Parasitol Res (1988) 74:221-227 Parasitnlngy Research �9 Springer-Verlag 1988

Light and electron microscope observation of virus-induced Tetrahymena pyriformis in newborn mice (Mus musculus Mbinicus) brain*

J. Teras 1, R. Entzeroth 2, E. Scholtyseck 2' **, L. Kesa 1, and I. Schrauf 2 1 Protozoology Department, Experimental Biology Institute, Academy of Sciences of the Estonian S.S.R. P.O. Box 85, 200001 Tallinn, UDSSR 2 Zoologisches Institut der Universitfit Bonn, Poppelsdorfer SchloB, D-5300 Bonn 1, Federal Republic of Germany

Abstract. Newborn mice were infected intracere- brally with abacterial cultures of the GL strain of Tetrahymena pyriformis, induced experimentally six years ago in vitro with Coxsackie B-5 virus. In the brain of the test animals there were patho- logic changes similar to those found in the primary investigation of the pathogenicity of this strain, i.e., after 96 h of contact with the virus. Thus, the pathogenicity acquired by T. pyriformis, as well as the persistence of Coxsackie B-5 virus in this ciliate, can be considered stable. Despite such spe- cific changes in the biological properties of T. pyri- formis, the changes were not reflected in the mor- phology of the protozoon, which was investigated by means of light and electron microscopy.

Proceeding from the hypothesis of the possible role of free-living protozoa as natural reservoirs of mammalian viruses by the Swedish virologist Kling and co-workers (1942a, b), and drawing upon the experimental data on the protozoa-virus interac- tion by Kovfics et al. (1966, 1967a, b, 1969), inves- tigations in this direction were begun in 1970 in the Protozoology Department of the Experimental Biology Institute of the Estonian Academy of Sciences.

In the first stage of this work (1970-1974), ex- periments were designed to discover a suitable

* This investigation was supported in part by the Academy of Sciences of the Estonian SSR and by a grant to J. Teras from the Deutsche Forschungsgemeinschaft, Federal Republic of Germany, and carried out thanks to the support of Prof. Dr. H. Schneider, Director of the Zoology Institute, University of Bonn ** Prof. Dr. Dr. hc. E. Scholtyseck died during the time this investigation was carried out

Reprint requests to: J. Teras

model of protozoa-virus interaction as well as to elucidate possible forms of such interaction. For this purpose 130 protozoa-virus systems were es- tablished experimentally in vitro, using 26 species of protozoa (20 were free-living and 6 were parasit- ic) and 5 types of viruses (Teras et al. 1974, 1977; Teras 1981). The results revealed that most of the studied protozoa were indifferent to the viruses and, vice versa, in only 25 protozoa-virus associa- tions did penetration of protozoa by the viruses occur. For the main model of the experiments, Te- trahymena pyriformis induced with the Coxsackie B-5 virus was chosen, and attempts were made to find out how long the viruses can persist in the protozoan organism and which alterations, if any, may be caused in the host by the viruses. The de- cisive reason for this selection was the abundance of information available in the literature on T. pyriformis and the Coxsackie B-5 virus, which en- abled us to differentiate better the arising alter- ations of their biological properties.

It was confirmed that the Coxsackie B-5 virus, having penetrated into T. pyriformis, can replicate and most likely persist there: it was reisolatable as long as 6 years after the establishment of this protozoan system, which was prepared from the lysates of cultures of T. pyriformis (Teras and Kesa 1981a). Within this period, no important devia- tions either in intensity of replication or in mor- phology could be observed under electron micros- copy, compared with the normal cultures of the protozoa that were not induced by viruses. Not- withstanding repeated electron microscopic inves- tigation, neither viruses nor virus-like particles were detected in the protozoa. Notable differences, however, were observed in the antigenic properties of these populations (Teras and Kesa 1981 b) and, yet more important, in their pathogenicity (Kesa and Teras 1981).

