Trypanosoma brucei: Infectivity and immunogenicity of cultured parasites

EXPERIMENTAL

PARASITOLOGY

60, 150-154 (1985)

Trypanosoma

brucei: Infectivity and lmmunogenicity of Cultured Parasites

MRAMBA

NYINDO

AND

BRUCET.

WELLDE*

The lnrernational Center qf Insect Physiology and Ecology (ICIPE), P.O. Box 30772, Nairobi, and *The Walter Reed Project, Veterinary Reseurch Laborutorq, Kubete, Kenycl

(Accepted for publication 8 January 1985) NYINDO, M., AND WELLDE, B. T. 1985. Trypunosomu brucei: Infectivity and immunogenicity of cultured parasites. Experimental Parasitology 60, 150- 154. Ttypanosoma brucri brucei, derived from the salivary glands of infected tsetse flies (Glossina morsitans morsirans) and maintained in culture for over 4 years, were infective to both albino rats and tsetse flies. Virulence was markedly enhanced during the first passage in albino rats or tsetse flies. Irradiated cultured trypanosomes induced immunity to homologous challenge but not to tsetse fly or blood-induced challenge with the same stock. D 1985 Academic Pre,,. Inc. INDEX DESCRIPTORS: Trypanosoma brucei brucei; Hemoflagellate; Protozoa, parasitic; Culture; Infectivity; Immunogenicity.

containing 2.5% glucose and 10% fetal calf serum for blood forms. The in vilvo cultivation of metacyclic TryRandomly bred male albino rats (Chester-Beatty panosoma brucei brucei from tsetse sali- strain) raised at the Veterinary Research Laboratories, vary glands at 25 to 30 C has been reported weighing between 75 and 125 g at the beginning of (Nyindo et al. 1979). Further studies on the experiments, were used in these studies. Animals were monitored for parasitemia by wet blood smears obcultured parasites revealed that they were tained from the tip of the tail. antigenically stable at temperatures of culTrypanosomes in culture medium were placed in an tivation but that changes in surface coat ice-water bath and irradiated in a Lisa IA cesium composition occurred when parasites were source (La Garenne, Colombes, France). Irradiated incubated at 37 C (Nyindo and Rurangirwa trypanosomes were administered to rats by the intra1981). We now report on studies in rats peritoneal route. Controls included rats injected with irradiated, noninfected culture medium and noninusing parasites which had been cultured for jetted normal rats. Challenges with nonirradiated culover 4 years. The infectivity of the parasites ture or blood form trypanosomes were given introperwas tested for both laboratory rats and itoneally in l-ml volumes containing 1 X IO4to I x tsetse flies and attempts were made to im- IO5 trypanosomes. One hundred and fifty newly munize rats using irradiated cultured try- emerged laboratory reared Glossina morsirans morsitans were fed once on five rats during the initial panosomes. rising parasitemia after inoculation with cultured trypanosomes. Infected flies were determined by alMATERIALS AND METHODS lowing the flies to probe warmed (30 C) microscope Parasite stock and methods of culture have been slides which were then examined for the presence of described in detail (Nyindo et al. 1979). Briefly, Try- metacyclic trypanosomes. Experimental rats were panosoma brucei brucei stock EATRO 1969 from challenged by the bite of one infected tsetse fly. Serum was collected from two immune rats on Day tsetse fly salivary glands was maintained in culture in bovine embryonic spleen cells in supplemented RPM1 30 after challenge, from two rats injected with unin1640medium at 25 to 30 C. Parasites were subcultured fected culture medium, and from two normal rats. Trythree times a week and, at the beginning of this study, panosomes from culture and those derived from inthey were on Day 1600 of cultivation. Trypanosomes fected rat blood were tested by a neutralization test were counted in a Neubauer chamber, and their num- (Soltys 1957). Three different levels of trypanosomes bers were adjusted by dilution in culture medium (cul- (6 X 106, 6 x IO’, 6 x IO’) were incubated in antiture forms) or in phosphate buffered saline (pH 7.8) serum, control, and normal serum for 1 hr at room

0014-4894/85$3.00

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

150

Trypanosoma

brucei: INFECTIVITY,IMMUNOGENICITY AFTERCULTURE

temperature, and each mixture was subsequently injected intraperitoneally into two normal rats.

