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Trichinella spp. biomass has increased in raccoon dogs (Nyctereutes procyonoides) and red foxes (Vulpes vulpes) in Estonia

  • Age Kärssin1, 2Email author,
  • Liidia Häkkinen1,
  • Enel Niin3,
  • Katrin Peik1,
  • Annika Vilem1,
  • Pikka Jokelainen2, 4, 5 and
  • Brian Lassen2, 6
Parasites & Vectors201710:609

https://doi.org/10.1186/s13071-017-2571-0

Received: 14 April 2017

Accepted: 5 December 2017

Published: 16 December 2017

Abstract

Background

Raccoon dogs and red foxes are well-adapted hosts for Trichinella spp. The aims of this study were to estimate Trichinella infection prevalence and biomass and to investigate which Trichinella species circulated in these indicator hosts in Estonia.

Methods

From material collected for evaluating the effectiveness of oral vaccination program for rabies eradication in wildlife, samples from 113 raccoon dogs and 87 red foxes were included in this study. From each animal, 20 g of masseter muscle tissue was tested for the presence of Trichinella larvae using an artificial digestion method. The Trichinella larvae were identified to species level by multiplex polymerase chain reaction method.

Results

The majority of tested animals were infected with Trichinella spp. The parasite species identified were T. nativa and T. britovi. The apparent infection prevalence was 57.5% in raccoon dogs and 69.0% in red foxes, which were higher than previous estimates. In addition, the larval burden had also increased in both hosts. We estimated that in 2011–2012, the Trichinella spp. biomass was more than 15 times higher in raccoon dogs and almost two times higher in red foxes than in 1992–2000 (based on mean larval burden), and almost 20 times higher in raccoon dogs and almost five times higher in red foxes than in 2000–2002 (based on median larval burden).

Conclusions

Raccoon dogs and red foxes are relevant reservoirs for Trichinella spp. in Estonia. The biomass of Trichinella circulating in sylvatic cycles was substantial and had increased: there is substantial infection pressure in the sylvatic cycle.

Keywords

Trichinella infection Trichinella nativa Trichinella britovi PrevalenceSylvaticZoonosis

Background

Trichinella spp. are zoonotic parasitic nematodes transmitted by carnivorism. Sylvatic Trichinella infections are endemic in Estonia, a EU country located in north-eastern Europe that is bordered by Latvia in the south and Russia in the east [15]. For example, while anti-Trichinella antibodies were not detected in the domestic pigs investigated in our recent study, a substantial proportion of hunted wild boars were Trichinella seropositive [5]. Assessment of the sylvatic component and awareness about it are important because there is a risk of spill-over to domestic animals and humans [5].

The raccoon dog (Nyctereutes procyonoides) is a suitable indicator species and well-adapted reservoir host for all four Trichinella species circulating in Europe, and the red fox (Vulpes vulpes) particularly for T. spiralis and T. britovi [4, 610]. The invasive raccoon dog [11], and the native red fox are common and numerous sylvatic carnivores in Estonia [12]. A total of 12,577 raccoon dogs and 7144 red foxes were hunted in Estonia during the hunting season 2011–2012 [13].

The most recent epidemiological data on Trichinella infections in raccoon dogs and red foxes in Estonia were based on material collected in 2000–2002 [4]. The apparent prevalence of Trichinella spp. was 42.0% in raccoon dogs and 40.6% in red foxes, which did not differ significantly from estimates from 1992 to 2000 [14]. In both earlier studies, T. nativa and T. britovi were identified in the target hosts. Our study aimed to update the Trichinella infection prevalence estimates in raccoon dogs and red foxes in Estonia and to identify the Trichinella species causing the infections. We compared the findings with the two previous estimates and with those reported from other European countries and estimated how the biomass of Trichinella has changed in Estonia.

Methods

For the evaluation of the effectiveness of the oral vaccination program for rabies eradication in wildlife [15], head samples from 1214 raccoon dogs and 625 red foxes were collected from whole Estonian territory (average density 4.3 animals per 100 km2) from August 2011 to March 2012. The animals sampled were apparently healthy hunted animals, rabies indicator animals killed due to abnormal behavior near human settlements, and animals killed in traffic or found dead. We could investigate muscle samples from 200 of these heads (113 raccoon dogs and 87 red foxes), which was evaluated to be a sufficient sample size to estimate the infection prevalence with 80% confidence level.

To obtain a geographically representative sample for this study, the number of samples from each county was adjusted according to the surface area of the county, and a random sample was drawn from the samples available from there. Data on the estimated age (less than 1 year old = juvenile, at least 1 year old = adult) and gender of each animal had been collected on the submission forms. Age group was unknown for 31 animals and gender for 69 animals.

