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Sarcocystis crotali sp. n. with the mojave rattlesnake (Crotalus scutulatus scutulatus) — Mouse (Mus musculus) cycle
Arch. Protistenk. 129 (1985): 19-23
Zoologisches Institut der Universitat Bonn, BRD; Andrews University of Michigan, USA
Sareocueii» crotali Sp. n. with the Mojave Rattlesnake [Crotalus scuiulatu« scutulatus) Mouse (MU8 musculus] Cycle By ROLF ENZEROTH, BILL CHOBOTAR and ERICH SCHOLTYSECK With 4 Figures
Summary Sporulated oocysts (11.0 x 17.9 ,urn) and sporocysts (7.9 X 10.8 ,urn) were recovered from the intestine of a rattlesnake (Crotalus scutulatus scutulatus} and fed to mice (Mus musculus). All 6 experimental mice produced cysts in skeletal muscle typical of Sarcocystis species. The cysts (0.5 X 4 mm) had a non-st.riated thin and highly folded primary cyst wall, a prominent ground substance from which extended septa dividing the cyst into compartments. The parasite described herein is a reptile-rodent combination not previously reported and is named Sarcocystis crotali sp. n.
Introduction The developmental cycle of Sarcocystis has become adapted to the predator-prey relationship occurring between their hosts. The cycle is obligatorily heteroxenous with asexual development in the prey animal and the sexual in a predator. This has been confirmed by numerous studies involving a wide range of predator-prey combinations (DUBEY 1977; MEHLHORN and HEYDORN 1978; LEVINE and TADROS 1980). In the present study we report a combination involving the rattlesnake (Crotalus scuiulatue scuiulatue} and the mouse (Mus musculus},
Materials and Methods The rattlesnake was captured in San Bernadino County, California, and maintained at 23°C and fed coccidia-free laboratory reared Mus musculus for approximately 3 weeks after capture. At necropsy the feces were removed and mucosal scrapings taken for observation. The material was examined with a Zeiss photomicroseope II by preparation of wet-mount slides. The parasites were washed in a 2.5 % w(v aqueous potassium dichromate solution and stored at 4°C in water for several days before use. Six Swiss Webster commercially purchased coccidia-free mice were inoculated by gavage with approximately 5,000 sporulated sporocysts; two additional mice were maintained as uninoculated controls. One of the experimental mice died 67 d postinoculation (PI); the remainder were necropsied at 203 d PI (3 mice) and at 280 d PI (2 mice and the 2 controls). Muscle samples from the abdomen and flanks were fixed in phosphate buffered 3 % glutaraldehyde (0.2 M, pH 7.4) for 4 h. The tissue was washed in buffer, post-fixed in 2.5 % OS04 for 2 h, dehydrated in ethanol, double-stained in uranyl-acetate and phosphotungstic acid (1 % in 70 % ethanol). After complete dehydration and dearing in propylene oxide the material was embedded in Dow epoxy resin. Ultrathin sections were cut with glass knives on a Reichert ultramicrotome. The sections were stained with lead citrate and uranyl acetate and examined with a Zeiss EM 9 82 electron microscope. 2*
Figs. 1-3. Electron micrographs of a muscle cysts of Sarcocystis crotali sp. n. Fig. 1. Peripheral portion of a cyst cut tangentially showing uneven nature of the primary cyst wall (PW), the ground substance (GS), a septum (SE), a metrocyte (MC) with two developing daughter cells (DC) and merozoites with numerous micronemes (MN) and a nucleus (N). X 5,800. Figs. 2, 3. Interface between the host cell (HC) and the cyst wall (PW) showing details of the vesicles and stalks (arrows) of the primary cyst wall and the ground substance (GS). Note the wavey nature of wall in an earlier stage (Fig. 2, 203 d PI) which became relatively straight in later stages (Fig. 3, 280 d PI). X 22,000.
Sarcocystis crotali
21
Fig. 4. Light micrograph of an oocyst (OW) with two sporocysts (81') inside, and free sporocysts from the intestinal contents of the rattlesnake. X 1,450.
