CJap. J. Parasit., Vol. 31, No. 6, 561-568, Dec, 1982]

Protective Effects of Immunization with Tetrahymena pyriformis on Murine Toxoplasmosis

Asao MAKIOKA*, Akio KOBAYASHI* and Kojiro SHICHIJOt

(Received for publication; September 10, 1982)

Key words: Tetrahymena pyriformis, nonspecific immunization, protective effects, toxoplas mosis

organisms (Ruskin and Remington, 1968 a, Introduction b). However, there has, so far, been little A significant body of evidence has been attempt to elucidate the efficacy of free-living accumulated which documents that a num organisms as an immunological adjuvant. ber of bacteria, among them Mycobacterium Tetrahymena pyriformis, a free-living cili- bo vis BCG and Propionibacterium acnes ate protozoan, is widely and extensively (Corynebacterium parvum), confer upon studied in the fields of cell biology and bio injection an increased nonspecific resistance chemistry. We chose this protozoan from in the host against a variety of viral (Larson among many free-living organisms because it et al, 1971), bacterial (Senterfitt and was known that the organism proliferatesas Shands, 1970; Adlam et al, 1972) and pro- fast as bacteria and can be easily cultured tozoal infections (Nussenzweig, 1967; Ortiz- in axenic condition, and numerous informa Ortiz et al, 1975; Tabbara et al, 1975; tions with regard to morphological and bio Clark et al, 1976; Clark et al, 1977; chemical characteristics of it have been ac Smrkovski and Larson, 1977; Smrkovski and cumulated. Strickland, 1978). They are most widely We considered it of interest to determine recognized as immunostimulators and non whether immunization of mice with this specific resistance by their administration is organism could induce nonspecific resistance associated with cell-mediated immunity. On against Toxoplasma challenge infection. the other hand, it has also been shown that infections with intracellular protozoa such Materials and Methods as Toxoplasma gondii and Besnoitia jellisoni provide resistance in mice against challenge Mice: Six-to 8-week-old female outbred with phylogenetically unrelated intracellular ddY mice were used in most experiments. BALB/c derived nu/nu and nu/+ mice, 6-

This work was supported by a Grant-in-Aid for to 8-week-old female, were also used in some Scientific Research (No. 56570165) from the experiments. All micewere purchased from Ministry of Education, Science and Culture of the Shizuoka Agricultural Cooperative As Japan. sociation for Laboratory Animals, Hama- * Department of Parasitology, Jikei University matsu, Japan. School of Medicine, Minato-ku, Tokyo 105, BCG vaccine: Lyophilized BCG vaccine Japan of Japanese substrain was obtained from Dr. t First Department of Internal Medicine, School of Medicine, Gunma University, Maebashi, Japan T. Tokunaga, the National Institute of

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Health of Japan, which was a product of the zoites which were equivalent to those within Japan BCG Laboratory. Tokyo. It was sus one cyst of mean size. This number of pended in sterile saline and adjusted to an bradyzoites usually caused death of 80% or appropriate concentration before use. Heat- more of normal mice when inoculated i.p.. killed BCG was prepared by heating viable Besides i.p. challenge, intravenous (i.v.), per organisms for 1 hr at 70 C. oral (p.o.) and subcutaneous (s.c.) chal Tetrahymena: The W-strain of Tetra lenges were also performed in some experi hymena pyriformis was obtained from Dr. ments. The mice were observed until 1 Y. Watanabe, Institute of Biological Sciences, month after the challenge and their mortality Tsukuba University, and cultivated in 3 1 and average survival time were assessed. The Fernbach flasks each containing 300 ml of dead mice in the experiment were each PYD medium (1% proteose peptone, 0.5% checked for the presence of toxoplasmas. yeast extract, and 0.8% dextrose) under Assay of Toxoplasma antibodies: An in sterile conditions (Watanabe and Ikeda, direct latex agglutination (LA) test (Toxo- 1965). The culture flasks were kept shaken test-MT, Eiken Co.) was used to determine horizontally. The organisms were harvested the serum level of Toxoplasma antibodies by centrifugation at 850 X g for 5 min and (Kobayashi et al, 1978). washed once with inorganic medium (NaCl