The samples were kept refrigerated until analysis, but few samples were or could have been frozen (n = 3 from raccoon dogs and n = 2 from foxes arrived frozen). From each animal, 20 g of masseter muscle tissue was analyzed for the presence of Trichinella spp. larvae using the European Union reference method, i.e. magnetic stirrer method, for artificial digestion [16]. The mean time between sampling and digestion was ten days (range: 1–92 days).

Larvae from each positive sample were evaluated morphologically and then counted, rinsed with water, collected, and stored in ethanol at room temperature until identification to species level. The species of Trichinella were identified using a previously described multiplex polymerase chain reaction method [17].

The sample size assessment and preliminary statistical calculations were done with OpenEpi software [18]. The confidence intervals (CI) of the prevalence estimates were calculated using Mid-P exact. Comparisons with the prevalence estimates, by host species and Trichinella species, from other European countries and previous Estonian studies were done using two by two tables. Two-tailed P-values (Mid-P exact) < 0.05 were considered statistically significant.

Logistic regression models were built with STATA 13.0 (Stata Corporation, College Station, Texas, USA) software for three outcomes: testing positive for Trichinella spp., testing positive for T. nativa, and testing positive for T. britovi. The variables we evaluated were ‘host species’ (raccoon dog or red fox), ‘age’ (juvenile or adult), ‘gender’ (female or male), ‘county’ (the 15 counties included as dummy variables i.e. allocated numbers that do not indicate any particular order), and ‘cause of death’ (whether the animal had been hunted, killed due to abnormal behavior, killed in traffic, or found dead). The variables with P-value ≤ 0.25 in univariable analysis were included in a multivariable model, followed by a stepwise backward elimination of those with P ≥ 0.05 that did not act as confounders.

Trichinella spp. biomass was quantitatively estimated for 1000 host animals and for the hunting bag, using estimate of weight of the host, estimate of proportion of muscle of the host weight (based on information available for small mammals of similar size), point estimate of Trichinella spp. prevalence, and mean or median larvae per gram of muscle tissue.

Results

The majority (62.5%, 125/200, 95% CI: 55.6–69.0) of the animals tested were infected with Trichinella spp. (Table 1). The apparent Trichinella spp. infection prevalence was 57.5% (65/113, 95% CI: 48.3–66.2) in raccoon dogs and 69.0% (60/87, 95% CI: 58.6–77.7) in red foxes. The prevalence was not significantly higher in red foxes than in raccoon dogs.
Table 1

Trichinella species identified in raccoon dogs (Nyctereutes procyonoides) and red foxes (Vulpes vulpes) in 2011–2012 in Estonia

Trichinella species

Raccoon dog (n = 113)

Red fox (n = 87)

Positive (n)

Prevalence (95% CI)a (%)

% of Trichinella-positive (95% CI)

Range of lpg

Mean (median) lpg

Positive (n)

Prevalence (95% CI)a (%)

% of Trichinella-positive (95% CI)

Range of lpg

Mean (median) lpg

T. nativa only

23

20.4 (13.7–29.0)

35.4 (24.5–47.5)

0.5–631.6

158.4 (135.0)

19

21.8 (14.1–31.4)

31.7 (20.9–44.2)

0.1–636.8

82.4 (13.8)

T. britovi only

15

13.3 (7.9–20.5)

23.1(14.1–34.5)

0.6–486.0

123.5 (58.8)

23

26.4 (18.0–36.4)

38.3 (26.7–51.1)

0.1–409.5

44.7 (10.5)

T. nativa and T. britovi

13

11.5 (6.6–18.4)

20.0 (11.6–31.8)

26.2–800.0

209.2 (98.0)

8

9.2 (4.4–16.7)

13.3 (6.4–23.8)

2.3–28.6

9.1 (7.1)

T. nativa, totalb

36

31.9 (23.8–40.9)

55.4 (43.2–67.1)

0.5–800.0

176.7 (130.0)

27

31.0 (22.0–41.3)

45.0 (32.8–57.7)

0.1–636.8

60.7 (8.4)

T. britovi, totalb

28

24.8 (17.5–33.4)

43.1 (31.5–55.3)

0.6–800.0

163.3 (83.2)

31

35.6 (26.1–46.1)

51.7 (39.1–64.1)

0.1–409.5

8.2 (35.5)

Species-level result

51

45.1 (36.1–54.4)

78.5 (67.3–87.2)

0.5–800.0

161.1 (101.4)

50

57.5 (46.9–67.5)

