ULTRASTRUCTURAL STUDY OF THE DEVELOPMENT
OF SARCOCYSTIS SINGAPORENSIS SARCOCYSTS IN THE MUSCLES OF ITS RAT HOST
PAPERNA I.*
Summary :
Laboratory rats fed sporocysts of Sarcocystis singaporensis (Zaman
& Colley, 1 975) Zaman & Colley, 1 9 7 6 originating from Singapore were euthanized 22, 23, 33 and 80 days later.
Sporocysts were extracted from feces of either naturally or laboratory-infected Python reticulatus. Electron microscopically examined tongue and esophageal muscles yielded images of successive developing stages of sarcocysts. The primary wall evolved from a continuous thin layer into folds and later, into villar protrusions. At all stages the wall was interrupted by pinocytotic- like indentations. Young sarcocysts contained only metrocytes, they divided by endodyogeny into daughter metrocytes. The first bradyzoites appeared only 33 d.p.i. Sarcocysts by 80 d.p.i.
were enclosed in a fully differentiated primary wall and contained almost entirely bradyzoites.
KEY WORDS : ultrastructure, Sarcocystis singaporensis, white rat, muscles, sarcocysts, development.
R é s u m é : ÉTUDE ULTRASTRUCTURALE DU DÉVELOPPEMENT DES SARCOCYSTES DE SARCOCYSTIS SINCAPORINSIS DANS LES MUSCLES DU RAT HÔTE
Des rats de laboratoires infestés avec des sporocystes de Sarcocystis singaporinsis (Zaman & Colley, 1975) Zaman &
Colley, 1976, provenant de Singapour, ont été sacrifiés 22, 23, 33 et 80 jours plus tard. Les sporocystes ont été extraits de fèces de Python reticulatus naturellement ou expérimentalement infesté.
L'examen en microscopie électronique des muscles de la langue et de l'œsophage a montré des sarcocystes aux différents stades de leur développement. La paroi se développe initialement en une mince couche avec des plis et plus tard sous la forme de protrusions villeuses. A tous les stades, la paroi est interrompue par des échancrures. Les sarcocystes jeunes ne renferment que des metrocystes qui se divisent en interne. Les premiers bradyzoites n'apparaissent qu'à partir du 33ème jour. Au 80ème jour, les sarcocystes sont entourés d'une paroi entièrement différenciée et sont composés presque en totalité de bradizoïtes.
MOTS CLÉS : ultrastructure, Sarcocystis singaporinsis, rat de laboratoire, muscle, sarcocystes, développement.
I N T R O D U C T I O N
S
a r c o c y s t s o f Sarcocystis s p e c i e s b e c o m e e s t a b l i s h e d in t h e m u s c l e , initially as a single cell entity, t h e u n i - z o i t e ( M e h l h o r n et al., 1 9 7 5 ) . A s t h e y differentiate, t h e s a r c o c y s t t h e p r i m a r y w a l l d e v e l o p s f r o m a s i m p l e t o a usually e l a b o r a t e structure. E l e c t r o n m i c r o s c o p i c i m a g e s o f e a r l y - s t a g e s a r c o c y s t s o f a f e w s p e c i e s o f Sar- cocystis, S. cruzi ( b y P a c h e c o , S h e f f i e l d & F a y e r , 1 9 7 8 ) , a n d S. muriviperae ( P a p e r n a & F i n k e l m a n , 1 9 9 6 ) , h a v e b e e n p u b l i s h e d . D u b e y , S p e e r & F a y e r ( 1 9 8 9 ) p r e s e n t e d i m a g e s o f e a r l y d e v e l o p i n g s a r c o c y s t s o f S. rauschorum a n d S. singaporensis t o illustrate t h e p r o g r e s s i o n o f prim a r y w a l l structural d e v e l o p m e n t . T h e p u r p o s e o f t h e p r e s e n t c o m m u n i c a t i o n is t o d e s c r i b e t h e c o u r s e o f t h e fine structural d e v e l o p m e n t o f S. singaporensis s a r c o c y s t s in e x p e r i m e n t a l l y i n f e c t e d w h i t e rats (Rattus norvegicus):
t h e primary wall e v o l u t i o n a n d t h e transition f r o m m e t r o - c y t e s t a g e t o b r a d y z o i t e s .
