Veterinary Parasitology 175 (2011) 60–65

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Veterinary Parasitology

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Toxoplasma gondii in feral American minks at the Maullín river,

Maximiliano A. Sepúlveda a,b,∗, Claudia Munoz-Zanzi˜ a,c, Carla Rosenfeld a, Rocio Jara a, Katharine M. Pelican b, Dolores Hill d a Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Isla Teja s/n, Casilla 567, , Chile b Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, 1365 Gortner Avenue, St. Paul, MN 55108, USA c Division of Epidemiology and Community Health, School of Public Health, University of Minnesota, 1300 S Second St. Suite 300, Minneapolis, MN 55454, USA d United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705, USA article info abstract

Article history: American mink (Neovison vison) is a widely distributed invasive species in southern Chile. Received 30 October 2008 Thirty four feral minks were trapped at two distinct sites (rural and peri-urban), diet ana- Received in revised form 4 August 2010 lyzed and Toxoplasma gondii exposure compared using PCR and specific antibodies. Serum Accepted 20 September 2010 samples were evaluated using a commercial latex agglutination test where a titer ≥1:32 was considered positive. Of 30 mink analyzed, 21 (70%) were positive to T. gondii antibodies, Keywords: with titers ranging from 1:32 to 1:2048. As expected, adult mink showed higher seropreva- American mink lence of exposure to T. gondii (18/21) than young mink (3/9) (P = 0.008). There was not Chile Neovison vison statistically significant difference between sex groups (P = 0.687). Differences in seropreva- PCR lence were observed between the two sample sites with a higher proportion of positive Toxoplasma gondii individuals in the peri-urban area, and therefore, closer to human settlements (35.7% vs. Seroprevalence 100%, P = 0.0001). Individuals positive to T. gondii using PCR and/or serology showed simi- lar differences by site with higher infected individuals in peri-urban areas (58.8% vs. 100%, P = 0.007). Diet of American mink based in fecal composition analyses was mainly based on crustaceans (frequency of occurrence: crustaceans = 100%, birds and rodents < 7%), suggest- ing that the high observed prevalence of T. gondii infection might be more associated with its aquatic behavior (e.g. ingestion of oocysts in contaminated fresh water) than with their trophic behavior (e.g. preying over species that can have T. gondii cysts in their tissues). As an invasive species potentially subject to routine culling to maintain population sizes under control, minks could be used as a sentinel species to monitor pathogens of public and wildlife health importance, such as T. gondii, in aquatic environments. © 2010 Elsevier B.V. All rights reserved.

1. Introduction mink farm escapees were first reported (Servicio Agrícola y Ganadero, 1988). At present, the species is considered The American mink (Neovison vison), a mustelid car- an invasive species and is widely distributed throughout nivore species, was first brought to Chile in the 1930s southern Chile including Tierra del Fuego Island (38◦35Sto for the pelt industry. In the 1970s feral populations of 55◦S) and adjacent archipelagos (Rozzi and Sherriffs, 2003). Research on the American mink in this neotropical area has focused mainly on diet, distribution, and aspects of inter- specific competition and predation (Medina, 1997; Jaksic et ∗ Corresponding author at: Department of Veterinary Population al., 2002; Rozzi and Sherriffs, 2003; Schuttler et al., 2008; Medicine, College of Veterinary Medicine, University of Minnesota, 1365 Fasola et al., 2009; Ibarra et al., 2009). Monitoring of specific Gortner Avenue, St. Paul, MN 55108, USA. Tel.: +1 612 625 8561; fax: +1 612 624 4906. pathogens in free-ranging carnivores can provide estimates E-mail address: [email protected] (M.A. Sepúlveda). of environmental contamination because of the high risk of

