THE EUROPEAN BADGER (CARNIVORA : MUSTELIDAE) AS INTERMEDIATE HOST OF FURTHER THREE SARCOCYSTIS SPECIES (SPOROZOA)
ODENING K., STOLTE M., WALTER G., BOCKHARDT I.*
Three species of sarcocysts are described by light and electron microscopy from the European badger (Meles meles) : Sarcocystis hofmanni n. sp. (a species otherwise occurring in roe deer, Capreolus capreolus), S. sp., cf. sebeki (a species usually parasiti
zing certain murids), and S. melis η. sp. (a species presumably specific for the badger).
KEY WORDS : Meles meles. Capreolus capreolus. Sarcocystis hofmanni n.
sp. Sarcocystis sp., cf. sebeki. Sarcocystis melis η. sp. intermediate host specificity.
MOTS CLES : Meles meles. Capreolus capreolus. Sarcocystis hofmanni n.
sp. Sarcocystis sp., cf. sebeki. Sarcocystis melis n. sp. spécificité parasitaire (hôte intermédiaire).
Résumé :LE BLAIREAU EUROPÉEN (CARNIVORA : MUSTELIDAE), HOTE INTERMÉDIAIRE DE TROIS ESPECES SUPPLÉMENTAIRES DE SARCOCYSTIS (SPO- ROZOA).
Trois espèces de sarcocystes sont décrites au microscope optique et électronique chez le blaireau européen (Meles meles) : Sarcocystis hofmanni n. sp. (espèce par ailleurs rencontrée chez le chevreuil, Capreolus capreolus), S . sp., cf. sebeki (espèce parasitant habituel- lement certains muridés), et S. melis n. sp. (espèce probablement spécifique du blaireau).
Am o n g o t h e r s t h e f o l l o w i n g q u e s t i o n s are connected with findings o f sarcocysts in carni- vores : Are there some less known predator- prey relations, in which carnivores act as totally normal intermediate hosts o f Sarcocystis ? Or can carnivores b e hosts also o f sarcocysts, which otherwise ("usually") are found in herbivores, thus breaking the intermediate host specificity which as a rule is considered as being narrow. Quite recently the first record o f sarcocysts f r o m t h e E u r o p e a n b a d g e r h a s b e e n p u b l i s h e d (Odening et al., 1994), which very likely refers to a spe- cies usually parasitizing roe deer. Subsequent to this unusual finding three further Sarcocystis species have b e e n found in badgers (for survey see Table I ) , which are described and discussed in the following. Again one of these species is veiy similar to (and considered iden- tical with) a species o f roe deer, another is similar to a species from small mammals, whereas the third shows peculiar features not yet described.
MATERIALS AND METHODS
rom S e p t e m b e r 1992 to S e p t e m b e r 1 9 9 3 nine E u r o p e a n badgers
(Meles meles]w e r e investi- gated, all o f them victims o f traffic accidents
* Institute for Zoo Biology and Wildlife Research, PF 1103.
D-10252 Berlin, Germany.
in Land B r a n d e n b u r g a n d Berlin. S e v e n o f them sho- w e d slight infection with Sarcocystis in the muscula- ture (Table I ) . Samples o f m u s c l e tissue from tongue, diaphragm, o e s o p h a g u s , heart a n d skeletal m u c u l a - t u r e ( t h i g h , l o i n , t h o r a x , r i b s ) w e r e i n v e s t i g a t e d , usually in the fresh state. T h e sarcocysts found indivi- dually in the m u s c u l a t u r e w e r e e x t r a c t e d from the m u s c u l a r fibres u n d e r a d i s s e c t i n g m i c r o s c o p e for fresh-state examination a n d / o r p r o c e s s e d for histolo- gical and transmission electron m i c r o s c o p i c a l ( Т Е М ) investigations. F o r histological investigation 3-5 μητ paraffin sections w e r e m a d e a n d stained with H / E . A 4 % solution o f formaldehyde w a s u s e d for fixation.
Using the semithin sections or fresh preparations, the size o f the bradyzoites ( c y s t o z o i t e s ) was determined.
