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J. Parasitol., 94(1), 2008, pp. 42–45 ᭧ American Society of Parasitologists 2008

ISOLATION AND GENETIC CHARACTERIZATION OF TOXOPLASMA GONDII FROM ( LOTOR), ( DOMESTICUS), STRIPED ( MEPHITIS), BLACK ( AMERICANUS), AND ( CONCOLOR) FROM CANADA

J. P. Dubey, T. Quirk*, J. A. Pitt†, N. Sundar, G. V. Velmurugan, O. C. H. Kwok, D. Leclair‡, R. Hill§, and C. Su§ United States Department of Agriculture, Agricultural Research Service, and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350. e-mail: [email protected]

ABSTRACT: Viable Toxoplasma gondii was isolated by bioassay in mice from tissues of 2 feral cats (Felis domesticus), 2 raccoons (Procyon lotor), a skunk (Mephitis mephitis) trapped in remote locations in Manitoba, Canada, and a black bear (Ursus ameri- canus) from Kuujjuaq, northern Quebec, Canada. Genotyping of these T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico revealed 4 genotypes. None of the isolates was clonal archetypal Types I, II, and III found in the United States. These results are in contrast with the Type II genotype that is widespread in domestic and throughout the United States and Europe. This is the first genotyping of T. gondii isolates from this part of North America.

Toxoplasma gondii infections are widely prevalent in from Canada and clarify the confusion about the origin of the beings and animals worldwide (Dubey and Beattie, 1988). Hu- isolates. mans become infected postnatally by ingesting tissue cysts from undercooked meat or by consuming food and drink contami- MATERIALS AND METHODS nated with oocysts. Only a small percentage of exposed adult Serological examination humans develop clinical toxoplasmosis. Whether the severity of Sera of carnivores were tested for T. gondii antibodies using 2-fold toxoplasmosis in immunocompetent persons is due to the par- serum dilutions from 1:20 to 1:640 with the modified agglutination test asite strain, host variability, or other factors is largely unknown. (MAT) as described by Dubey and Desmonts (1987). Toxoplasma gondii isolates have been classified in 3 genetic Types (I, II, III) based on restriction fragment length polymor- Bioassay for T. gondii phism (RFLP) (Howe and Sibley, 1995; Howe et al., 1997). For bioassay in mice, 50 g of brain, heart, and muscle were homog- Until recently most isolates of T. gondii were considered clonal, enized, digested by an acidic pepsin solution (Dubey, 1998), neutral- with little genetic diversity (Lehmann et al., 2006), although ized, and washed; the homogenate was inoculated subcutaneously (SC) most of this information is derived from isolates from domestic into 5 outbred female Swiss Webster mice obtained from Taconic Farms, Germantown, New York, U.S.A., as previously described (Du- animals and human patients. Little is known of the prevalence bey et al., 2002) (Table I). Imprints of lungs or brains of mice that died and distribution of genotypes of T. gondii in wildlife in were examined for T. gondii tachyzoites or tissue cysts. Survivors were North America. There are a few reports of T. gondii genotypes bled on day 40–42 post-inoculation (PI), and a 1:25 dilution of serum in land (Dubey, Graham et al., 2004; Dubey, Parnell et al., from each mouse was tested for T. gondii antibodies with the MAT. Mice were killed 6 wk PI, and brains of all mice were examined for 2004; Dubey, Sundar, Nolden et al., 2007) and marine (Cole et tissue cysts as described (Dubey and Beattie, 1988). The inoculated al., 2000; Miller et al., 2004; Conrad et al., 2005) in mice were considered infected with T. gondii when tachyzoites or tissue the United States. Little is known of genotypes of T. gondii cysts were found in tissues. circulating in humans or animals in Canada. There is only brief information on an isolate of T. gondii (called Cougar isolate) Toxoplasma gondii isolates from raccoons, cats, and skunk from the oocysts shed by a naturally infected cougar from Van- As part of a larger study of wild carnivores in Manitoba, 3 raccoons, couver (Aramini et al., 1998; Lehmann et al, 2000; Su et al., 2 cats, and 1 skunk were available for T. gondii isolation (Table I). The cats and raccoons were collected near Minnedosa, Manitoba (50Њ10ЈN, 2006), and a human isolate (Lehmann et al., 2000) from an 99Њ47ЈW), and the skunk was collected at Delta Beach along the south undefined location in Canada. shore of Lake Manitoba (50Њ11ЈN, 99Њ19ЈW). The animals were killed In the present paper we describe the genetic and biologic with an intravenous inoculation of a euthanasia solution (T-61௢ 0.3 mg/ characteristics of isolates of T. gondii from (Me- kg; Hoechst Animal Health Benelux, Brussels, Belgium). Samples of phitis mephitis), raccoons (Procyon lotor), feral cats (Felis do- blood, heart, brain, and skeletal muscle were collected and shipped cold to the Animal Parasitic Diseases Laboratory (APDL), Beltsville, Mary- mesticus), and a black bear (Ursus americanus) from remote land for T. gondii examination. areas of Canada. We also properly designate cougar isolates Toxoplasma gondii isolate from a black bear Received 6 June 2007; revised 9 July 2007; accepted 10 July 2007. A young (2- to 3-yr-old) male black bear was killed near Kuujjuaq * Department of Biology, University of Saskatchewan, Saskatoon, SK, (58Њ5ЈN, 68Њ25ЈW), northern Quebec on 31 October 1999, and samples S7N5E2, Canada. of blood, heart, and tongue were collected soon after the kill. The bear † Department of Biological Sciences, University of Alberta, Edmonton, samples were stored in refrigerator (4 C) for 1 wk in the laboratory of AB T6G 2E9, Canada. the Nunavik Research Center in Kuujjuaq and were kept in a cooler for ‡ Nunavik Research Center, Makivik Corporation, Kuujjuaq Quebec 1 wk during transit from Canada to the United States. The samples were JOM 1MO, Canada. Present address: Food Safety Division, Canadian foul smelling when received at the Animal Parasitic Diseases Labora- Food Inspection Agency, 159 Cleopatra Drive, Ottawa, ON K1A 0Y9, tory (APDL), Beltsville, Maryland, on 15 November 1999. A serolog- Canada. ical examination revealed that the bear had antibodies to T. gondii § Department of Microbiology, University of Tennessee, Knoxville, (MAT titer 1:200). On November 20 (20 days from the kill) 50 g of Tennessee 37996-0845. heart and 35 g of tongue were homogenized separately and digested in

