Parasitenkunde. (Odocoileus virginianus ) Ultrastructure of Sarcocystis sp. from the Muscle of a White-Tailed Deer

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1 Z Parasitenkd (1982) 68 : Zeitschrift for Parasitenkunde Parasitology Research 9 Springer-Verlag 1982 Ultrastructure of Sarcocystis sp. from the Muscle of a White-Tailed Deer (Odocoileus virginianus ) R. Entzeroth 1, B. Chobotar 2 and E. Scholtyseck 1 1 Abteilung f/it Protozoologie, Zoologisches Institut der Universit/it Bonn, Poppelsdorfer Schlo6, D-5300 Bonn 1, Federal Republic of Germany 2 Department of Biology, Andrews University, Berrien Springs, Michigan 49104, USA Abstract. Sarcocystis sp. from the muscle of naturally infected whitetailed deer (Odocoileus virginianus) was examined by transmission electron microscopy. The primary cyst wall forms regularly spaced protrusions filled with electron-lucent ground substance; no fibrils are present in the protrusions. The cysts are divided by septa into compartments containing typical coccidian metrocytes and merozoites. Taxonomy of the protozoon from the white-tailed deer-dog is discussed. Introduction Since 1969 several reports have been published on Sarcocystis of the whitetailed deer (Karstad and Trainer 1969; Pond and Speer 1979; Mahrt et al. 1980; Crum et al ; Entzeroth et al. 1982). Many species of Sarcocystis have been described from the cervid family in North America, Africa, Asia, and Europe (Crum et al ; Dubey 1980; Kaliner et al. 1975; Levchenko 1963; Ippen and Hilgenfeld 1961). The criteria most commonly used for the differentiation of species have been cyst size and cyst wall structure. This has led to the description of up to three species in a single host, either a wild or a domestic herbivore (Erber etal. 1978; Heydorn etal. 1975). Ultrastructural studies give more detailed information on the wall structure and cyst contents (Sanaud 1967; Mehlhorn et al. 1976; S6naud and Cernfi 1978; Entzeroth 1982). At the present time there is only one electronmicroscope study of a Sarcocystis from a North American cervid (Colwell and Mahrt 1981). The present paper describes the ultrastructure of a species of Sarcocystis from a naturally infected white-tailed deer (Odocoileus virgin- Janus). Offprint requests to: R. Entzeroth /82/0068/0033/$01.20

2 34 R. Entzeroth et al. Materials and Methods Samples of tongue, diaphragm, and abdominal muscle were collected from a recently shot white-tailed deer in Michigan [the same material that was used in the white-tailed deer-dog life cycle studies (Entzeroth et al. 1982)]. The tissues were fixed in 2.5% glutaraldehyde in phosphate buffer, ph 7.4. Samples found to be positive for Sarcocystis with the aid of frozen sections were washed in S6rensen's phosphate buffer, postfixed in 2.5% OsO 4 for 2 h, dehydrated in ethanol, then double-stained with uranyl acetate and phosphotungstic acid in 70% ethanol. After final dehydration in ethanol, the material was embedded in Dow Epoxy Resin (DER). Ultrathin sections were cut with glass knives on a Porter Blum ultramicrotome and examined in a Zeiss EM 9 $2 electron microscope. Results Esophagus, tongue, diaphragm, and abdominal muscle of white-tailed deer were found to be infected with Sarcocystis which had a preference for tongue muscle. All cysts were spindle-shaped and measured gm. Cyst wall Electron micrographs show that the cysts lodged within muscle cells are limited by a primary wall which ranges in thickness from 15 to 20 ~tm and consists of a membrane reinforced by osmiophilic material. This wall bears regularly spaced protrusions, measuring x 1.18 ~tm (Figs. 1, 2). In the distal part of the protrusions bordering the sarcoplasm of the host cell, the limiting primary cyst wall is underlined by a narrow band of electron-dense material. Distinct invaginations and folds of the cyst wall are located at the base of the protrusions. In this area the wall measures gm and forms a root-like pattern. The protrusions are filled with electron-lucent granular material which constitutes a part of the cyst ground substance. This substance forms also a 0.4 to 1.0 gm wide layer in the peripheral part of the cyst and gives rise to septa which divide the cyst into several compartments. The compartments are tightly packed with numerous merozoites; they contain also a few metrocytes. Merozoites and Metrocytes The banana-shaped merozoites fill the cyst compartments. They measure x ~tm and contain large numbers of mlcronemes and several rhoptries in their anterior third. A prominent nucleus, a mitochondrion, amylopectin granules, and lamellae of the rough endoplasmic reticulum are found in the posterior third of the cell. The merozoites are limited by a three-membrane pellicle. The spherical metrocytes are located most often at the periphery of the cyst. Uninucleate metrocytes are characterized by electron-lucent cytoplasm. In Fig. 3 we see a section through a binucleate metrocyte. The two nuclei are situated in granular cytoplasm which contains remnants of micronemes and rhoptries. Each metrocyte, limited by a three-membrane pellicle, gives rise to two merozoites by endodyogeny.