M A T E R I A L S A N D M E T H O D S
S
p o r o c y s t s o f Sarcocystis singaporensis w e r e o b t a i n e d f r o m f e c e s o f a ) a n a t u r a l l y i n f e c t e d Python reticulatus f r o m S i n g a p o r e ; b ) a P. reticulatus o f S i n g a p o r e o r i g i n , b o r n in c a p t i v i t y a n d f e d o n w h i t e rats e x p e r i m e n t a l l y i n f e c t e d w i t h S i n g a p o r e - o r i g i n p a r a sites. T h e s p e c i f i c i d e n t i t y o f t h e i n f e c t i o n m a t e r i a l w a s v e r i f i e d f r o m h i s t o l o g i c a l l y e x a m i n e d w h i t e rats e x p o s e d t o s p o r o c y s t s e x t r a c t e d f r o m t h e s a m e f e c a l s a m p l e s , s a c r i f i c e d 4 5 t o 9 0 d a y s p o s t i n f e c t i o n ( d . p . i . ) . All s a r c o c y s t s in t h e m u s c l e s o f t h e s e rats ( 1 4 e x p o s e d t o s o u r c e a a n d f o u r t o s o u r c e b ) w e r e o f S. singaporensis.T h e s p o r o c y s t s w e r e e x t r a c t e d f r o m s n a k e f e c e s b y w a s h i n g t h r o u g h r e p e a t e d c e n t r i f u g a t i o n s , a n d u s e d i m m e d i a t e l y ( s o u r c e a ) o r after b e i n g s t o r e d in 2 % k a l i u m d i c h r o m a t e u n d e r refrigeration (in 4 ° C, s o u r c e b ) . S p o r o c y s t s w e r e w a s h e d o f f t h e k a l i u m d i c h r o m a t e b y r e p e a t e d c e n t r i f u g a t i o n . W h i t e rats w e r e i n f e c t e d b y f e e d i n g o n s p o r o c y s t d r o p p e d o n t o a small p i e c e o f b r e a d .
* Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences of the Hebrew University of Jeru- salem, Rehovot 76-100, Israel.
Correspondence: I. Paperna.
Tel.: 972 8 9488945 - Fax: 972 8 9465763.
E-mail: paperna@agri.huji.ac.il
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2002092161
Days post-
infection Rat no.
Sporocyst
dose Source Tissue studied
11 R21 22.000* a Esophagus
23 R8000 8.000 b Tongue & esophagus
3 3 R17 2.000 a Esophagus
80 R27 2.200 a Tongue
* Rats usually did not survive this dose; apparently, part of the applied sporocysts lost ability to infect, lowering the actual admi
nistrated infective dose.
Table I. – List of studied rats.
Rats were euthanized 22, 23, 33 and 80 d.p.i., doses and organs sampled for transmission electron micro
scopy (TEM) are detailed in Table I.
Light microscopic examination of tissue squashes veri
fied the presence of a sufficient concentration of sar
cocysts to ensure detection in semithin sections. For TEM, segments of infected muscles were fixed in 2.5 % glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) for 24 h at 4° C, rinsed repeatedly in the same buffer, post-fixed in 1.0 % osmium tetroxide in the cacodylate buffer for 1 h and, after rinsing in the same buffer, dehydrated in graded ethyl a l c o h o l s a n d e m b e d d e d in Agar 100 medium (Agar Scientific, Ltd., UK). Thin sections, cut on a Reichert Ultracut micro
tome with a diamond knife were stained on-grid with uranyl acetate and lead citrate, and examined with a Jeol 100CX TEM.
RESULTS
SARCOCYSTS IN RATS 2 2 AND 2 3 D.P.I
E
arliest-stage sarcocysts, seen in both 22 and 23 d.p.i. rats, were enclosed in a lighly undulating primary wall (PW) (Figs 1, 2) comprised of a continuous layer of parasitophorus vacuole (PV) membrane underscored by an electron-dense thickened layer, regularly interrupted by the so called pinocytotic- like indentations (Dubey et al, 1989) (Fig. 3).
Sarcocyst in longitudinal section (13-6 x 5.8 μm), dis
closed two metrocytes in early endodyogeny. Another showed four metrocytes in cross section, all at the onset of endodyogeny (Fig. 2).
Longitudinal sections (13.6 x 5.8 & 14.6 x 7.6 pm in size) of a seemingly more differentiated sarcocysts included eight metrocytes (Fig. 4), while the cross sec
tion (9 x 9.1 μm in size) again showed four metrocytes, at variable stages of endodyogeny. A metrocyte with fairly differentiated metrocyte progeny is shown in Figure 5. The PW of sarcocysts at this stage already s h o w e d s o m e w h a t m o r e p r o n o u n c e d undulation;
(enlarged view is shown in Figure 6).
