1264 J. Parasitol., 94(6), 2008, pp. 1264–1268  American Society of Parasitologists 2008 NOVEL AND CANINE GENOTYPES OF GIARDIA DUODENALIS IN HARBOR SEALS (PHOCA VITULINA RICHARDSI ) J. K. Gaydos, W. A. Miller*, C. Johnson†, H. Zornetzer*, A. Melli*, A. Packham*, S. J. Jeffries‡, M. M. Lance‡, and P. A. Conrad*† University of California, Davis Wildlife Health Center–Orcas Island Office, School of Veterinary Medicine, 942 Deer Harbor Road, Eastsound, Washington 98245. e-mail: jkgaydos@ucdavis.edu ABSTRACT: Feces of harbor seals (Phoca vitulina richardsi) and hybrid Glaucous-winged/Western gulls (Larus glaucescens/ occidentalis) from Washington State’s inland marine waters were examined for Giardia spp. and Cryptosporidium spp. to deter- mine whether genotypes carried by these wildlife species were the same as those that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% (41/97) of seal fecal samples. Giardia spp.–positive samples came from 90% (9/10) of the sites, and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with 400 animals were 4.7 times more likely to have Giardia spp. cysts than were samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% (3/78) of fecal samples. Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 base pair (bp) segment of Giardia duodenalis DNA at the glutamate dehydrogenase (GDH) locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan polymerase chain reaction (PCR) amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences that could not be evaluated. Species of Giardia and Cryptosporidium, protozoans once thought to be primarily associated with freshwater and terres- trial hosts, have been identified in a variety of wildlife species, including marine-foraging river otters (Lontra canadensis; Gay- dos et al., 2007), California sea lions (Zalophus californianus; Deng et al., 2000), ringed seals (Phoca hispida; Olson et al., 1997; Fayer et al., 2004; Hughes-Hanks et al., 2005; Santin et al., 2005), north Atlantic right whales (Eubalaena glacialis), and bowhead whales (Balaena mysticetus) (Hughes-Hanks et al., 2005). Additionally, Giardia spp. have been identified in harp seals (Phoca groenlandica), grey seals (Halichoerus gry- pus), and 1 harbor seal (Phoca vitulina) from eastern coastal Canada (Measures and Olson, 1999), and Cryptosporidium spp. oocysts were identified in dugong (Dugong dugon; Hill et al., 1997). Very little has been done, however, to characterize the genotypes of Giardia spp. and Cryptosporidium spp. infecting marine wildlife. Understanding the molecular epidemiology of these proto- zoans in marine wildlife is important for evaluating human health risks that could be associated with marine ecosystems. Oocysts of Cryptosporidium parvum can survive for at least a year in salt water (Tamburrini and Pozio, 1999) and can be concentrated by filter-feeding bivalves, including mussels (Tam- burrini and Pozio, 1999) and oysters (Fayer et al., 1999). Sim- ilarly, G. duodenalis (synonymous with G. lamblia and G. in- testinalis) cysts can persist in marine environments and are con- centrated by bivalves such as mussels and clams (Graczyk et al., 2003). Washington State is the nation’s largest producer of farmed shellfish (U.S. Department of Agriculture, 2006) and, depending on the species and genotypes carried by wildlife, Received 14 May 2007; revised 14 January 2008, 9 April 2008; ac- cepted 10 April 2008. * Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, One Shields Ave., Davis, California 95616. † University of California, Davis Wildlife Health Center, School of Vet- erinary Medicine, One Shields Ave., Davis, California 95616. ‡ Washington Department of Fish and Wildlife, Wildlife Science Pro- gram, 7801 Phillips Rd. S.W., Lakewood, Washington 98498. contamination of shellfish-growing areas by marine wildlife could present a threat to human health. The molecular epidemiology of Giardia spp. and Cryptospo- ridium spp. also can provide insights as to whether marine wild- life species are being infected by protozoans of human or do- mestic animal origin. Human, companion animal, and agricul- ture-related fecal material is discharged, dumped, or carried in runoff into marine waters all over the world (Fayer et al., 2004). For example, in the inland marine waters shared by Washington State (United States) and British Columbia (Canada), untreated sewage effluent from an estimated 210,000 people living on the south end of Vancouver Island (Canada) is discharged from 2 marine outfalls, averaging 80,000 m 3 /day and 50,000 m 3 /day (Hodgins et al., 1998). Human and domestic animal feces from untreated sewage, failing septic systems, and storm water runoff have the potential to infect and impact marine wildlife species. The shared inland marine waters of Washington and British Columbia (N4830, W12340) is a highly productive marine ecosystem. Nearly 7 million people reside on the shoreline of this ecosystem where humans, wildlife, and domestic animals share habitat and marine resources (Fraser et al., 2006). Harbor seals (P. vitulina richardsi) and hybrid Glaucous-winged/West- ern gulls (L. glaucescens/occidentalis) are some of the most common marine mammal and marine bird species found throughout this region (Angell and Balcomb, 1982; Jeffries et al., 2003). We tested seal and gull fecal samples from Wash- ington’s inland marine waters to determine whether they were infected with Giardia spp. and Cryptosporidium spp. and to molecularly characterize isolates from these common wildlife species. MATERIALS AND METHODS Between March and August 2005, fresh harbor seal fecal samples were collected from 10 seal haulout sites widely distributed throughout the Puget Sound region (Fig. 1). Fecal samples were individually col- lected and identified as harbor seal based on size, conformation, content, and location. Only samples less than 24-hr old were collected, as de- termined by location on haulout site, tidal heights over the last 24 hr,