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The Pine marten (Martes martes) and the Stone marten (Martes foina) as possible wild reservoirs of Toxoplasma gondii in the Baltic States
Accepted Manuscript The Pine marten (Martes martes) and the Stone marten (Martes foina) as possible wild reservoirs of Toxoplasma gondii in the Baltic States
Gunita Deksne, Zanda Segliņa, Bettija Ligere, Muza Kirjušina PII: DOI: Reference:
S2405-9390(16)30207-6 doi: 10.1016/j.vprsr.2017.05.004 VPRSR 101
To appear in:
Veterinary Parasitology: Regional Studies and Reports
Received date: Revised date: Accepted date:
15 September 2016 17 May 2017 19 May 2017
Please cite this article as: Gunita Deksne, Zanda Segliņa, Bettija Ligere, Muza Kirjušina , The Pine marten (Martes martes) and the Stone marten (Martes foina) as possible wild reservoirs of Toxoplasma gondii in the Baltic States, Veterinary Parasitology: Regional Studies and Reports (2017), doi: 10.1016/j.vprsr.2017.05.004
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ACCEPTED MANUSCRIPT The Pine marten (Martes martes) and the Stone marten (Martes foina) as possible wild reservoirs of Toxoplasma gondii in the Baltic States
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Gunita Deksnea*, Zanda Segliņaa, Bettija Ligerea, Muza Kirjušinab
Institute of Food Safety, Animal Health and Environment BIOR, Riga, Latvia.
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Institute of Life Sciences and Technology, Daugavpils University, Daugavpils, Latvia.
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* Corresponding author: Gunita Deksne, Institute of Food Safety, Animal Health and
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Environment BIOR, Lejupes street 3, Riga, Latvia, LV-1076; Tel.: +371 676 207 18;
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fax.: +371 676 204 34; e-mail: [email protected]
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Abstract Toxoplasma gondii is an important zoonotic parasite that infects a wide spectrum of mammals. Moreover, its presence in wild carnivores is indicative of environmental
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contamination by the parasite. A total of 186 marten (152 pine marten; 34 stone marten)
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meat juice samples from Latvia and Lithuania were tested for T. gondii seroprevalence.
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Samples were tested for T. gondii specific antibodies by a commercial ELISA and antibodies were found in 121 (65.1%) samples. Higher prevalence (67.8%) occurred in
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pine martens compared to that of stone martens (52.9%). Adult pine martens had a 2.0
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(95% CI 0.9-4.7) times higher odds ratio to test seropositive than juvenile Pine martens. In addition, a significant positive correlation (r=0.75; P=0.05) was observed between T.
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gondii seroprevalence in martens and the number of estimated Eurasian lynx as a possible
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definitive host within the game administrative unit. The present study suggests that the high seroprevalence of T. gondii in Pine and Stone martens could indicate a high
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incidence of the parasite in the intermediate and definitive host population and in the
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environment.
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Keywords: Toxoplasma gondii, seroprevalence, martens, the Baltic States
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ACCEPTED MANUSCRIPT 1. Introduction The obligate coccidian parasite Toxoplasma gondii is an important zoonotic parasite that infects a wide spectrum of warm-blooded animals. This includes mammals, birds, game and none-game animals. The members of the cat family (Felidae) are the
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only known definitive hosts for the parasite that release parasite oocysts in their feces
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(Dubey, 2010). The Eurasian lynx (Lynx lynx) is the largest wild representative of the cat
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family (Felidae) in Europe and the only wild species of cats in Latvia and Lithuania (Balčiauskas et al. 2010; Ozoliņš et al. 2002). Latvia hosts 37 % of the Baltic lynx
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population (Boitani et al. 2015). In intermediate hosts, theinfection prevalence may vary
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in terms of feeding habits, geographical region and mutual species sharing the same ecosystem. Wild felids share some common areas in the wild with other animals that
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allow the dissemination of the T. gondii into the wild animal population (da Silva et al.,
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2014). The wild fauna may be a marker of environmental contamination with T. gondii and also an indicator of the transmission risk to humans and livestock (Dubey, 2010;
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Gennari et al., 2004). Wildlife can serve as a source of human infection if infected tissues
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are ingested, from poor hygiene when dressing killed game, or from fur bearing animals. Discarded meat scraps and viscera can contain T. gondii and result in tissue infection
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when ingested by scavengers (Briscoe et al., 1993; Dubey, 2010). The infection prevalence and effects of T. gondii in Martens has received little attention. In Europe, the prevalence of T. gondii was studied in several species of the family Mustelidae. Toxoplasma gondii antibodies and/or DNA was found in the European badger (Meles meles), Stone marten (Martes foina), Pine marten (Martes martes), Stoat (Mustela erminea), Least Weasel (Mustela nivalis), Polecat (Mustela
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ACCEPTED MANUSCRIPT putorius), Ferret (Mustela putorius furo) and the Eurasian otter (Lutra lutra) (Hejlíček et al. 1997; Anwar et al., 2006; Hůrková and Modrý, 2006; Sobrino et al., 2007; Lopes et al., 2011; Turčeková et al., 2014). High T. gondii seroprevalence was observed in Pine martens (62.5%, n=64; 100%, n=4) in Germany and in Stone martens (85.0%, n=20) in
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Spain (Weiland and Geisel, 1981; Sorbino et al., 2007). In addition to higher
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environmental pressure of infection, there is a cumulative effect of age in many wild and
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domestic animals which results in very high prevalence of T. gondii infection adult animals (Tenter et al. 2000). The data on T. gondii seroprevalence in wild animals in the
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Baltic States is limited and in many species remains unknown (Deksne et al., 2013a;
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Jokelainen et al., 2015). Even less is known about the T. gondii effects on population dynamics and epidemiological factors influencing T. gondii infection in martens.
