Zeitschrift for

Z Parasitenkd (1982)66:281-292 Parasitenkunde Parasitology Research

�9 Springer-Verlag 1982

A Comparative Light and Electron Microscope Study of the Cysts of Sarcocystis Species of Roe Deer (Capreolus capreolus)

Rolf Entzeroth* Abteilung fiir Protozoologie, Zoologisches Institut der Universit~t Bonn, Poppelsdorfer Schloss, D-5300 Bonn, Federal Republic of Germany

Abstract. Sarcocystis muscle cysts of naturally infected roe deer were exam- ined by light and electron microscopy. Two types of thin- and thick-walled cysts could be distinguished by light microscopy, whereas by electron micros- copy six types of cyst walls could be differentiated on the basis of the size and shape of protrusions of the primary cyst wall and by the presence or absence of fibrillar elements. The paper also discusses whether the cyst wall types are species related or represent cysts of different ages. The fine structure of merozoites and metrocytes of Sarcocystis of roe deer resembles that of other Sarcocystis species.

Introduction

Since the discovery of the coccidian nature of Sarcocystis species much of the experimental work has been done with domestic animals (Fayer 1972; Rom- mel et al. 1972; Fayer and Johnson 1973; Heydorn et al. 1975). In contrast, relatively few studies have involved game animals (Kaliner et al. 1971 ; Hudkins and Kistner 1977). The subject of the present study, Sarcocystis of roe deer muscles, was first described by von Hessling in 1854 and called Sarcocystis gracilis by Ratz in 1909. More recently, light and electron microscope studies on roe deer sarcocysts have shown that there are at least three types of cysts in the musculature of these hosts (Erber et al. 1978; Bergmann and Kinder 1976; Schramlov/t and Bla2ek 1978). Experimental studies on the life cycle of these parasites, however, have so far revealed only Canidae (dog, fox) as the final host (Entzeroth et al. 1978; Erber et al. 1978; Bla~ek et al. 1978).

Earlier studies have shown that one intermediate host can be infected by more than one Sarcocystis species (Gestrich et al. 1975). In the present paper we compare the ultrastructure of Sarcocystis muscle cysts of roe deer and discuss

* Present address : Department of Biology, Andrews University, Berrien Springs, Springs, Michigan 49103, USA

Supported by a Feodor Lynch Stipend of the Alexander von Humboldt Foundation

0044-3255/82/0066/0281/$02.40

282 R. Entzeroth

whether the morphological variations of these cysts are species related or repre- sent the different developmental stages of one species.

Materials and Methods

Samples of tongue, heart, diaphragm and skeletal musculature of 103 roe deer were fixed in 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) and examined for natural Sarcocystis infection by aid of cryosections which were stained by hematoxylin-eosin. For electron microscopy heavily infected samples were washed in cacodylate buffer for 4~6 h, dehydrated with acetone and pre-stained with uranyl acetate and phosphotungstic acid in 70% acetone. After final dehydration in acetone the material was embedded in Vestopal W or ERL-4206 (Spurr 1969).

For light microscopic studies samples of infected animals were dehydrated in isopropanol and embedded in Paraplast. Sections cut with a Leitz microtome were stained with hematoxylin-eosin. Semi-thin sections of Vestopal W or ERL-4206 embedded material were stained with Azur II and methylene blue. Semi- and ultrathin sections were cut with glass knives using a Reichert Ultracut Ultramicrotome and examined with a Zeiss EM 9 $2 electron microscope.

Results

1. Light Microscopy

Of the 103 roe deer examined 74 were naturally infected with sarcocysts. Of the animals more than 3 years old 95% were infected. Two types of sarcocysts could be differentiated on the basis of cyst wall morphology. Type 1 sarcocysts had thick striated walls (Figs. 1, 2), and type 2 had thin, non-striated walls (Fig. 3). The cysts measured 250-1000 gm in length and 50-200 ~tm in width. Both types of cysts were septated and contained metrocytes and merozoites. The merozoites were banana-shaped, slightly pointed at the posterior pole, with a' size range of 8-14 to 2-3 gin.

