J. Parasitol., 94(2), 2008, pp. 496–499 ᭧ American Society of Parasitologists 2008

A NEW OF SARCOCYSTIS (: ) FROM THE BLACK (URSUS AMERICANUS)

J. P. Dubey, J. G. Humphreys*, and D. Fritz† Animal Parasitic Diseases Laboratory, Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, BARC-East, Building 1001, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350. e-mail: [email protected]

ABSTRACT: Infection with Sarcocystis species is common in herbivores but is rare in . Histological sections of 374 black bears (Ursus americanus) from Pennsylvania were examined for sarcocysts. In total, 3 sarcocysts were found in 3 bears, with 1 sarcocyst per section. Sarcocysts from 2 bears were considered a new species, Sarcocystis ursusi. Sarcocysts of S. ursusi n. sp. were microscopic and contained only bradyzoites. By light microscopy, the sarcocyst wall was thin (Ͻ0.5 ␮m thick) and had minute serrations. Ultrastructurally, the serrations on the sarcocyst wall consisted of villar protrusions (Vp) that were mostly 0.5 ␮m long. The Vp had bundles of electron-dense microtubules that were as wide as long; these microtubules extended deep into the ground substance layer, a feature that distinguished this species from unnamed sarcocysts from black bear. Bradyzoites were 4.8–6.0 ␮m long. The sarcocyst from the third bear was structurally different from S. ursusi; its sarcocyst wall was approximately 2 ␮m thick and had finger-like villi on the cyst wall giving the sarcocyst wall a striated appearance.

Parasites belonging to species of Sarcocystis (Apicomplexa) no. 3 (ear tag BV 13) was from Venango Township, Clinton have a 2-host life cycle (Dubey et al., 1989). The definitive County. Only 1 sarcocyst was found in each bear. Sarcocysts host becomes infected by ingesting the asexual stage (sarcocyst) were microscopic. In bear no. 1, the sarcocyst was cut longi- encysted in the intermediate host’s tissues, whereupon the sex- tudinally in 3 parts that probably belonged to a single sarcocyst ual cycle may commence in the lamina propria of the small (the myocyte was cut unevenly, and thus the sarcocyst appeared intestine of the . Typically, species of Sarcocystis ex- to be 3 separate sarcocysts). The total length of the sarcocyst clusively parasitize a single intermediate host species. Little is was 700 ϫ 50 ␮m. The sarcocyst from bear no. 2 measured known of the species of Sarcocystis in bears. Previously sar- 250 ϫ 75 ␮m in size and its sarcocyst wall (Fig. 1B) was cocysts of an unnamed species were found in tissue sections of identical to the sarcocyst in bear no. 1. We were unable to locate 6 of 53 black bears (Ursus americanus) from the southeastern this sarcocyst in sections made for transmission electron mi- and northwestern United States (Crum et al., 1978), i.e., 1 of croscopy (TEM). A new name is proposed for the sarcocysts 92 in North Carolina (Dubey et al., 1998), 1 of 132 from Florida in bears nos. 1 and 2. (Cheadle et al., 2002), and 2 of 46 in Oregon (Foreyt et al., 1999). However, sarcocysts from the black bear have not been DESCRIPTION named. In the present paper sarcocysts from black bears from Sarcocystis ursusi n. sp. Pennsylvania are described. (Figs. 1–3) ␮ MATERIALS AND METHODS Diagnosis: In 5 m section stained with H and E; sarcocyst wall thin (Ͻ1 ␮m) with minute serrations (Fig. 1A, B). Sarcocyst interior packed Samples of from 374 black bears, legally shot in with slender bradyzoites; difficult to measure because boundaries indis- November 1997, were fixed in 10% buffered formalin 1–4 days after tinct. Ultrastructurally, outer layer of sarcocyst (parasitophorous vacu- death. Attempts were made to collect muscle away from gunshot olar membrane [PVM]), wavy in outline, with minute undulations that wounds. Routine histologic examination was performed on paraffin- did not invaginate toward sarcocyst interior (Fig. 2). Undulations oc- embedded sections (5 ␮m) stained with hematoxylin and eosin (H and curred at irregular intervals. Interior of undulations electron-dense, E). thinned out at irregular distances. Villar protrusions (VPs) on PVM For transmission electron microscopy, a paraffin-embedded section located at irregular distances. VPs up to 3.8 ␮m long depending on from 1 bear was postfixed in 1% osmium tetroxide in Millonig’s phos- plane of section, most 0.5 ␮m long (Fig. 2). VPs with tufts of prominent phate buffer, rinsed in the same buffer, dehydrated in ethanol, and em- electron-dense microtubules continuing into ground substance (GS) lay- bedded in epoxy resin. Semithin sections were stained with Toluidine er. GS 0.3–1.0 ␮m thick; thickest at origin of septa (Fig. 2). GS con- blue in 1% sodium tetraborate. The ultrathin sections were contrasted tinued into interior of sarcocyst as septa (Fig. 2). Both sarcocysts ma- with uranyl acetate and lead citrate before examination in a transmission ture, containing fully formed bradyzoites; metrocytes absent. Groups of electron microscope. Sections of 1 sarcocyst from bear no. 1 were ex- bradyzoites separated by septa. Bradyzoites butted against GS. Longi- amined ultrastructurally. tudinally cut bradyzoites measured 4.8–6.0 ϫ 1.4–1.8 ␮m(nϭ 8). Bradyzoites with conoid, micronemes, 1–2 rhoptries per section, and RESULTS posteriorly located nucleus (Fig. 3). Rhoptries with long neck, often looped so that blunt end directed toward conoid (Fig. 3). Micronemes Sarcocysts were found in 3 bears. All 3 infected bears were numerous, located mostly at conoidal end of bradyzoite (Fig. 3). Nu- adult females (51, 80, 90 kg). Bear no. 1 (ear tag 97-1035) was cleus located in posterior half of parasite. Amylopectin granules present throughout bradyzoites. from Houston Township, Clearfield County; bear no. 2 (ear tag 97-1626) was from Foster Township, McKean County; and bear Taxonomic summary Type host: Black bear (Ursus americanus). Received 1 August 2007; revised 14 September 2007; accepted 24 Other hosts: Unknown. September 2007. Type locality: Clearfield County, McKean County, Clinton County, * Department of Biology, Indiana University of Pennsylvania, Weyandt Pennsylvania. Hall, Indiana, Pennsylvania 15705. Specimens deposited: Two histological sections stained with H and † Department of Pathology, United States Army Research Institute of E were deposited as syntypes for bears nos. 1 and 2 in the United States Infectious Diseases (USAMRIID), Fort Detrick, 1425 Porter Street, National Parasite Collection (USNPC nos. 10101 and 10102), United Frederick, Maryland 21702. States Department of Agriculture, Beltsville, Maryland. One H and E