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The Pine marten is an essential fur game animal species located throughout the
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Baltic States. Its population is increasing because of decreased hunting pressure due to a significant reduction in fur prices (Ozolinš and Pilāts, 1995; Prolux et al., 2005). On the
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other hand, the Stone marten is not as common as the Pine marten, and its distribution is
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patchy at best. The greatest densities are found in the south of Lithuania. In Latvia, however, the Stone marten is rare, and considered to be at the periphery of its habitat
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(Ozolinš and Pilāts, 1995; Prolux et al., 2005). The Stone marten is better adapted to warm climates and lacks morphological adaptations (meaning its fur is less dense and its feet are hairless) to survive severe winters with deep snow (Bakeyev, 1994). Pine marten populations reach higher densities in mature or old coniferous, deciduous or mixed forests. Stone marten frequents forests, cork oak, woodlands, rocky areas, fields, and is well adapted to the human habitat. Moreover, it continues to expand its range in suburban
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ACCEPTED MANUSCRIPT and urban areas (Prolux et al., 2005). Pine marten is most often characterized as a food generalist, and squirrels, shrews, cervid carcasses, birds, invertebrates, and berries may all be important prey. It feeds opportunistically and its diet differs among seasons, years, and areas (Helndin 2000). Stone martens feed on fruits most frequently followed by
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rodents, birds and insects (Posłuszny et al., 2007).
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The sylvatic life cycle is important in the epizootology of the T. gondii infection.
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However, it is unknown whether or not martens (Martes spp.) are a potential source of toxoplasmosis for other animals exposed to infected marten tissues. This study was
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conducted in order to estimate the seroprevalence of T. gondii in martens in Latvia and
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Lithuania. 2. Materials and methods
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2.1. Collection of samples
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Between 2011 and 2014, a total of 186 martens (152 Pine martens; 34 Stone martens) were collected in Latvia and Lithuania. Hunters were invited to provide marten
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carcasses on a voluntary basis from different hunting associations from the two countries.
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Overall, hunters from five game administrative units (Dienvidkurzeme, Riga and Zemgale in Latvia; Kauno and Šiaulių in Lithuania) provided martens which were hunted
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according to the hunting regulations of Latvia and Lithuania. The hunters provided the hunting site of each killed animal, its sex and its weight (g). The age group (juvenile/adult) of Pine martens (n=113) was determined by the weight of the animal according to study of Helldin (1999). A muscle sample of 50 g was cut from the front and/or hind legs of each animal and placed into a plastic container and then stored at -20° C. Meat juice was obtained in two ml Eppendorf tubes upon thawing the frozen muscle
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ACCEPTED MANUSCRIPT tissue directly from containers where the meat samples were stored, and then kept frozen at -20° C until analyzed. The data regarding the Eurasian lynx (Lynx lynx) estimated population size was collected from the database of the State Forest Service in Latvia and the Ministry of
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2.2. Serological enzyme-linked immunosorbent assay (ELISA)
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Environment in Lithuania.
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Toxoplasma-specific IgG antibodies were measured by use of a commercial kit for multiple species, including ruminants, swine, dogs, cats and others for serum, plasma or
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meat juice samples (ELISA ID Screen® Toxoplasmosis Indirect Multi-species, ID VET,
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Montpellier, France). The kit uses anti-multi-species peroxidase as a non-species specific protein conjugate. The ELISA was performed according to the manufacturer`s
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instructions and all the required materials and reagents provided only by manufacturer
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was used. Briefly, 50 µl of meat juice at a dilution of 1:2 of dilution buffer was dispensed into microtiter plates coated with recombinant T. gondii surface antigen P30. Microtiter
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plates were incubated for 45 minutes at room temperature (21° C ± 5° C) then washed
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three times with washing buffer; next, 100 µl anti-multi-species IgG-HRP conjugate was added and incubated for 30 minutes at room temperature and 100 µl peroxidase substrate
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was added to each well. Following incubation at room temperature for 15 minutes, the reaction was stopped with 100 µl stop solution. The optical density (OD) of each well was measured at 450 nm. Samples were analyzed in duplicate. In order to correct plateto-plate variation, positive and negative controls provided by manufacturer were included on every plate.
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ACCEPTED MANUSCRIPT For interpretation of the results a sample presenting percentage (S/P %) was calculated as: S/P% = (OD450 value of the sample – OD450 value of the negative control) x 100. Any sample with S/P% ≤ 40%, ≥ 50% and 40% - 50% was considered as negative, positive or doubtful, respectively. The doubtful results were retested. The test
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was validated if the mean value of the positive control OD (ODPC) was greater than 0.350
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(ODPC and ODNC) was greater than 3.5 (ODPC / ODNC > 3.5).
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(ODPC > 0.350) and the ratio of the mean OD values of the positive and negative controls
To compare the results of the ELISA on marten meat juice samples, a subset of the
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samples (n=44) from 186 was also analyzed using commercially available Direct
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Agglutination Test (DAT, bioMérieux, Marcy l’Etoile, France). The DAT was performed according to the manufacturer’s instructions and samples were tested in dilution 1:40 and
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1:4000 and meat juice positive at both dilution were considered positive.
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2.3. Data analyses
Statistical analyses were performed with SPSS Statistics Version 21 (IBM
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Corporation, Chicago, Illinois). Prevalence differences between different groups were
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compared by Chi-square and Fisher`s exact test as appropriate, and 95% confidence intervals (CI) were calculated. The prevalence differences for sexes were compared by
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combining both marten species, because the stone marten female and male sample size was low, 10 and 24 animals, respectively. The prevalence between different age groups was compared only for Pine martens. Spearman`s rank-order correlation was calculated for the T. gondii seroprevalence and estimated number of Eurasian lynx within the game administrative unit. P < 0.05 was considered significant.
