Seroprevalence of Toxoplasma gondii in wild boar and deer in Brandenburg, Germany

Consumption of game in Germany has increased during the past 10 years. Wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) are the most frequently hunted and consumed game animals in Germany, yet information on the occurrence of zoonotic pathogens in these animal species is scarce. To better estimate the public health risk emanating from handling and consumption of game, this study investigated seroprevalences of Toxoplasma gondii in game hunted in the German federal state of Brandenburg during two hunting seasons from 2017 to 2019. Toxoplasma gondii‐specific antibodies were detected in 24.4% (44/180, 95% CI: 18.4%–31.4%) of wild boar, 12.8% (16/125, 95% CI: 7.5%–20%) of roe deer and 6.4% (3/47, 95% CI: 1.3%–17.5%) of red deer using a commercial ELISA kit. Seroprevalences were similar in the two hunting seasons. Correlation between sex and seropositivity could not be observed. A rise in seroprevalence was seen with increasing age in all studied game species. Observed seroprevalences suggest that T. gondii is endemic in the sylvatic environment in the German federal state of Brandenburg and imply that game could represent a relevant source for human T. gondii infection.

undercooked meat containing infective tissue cysts or of raw plantbased food or water contaminated with sporulated oocysts. Scallan et al. (2011) estimated that 50% of the T. gondii infections in the USA are foodborne. Due to the high disease burden associated with human toxoplasmosis, T. gondii ranks among the top of the most important foodborne pathogens in the USA and the Netherlands (Havelaar et al., 2012;Scallan, Hoekstra, Mahon, Jones, & Griffin, 2015;WHO, 2015). In Europe, consumption of raw or undercooked meat, including game, has been identified as one of the most important risk factors for toxoplasmosis (Cook et al., 2000;Kapperud et al., 1996). Indeed, consumption of raw venison and meat of wild boar has already been identified as probable infection source in cases of acute toxoplasmosis (Carme et al., 2002;Choi et al., 1997;Ross et al., 2001;Sacks, Delgado, Lobel, & Parker, 1983). In addition, potential smear infections during or after evisceration of carcasses or handling of raw meat have to be considered (Dubey, 1991;McDonald et al., 1990).
According to the German hunting association, consumption of game in Germany has increased during the past 10 years (German Hunting Association, 2019a) and as in other European countries, there is a growing trend towards consumption of undercooked game with a pink or bloody core . Wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) are the most frequently hunted and consumed game animals in Germany, yet information on the prevalence of T. gondii in these species is scarce. To better estimate the public health risk emanating from handling and consumption of game, this study aimed to assess the prevalence of T. gondii in wild boar, roe deer and red deer in the German federal state of Brandenburg.  (Heddergott, Steinbach, Pohl, & Frantz, 2018), 25% for wild boar (Lutz, 1997) and 24% for red deer (Witkowski et al., 2015)  All animals were legally hunted for human consumption and made available for sampling post-mortem as part of the cooperation with the German Federal Forest Service. No animal was killed for the purpose of providing samples. Animal age was determined by hunters based on tooth eruption, tooth replacement, tooth wear and where available on antler development and physical appearance. In this study, the animals were divided into three age groups: juveniles (<1 years old), yearlings (1-2 years old) and adults (>2 years old).

| M ATERI A L S A N D M E TH O DS
Blood samples were obtained from the abdominal or thoracic cavity in 50 ml centrifugation tubes without additives and stored for 24-72 hr at 4°C for coagulation. After centrifugation at 1,600 g for 10 min at 4°C, sera were collected and stored at −20°C until analysis. Most sera were slightly haemolytic. T. gondii-specific antibodies were detected using the commercially available ELISA kit 'ID Screen® Toxoplasmosis Indirect Multi-species' (ID.Vet), which has been approved for the detection of T. gondii-specific antibodies in livestock and pets by the Licensing Authority of the Friedrich-Loeffler-Institut, Germany (FLI-B 550). It has also been utilized in T. gondii seroprevalence studies in wildlife, including wild boar, roe deer and red deer (Racka, Bartova, Budikova, & Vodrazka, 2015;Witkowski et al., 2015). The ELISA was performed and analysed in accordance with the manufacturer's instructions. The detected optical densities (OD 450nm ) were converted to S/P ratios (S/P% = [OD sample − OD negative control )/(OD positive control − OD negative control ] × 100) and interpreted as follows: S/P ≤ 40% negative, S/P ≥ 50% positive, 40% < S/P < 50% doubtful. To assess a possible association between seroprevalence and sex, age or hunting season, the Fisher's exact test was performed using the software
Seroprevalences in both hunting seasons were similar and no statistical significant differences were observed (Table 1; p = .43-1).
There was no correlation between sex and seropositivity (Table 1; p = .26-1). In all examined game species, seroprevalences increased with age. However, the increase in seroprevalence with age was only statistically significant for roe deer (Table 1; p = .004).