83.3 (72.3–91.2)

0.1–636.8

53.3 (9.4)

No species-level result

14

12.4 (7.2–19.5)

21.5 (12.8–32.8)

0.1–576.0

161.1 (43.6)

10

11.5 (6.0–19.5)

16.7 (8.8–27.7)

0.1–142.9

21.4 (2.4)

Total

65

57.5 (48.3–66.4)

100 (95.5–100)

0.1–800.0

161.1 (98.0)

60

69.0 (58.7–78.0)

100 (95.1–100)

0.1–636.8

48.0 (8.2)

Abbreviation: lpg, larvae per gram of muscle tissue

a95% confidence interval, Mid-P exact

bWith this particular Trichinella species, either as the only species or in mixed infection

The Trichinella species present were successfully identified from 80.8% of the animals that had larvae (Table 1). The success rate of Trichinella species identification was 82.7% (91/110, 95% CI: 74.8–89.0) from larvae from samples that were digested within the recommended 21 days after sampling [9], and 66.7% (10/15, 95% CI: 40.8–86.6) from samples stored longer. However, the difference was not significant.

The presence of two sylvatic species, T. nativa and T. britovi, was confirmed (Table 1). Trichinella nativa was detected as the only species present or in mixed infections in 31.9% of raccoon dogs and 31.0% of red foxes, and T. britovi was detected as the only species present or in mixed infections in 24.8% of raccoon dogs and 35.6% of red foxes. Of those animals that hosted Trichinella spp. larvae that were determined to the species level, T. nativa was detected as the only species present or in mixed infection in 70.6% of raccoon dogs and 54.0% of red foxes. Of those animals that hosted Trichinella spp. larvae that were determined to the species level, T. britovi was detected as the only species or in mixed infection in 54.9% of raccoon dogs and 62.0% of red foxes. The prevalence of mixed infections had increased in red foxes from the estimate of the previous Estonian study (Table 3) [4].

The apparent Trichinella spp. infection prevalences estimated from the samples from 2011 to 2012 were higher than those from 2000 to 2002 in both raccoon dogs and red foxes (Tables 2 and 3) [4]. Moreover, the prevalence estimated from samples from 2011 to 2012 was higher than the one from 1992 to 2000 in red foxes; however, the estimate from 2011 to 2012 did not differ significantly from the estimate from 1992 to 2000 in raccoon dogs (Tables 2 and 3) [14].
Table 2

Prevalence of Trichinella spp. in raccoon dogs (Nyctereutes procyonoides) in European countries and comparison with the present study

Country

Sampling period

Samples (n)

Prevalence of Trichinella spp. (95% CI) (%)

Prevalence of Tn (total) (95% CI) (%)

Prevalence of Tb (total) (95% CI) (%)

Prevalence of Ts (total) (95% CI) (%)

Prevalence of Tp (total) (95% CI) (%)

Prevalence of mixed infections (95% CI) (%)

Reference

Estonia

2011–2012

113

57.5 (48.3–66.4)

31.9 (23.8–40.9)

24.8 (17.5–33.4)

0.0 (0.0–2.6)

0.0 (0.0–2.6)

11.5 (7.9–20.5)

Present study

Estonia

2000–2002

157

42.0* (34.5–49.9)

15.9** (10.8–22.3)

15.9 (10.8–22.3)

0.0 (0.0–5.1)

0.0 (0.0–5.1)

5.1 (2.4–9.4)

[4]

Estonia

1992–2000

33

45.5 (29.2–62.5)

15.2 (5.8–30.4)

27.3 (14.2–44.2)

0.0 (0.0–8.7)

0.0 (0.2–8.7)

3.0 (0.2–14.1)

[3, 14, 57]

Finland

1999–2005

662

28.1*** (24.8–31.6)

     

[19]

Finland

1996–1998

199

37.7*** (31.2–44.6)

     

[8]

Germany

2006–2007

146

4.8*** (2.1–9.3)

0.0*** (0.0–2.0)

0.0*** (0.0–2.0)

4.1* (1.7–8.3)

2.7 (0.9–6.5)

1.4*** (0.2–4.5)

[20]

Germany

2000–2014

1527

1.9*** (1.3–2.7)

0.0*** (0.0–0.2)

0.1*** (0.0–0.3)

1.7 (1.1–2.4)

0.1 (0.0–0.3)

0.0 (0.0–0.2)***

[21]

Latvia

2010–2014

394

37.3*** (32.6–42.2)

2.8*** (1.5–4.8)

35.0* (30.4–39.9)

0.5 (0.1–1.7)

0.0 (0.0–0.8)