100 mg, KC1 4mg, CaCl2 6 mg in 1 1 of Results distilled water) (Watanabe and Ikeda, 1965). For lysis, the organisms in inorganic Effect of immunization with various doses medium were frozen-thawed twice. This en of Tetrahymena against Toxoplasma chal tire preparation was referred to lysed Tetra lenge hymena antigen. To establish the minimum effective dose of Tetrahymena, mice were immunized i.p. Immunization: Mice were injected intra- peritoneally (i.p.) with live Tetrahymena or its lysed antigen. Live or heat-killed BCG Table 1 Effect of immunization with various were injected similarly into mice as positive doses of Tetrahymena against Toxoplasma control. Dose and immunization schedules challenge* of Tetrahymena and BCG are described in No. mice dead/ Average particular experiments. Immunization! Dose no. inoculated days Challenge with Toxoplasma; Immunized (%) survival %

and control mice were challenged with 0 6/10( 90) 12.2 bradyzoites of the weak virulent Beverley Th§(live) 1X105 6/10( 60) 11.7 strain of T. gondii. The inoculum of Toxo 1X106 l/10( 10) 14 plasma was prepared as follows. Brains of 1X107 1/10( 10) 13

the chronically infected mice were removed 0 10/10(100) 11.7 and triturated in 5 ml of phosphate-buffered Th (lysed) 1X105 10/10(100) 17.3 saline at pH 7. 2 (PBS) with mortar and 1X106 5/10( 50) 15.7 pestle. The number of cysts in a unit volume 1X107 5/10( 50) 19.0 of the brain suspension was estimated. The * Mice were challenged i.p. with 5,000 brady supension was treated with 0.25% trypsin zoites of the Beverley strain of Toxoplasma for 10 min and released bradyzoites wrere and investigated up to 30 days after the challenge. washed twice in PBS, centrifuged at 700 Xg t Immunization was made by i.p. route 7 days for 10 min and resuspended in PBS. The before Toxoplasma challenge infection. inoculum for the challenge infection was ad X Average surviving days of mice that died. justed to contain approximately 5.000 brady § Th, Tetrahymena.

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564 mice once or twice. For two immunizations, Table 4 Comparison of protective effects of the interval was 14 days. Mice non-treated Tetrahymena and BGG against Toxoplasma or inoculated with inert substances such as challenge glycogen and sheep erythrocytes (SRBC) No. mice dead/ Average were served as controls. Toxoplasma chal Immunization* no. inoculated days (%) survival lenge was performed 7 or 4 days after a None 52/54(96.3) 11.1 single or two injections with Tetrahymena Glycogen 10/10(100) 11.1 or BCG. Mice that received 0.5 ml of 0.1% SRBG 10/10(100) 14.1 glycogen or lxlO9 SRBC were challenged Th(live), once 4/40(10.0) 18.3 1 day and 7 days after inoculation, respec Th(lysed), once 14/30(46.7) 12.8 tively. The results are shown in Table 4. BCG (live), once 6/30(20.0) 16.3 All groups of immunized mice were signifi BCG (heat-killed), once 6/30(20.0) 12.8 cantly protected from death, i.e., smaller Th(live), twice! 3/15(20.0) 14.7 mortal rate as compared with control mice. Th(lysed), twice 18/28(64.3) 13.4 BCG (live), twice The degree of protection afforded by live 6/28(21.4) 14.3 BCG (heat-killed), twice 1/15 ( 6.7) 19 Tetrahymena was comparable to that in duced by live or heat-killed BGG, but lysed * 1X107 organisms for both Th and BCG, 0.5 Tetrahymena antigen conferred lesser degree ml of 0.1% glycogen or 1X109 sheep eryth rocytes (SRBC) were each injected i.p. into of resistance than did both live and heat- mice. Mice were challenged i.p. with Toxo killed BCG vaccines. There was no signifi plasma 7 days after a single injection or 4 cant difference in mortality between the days after second injection with Th or BCG. groups of mice immunized with a single in Mice that received glycogen or SRBC were jection and with two injections of Tetra challenged 1 day and 7 days after inoculation, hymena or BCG. respectively. For Toxoplasma challenge see Effect of immunization with Tetrahymena footnote of Table 1. against Toxoplasma challenge by various t Interval between two immunizations was 14 days. routes To evaluate the effect of immunization with Tetrahymena against Toxoplasma chal i.v. challenge, whereas considerable degree lenge by various routes, mice were injected of reduction in mortality was observed in i.p. with 1 X 107 Tetrahymena and challenged Tetrahymena-irnrnvLXiized mice. The result i.p., i.v., p.o. or s.c. with Toxoplasma. For of i.p. challenge with Toxoplasma was simi the comparison, BCG was also used as an lar to that obtained in the previous experi immunostimulant. Results are shown in ment; the degree of protection afforded by Table 5. All control mice died from i.p. or live Tetrahymena was greater than that by