0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.09.020 M.A. Sepúlveda et al. / Veterinary Parasitology 175 (2011) 60–65 61 exposure associated with their trophic position (Smith and a total human population of 273,256 inhabitants (INE, Frenkel, 1995). However, there are no published studies on 2002). Within this basin, one peri-urban site and one rural the infections present in feral mink populations in South site were selected for mink captures. The peri-urban site America. was located 3.2 km from (22,309 habitants), The protozoan parasite Toxoplasma gondii is a 7.6 km from Llanquihue (12,728 habitants) and 12 km widespread pathogen of warm-blooded animals, including from (155,895 habitants). The rural site was humans. This parasite has a two-host life cycle. Felids located 16.2 km, 19.3 km and 17.3 km, respectively, from are the definitive hosts and the only animals known to the previously mentioned urban areas, as well as 23.1 km shed oocysts in their feces. They can be infected through from (5,707 habitants) (INE, 2002)(Fig. 1). oocyst exposure or consumption of infected intermediate hosts. Many animals, including mice, birds, domestic 2.2. American minks livestock and humans can serve as intermediate hosts, where invasive stages of T. gondii may spread throughout American minks were trapped between March and the muscles, nervous system and other tissues, forming October of 2007 using Tomahawk traps. Eighteen traps long-lived tissue cysts (Frenkel and Dubey, 1972). Inter- per site were distributed homogeneously over a 3.8 km mediate hosts can be infected via ingestion of feline fecal area in the peri-urban site and a 2 km area in the rural matter contaminated with sporulated oocysts, ingestion of site. Captured minks were anaesthetized with a combi- tissue cysts or tachyzoites in meat from infected animals, nation of Ketamine (Imalgene®, 10 mg/kg; Merial) and or congenitally due to maternal infection during gestation Xylazine (Rompun®, 0.5 mg/kg; Bayer). Blood samples for (Dubey and Beattie, 1988). serum were collected by cardiac puncture and stored at In immune competent humans, acute infection with ◦ −20 C until testing. Data collected included sampling site, T. gondii is usually asymptomatic or causes mild disease; age, sex, weight, and length. Age category was estimated however, it can cause devastating disease in congenitally by teeth condition as follows: juvenile (adult teeth with- infected children and in people with depressed immu- out abrasion and tartar) or adult (teeth abraded and with nity. Similarly, clinical and subclinical toxoplasmosis has tartar) (modified from Fournier-Chambrion et al., 2004). been reported in many host wild species and serologic Body condition or relative weight (K) was estimated for surveys indicate that T. gondii infections are common in adult individuals only as K = W/(aLn), where W = weight (g), free-ranging carnivores (Hill et al., 2005). The critical role L = body length (cm), and a and n are constants (Kruuk of T. gondii in mortality of southern sea otters (Enhydra lutris and Conroy, 1991). Values for males were a = 0.0252 and nereis)(Kreuder et al., 2003), where it is considered a sig- n = 2.8833 and values for females were a = 0.0524 and nificant cause of encephalitis (Cole et al., 2000; Lindsay et n = 2.6594 (Sidorovich et al., 1999). K values close to 1 al., 2001; Miller et al., 2004), is of particular relevance for indicate adequate body condition, less than 1 indicate this study. Miller et al. (2002) has also documented a high poor body conditions, and values higher than 1 indi- prevalence of infection in sea otters and concurrent popu- cate overweight (Kruuk, 1995). After data collection and lation declines along the Pacific coast of the U.S., probably under sedation, all individuals were sacrificed by cardiac the result of surface water run-off contaminated with T. puncture with a commercial euthanasia solution (T61®; gondii oocysts. 0.5 ml/mink; Intervet, Canada). Brain tissue was harvested The objective of this study was to describe T. gondii ◦ and stored at −20 C for PCR testing. The National Agricul- infection in American mink in Chile and to identify fac- tural and Livestock Service (Servicio Agricola y Ganadero) tors associated with infection, most importantly, proximity from Chile provided the required capture permit for this to human populations. Secondary objectives included research (legal permit no. 3206 from July 14, 2006 and its description of dietary aspects and abundances. modification no. 904 from February 23, 2007).