3.0*** (1.7–5.1)

[22]

Latvia

2000–2002

17

35.3 (15.7–59.5)

     

[4]

Lithuania

2001–2006

75

29.3*** (19.9–40.4)

     

[23]

Lithuania

2000–2002

83

32.5*** (23.1–43.1)

2.4*** (0.4–7.7)

25.3 (16.8–35.5)

4.8* (1.6–11.2)

0.0 (0.0–3.5)

4.8 (1.6–11.2)

[4]

Poland

2012

39

5.1*** (0.9–15.9)

0.0*** (0.0–7.4)

0.0*** (0.0–7.4)

5.1 (0.9–15.9)

0.0 (0.0–7.4)

0.0* (0.0–7.4)

[24]

Abbreviations: Tn, Trichinella nativa; Tb, Trichinella britovi; Ts, Trichinella spiralis; Tp, Trichinella pseudospiralis

*P < 0.05, **P < 0.01, ***P < 0.001

Table 3

Prevalence of Trichinella spp. in red foxes (Vulpes vulpes) in European countries and comparison with the present study

Country

Sampling period

Samples (n)

Prevalence of Trichinella spp. (95% CI) (%)

Prevalence of Tn (total) (95% CI) (%)

Prevalence of Tb (total) (95% CI) (%)

Prevalence of Ts (total) (95% CI) (%)

Prevalence of Tp (total) (95% CI) (%)

Prevalence of mixed infections (95% CI) (%)

Reference

Estonia

2011–2012

87

69.0 (58.7–78.0)

31.0 (22.0–41.3)

35.6 (26.1–46.1)

0.0 (0.0–3.4)

0.0 (0.0–3.4)

9.2 (4.4–16.7)

Present study

Estonia

2000–2002

446

40.6*** (36.1–45.2)

16.6** (13.4–20.3)

13.5*** (10.5–16.7)

0.0 (0.0–0.7)

0.0 (0.0–0.7)

2.9*(1.6–4.8)

[4]

Estonia

1992–2000

21

42.9* (23.3–64.3)

14.3 (3.8–34.1)

28.6 (12.5–50.2)

0.0 (0.0–13.3)

0.0 (0.0–13.3)

4.8 (0.2–21.3)

[3, 14, 57]

Austria

 

1546

1.6*** (1.0–2.3)

0.0*** (0.0–0.2)

1.6*** (1.0–2.3)

0.0 (0.0–0.2)

0.0 (0.0–0.2)

0.0*** (0.0–0.2)

[27]

Belgium

1996–2000

818

0.0*** (0.0–0.4)

0.0*** (0.0–0.4)

0.0*** (0.0–0.4)

0.0 (0.0–0.4)

0.0 (0.0–0.4)

0.0*** (0.0–0.4)

[28]

Denmark

1995–1996

3133

0.1*** (0.0–0.3)

     

[29]

Denmark

1997–1998

3008

0.0*** (0.0–0.1)

0.0*** (0.0–0.1)

0.0*** (0.0–0.1)

0.0 (0.0–0.1)

0.0 (0.0–0.1)

0.0*** (0.0–0.1)

[29]

Finland

1999–2005

1010

18.7*** (16.4–21.2)

     

[19]

Finland

1996–1998

158

36.7*** (29.5–44.4)

     

[8]

France

2006–2009

108

2.8*** (0.7–7.4)

0.0*** (0.0–2.7)

2.8*** (0.7–7.4)

0.0 (0.0–2.7)

0.0 (0.0–2.7)

0.0*** (0.0–2.7)

[29]

France

2006–2008

74

0.0*** (0.0–4.0)

0.0*** (0.0–4.0)

0.0*** (0.0–4.0)

0.0 (0.0–4.0)

0.0 (0.0–4.0)

0.0*** (0.0–4.0)

[31]

Germany

2011–2012

3154

0.3*** (0.2–0.6)

     

[25]

Germany

2006–2007

100

1.0*** (0.1–4.8)

0.0*** (0.0–3.0)

0.0*** (0.0–3.0)

0.0 (0.0–3.0)

0.0 (0.0–3.0)

0.0*** (0.0–3.0)

[20]

Hungary

2008–2013

3304

2.1*** (1.6–2.6)

0.0*** (0.0–0.1)

1.8*** (1.4–2.3)

0.2 (0.1–0.5)

0.0 (0.0–0.1)

0.0*** (0.0–0.1)

[32]

Hungary

2007–2008

2116

1.7*** (1.2–2.3)

0.0*** (0.0–0.1)

1.4*** (1.0–2.0)