T h e length o f the bradyzoites w a s taken by measu
ring the m o r e or less b e n t median line from p o l e to pole. T h e width w a s m e a s u r e d at the widest diame
For ТЕМ investigation the sarcocysts w e r e fixed accor
ding to P o s p i s c h i l a n d v. B o m h a r d . After r e p e a t e d w a s h i n g with 0.1 m o l p h o s p h a t e buffer, they w e r e post-fixed or p r e - c o n t r a s t e d in 2 % o s m i u m tetroxid s o l u t i o n , d e h y d r a t e d in e t h a n o l a n d e m b e d d e d in E p o n 8 1 2 . After polymerisation for 3 days semithin and ultrathin sections w e r e made. T h e semithin sec
tions w e r e stained after Richardson. T h e ТЕМ investi
gations w e r e carried out using an EM 9 0 2 A (Zeiss).
Parasite, 1 9 9 4 , 1, 2 3 - 3 0 23
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1994011023
ODENING К., STOLTE M., WALTER G., BOCKHARDT I,
T a b l e I. - S u r v e y o f s i n g l e a n d m i x e d i n f e c t i o n o f E u r o p e a n b a d g e r s f r o m L a n d B r a n d e n b u r g a n d B e r l i n b e t w e e n S e p t e m b e r 1 9 9 2 a n d S e p t e m b e r 1 9 9 3 .
DESCRIPTION AND DISCUSSION OF THE SPECIES
SARCOCYTIS HOFMANNI N. SP. (Figs. 1-3) Synonyms
Sarcocystis sp. "type Rh 1" Bergmann and Kinder, 1976.
Sarcocystis s p . " t h i c k - w a l l e d cyst" S c h r a m l o v á a n d Blazek, 1 9 7 8 .
Sarcocystis sp. Erber et al., 1 9 7 8 .
Sarcocystis sp. "type 1, 2, 3" Entzeroth, 1 9 8 2 .
Sarcocystis gracilis in Entzeroth ( 1 9 8 5 ) and D u b e y et al. ( 1 9 8 9 ) , пес Rátz, 1 9 0 9 .
T h e n a m e o f t h e n e w s p e c i e s is a d e d i c a t i o n to Professor Dr. Reinhold R. Hofmann, to w h o m wildlife b i o l o g y is g r e a t l y i n d e b t e d for his r e s e a r c h w o r k , n a m e l y in r o e d e e r and E u r o p e a n badger.
Near Fürstenberg, about 7 0 k m NNW o f Berlin.
№ 2 : F e m a l e ; 1 1 . 8 k g in w e i g h t ; d e a d o n S e p t e m b e r 7, 1992 ; deep-frozen ; post-mortem e x a mination o n February 19, 1993
Heart mLtscle Description
T h e sarcocysts were 0.8-1.2 mm long in fresh state and 78 μιτι wide in formalin-fixed histological sections. T h e bradyzoites were elongated with semicircular poles, in part curved ; in semithin section they were 8.3-9·9μτη
long ( χ = 9-3 μπι, s = 0 . 6 1 ) and 2.3-3-6 μηι wide ( χ = 2.3 μπι, s = 0 . 3 0 ) ; in the transmission electron micro
graphs their size was 9-4-11.9 μιτι χ 2.3-3-6 μπι.
ТЕМ results : Cyst wall (without protrusions) 0.7-0.9 μπι wide. The surface o f the cyst wall showed palisade-like arranged finger-shaped protrusions. Their diameter was 1.1-1.4 μπι at the base, their length 6.3-6.6 μπι. The dis
tance between the protrusions was 0.2-0.3 μπι at their base. The core of the protmsions consisted of a granular substance, streaked with numerous filaments. The small elevations and fossule-like invaginations with underlying osmiophilic layer covered the-space between the finger- shaped protrusions. They were distinctly visible in the lower (proximal) third of the protrusions in our prepara
tions. The protrusions broadly arose from the cyst wall surface. They were rhombic to triangular in cross-section.
