end they solicit information and comment from users. The publication is in two parts: the first and largest (334 pp.) consists of 21 tabulated hostpathogen lists for the various arthropod groups concerned. Each such list is followed by an annotat-

II

ed bibliography (up to 1974-75, generally). The bibliographies are particularly well served by short but very adequate and succinct abstracts (prepared in the main by Mary Ann Strand, who clearly has a gift for the art). The abstracts indicate nlainlv the ielevance of each citation and give outline facts and figures where appropriate. An interpolation among the host-pathogen lists is a section on “Synonomy and host-records of Microsporida affecting Culicidae” (by E. I. Hazard) which the Editors rightly consider to be a necessary adjunct to the section on Microsporidan pathogens of mosquitoes which precedes it. In succession the host-pathogen list sections cover the following areas: pathogens of phlebotomine sandflies (Psvchodidae)-viruses, bacteria, Microsporida, protozoa other’ than Microsporida; Coelomomyces, other fungal pathogens, and pathogenic nematodes affecting Culicidae; pathogens of Ceratopogonidae, Simuliidae, successivelv Tabanidae; Muscu domes&u and M. autumnalis, Stomoxvs culcitruns. Glossinu. Sinhonavtera, Blattidae, Anoplura and* Mallophaga,- Cimicidae; Reduviidae (Triatominae), Acarina (one section for mites and another for ticks). A second and shorter part (77 pp.) consists of nine pathogen-host lists, each with appropriate cross-reference to the earlier bibliographical sections. The pathogen-host lists are grouped under the following headings : viruses, rickettsiae, bacteria, protozoa other than Microsporida, Microsporida, Coelomomyces, fungi other than Coelomomyces, nematodes and “Others”. The brief final “Other” category includes mainly helminths, unidentified organisms and miscellaneous inclusions observed in the arthropods concerned. The volume is cross-references throughout simply and is hence easy to use; it constitutes a valuable source of reference to all interested in the subject of control of arthropod vectors and pest species. DONALD M.MINTER