2. Material and methods 2.3. T. gondii antibody detection 2.1. Study area Samples were evaluated for IgG antibodies against All mink captures were located within the Maullín T. gondii using a commercial latex agglutination test ® river basin of the Llanquihue , Lake District in (Toxotest-MT , Eiken Chemical Company, Ltd, Tokyo, Chile (41◦26S; 73◦07W). The basin is characterized by Japan) performed at the Preventive Medicine Institute mesotemplate hyper-humid climate, with yearly aver- laboratory, Universidad Austral de Chile following the age precipitation of 2021 mm and average temperature of manufacturer’s protocol. Briefly, this test uses inactivated T. 10.7 ◦C(Luebert and Pliscoff, 2006). The vegetation asso- gondii antigen coated latex particles that agglutinate when ciated with the river is primarily swamp forest which exposed to T. gondii specific antibodies. Sera were diluted is characterized by native species such as Myrceugenia in duplicate wells from 1:16 to 1:2408 in buffer solution exsucca, Blepharocalyx cruckshanksii, Luma gayana, Drimys to determine antibody titers. Based on the manufacturer’s winteri, Tepualia stipularis, Blechnum chilense, Chusquea recommendation, a titer ≥1:32 was considered positive. A quila, Boquila trifoliata and Cissus striata (Hauenstein et al., positive result is indicative of past exposure to T. gondii.In 2005). The river adjacent area is mostly used for agricul- order to increase reliability of results, all samples were re- tural purposes such as livestock and dairy farming. The tested at the Minnesota Veterinary Diagnostic Laboratory total surface of the Maullín basin covers 397,200 ha with using a similar latex agglutination test and interpretation. 62 M.A. Sepúlveda et al. / Veterinary Parasitology 175 (2011) 60–65

Fig. 1. Hydrographic basin map of the Maullín river watershed in the Lakes District of southern Chile. The black arrows on the left map indicate the position of sampling sites (left: rural; right: peri-urban).

2.4. T. gondii DNA detection stray hairs ingested while grooming (Chehébar and Benoit, 1988; Bonesi et al., 2004). After collection, feces were DNA was extracted from all available brain tissues washed, passed through a sieve, dried and analyzed under using a commercial kit (DNeasy® Blood and Tissue Blood the microscope. Undigested prey remains were classified and Tissue, Qiagen Inc., Valencia, CA) and following the using reference material obtained at the Zoology Institute manufacture’s protocol. Extracted DNA was shipped to of the Universidad Austral de Chile, expert consults and the Animal Parasitic Diseases Laboratory of USDA-ARS keys from Retamal (1981) for crustaceans and Chehébar on FTA® cards (Whatman Inc., Kent, UK) for PCR test- and Martín (1989) for mammals. The diet was described ing using a published protocol (Hill et al., 2006). PCR as: (1) the prey occurrence (the number of feces in which was performed using direct and semi-nested methods to the prey class was identified) and (2) frequency of occur- amplify segments of the 35-fold repetitive DNA region B1 rence (number of occurrences of a prey class in relation to of T. gondii. Direct PCR was performed on individual sam- the sum of occurrences of all prey classes). ples using the primer pair forward: 5GGAACTGCATCCGT Relative abundances of mink in the study area were TCATGAG-3 and reverse: 5-TCTTTAAAGCGTTCGTGGTC-3. inferred by trapping (Bonesi et al., 2004) while calibrat- Semi-nested PCR was performed using 1 ml of the ampli- ing the difference in sampling effort between the two sites cons resulting from the first round direct PCR as the target (rural and urban). Sampling effort was calculated as num- DNA, the forward primer 5-TGCATAGGTTGCAGTCACTG-3, ber of traps night/number of minks trapped. and the reverse primer from the first round PCR. Cycling conditions for both the direct and semi-nested PCR were denaturation at 94 ◦C for 5 min, followed by 40 cycles of 2.6. Statistical analysis 94 ◦C for 1 min, 60 ◦C for 1 min, and 72 ◦C for 2 min. Prod- ucts from the direct and semi-nested PCR were analyzed Seroprevalence of T. gondii infection was defined as on pre-poured 2.0% agarose E-gels along with Hae III DNA the proportion of individuals with a positive ELISA result. markers (Invitrogen, Carlsbad, CA). Because of the known likelihood of false negative results with PCR (Hill et al., 2006) an overall estimation of preva- lence of infection was also calculated defining infected 2.5. Mink diet and abundances individuals as those who were positive to either T. gondii- specific antibodies or PCR. Confidence intervals (CI) for Fecal samples were collected from the same transects the estimates were calculated using exact methods. Preva- used for mink trapping. Mink feces were recognized based lences were compared by location (rural, peri-urban), age on a combination of size, appearance and the presence of class (adult, juvenile), and sex using Fisher’s exact test. M.A. Sepúlveda et al. / Veterinary Parasitology 175 (2011) 60–65 63