The specimen depicted in Fig. 2. Collection o f Protozoa, Institute for Zoo Biology and Wildlife Research, Alfred- Kowalke-Str. 17, D-10315 Berlin, Germany, No. В 2 / 1 9 9 3 (part of a sarcocyst embedded in Epon)
W e regard the E u r o p e a n b a d g e r as a devious inter
mediate host o f a s p e c i e s in this c a s e normally using r o e d e e r as intermediate host. W e n a m e this s p e c i e s here as a n e w o n e (S. hofmanni n. s p . ) , since it has n o valid n a m e either as a parasite o f r o e deer. Rátz ( 1 9 0 9 ) described a Sarcocystis s p e c i e s from r o e d e e r as S. gracilis. This n a m e is c o n n e c t e d with a compli
cated history. Babudieri ( 1 9 3 2 ) , in a review p a p e r o n Sarcocystis, e r r o n e o u s l y a s c r i b e d red d e e r (Cervus elaphus) as host to this s p e c i e s . As an o u t c o m e o f this mistake MachuPskij ( 1 9 4 7 ) and L e v c h e n k o ( 1 9 6 3 ) d e s c r i b e d n e w Sarcocystis s p e c i e s from r o e d e e r , Capreolus capreolus, in Central Siberia (S. sibirica)
T H E E U R O P E A N B A D G E R (CARNIVORA : MUSTELIDAE)
Figs. 1. et 2. - Sarcocystis hofmanni n. sp. from Meles meles.
1. Semithin section (cross-section) of a mature sarcocyst with cystozoites, showing the palisade-like cyst wall. Stain after Richardson 2. TE micrograph of cystozoites.
Mémoire 2 5
Fig. 3. - Sarcocystis hofmanni n. s p . f r o m Meles meles.
3. T E m i c r o g r a p h s o f t h e f i n g e r - s h a p e d p r o t r u s i o n s w i t h s m a l l e l e v a t i o n s o n t h e s u r f a c e ( a r r o w s ) . T h e p r o t a i s i o n s a p p e a r in t h e c r o s s - s e c t i o n a s r h o m b s ( b ) . C o r e o f t h e p r o t r u s i o n s with m a n y fine g r a n u l a ( g ) a n d l o n g i t u d i n a l l y r u i n i n g f i l a m e n t s ( F ) .
and Kirghizia (S. capreoli), because they believed there w o u l d n o t h a v e b e e n d e s c r i b e d a n y s p e c i e s o f Sarcocystis from Capreolus yet. T h e incorrect ascription of the host for S. gracilis was repeated also by Kalyakin and Zasukhin (1975) and Levine and Tadros (1980) in important review papers on Sarcocystis and corrected only by Levine (1986, 1988) (as a mistake o f the summa- rizing literature). In the original literature this mistake was not m a d e (with the e x c e p t i o n o f Blazek et al., 1978). There, however, another faulty development took p l a c e instead. Erber et al. ( 1 9 7 8 ) stated 3 s p e c i e s o f Sarcocystis in roe d e e r in Europe, already distingui- shable by light microscopy (meanwhile 4 species are known to occur in roe deer). They established one o f these species, completely justified, as S. gracilis [accor- ding to article 17 ( 2 ) o f the International C o d e for Zoological Nomenclature, ICZN], named another one as new (S. capreolicanis) and designated the third in "open nomenclature" as S. sp., because they could not ascer- tain the definitive host. After that Entzeroth (1985) desi- gnated this third species (S. sp.) as S. gracilis, without any reference to the decision by Erber et al. ( 1 9 7 8 ) . Unfortunately, Dubey et al. (1989) followed this procee- ding. T h e establishment o f S. gracilis by Erber et al.
