Pathogenic New Species of Eimeria from the Pygmy Rabbit, Brachylagus idahoensis, in Washington and Oregon, with Description of the Sporulated Oocyst and Intestinal Endogenous Stages

In January 2003, fecal samples from 13 live pygmy rabbits, Brachylagus idahoensis (Merriam, 1891), were collected at the Oregon Zoo in Portland, Oregon, and sent to the University of New Mexico (UNM), Albuquerque, New Mexico, to be examined for coccidia. In July 2004, 14 more fecal samples were collected and sent to UNM, 6 from some of the same rabbits and 8 from 16 other rabbits (4 were pooled samples from siblings). In addition, tissue sections from 3 dead rabbits (2 from the Oregon Zoo, 1 from Washington State University) also were examined. Two of 4 (50%) pooled fecal samples and 8 of 17 (47%) 1-rabbit samples were positive for a single species of Eimeria, which we describe here as a new species. Sporulated oocysts were subspheroidal, 25.6 3 23.8 (22–28 3 21–27) mm, with a length:width (L:W) ratio of 1.1 (1.0–1.2). A micropyle (;2 mm wide) and 0–1 polar granules were present, but an oocyst residuum was absent. Sporocysts were ellipsoidal, 13.4 3 8.1 (11– 16.5 3 7.5–9) mm, with a L:W ratio of 1.7 (1.3–2.2), and they had a Stieda body and sporocyst residuum. Tissue sections showed a heavy infection of the villous epithelial cells of the proximal and mid-small intestine with coccidial endogenous stages, but no stages were found in liver hepatocytes. Meronts with approximately 46 (26–70) merozoites per infected cell appeared to be fully developed and were subspheroidal, 14.8 3 13.9 (13–18 3 10.5–16.5) mm. Developing macroand microgamonts were indistinguishable from each other and were spheroidal to subspheroidal, 10.4 3 9.5 (9–11 3 7.5–10.5) mm. Mature macrogamonts were spheroidal to subspheroidal, 14.2 3 13.7 (12–17 3 11–16) mm, and mature microgamonts were smaller and subspheroidal, 11.9 3 10.8 (10.5–13 3 9–12) mm. This eimerian seems to be extremely pathogenic to young pygmy rabbits, and given the precarious nature of this unique genetic population, it appears to be an emerging pathogen that deserves immediate further study. Relatively little is known about the biology of the pygmy rabbit, Brachylagus idahoensis (Merriam, 1891). Weighing approximately 400 g as adults, they are the only species in Brachylagus, are restricted to the Great Basin of the western United States, and are found only in isolated populations in northeastern California, southern Idaho, southwestern Montana, northern Nevada, eastern Oregon, western Utah, western Wyoming, and southeastern Washington (Green and Flinders, 1980; Campbell et al., 1982; Nowak, 1991). The population in Washington State is confined to the Columbia Basin and thought to have been geographically isolated from other populations of the species for thousands of years (Lyman, 1991, 2004; K. Warheit, pers. comm.). Pygmy rabbits display several traits that set them apart from species of cottontails (Sylvilagus), jackrabbits (Lepus), and Old World rabbits (Oryctolagus). They are the only leporids in the continental United States that dig their own extensive and intercommunicating burrows. They give alarm calls and other vocalizations. They move by a low, scampering gait (not leaping). They are almost totally dependent on sagebrush (Artemisia tridentata) for their diet, especially during winter months, when it may comprise 98–99% of their food intake (Nowak, 1991). During the last decade, populations have been declining in Washington, Oregon, and California, where sagebrush habitat has been burned, converted to agriculture, or cleared from large areas and replaced with bunch grasses to improve livestock forage. Studies by the Washington Department of Fish and Wildlife (WDFW) in 2001 determined that the Columbia Basin population of pygmy rabbits (Douglas County, Washington) is genetically distinct and has been isolated from the Idaho and Oregon populations for at least 7,000 yr. The population has declined precipitously from approximately 150 pygmy rabbits Received 16 July 2004; revised 11 October 2004; accepted 12 October 2004. * Oregon Zoo, 4001 SW Canyon Road, Portland, Oregon 97221. † Northwest ZooPath, 18210 Waverly Drive, Snohomish, Washington 98296. in 1995 (WDFW, 1995) to less than 30 in 2001 (Hays, 2001). Because no one had bred this species in captivity successfully, the WDFW initiated a captive-breeding program in cooperation with Washington State University and the Oregon Zoo. It started in December 2000, when 4 pygmy rabbits were brought from the Lemhi Valley, Idaho, to the Oregon Zoo to help develop pygmy rabbit husbandry protocols. Later, 16 Columbia Basin pygmy rabbits were captured as an initial source for captivebreeding efforts, and in 2002, both facilities began breeding the endangered Columbia Basin rabbits for eventual reintroduction to a protected habitat in central Washington. The U.S. Fish and Wildlife Service listed the Columbia Basin pygmy rabbit population under emergency provisions of the Endangered Species Act on 30 November 2001 (USFWS, 2001) and provided the population with full listing status on 5 March 2003 (USFWS, 2003). As of spring 2004, 22 Columbia Basin pygmy rabbits were in captive-breeding programs in Washington and Oregon, but the program did not produce many offspring during the first 2 yr. In addition, in May 2002, 4 captive-bred young rabbits died in 5 days, apparently because of gastrointestinal coccidiosis. Here, we describe the species responsible for these deaths as a new and potentially pathogenic species of Eimeria, and we document the structure of the sporulated oocyst and some of the endogenous stages and histopathology. MATERIALS AND METHODS Fecal samples from 13 live pygmy rabbits were collected at the Oregon Zoo in Portland, Oregon, in January 2003 and sent to the University of New Mexico (UNM), Albuquerque, New Mexico. These rabbits were all of Washington origin, and they currently are captivehoused at the Oregon Zoo. In July 2004, 14 additional fecal samples (10 from individual rabbits and 4 pooled samples from siblings) also were collected and sent to UNM for analysis. Fecal material was placed in vials containing 2% (w/v) aqueous potassium dichromate (K2Cr2O7) solution, mixed thoroughly, and refrigerated for 2–3 days before being shipped to UNM for examination. On receipt, the samples were stored at ambient temperature for approximately 1–2 mo, until they could be processed and screened for coccidia as detailed by Duszynski and WilDUSZYNSKI ET AL.—A NEW EIMERIAN FROM PIGMY RABBITS 619 TABLE I. Fecal samples from 29 captive-born (C) and wild-caught (W) pygmy rabbits, Brachylagus idahoensis, examined for coccidia and those found to be infected with Eimeria brachylagia. All samples were collected when animals were at the Oregon Zoo. Rabbit no. Sex C or W Birth date Death date E. brachylagia (1/2) and fecal sample date(s) 01/03/04 07/13/04 Housed* Subspecies†