I51

When immunized rats were rechallenged 80 days after immunization, they were refractive to infection although all four control RESULTS rats became patent and succumbed to the In order to compare the infectivity of cul- infection. Three immunized rats challenged tured Ttypanosoma brucei brucei with that 119 days after immunization showed a parof blood form parasites, graded doses of tial immunity. Another experiment, using two immunizations with a total of 5.0 x lo6 trypanosomes were injected intraperitoneally into rats. Table I summarizes the re- irradiated culture forms, confirmed the imsults of Experiment I. All rats with the ex- munogenic effect against homologous chalception of one (Culture form, 1 x 10’) be- lenge (Table III), and immunity was detectcame infected; however, prepatent periods able but appeared to be waning at 124 days and survival times were markedly pro- after immunization. Three rats immunized longed in rats inoculated with cultured try- with culture forms and challenged with panosomes, especially in the lower dos- blood form trypanosomes of the same ages. All rats whose blood became patent stock, however, showed no resistance to insubsequently died. Five of 150 tsetse flies fection, with prepatent periods and survival allowed to feed on infected rats subse- times being shorter than those of control quently developed salivary gland infec- rats. When infected tsetse flies were allowed to feed on immunized rats which tions. In Experiment 2, six groups of rats, with were immune to culture form challenge, no three animals per group, received irradiated resistance to the resulting infection was apcultured trypanosomes at levels of 0, 5, 10, parent (Table III). All four immunized rats 20, 40, and 80 kr, respectively. Three con- developed parasitemia and died, while three trol rats (0 kr) and two of three rats in- of five control rats succumbed to the infecjected with trypanosomes irradiated at the tion. Decreased prepatent periods and sur5-kr level became infected. No other rats vival times were observed in immunized receiving trypanosomes irradiated at higher rats challenged by tsetse bite when comlevels became patent. Irradiation within the pared to controls. Cultured trypanosomes, which were levels tested appeared to have no effect on mixed with serum collected from immune motility (Table II). Immunization experiments were carried rats and incubated at room temperature for out to determine if irradiated culture forms 1 hr, were not infective to normal rats at any could induce a protective immunity to var- of the three dose levels. The immune serum ious forms of challenge. Thirteen rats had no effect on blood form trypanosomes which had survived Experiment 2 were in- from an infected rat. Normal and cultureoculated with a total of 5.4 x lo6 cultured control rat serum had no effect on the intrypanosomes in two doses irradiated at 20 fectivity of cultured or blood form trypankr and challenged with nonirradiated cul- osomes, and all rats inoculated with these tured parasites. Seven of the ten immu- mixtures succumbed to the infection. nized animals challenged remained apatent DISCUSSION throughout the experiment. Extended prepatent periods and survival times were The possibility of protecting animals noted in the other three immunized rats. against infection with African trypanoRats injected with noninfected irradiated somes by immunization has been frustrated culture medium did not differ from normal by the phenomenon of antigenic variation rats in response to challenge, and these two inherent in trypanosomes (Gray 1967). Recontrol groups were combined in Table III. sults of several experiments have been pub-

152

NYINDO

AND

WELLDE

lished which show that African trypanosomes derived from the blood of infected animals and subjected to varying doses of gamma or ultraviolet radiation induced protective immunity (Wellde et al. 1973, 197.5; Charoenvit and Campbell 1981; Morrison et al. 1982). However, some of these experiments also showed that the immunity was variant specific and was only possible to homologous challenge. Self-cure or drug cure of infected animals has been shown to produce persisting immunity to both blood and tsetse fly challenge in cattle. Again, immunity was expressed only to the infecting stock of trypanosomes (Wellde et al. 1981). Metacyclic Trypanosoma brucei brucei which was grown in vitro was shown to exhibit antigenic stability at 29 C (Nyindo et al. 1981). Results of our immunization and challenge experiments (Table III) tend to support this finding, at least for the period during which this study was done. No similar antigenic stability was noted with blood stream form parasites grown in vitro (Doyle et al. 1980). In our immunization experiments employing culture form challenge, most of the immunized animals did not develop parasitemia while the others had extended prepatent periods and survival times. The immunity was persistent, and immunized animals were resistant to challenge for at least 4 months after immunization, although at this time the immunity appeared to be waning. The immunoprotective effect was at least in part serum mediated as shown by a serum neutralization experiment. Animals immunized with the irradiated cultured parasites, however, appeared to be somewhat more susceptible to fly challenge and also to blood form challenge of the same stock. The two latter experiments indicate that parasites of fly and blood stream origin had modulated their surface coat antigens and differed antigenically from the cultured parasites used for immunization. The unknown number of trypanosomes injected during tsetse challenge could possibly have overwhelmed a

Trypanosoma

brucei: INFECTIVITY,

IMMUNOGENICITY

AFTER CULTURE

TABLE II Effect of Gamma Radiation on the Infectivity of Cultured Ttypanosomu Kr

Motility

0

+

5 10 20 40 80

+ + + + +

153

brucei brucei”

No. infected/ no. injected

PPP (Range)b

MST (range)’

313 213 013 013 o/3 o/3

7 23 (12->80) >80 >80 >80 >80 -

36 (21-36) 49 (43->80) >80 >80 >80 >80 -

a 1.4 x lo6 trypanosomes in 1 ml. b Median prepatent period in days, - indicates no range c Median survival time in days.

partial immunity in the experimental rats since tsetse challenge was delayed. The same rats, however, had resisted culture form challenge 40 days previously. Another possibility is that in our culture system some degree of antigenic variation has occurred during the 4-year period of culture. As opposed to our findings, Jenni and Brun (1981) were able to induce protective immunity against infective tsetse transmission

in mice inoculated with gamma-irradiated parasites obtained from short term cultures. A marked increase in virulence of Trypanosoma brucei brucei occurred during the first passage of cultured parasites in rats, and fly infective trypanosomes were also present in the first rat passage of trypanosomes which had been in continuous culture for over 4 years.