42 DUBEY ET AL.—TOXOPLASMA GONDII FROM WILDLIFE IN CANADA 43

TABLE I. Isolation of Toxoplasma gondii from cats, raccoons, and a RESULTS skunk in Manitoba, Canada. Toxoplasma gondii isolates from raccoons, cats, and T. gondii Isolation in mice* MAT strain Toxoplasma gondii was isolated from 2 cats, 2 raccoons, and Host titer Brain Heart Muscle designation 1 skunk (Table I). It is noteworthy that T. gondii was not iso- 1 100 0 0 4 (16)† TgCatCa1 lated from the brain of any animal (Table I). The 1 Cat 2 800 0 4 5 TgCatCa2 isolate was obtained from the muscle of the animal; 1 of the 5 Raccoon 1 100 0 0 1 (11) TgRcCa1 mice inoculated died of acute toxoplasmosis, and tachyzoites Raccoon 3 100 0 2 (43) 0 TgRcCa2 were found in its lungs. The T. gondii isolate from raccoon 1 Skunk 400 Not done 5 5 (13, 15) TgSkCa1 was lost and was not available for DNA characterization. * Five mice were inoculated with each tissue. † Day of death of individual mouse. Toxoplasma gondii isolate from a black bear Toxoplasma gondii was isolated from the tongue but not from the heart of the bear. One of the 5 mice died on day 26 PI with pepsin, before being inoculated into mice (5 for the tongue, and 10 for tongue of the bear isolate. Toxoplasma gondii was not found in the heart). The mice inoculated with bear tissues were examined for T. gondii infection. smears of the pneumonic lung because of autolysis; therefore, a homogenate of the lung of this mouse was inoculated SC into Toxoplasma gondii isolates from 2 mice. The recipient mice died of acute toxoplasmosis 11 and 14 days PI, and a homogenate of the lung containing tachy- During an epidemiological study of a waterborne outbreak of toxo- zoites was inoculated into 3 mice that were medicated with plasmosis in humans, T. gondii was isolated from feces (samples A and B) from 2 naturally infected cougars from Vancouver Island, British sulfadiazine sodium in drinking water (1 mg per 100 ml of Columbia, Canada (Aramini et al., 1998). Sample A was obtained from drinking water) from day 7 to 12 PI. Two of these 3 mice died the rectum of a 1.5-yr-old male that was killed; it had a serum MAT of toxoplasmosis in spite of therapy. Numerous tissue cysts titer of 1:500 (Aramini et al., 1998). Mice orally inoculated with oocysts were found in the brain of the mouse when killed 49 day PI. from both cougars developed toxoplasmosis, and the strains were cryo- Bradyzoites released from the tissue cysts from this mouse were preserved in liquid nitrogen. These strains had no specific designation, until now. The strain from sample A is now designated as TgCgCa1 cryopreserved in liquid nitrogen. For genetic characterization, and the strain from sample B as TgCgCa2. For the present study both a vial containing the bear isolate frozen for 7 yr was thawed, isolates were successfully revived after a 10-yr storage in liquid nitro- and half of the contents were inoculated SC into 2 mice and gen. the remainder half was processed for DNA extraction. The There is a considerable amount of published information on the ge- netics of the TgCgCA1 isolate, because it was found to be different strain was revived successfully because the 2 mice inoculated from the typical Types I, II, and III (Lehmann et al., 2000; Su et al., with frozen bradyzoites died of acute toxoplasmosis on day 18 2003, 2006; Fux et al., 2007); however, the isolate has been named and 21 PI. Cougar2, Cougar, or COUG, but never clearly identified in these pub- lications. Genetic characterization Genetic characterization Genotyping was performed on 5 of the 6 T. gondii strains Toxoplasma gondii DNA was extracted from the tissues of infected isolated in the present study (isolate from raccoon 1 was lost) mice from each group, and strain typing was performed using PCR- and from the cougar from sample B of Aramini et al. (1998). RFLP genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29- Five different genotypic groups were recognized (Table II). 2, L358, PK1, and Apico (Su et al., 2006; Dubey, Sundar, Gennari et None of the isolates belong to clonal archetypal Type I, II, III al., 2007). Allele types for all isolates were determined based on the lineage. Two isolates (TgCatCa2 from cat 2 and TgSkCa1 from RFLP patterns of 6 reference strains including RH88, PTG, CTG, COU- GAR (TgCgCA1), MAS, and TgCatBr5 (Su et al., 2006). These refer- the skunk) were similar by all markers tested. The 5 genotypes ence strains allow us to capture most known alleles for each marker had a combination of Type I and II, I and III, and I, II, and III and to identify potential unique alleles in new samples. (Table II).

TABLE II. Genotype of T. gondii isolates from wild animals from Canada.

Host T. gondii isolate SAG1 SAG2* SAG2† SAG3 BTUB GRA6 c22-8 c29-2 L358 PK1 Apico

Cat 1 TgCatCa1 II or III III III I III III II I III III III Cat 2 TgCatCa2 u-1 II II II II II II II I II I Raccoon 3 TgRaCa2 u-1 II II II II II II II I II I Skunk TgSkCa1 u-1 II II II II II II II I II I Black bear TgBBeCa1 I I I III III III III III III I III Cougar‡ TgCgCa2 I II II III II II II u-1 I u-2 I

* SAG2 marker based on 5Ј- and 3Ј-ends of the gene sequence (Howe et al., 1997). † A new SAG2 marker based on the 5Ј-end of the gene sequence (Su et al., 2006). ‡ Sample B of Aramini et al. (1998). 44 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 1, FEBRUARY 2008