Results An estimated several 100,000 sporulated occysts and sporocysts were found in the feces and mucosal scrapings of the rattlesnake. The oocysts were ellipsoid with a thin, smooth, colourless, single-layered wall and each contained 2 sporocyst (Fig. 4). The oocysts measured 11.0 X 17.9.um (10.0-11.7 X 17.3-19.3) with the length-width ratio of 1.49 (1.2-1.73). The ellipsoid sporocysts had a smooth wall, no Stieda body, a residuum composed of numerous fine globules, and were mostly free of the oocyst walls (Fig. 4). Ten sporocysts measured 7.9 X 1O.8.um (7.6-10.4 X 10.4-11.7). The sporozoites were typically banana-shaped with one end more pointed than the other; a prominent refractile body was present near the rounded end. 5 sporozoites measured 2.4 x 7.4 ftm (2.2-2.7 x 6.9-8.2). All 6 of the experimental mice including the one that died 67 d PI were positive for sarcosysts. These were visible macroscopically as whitish opaque streaks running parallel with skeletal muscles fibers. Macroscopic and microscopic examination of the control mice revealed no sarcocysts, The cysts observed at necropsy were approximately 0.5 X 4 mm and had an overall smooth appearance with no striations. At ultrastructural levels the primary cyst wall consisted of an extensively folded dense membrane. These folds formed the lining of numerous spherical vesicles (about 40 nm in diameter) on short stalks (about 70 nm long (Figs. 1-3). The internal contents of the vesicles and stalks, and a short distance below formed a prominent electrondense zone at the periphery of the ground substance (Figs. 1-3). In somewhat younger cysts (203 d PI) the electron-dense zone followed an undulating or wavey course (Figs. 1, 2), whereas in the oldest cysts (280 d PI) this zone had apparently become stretched and was relatively smooth (Fig. 3). The ground substance was homogeneous and granular with a thickness averaging 1.0.um. At numerous places the ground substance branched into the interior of the cyst as fine septa forming numerous compartments occupied by the parasites (Fig. 1). Merozoites, the predominant form within the cysts, had typical ultrastructural features of coccidian sporozoites and rnerozoites. These included the pellicle of 3 membranes, the conoid, polar rings, numerous rnicronemes, rhoptries, subpellicular microtubules, polysaccharide granules, micropores, and a nucleus (Fig. 1). The occasional me-
22
R . ENTZEROTH et a l.
trocytes were at t he periphery of t he cysts with an elect ron-pale cytoplas m conta ining scat tered micron emes, several polysaccharide gra nules, an d usually 2 daugh t er cells developing by endody ogeny.
Discussion Several species of Sa rcocystis wit h a snake -rodent life cycle ha ve alread y been repor ted. These includ e rat -pyth on (R ZEPCZYK 1974 ; ZAMAN and COLLEY 1975, 1976) and deer mouse-gopher sna ke cycles (BLEDSOE 1980). I n t he pr esent st udy th e presence of sporulated oocysts and sporocysts in t he feces and intestina l tissues of t he sna ke, and t he resultan t sarcocysts produ ced in mice fed t he sna ke parasites is sufficient evidence for placing t his parasite in t he genus Sarcocystis. Alt hough we used la boratory mice, the host combi nation in our st udy is logical beca use small rodents found in t he snake habi tat likely serve as prey an imals. To t he best of our kno wledge th e host combination used by t he parasite in this study has not been previously re ported and represents a new species designated as Sar cocystis crotali sp. n. Th e fin structure of sarcocyst s has revealed consid era ble morphological variations a mong the species, and at different stages of cyst maturity. Basically muscle cysts are defined as " thick-walled " when finger-like projecti ons are pr esent, or "thin-walled " when t hese projections are absent (MEHLHORN et al. 1976 ; MEHLHORN and HEYDORN 1978; E NTZEROTH 1982). Cysts in t he present st udy a re t hin-walled a nd resemble those of S. muris , a nd S arcocystis sp. from t he whale and rhesus monkey (MEHLHORN et al. 1976; SHEFFIELD et a t. 1977; MEHLHORK and HEYDORN 1978). Th e study of S. idahoensis did not includ e an ultrastructural analysis, but high magn ification light mic rogra phs st rongly suggest a cyst morphology similar to t hat in or st udy (BLEDSOE 1980).