0.2 (0.1–0.5)

0.0 (0.0–0.2)

0.0*** (0.0–0.1)

[33]

Italy

2010–2014

153

8.5*** (4.8–13.8)

0.0*** (0.0–1.9)

8.5*** (4.8–13.8)

0.0 (0.0–1.9)

0.0 (0.0–1.9)

0.0*** (0.0–1.9)

[34]

Italy

2004–2014

480

5.0*** (3.3–7.2)

     

[35]

Italy

2001–2004

229

3.1*** (1.3–6.0)

0.0*** (0.0–1.3)

3.1*** (1.3–6.0)

0.0 (0.0–1.3)

0.0 (0.0–1.3)

0.0*** (0.0–1.3)

[36]

Italy

2001–2004

227

3.5*** (1.7–6.6)

0.0*** (0.0–1.3)

3.5*** (1.7–6.6)

0.0 (0.0–1.3)

0.0 (0.0–1.3)

0.0*** (0.0–1.3)

[37]

Italy

1997–2003

172

1.2*** (0.2–3.8)

     

[38]

Ireland

2002

454

0.9*** (0.3–2.1)

0.0*** (0.0–0.7)

0.0*** (0.0–0.7)

0.9 (0.3–2.1)

0.0 (0.0–0.7)

0.0*** (0.0–0.7)

[39]

Latvia

2010–2014

668

50.6** (46.8–54.4)

1.5*** (0.8–2.7)

40.9 (37.2–44.6)

0.0 (0.0–0.4)

0.0 (0.0–0.4)

1.0*** (0.5–2.1)

[22]

Latvia

2000–2002

1112

28.9*** (26.3–31.6)

2.2*** (1.4–3.1)

10.3*** (8.6–12.1)

0.4 (0.2–1.0)

0.0 (0.0–0.3)

1.3*** (0.7–2.1)

[4]

Lithuania

2001–2006

206

46.6*** (39.9–53.4)

     

[23]

Lithuania

2000–2002

567

40.0*** (36.1–44.1)

0.9*** (0.3–1.9)

23.3* (19.9–26.9)

4.8* (3.2–6.8)

0.2 (0.0–0.9)

3.2* (2.0–4.9)

[4]

Netherlands

2010–2013

369

0.3*** (0.0–1.3)

     

[40]

Netherlands

1996–1997

276

4.0*** (2.1–6.8)

0.0*** (0.0–1.1)

4.0*** (2.1–6.8)

0.0 (0.0–1.1)

0.0 (0.0–1.1)

0.0*** (0.0–1.1)

[41]

Norway

1994–1995, 2002–2005

393

4.8*** (3.0–7.3)

4.6*** (2.8–7.0)

0.3*** (0.0–1.2)

0.0 (0.0–0.8)

0.0 (0.0–0.8)

0.0*** (0.0–0.8)

[42]

Poland

2010–2015

1447

10.0*** (8.6–11.7)

0.0*** (0.0–0.2)

7.2*** (5.9–8.6)

1.1 (0.7–1.8)

0.1 (0.0–0.3)

0.0*** (0.0–0.2)

[26]

Poland

2011–2012

1634

2.7*** (2.0–3.6)

0.1*** (0.0–0.3)

2.0*** (1.4–2.7)

0.6 (0.3–1.0)

0.0 (0.0–0.2)

0.1*** (0.0–0.3)

[25]

Portugal

2008–2010

47

2.1*** (0.1–10.1)

0.0*** (0.0–6.2)

2.1*** (0.1–10.1)

0.0 (0.0–6.2)

0.0 (0.0–6.2)

0.0*** (0.0–6.2)

[43]

Romania

2012–2014

121

21.5*** (14.9–29.5)

0.0*** (0.0–2.4)

19.8* (13.5–27.7)

0.8 (0.0–4.0)

0.0 (0.0–2.4)

0.0*** (0.0–2.4)

[44]

Romania

2000–2005

71

7.0*** (2.6–14.9)

0.0*** (0.0–4.1)

5.6*** (1.8–13.0)

1.4 (0.1–6.7)

0.0 (0.0–4.1)

0.0** (0.0–4.1)

[45]

Serbia

2009–2010

57

12.3*** (5.5–22.8)

0.0*** (0.0–5.1)

3.5*** (0.6–11.1)

8.8** (3.3–18.4)

0.0 (0.0–5.1)

3.5 (0.6–11.1)

[46]

Slovakia

2000–2007

5270

11.5*** (10.7–12.4)

     

[47]

Slovakia

2007

601

20.3*** (17.2–23.7)

0.0*** (0.0–0.5)