DISCUSSION genetic characterization has been limited essentially to isolates from patients ill with toxoplasmosis. There is very little infor- The role of wildlife in the epidemiology of T. gondii infec- mation regarding the genetic diversity of T. gondii isolates cir- tions in livestock or humans is unknown. Toxoplasma gondii culating in the general human population. Therefore, we must infections are widely prevalent in white-tailed deer, raccoons, be cautious in claiming a linkage between parasite genotypes and black in the United States (reviewed in Dubey, Gra- and disease presentations without the good knowledge of par- ham et al., 2004). Meat of many wildlife species is consumed asite biology in the human population and the environment. by humans, and occasionally human cases of clinical toxoplas- mosis have been linked to eating uncooked venison (Sacks et ACKNOWLEDGMENTS al., 1983; McDonald et al., 1990). A large outbreak of toxo- This study was supported in part by the Delta Waterfowl Research plasmosis in humans in Vancouver, British Columbia, Canada, Foundation. was epidemiologically linked to the contamination of a water reservoir by oocysts (Bowie et al., 1997); however, attempts to LITERATURE CITED isolate T. gondii from water samples were unsuccessful (Isaac- ARAMINI, J. J., C. STEPHEN, AND J. P. DUBEY. 1998. Toxoplasma gondii Renton et al., 1998). It was suspected that feces of infected cats in Vancouver Island cougars (Felis concolor vancouverensis): Se- (domestic, or feral Felis domesticus, or cougars) entered the rology and oocyst shedding. Journal of Parasitology 84: 438–440. reservoir or 1 of its streams, resulting in contamination of the BOWIE, W. R., A. S. KING,D.H.WERKER,J.L.ISAAC-RENTON,A.BELL, water supply with T. gondii oocysts (Aramini et al., 1998). Cou- S. B. ENG, AND S. A. MARION. 1997. Outbreak of toxoplasmosis associated with municipal drinking water. Lancet 350: 173–177. gars are the only wild felid found on Vancouver Island. One COLE, R. A., D. S. LINDSAY,D.K.HOWE,C.L.RODERICK,J.P.DUBEY, cougar was known to be in the vicinity of the Victoria water- N. J. THOMAS, AND L. A. BAETEN. 2000. Biological and molecular shed, and sample A was derived from this cougar. Sample B characterizations of Toxoplasma gondii strains obtained from was presumed to be of cougar origin (see Materials and Meth- southern sea (Enhydra lutris nereis). Journal of Parasitology ods). Results of the present study indicate that T. gondii strains 86: 526–530. CONRAD,P.A.,M.A.MILLER,C.KREUDER,E.R.JAMES,J.MAZET,H. from sample A and B are genetically identical based on the DABRITZ,D.A.JESSUP,F.GULLAND, AND M. E. GRIGG. 2005. Trans- results of 10 PCR-RFLP markers, and both might have come mission of Toxoplasma: Clues from the study of sea otters as sen- from the same cougar or derived from the ancestor strain. To tinels of Toxoplasma gondii flow into the marine environment. In- our knowledge, there is no published information on the genetic ternational Journal for Parasitology 35: 1155–1168. DUBEY, J. P. 1998. Refinement of pepsin digestion method for isolation makeup of the isolates of T. gondii from humans from the Van- of Toxoplasma gondii from infected tissues. Veterinary Parasitol- couver outbreak. Results of the cougar isolate are documented ogy 74: 75–77. here to exclude or incriminate the cougar with the human out- ———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man. break. CRC Press, Boca Raton, Florida, 220 p. Recently we determined that 4 of the 5 isolates from rac- ———, AND G. DESMONTS. 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337– coons and 1 skunk isolate from Wisconsin belonged to 5 T. 339. gondii genotypes (Dubey, Sundar, Nolden et al., 2007); these ———, J. A. CORTE´ S VECINO,J.J.VARGAS-DUARTE,N.SUNDAR,G.V. genotypes are different from genotypes from the Canadian rac- VELMURUGAN,L.M.BANDINI,L.J.POLO,L.ZAMBRANO,L.E. coon and the skunk in the present study. The 4 T. gondii ge- MORA,O.C.H.KWOK,T.SMITH, AND C. SU. 2007. Prevalence of Toxoplasma gondii in from Colombia, and notypes identified in this study are also different from previ- genetic characterizaation of T. gondii isolates. Veterinary Parasi- ously published results on isolates of chickens, dogs, and cats tology 145: 45–50. from Brazil (Su et al., 2006; Dubey, Gennari et al., 2007; Du- ———, D. H. GRAHAM,C.R.BLACKSTON,T.LEHMANN,S.M.GENNARI, bey, Sundar et al., 2007), dogs from Colombia (Dubey, Corte´s A. M. A. RAGOZO,S.M.NISHI,S.K.SHEN,O.C.H.KWOK,D.E. HILL, AND P. T HULLIEZ. 2002. 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