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ACCEPTED MANUSCRIPT Repeatability of ELISA was evaluated by calculating the coefficient of variation (CV) for 96 repetitions of the positive control, and 96 repetitions of weak positive (S/P/% close to 50%) samples. The CV was expressed as a percentage. To evaluate the agreement among different tests, inert-rater agreement (kappa) was
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calculated and kappa values (k) were considered as follows: poor agreement (k<0.20);
3. Results
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(k=0.61-0.80); or very good agreement (k=0.81-1.00).
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fair agreement (k=0.21-0.40), moderate agreement (k=0.41-0.60); good agreement
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From 186 martens analyzed by ELISA, 121 (65.1%, 95% CI: 58.0-71.7%) were
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seropositive for T. gondii antibodies. Slightly higher prevalence (67.8%) occurred in Pine martens than in Stone martens (52.9%)(Table 1). Toxoplasma gondii seropositive animals
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were detected in animals originated from restricted areas (Fig. 1.). However, martens
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from Latvia had 2.9 (95% CI 1.5-6.0) times higher odds (OR) to test seropositive than the martens from Lithuania (Table 1). A significant positive correlation (r=0.75; P=0.05) was
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observed between T. gondii seroprevalence and the number of estimated Eurasian lynxes
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within the game administrative unit (Fig. 2.). There was no significant difference in the seroprevalence between female and male martens (Table 1). However, significantly
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(P=0.04) higher seroprevalence was observed for adult Pine martens in comparison to juvenile martens (Table 2). Adults had a 2.0 (95% CI 0.9-4.7) time higher OR to test seropositive than juvenile Pine martens. The ELISA kit was evaluated by comparing the obtained results of marten meat juice samples to the results obtained by testing the subset of samples in DAT performing
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ACCEPTED MANUSCRIPT inter-rater agreement analysis (Table 3). Calculated kappa value was 0.743 (P<0.01) representing a good agreement. For the evaluation of ELISA repeatability, the CVs obtained found to be between 6% and 10%.
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4. Discussion
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This study, based on the data collected over the past four years (2011-2014), is the
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first study on the circulation of T. gondii in martens of the two Baltic States (Latvia and Lithuania). The overall estimate of T. gondii seroprevalence in martens was high (65.1%)
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and it was higher compared to the omnivorous wild boar (24-33.2%, Deksne et al.,
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2013a, Jokelainen et al., 2015) and herbivorous sheep (12.3-42.1%, Stimbirys et al., 2006, G. Deksne pers. comm.) in the Baltic States. The results form present study where
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animals were collected from nearby locations are similar to those of Heljiček et al.
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(1997) who hypothesized that due to the feeding habits of the species, carnivores have a higher prevalence of antibodies against T. gondii due to their accumulative ingestion of
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infected meat from other animals. Martens found with antibodies to T. gondii indicated
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an exposure and a probable tissue infection (Tocidlowski et al., 1997). The presence of T. gondii in wild carnivores indicates the presence of T. gondii in
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carnivore prey base, for example in small rodents (Heljiček et al., 1997). The higher T. gondii seroprevalence in Pine marten compared to seroprevalence in Stone martens can be explained by their different feeding habits. The basis of the Pine marten diet is formed by rodents, birds and vegetable food during the warm season. In cold season, the Pine marten feeds mainly on mammals which constitute nearly 90% of its consumed biomass, in which rodents make up to 35.6% and carrion of wild ungulates constitute 45% of the
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ACCEPTED MANUSCRIPT consumed biomass (Baltrūnaitė, 2002). Stone martens feed on fruits most frequently followed by rodents, birds and insects (Posłuszny et al., 2007). Diet overlap varied seasonally and was highest in summer when the supply of different type of feed: rodents, birds, insects and fruits were the steadiest. This may suggest that the similarity of feeding
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niches results from opportunistic foraging strategies of both species (Posłuszny et al.,
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2007). Small rodents are considered an important reservoir of T. gondii for predators
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(Dabritz et al., 2008). The large survey of 17 small mammal species from the Czech Republic reported viable T. gondii in 0.9% from 5,166 tested animals (Hejlíček and
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Literák, 1998). The potential for small rodents to act as reservoirs has been emphasized
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by the discovery that there were extremely efficient vertical transmission in species such as the Wood mouse (Apodemus sylvaticus) and House mouse (Mus musculus) (Owen and
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Trees, 1998). Therefore, rodents represented a significant reservoir of infection for wild
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and domestic animals.
Significantly more seropositive martens were found in Latvia compared to martens
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from Lithuania. Moreover, a close relation of T. gondii seropositive martens and the
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number of estimated Eurasian lynx within the game administrative unit was observed in this study (Fig. 2.). A significantly higher mean number (1,696 lynx) of estimated
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Eurasian lynx in Latvia during 2010-2014 was reported compared to the mean number (544) of estimated Eurasian lynx in Lithuania (unpublished data from the State Forest Service in Latvia and the Ministry of Environment in Lithuania). There were no T. gondii seroprevalence studies of the Eurasian lynx in Latvia and Lithuania. Even so, a study in Finland showed a high T. gondii seroprevalence (86.1%) among lynxes (Jokelainen et al., 2013). On the other hand no evidence of an ongoing contribution of the environmental
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ACCEPTED MANUSCRIPT oocyst burden was found in Finnish lynx due to the lynx age distribution of animals examined (Jokelainen et al., 2013). Meanwhile, previous studies on T. gondii prevalence in domestic cats from urban areas in Latvia shows a high seroprevalence 51.6% (95 % CI: 45.4 – 57.9%) and 2.5% (95% CI: 0.4 – 8.0%) of examined cats were found to shed
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Toxoplasma-like oocysts (Deksne et al., 2013b). While pine martens are strongly
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associated with forest habitats and they avoid open areas such as fields and meadows,
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stone martens are closely associated with human settlements and their close surroundings; they live in villages, towns and cities (Prolux et al. 2005). Because of this,
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urban cats could be considered as a potential source of infection for martens.