Zusammenfassung Sporuli erte Oc cy sben (11 ,0 x 17,9 Ilm ) u n d Sporocyst.en (7,9 x 10,8 /1m ) wurde n aus d em D a r minhale eine r Klapper schl ange ( Crotalus scu tulatus scutulatus ] gesa mmelt und a n Mause (Mus musculus ] verfiittert. Ail e 6 f u r diesen Versuch verwendeten Miiu se ha t t en typische SarkosporidienCy s t en in ih rer S ke lettm usk ulatur. D ie Cy sten (0,5 x 4 m m) b esaBen eine dimne , s tar k gefaltete p rimiire Cys tenwan d und eine a uffa llende Grun ds u bstan z. di e Septen bi ldete und di e Cyste in Kammer n a u ft eilte. In d iesen befanden sic h typ isc he Coecidien-Merozoiten u nd einige we nige Met rocyten . Di e vorliegen d e Arbeit berichtet uber eine weiter e R eptil -N ager -W irtkombina t ion n eb en den bere its bekannten, So mit liegt h ie r mit grofler Wahrsch einl ich k eit eine neue S ar cocystisArt v or, die den Namen Sarcocystis crotali sp . n. erhalt.
Acknowledgement S u pported in part b y a F eod or L ynen F ell ow ship of the Alex ander vo n Humboldt Foun d ation (t o R . ENTZEROTH) an d a F a cult y Development Grant to An d rew s U niversity by t he Merck F oundation, R ahwa y , N ew J ersey. W e a re grateful to Mr . D AVID REIH~;R who s up p lied t he rattl esn a ke for study.
Literature BLE DSOE, B. : Sarcocystis idahoensis sp, n . in d eer mi ce P erom qscus maniettlatus (WAGNER) a n d gophe r snakes P it uophis melanoleucus (DAUDIN). J. Prot oz ool. 27 (1980) : 93- 102. DUB EY, J . P.: T ox opl asm a, H am m ondia , B esn oitia , S arcocystis, and other Tissue Cyst-for ming Coccidia of Man a n d An imal s. In: .J. P. KR EIER (ed.): P ara siti c P rotozoa V ol. III. N ew Y ork
1977, 101-237.
Sarcocystis crotali
23
ENTZEROTH, R.: A comparative light and electron microscope study of cysts of Sarcocystis species of Roe deer (Capreolus capreolus). Z. Parasitenk. 66 (1982): 281~292. LEVINE, N. D., and TADROS, W.: Named species and hosts of Sarcocystis (Protozoa: Apicomp1exa: Sarcocystidae). Sys. Parasitol. 2 (1980): 41~59. MEHLHORN, H., HARTLEY, W. ,T., and HEYDORN, A. 0.: A comparative ultrastructural study of the cyst wall of 13 Sarcocystis species. Protistologica 12 (1976): 451~467. MEHLHORN, H., and HEYDORN, A. 0.: The Sarcosporidia (Protozoa, Sporozoa): Life cycle and fine structure. Adv. Parasitol. 16 (1978): 43~93. RZEPCZYK, C.: Evidence for a rat- snake life cycle for Sarcocystis. Int. ,J. Parasitol. 4 (1974): 447 to 449. SHEFFIELD, H. G., FRENKEL, J. K., and RUIZ, A.: Ultrastructure of the cyst of Sarcocystis muris. ,1. Parasitol. 63 (1977): 629-641. ZAMAN, V., and COLLEY, F. C.: Light and electron microscopic observations of the life cycle of Sarcocystis orientolis sp. n. in the rat [Iiattus norceqicus] and the Malaysian reticulated python (Python reticulatus}, Z. Parasitenk. 47 (1975): 169~185. ZAMAN, V., and COLLEY, F. C.: Replacement of Sarcocystis orientolis ZAMAN and COLLEY, by Sarcoeustis sinqa/porensis sp. n. Z. Parasitenk. 51 (1976): 137. Authors' addresses: Dr. ROLF ENTZEROTH and Dr. ERICH SCHOLTYSECK, Zoologisches Institut del' Universitnt Bonn, Abteilung fur Protozoologie, Poppelsdorfer SchloB, D . 5300 Bonn; Dr. BILL CHOBOTAR, Department of Biology, Andrews University, Berrien Springs, Michigan 49104, USA.
