First report on the sero-epidemiology of Toxoplasma gondii infection in German roe deer (Capreolus capreolus).

While the roe deer (Capreolus capeolus) is the most important game species in Germany and its venison is popular, there is limited knowledge about the prevalence of Toxoplasma gondii in this animal population in the country, and in wild ungulates in Germany generally. Between 2013 and 2015, we collected 295 blood samples from roe deer belonging to a central German population. Sera were analysed using a modified agglutination test (MAT, cut-off 1:20), and antibodies were detected in 86 of the 295 samples (29%). Seroprevalence values differed significantly between the different age classes, with antibodies more frequently observed in adults. In contrast, seroprevalence did not differ significantly between the sexes or collection years. Venison is frequently consumed raw or undercooked and may be a potential source of human infection with T. gondii. © M. Heddergott et al., published by EDP Sciences, 2018.

Introduction

Toxoplasma gondii is an obligate intracellular protozoan and the causative agent of toxoplasmosis [9]. Unsporulated oocysts are shed into the environment by felids, which are the only definitive hosts [8]. Most mammals can become intermediate hosts after consuming raw or undercooked meat containing T. gondii tissue cysts, or food and drink with oocysts [6, 9, 21]. Meat-derived products from domestic animals and game species may represent a potential source of human infection with T. gondii and the European Food Safety Authority (EFSA) recommends the monitoring of toxoplasmosis in humans, animals and foodstuffs [11].

The roe deer (Capreolus capreolus) is the most important game species in Germany [32]. According to the German Hunting Federation (DJV), around 1.2 million animals have been harvested annually countrywide in recent years (www.jagdverband.de). Despite the popularity of venison and the associated processed meats, there is currently no surveillance of T. gondii infection in German roe deer populations and little knowledge about the prevalence of the parasite in wild ungulates in Germany generally [17, 23, 29].

Here, we aim to assess the seroprevalence of T. gondii in a free-living German population of roe deer by sampling carcases that were intended for human consumption.

Material and methods

Ethics

Roe deer are a legal game species in Germany that licensed hunters can harvest outside the closed season. No animals were killed in order to provide samples for this study. All animals were legally shot and the carcases made available to the authors.

Sample collection

The study was performed in the west of the German federal state of Thuringia. The total size of the study area was roughly 1800 km2 comprising the Eichsfeld, the western part of the Unstrut-Hainich and the northern part of the Wartburg administrative districts. Between 2013 and 2015, local hunters collected blood from the heart of 295 legally hunted roe deer. Animals were sampled in hunting areas across the whole study area. After centrifuging samples for 10 min at 1000g using an EBA 200 (Hettich, Tuttlingen, Germany), the sera were stored at −20 °C until analysis. The sex, age and year of sample collection were recorded for each animal. Based on the dentition of the lower jaw, animals were classified as juveniles (≤1 year), yearlings (1–2 years), or adults (≥2 years) [19, 32].

Determination of antibodies to T. gondii

A commercial kit (Toxo-Screen DA®, bioMérieux, Lyon, France) was used to perform a modified agglutination test (MAT) to analyse sera for the presence of T. gondii immunoglobulin G (IgG) antibodies. Positive and negative controls employed formalin-fixed tachyzoites as antigens. Serum samples were tested at dilutions of 1:20, 1:400, 1:1600 and 1:3200. The sensitivity and specificity of the test were maximized by using a cut-off titre of 1:20 [10]. Of all the available serological tests, the MAT is considered to be the most reliable in terms of detecting antibodies to T. gondii, especially in latently infected animals [9].

Statistical analysis

We performed a χ2-test in SPSS v.22 (SPSS Inc., Chicago, Illinois, USA) to assess the effect of sex, age class and collection year on T. gondii seroprevalence. Odds ratios (ORs) and their 95% confidence intervals (95% Cls) were calculated to assess the strength of the association between the presence of antibodies and the explanatory variables.

Results

Toxoplasma gondii antibodies were detected in 86 of the 295 analysed roe deer (29.15%, 95% CI: 24.10–34.75). Positive results were recorded at titres between 1:20 (34.88%), 1:400 (51.16%), 1:1600 (11.63%), and 1:3200 (2.33%). The difference in seroprevalence between males and females was not statistically significant (Table 1; p = 0.328). Also, the difference in seroprevalence between collection years was not significant (Table 1; p = 0.279). In contrast, there was a significant difference in seroprevalence between the different age classes (p < 0.001), with antibodies to T. gondii more frequently detected in adults (Table 1).

Table 1.

Seroprevalence of Toxoplasma gondii in roe deer by gender, age, and collection year.