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Toxoplasma gondii infected lynx carcasses may be eaten by other hosts mainly by scavengers such as red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes
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procyonoides) (Jakubek et al., 2001). In Latvia, there is an increasing population of red
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foxes and raccoon dogs that may have been exacerbated by the common habit of hunters to leave different animal carcasses in the field after killing or skinning them, or removing
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and discarding the entrails. This has been demonstrated to strongly increase the
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probability of transmitting other important zoonotic parasites such as Trichinella spp. in the wild (Pozio et al., 2001). Preliminary studies of T. gondii seroprevalence in red foxes
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(29.7%, 95%CI 24.9-34.9%, n=323) and raccoon dogs (14.8%, 95%CI 11.4-18.9%, n=344) in Latvia shows an exposure and possible tissue infection also in these abundant wild animals (G. Deksne unpublished data). The diet composition studies showed that mostly red foxes in North Europe consumed carrion (e.g., wild boar) (64.8%) and small mammals (53.4%) while raccoon dogs mainly chose plants (82.5%), following carrion (48.5%) and small mammals (28.6 %) (Süld et al. 2014). This shows that martens and
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ACCEPTED MANUSCRIPT small predators share the prey base and may get infected with T. gondii from infected prey rodents. In Pine martens sexual dimorphism appears to be related to food acquisition and processing, suggesting that males are more specialized to actively capture and kill prey,
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while females rely on easier-to-catch prey (Loy et al., 2004). However, there was no
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significant difference in the T. gondii seroprevalence between female and male martens.
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The present study shows that the high seroprevalence of T. gondii in Pine and Stone martens implicitly could refer to high incidence of the parasite in the intermediate and
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definitive host population and in the environment. Martens could serve as a marker of
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environmental contamination with T. gondii and also as an indicator of the transmission risk to humans by skinning them for the pelts and livestock by ingesting discarded meat
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scraps and viscera that contain T. gondii (Dubey, 2010; Gennari et al., 2004). This is
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especially because open air farming constitutes a risk situation and some livestock species are reared more extensively and thus, are more prone to share diseases with
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wildlife. Although, further studies are needed to provide a confirmation on actual marten
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infection with the T. gondii parasite and its importance in T. gondii sylvatic cycle as possible a reservoir and source of infection to other wildlife hosts.
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Conflict of interests
None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the work presented herein. Acknowledgements We would like to thank the hunters for their cooperation and positive attitude toward studying marten parasites. The authors would also like to thank the staff of the
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ACCEPTED MANUSCRIPT State Forest Service (Latvia) and the Ministry of Environment (Lithuania) for providing information and estimates on the carnivore mammal population size. We are also grateful to Māris Nitcis of the Institute of Life Sciences and Technology, Daugavpils University
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for creating the map.
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Dubey, J.P., Almeria, S., 2007. Seroprevalence of Toxoplasma gondii antibodies in wild carnivores from Spain. Vet. Parasitol. 148, 187–192. Stimbirys, A., Bagdonas, J., Gerulis, G., Russo, P., 2006. A serological study on the prevalence of Toxoplasma gondii in sheep of Lithuania. Pol. J. Vet. Sci. 10, 83–87.
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ACCEPTED MANUSCRIPT Süld, K., Valdmann, H., Laurimaa, L., Soe, E., Davison, J., Saarma, U. 2014. An invasive vector of zoonotic diseases sustained by anthropogenic resources: the raccoon dog in Northern Europe. PLoS ONE 9(5): e96358. Tenter, A. M., Heckeroth, A. R., Weiss, L. M. 2000. Toxoplasma gondii: from animals
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to humans. Int. J. Parasitol. 30, 1217-1258.
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Tocidlowski, M.E., Lappin, M.R., Sumner, P.W., Stoskopf, M.K., 1997. Serological
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survey for Toxoplasmosis in River Otter. J. Wildl. Dis. 33, 649–652. Turčeková, Ľ., Hurníková, Z., Spišák, F., Miterpáková, M., Chovacová, B., 2014.
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Toxoplasma gondii in protected wildlife in the Tatra National Park (TANAP),
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Slovakia. Ann. Agric. Environ. Med. 21, 235–238.
Weiland, G., Geisel, O., 1981. Parasitological and histopathological studies of
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Wochenschr. 94, 246–248.
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Toxoplasma infections in stone martens (Martes foina). Berl. Munch. Tierarztl.
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ACCEPTED MANUSCRIPT Fig. 1. Map of Latvia and Lithuania showing the origin of individuals from both marten species Martes spp. tested for antibodies specific to Toxoplasma gondii. The samples were collected from five game administrative units (Dienvidkurzeme, Riga and Zemgale in Latvia; Kauno and Šiaulių in Lithuania).
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Fig. 2. Seroprevalence of Toxoplasma gondii combined in individuals from both marten
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species and number of estimated Eurasian lynxes per game administrative unit in Latvia
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and Lithuania (r=0.75; P=0.05).
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ACCEPTED MANUSCRIPT Table 1 Seroprevalence of Toxoplasma gondii in martens Martes spp. associated with variable factors. No. of investigated animals/
Prevalence % /
No. of seropositive animals
95% CI
Country**
Stone marten
34/18
Female
79/48
Male
107/73
Latvia
142/101
Lithuania
44/20
45.5/ 31.7-59.9
186/121
65.1/ 58.0-71.5
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TOTAL
67.8/ 60.0-74.7
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152/103
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Sex*
Pine marten
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Species
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Factors
52.9/ 36.7-68.6 60.8/ 49.7-70.8 68.2/ 58.9-76.3 71.3/ 63.2-78.0
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* The prevalence differences for sexes were compared by combining both marten species
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different countries.