222 J. Teras et al. : Microscope observation of virus-induced Tetrahymena pyriformis

Figs. 1-6. Light micrographs of semithin sections of brain tissue from a newborn mouse killed on the second day after intracerebral infection with living ciliates of a subculture (125th passage) of the system culture Tetrahymena pyriformis + Coxsackie B-5 virus

Fig. 1. Necrotic tissue in the center of the brain with invasive T. pyriformis (T, arrow) in the nuclear layer (nO and in the underlying tissue, x 130

J. Teras et al. : Microscope observation of virus-induced Tetrahymena pyriformis 223

After injection of living cultures of T. pyrifor- mis induced by Coxsackie B-5 virus into the brain of newborn white mice, the test animals usually died within 72 h at the latest. Examination of the test animals revealed changes in striated muscles and in brown fat, characteristic of the Coxsackie B-5 virus, and extensive necrotic foci with lysed centers in the brain, where large numbers of ciliates could be seen. The latter were also often detected in inflamed areas of the meninges. Since the injec- tion of the normal culture of the same strain of T. pyriformis, i.e., the one not induced by viruses, in the brain of the test animals did not cause simi- lar pathologic changes, and as the latter became normal and viable white mice capable of multiply- ing, the authors drew the conclusion (Kesa and Teras 1981) that, simultaneously with becoming a permanent reservoir of Coxsackie B-5 virus, T- pyriformis also acquired pathogenic properties. The aim of our common investigation was to learn if the observed pathogenicity was as stable and prolonged as the ability of the virus to persist in T. pyriformis. In addition to using light microsco- py, we also examined the virus-induced protozoa in the brain of the test animals by means of elec- tron microscopy.

Materials and methods

The initial culture of the GL strain of T. pyriformis came from the Laboratory of Unicellular Organisms, Cytology Institute, Academy of Sciences of the USSR (Leningrad). This strain was cultivated before induction with Coxsackie B-5 virus at a temperature of 28~ in Tp-culture medium consisting of equal amounts of phosphate buffer (pH 7.2-7.4) and liver infu- sion, to which 0.6% (by vol.) aminopeptide was added. From the culture of T. pyriformis synchronized by the thermal shock method of Anderson and Zeuthen (1971), 30 min after the first synchronized division, 300,000 protozoa were put into 27 ml Tp-medium, to which 3 ml Coxsackie B-5 virus suspension had been added. The virus strain, which had been isolated and cloned in Tallin (Estonian SSR), was multiplied in HEp-2 cell cultures. The latter were cultivated in a nutrient solution con- taining (by vol.) 60% medium 199 and 30% Eagle's medium,

to which 10% human blood serum had been added. The titer (TCIDs0/ml) of the virus used for inducing T. pyriformis was determined with the HEp-2 cell culture. The culture of ciliates induced by Coxsackie B-5 virus was incubated for 96 h at 28 ~ C, and thereafter the protozoa were separated by centrifugation (15 rain, 1500 rpm), washed 3 times with 0.85% NaC1 solution, and inoculated into Tp-medium. The amount of inoculum in the original culture as well as in each of the following passages was 10,000 protozoa per ml. In the present work we used the strain of T. pyriformis induced with Coxsackie B-5 virus in the Protozoology Department, Experimental Biology Institute, Estonian Academy of Sciences (Teras and Kesa 1981a). After 96 h contact with the virus, it was passaged 50 times by the methods described above in the Tp-medium, and thereafter, over a 6-year period, with regular monthly passages in the so- called conserving medium containing 10 ml phosphate buffer with 0.5 g crushed mouse liver.