In the rat euthanized 23 d.p.i., together with the early- stage sarcocysts, the tongue and esophagus muscles contained more advanced-stage sarcocysts with PW demonstrating the entire range of development, from folds (Figs 7, 8 ) to prolonged protrusions (Figs 9-12):
the PW folding intensified and deepened, while their surface b e c a m e increasingly interrupted by the regu
larly spaced invaginations the pinocytotic-like inden
tations (Fig. 8). In the more developed sarcocysts, PW consolidated into repeated elevations (Fig. 9) and ulti
mately into regularly spaced protrusions (Figs 10, 11).
Some sarcocysts PW in the 23 d.p.i. rat already demons
trated fully differentiated, elongated villar protrusions (Fig. 12), the entire length of which was filled with densely spaced pinocytotic-like indentations. With dif
ferentiation, sarcocysts increased in size and enlarged their metrocyte contents. Longitudinal sections of those with a folded surface reached 24 x 12 μm, the ones with indented PW reached 38 x 10 μm, and those already showing regular protrusions 72 x 11 μm. All sarcocysts at 22 and 23 d.p.i. contained only metro
cytes (Fig. 10); an endodyogeny yielding daughter metrocytes is shown in Figure 6. T h e sizes o f the sar
cocysts already exhibiting extended villar protrusions (Figs 10-12) were too large to b e evaluated from ultra- thin material.
SARCOCYSTS IN A 3 3 D.P.I. RAT
The villar protrusions already showed the characteristic separation into basal thickened indented wall, inter
rupted by numerous pinocytotic-like indentations, and a distal, thin-walled, vesicle-like extension containing only a few indentations (Fig. 13). Such sarcocysts still contained mainly undivided metrocytes (Fig. 14), but a few were yielding bradyzoites by e n d o d y o g e n y (Fig. 15). One, apparently a bradyzoite, with rhoptries and micronemes, appeared to contain primordia of endodyogeny progeny (Fig. 15).
SARCOCYSTS IN AN 8 0 D.P.I RAT
T h e s a r c o c y s t a p p e a r e d fully differentiated and contained predominantly bradyzoites, separated by septa into compartments (Fig. 16). The PW was com
prised of fully formed villar protmsions with a thin- walled vesicle-like projection arising from a thick- walled, indented stalk. T h e protrusions contained coarse granular substance (Fig. 17).
DISCUSSION
B
eaver & Maleckar (1981) and Brehm & Frank (1980) reported first detection of S. singapo- rensis sarcocysts on 22 and 25 d.p.i., respecti-Figs 1-5. - Young-stage sarcocysts from esophageal muscle of a 22 d.p.i. rat. Figs 1, 2. Sarcocysts containing metrocytes (*) enclosed in a shallow primary wall (PW, bold arrow); n, nucleus, fine arrow : next generation primordium; x 11,600 and x 12,000. Fig. 3. Enlarged view of the primary wall, arrowhead: pinocytotic-like indentation; x 18,460. Fig. 4. Longitudinal section of a sarcocyst with metrocytes under
going endodyogeny (fine arrows, emerging primordia), the primary wall (bold arrow) already with pronounced folds; x 8,580. Fig. 5. Sar
cocysts containing a metrocyte with developing daughter metrocyte; M, mitochondrium; n, nucleus; fine arrow: anterior end of the metro
cyte primordium. primary wall (bold arrow) in same stage as in Figure 4; x 11,500.
Figs 6-12. - Young-stage sarcocysts from tongue muscle of a 23 d.p.i. rat. Fig. 6. Enlarged view of a sarcocyst with increasingly folded pri
mary wall, x 18,460. Fig. 7. Sarcocyst containing daughter metrocyts and enclosed by primary wall (bold arrow) of deepened protrusions (fine arrow: apical region); x 9,960. Fig. 8. Enlarge view of a sarcocysts primary wall with increasingly deepening protrusions; x 18,460.
Fig. 9. Sarcocyst primary wall (bold arrow) already organized into rudimentary villar protrusions; x 10,500. Fig. 10. Sarcocyst with metro
cyte enclosed in a primary wall with evolved protrusion (bold arrow); x 8,828. Figs 11, 12. Close-up view of sarcocysts’ primary wall with already elaborate villar protrusions dotted with pinocytotic-like indentations (arrows); xx 14,520 and x 42,830 respectively.