Table 1 Results of risk factors associated with T. gondii antibodies in serum samples from N. vison using agglutination in latex test in Maullín river, Chile.

Positive (%) Negative (%) Total (%) OR (95% IC) P-values

Location Peri-urban 16 (100%) 0 (0%) 16 (53.3%) NC P = 0.0001a Rural 5 (35.7%) 9 (64.3%) 14 (46.7%) Sex Male 14 (73.67%) 5 (26.3%) 19 (63.3%) 1.6 (0.32–7.9) P = 0.687a Female 7 (63.6%) 4 (36.4%) 11 (36.7%) Age Adult 17 (81%) 4(19%) 21 (70%) 4.9 (0.9–30) P = 0.081a Juvenile 4 (44.4%) 5 (55.6%) 9 (30%)

Total 21 (70%) 9 (30%) 30 (100%)

NC: not calculated. a Fisher exact test.

Table 2 Distribution of T. gondii antibody titers (<16, 32, 64, 128, 256, 512, 1024, 2048) by age group and location among American minks from Maullín river, Chile.

Titer dilution

<1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048

Total 8 1 3 1 2571 2 Rural 8 1 1 0 0 2 0 1 1 Peri-urban 0 0 2 1 2 3 7 0 1 Juvenile 5 1 1 0 0 1 0 1 0 Adult 3 0 2 1 2 4 7 0 2

Stratified analysis was used to adjust for age and for detect- this limitation, the association between site and serosta- ing effect modification (Agresti, 2002). In addition, t-test tus was examined among adults only which resulted in was used to compare body condition score (K) between the same type of positive association between peri-urban positive and negative animals and Pearson correlation coef- site and seroprevalence. Considering adults only, sero- ficient was used to examine a relation between ln(antibody prevalence was 100% (16/16, 90% CI: 82.9–100%) for the titer) against T. gondii in positive individuals and body con- peri-urban site and 20% (1/5, 90% CI: 1–65.7%) for the rural dition score. Due to the small sample size, for all analysis, a site (P = 0.0008). Considering either seropositive or PCR P < 0.1 was considered statistically significant. All analyses positive as an indication of T. gondii infection, adults were were performed using R 2.4.1 (R Development Core Team, more likely to be infected (19/22 = 86.3%) compared with 2005). juveniles (7/11 = 63.6%), though not statistically signifi- cant (P = 0.18). Similarly to serology only results, animals from the peri-urban site (16/16 = 100%) were more likely 3. Results to be infected compared with animals from the rural site (10/17 = 82%, P = 0.007), even controlling by age (P = 0.012). A total of 34 American minks (17 individuals at each Among adults, body condition score ranged from 0.33 site) were captured, using 270 and 188-traps night at the to 1.10 with a mean of 0.82. Seropositive animals (mean rural and peri-urban sites, respectively. Relative abundance K = 0.89) had a statistically significant higher score than calculated by capture and removal was 8.5 minks/km in seronegative animals (mean K = 0.54) (P = 0.0009). How- the rural site and 4.5 minks/km at the peri-urban site. All ever, there was no correlation between antibody titers and animals appeared to be healthy on physical examination. body condition score (r = 0.25, P = 0.33). There was no sta- Serum samples were obtained from 30 mink, 16 from the tistically significant association between serostatus and sex peri-urban site and 14 from the rural site, and 21 were (P = 0.687) (Table 1). classified as seropositive for T. gondii (Table 1) by both In relation to mink’s diet, sixty fecal samples were col- laboratories. Serum titers for seropositive animals ranged lected and the main diet item present in all feces was ≥ from 1:32 to 1:2048 (Table 2). Overall seroprevalence was crustaceans, other items represented in less than 7% fre- 70% (90% CI: 53.5–83.4%). Serostatus differed (P = 0.08) with quency occurrence were mammals, birds and fish (Table 3). age where 81.0% (17/21) of adults were seropositive com- pared to 44.4% (4/9) of juveniles. The age distribution of captured mink differed by site where 36% (5/14) of minks 4. Discussion in the rural site were adults and 100% (16/16) of minks in the peri-urban site were adults. Seroprevalence in minks To our knowledge this is the first study of T. gondii preva- from the rural site was 35.7% (5/14). In contrast, all 16 indi- lence in feral minks over a small spatial and temporal scale. viduals (100%) from the peri-urban site were classified as Previous reports were based on samples collected from seropositive (P = 0.001). Because of this markedly differ- larger regions and over long time intervals (Tizard et al., ent age distribution, the true association between site and 1976; Smith and Frenkel, 1995). Even though the number serostatus was confounded by age. In order to overcome of tested individuals included in our study was limited, we 64 M.A. Sepúlveda et al. / Veterinary Parasitology 175 (2011) 60–65