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** Statistically significant differences between the seroprevalence of the martens from
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ACCEPTED MANUSCRIPT Table 2 Age group related seroprevalence of Toxoplasma gondii in pine martens Martes martes. Prevalence, %
No of positive
(95% CI)
Juvenile
46/28
60.9 (46.4-73.7)
Adult
67/51
76.1 (64.6-84.8)
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No of analyzed/
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Age group
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ACCEPTED MANUSCRIPT Table 3 The comparison the obtained results of marten meat juice samples by ELISA and DAT for the subset of samples (n=44).
DAT
Negative
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2
Positive
3
ELISA
27
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15
29
14 30 44
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Total
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Positive
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Negative
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Total
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Fig. 1
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Fig. 2
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ACCEPTED MANUSCRIPT Highlights: A high seroprevalence of T. gondii was detected in both marten species
Higher seroprevalence occurred in pine martens than in stone martens
The seroprevalence was higher in adult pine martens than in juvenile
Seroprevalence in martens positively correlated with a number of Eurasian lynx
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Gunita Deksne, Zanda Segliņa, Bettija Ligere, Muza Kirjušina PII: DOI: Reference:
S2405-9390(16)30207-6 doi: 10.1016/j.vprsr.2017.05.004 VPRSR 101
To appear in:
Veterinary Parasitology: Regional Studies and Reports
Received date: Revised date: Accepted date:
15 September 2016 17 May 2017 19 May 2017
Please cite this article as: Gunita Deksne, Zanda Segliņa, Bettija Ligere, Muza Kirjušina , The Pine marten (Martes martes) and the Stone marten (Martes foina) as possible wild reservoirs of Toxoplasma gondii in the Baltic States, Veterinary Parasitology: Regional Studies and Reports (2017), doi: 10.1016/j.vprsr.2017.05.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The Pine marten (Martes martes) and the Stone marten (Martes foina) as possible wild reservoirs of Toxoplasma gondii in the Baltic States
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Gunita Deksnea*, Zanda Segliņaa, Bettija Ligerea, Muza Kirjušinab
Institute of Food Safety, Animal Health and Environment BIOR, Riga, Latvia.
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Institute of Life Sciences and Technology, Daugavpils University, Daugavpils, Latvia.
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a
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* Corresponding author: Gunita Deksne, Institute of Food Safety, Animal Health and
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Environment BIOR, Lejupes street 3, Riga, Latvia, LV-1076; Tel.: +371 676 207 18;
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fax.: +371 676 204 34; e-mail: [email protected]
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ACCEPTED MANUSCRIPT
Abstract Toxoplasma gondii is an important zoonotic parasite that infects a wide spectrum of mammals. Moreover, its presence in wild carnivores is indicative of environmental
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contamination by the parasite. A total of 186 marten (152 pine marten; 34 stone marten)
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meat juice samples from Latvia and Lithuania were tested for T. gondii seroprevalence.
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Samples were tested for T. gondii specific antibodies by a commercial ELISA and antibodies were found in 121 (65.1%) samples. Higher prevalence (67.8%) occurred in
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pine martens compared to that of stone martens (52.9%). Adult pine martens had a 2.0
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(95% CI 0.9-4.7) times higher odds ratio to test seropositive than juvenile Pine martens. In addition, a significant positive correlation (r=0.75; P=0.05) was observed between T.
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gondii seroprevalence in martens and the number of estimated Eurasian lynx as a possible
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definitive host within the game administrative unit. The present study suggests that the high seroprevalence of T. gondii in Pine and Stone martens could indicate a high
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incidence of the parasite in the intermediate and definitive host population and in the
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environment.
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Keywords: Toxoplasma gondii, seroprevalence, martens, the Baltic States
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ACCEPTED MANUSCRIPT 1. Introduction The obligate coccidian parasite Toxoplasma gondii is an important zoonotic parasite that infects a wide spectrum of warm-blooded animals. This includes mammals, birds, game and none-game animals. The members of the cat family (Felidae) are the
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only known definitive hosts for the parasite that release parasite oocysts in their feces
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(Dubey, 2010). The Eurasian lynx (Lynx lynx) is the largest wild representative of the cat
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family (Felidae) in Europe and the only wild species of cats in Latvia and Lithuania (Balčiauskas et al. 2010; Ozoliņš et al. 2002). Latvia hosts 37 % of the Baltic lynx
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population (Boitani et al. 2015). In intermediate hosts, theinfection prevalence may vary
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in terms of feeding habits, geographical region and mutual species sharing the same ecosystem. Wild felids share some common areas in the wild with other animals that
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allow the dissemination of the T. gondii into the wild animal population (da Silva et al.,
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2014). The wild fauna may be a marker of environmental contamination with T. gondii and also an indicator of the transmission risk to humans and livestock (Dubey, 2010;
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Gennari et al., 2004). Wildlife can serve as a source of human infection if infected tissues
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are ingested, from poor hygiene when dressing killed game, or from fur bearing animals. Discarded meat scraps and viscera can contain T. gondii and result in tissue infection
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when ingested by scavengers (Briscoe et al., 1993; Dubey, 2010). The infection prevalence and effects of T. gondii in Martens has received little attention. In Europe, the prevalence of T. gondii was studied in several species of the family Mustelidae. Toxoplasma gondii antibodies and/or DNA was found in the European badger (Meles meles), Stone marten (Martes foina), Pine marten (Martes martes), Stoat (Mustela erminea), Least Weasel (Mustela nivalis), Polecat (Mustela
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ACCEPTED MANUSCRIPT putorius), Ferret (Mustela putorius furo) and the Eurasian otter (Lutra lutra) (Hejlíček et al. 1997; Anwar et al., 2006; Hůrková and Modrý, 2006; Sobrino et al., 2007; Lopes et al., 2011; Turčeková et al., 2014). High T. gondii seroprevalence was observed in Pine martens (62.5%, n=64; 100%, n=4) in Germany and in Stone martens (85.0%, n=20) in
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Spain (Weiland and Geisel, 1981; Sorbino et al., 2007). In addition to higher
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environmental pressure of infection, there is a cumulative effect of age in many wild and
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domestic animals which results in very high prevalence of T. gondii infection adult animals (Tenter et al. 2000). The data on T. gondii seroprevalence in wild animals in the
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Baltic States is limited and in many species remains unknown (Deksne et al., 2013a;
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Jokelainen et al., 2015). Even less is known about the T. gondii effects on population dynamics and epidemiological factors influencing T. gondii infection in martens.
