Acta Parasitologica, 2004, 49(2), 93–101; ISSN 1230-2821 Copyright © 2004 W. Stefañski Institute of Parasitology, PAS Sarcocystis ramphastosi sp. nov. and Sarcocystis sulfuratusi sp. nov. (Apicomplexa, Sarcocystidae) from the keel-billed Stefański toucan (Ramphastos sulfuratus) J.P. Dubey1*, Emily Lane2 and Erna van Wilpe3 1Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, BARC-East, Building 1001, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA; 2P.O. Box 556, Derdepark, South Africa; 3Electron Microscope Unit, Faculty of Veterinary Science, University of Pretoria, South Africa Abstract Two new species of Sarcocystis, Sarcocystis ramphastosi sp. nov. and Sarcocystis sulfuratusi sp. nov. are described from a nat- urally infected keel-billed toucan (Ramphastos sulfuratus). Only sarcocysts were found and they were mature. Sarcocysts of S. ramphastosi were up to 3 mm long and up to 1 mm wide. The sarcocyst wall was smooth. The villar protrusions on the sar- cocyst wall of S. ramphastosi were up to 6.5 µm long and up to 3 µm wide; they were folded over the sarcocyst wall giving a thin-walled appearance. The microtubules in villar protrusions were smooth and confined to villar protrusions. Bradyzoites in sections were 4–4.5 × 1.3–1.6 µm in size. Sarcocysts of S. sulfuratusi were up to 900 µm long and up to 200 µm wide. The sarcocyst wall was smooth and thin-walled. The villar protrusions were up to 4.3 µm long and 1.4 µm wide. The microtubules in villar protrusions extended deeper into the granular layer of the sarcocyst wall and those in the granular layer were more elec- tron-dense than in the villar protrusions. Bradyzoites were 5.0–7.0 × 1.5–2.2 µm in size. Key words Sarcocystis ramphastosi, Sarcocystis sulfuratusi, Protozoa, toucan, Ramphastos sulfuratus Introduction Skóra sum (Didelphis virginianus) as the definitive host (Cawthorn et al. 1981; Box et al. 1984; Dubey et al. 1989, 2003). We re- Sarcocystis species are protozoan parasites with prey-predator port two new species of Sarcocystis, S. ramphastosi and S. sul- 2-host life cycles (Dubey et al. 1989). Herbivores (prey) are furatusi from the keel-billed toucan (Ramphastos sulfuratus). intermediate hosts and carnivores (predator) are the definitive hosts. The definitive host becomes infected by ingesting the asexual stage (sarcocyst) encysted in the tissues (muscles) of Materials and methods the intermediate host. The sexual cycle occurs only in the car- nivore host and it is restricted to the intestinal lamina propria. An adult toucan, which had been imported from Central Amer- Species of Sarcocystis are generally host-specific for their ica nine years ago died in captivity in Hillcrest, South Africa intermediate hosts. There are more than 100 species of Sarco- of iron storage disease. The bird was examined at necropsy cystis but life cycles of only a few of them are known (Dubey one day after death and tissues had undergone mild autolysis. et al. 1989). Multiple sections were immersed in 10% buffered neutral for- Little is known of the species of Sarcocystis in birds. There malin and routine histologic examination was performed on are 2 well known avian species, Sarcocystis rileyi with ducks paraffin-embedded sections (6 µm), stained with hematoxylin (Anas clypeata, Anas sp.) as intermediate host and the skunk and eosin (H and E). (Mephitis mephitis) as the definitive host and Sarcocystis fal- For transmission electron microscopy, formalin-fixed catula with passerine birds as intermediate hosts and the opos- muscle from neck was post-fixed in 1% osmium tetroxide in *Corresponding author: [email protected] 94 J.P. Dubey et al. Śląski Millonig’s phosphate buffer, rinsed in the same buffer, dehy- rate. The ultrathin sections were contrasted with uranyl ace- drated in ethanol and embedded in epoxy resin. Semithin sec- tate and lead citrate before examination in a Philips CM10 tions were stained with toluidine blue in 1% sodium tetrabo- transmission electron microscope operated at 80 kV. Fig. 1. Sarcocysts of Sarcocystis ramphastosi sp. nov. in histologic section of skeletal muscle of toucan. A and B. 5-µm section stained with hematoxylin and eosin. C-E. 1-µm section stained with toluidine blue. The thickness of the sarcocyst wall is indicated by opposing arrow- heads. A and B. Note thin sarcocyst wall (opposing arrowheads), a peripheral area with intact bradyzoites (small arrows) and a central degen- erating area (large arrows). C and D. Note variability in the thickness of the sarcocyst wall (opposing arrowheads and arrows) in the same sarcocyst and septa (se). E. A group of bradyzoites (b) separated by septa (se). Opposing arrows point to longitudinally cut bradyzoites Sarcocystis species in toucan Zdzisław 95 Stanisła Results reaction. In the H and E-stained sections two sizes of sarco- cysts were recognized, the large ones with a diameter of up