Sarcocystis ramphastosi sp. nov. and Sarcocystis sulfuratusi sp. nov ...

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 naturally 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

Sarcocystis species are protozoan parasites with prey-predator 2-host life cycles (Dubey et al. 1989). Herbivores (prey) are 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 the intermediate host. The sexual cycle occurs only in the carnivore host and it is restricted to the intestinal lamina propria. Species of Sarcocystis are generally host-specific for their intermediate hosts. There are more than 100 species of Sarcocystis but life cycles of only a few of them are known (Dubey et al. 1989). Little is known of the species of Sarcocystis in birds. There are 2 well known avian species, Sarcocystis rileyi with ducks (Anas clypeata, Anas sp.) as intermediate host and the skunk (Mephitis mephitis) as the definitive host and Sarcocystis falcatula with passerine birds as intermediate hosts and the opos-

*Corresponding

sum (Didelphis virginianus) as the definitive host (Cawthorn et al. 1981; Box et al. 1984; Dubey et al. 1989, 2003). We report two new species of Sarcocystis, S. ramphastosi and S. sulfuratusi from the keel-billed toucan (Ramphastos sulfuratus).

Materials and methods An adult toucan, which had been imported from Central America nine years ago died in captivity in Hillcrest, South Africa of iron storage disease. The bird was examined at necropsy one day after death and tissues had undergone mild autolysis. Multiple sections were immersed in 10% buffered neutral formalin and routine histologic examination was performed on paraffin-embedded sections (6 µm), stained with hematoxylin and eosin (H and E). For transmission electron microscopy, formalin-fixed muscle from neck was post-fixed in 1% osmium tetroxide in

author: [email protected]

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Śląski Millonig’s phosphate buffer, rinsed in the same buffer, dehydrated in ethanol and embedded in epoxy resin. Semithin sections were stained with toluidine blue in 1% sodium tetrabo-

rate. The ultrathin sections were contrasted with uranyl acetate and lead citrate before examination in a Philips CM10 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 degenerating 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

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Sarcocystis species in toucan

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Results Grossly visible sarcocysts measuring approximately 3 × 1 mm were seen in cervical musculature, unassociated with any host

reaction. In the H and E-stained sections two sizes of sarcocysts were recognized, the large ones with a diameter of up to 750 µm, and the small ones with a diameter of 200 µm. Because 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

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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)


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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

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bradyzoites they were considered one species. However, under oil immersion differences could be detected between the two types of sarcocysts. Electron microscopy revealed ultrastructural differences. New names are proposed for these two types of sarcocysts because they are structurally distinct from known species of avian Sarcocystis species.

Sarcocystis ramphastosi sp. nov. (Figs 1–4) Description: Sarcocysts were 3 × 1 mm in size. In 5-µm sections stained with H and E the sarcocyst had a thin (< 2 µm) and smooth sarcocyst wall (Fig. 1A, B). The interior of the 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)


Two distinct zones were recognized, an outer zone containing intact bradyzoites and an inner central area with degenerated material (Fig. 1A, B). In 1-µm sections stained with toluidine blue, the sarcocyst wall was approximately 2–7 µm thick, depending on the plane of section. Two components of the sarcocyst wall were recognized in 1-µm sections. The outer component consisted of villar protrusions (vp) that were folded

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over the sarcocyst surface. The inner component consisted of a smooth granular layer (gl) which was approximately 1 to 5 µm thick (Fig. 1C, D). Bradyzoites were 3–5 µm long (Fig. 1E). Two sarcocysts were examined ultrastructurally. The outer layer of the sarcocyst, the parasitophorous vacuolar membrane (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 tangentially in C. The microtubules are more electron-dense than in the granular layer (gl) (large arrows) than in the vp