The protozoa necessary for intracerebral injection into newborn mice were obtained from the 96-h culture cultivated at 28 ~ C in Tp-medium, from which the ciliates were separated by centrifugation (1500 rpm for 15 min), followed by 3 washes in 0.85% NaC1 solution. This culture, containing 2 million liv- ing ciliates per ml, was injected into the brain of each of the test animals through the calvaria center at the great fontanelle in amounts of 0.02 ml (i.e., 40,000 protozoa). The same amount of ciliates was injected in the same way into the brains of other newborn white mice, but with the normal GL strain cultures of T. pyriformis, i.e., those not induced with viruses. A part of the test animals were analogically infected with the suspen- sion of Coxsackie B-5 virus multiplied in HEp-2 cell cultures, of which 0.02 ml was injected into the brain of each mouse. All the test animals were decapitated 48 h after injection, and the brains were quickly removed. Part of the brain was placed in Tp-medium for reisolation of T. pyriformis, and another part was fixed in a 2.5% solution of glutaraldehyde in phos- phate buffer (pH 7.2) for histologic and electron microscopic investigations. For histologic study, striated muscles and inter- nal organs were also taken from each of the test animals.

After fixation in glutaraldehyde, the material used for light and electron microscopic investigation was treated with 100 m M cacodylate (pH 7.4) for at least 1 h, dehydrated in graded ethanol and propylene oxide, and embedded in Epon. Semithin sections were stained with methylene blue-azure II. Ul t ra thin sections stained with uranyl acetate and lead citrate were viewed in a Zeiss electron microscope (EM 9S2). In the test animals infected with normal cultures of the GL strain of T. pyriformis (not induced with virus), we did not find any pathologic or histologic changes either in the brain or in other organs. Therefore, only the brains of the mice infected with the culture induced with Coxsackie B-5 virus were investigated electron microscopically.

Fig. 2. Section through the brain meninges, showing 2 T. pyr(formis (1) in the lysed and erythrocyte-containing (e) tissue underneath the nuclear layer (n/). • 370

Fig. 3. Section showing T. pyriformis (T) surrounded by inflammatory infiltrate (/J) and necrotic cells (nt) within the lysed area of the brain. • 150

Fig. 4. Dividing form of T. pyriformis (T) at the periphery of necrotic cells (nt) in the lysed space of the brain tissue. • 370

Fig. 5. T. pyriformis (7) in leukocyte-infiltrated brain tissue. Note the specimen containing erythrocytes (e) in the cytoplasm of the ciliate, x 300

Fig. 6. T. pyriformis (T) in the brain tissue, surrounded by erythrocytes (e). Note the food vacuoles (/b) with partly digested erythrocytes, x 520

224 J. Teras et al. : Microscope observation of virus-induced Tetrahymena pyriformis

Results

In spite of the fact that 6 years have already passed since the induction of the GL strain of T. pyrifor- mis used for infecting test animals with Coxsackie B-5 virus, we found the same kind of pathologic changes in the newborn white mice as had been established in the first study of the pathogenicity of the strain (Teras et al. 1977; Kesa and Teras 1981). In the present study, symptoms of limb pa- ralysis appeared within 24 h as a result of the infec- tion; in the following 24 h the symptoms percepti- bly increased.

In all of the wet smears prepared from the brains, large numbers of actively moving ciliates could be seen, which under light microscopy dif- fered in no way from the individuals used for in- fecting or from the normal population of T. pyri- formis. The ciliates reisolated from the inoculum of brain tissue with the aid of Tp-medium had the same morphology. In the histologic investiga- tion (Figs. 1-6) of the brain, empty spaces were observed, evidently caused by lysis of the necrotic tissue (Figs. 1 and 2). In some places the cerebral membranes were thickened and infiltrated with leukocytes (Fig. 3). In the necrotic foci and the lysed area, abundant intensively stained protozoa with easily distinguishable nuclei could be seen, dividing ciliates among them (Fig. 4). In the cyto- plasm of the established protozoa, there were nu- merous food vacuoles that often contained a thick, basophilic substance (Figs. 5 and 6). In addition to the described changes, foci of interstitial inflam- mation characteristic of the Coxsackie B-5virus, but comparatively weakly expressed, as well as changes in brown fat were found in the histologic investigation. These alterations were more promi- nent in skeletal muscles and in brown fat of the test animals infected with the Coxsackie B-5 virus; the brain contained only a few small groups of necrotic cells and round foci of infiltration.