(1978) was definite (in Rátz, 1909 obviously the figure and the Hungarian text, i. e. the full text, refer to diffe- rent species, at which the decision by Erber et al, 1978 relates to the figure). Moreover, it was completed by the reference to the ultrastructure o f the sarcocyst wall in the presentation by Bergmann and Kinder (1976), which is diagnostically important in the distinction o f species (it refers to type 10 of the cyst wall ultrastructure accor- ding to the classification by Dubey et al, 1989). A rene- wed subsequent ascription o f establishments once made is not admissible [provided these establishments were made in the sense o f article 17 ( 2 ) o f ICZN],
Erbel et ¿¡¡/.,(1978) did not n a m e Sarcocystis s p . o r
"type 3" from r o e d e e r w h i c h is morphologically not d i s c e r n a b l e from S. hofmanni n. sp., b e c a u s e t h e y b e l i e v e d that establishing o f the definitive host w o u l d b e n e c e s s a r y for the description o f a n e w Sarcocystis s p e c i e s ( w h i c h is, h o w e v e r , not true).
SARCOCYSMSW, CF. S. SEBEKI (TADROS AND LAARMAN,
1976) (Figs. 4-5) Places
Rhinow, about 7 5 km W N W o f Berlin ( b a d g e r n° 3 ) Near Fürstenberg, about 7 0 km NNW o f Berlin ( b a d - ger n° 2 )
M o n c a p r i c e , a b o u t 4 5 k m NNW o f B e r l i n ( b a d g e r n° 6 )
Milmersdorf, about 7 0 km NNO o f Berlin (badger n° 9 ) Hosts
№ 3 : Male ; 11.5 kg in w e i g h t ; d e a d o n J u n e 5, 1 9 9 2 ; d e e p - f r o z e n ; p o s t - m o r t e m e x a m i n a t i o n o n February 19, 1 9 9 3
№ 2 : F e m a l e , 11.8 kg in weight ; d e a d o n S e p t e m b e r 7, 1 9 9 2 ; deep-frozen, p o s t - m o r t e m e x a m i n a t i o n o n February 19, 1 9 9 3
№ 6 : F e m a l e , 11 kg in w e i g h t , d e a d o n J u n e 6, 1993 ; deep-frozen ; post-mortem e x a m i n a t i o n o n J u l y 2 8 / 2 9 , 1 9 9 3
№ 9 : F e m a l e , 17.8 kg in weight ; dead o n August 3 1 , 1 9 9 3 ; d e e p - f r o z e n ; p o s t - m o r t e m e x a m i n a t i o n o n S e p t e m b e r 24, 1 9 9 3
Thigh, loin, thorax, t o n g u e
26 M É M O I R E
ODENING К . , STOLTE M., WALTER G., BOCKHARDT I.
THE EUROPEAN BADGER (CARNIVORA : MUSTE1IDAE)
Figs. 4, 5- - Sarcocystis sp., cf. sebeki from Meles meles.
4. Semithin section (cross-section) of a mature sarcocyst with cystozoites, showing a "thin" cyst wall and empty compartments in the centre. Stain after Richardson. 5. TE micrographs of the cyst wall with the small elevations on the surface (arrows).
Substance in the core of the small elevations more compact than the ground substance of the cyst wall.
Sarcocysts 6.5-9-0 m m long and 1 7 2 - 2 0 0 μπι wide in fresh state. Bradyzoites squatly fusiform, in fresh state 6.1-7.2 μπι long ( χ = 6.9 vim, s = 0 . 2 6 ) and 1.6-2.2 μπι w i d e ( χ = 1.9 pm, s = 0 . 2 4 ) ; in semithin section 5.7-6.3 p m long ( χ = 5.9 μιτι, s = 0 . 2 9 ) and 1.7-2.2 Vim w i d e ( χ = 2.0 μιτι, s = 0 . l 6 ) .
ТЕМ results: T h e cyst wall was 0.9-1-4 μπι wide. There were n o protnisions. T h e small elevations o f the pri
mary cyst wall w e r e 0.08-0.09 μιτι long, the fossule-like invaginations in b e t w e e n had a maximum diameter o f
0.05 μπι. T h e i r distance from e a c h other was about 0.05 μπι. T h e small elevations and invaginations were underlayed with an osmiophilic layer. T h e ground sub
stance (granular layer) o f the cyst wall was fine-grai
ned and c o n d e n s e d within the small elevations.