3 Ultrastructure of Sarcocystis sp. from the muscle of a white-tailed deer 35 Fig. 1. Section through the marginal part of a cyst showing the protrusions of the cyst wall (PR) adjacent to the sarcoplasm (SP) of the host cell. Note also the merozoites (ME) containing a mitochondrion (M/), micronemes (M/V), and a nucleus (N). They are situated in compartments formed by the septa (SE) of the cyst ground substance (GS). x 8,300 All electron micrographs represent sections through cysts of Sarcocystis sp_ from the tongue muscle of the white-tailed deer Discussion In this report the ultrastructure of Sarcocystis sp. from the white-tailed deer is described for the first time, The cysts found in muscle tissue appear thin-waited in the Iight microscope (Entzeroth et at. I982). The etectron

4 36 R. Entzeroth et al. Fig. 2. Tangential section through the cyst wall and host cell (SP). The root-like branching of the primary cyst wall (PW) is clearly evident at the base of the protrusions (PR). Note amylopectin (A) granules in the merozoite embedded in the ground substance (GS). x 19,400 Fig. 3. Binucleate (N) metrocyte with the remnants of the micronemes (MAr) and rhoptries (RH). x 24,400

5 Ultrastructure of Sarcocystis sp. from the muscle of a white-tailed deer 37 microscope observations revealed characteristic protrusions of the primary cyst wall similar to those of Sarcocystis sp. from Rattusfuscipes (Rzepczyk and Scholtyseck 1976), and Sarcocystis sp. from the roe deer (Schramlovfi and Bla~ek 1978; Entzeroth 1981). The apical part of the cyst wall protrusions is bounded by two layers. This unusual situation has not been reported from other species of Sarcocystis. The outer layer represents the original membrane of the parasitophorous vacuole which is strenghtened by electron-dense material. The origin of the inner layer is uncertain. At the basal part of the protrusions, the primary wall forms a root-like pattern which represents an increased surface as observed in other species of Sarcocystis (Mehlhorn et al. 1976). The merozoites of the presently examined Sarcocystis have the characteristics typical of all members of this genus (Scholtyseck 1979; Mehlhorn and Heydorn 1978; Porchet and Torpier 1977; S6naud 1967). The size of these stages corresponds to that of merozoites of Sarcocystis sp. from the goat (Chhabra and Mahajan 1978) and Sarcocystis sp. of the moose (Colwell and Mahrt 1981). A very important question which cannot be answered at the present time is whether the ultrastructure of the cyst wall of the Sarcocystis sp. from the white-tailed deer is related to that of other species of this genus. The cyst wall from the presently examined host is, however, very different from those reported from the moose (Colwell and Mahrt 1981) or the roe deer (Entzeroth 1982). In natural infections the intermediate host can harbor simultaneously several species of Sarcocystis (Mehlhorn and Heydorn 1978). Only further studies can reveal whether the fine structure of the cysts described in this paper is identical with that of Sarcocystis odocoileocanis (Crum et al. 1981) or with that of the second Sarcocystis sp., known from the white-tailed deer, that has the cat as the final host (Crum et al. 1981). There exists also the possibility that there are more species of Sarcocystis with the same intermediate-final host combination in the white-tailed deer, as reported from Sarcocystis of the roe deer (Erber et al. 1978) and of the horse (Dubey et al. 1977; Rommel and Geisel 1975). It will have to be ascertained also by transmission experiments if relationships exist between the cysts of Sarcocyxtis from white-tailed deer of the presently studied species and that of Sarcocystis hemionilatrantis from the mule deer (Hudkins and Kistner 1977). Acknowledgements. This investigation was supported by a Feodor Lynen Fellowship, from the Alexander von Humboldt Foundation (to R. Entzeroth, while at Andrews University) and by an Andrews University Research Grant and by a Faculty Development Grant from the Merck Foundation, Rahway, New Jersey (to B. Chobotar). References Chhabra MB, Mahajan RC (1978) Sarcocystis sp. from the goat in India. Vet Rec 103 : 56~563 Colwell DD, Mahrt JL (1981) Ultrastructure of the cyst wall and merozoites of Sarcocystis from moose (Alces alces) in Alberta, Canada. Z Parasitenkd 65:31~329