The ultrastructure of the ciliates, in their changed environment within the spaces of the ne- crotic brain tissue, appeared normal in most details (Figs. 7-10). The cells appeared well fed, with many food vacuoles, and the nucleoli did not look like fusion bodies (Fig. 9). Sections through the pharyngeal pouch of T. pyriformis showed cilia and mitochondria in the periphery of the proto- zoan cell (Fig. 8). The mitochondria, mostly lo- cated under the cell surface, were as numerous as normal and well structured, the tubular cristae un- damaged (Fig. 10). In Comparison to uninfected T. pyriformis, the number of mucocysts was ex- tremely low. Moreover, the macronuclei sometimes

had a somewhat unusual appearance (Fig. 9). In some macronuclei, areas free of chromatin bodies were observed, and numerous small nucleoli were found in central areas rather than close to the nu- clear envelope (Fig. 9), the latter location being typical for this organism under good conditions (Nilsson 1981).

Discussion

The GL strain of T. pyriformis that had been in brief contact with Coxsackie B-5 virus in vitro caused serious and extensive, lethal damage in the brain of newborn white mice after as long as 6 years of passaging in a virus-free medium. This is a further confirmation of the conclusion (Teras et al. 1977; Kesa and Teras 1981) that viruses, hav- ing penetrated into the organism and having prob- ably been incorporated into the macronuclear ge- nome of this protozoon, cause changes in the bio- logical properties of the new host, and that these changes persist and may be irreversible. Special attention must be given to the fact that T. pyrifor- mis, since its initial isolation in 1922 by Lwoff, has been considered fully harmless to warm- blooded animals. However, this study shows that genetically stable hosts (Tetrahymena), after con- tact with mammalian viruses, acquire strong pathogenicity, especially to mice. As we know, the pathogenic effect and facultative parasitism of some strains of T. pyriforrnis have hitherto been asserted only in insects (Lwoff 1924; Janda and Jirovec 1937; McCaul 1956; Thompson 1958; Clark and Brandl 1976) in snails (Kozloff 1956) and in turbellarians (Wright 1981).

It is likely that the pathogenicity of T. pyrifor- mis and other free-living protozoa can be in- fluenced not only by Coxsackie B-5 virus but also by other mammalian viruses capable of penetrating into protozoan organisms. Unfortunately, the morphology and ultrastructure of the pathogenic strain of T. pyriformis do not essentially differ, ei- ther in vitro or in vivo, from the normal strains. Therefore, it seems highly possible that the virus genome has been incorporated into the host ge- nome, which can be established only by molecular hybridization. At present we cannot be sure what caused the ultrastructural peculiarities in the ciliate cells observed in the brain tissue. In particular, it is unknown whether the unusual fine structure of macronuclei observed in some ciliates is a result of the virus infection or of the specific environment in the brain. It is therefore necessary to investigate repeatedly the ultrastructure of virus-infected T. pyriformis in vitro as well as in vivo. The cause

J. Teras et al. : Microscope observation of virus-induced Tetrahyrnena pyriformis 225

Fig. 7-10. Electron micrographs of T. pyriformis in the brain of newborn mice

Fig. 7. Section through T. pyriformis containing numerous food vacuoles (fv) and surrounded by host cells (hc) and erythrocytes (e) x 6,000

Fig. 8. Section through cytopharyngeal pouch (cy) of T. pyriformis, showing cilia (c) and mitochondria (rn 0 in the periphery of the cytoplasm of the cell. x 11,500

Fig. 9. Section through the macronucleus (rna) of T. pyriformis with numerous small nucleoli in the central area (arrow). x 9,600

Fig. 10. Section through the cortex of T. pyriformis with cilia (c) in longitudinal and cross section. Undernea th the pellicle (pe), mitochondria (m0 and basal bodies (bb) are visible, x 2,000

