Sarcocystis Ramphastosi Sp. Nov. and Sarcocystis Sulfuratusi Sp. Nov

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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 sarcocyst 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 electron-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 arrowheads. 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 sarcocysts 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 microtubules 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.

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Fig. 3. TEM of a villar protrusions (vp) of Sarcocystis ramphastosi sp. nov. Higher magnification of villus no. 2 from Figure 2A. Note electron-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 electron-dense thickening (arrowheads) at the conoidal ends, few rhoptries (r) with long neck (double arrows), few micronemes (mi), subpellicular 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 undulations (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. and E (USNPC no. 094501) and 4 sections stained with tolu- Both sarcocysts were mature and contained fully formed idine blue (USNPC no. 094502). bradyzoites (Fig. 4). Longitudinally cut bradyzoites were 4.0–4.5 × 1.3–1.6 µm (n = 10) in size. Bradyzoites contained Sarcocystis sulfuratusi sp. nov. (Figs 5–8) a conoid, few micronemes, few rhoptries (1–2 per section), subpellicular microtubules, and a posteriorly located nucleus. Description: In H and E-stained sections sarcocysts were up to The pellicle was thickened towards the conoidal part of the 900 µm long and up to 200 µm wide. Sarcocyst walls were bradyzoite (Fig. 4). thin-walled (< 2 µm wide) and smooth. In 1-µm toluidine

Fig. 8. TEM of bradyzoites of Sarcocystis sulfuratusi sp. nov. A. From sarcocyst no. 3. B. From sarcocyst no. 7. Note conoid (c) pellicular thickening (arrowheads) at the conoidal end, few micronemes (mi), rhoptries (r) with a long neck (double arrows), a terminal nucleus (n) and subpellicular microtubules (mt) Sarcocystis species in toucan 101

blue-stained sections the sarcocyst wall had dark-stained areas distinctive microtubules that extend half way in to the granu- (Fig. 5A, B) that were found to be microtubules by transmis- lar layer and they are more electron-dense in the granular layer sion electron microscopy (TEM). than in vp. Five sarcocysts were examined ultrastructurally. The pvm was undulated and lined by a 32 nm electron-dense layer (edl). Acknowledgements. The authors thank Mr. William Horsfield of The vp were up to 4.3 µm long and up to 1.4 µm wide and Amazona, and Dr Chris Kingsley of Dendravia Consulting Room for providing the case material, and the staff at the Histology Laboratory, were folded over the sarcocyst surface (Fig. 6). The micro- Faculty of Veterinary Science, University of Pretoria for their help. tubules in the vp were smooth, they extended deeper into the granular layer (Fig. 7) and their structure varied depending on the location. The mt in the granular layer (gl) were more elec- References tron-dense than in the villar protrusions. The gl was smooth. All sarcocysts were mature and contained bradyzoites. Box E.D., Meier J.L., Smith J.H. 1984. Description of Sarcocystis The bradyzoites were 5.0–7.0 × 1.5–2.2 µm (n = 10) in size falcatula Stiles, 1893, a parasite of birds and opossums. Jour- nal of Protozoology, 31, 521–524. (Fig. 8). Bradyzoites contained a conoid, a few rhoptries and Cawthorn R. J., Rainnie D., Wobeser G. 1981. Experimental trans- micronemes, subpellicular microtubules, amylopectin and a mission of Sarcocystis sp. (Protozoa: Sarcocystidae) between terminal nucleus. The pellicle at the conoidal end was more the shoveler duck (Anas clypeata) and the striped skunk (Me- electron-dense (Fig. 8). phitis mephitis). Journal of Wildlife Diseases, 17, 389–394. The information on hosts, location, and specimens depo- Drouin T.E., Mahrt J.L. 1980. The morphology of cysts of Sarco- sited is the same as stated for S. ramphastosi. Both species of cystis infecting birds in western Canada. Canadian Journal of Zoology, 58, 1477–1482. Sarcocystis were present in same histologic sections. Dubey J.P., Odening K. 2001. Toxoplasmosis and related infections. In: Parasitic diseases of wild mammals (Eds. W.M. Samuel, M.J. Pybus and A.A. Kocan). Iowa State University Press, Discussion Ames, 478–519. Dubey J.P., Cawthorn R.J., Speer C.A., Wobeser G.A. 2003. Rede- scription of the sarcocysts of Sarcocystis rileyi (Apicom- Complete life cycles of Sarcocystis are known for only a few plexa: Sarcocystidae). Journal of Eukaryotic Microbiology, species of animals, mostly those in livestock (Dubey et al. 50, 476–482. 1989). Most Sarcocystis species have been named based on Dubey J.P., Garner M.M., Stetter M.D., Marsh A.E., Barr B.C. their host occurrence and structure. Among all taxonomic cri- 2001a. Acute Sarcocystis falcatula-like infection in a carmine teria, the structure of the sarcocyst wall is considered the most bee-eater (Merops nubicus) and immunohistochemical cross reactivity between Sarcocystis falcatula and Sarcocystis neu- valuable for dividing species within a given host. Dubey et al. rona. Journal of Parasitology, 87, 824–831. (1989) and Dubey and Odening (2001) recognized 35 types of Dubey J.P., Johnson G.C., Bermudez A., Suedmeyer K.W., Fritz D.L. sarcocyst walls based on their structure. The two types of sar- 2001b. Neural sarcocystosis in a straw-necked ibis (Carphibis cocysts found in the present study were structurally distinct spinicollis) associated with a Sarcocystis neurona-like organ- from those described previously. They are different from two ism and description of muscular sarcocysts of an unidentified Sarcocystis species. Journal of Parasitology, 87, 1317–1322. named avian species, S. rileyi and S. falcatula. The sarcocyst Dubey J.P., Lindsay D.S., Rezende P.C.B., Costa A.J. 2000. Char- wall in S. rileyi is type 21 with cauliflower-like anastomosing acterization of an unidentified Sarcocystis falcatula-like par- villar protrusions (Dubey et al. 1989, 2003). The sarcocyst asite from the South American opossum, Didelphis albiven- wall in S. falcatula is thick, striated, with finger-like villar tris from Brazil. Journal of Eukaryotic Microbiology, 47, protrusions (Drouin and Mahrt 1980; Box et al. 1984; Dubey 538–544. Dubey J.P., Rosenthal B.M., Speer C.A. 2001c. Sarcocystis lindsayi et al. 2000, 2001a, b, c; Luznar et al. 2001). n. sp. (Protozoa: Sarcocystidae) from the South American The villar protrusions in S. ramphastosi resemble sarco- opossum, Didelphis albiventris from Brazil. Journal of Eu- cyst wall type 9 of Dubey et al. (1989). However, in S. ram- karyotic Microbiology, 48, 595–603. phastosi the villar protrusions are at irregular distances, the Dubey J.P., Speer C.A., Fayer R. 1989. Sarcocystosis of animals and microtubules do not contain electron-dense granules and do man. CRC Press, Boca Raton, Florida, 1–215. Luznar S.L., Avery M.L., Dame J.B., MacKay R.J., Greiner E.C. not extend into the granular layer whereas the vp in type 9 2001. Development of Sarcocystis falcatula in its intermedi- either have electron-dense granules or extend deeper into the ate host, the brown-headed cowbird (Molothrus ater). granular layer. The villar protrusions in S. sulfuratusi have Veterinary Parasitology, 95, 327–334.

(Accepted February 24, 2004)