to Grossly visible sarcocysts measuring approximately 3 × 1 mm 750 µm, and the small ones with a diameter of 200 µm. Be- were seen in cervical musculature, unassociated with any host cause both types of sarcocysts had thin walls and similar sized Fig. 2. TEM of sarcocyst walls of Sarcocystis ramphastosi sp. nov. A. Note three (1–3) villar protrusions (vp) at irregular distance, absence of vp in an area indicated by arrows, granular layer (gl) devoid of microtubules, septum (s) and bradyzoites (b). Sarcocyst no. 1. B. Higher magnification of a villar protrusion, showing parasitophorous vacuolar membrane (pvm), microtubules (mt), and granular layer (gl). The mi- crotubules at the villar tip are less electron-dense than at the base of the vp, and they do not extend into the granular layer. Sarcocyst no. 2 96 J.P. Dubey et al. Roborzyński rosbśźćv fjad kadsææ¿æ Fig. 3. TEM of a villar protrusions (vp) of Sarcocystis ramphastosi sp. nov. Higher magnification of villus no. 2 from Figure 2A. Note elec- tron-dense layer (edl) of varying thickness lining the parasitophorous vacuolar membrane (pvm). Note undulations on pvm (arrowheads). The microtubules (mt) are more dense at base (double arrows) than at the tip (single arrow) of the villus. There are no mt in the granular layer (gl) Sarcocystis species in toucan 97 Fig. 4. Bradyzoites of Sarcocystis ramphastosi sp. nov. A. From sarcocyst no. 1. B. From sarcocyst no. 2. Note conoid (c), pellicle with elec- tron-dense thickening (arrowheads) at the conoidal ends, few rhoptries (r) with long neck (double arrows), few micronemes (mi), subpellic- ular microtubules (mt) and a terminal nucleus (n) Fig. 5. Sarcocysts of Sarcocystis sulfuratusi sp. nov. from the toucan. Toluidine blue stain. Note extension of microtubules (arrowheads) in the granular layer of the sarcocyst 98 J.P. Dubey et al. bradyzoites they were considered one species. However, un- Sarcocystis ramphastosi sp. nov. (Figs 1–4) der oil immersion differences could be detected between the two types of sarcocysts. Electron microscopy revealed ultra- Description: Sarcocysts were 3 × 1 mm in size. In 5-µm sec- structural differences. New names are proposed for these two tions stained with H and E the sarcocyst had a thin (< 2 µm) types of sarcocysts because they are structurally distinct from and smooth sarcocyst wall (Fig. 1A, B). The interior of the known species of avian Sarcocystis species. sarcocyst was divided into compartments by prominent septa. Fig. 6. TEM of three sarcocysts of Sarcocystis sulfuratusi sp. nov. from the toucan. A. Sarcocyst no. 3. B. Sarcocyst no. 4. C. Sarcocyst no. 5. Note villar protrusions (arrowheads) extending into the granular layer (arrows), septa (s), and longitudinally cut bradyzoites (b) Sarcocystis species in toucan 99 Two distinct zones were recognized, an outer zone containing over the sarcocyst surface. The inner component consisted of intact bradyzoites and an inner central area with degenerated a smooth granular layer (gl) which was approximately 1 to 5 material (Fig. 1A, B). In 1-µm sections stained with toluidine µm thick (Fig. 1C, D). Bradyzoites were 3–5 µm long (Fig. blue, the sarcocyst wall was approximately 2–7 µm thick, de- 1E). pending on the plane of section. Two components of the sar- Two sarcocysts were examined ultrastructurally. The outer cocyst wall were recognized in 1-µm sections. The outer com- layer of the sarcocyst, the parasitophorous vacuolar mem- ponent consisted of villar protrusions (vp) that were folded brane (pvm) was wavy in outline and undulated (Figs 2 and 3). Fig. 7. TEM of villar protrusions (vp) of Sarcocystis sulfuratusi sp. nov. A. Sarcocyst no. 4. B. Sarcocyst no. 6. C. Sarcocyst no. 5. Note undu- lations (arrowheads) on parasitophorous vacuolar membrane (pvm), microtubules (mt) cut longitudinally in A, in cross-section in B and tan- gentially in C. The microtubules are more electron-dense than in the granular layer (gl) (large arrows) than in the vp 100 J.P. Dubey et al. The pvm was lined by a 40–50 nm thick electron-dense layer Intermediate host: Keel-billed toucan (Ramphastos sulfu- (edl) that was thicker at the undulated areas. The vp were ratus). spaced at irregular distances (Fig. 2A), were narrow at the tip Definitive host: Unknown. and wider in the basal half. The vp were up to 6.5 µm long and Distribution: Unknown. up to 3 µm wide. They contained smooth microtubules (mt) Specimens (syntypes) deposited: Histologic sections of that converged towards the base of the vp and that were more muscle deposited in the United States National Parasite electron-dense at the base than at the tip of the vp (Figs 2 and 3). Collection (USNPC) United States Department of Agriculture Microtubules were restricted to vp. The granular layer was Beltsville, Maryland 20705, USA. One section stained with H smooth and continued into the sarcocyst as septa.