226 J. Teras et al. : Microscope observation of virus-induced Tetrahymena pyriformis

of the reduction in the number of mucocysts is also unknown. The low number could indicate that the creation of mucocysts was reduced or that they were discharged. It is known that the discharge of mucocysts can be triggered by various means (Tiedtke 1976; Hausmann 1978). Possibly the ci- liates discharge all the mucocysts, as soon as they become mature, in response to the surrounding mouse tissue. As a result, the excreted contents of mucocysts could form a mucous layer surround- ing the ciliates. Thus, viability can be demonstrated only experimentally, and a variability in the two strains of T. pyriformis must be taken into account. The fact that the viruses persisting in the proto- zoan organism do not influence the morphology or the ultrastructure of the host has also been ob- served by Diamond and Mattern (1976), who were the first to report on the so-called endosymbiotic protozoan viruses. According to these authors, these kinds of virus do not influence the pathogeni- city of the host (Entamoeba histolytica) but play, however, an important role in the life cycle of pro- tozoa.

Thus, whether the protozoon has been pene- trated by mammalian viruses or already contained persisting, endosymbiotic protozoan viruses, it will still be reflected in one way or the other in the biological properties of the host. Consequently, be- sides other possible causes, the effect of the viruses has to be taken into account, particularly in the free-living species of protozoa whose biological properties have changed. This has become especially critical because of constantly increasing viral contamination of sewage as well as natural waters, which has created very favorable condi- tions for contact of free-living protozoa with the various types of mammalian virus. The importance of these kinds of investigation is expressed not only by the pathogenicity acquired by one species of the free-living protozoa described by us, but also through the discovery of an increasing number of pathogenic forms of amebas of the so-called limax group in different parts of the world (Martinez and De Jonckheere 1981 ; John 1982).

Acknowledgements. The authors gratefully acknowledge the ad- vice of Prof. Dr. Hans-Dieter G6rtz, Zoologisches Institut, Mfinster, for interpretation of the results, and Prof. Dr. Bill Chobotar, Biology Department, Andrews University, Michigan (USA), for constructive comments and his help with the English text.

References

Andersen HA, Zeuthen E (1971) D N A replication sequence in Tetrahymena is not repeated from generation to genera- tion. Exp Cell Res 68:309-314

Clark TB, Brandl R (1976) Observation on the infection of Aedes sierrensis by a Tetrahymena ciliate. J Invertebr Pathol 28 : 341-349

Diamond LS, Mattern CFT (1976) Protozoal viruses. Adv Vi- rus Res 20:87 112

Hausmann K (1978) Extrusive organelles in protists. Int Rev Cytol 52:197-276

Janda V, Jirovec O (1937) fJber kfinstlich hervorgerufenen Pa- rasitismus eines freilebenden Ciliates Glaucoma pyriformis und Infektionsversuche mit Euglena gracilis und Spirochaete biflexa. Mem Soc Zool Tch6c Prag 5 : 34-58

John DT (1982) Primary amebic meningoencephalitis and the biology of Naegleria fowleri. Ann Rev Microbiol 36:10/ - 123

Kesa L, Teras J (1981) The experimental study on the relation- ships between Tetrahymena pyriformis and RNA- and DNA-viruses. II. On the acquisition of pathogenicity by T. pyriformis after the interaction with Coxsackie B-5 virus. In: The interaction between protozoa and viruses. Proto- zoology series, vol 6. USSR Academy of Science, Len- ingrad, pp 96-111 (in Russian, English summary)

Kling C, Olin G, Fahraeus J, Norlin G (/942a) Sewage as a carrier and disseminator of poliomyelitis virus. I. Search- ing for poliomyelitis virus in Stockholm sewage. Acta Med Scand 112: 217-249

Kling C, Olin G, Fahraeus J, Norlin G (1942b) Sewage as a carrier and disseminator of poliomyelitis virus. II. Studies on the conditions of life of poliomyelitis virus outside the human organism. Acta Med Scand 112:250-266