Figs 13-15. - Sarcocysts from esophageal muscles of a 33 d p i . rat. Fig. 13. Details of villar protrusions with their basal portions (arrow
heads) and distal villi (arrow); x 9,000. Fig. 14. A sarcocyst with metrocytes dividing into daughter metrocytes; primary wall same as in Figure 13 ; x 7,260. Fig. 15. Sarcocysts with a mixed population of metrocytes (M) and bradyzoites (b); one zoite (T) with rhoptries and micronemes (x) is seemingly dividing by endodyogeny (arrowheads); x 5,940.
Figs. 16, 17. – Sarcocysts in the tongue muscle of a 80 d.p.i. rat. Fig. 16. View of the sarcocyst primary wall, and the bradyzoite contents compartmentalized by septa (arrowheads), x 4,620. Fig. 17. Enlarged view of the PW : v, vesicle-like projection; s, stalk; M, metrocyte (dege
nerate?) ; b, bradyzoite ; x 9,000.
vely. Jäkel et al. (2001) report first appearance o f sar
cocysts not before 30 d.p.i. In mice infected with S. muriviperae, sarcocysts were already observed 18 to 21 pi (Paperna & Finkelman, 1996). T h e merogony prior to the sarcocyst stage in Sarcocystis species deve
loping in larger intermediate hosts (ruminants) is longer ( 2 9 - 4 9 clays), but with similar pattern o f events (Pacheco et al, 1978, Dubey et al, 1989).
The unizoite shown by Mehlhorn et al. (1975), by Caw- thorn (in Dubey et al, 1989) and Paperna & Finkel
man (1996) was not seen in the presently studied mate
rial, and could have b e e n formed prior 22 d.p.i.
Sarcocysts in 23 and 33 d.p.i. rats contained metrocytes.
Initial bradyzoites formation was detected in some sarcocysts as early as 33 d.p.i. Sarcocysts in rats 80 d.p.i.
contained predominantly bradyzoites. Metrocytes pro
duced several generations of daughter metrocytes, and later also a transitional bradyzoite-like stage which pro
ceeded to divide by endodyogeny.
Worth noting is the asynchronous appearance of sar
cocysts at different stage of differentiation in rats fed a single dose of sporocysts. By day 23 there were already considerable discrepancies in the develop
mental stage o f sarcocysts, as evidenced by the varia
tions in the state o f differentiation o f their PW. This also suggests the onset o f sarcocyst development in muscles to b e earlier than 22 to 23 d.p.i.
Dubey et al. (1989) suggested that the PW o f S. sin- gaporensis sarcocysts requires nearly 200 days to b e c o m e fully formed. A prepatent period of 200 days seems to narrow the chances for effective transmission if the life span of brown (R. norvegiens) and house (R. rattus) rats in Singapore is comparable to the mean life expectancy o f 3 to 3.5 month estimated for Rattus species in Malaysia (Harrison, 1956; Medway, 1978).
It is not certain whether a full differentiation of the PW is a prerequisite for the infectivity of the sarcocyst's bra
dyzoites. In our experiments w e were already able to infect pythons with rats that were three months p.i. Our histological data from experimentally infected white rats show sarcocyst contents to b e dominated by bradyzoites as early as 47 d.p.i., compared with the predominantly metrocyte contents in sarcocysts from infections 33 d.p.i. and less (Paperna & Barlev, unpu
blished). In enzootic regions, the efficacy of transmis
sion appears to b e high. In Singapore, in forested habi
tats (where reticulated pythons are c o m m o n ) 74 % o f R. rattus were infected, (62 % o f rats < 100 g, and 87-
100 % > 100 g body mass; Paperna & Martelli, 2001).
ACKNOWLEDGEMENTS
T his study was supported by a fund from the Ministry o f Science o f the State Baden-Würtem- berg to the University o f Hohenheim and the
Hebrew University of Jerusalem's Faculty o f Faculty o f Agricultural, Food and Environmental Quality Sciences.
I wish to thank Ms Maya Ruso for technical assistance and Dr Paolo Martelli from the Veterinary Hospital o f Singapore Zoological Gardens for providing study material.
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Reçu le 2 août 2 0 0 1 Accepté le 1 2 novembre 2 0 0 1