| D I SCUSS I O N
Consumption of game in the German population has been on the rise in the past 10 years (German Hunting Association, 2019a).
However, information on the occurrence of zoonotic pathogens in wild animals that are frequently hunted for human consumption is generally scarce in Germany.
In this study, observed seroprevalences of T. gondii in the investigated game species varied between 6.4% in red deer to 24.4% in wild boar. These results are in line with other European studies, where seroprevalences between seven and 60% in cervids and five and 57% in wild boar have been observed in the past (European Food Safety Authority, 2013; Heddergott et al., 2018;Jokelainen, Velstrom, & Lassen, 2015;Racka et al., 2015;Wallander, Frossling, Vagsholm, Uggla, & Lunden, 2015), further implying the endemic nature of T. gondii in European sylvatic environments.
The observed seroprevalence of 24.4% among wild boars in this study falls into the mid-range of seroprevalence estimates reported in former German studies (15%-33%) (Lutz, 1997;Rommel, Sommer, & Janitschke, 1967;Tackmann, 1999). Moreover, it is also comparable to the pooled estimated seroprevalences published for the Northern-Baltic region (33%), Europe (26%) and also globally (23%) TA B L E 1 Seroprevalence of Toxoplasma gondii in wild boar, roe deer and red deer in total as well as by hunting season, age and sex Note: For statistical analyses, the Fisher's exact test was performed. Statistical analyses considering age were performed using three age groups (<1, 1-2, >2 years old). The age of four wild boar and two roe deer, as well as sex of four wild boar, one red deer and one roe deer was not recorded and could therefore not be included in the analysis. a The Haldane-Anscombe correction was used for odds ratios considering age of red deer.
To our knowledge, this is the first study reporting the occurence of T. gondii-specific antibodies in red deer in Germany.
The observed seroprevalence of 6.4% is similar to the reported seroprevalence in Norway (7.7%) (Vikoren, Tharaldsen, Fredriksen, & Handeland, 2004), but relatively low compared to other European studies from Poland and the Czech Republic, where 24.1%, (Witkowski et al., 2015) and 45% (Bartova, Sedlak, Pavlik, & Literak, 2007) of the animals have been found to be seropositive, respectively. However, the relatively small sample size of red deer in this study resulted in a large confidence interval and more animals should be investigated to obtain more representative data. Sampling will continue during future hunting seasons.
Noteworthy, irrespective of the sample size, we obtained comparable prevalence in each hunting season.
In general, seroprevalences for all game species reported in different European studies vary substantially. A variety of factors can influence T. gondii seroprevalence such as local differences in the environmental oocyst contamination, which is also dependent on the density of felids in the sampled area. Further, the sampling strategy and application of different serological test systems, cut-off values and sample types, can be a source for heterogeneity (Olsen et al., 2019;Rostami et al., 2017).
All animals investigated in this study were intended for human consumption. However, game meat is not examined for the presence of T. gondii during official meat inspections in Germany and many other countries and could therefore represent a relevant source for human T. gondii infection, especially if consumed frequently and not cooked thoroughly. As precautionary measures, the German Federal Institute for Risk Assessment recommends that game and raw game meat products made from it should therefore undergo proper heating (core temperature of at least 72°C for 2 min) prior to consumption. As potential smear infections and cross-contamination during evisceration of carcasses by the hunters as well as during processing of raw game meat by consumers are conceivable (Kapperud et al., 1996), general hygienic procedures should be followed.
Future studies on T. gondii seroprevalence in game should be extended to other regions in Germany to investigate temporal and spatial distribution patterns as have already been performed in Finland (Jokelainen et al., 2010), Sweden (Wallander et al., 2015) and Estonia (Remes et al., 2018). As game becomes a more and more popular meat source, an increase in the number of farmed game can be expected and should therefore also be included in the studies. To better emanate the public health risk from T. gondii in game, molecular methods should additionally be applied for the direct detection of the parasite and to identify prevailing genotypes. Related Evaluation Methods, One Health Approaches for providing assistance and support for this study.

CO N FLI C T O F I NTE R E S T
The authors declare they have no conflicts of interest.

E TH I C A L S TATE M E NT
The study only involved animals killed by licensed hunters according to German regulations (German Hunting Act) with permission of the German Federal State of Brandenburg and the corresponding hunting authority. All animals were killed during the established hunting season on a regular wildlife management basis (population control).
No animal was killed for the purpose of providing samples.