20.3*** (17.2–23.7)

0.3 (0.1–1.1)

0.2 (0.0–0.8)

0.5*** (0.1–1.4)

[47]

Slovakia

2000–2006

4669

10.4*** (9.5–11.3)

0.0*** (0.0–0.1)

8.3*** (7.6–9.2)

0.1 (0.0–0.2)

0.0 (0.0–0.1)

0.0*** (0.0–0.1)

[48]

Slovakia

2000

545

6.1*** (4.3–8.3)

0.0*** (0.0–0.5)

6.1*** (4.3–8.3)

0.0 (0.0–0.5)

0.0 (0.0–0.5)

0.0*** (0.0–0.5)

[47]

Spain

400

15.5*** (12.2–19.3)

 

15.3*** (12.0–19.0)

   

[49]

Spain

1997–1999

67

8.9*** (3.7–17.7)

     

[50]

Spain

1985–1997

227

2.6*** (1.1–5.4)

0.0*** (0.0–1.3)

1.8*** (0.6–4.2)

0.9 (0.1–2.9)

0.0 (0.0–1.3)

0.0*** (0.0–1.3)

[51]

Spain

1989–1993

84

1.2*** (0.1–5.7)

     

[52]

Sweden

1985–2003

1800

4.5*** (3.6–5.5)

     

[53]

Switzerland

2006–2007

1289

1.6*** (1.0–2.4)

0.0*** (0.0–0.2)

1.6*** (1.0–2.4)

0.0 (0.0–0.2)

0.0 (0.0–0.2)

0.0*** (0.0–0.2)

[54]

United Kingdom (Great Britain)

2003–2007

1144

0.0*** (0.0–0.3)

0.0*** (0.0–0.3)

0.0*** (0.0–0.3)

0.0 (0.0–0.3)

0.0 (0.0–0.3)

0.0*** (0.0–0.3)

[55]

United Kingdom (Northern Ireland)

2003–2004; 2006–2007

443

0.2*** (0.0–1.1)

0.0*** (0.0–0.7)

0.0*** (0.0–0.7)

0.2 (0.0–1.1)

0.0 (0.0–0.7)

0.0*** (0.0–0.7)

[56]

Abbreviations: Tn, Trichinella nativa; Tb, Trichinella britovi; Ts, Trichinella spiralis; Tp, Trichinella pseudospiralis

*P < 0.05, **P < 0.01, ***P < 0.001

The number of Trichinella spp. larvae recovered per gram muscle tissue (lpg) was higher in raccoon dogs (median: 98.0, mean: 161.1, range: 0.1–800.0 lpg) than in red foxes (median: 8.2, mean: 48.0, range: 0.1–636.8 lpg), and varied by Trichinella species (Table 1). The highest larval burden, 800 lpg, was detected in a raccoon dog with mixed infection.

The median larval burden had increased in both raccoon dogs and red foxes from those reported in the previous study: from 7.2 lpg to 98.0 lpg in raccoon dogs and from 3.0 lpg to 8.2 lpg in red foxes [4]. Furthermore, the proportion of animals with low larval burden (< 1 lpg) had decreased from 18.1% to 7.7% in raccoon dogs, and from 23.7% to 11.7% in red foxes, further indicating that the circulating parasite biomass of Trichinella larvae had increased [4]. The Trichinella spp. biomass was estimated to have increased 18.6-fold in raccoon dogs and 4.6-fold in red foxes (based on median larval burden) (Table 4).
Table 4

Calculation of the change in Trichinella spp. biomass in raccoon dogs and red foxes in Estonia

 

Raccoon dog

Red fox

Reference

Hunting bag 1995, n animals

1723

3326

[67]

Hunting bag 2001, n animals

4259

6628

[67]

Hunting bag 2011, n animals

12,577

7144

[13]

With Trichinella larvae (%)

1992–2000

45.5

42.9

[14]

2000–2002

42.0

40.6

[4]

2011–2012

57.5

69.0

Present study

Mean body weight of host, g

4830

4890

[68, 69]

Muscle tissue of body weight, %

60

60

[70]

Median (mean) Trichinella lpg

1992–1996

nd (13.4)

nd (43.1)

[2]

2000–2002

7.2 (nd)

3.0 (nd)

[4]

2011–2012

98.0 (161.1)

8.2 (48.0)

Current study

Trichinella biomass 1992–2000, median (mean) n larvae

in 1000 animals

nd (17,669,106)

nd (54,249,367)

 

in the hunting baga

nd (30,443,870)

nd (180,433,393)

 

Trichinella biomass 2000–2002, median (mean) n larvae

in 1000 animals

8,763,552 (nd)