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The Pine marten is an essential fur game animal species located throughout the
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Baltic States. Its population is increasing because of decreased hunting pressure due to a significant reduction in fur prices (Ozolinš and Pilāts, 1995; Prolux et al., 2005). On the
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other hand, the Stone marten is not as common as the Pine marten, and its distribution is
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patchy at best. The greatest densities are found in the south of Lithuania. In Latvia, however, the Stone marten is rare, and considered to be at the periphery of its habitat
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(Ozolinš and Pilāts, 1995; Prolux et al., 2005). The Stone marten is better adapted to warm climates and lacks morphological adaptations (meaning its fur is less dense and its feet are hairless) to survive severe winters with deep snow (Bakeyev, 1994). Pine marten populations reach higher densities in mature or old coniferous, deciduous or mixed forests. Stone marten frequents forests, cork oak, woodlands, rocky areas, fields, and is well adapted to the human habitat. Moreover, it continues to expand its range in suburban
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ACCEPTED MANUSCRIPT and urban areas (Prolux et al., 2005). Pine marten is most often characterized as a food generalist, and squirrels, shrews, cervid carcasses, birds, invertebrates, and berries may all be important prey. It feeds opportunistically and its diet differs among seasons, years, and areas (Helndin 2000). Stone martens feed on fruits most frequently followed by
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rodents, birds and insects (Posłuszny et al., 2007).
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The sylvatic life cycle is important in the epizootology of the T. gondii infection.
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However, it is unknown whether or not martens (Martes spp.) are a potential source of toxoplasmosis for other animals exposed to infected marten tissues. This study was
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conducted in order to estimate the seroprevalence of T. gondii in martens in Latvia and
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Lithuania. 2. Materials and methods
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2.1. Collection of samples
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Between 2011 and 2014, a total of 186 martens (152 Pine martens; 34 Stone martens) were collected in Latvia and Lithuania. Hunters were invited to provide marten
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carcasses on a voluntary basis from different hunting associations from the two countries.
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Overall, hunters from five game administrative units (Dienvidkurzeme, Riga and Zemgale in Latvia; Kauno and Šiaulių in Lithuania) provided martens which were hunted
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according to the hunting regulations of Latvia and Lithuania. The hunters provided the hunting site of each killed animal, its sex and its weight (g). The age group (juvenile/adult) of Pine martens (n=113) was determined by the weight of the animal according to study of Helldin (1999). A muscle sample of 50 g was cut from the front and/or hind legs of each animal and placed into a plastic container and then stored at -20° C. Meat juice was obtained in two ml Eppendorf tubes upon thawing the frozen muscle
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ACCEPTED MANUSCRIPT tissue directly from containers where the meat samples were stored, and then kept frozen at -20° C until analyzed. The data regarding the Eurasian lynx (Lynx lynx) estimated population size was collected from the database of the State Forest Service in Latvia and the Ministry of
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2.2. Serological enzyme-linked immunosorbent assay (ELISA)
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Environment in Lithuania.
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Toxoplasma-specific IgG antibodies were measured by use of a commercial kit for multiple species, including ruminants, swine, dogs, cats and others for serum, plasma or
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meat juice samples (ELISA ID Screen® Toxoplasmosis Indirect Multi-species, ID VET,
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Montpellier, France). The kit uses anti-multi-species peroxidase as a non-species specific protein conjugate. The ELISA was performed according to the manufacturer`s
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instructions and all the required materials and reagents provided only by manufacturer
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was used. Briefly, 50 µl of meat juice at a dilution of 1:2 of dilution buffer was dispensed into microtiter plates coated with recombinant T. gondii surface antigen P30. Microtiter
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plates were incubated for 45 minutes at room temperature (21° C ± 5° C) then washed
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three times with washing buffer; next, 100 µl anti-multi-species IgG-HRP conjugate was added and incubated for 30 minutes at room temperature and 100 µl peroxidase substrate
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was added to each well. Following incubation at room temperature for 15 minutes, the reaction was stopped with 100 µl stop solution. The optical density (OD) of each well was measured at 450 nm. Samples were analyzed in duplicate. In order to correct plateto-plate variation, positive and negative controls provided by manufacturer were included on every plate.
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ACCEPTED MANUSCRIPT For interpretation of the results a sample presenting percentage (S/P %) was calculated as: S/P% = (OD450 value of the sample – OD450 value of the negative control) x 100. Any sample with S/P% ≤ 40%, ≥ 50% and 40% - 50% was considered as negative, positive or doubtful, respectively. The doubtful results were retested. The test
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was validated if the mean value of the positive control OD (ODPC) was greater than 0.350
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(ODPC and ODNC) was greater than 3.5 (ODPC / ODNC > 3.5).
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(ODPC > 0.350) and the ratio of the mean OD values of the positive and negative controls
To compare the results of the ELISA on marten meat juice samples, a subset of the
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samples (n=44) from 186 was also analyzed using commercially available Direct
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Agglutination Test (DAT, bioMérieux, Marcy l’Etoile, France). The DAT was performed according to the manufacturer’s instructions and samples were tested in dilution 1:40 and
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1:4000 and meat juice positive at both dilution were considered positive.