Zoologisches Institut der Universitat Bonn, BRD; Andrews University of Michigan, USA
Sareocueii» crotali Sp. n. with the Mojave Rattlesnake [Crotalus scuiulatu« scutulatus) Mouse (MU8 musculus] Cycle By ROLF ENZEROTH, BILL CHOBOTAR and ERICH SCHOLTYSECK With 4 Figures
Summary Sporulated oocysts (11.0 x 17.9 ,urn) and sporocysts (7.9 X 10.8 ,urn) were recovered from the intestine of a rattlesnake (Crotalus scutulatus scutulatus} and fed to mice (Mus musculus). All 6 experimental mice produced cysts in skeletal muscle typical of Sarcocystis species. The cysts (0.5 X 4 mm) had a non-st.riated thin and highly folded primary cyst wall, a prominent ground substance from which extended septa dividing the cyst into compartments. The parasite described herein is a reptile-rodent combination not previously reported and is named Sarcocystis crotali sp. n.
Introduction The developmental cycle of Sarcocystis has become adapted to the predator-prey relationship occurring between their hosts. The cycle is obligatorily heteroxenous with asexual development in the prey animal and the sexual in a predator. This has been confirmed by numerous studies involving a wide range of predator-prey combinations (DUBEY 1977; MEHLHORN and HEYDORN 1978; LEVINE and TADROS 1980). In the present study we report a combination involving the rattlesnake (Crotalus scuiulatue scuiulatue} and the mouse (Mus musculus},
Materials and Methods The rattlesnake was captured in San Bernadino County, California, and maintained at 23°C and fed coccidia-free laboratory reared Mus musculus for approximately 3 weeks after capture. At necropsy the feces were removed and mucosal scrapings taken for observation. The material was examined with a Zeiss photomicroseope II by preparation of wet-mount slides. The parasites were washed in a 2.5 % w(v aqueous potassium dichromate solution and stored at 4°C in water for several days before use. Six Swiss Webster commercially purchased coccidia-free mice were inoculated by gavage with approximately 5,000 sporulated sporocysts; two additional mice were maintained as uninoculated controls. One of the experimental mice died 67 d postinoculation (PI); the remainder were necropsied at 203 d PI (3 mice) and at 280 d PI (2 mice and the 2 controls). Muscle samples from the abdomen and flanks were fixed in phosphate buffered 3 % glutaraldehyde (0.2 M, pH 7.4) for 4 h. The tissue was washed in buffer, post-fixed in 2.5 % OS04 for 2 h, dehydrated in ethanol, double-stained in uranyl-acetate and phosphotungstic acid (1 % in 70 % ethanol). After complete dehydration and dearing in propylene oxide the material was embedded in Dow epoxy resin. Ultrathin sections were cut with glass knives on a Reichert ultramicrotome. The sections were stained with lead citrate and uranyl acetate and examined with a Zeiss EM 9 82 electron microscope. 2*
Figs. 1-3. Electron micrographs of a muscle cysts of Sarcocystis crotali sp. n. Fig. 1. Peripheral portion of a cyst cut tangentially showing uneven nature of the primary cyst wall (PW), the ground substance (GS), a septum (SE), a metrocyte (MC) with two developing daughter cells (DC) and merozoites with numerous micronemes (MN) and a nucleus (N). X 5,800. Figs. 2, 3. Interface between the host cell (HC) and the cyst wall (PW) showing details of the vesicles and stalks (arrows) of the primary cyst wall and the ground substance (GS). Note the wavey nature of wall in an earlier stage (Fig. 2, 203 d PI) which became relatively straight in later stages (Fig. 3, 280 d PI). X 22,000.
Sarcocystis crotali
21
Fig. 4. Light micrograph of an oocyst (OW) with two sporocysts (81') inside, and free sporocysts from the intestinal contents of the rattlesnake. X 1,450.