3,573,612 (nd)

 

in the hunting bagb

37,323,968 (nd)

23,685,900 (nd)

 

Trichinella biomass 2011–2012, median (mean) n larvae

in 1000 animals

163,302,300 (268,448,985)

16,600,572 (97,174,080)

 

in the hunting bagc

2,053,853,027 (3,376,282,884)

118,594,486 (694,211,628)

 

Increase in Trichinella biomass from 1992 to 2000 to 2011–2012, calculated from median (mean) n larvae

in 1000 animals

nd (15.2-fold)

nd (1.8-fold)

 

in the hunting bag

nd (110.9-fold)

nd (3.8-fold)

 

Increase in Trichinella biomass from 2000 to 2002 to 2011–2012, calculated from median (mean) n larvae

in 1000 animals

18.6-fold (nd)

4.6-fold (nd)

 

in the hunting bag

55.0-fold (nd)

5.0-fold (nd)

 

Abbreviations: nd, no data; lpg, larvae per gram of muscle tissue

a n larvae = n animals 1995 × % with larvae 1992–2000 × (mean body weight of host, g × muscle tissue of body weight, %) × median (mean) lpg (1992–1996)

b n larvae = n animals 2001 × % with larvae 2000–2002 × (mean body weight of host, g × muscle tissue of body weight, %) × median (mean) lpg (2000–2002)

c n larvae = n animals 2011 × % with larvae 2011–2012 × (mean body weight of host, g × muscle tissue of body weight, %) × median (mean) lpg (2011–2012)

Trichinella nativa was not detected in samples from the large islands Saaremaa and Hiiumaa, nor the most southeastern county Võrumaa, while T. britovi was found in samples collected from all counties (Fig. 1).
Figure 1
Fig. 1

Trichinella spp. in raccoon dogs (Nyctereutes procyonoides) (a) and red foxes (Vulpes vulpes) (b) in 2011–2012 in Estonia, by counties. Key: yellow dot, T. britovi; green dot, T. britovi + T. nativa; blue dot, T. nativa; grey dot, Trichinella spp. (no species-level result); black dot, no larvae detected

None of the variables were significant factors for testing positive for Trichinella spp. in either of the hosts nor in both hosts together. The final model for testing positive for T. nativa had two variables, ‘age’ and ‘county’, and the area under the receiver operating characteristic (ROC) curve was 0.72. The odds of testing positive for T. nativa were 3.6 times (P = 0.009, 95% CI: 1.4–9.3) higher in adults than in juveniles, and higher in counties Põlvamaa and Pärnumaa when compared with Harjumaa where the capital is located (P = 0.009, OR = 15.2, 95% CI: 2.0–117.3, and P = 0.029, OR = 7.6, 95% CI: 1.2–47.1, respectively). The final model for testing T. britovi positive included only the variable ‘county’, and the area under the ROC curve was 0.68. The odds of an animal testing T. britovi positive were higher in the counties Valgamaa, Saaremaa, Läänemaa, and Pärnumaa (P = 0.019, OR = 16.8, 95% CI: 1.6–176.2; P = 0.023, OR = 14.0, 95% CI: 1.4–137.3; P = 0.040, OR =11.7, 95% CI: 1.1–122.4; and P = 0.043, OR = 9.7, 95% CI: 1.1–87.4, respectively) than in the reference county Harjumaa.

Discussion

We summarized the results of European studies on Trichinella spp. infection prevalence in raccoon dogs (Table 2) and red foxes (Table 3). Lower prevalences than our estimate from Estonia have been observed in both hosts in Finland, Latvia, Lithuania, Poland and Germany (Tables 2 and 3) [1926]. Moreover, in red foxes, the Trichinella spp. infection prevalence was higher in Estonia than what has been reported in Austria, Belgium, Denmark, France, Great Britain, Hungary, Italy, Ireland, Netherlands, Norway, Northern Ireland, Portugal, Romania, Serbia, Slovakia, Spain and Switzerland (Table 3) [2656]. However, as different sampling schemes, sample sizes, sample material, and detection methods were used, these studies are not all directly comparable with our study.

In Europe, according to the International Trichinella Reference Centre [57], the northern species T. nativa has been found in raccoon dogs in Estonia, Finland, Latvia, Russia, and Sweden; and in red foxes in Estonia, Finland, Germany, Latvia, Norway, Poland, Sweden and Ukraine. The published studies on T. nativa in raccoon dogs and red foxes report lower prevalences (single and mixed infections included) in Latvia, Lithuania and Norway than our estimate from Estonia [4, 22, 42]. In Poland and Germany, T. nativa has been found in red foxes (Table 3) [20, 21, 2426]. When comparing the result of our study with that from the previous Estonian study, the T. nativa infection prevalence had increased in both raccoon dogs and red foxes (Tables 2 and 3) [4].