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2.3. Data analyses
Statistical analyses were performed with SPSS Statistics Version 21 (IBM
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Corporation, Chicago, Illinois). Prevalence differences between different groups were
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compared by Chi-square and Fisher`s exact test as appropriate, and 95% confidence intervals (CI) were calculated. The prevalence differences for sexes were compared by
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combining both marten species, because the stone marten female and male sample size was low, 10 and 24 animals, respectively. The prevalence between different age groups was compared only for Pine martens. Spearman`s rank-order correlation was calculated for the T. gondii seroprevalence and estimated number of Eurasian lynx within the game administrative unit. P < 0.05 was considered significant.
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ACCEPTED MANUSCRIPT Repeatability of ELISA was evaluated by calculating the coefficient of variation (CV) for 96 repetitions of the positive control, and 96 repetitions of weak positive (S/P/% close to 50%) samples. The CV was expressed as a percentage. To evaluate the agreement among different tests, inert-rater agreement (kappa) was
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calculated and kappa values (k) were considered as follows: poor agreement (k<0.20);
3. Results
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(k=0.61-0.80); or very good agreement (k=0.81-1.00).
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fair agreement (k=0.21-0.40), moderate agreement (k=0.41-0.60); good agreement
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From 186 martens analyzed by ELISA, 121 (65.1%, 95% CI: 58.0-71.7%) were
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seropositive for T. gondii antibodies. Slightly higher prevalence (67.8%) occurred in Pine martens than in Stone martens (52.9%)(Table 1). Toxoplasma gondii seropositive animals
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were detected in animals originated from restricted areas (Fig. 1.). However, martens
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from Latvia had 2.9 (95% CI 1.5-6.0) times higher odds (OR) to test seropositive than the martens from Lithuania (Table 1). A significant positive correlation (r=0.75; P=0.05) was
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observed between T. gondii seroprevalence and the number of estimated Eurasian lynxes
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within the game administrative unit (Fig. 2.). There was no significant difference in the seroprevalence between female and male martens (Table 1). However, significantly
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(P=0.04) higher seroprevalence was observed for adult Pine martens in comparison to juvenile martens (Table 2). Adults had a 2.0 (95% CI 0.9-4.7) time higher OR to test seropositive than juvenile Pine martens. The ELISA kit was evaluated by comparing the obtained results of marten meat juice samples to the results obtained by testing the subset of samples in DAT performing
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ACCEPTED MANUSCRIPT inter-rater agreement analysis (Table 3). Calculated kappa value was 0.743 (P<0.01) representing a good agreement. For the evaluation of ELISA repeatability, the CVs obtained found to be between 6% and 10%.
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4. Discussion
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This study, based on the data collected over the past four years (2011-2014), is the
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first study on the circulation of T. gondii in martens of the two Baltic States (Latvia and Lithuania). The overall estimate of T. gondii seroprevalence in martens was high (65.1%)
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and it was higher compared to the omnivorous wild boar (24-33.2%, Deksne et al.,
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2013a, Jokelainen et al., 2015) and herbivorous sheep (12.3-42.1%, Stimbirys et al., 2006, G. Deksne pers. comm.) in the Baltic States. The results form present study where
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animals were collected from nearby locations are similar to those of Heljiček et al.
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(1997) who hypothesized that due to the feeding habits of the species, carnivores have a higher prevalence of antibodies against T. gondii due to their accumulative ingestion of
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infected meat from other animals. Martens found with antibodies to T. gondii indicated
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an exposure and a probable tissue infection (Tocidlowski et al., 1997). The presence of T. gondii in wild carnivores indicates the presence of T. gondii in
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carnivore prey base, for example in small rodents (Heljiček et al., 1997). The higher T. gondii seroprevalence in Pine marten compared to seroprevalence in Stone martens can be explained by their different feeding habits. The basis of the Pine marten diet is formed by rodents, birds and vegetable food during the warm season. In cold season, the Pine marten feeds mainly on mammals which constitute nearly 90% of its consumed biomass, in which rodents make up to 35.6% and carrion of wild ungulates constitute 45% of the
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ACCEPTED MANUSCRIPT consumed biomass (Baltrūnaitė, 2002). Stone martens feed on fruits most frequently followed by rodents, birds and insects (Posłuszny et al., 2007). Diet overlap varied seasonally and was highest in summer when the supply of different type of feed: rodents, birds, insects and fruits were the steadiest. This may suggest that the similarity of feeding
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niches results from opportunistic foraging strategies of both species (Posłuszny et al.,
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2007). Small rodents are considered an important reservoir of T. gondii for predators
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(Dabritz et al., 2008). The large survey of 17 small mammal species from the Czech Republic reported viable T. gondii in 0.9% from 5,166 tested animals (Hejlíček and
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Literák, 1998). The potential for small rodents to act as reservoirs has been emphasized
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by the discovery that there were extremely efficient vertical transmission in species such as the Wood mouse (Apodemus sylvaticus) and House mouse (Mus musculus) (Owen and
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Trees, 1998). Therefore, rodents represented a significant reservoir of infection for wild
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and domestic animals.
Significantly more seropositive martens were found in Latvia compared to martens
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from Lithuania. Moreover, a close relation of T. gondii seropositive martens and the
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number of estimated Eurasian lynx within the game administrative unit was observed in this study (Fig. 2.). A significantly higher mean number (1,696 lynx) of estimated
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Eurasian lynx in Latvia during 2010-2014 was reported compared to the mean number (544) of estimated Eurasian lynx in Lithuania (unpublished data from the State Forest Service in Latvia and the Ministry of Environment in Lithuania). There were no T. gondii seroprevalence studies of the Eurasian lynx in Latvia and Lithuania. Even so, a study in Finland showed a high T. gondii seroprevalence (86.1%) among lynxes (Jokelainen et al., 2013). On the other hand no evidence of an ongoing contribution of the environmental
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ACCEPTED MANUSCRIPT oocyst burden was found in Finnish lynx due to the lynx age distribution of animals examined (Jokelainen et al., 2013). Meanwhile, previous studies on T. gondii prevalence in domestic cats from urban areas in Latvia shows a high seroprevalence 51.6% (95 % CI: 45.4 – 57.9%) and 2.5% (95% CI: 0.4 – 8.0%) of examined cats were found to shed
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Toxoplasma-like oocysts (Deksne et al., 2013b). While pine martens are strongly
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associated with forest habitats and they avoid open areas such as fields and meadows,
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stone martens are closely associated with human settlements and their close surroundings; they live in villages, towns and cities (Prolux et al. 2005). Because of this,
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urban cats could be considered as a potential source of infection for martens.