2. Electron Microscopy

Cyst Wall

Thin-walled and thick-walled muscle cysts of roe deer were examined by electron microscopy. While the light microscope studies revealed only two types of cyst

Abbreviations

A Polysaccharide granules (amylopectin) ME Merozoite B Granular-like thickenings MF Myofibrils

of the primary wall MI Mitochondrion BU Bubble-like projections of the primary MIH Host cell mitochondrion

cyst wall MN Micronemes CW Cyst wall N Nucleus DC Daughter cell PE Pellicle ER Endoplasmic reticulum PR Protrusion of the primary cyst wall FE Fibrillar elements PW Primary cyst wall G Granules in the cyst ground substance SE Septa GS Cyst ground substance SP Sarcoplasm MC Metrocyte Z Z-line of sarcomeres of the muscle cyst

Fig. 1. Sarcocystis sp. from the tongue muscle of a roe deer. The cyst is thick-walled (CW) and divided into chambers through septa (SE) with numerous merozoites (ME). Light micrograph, toluidine blue, Paraplast. • 530

Fig. 2. Sarcocystis sp. from the esophagus of roe deer. Higher magnification of host cell-parasite contact zone with protrusions (PR) and the cyst contents of merozoites (ME) and metrocytes (MC). SP, sarcoplasm. Light micrograph, hematoxylin-eosin, Paraplast. xl,600

Fig. 3. Sarcocystis sp. from the esophagus of a roe deer. Section through a thin-wailed cyst (CW) with groups of merozoites (ME) divided through septa (SE). Light micrograph, hematoxylin-eosin, Paraplast. x 1,400

284 R. Entzeroth

wall, on the ultrastructural level six different types could be distinguished on the basis of their morphology. The cyst wall consists of a primary wall that is derived from the limiting membrane of the former parasitophorous vacuole formed by the host cell. The primary cyst wall forms various protrusions towards the host cell. Cyst walls of types 1-3 were characterized by the presence of protrusions that are the cause of the striated appearance of the cyst wall by light microscopy. Under the primary wall an amorphous granular material forms the cyst ground substance in which the cyst contents (merozoites and metrocytes) are embedded.

Type 1

The protrusions of this type have a finger-like appearance with a length of 2.6 3.6 ~tm and width of 0.5-0.7 gm (Fig. 4). The protrusions stand upright and contain multiple fibrillar elements that run parallel from the tip of the protrusions towards the cyst inside. The cyst ground substance appears electron- pale and surrounds numerous metrocytes.

Type 2

This kind of cyst is characterized by tube-like protrusions (Fig. 5) with a length of 5-8 ~tm and a base width of 1.2-1.5 ~tm. The upper part of the protrusion becomes slender and is bent sidewards at the point of contact with the host cell cytoplasm and mitochondria. The protrusions are filled with ground sub- stance that contains fibrillar elements and tiny electron-dense granules. At the distal end of the protrusions the limiting membrane has a wavy appearance. At the base of the protrusions granular-like thickenings are present. Between the thickenings the primary wall is thin consisting of a unit membrane derived from the parasitophorous vacuole.

Type 3

A section through a cyst wall with cylindrical, blunt-tipped protrusions is shown in Fig. 6. The protrusions measure 4-6 ~tm by 1.2-1.5 gm. The ground substance inside the protrusion is characterized by the presence of fibrillar elements which run parallel towards the inner margin of the cyst. The primary wall is folded and has granule-like thickenings at the base of the protrusions.

Type 4

The next three types of cyst walls are characterized by the absence of fibrillar structures in the cyst ground substance and thus appear smooth in the light microscope. Figure 8 shows a section through a thin-walled cyst of type 4 with merozoites. The host cell cytoplasm seems to be degenerated adjacent to the cyst wall (Fig. 8). Higher magnification of the cyst wall (Fig. 9) shows that the primary wall forms overlapping bands 1.2 btm long and 20-30 nm wide. In this type of cyst the primary wall consists of a unit membrane; no

Fig. 4. Sarcocystis sp. from skeletal muscle of a roe deer. Electron micrograph of the cyst wall of type 1 with finger-like protrusions (PR) which contain fibrillar elements (FE). Underneath, a metrocyte (MC) with nucleus (N) and electron-pale cytoplasm PE, pellicle; GS, cyst ground substance; MI, mitochondrion. Glutaraldehyde, OsO4, Vestopal W. x 15,000

Fig. 5. Sarcocystis sp. from the esophagus muscle of a roe deer. The cyst wail of type 2 is characterized by tube-like protrusions (PR) which contain fibriilar elements (FE). B, granuIar-like thickenings of primary wall (PW); Glutaraldehyde, OsO4, Vestopal W. x 14,000