Results An estimated several 100,000 sporulated occysts and sporocysts were found in the feces and mucosal scrapings of the rattlesnake. The oocysts were ellipsoid with a thin, smooth, colourless, single-layered wall and each contained 2 sporocyst (Fig. 4). The oocysts measured 11.0 X 17.9.um (10.0-11.7 X 17.3-19.3) with the length-width ratio of 1.49 (1.2-1.73). The ellipsoid sporocysts had a smooth wall, no Stieda body, a residuum composed of numerous fine globules, and were mostly free of the oocyst walls (Fig. 4). Ten sporocysts measured 7.9 X 1O.8.um (7.6-10.4 X 10.4-11.7). The sporozoites were typically banana-shaped with one end more pointed than the other; a prominent refractile body was present near the rounded end. 5 sporozoites measured 2.4 x 7.4 ftm (2.2-2.7 x 6.9-8.2). All 6 of the experimental mice including the one that died 67 d PI were positive for sarcosysts. These were visible macroscopically as whitish opaque streaks running parallel with skeletal muscles fibers. Macroscopic and microscopic examination of the control mice revealed no sarcocysts, The cysts observed at necropsy were approximately 0.5 X 4 mm and had an overall smooth appearance with no striations. At ultrastructural levels the primary cyst wall consisted of an extensively folded dense membrane. These folds formed the lining of numerous spherical vesicles (about 40 nm in diameter) on short stalks (about 70 nm long (Figs. 1-3). The internal contents of the vesicles and stalks, and a short distance below formed a prominent electrondense zone at the periphery of the ground substance (Figs. 1-3). In somewhat younger cysts (203 d PI) the electron-dense zone followed an undulating or wavey course (Figs. 1, 2), whereas in the oldest cysts (280 d PI) this zone had apparently become stretched and was relatively smooth (Fig. 3). The ground substance was homogeneous and granular with a thickness averaging 1.0.um. At numerous places the ground substance branched into the interior of the cyst as fine septa forming numerous compartments occupied by the parasites (Fig. 1). Merozoites, the predominant form within the cysts, had typical ultrastructural features of coccidian sporozoites and rnerozoites. These included the pellicle of 3 membranes, the conoid, polar rings, numerous rnicronemes, rhoptries, subpellicular microtubules, polysaccharide granules, micropores, and a nucleus (Fig. 1). The occasional me-
22
R . ENTZEROTH et a l.
trocytes were at t he periphery of t he cysts with an elect ron-pale cytoplas m conta ining scat tered micron emes, several polysaccharide gra nules, an d usually 2 daugh t er cells developing by endody ogeny.
Discussion Several species of Sa rcocystis wit h a snake -rodent life cycle ha ve alread y been repor ted. These includ e rat -pyth on (R ZEPCZYK 1974 ; ZAMAN and COLLEY 1975, 1976) and deer mouse-gopher sna ke cycles (BLEDSOE 1980). I n t he pr esent st udy th e presence of sporulated oocysts and sporocysts in t he feces and intestina l tissues of t he sna ke, and t he resultan t sarcocysts produ ced in mice fed t he sna ke parasites is sufficient evidence for placing t his parasite in t he genus Sarcocystis. Alt hough we used la boratory mice, the host combi nation in our st udy is logical beca use small rodents found in t he snake habi tat likely serve as prey an imals. To t he best of our kno wledge th e host combination used by t he parasite in this study has not been previously re ported and represents a new species designated as Sar cocystis crotali sp. n. Th e fin structure of sarcocyst s has revealed consid era ble morphological variations a mong the species, and at different stages of cyst maturity. Basically muscle cysts are defined as " thick-walled " when finger-like projecti ons are pr esent, or "thin-walled " when t hese projections are absent (MEHLHORN et al. 1976 ; MEHLHORN and HEYDORN 1978; E NTZEROTH 1982). Cysts in t he present st udy a re t hin-walled a nd resemble those of S. muris , a nd S arcocystis sp. from t he whale and rhesus monkey (MEHLHORN et al. 1976; SHEFFIELD et a t. 1977; MEHLHORK and HEYDORN 1978). Th e study of S. idahoensis did not includ e an ultrastructural analysis, but high magn ification light mic rogra phs st rongly suggest a cyst morphology similar to t hat in or st udy (BLEDSOE 1980).