6 38 R. Entzeroth et al. Crum JM, Fayer R, Prestwood AK (1981) Sarcocystis spp. in white-tailed deer I. Definitive and intermediate host spectrum with a description of Sarcocystis odoeoileocanis n. sp. J Wildl Dis 17 : Dubey JP, Streitel RH, Stromberg PC, Toussant MJ (1977) Sarcocystis fayeri n. sp. from the horse. J Parasit 63 : Dubey JP (1980) Coyote as a final host for Sareocystis species of goats, sheep, cattle, elk, bison and moose in Montana. Am J Vet Res 41 : Entzeroth R (1981) Untersuchungen an Sarkosporidien (Mieschersche Schl/iuche) des einheimischen Rehwitdes (Capreolus capreolus L.). Z Jagdwiss 27 : Entzeroth R (1982) A comparative light and electron microscope study of the cysts of Sarcocystis species of Roe deer (Capreolus capreolus). Z Parasitenkd 66: Entzeroth R, Stuht N, Chobotar B, Scholtyseck E (1982) Sarcocystis of white-tailed deer (Odocoileus virginianus) and its transmission to the dog (Canis familiaris). Tropenmed Parasit 33: Erber M, Boch J, Barth D (1978) Drei Sarkosporidienarten des Rehwildes. Berl Mfinch Tier- 5xztl Wschr 91: Heydorn AO, Gestrich R, Mehlhorn H, Rommel M (1975) Proposal for a new nomenclature of Sarcosporidia. Z Parasitenkd 48 : Hudkins GG, Kistner TP (1977) Sarcocystis hemionilatrantis (sp. nov.) life cycle in mule deer and coyote. J Wildl Dis 13:80-84 Ippen R, Hilgenfeld M (1961) Ergebnisse der reihenm/ibigen Untersuchung des erlegten Rehwildes aus dem Wildforschungsgebiet Hakel bei Gatersleben. Beitr/ige zur Jagd- und Wildforschung I : Kaliner G (1975) Observations on the histomorphology of sarcosporidian cysts of some East African game animals (Artiodactyla). Z Parasitenkd 46:13-23 Karstad L, Trainer DO (1969) Sarcocystis in white-tailed deer. Bull Wildl Dis Ass:25-26 Levchenko NG (1963) Ein Fall yon Sarkosporidiose beim Rehwild (Capreolus capreolus) [Auf russisch]. Tr Inst Zool AN Kaz SSR 19: Mahrt JL, Colwell DD (1980) Sarcocystis in wild ungulates in Alberta~ J Wildl Dis 16: Mehlhorn H, Hartley WJ, Heydorn AO (1976) A comparative ultrastructural study of the cyst wall of 13 Sarcocystis species. Protistologica 12: Mehlhorn H, Heydorn AO (1978) The Sarcosporidia (Protozoa, Sporozoa): life cycle and fine structure. Adv. Parasitol. 16:43-93 Pond DB, Speer CA (1979) Sareocystis in free-ranging herbivores on the National Bison Range. J Wildl Dis 15:51-53 Porchet E, Torpier G (1977) Etude du germe infectieux de Sarcocystis tenella et Toxoplasma gondii par la technique du cryod6capage. Z Parasitenkd 54: Rommel M, Geisel O (1975) Untersuchungen fiber die Verbreitung und den Lebenszyklus einer Sarkosporidienart des Pferdes (Sarcoeystis equicanis n. spec.). Berl Miinch Tier~irztl Wschr 88: Rzepczyk C, Scholtyseck E (1976) Light and electron microscope studies on the Sarcocystis of Rattusfuscipes, an Australian rat. Z Parasitenkd 50: Scholtyseck E (1979) Fine Structure of Parasitic Protozoa. An Atlas of micrographs, drawings and diagrams. Berlin Heidelberg New York: Springer Schramlovfi J, Bla~ek K (1978) Ultrastruktur der Zystenwand der Sarkosporidien des Rehes (Capreolus capreolus L.). Z Parasitenkd 55:43-48 S+naud J (1967) Contribution fi l'6tude des Sarcosporidies et des Toxoplasmes (Toxoplasmea). Protistologica 3: S6naud J, Cernfi Z (1978) Le cycle de d6veloppement asexu~ de Sarcocystis dispersa (Cerna, Kolarova et Sulc 1977) chez la souris: Etude au microscope 61ectronique. Protistologica 14: Accepted May 10, 1982