This s p e c i e s s h o w s type 1 o f the cyst wall ultrastruc
ture (according to the classification b y D u b e y et al., 1 9 8 9 ) . W e assign it n e a r S. sebeki (cf. T a d r o s and Laarman 1 9 7 6 , 1 9 7 8 , 1 9 7 9 , 1 9 8 0 , 1 9 8 2 ) , b e c a u s e it is
Mémoire - 2 7
O D E N I N G К . , S T O L T E M . , W A L T E R G . , B O C K H A R D T I .
Figs. 6, 7. - Sarcocystis melis η. sp. from Meles meles.
6. Two interwined sarcocysts in the fresh state, for the most part extracted from the muscular fibres 7. Released cystozoites in the fresh state.
FIGS. 8, 9. - Sarcocystis melis η. sp. from Meles meles.
Η. ΤΗ micrograph of a bradyzoite.
9. TE micrographs of the cyst wall with the filiform protrusions (arrows), which are streaked with parallel running fibrillary elements (F).
m o r p h o l o g i c a l l y v e r y s i m i l a r t o t h i s s p e c i e s a n d b e c a u s e it w a s already earlier s u p p o s e d that S. sebeki c o u l d i n f e c t Mustela nivalis a s w e l l , a m u s t e l i d , b e s i d e its "normal" intermediate hosts f Apodem us syl- vaticus and Mus musculus; the definitive host is Strix aluco) ( T a c h o s a n d L a a r m a n , 1 9 7 9 ) . T a c h o s a n d Laarman ( 1 9 7 9 ) found sarcocysts in the musculature o f a E u r o p e a n w e a s e l , d e s c r i b e d t h e m by light micro
s c o p y and fed t h e m to a tawny owl, in w h i c h a w e a k infection was obtained.
SARCOCYSW MELIS N. SP. (Figs. 6-9)
Genitive singular o f meles (Latin = b a d g e r ) Place
Berlin ( G r u n e w a l d )
THE EUROPEAN BADGER (CARNIVORA : MUSTELIDAE)
ODENING К., STOLTE M., WALTER G., BOCKHARDT I.
o f Sarcosporidia infecting roe deer. Foi. Parasito!.
(Praha), 1978, 25. 95-96.
Dubey J . P.. Speer С. Α., Fayer R. : Sarcocystosis of animals and man. CRC Press. Inc.. Boca Raton. Florida, 1989. 215 p.
Entzeroth R. : A comparative light and electron microscope study of the cysts o f Sarcocystis species o f roe deer (Capreolus capreolus). Z. Parasitenkd., 1982. 66. 281-292.
Entzeroth R. : Light-, scanning-, and transmission electron microscope study of the cyst wall of Sarcocystis gracilis Rátz, 1909 ( S p o r o z o a , C o c c i d i a ) from the roe deer (Capreolus capreolus L.). Arch. Protistenkd., 1985. 129.
Erber M., Boch J . , Barth D. : Drei Sarkosporidienarten des Rehwildes. Beri. Münch. Tierãrztl. Wochenschr., 1978, 91. 482-486.
Kalyakin V. N.. Zasukhin D. N. : Distribution of Sarcocystis (Protozoa: S p o r o z o a ) in vertebrates. Pol. I'arasitol.
rPrahaA 1975. 22. 289-307.
Levchenko N. G. : A case of sarcosporidiosis in roe deer Capreolus capreolus /in Russian!. Trudy Instituía Zoologii Akad. Nauk Kazakh. SSR (Alma-Ata), 1963. 19. 244-245.
Levine N. D. : The taxonomy o f Sarcocystis (Protozoa, Apicomplexa) species./. Parasitai., 1986, 72, 372-382.
Levine N. D.: The protozoan phylum Apicomplexa. Vol. II.
CRC Press. Inc.. Boca Raton. Florida. 1988, 154 p.
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Parasitol., 1980. 2. 41-59.
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Inst. , 1947. 3. 87-92.