Zusammenfassung Sporuli erte Oc cy sben (11 ,0 x 17,9 Ilm ) u n d Sporocyst.en (7,9 x 10,8 /1m ) wurde n aus d em D a r minhale eine r Klapper schl ange ( Crotalus scu tulatus scutulatus ] gesa mmelt und a n Mause (Mus musculus ] verfiittert. Ail e 6 f u r diesen Versuch verwendeten Miiu se ha t t en typische SarkosporidienCy s t en in ih rer S ke lettm usk ulatur. D ie Cy sten (0,5 x 4 m m) b esaBen eine dimne , s tar k gefaltete p rimiire Cys tenwan d und eine a uffa llende Grun ds u bstan z. di e Septen bi ldete und di e Cyste in Kammer n a u ft eilte. In d iesen befanden sic h typ isc he Coecidien-Merozoiten u nd einige we nige Met rocyten . Di e vorliegen d e Arbeit berichtet uber eine weiter e R eptil -N ager -W irtkombina t ion n eb en den bere its bekannten, So mit liegt h ie r mit grofler Wahrsch einl ich k eit eine neue S ar cocystisArt v or, die den Namen Sarcocystis crotali sp . n. erhalt.
Acknowledgement S u pported in part b y a F eod or L ynen F ell ow ship of the Alex ander vo n Humboldt Foun d ation (t o R . ENTZEROTH) an d a F a cult y Development Grant to An d rew s U niversity by t he Merck F oundation, R ahwa y , N ew J ersey. W e a re grateful to Mr . D AVID REIH~;R who s up p lied t he rattl esn a ke for study.
Literature BLE DSOE, B. : Sarcocystis idahoensis sp, n . in d eer mi ce P erom qscus maniettlatus (WAGNER) a n d gophe r snakes P it uophis melanoleucus (DAUDIN). J. Prot oz ool. 27 (1980) : 93- 102. DUB EY, J . P.: T ox opl asm a, H am m ondia , B esn oitia , S arcocystis, and other Tissue Cyst-for ming Coccidia of Man a n d An imal s. In: .J. P. KR EIER (ed.): P ara siti c P rotozoa V ol. III. N ew Y ork
1977, 101-237.
Sarcocystis crotali
23
ENTZEROTH, R.: A comparative light and electron microscope study of cysts of Sarcocystis species of Roe deer (Capreolus capreolus). Z. Parasitenk. 66 (1982): 281~292. LEVINE, N. D., and TADROS, W.: Named species and hosts of Sarcocystis (Protozoa: Apicomp1exa: Sarcocystidae). Sys. Parasitol. 2 (1980): 41~59. MEHLHORN, H., HARTLEY, W. ,T., and HEYDORN, A. 0.: A comparative ultrastructural study of the cyst wall of 13 Sarcocystis species. Protistologica 12 (1976): 451~467. MEHLHORN, H., and HEYDORN, A. 0.: The Sarcosporidia (Protozoa, Sporozoa): Life cycle and fine structure. Adv. Parasitol. 16 (1978): 43~93. RZEPCZYK, C.: Evidence for a rat- snake life cycle for Sarcocystis. Int. ,J. Parasitol. 4 (1974): 447 to 449. SHEFFIELD, H. G., FRENKEL, J. K., and RUIZ, A.: Ultrastructure of the cyst of Sarcocystis muris. ,1. Parasitol. 63 (1977): 629-641. ZAMAN, V., and COLLEY, F. C.: Light and electron microscopic observations of the life cycle of Sarcocystis orientolis sp. n. in the rat [Iiattus norceqicus] and the Malaysian reticulated python (Python reticulatus}, Z. Parasitenk. 47 (1975): 169~185. ZAMAN, V., and COLLEY, F. C.: Replacement of Sarcocystis orientolis ZAMAN and COLLEY, by Sarcoeustis sinqa/porensis sp. n. Z. Parasitenk. 51 (1976): 137. Authors' addresses: Dr. ROLF ENTZEROTH and Dr. ERICH SCHOLTYSECK, Zoologisches Institut del' Universitnt Bonn, Abteilung fur Protozoologie, Poppelsdorfer SchloB, D . 5300 Bonn; Dr. BILL CHOBOTAR, Department of Biology, Andrews University, Berrien Springs, Michigan 49104, USA.