SHORT COMMUNICATION Toxoplasma gondii Isolates from Mouflon Sheep (Ovis ammon) from Hawaii, USA Shiv K. Verma a , Chunlei Su b & Jitender P. Dubey a a United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland, 20705-2350 b Department of Microbiology, The University of Tennessee, Knoxville, Tennessee, 37996-0845 Keywords Bioassay; genotype; mouflon sheep. PCR-RFLP. Correspondence J. P. Dubey, USDA-ARS, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA Telephone number: +1-301-504-8128; FAX number: +1-301-504-9222; e-mail: jitender.dubey@ars.usda.gov Received: 3 June 2014; revised 24 June 2014; accepted June 24, 2014. doi:10.1111/jeu.12151 ABSTRACT Little is known of Toxoplasma gondii isolates circulating in wildlife. The mou- flon (Ovis ammon) is very popular game animal, hunted for its trophy horns. Here, we report the isolation and genetic characterization of T. gondii from two mouflons from Hawaii, USA. Both sheep had antibodies titers of 1:800 or higher. Viable T. gondii were isolated and nested PCR-RFLP genotyping revealed two genotypes, a clonal Type III (designated TgMouflonUS1), and a new genotype (designated TgMouflonUS2, and ToxoDB PCR-RFLP genotypes #249). This is the first report of T. gondii infection, isolation and genetic char- acterization in mouflons from the USA. TOXOPLASMOSIS is a worldwide zoonosis. The consump- tion of undercooked meat infected with Toxoplasma gondii is a major risk factor of infection in humans (Dubey 2010). Among food animals, pigs, sheep, and goats are commonly infected with T. gondii. Infected game meat is also a source of human infection. Among the game animals in the USA, white-tailed deer and bears have a high T. gondii infection and viable T. gondii has been isolated from these animals. The mouflon (Ovis ammon) is very popular game animal, hunted for their trophy horns, especially in Hawaii. Nothing is known of T. gondii infection in mouflon in the USA. Here, we report the isolation and genetic characterization of T. gondii from mouflons from Hawaii, USA. MATERIALS AND METHODS Naturally infected Mouflon sheep As part of an epidemiological surveillance of T. gondii infection in the USA wildlife, hearts, and serum samples of two mouflon sheep (O. ammon) were submitted to the Animal Parasitic Diseases Laboratory (APDL), United States Department of Agriculture in Beltsville, Maryland for T. gondii examination. These samples were collected in August 2013, by USDA’s Animal and Plant Health Inspection Service, Wildlife Services unit (APHIS-WS) from Honolulu, Hawaii. Both mouflon sheep were adult and one was male while another female. These wild Moufflon were caught on the island of Oahu, near the town Hawaii Kai (southeast Oahu). Sera were diluted two-fold serially from 1:25 to 1:3,200 and tested for antibodies to T. gondii by the modified agglutination test (MAT) as described previously (Dubey and Desmonts 1987). Bioassay in mice The 50 g myocardium of both sheep were homogenized individually, and digested in acidic pepsin, washed, and aliquots of homogenates were inoculated subcutaneously into three outbred Swiss Webster (SW) mice and two gamma interferon gene knockout mouse (Dubey 2010). Mice were bled on day 45 postinoculation (p.i) and a 1:25 dilution of serum was tested for T. gondii antibod- ies by MAT. Mice that died or killed on day 70 p.i. were examined for T. gondii tachyzoites or tissue cysts in their tissue imprint of lung and brain (Dubey 2010). The inoculated mice were considered infected with T. gondii when antibodies to T. gondii were demonstrable in their Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Eukaryotic Microbiology 2014, 0, 1–3 1 Journal of Eukaryotic Microbiology ISSN 1066-5234 Published by the International Society of Protistologists Eukaryotic Microbiology The Journal of