496 DUBEY ET AL.—SARCOCYSTIS URSUSI N. SP. 497

FIGURE 1. (A, B) Sarcocysts of Sarcocystis ursusi n. sp. in skeletal muscles of the naturally infected black bears. Note thin sarcocyst wall with minute serrations on sarcocyst wall (arrowheads) and septa (s). (C) Sarcocyst of Sarcocystis sp. Note thick sarcocyst wall with finger-like villi.

stained section from bear no. 3 was deposited as voucher specimen from REMARKS bear no. 3 (USNPC no. 10103). Complete life cycles of Sarcocystis spp. are known for only a few species of animals, mostly those in livestock (Dubey et al., 1989). Most Sarcocystis sp. Sarcocystis species have been named based on their intermediate host occurrence and their sarcocyst structure. Dubey et al. (1989) and Dubey Diagnosis: Sarcocyst in bear no. 3 cut in cross section; measured 75 and Odening (2001) recognized 35 types of sarcocysts based on their ϫ 50 ␮m. Sarcocyst wall (Fig. 1C) distinct from those in bears nos. 1 structure. and 2. Its sarcocyst wall approximately 2 um thick with finger-like villi Morphologically, the sarcocysts from black bears nos. 1 and 2 were on sarcocyst wall giving it a striated appearance. Unable to locate sar- distinct from the unnamed species previously reported from black bear. cocyst in sections made for TEM. Prior to the present report, only 1 sarcocyst from a black bear was 498 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 2, APRIL 2008

FIGURE 2. TEM of the sarcocyst of Sarcocystis ursusi n. sp. with details of the sarcocyst wall cut at different angles (A, B, C). Note villar protrusions (V) on the sarcocyst wall. Villar protrusions have prominent microtubules (T) that extend (arrow) into the ground substance layer (GS). The parasitophorous vacuolar membrane has undulations (arrowheads). Bradyzoites (B) are located just beneath the sarcocyst wall. Also note prominent septum (S). DUBEY ET AL.—SARCOCYSTIS URSUSI N. SP. 499

studied ultrastructurally (Dubey et al., 1998). In that sarcocyst the sar- cocyst wall had 2-␮m–long villar protrusions that lacked microtubules. The sarcocyst from bear no. 3 resembled the sarcocyst from black bear from North Carolina (Dubey et al., 1998), but in the absence of electron microscopic examination it was difficult to compare them. Recently a new species, Sarcocystis arctosi, was described from brown bear (Ursus arctos) from Alaska (Dubey et al., 2007). Sarcocysts of S. arctosi are structurally distinct from S. ursusi; sacocysts of S. arctosi have minute undulations on the sarcocyst wall instead of long villar protrusions on S. ursusi.

ACKNOWLEDGMENTS We would like to thank Kathy Kuehl for her electron microscopy work.

LITERATURE CITED

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