Table 5 Effect of immunization with Tetrahymena against Toxoplasma challenge by various routes

No. mice dead/no, inoculated (%) and average days survival Immunization* after Toxoplasma challenge by routet of (i.p.) i.p. p.o.

None 10/10(100)11.3 10/10(100)12.9 6/10( 60)14.8 6/10 ( 60)16.0 Th(live) l/10( 10)17 6/10( 60)15.2 3/10( 30)19.7 6/10( 60)18.0 Th (lysed) 6/10( 60)11.8 3/10( 30)16.3 4/10( 40)13.8 3/10( 30)19.7 BCG (live) 2/10( 20)19.3 6/10( 60)14.5 l/10( 10)14 3/10( 30)19.7

* For immunization and Toxoplasma challenge see footnotes of Table 1 and 4. t Challenge route: i.v. = intravenous, p.o. = per oral, s.c. = subcutaneous.

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Table 6 Effect of immunization with Tetra- tection against a subsequent Toxoplasma hymena against Toxoplasma challenge challenge infection. It is clear from the re in nude mice sult that an immunization of mice with No. mice dead/ Average Tetrahymena prior to Toxoplasma challenge Mice no. inoculated days zation* (%) survival is requisite for development of protective effect against the infection. Several condi None 10/10(100) 12.6 Athymic Th(live) 10/10(100) 16.5 tions for elicitation of the protective effect by nu/nu Th(lysed) 10/10(100) 14.6 Tetrahymena have been clarified. The mini BGG(live) 10/10(100) 17.0 mum effective dose was 1X106 organisms Littermate None 10/10(100) 10.3 and equivalent for both live and lysed Tetra nu/ + Th(live) 7/10( 70) 15.8 hymena antigens and the protective effect

* For immunization and Toxoplasma chal- persisted for 2—3 weeks with decrease after lenge see footnotes of Table 1 and 4. ward. Tetrahymena antigens behaved differently from inert subsances. Not only glycogen, lysed antigen and was comparable to that which is often used to induce peritoneal by BCG. Somewhat greater degree of resist macrophages, but also high dose of SRBC ance appeared in mice that received lysed could not confer any protection against Toxo Tetrahymena than in those immunized with plasma, whereas Tetrahymena could elicit live Tetrahymena or BCG when challenged significant protection. This suggests that i.v.. In contrast to challenge by i.p. or i.v., Tetrahymena is quite different from the mere p.o. or s.c. challenge produced lower mor inert substances like glycogen and SRBC as tality in normal mice, where small reduction immunostimulant. in mortality was observed in most of the immunized mouse groups than in the controls. It was also demonstrated that there were Effect of immunization with Tetrahymena differences in degree of resistance conferred against Toxoplasma challenge in nude mice by live Tetrahymena and lysed one depend To determine whether protective effect of ing on the challenge route of Toxoplasma. Tetrahymena against Toxoplasma challenge Live Tetrahymena produced greater degree is elicited in nude mice, nu/nu and nu/ + of protection than did lysed antigen against mice were each inoculated i.p. with lxlO7 i.p. challenges, while somewhat lesser resist live or lysed Tetrahymena, or live BCG and ance in mice when challenged i.v. or s.c. they were challenged i.p. with Toxoplasma There was no marked difference in the 7 days later. The results are shown in Table degree of protection by those antigens against 6. In nu/nu mice, all were killed by Toxo p.o. challenge. Live Tetrahymena inoculated plasma infection, although there could be into mice may soon be killed and eventually observed a tendency of prolonged survival in digested since they are not parasitic. It re the immunized groups than in the control. mains to be elucidated how the live and lysed In contrast, nu/ + mice revealed certain re antigens behave differently as immunostimu sistance to the challenge infection when im lant and also why the protective effect by munized with live Tetrahymena: 30% of Tetrahymena varies depending on route of them survived. the challenge. The facts that i.p. inoculation of Tetrahymena could also elicit certain pro tection against Toxoplasma challenge by Discussion various routes may suggest that the protective The results indicate that when Tetra effect occurred not only locally but also some hymena, a free-living ciliate, is inoculated what systemically. i.p. into mice, it does produce a potent pro In this study, BCG was also used as refer-