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The pvm was lined by a 40–50 nm thick electron-dense layer (edl) that was thicker at the undulated areas. The vp were spaced at irregular distances (Fig. 2A), were narrow at the tip and wider in the basal half. The vp were up to 6.5 µm long and up to 3 µm wide. They contained smooth microtubules (mt) that converged towards the base of the vp and that were more electron-dense at the base than at the tip of the vp (Figs 2 and 3). Microtubules were restricted to vp. The granular layer was smooth and continued into the sarcocyst as septa. Both sarcocysts were mature and contained fully formed bradyzoites (Fig. 4). Longitudinally cut bradyzoites were 4.0–4.5 × 1.3–1.6 µm (n = 10) in size. Bradyzoites contained a conoid, few micronemes, few rhoptries (1–2 per section), subpellicular microtubules, and a posteriorly located nucleus. The pellicle was thickened towards the conoidal part of the bradyzoite (Fig. 4).

Intermediate host: Keel-billed toucan (Ramphastos sulfuratus). Definitive host: Unknown. Distribution: Unknown. Specimens (syntypes) deposited: Histologic sections of muscle deposited in the United States National Parasite Collection (USNPC) United States Department of Agriculture Beltsville, Maryland 20705, USA. One section stained with H and E (USNPC no. 094501) and 4 sections stained with toluidine blue (USNPC no. 094502). Sarcocystis sulfuratusi sp. nov. (Figs 5–8) Description: In H and E-stained sections sarcocysts were up to 900 µm long and up to 200 µm wide. Sarcocyst walls were 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)


blue-stained sections the sarcocyst wall had dark-stained areas (Fig. 5A, B) that were found to be microtubules by transmission electron microscopy (TEM). Five sarcocysts were examined ultrastructurally. The pvm was undulated and lined by a 32 nm electron-dense layer (edl). The vp were up to 4.3 µm long and up to 1.4 µm wide and were folded over the sarcocyst surface (Fig. 6). The microtubules 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 electron-dense than in the villar protrusions. The gl was smooth. All sarcocysts were mature and contained bradyzoites. The bradyzoites were 5.0–7.0 × 1.5–2.2 µm (n = 10) in size (Fig. 8). Bradyzoites contained a conoid, a few rhoptries and micronemes, subpellicular microtubules, amylopectin and a terminal nucleus. The pellicle at the conoidal end was more electron-dense (Fig. 8). The information on hosts, location, and specimens deposited is the same as stated for S. ramphastosi. Both species of Sarcocystis were present in same histologic sections.

Discussion Complete life cycles of Sarcocystis are known for only a few species of animals, mostly those in livestock (Dubey et al. 1989). Most Sarcocystis species have been named based on their host occurrence and structure. Among all taxonomic criteria, the structure of the sarcocyst wall is considered the most valuable for dividing species within a given host. Dubey et al. (1989) and Dubey and Odening (2001) recognized 35 types of sarcocyst walls based on their structure. The two types of sarcocysts found in the present study were structurally distinct from those described previously. They are different from two named avian species, S. rileyi and S. falcatula. The sarcocyst wall in S. rileyi is type 21 with cauliflower-like anastomosing villar protrusions (Dubey et al. 1989, 2003). The sarcocyst wall in S. falcatula is thick, striated, with finger-like villar protrusions (Drouin and Mahrt 1980; Box et al. 1984; Dubey et al. 2000, 2001a, b, c; Luznar et al. 2001). The villar protrusions in S. ramphastosi resemble sarcocyst wall type 9 of Dubey et al. (1989). However, in S. ramphastosi the villar protrusions are at irregular distances, the microtubules do not contain electron-dense granules and do not extend into the granular layer whereas the vp in type 9 either have electron-dense granules or extend deeper into the granular layer. The villar protrusions in S. sulfuratusi have

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distinctive microtubules that extend half way in to the granular layer and they are more electron-dense in the granular layer than in vp. Acknowledgements. The authors thank Mr. William Horsfield of Amazona, and Dr Chris Kingsley of Dendravia Consulting Room for providing the case material, and the staff at the Histology Laboratory, Faculty of Veterinary Science, University of Pretoria for their help.

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