Table 3 ingestion (Munoz-Zanzi˜ et al., 2010). These results are Diet of American mink based in 60 fecal samples at Maullín river, Chile. consistent with our observations of high prevalence of Occurrence (O) and frequency of occurrence (FO). infection in minks and its association with populated areas Prey class O FO (%) which could be explained by higher abundances of cats in Birds 3 5.0 urban or peri-urban areas than in rural areas. The humid Crustaceans 60 100 environmental conditions in this southern part of Chile are Samasthacus spinifrons 60 100 also favorable for the survival of the oocysts in the envi- Aegla spp. 13 21.7 ronment. At present, there have been no studies on cat Mammals Abrothrix longipilis 4 6.7 ecology and management in the south of Chile. Such stud- Fish 2 3.3 ies could help understand the epidemiology of T. gondii in this ecosystem, including the inter-relationships between the urban and wildlife cycles. observed a significant difference in exposure to T. gondii between the peri-urban and rural sites. This difference was Acknowledgements striking considering the relatively short distances between the two sites (15 km). As expected with serologic results The authors wish to thank Ignacio Rodriguez, Jorge Ruiz, and chronic infections, we observed that increasing mink and Tatiana Proboste for logistic support and the landown- age increased the odds of positivity to T. gondii probably as ers of the trapping sites. This project was financed by a result of higher probability of life time exposure in older personal funds (M.A.S.), Universidad Austral de Chile Grad- animals. In the case of sex, we did not find a difference in uate School, the Southern River Otter Project CODEFF-FZS the seroprevalence of T. gondii antibodies in mink. If that is and a Fulbright-CONICYT scholarship (M.A.S.). the case, it could indicate that both male and female have similar probabilities of acquiring infection with T. gondii, References despite the known differences in spatial ecology and move- ments between sexes in these mustelids (Dunstone, 1993). Agresti, A., 2002. Categorical Data Analysis. Wiley-Interscience, New York, Diet plays a key role in T. gondii transmission. Previous 710 pp. studies of T. gondii in mammals have reported higher preva- Bonesi, L., Chanin, P., Macdonald, D.W., 2004. Competition between Eurasian otter Lutra lutra and American mink Mustela vison proved lences in carnivore species than omnivores and herbivores by niche shift. Oikos 106, 19–26. (Smith and Frenkel, 1995; Hejlicek et al., 1997; Sobrino et Chehébar, C.E., Benoit, I., 1988. 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