Odening K., Stolte M., Walter G.. Bockhardt I., Jakob. W.: Sarcocysts (Sarcocystis sp.: Sporozoa) in the European badger Meles meles. Parasitology, 1994, 108, in press.
Rátz I. : Die Sarcosporidien und ihre in Ungarn vorkom- menden Arten [in Hungarian]. Allattani Közleinények.
1909, 8, 1-37, Tab. I-III ; 91-95 [German review].
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Parasitenkd.. 1978, 55. 43-48.
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1976, 44. 1-107.
Tadros W., Laarman J. J.: A comparative study of the light and electron microscopic structure of the walls of the muscle cysts of several species of sarcocystid eimeriid Coccidia.
Рте. К. Nederl. Akad Wet.. Ser. С 1978, 81, 469-491.
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Tadros W.. Laarman J . J . : Further investigations on the life history and t r a n s m i s s i o n o f Sarcocystis sebeki o f Apodemus sylraticus. Trop. Geograph. Med., 1980, 32. 362.
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Accepté le 2 décembre 1993
3 0 Mémoire
№ 4 : F e m a l e ; 8 kg in weight ; dead on March 3, 1 9 9 3 ; post-mortem examination on March 22, 1 9 9 3 . Localisation
Sarcocysts 2-5 mm long in fresh state and 51-71 μιη wide in histological sections ( 6 0 μιη in semithin sec
t i o n s ) . C o m p a r t m e n t s p o l y h e d r a l , with m a x i m u m 7 sides, 9.3-11.0 μιη wide (x = 9.6 μηι, s = 1.42) and 7 . 1 - 13.8 μηι d e e p (χ = 11.6 μηι, s = 2.33). Bradyzoites fusi
form, 6.9-8.2 μιη long (χ = 7.4 μιη, s = 0 . 5 4 ) and 0.8-1.7 μηι wide ( χ = 1.4 μηι. s = 0.28) in semithin sections.
ТЕМ results: Cyst wall 0.4-0.6 μπι wide. Small "eleva
tions" and fossule-like invaginations (diameter about 0.05 μηι) of the primary cyst wall poorly pronounced.
The primary cyst wall consisted proximally (internally) of an osmiophilic layer. Peculiarly formed filiform pro
trusions arose from the surface in irregular, mostly grea
ter distances. They lay close to the primary cyst wall or overlapped it. O n e o f them, well visible, was 0.5 μιη long and at its base 0.07 μηι wide. The filiform protru
sions were streaked with several parallel, longitudinally running fibrillar elements, which partially p r o c e e d e d into the ground substance o f the cyst wall.
T h e s p e c i m e n l y i n g b e h i n d Fig. 8. C o l l e c t i o n o f P r o t o z o a , I n s t i t u t e for Z o o B i o l o g y a n d W i l d l i f e R e s e a r c h . A l f r e d - K o w a l k e - S t r . 17, D - 1 0 3 1 5 B e r l i n . Germany, No. В 3 / 1 9 9 3 (part o f a sarcocyst e m b e d ded in E p o n ) .
S. melis η. sp. has an ultrastructure o f the cyst wall h i t h e r t o n o t d e s c r i b e d ( c f . D u b e y et al.. 1 9 8 9 ) . Therefore, it could refer to a s p e c i e s specific to the European badger.
It could b e that the characteristic filiform protrusions are longer than stated in our description, if our figure refers to first cuts o f folded protrusions, or if longer hair-like formations should be broken off. Both would not call into question the peculiarity of the structures described.
Babudieri Β. : I sarcosporidi e le sarcosporidosi. (Studio monografico). Arch. Protistenkd., 1932, 76. 421-580.
Bergmann V., Kinder E. : E l e k t r o n e n m i k r o s k o p i s c h e Untersuchungen zur Wandstruktur von Sarkozysten in der Skelettmuskulatur von Wildschwein unci Reh. Mon.h.
Vet.med.. 1976, 3 1 . 785-788.
Blazek K... Schramlová J . . Ippen R.: Dog as definitive host