Kov~cs E, Kolompar G (1969) Hemadsorption reaction in Te- trahymena pyriformis incubated with measles virus. Life Sci 8 : 1089-1097

Kov/tcs E, Bucz B, Kolompar C (1966) Propagation of mamma- lian viruses in Protista. I. Visualization of fluorochrome labelled EBC virus in yeast and Tetrahymena. Life Sci 5:2117-2/26

Kovfics E, Bucz B, Kolompar C (1967a) Propagation of mam- malian viruses in Protista. II. Isolation of complete virus from yeast and Tetrahymena experimentally infected with picorna viral particles and their infectious RNA. Life Sci 6 : 347-358

Kovfics E, Bucz B, Kolompar C (1967b) Propagation of mam- malian viruses in Protista. III. Change in population densi- ties, viability and multiplication of yeast and Tetrahymena experimentally infected with encephalomyocarditis virus. Life Sci 6:2359-2371

Kozloff EN (1956) Experimental infection of the grey garden slug, Deroceras reticulatum (Mfiller), by the holotrichous ciliate Tetrahymena pyriformis (Ehrenberg). J Protozool 3:17-19

Lwoff A (1923) Sur la nutrition des infusoires. Compt Rend 176: 928-930

Lwoff A (1924) Infection experimentale a Glaucoma pyriformis chez Galleria mellonella (Lepidoptere). Compt Rend 178:1106-1108

Martinez AJ, De Jonckheere JF (1981) Les infections par les amibes libres. Bull Inst Pasteur Paris 79:171-205

McCaul WE (1956) An experimental attempt to parasitize mammals with the free-living ciliate Tetrahymena pyrifor- mis. J Protozool 3 (Suppl) : 11

Nilsson JR (1981) On cell organelles in Tetrahymena. With spe- cial reference to mitochondria and peroxisomes. Carlsberg Res Commun 46:279 304

Teras J (1981) Forms of interaction between protozoa and vi- ruses. In: The interaction between protozoa and viruses. Protozoology series, vol 6. USSR Academy of Sciences, Leningrad, pp 31-55 (in Russian, English summary)

J. Teras et al. : Microscope observation of virus-induced Tetrahymena pyriformis 227

Teras J, Kesa L (1981 a) The experimental study on the relation- ships between Tetrahymena pyriformis and RNA- and DNA-viruses. I. Persistence and replication of picorna- and adenovirus in T. pyr(forrnis. In: The interaction between protozoa and viruses. Protozoology series, vol 6. USSR Academy of Sciences, Leningrad, pp 73 96 (in Russian, En- glish summary)

Teras J, Kesa L (1981 b) The experimental study on the relation- ship between Tetrahymena pyriformis and RNA- and DNA- viruses. III. A comparative study o f antigenic properties of T. pyriformis before and after the interaction with Cox- sackie B-5 virus. In: The interaction between protozoa and viruses. Protozoology series, vol 6. USSR Academy of Sciences, Leningrad, pp 112-125 (in Russian, English sum- mary)

Teras J, J6giste A, Kallas E (1974) Theoretical aspects of the pathogenicity mechanisms of free-living protozoa. Proceed-

ings of the Third International Congress of Parasitology Munich 25-31 August 1974, vol 1 : 185

Teras J, Kesa L, Kallas E, J6giste A (1977) On the relationship between some free-living and parasitic protozoa and the RNA- and DNA-viruses. Abstracts of papers read at the Fifth International Congress on Protozoology New York 26 June-2 July 1977:446

Thonapson JC (1958) Experimental infections of various animal strains of the genus Tetrahymena. J Protozool 5 : 203-205

Tiedtke A (1976) Capsule shedding in Tetrahymena. Natur- wissenschaften 63 : 93

Wright JF (1981) Tetrahymena pyriformis (Ehrenberg) and T. corlissi (Thompson) parasitic in stream-dwelling triclads (Platyhelminthes turbellaria). J Parasitol 62:131-133

Accepted September 16, 1987