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 examined by light and electron microscopy. Two types of thin- and thick-walled
cysts could be distinguished by light microscopy, whereas by electron microscopy 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; R o m 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 represent 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
B
BU
CW
DC
ER
FE
G
GS
MC
Polysaccharide granules (amylopectin)
Granular-like thickenings
of the primary wall
Bubble-like projections of the primary
cyst wall
Cyst wall
Daughter cell
Endoplasmic reticulum
Fibrillar elements
Granules in the cyst ground substance
Cyst ground substance
Metrocyte
ME
MF
MI
MIH
MN
N
PE
PR
PW
SE
SP
Z
Merozoite
Myofibrils
Mitochondrion
Host cell mitochondrion
Micronemes
Nucleus
Pellicle
Protrusion of the primary cyst wall
Primary cyst wall
Septa
Sarcoplasm
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 electronpale 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 substance 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. Glutaraldehyde, 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 MicroscopeStudies 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 osmiophilic 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 threemembranous 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 threemembranous pellicle. At the anterior pole numerous osmiophilic structures,
micronemes, dominate. A conoid with preconoidal rings, polar rings and ductules 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 protrusions 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 confirmed 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 protrusions 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 microscopy, 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) described thin-walled roe deer Sarcocystis muscle cysts with protrusions similar
to type 4 shown in this study. So far there are no other ultrastructural descriptions 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 c y s t g r o u n d s u b s t a n c e a r e t h o u g h t t o give
a g r e a t e r s t a b i l i t y t o t h e c y s t s ( 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 y p e s 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 i b l e 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 c y s t
ground substance underwent disintegration during an advanced stage of developm e n t o r w h e t h e r it 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.
References
Bergmann V, Kinder E (1976) Elektronenmikroskopische Untersuchungen zur Wandstruktur yon
Sarkozysten in der Skelettmuskulatur yon Wildschwein und Reh. Mh Vet Med 31:785 788
Bla2ek K, Schramlovfi J, Ippen R (1978) Dog as definitive host of Sarcosporidia infecting roe
deer. Fol Parasitol 25 : 95-96
Entzeroth R, Scholtyseck E, Greuel E (1978) The roe deer intermediate host of different coccidia.
Naturwissenschaften 65:395
Erber M, Boch J, Barth D (1978) Drei Sarkosporidienarten des Rehwildes. Berl Mfinch Tierfirztl
Wschr 91:482-486
Fayer R (1972) Gametogony of Sarcocystis sp. in ceil culture. Science I75:65-67
Fayer R, Johnson AJ (1973) Development of Sarcocystisfusiformis in calves infected with sporocysts
from dogs. J Parasitol 59(6) : 1135-1137
Gestrich R, Mehlhorn H, Heydorn AO (1975) Licht- und elektronenmikroskopische Untersuchungen
an Cysten von Sarcocystis fusiformis in der Muskulatur yon Kfilbern nach experimenteller
Infektion mit Oocysten und Sporocysten der groBen Form yon Isospora bigemina der Katze.
Zbl Bakt Hyg I Abt Orig A 233(2):261-276
Hessling Th v (1854) Histologische Mittheilungen. Zeitschrift f/fir wissensch Zoologie 5:189 199
Taf X Figg 9 u 10 Mit Zusatz von v Siebold, 199-200
Heydorn AO, Mehlhorn H, Gestrich R (1975) Licht- und elektronenmikroskopische Untersuchungen
an Cysten von Sarcocystis fusiformis in der Muskulatur yon KS.lbern nach experimenteller
Infektion mit Oocysten und Sporocysten von Isospora hominis (Railliet et Lucet 1891). 2. Die
Feinstruktur der Metrocyten und Merozoiten. Zbl Bakt Hyg I Abt Orig A 232:373-391
Hudkins GG, Kistner TP (1977) Sarcocystis hemionilatrantis (sp nov); life cycle in mule deer
and coyote. J Wild1 Dis 13:80 84
Kaliner G, Sachs R, Fay LD, Schiemann B (1971) Untersuchungen fiber das Vorkommen von
Sarcosporidien bei ostafrikanischen Wildtieren. Z Tropenmed Parasitoi 22:156 164
Kepka O, Scholtyseck E (1970) Weitere Untersuchungen der Feinstruktur yon Frenkelia sp. ( = MOrganismus, Sporozoa). Protistologica 5(3):249 266
Mehlhorn H, Scholtyseck E (1973) Elektronenmikroskopische Untersuchungen an Cystenstadien
von Sarcocystis tenella aus der Oesophagusmuskulatur des Schafes. Z Parasidenkd 41:291 310
Mehlhorn H, Hartley WJ, Heydorn AO (1976) A comparative ultrastructural study of the cyst
wail of 13 Sareocystis species. Protistologica 12(3):45I 467
Mehlhorn H, Frenkel JK (1980) Ultrastructural comparison of cysts and zoites of Toxoplasma
gondii, Sarcocystis muris, and Hammondia hammondi in skeletal muscle of mice. J Parasitoi
66 : 59-67
Mehlhorn H, Heydorn AO (1978) The sarcosporidia (Protozoa, Sporozoa): life cycle and fine
structure. Advances in Parasitoiogy:43-93
Mehlhorn H, Heydorn AO, Gestrich R (1975) Light microscope and electron microscope studies
on cysts of Sarcocystis fusiformis in the muscles of calves infected experimentally with oocysts
and sporocysts of Isospora hominis. 1. The development of cyst and cyst wall. Zbl Bakt Hyg
I Abt Orig A 231(1-3):301-322
Ratz I (1909) Az izmokban +16sk6d6 v&gl6nyekrol 6s a magyar faunfib/m el6fordulo fajaik. Allattani
k6zlem&nyek 8:1 37
Rommel M, Heydorn AO, Gruber F (1972) Beitrfige zum Lebenszyklus der Sarkosporidien. I.
Die Sporozyste yon S. tenella in den Ffizes der Katze. Berl Mtinch TierS.rztl Wschr 85:101-105
292
R. Entzeroth
Rzepczyk C, Scholtyseck E (1976) Light and electron microscope study on the Sarcocystis of
Rattus fuscipes, an Australian rat. Z Parasitenkd 50:137-150
Sheffield HG (1968) Observations on the fine structure of the "cyst stage" of Besnoitia jellisoni.
J Protozool 15 : 685-693
Scholtyseck E (1979) Fine Structure of Parasitic Protozoa. An Atlas of micrographs, drawings
and diagrams. Springer, Berlin Heidelberg New York
Scholtyseck E, Mehlhorn H, M/iller BEG (1974) Feinstruktur der Cyste und Cystenwand von
Sarcocystis tenella, Beonoitia jellisoni, Frenkelia spec. und Toxoplasma gondii. J Protozool
21 (2) :284-294
Schramlovfi J, Bla~ek K (1978) Ultrastruktur der Zystenwand der Sarkosporidien des Rehes (Capreolus capreolus L.). Z Parasitenkd 55:4348
S6naud J (1967) Contribution /l l'6tude des Sarcosporidies et des Toxoplasmes (Toxoplasmea).
Protistologica 3 : 167-232
Spurr AR (1969) A iow-visocosity epoxy resin embedding medium for electron microscopy. J
Ultrastruct Res 26 : 31-43
Received June 16, 1981