TABLE III Trypanosoma

Group Immunized”

brucei brucei: Immunization and Challenge Exposure of Rats

Challenge’

Dayd

No. infected/ challenged

PrepatenV period

Survival’ time

>80 (12->80) 3 8 (6-11) 4 -

>80 (23%>80) 13 23 (20-36) 16 (14-22)

-

28 (28-30)

CF BF CF BF

8

3110 313 919 313

Immunized” Control

CF CF

80

014 414

Immunized0 Control

CF CF

119

l/3 313

Immunized” Control Immunizedb Control

TF TF CF CF

119

414 315 Oil0 lo/lo

3 (3-6) 6 (4->90) 8 (4-19)

10 (9- 16) 15 (12->90) 32 (22-36)

Immunizedb Control

CF CF

124

215 515

>40 (8->40) 8 (5- 17)

>40 (38~>40) 28 (23-35)

Control

9

9

>40 (34->40) 10 (8- 12)

>40 29 (25-32)

a Immunized on Days 0, 123, and 133 with 1 x 106, 1.5 x 106,and 3.9 x lo6 irradiated trypanosomes. b Immunized on Day 0 and 6 with 4 x lo6 and 1 x lo6 trypanosomes irradiated at 20 Kr, respectively. c CF, culture forms; BF, blood forms; TF, Tsetse flies. d Days after immunization. e Median (range) in days.

154

NYINDOANDWELLDE ACKNOWLEDGMENTS

We thank David Chumo and James Ebuga for their technical assistance, and Joyce Mburu for typing the manuscript. We also thank the Director, International Laboratory for Research on Animal Diseases, Nairobi, for allowing us to use the irradiation facility. These data are published with the approval of Dr. W. Njoroge, Director of Veterinary Services, Government of Kenya. This research was supported in part by Grant DAMD 17-83-G-9517, U.S. Army Medical Research and Development Command, Frederick, Maryland, USA. REFERENCES

C. A., ANDWELLS, P. W. 1982. Protective immunity and specificity of antibody responses elicited in cattle by irradiated Trypanosoma hrucei. Purasite Immunology

4, 395-407.

NYINDO, M., PATEL, N., DARJI, N., AND GOLDER, T. K. 1979. Trypanosoma brucei in vitro propagation of metacyclic forms derived from the salivary glands of Glossina morsitans. Journal of Parasitology 65, 751-755.

NYINDO, M., AND RURANGIRWA,F. R. 1981. Trypunosoma brucei: Continuous cultivation of antigenitally stable parasites at 29°C and induction of antigenie variants at 37°C. Insect Science and Application 2, 263-266.

SOLTYS,M. A. 1957. Immunity in Trypanosomiasis. I. Neutralization reaction. Parasitology 47, 375-389. WELLDE, B. T., DUXBURY, R. E., SADUN, E. H., LANGBEHN,H. R., LOTZSCH,R., DIENDLE, G., AND WARUI, G. 1973. Experimental infections with African Trypanosomes: IV. Immunization of cattle with gamma-irradiated Trypanosoma rhodesiense.

CHAROENVIT,Y., AND CAMPBELL,G. H. 1981. Immunization of mice with Trypano~oma rhodesiense exposed to ultraviolet irradiation. Americun Journa/ of Tropical Medicine and Hygiene 30, 1198- 1200. DOYLE,J. J., HIRUMI, H., HIRUMI, K., LLJ~TON,E. N., AND CROSS,G. A. M. 1980. Antigenic variation in clones of animal-infective Trypanosoma brucei deExperimental Parasifology 34, 62-68. rived and maintained in vitro. Parasitology 80, 359WELLDE, B. T., HOCKMEYER, W. T., KOVATCH, 369. R. M., BHOGAL,M. S., AND DIGGS,C. L. 1981. TryGRAY, A. R. 1967. Some principles of the immunology panosoma congolense: Natural and acquired reof trypanosomiasis. Bulletin of the World Health sistance in the bovine. Experimental Parasitology Organization 37, 177-193. 52, 219-232. JENNI, L., AND BRUN, R. 1981. In vitro cultivation of pleomorphic Trypanosomn hrucei stocks: A pos- WELLDE, B. T., SCHOENBECHLER, M. J., DICGS, sible source of variable antigens for immunization C. L., LANGBEHN,H. R., AND SADUN, E. H. 1975. studies. Transactions of the Royal Society of TropTrypanosoma rhodesiense: Variant specificity of imical Medicine and Hygiene 75, 150- 151. munity induced by irradiated parasites. ExperiMORRISON,W. I., BLACK, S. J., PARIS, J., HIDSON, mental Parasitology 37, 125-129