( 87 ) 566 ence immunostimulant to evaluate protective specific vaccines as well. Comparison of effect by Tetrahymena. Although some vari these specific vaccines with Tetrahymena in ations were observed in the effect of those respect to preventive effect is a matter of immunogens depending on Toxoplasma chal different scope. lenge routes, there were essentially no marked Production of anti-Toxoplasma antibodies differences in potency between Tetrahymena was negligible, if any, in both Tetrahymena- and BCG so far as judved by mortal rate of immunized and non-immunized mouse the mice. groups at least until days of their deaths so It is clear from the results that two injec far as tested by LA test. Therefore it may tions with Tetrahymena as well as BCG did be difficult to attribute the difference in mor not give increasing resistance as compared tality between the groups to different anti with a single immunization. Similar results body productions. It was also found in our have been reported by Smrkovski and Strick separate experiment that peritoneal macro land (1978), who demonstrated that multi phages from mice immunized with Tetra ple doses of BCG did not induce a greater hymena were markedly activated to kill degree of protection in mice against malaria Toxoplasma in vitro as compared with those than did a single inoculation. from control mice. Detailed informations on Several reports have been presented with the in vitro study will be presented in else regard to nonspecific resistance to Toxoplas where. Thus, cell-mediated immunity is con ma. Tabbara et al. (1975) have found sig sidered to play most important role for the nificant protection in rabbits against toxo- resistance against Toxoplasma, in which the plasmic retinochoroiditis when the animals activated macrophages should be regarded were immunized with BCG i.v. but not by as the essential cells as suggested earlier by inoculation into retrobulbar space. On the Remington and Krahenbuhl (1976). other hand, Dubey (1978) reported that the While the effect of immunization with pretreatment of hamsters i.v. or i.p. with Tetrahymena or BCG was equivocal in nu/ BCG at the single dose of 2xlO7 did not nu mice, nu/ + mice could elicit resistance afford any protection against oral challenge to Toxoplasma challenge in vivo test. A pos of Toxoplasma oocysts. Also, Swartzberg et sible participation of lymphocytes of mice in al. (1975) have demonstrated that the pre the immune system with Tetrahymena is treatment of mice with P. acnes activated under study. macrophages to kill Toxoplasma in vitro but Effectiveness of the immunizaiton with conferred only transient protection in vivo Tetrahymena for increasing resistance to against the C56 strain of Toxoplasma. These other parasitic infections and elucidation of reports cannot be compared directly with our the degree and extent of possible side effect study results because there exist many differ caused by Tetrahymena remain to be clari ences in experimental conditions, e.g., species fied. of experimental animal used, the virulence and form of Toxoplasma, and the route and Summary interval of immunization. It may be natural that the specific im Experiments were carried out to evaluate munization with live avirulent toxoplasmas the effect of immunization of mice with would give more intensive protection from Tetrahymena pyriformis, a free-living ciliate death caused by challenge infection with the protozoan, on protection against Toxoplasma virulent strain of Toxoplasma than vaccina gondii. Significant protections was elicited tions with the dead toxoplasmas (Krahen- when mice were injected with 1X106 or buhl et al., 1972) and possibly than the non more of Tetrahymena. Immunization with