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Toxoplasma gondii infected lynx carcasses may be eaten by other hosts mainly by scavengers such as red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes
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procyonoides) (Jakubek et al., 2001). In Latvia, there is an increasing population of red
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foxes and raccoon dogs that may have been exacerbated by the common habit of hunters to leave different animal carcasses in the field after killing or skinning them, or removing
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and discarding the entrails. This has been demonstrated to strongly increase the
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probability of transmitting other important zoonotic parasites such as Trichinella spp. in the wild (Pozio et al., 2001). Preliminary studies of T. gondii seroprevalence in red foxes
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(29.7%, 95%CI 24.9-34.9%, n=323) and raccoon dogs (14.8%, 95%CI 11.4-18.9%, n=344) in Latvia shows an exposure and possible tissue infection also in these abundant wild animals (G. Deksne unpublished data). The diet composition studies showed that mostly red foxes in North Europe consumed carrion (e.g., wild boar) (64.8%) and small mammals (53.4%) while raccoon dogs mainly chose plants (82.5%), following carrion (48.5%) and small mammals (28.6 %) (Süld et al. 2014). This shows that martens and
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ACCEPTED MANUSCRIPT small predators share the prey base and may get infected with T. gondii from infected prey rodents. In Pine martens sexual dimorphism appears to be related to food acquisition and processing, suggesting that males are more specialized to actively capture and kill prey,
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while females rely on easier-to-catch prey (Loy et al., 2004). However, there was no
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significant difference in the T. gondii seroprevalence between female and male martens.
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The present study shows that the high seroprevalence of T. gondii in Pine and Stone martens implicitly could refer to high incidence of the parasite in the intermediate and
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definitive host population and in the environment. Martens could serve as a marker of
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environmental contamination with T. gondii and also as an indicator of the transmission risk to humans by skinning them for the pelts and livestock by ingesting discarded meat
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scraps and viscera that contain T. gondii (Dubey, 2010; Gennari et al., 2004). This is
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especially because open air farming constitutes a risk situation and some livestock species are reared more extensively and thus, are more prone to share diseases with
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wildlife. Although, further studies are needed to provide a confirmation on actual marten
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infection with the T. gondii parasite and its importance in T. gondii sylvatic cycle as possible a reservoir and source of infection to other wildlife hosts.
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Conflict of interests
None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the work presented herein. Acknowledgements We would like to thank the hunters for their cooperation and positive attitude toward studying marten parasites. The authors would also like to thank the staff of the
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ACCEPTED MANUSCRIPT State Forest Service (Latvia) and the Ministry of Environment (Lithuania) for providing information and estimates on the carnivore mammal population size. We are also grateful to Māris Nitcis of the Institute of Life Sciences and Technology, Daugavpils University
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for creating the map.
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Wochenschr. 94, 246–248.
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Toxoplasma infections in stone martens (Martes foina). Berl. Munch. Tierarztl.
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ACCEPTED MANUSCRIPT Fig. 1. Map of Latvia and Lithuania showing the origin of individuals from both marten species Martes spp. tested for antibodies specific to Toxoplasma gondii. The samples were collected from five game administrative units (Dienvidkurzeme, Riga and Zemgale in Latvia; Kauno and Šiaulių in Lithuania).
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Fig. 2. Seroprevalence of Toxoplasma gondii combined in individuals from both marten
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species and number of estimated Eurasian lynxes per game administrative unit in Latvia
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and Lithuania (r=0.75; P=0.05).
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ACCEPTED MANUSCRIPT Table 1 Seroprevalence of Toxoplasma gondii in martens Martes spp. associated with variable factors. No. of investigated animals/
Prevalence % /
No. of seropositive animals
95% CI
Country**
Stone marten
34/18
Female
79/48
Male
107/73
Latvia
142/101
Lithuania
44/20
45.5/ 31.7-59.9
186/121
65.1/ 58.0-71.5
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TOTAL
67.8/ 60.0-74.7
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152/103
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Sex*
Pine marten
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Species
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Factors
52.9/ 36.7-68.6 60.8/ 49.7-70.8 68.2/ 58.9-76.3 71.3/ 63.2-78.0
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* The prevalence differences for sexes were compared by combining both marten species
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different countries.
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** Statistically significant differences between the seroprevalence of the martens from
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ACCEPTED MANUSCRIPT Table 2 Age group related seroprevalence of Toxoplasma gondii in pine martens Martes martes. Prevalence, %
No of positive
(95% CI)
Juvenile
46/28
60.9 (46.4-73.7)
Adult
67/51
76.1 (64.6-84.8)
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No of analyzed/
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Age group
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ACCEPTED MANUSCRIPT Table 3 The comparison the obtained results of marten meat juice samples by ELISA and DAT for the subset of samples (n=44).
DAT
Negative
12
2
Positive
3
ELISA
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15
29
14 30 44
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Total
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Positive
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Negative
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Total
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ACCEPTED MANUSCRIPT Highlights: A high seroprevalence of T. gondii was detected in both marten species
Higher seroprevalence occurred in pine martens than in stone martens
The seroprevalence was higher in adult pine martens than in juvenile
Seroprevalence in martens positively correlated with a number of Eurasian lynx
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