Variable	Category	No. tested	No. positive	Prevalence in % (95% CI)	p-value	OR (95% CI)
Gender	Male	155	49	31.61 (24.21–39.01)	0.328	Reference
	Female	140	37	26.43 (19.03–33.82)		0.78 (0.47–1.29)
Age	≤1 year	81	5	6.17 (0.82–11.53)	<0.001	Reference
	1–2 year	109	28	25.69 (11.35–34.02)		5.25 (1.93–14.31)
	≥2 year	105	53	50.24 (40.75–60.20)		15.49 (5.8–41.38)
Collection year	2013	86	22	25.58 (16.17–34.99)	0.279	Reference
	2014	113	39	34.51 (25.61–43.41)		1.53 (0.82–2.85)
	2015	96	25	26.04 (17.01–34.98)		1.05 (0.54–2.05)
Total		295	86	29.15 (23.94–34.37)

Discussion

This is the first study investigating the seroprevalence of T. gondii antibodies in German roe deer. Values reported from other European roe deer populations ranged from 13% to 63% (Table 2). These previous studies used at least six different diagnostic tests (Table 2). In addition to our MAT test, the direct agglutination test (DAT) and the enzyme-linked immunosorbent assay (ELISA) have also been used frequently in this context and it has been shown that the three tests produced congruent and comparable results [13, 14, 24, 34]. Seroprevalences reported using one of these three tests ranged from 13% to 52% (Table 2). Of these, studies performed in Spain and Poland often reported substantially lower prevalence values than the 29.15% reported here, while studies from Belgium and France reported substantially higher figures. Other studies presented estimates that were in line with the estimate from the present study (Table 2).

Table 2.

Seroprevalence of Toxoplasma gondii in roe deer from Europe.

State	Source	No. tested	Prevalence in %	Serological testa	References
Belgium	Wildlife	73	52.0	ELISA	De Craeyea et al. [7]
Czech Republic	Captive	4	50.0	IFAT	Sedlák and Bartová [28]
	Wildlife	95	13.0	DT	Hejlíček et al. [18]
	Wildlife	79	24.0	IFAT	Bárlová et al. [3]
France	Wildlife	33	36.4	MAT	Aubert et al. [2]
	Wildlife	245	46.4	ELISA	Candela et al. [5]
Germany	Wildlife	295	29.15	MAT	Present study
Italy	Wildlife	207	13.0	LAT	Gaffuri et al. [12]
Norway	Wildlife	760	33.9	DAT	Vikoren et al. [33]
Norway and Sweden	Wildlife	8	63.0	DT	Kapperud [22]
Poland	Wildlife	19	15.8	DAT	Sroka et al. [31]
	Wildlife	92	30.4	ELISA	Witkowski et al. [35]
Sweden	Wildlife	199	34.0	DAT	Malmsten et al. [25]
Spain	Wildlife	33	21.8	MAT	Gauss et al. [15]
	Wildlife	278	33.9	MAT	Gamarra et al. [13]
	Wildlife	160	13.7	DAT	Panadero et al. [27]
	Wildlife	84	25.0	ELISA	Morrondo et al. [26]
	Wildlife	22	13.6	MAT	Almería et al. [1]

DT – dye test; DAT – direct agglutination test; ELISA – enzyme-linked immunosorbent assay; IFAT – indirect fluorescent test; LAT – latex agglutination test; MAT – modified agglutination test.

There are two previous studies that investigated the T. gondii seroprevalence in wildlife from our study region. The values of 38.3% reported for raccoons (Procyon lotor) [16] and of 24.5% reported for the European mouflon (Ovis orientalis musimon) [17] were relatively high compared to values from other European studies in these species. These authors took this as evidence of high environmental contamination with oocysts as, in addition to the presence of feral, stray, and pet cats (Felis sylvestris domesticus), the study region was located within the core distribution area of the wildcat in central Germany [16, 17]. Beral et al. [4] found a positive link between higher T. gondii antibody levels in wild boar (Sus scrofa) and the occurrence of wildcats in France. Our results do not contradict this conclusion, as the T. gondii seroprevalence in the roe deer population in the area is comparable to the values observed in the other two species. While the roe deer value obtained here is not particularly high compared to other European results (Table 2), the wildcat also occurs in the study areas in France and Belgium where a high seroprevalence was observed in roe deer. Further research on the environmental factors associated with high T. gondii seroprevalence in European wildlife is clearly needed.

Our results suggest that older roe deer had a higher seroprevalence than younger animals. Other studies on roe deer came to a similar conclusion [25, 33]. T. gondii antibodies are frequently more prevalent in older animals, since the cumulative likelihood of exposure to T. gondii increases with age and the antibodies persist for a lifetime [1, 20]. We did not identify a significant difference in seroprevalence depending on sex and year of sample collection. For at least some part of the year, both sexes have overlapping home ranges [32] and a substantial difference in exposure risks between the two sexes seems unlikely. Seroprevalence did not significantly differ between years, implying that the environmental contamination with infective oocysts remained constant throughout the study, corroborating findings from the mouflon obtained for the same region and study period [17]. It has indeed been suggested that humidity and moderate temperatures promote the survival and sporulation of the oocysts [1, 9, 13, 30].

The high seroprevalence of T. gondii antibodies in a Central German population of roe deer highlights a potential source of human infection. German hunters frequently produce home-made sausages using raw or undercooked meat. Our results suggest that this may lead to an increased risk of food-borne transmission of T. gondii. Additional studies are required to assess infection levels in venison and derived products in order to assess the risk of transmitting T. gondii to humans.

Conclusions

We analysed the sero-epidemiology of T. gondii infection in roe deer from a central German study population. T. gondii antibodies were present in animals of all ages. Raw or undercooked venison and its derived products may be a potential source of human infection with T. gondii.

Conflict of interest

The authors declare that they have no conflicts of interest in relation to this article.