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Tetrahymena was effective only when per gondii infection on macrophage-mediated formed within 2-3 weeks before the chal cytostasis of tumor cells. Immunology, 31, lenge. Intraperitoneal immunization with 837-846. 7) Krahenbuhl, J. L., Ruskin, J. and Remington, 1 X 107 live tetrahymenas or the lysed antigen J. S .(1972): The use of killed vaccines in conferred a considerable protection in mice immunization against an intracellular para against intraperitoneal or intravenous chal site: Toxoplasma gondii. J. Immunol., 108, lenge with Toxoplasma, while little protec 425-431. tion against oral or subcutaneous challenge. 8) Larson, C. L., Ushijima, R. N., Florey, M. J., The degree of protection by Tetrahymena Baker, R. E. and Baker, M. B. (1971) : Ef was comparable to that by BCG so far as fect of BCG on Friend disease virus in mice. Nature (London) New Biol., 229, 243-244. the present experimental system is concerned. 9) Nussenzweig, R. S. (1967): Increased non Although no decrease in mortality was ob specific resistance to malaria produced by ad served in nude mice immunized with Tetra ministration of Corynebacterium parvum. hymena, longer survival time was observed Exp. Parasit., 21, 224-231. as compared with control mice. 10) Ortiz-Ortiz, L., Gonzalez-Mendoza, A. and Lamoyi, E. (1975): A vaccination proce dure against Trypanosoma cruzi in mice by Acknowledgement nonspecific immunization. J. Immunol., 114, We wish to thank Dr. T. Tokunaga, National 1424-1425. Institute of Health of Japan for his valuable advice 11) Remington, J. S. and Krahenbuhl, J. L. and discussion. (1976): Immunology of Toxoplasma infec- iton. In Immunology of parasitic infections, ed. by S. Cohen and C. Sadun, Blackwell References Scientific Publications, Oxford, 235-267.

1) Adlam, C, Broughton, E. S. and Scott, M. T. 12) Ruskin, J. and Remington, J. S. (1968a): (1972): Enhanced resistance of mice to in Immunity and intracellular infection: resist fection with bacteria following pretreatment ance to bacteria in mice infected with a proto with Corynebacterium parvum. Nature (Lon zoan. Science, 160, 72-74. don) New Biol., 235, 219-220. 13) Ruskin, J. and Remington, J. S. (1968b): 2) Clark, I. A., Allison, A. C. and Cox, F. E. Role for the macrophage in aquired immunity (1976): Protection of mice against Babesia to phylogenetically unrelated intracellular or and Plasmodium with BCG. Nature (Lon ganisms. Antimicrob. Agents Chemother., 8, don), 259, 309-311. 474-477. 3) Clark, I. A., Cox, F. E. G. and Allison, A. C. 14) Senterfitt, V. C. and Shands, J. W. (1970): (1977): Protection of mice against Babesia Salmonellosis in mice infected with Mycobac- spp. and Plasmodium spp. with killed Coryne terium bovis BCG. II. Resistance to infection. bacterium parvum. Parasitology, 74, 9-18. Infect. Immun., 1, 583-586. 4) Dubey, J. P. (1978) : A comparison of cross 15) Smrkovski, L. L. and Larson, C. L. (1977): protection between BCG, Hammondia ham- Effect of treatment with BCG on the course mondi, Besnoitia jellisoni and Toxoplasma of visceral leishmaniasis in BALB/c mice. gondii in hamsters. J. ProtozooL, 25, 382- Infect. Immun., 16, 249-257. 384. 16) Smrkovski, L. L. and Strickland, G. T. 5) Kobayashi, A., Watanabe, N., Suzuki, Y. and (1978) : Rodent malaria: BCG-induced pro Hirai, N. (1978): A simple mass-screening tection and immunosuppression. J. Immunol.. method for toxoplasmosis—Detection of the 121, 1257-1261. antibodies from blood-absorbed-filter-paper 17) Swartzberg, J. E., Krahenbuhl, J. L. and discs using the indirect latex agglutination Remington, J. S. (1975): Dichotomy be test. Jap. J. Parasit., 27, 483-487. tween macrophage activation and degree of 6) Krahenbuhl, J. L., Lambert, L. H. and Re protection against Listeria monocytogenes and mington, J. S. (1976): Effects of Coryne Toxoplasma gondii in mice stimulated with bacterium parvum treatment and Toxoplasma Corynebacterium parvum. Infect. Immun.,

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12, 1037-1043. Ophthalmol., 79, 641-647. 18) Tabbara, K. F., O'Connor, G. R. and Nozik, 19) Watanabe, Y. and Ikeda, M. (1965): Iso- R. A. (1975): Effect of immunization with lation and characterization of the division attenuated Mycobacterium bovis on experi- protein in Tetrahymena pyriformis. Exp. Cell mental toxoplasmic retinochoroiditis. Am. J. Res., 39, 443-452.

Tetrahymena pyriformis

Tetrahymena pyriformis (Th) tzifcit, Tp \ tf *^t, Th , bcg c. Th x 106 fflK±o Th Sri

Tp

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