Fig. 6. Sarcocystis sp. from the esophagus muscle of a roe deer. Note that the cube-like protrusions (PR) of the primary wall (PW) of type 3 contain fibrillar elements (FE) and fine granules (G). Glutaraldehyde, OSO4, Vestopal W. x 16,000

Fig. 7. Sarcocyst& sp. from the esophagus muscle of roe deer. Section through a metrocyte (MC) with 2 daughter cells (DC). Glutaraldehyde, OsO4, Vestopal W. x 18,000

Fig. 8. Sarcocystis sp. from the diaphragm of a roe deer. Electron micrograph of the periphery of a thin-walled cyst which contains groups of merozoites (ME) separated by septa (SE). The adjacent sarcoplasm (SP) of the host cell is disintegrating. Glutaraldehyde, OsO4, Vestopal W. x 6,000

Fig. 9. Sarcocystis sp. from the diaphragm of a roe deer. Higher magnification of a thin-walled cyst with overlapping band-like protrusions (PR) representing cyst wall type 4. Glutaraldehyde, OsO~, Vestopal W. x 25,000

Fig. 10. Sarcocystis sp. from the diaphragm of roe deer. The primary wall (PW) forms stubby-like protrusions (PR) with bubble-like folds (BU) inbetween and represents the cyst wall type 5. Glutaral- dehyde, OsO4, VestopaI W. x 23,000

Fig. 11. Sarcocystis sp. from the diaphragm of a roe deer. Electron micrograph of an ultrathin section through a thin-walled cyst of type 5 with numerous merozoites (ME) inside. Glutaratdehyde, OsO4, Vestopal W. x 4,600

Fig. 12. Electron micrograph of a longitudinal section through curled thread-like protrusions (PR) of type 6. Glutaraldehyde, OsO4, VestopaI W. x 14,000

Fig. 13. Cross section through the cyst wall of type 6 showing the protrusions (PR) curling round host cell mitochondria (MIH). Glutaraldehyde, OsO4, Vestopal W. x 14,000

Light and Electron Microscope Studies of Sarcocystis in Roe Deer 289

thickenings are present. There is little ground substance between the parasites and the cyst wall and within the protrusions.

Type 5

This type of cyst forms stubby protrusions 0.4 0.5 btm long and 0.3 I~m wide. Among the protrusions numerous bubble-like projections extending towards the cyst outside increase the surface of the cyst wall. The primary wall has a thickness of 20 nm. The ground substance consists of a homogeneous osmio- philic material and forms a zone 0.5 gm wide between the primary wall and the metrocytes and merozoites. Septa are thin and hardly visible with the electron microscope.

Type 6

This type of thin-walled cyst possesses thread-like protrusions 5 8 btm long and 0.3 I~m wide (Fig. 12). The ground substance is virtually absent in these cysts so that the parasites lie immediately beneath the primary cyst wall (Fig. 11). Typical septa as shown in types 1-3 are not present. Higher magnification of the cyst walls shows that the thread-like protrusions can be curled as shown in Fig. 12. Cross sections through the cyst wall protrusions reveal that the projections of the primary wall sometimes twist around the adjacent host cell mitochondria (Fig. 13).

Metrocytes

Metrocytes of all cyst types are mostly located at the cyst periphery close to the cyst wall as seen in Fig. 4. Their shape is round to ovoid and they measure 6 8 I~m in length and 3 6 btm in width. They are limited by a three- membranous pellicle. Young metrocytes are characterized by an electron-pale cytoplasm which contains mitochondria and rough endoplasmic reticulum. The nucleus is situated centrally and contains plaques of osmiophilic material. After a developmental phase called endodyogeny the merozoites give rise to two daugther cells (Fig. 7).

Merozoites

The merozoites of roe deer sarcocysts have a typical banana-shape with a pointed anterior pole (Fig. 11). In all cyst types they measure 8-14 gm in length and 2-3 gm in width. Electron micrographs reveal that they are limited by a three- membranous pellicle. At the anterior pole numerous osmiophilic structures, micronemes, dominate. A conoid with preconoidal rings, polar rings and duc- tules of rhoptries are present.

Discussion

Electron micrographs of Sarcocystis muscle cysts have shown that the cyst wall originates from the limiting membrane of the parasitophorous vacuole. During the development this membrane becomes strengthened by osmiophilic

290 R. Entzeroth

material and is then called "Prim/irhiille" or "primary cyst wall" .(Mehlhorn and Scholtyseck 1973; Scholtyseck et al. 1974; Mehlhorn et al. 1975). A primary cyst wall has been reported for all Sarcocystis species and also for Toxoplasma and Frenkelia cysts (Scholtyseck et al. 1974; S6naud 1967 ; Kepka and Scholtyseck 1970). It is, however, not present in Besnoitia (Sheffield 1968; S6naud 1969). In some Sarcocystis species the primary cyst wall may form characteristic protru- sions which are identical in all mature cysts of the same species wherever they are located (Mehlhorn et al. 1976). In the present study on Sarcocystis muscle cysts, six different types of cyst wall can be distinguished on the basis of their ultrastructural morphology. They can be separated into two groups by the size and shape of the protrusion and by the presence or absence of fibrillar elements inside the protrusions. Cyst wall type 2 is similar to that found by Schramlov/t and Bla2ek (1978) who described palisade-like protrusions in the primary cyst wall of roe deer. On stained Paraplast sections two types of cysts can be distinguished: thin-walled and striated cyst walls. This finding is con- firmed by Schramlovfi and Bla~ek (1978). Erber et al. (1978), however, described three different types of cyst wall visible by light microscopy, one thick-walled and two thin-walled cysts.

In the present study the cyst wall of type 1 with upright finger-like protru- sions may be interpreted as a young cyst because of the fact that metrocytes were the predominant intracystic form. Thus it is possible that the cyst walls of types 1-3 may belong to the same species at different stages of growth. Similar observations of an alteration of the cyst wall during aging of Sarcocystis fusiformis have been made by Mehlhorn et al. (1975).

Although no variation was obvious in the thin-walled cyst by light microsco- py, three further types of cysts (types 4-6) could be differentiated by electron microscopy. A cyst wall with stubby protrusions (similar to our type 5) has also been seen by Bergmann and Kinder (1976). Schramlovfi et al. (1975) de- scribed thin-walled roe deer Sarcocystis muscle cysts with protrusions similar to type 4 shown in this study. So far there are no other ultrastructural descrip- tions of roe deer sarcocysts which resemble cyst wall type 6 with the long thread-like protrusions. They might be identical with the roe deer sarcocysts S. capreolicanis examined by light microscopy by Erber et al. (1978). However, until further studies are done it is difficult to conclude whether the different types of thin-walled sarcocysts represent different dog-roe deer species as stated by Erber et al. (1978). It is possible that they represent cysts of a different developmental stage and age.

Metrocytes and merozoites of roe deer sarcocysts are similar in shape and fine structure to other Sarcocystis species (Scholtyseck 1979). They are embedded in the cyst ground substance, which is of granular homogeneous appearance and forms septa that lead to compartments. The ground substance is absent in Toxoplasma and Besnoitia, but present in Frenkelia (Scholtyseck et al. 1974). The peripheral zone of ground substance is considerably thicker in the cysts with fibrillar elements than in those without these structures. Mehlhorn et al. (1976), however, observed a wider zone of peripheral cyst ground substance in cysts with short cyst wall protrusions.

Light and Electron Microscope Studies of Sarcocystis in Roe Deer 291

T h e c o m p a r t m e n t s c o n s i s t i n g o f cys t g r o u n d s u b s t a n c e a re t h o u g h t to g ive

a g r e a t e r s t a b i l i t y to t h e cys ts ( M e h l h o r n a n d H e y d o r n 1978). C y s t s o f t ypes 5

a n d 6, h o w e v e r , h a v e h a r d l y a n y g r o u n d s u b s t a n c e i n s i d e a n d c o m p a r t m e n t s

a r e n o t v i s ib le w i t h t h e e l e c t r o n m i c r o s c o p e . I t is n o t k n o w n w h e t h e r t h e cys t

g r o u n d s u b s t a n c e u n d e r w e n t d i s i n t e g r a t i o n d u r i n g a n a d v a n c e d s t age o f d e v e l o p -

m e n t or w h e t h e r i t h a d n e v e r b e e n p r e s e n t . T h e r e f o r e , it m a y b e c h a r a c t e r i s t i c

o f t h e species .

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Received June 16, 1981