In Europe, T. britovi has been found in raccoon dogs in Estonia, Finland, Germany, Latvia and Lithuania (Table 2) [4, 57]. It is the most common Trichinella species in red foxes in Europe [10]. The prevalence of T. britovi we observed in raccoon dogs in single and mixed infections was similar to that reported from Lithuania, lower than that from Latvia, and higher than those from western Poland and Germany (Table 2) [4, 21, 22, 24]. The prevalence of T. britovi we observed in red foxes, including both single and mixed infections, was higher than those reported from Austria, France, Hungary, Norway, Poland, Portugal, Romania, Serbia, Slovakia and Switzerland (Table 3) [2124, 32, 33, 4548, 50, 5456]. A similar to our prevalence estimate for T. britovi was detected in red foxes in Latvia [22]. When comparing the result of our study with that from the previous Estonian study, the T. britovi infection prevalence had increased in red foxes (Table 3) [4]. Moreover, mixed infections were more common in our study than what was observed in raccoon dogs and red foxes in the neighboring country Latvia and in red foxes in Lithuania (Tables 2 and 3) [4, 22].

In this study, the odds of being Trichinella-infected were not significantly different in raccoon dogs and red foxes, whereas the mean larval burden was 3.2 times higher in raccoon dogs than in red foxes. In Latvia, red foxes had higher odds to test positive (P = 0.010, OR = 1.41, 95% CI: 1.08–1.83) than raccoon dogs, but raccoon dogs had 2.9 times higher mean larval burden than red foxes [22]. In Finland, both indicators were higher in raccoon dogs than in red foxes (P < 0.001, OR = 1.70, 95% CI: 1.35–2.14; 3.8 times higher mean larval burden) [19]. A higher larval burden in raccoon dogs than in red foxes has also been described in other studies [4, 8].

Despite the fact that we used 20 g of tissue for the digestion, our study likely underestimated the actual infection prevalence and larval burdens, because the available material was not optimal for finding Trichinella larvae [9, 58, 59]. In experimentally infected raccoon dogs, the T. nativa larval density in masseter muscle was about half of that in foreleg muscles [58]. The storage conditions and transport time could also affect the results [29, 40, 60].

The high Trichinella infection prevalence in raccoon dogs and red foxes, as well as the overall circulation of the parasites in the sylvatic cycle, may be supported by human behavior. For example, the local hunters use carcasses of hunted raccoon dogs as baits [61], which might help the transmission. According to winter tracking index and hunters’ estimations, after the rabies vaccination program started in 2005 [62], the red fox population size first increased, with a peak in 2009–2010, and then decreased [12]. The raccoon dog population size has increased since the second half of last century [62] and has relatively stabilized after 2011–2012 [12]. These changes are also reflected in the increased hunting bag sizes [13] and may have relevance beyond simply higher numbers. There was an association between the abundance index of raccoon dogs and the proportion of Trichinella-infected raccoon dogs and red foxes in Finland [19].

Estonia is located in the transition zone of maritime and continental climate [63]. The coldest months with mean air temperature below zero are December to February [64]. According to data covering these three months from six weather stations located in Harjumaa, Lääne-Virumaa, Pärnumaa, Saaremaa, Tartumaa and Võrumaa, the mean number of days with snow cover was 12.7% (from 4% in Pärnumaa to 26% in Saaremaa) higher in 2002–2011 than in 1992–2001 (data received on request from Estonian Environment Agency). The snow cover could reduce the destructive effect of freezing-thawing cycles on carcasses of infected animals and thus facilitate survival of Trichinella larvae [65, 66].

Raccoon dogs and red foxes act as reservoir hosts for Trichinella spp. in the sylvatic cycle, where the infection can spread to game animals, such as wild boars, that are hunted for human consumption. The Trichinella seroprevalence in wild boars is high in Estonia [5], and the odds of testing Trichinella-seropositive were higher if the wild boar was hunted in certain counties, including Pärnumaa and Saaremaa, when compared with Harjumaa. In this study, a similar comparison was made, with Harjumaa as the reference county. Raccoon dogs and red foxes had higher odds to test T. nativa positive in Pärnumaa, whereas the odds to test T. britovi positive were higher in Pärnumaa and Saaremaa. Moreover, the highest larval burden was detected in a young raccoon dog from Pärnumaa. This raccoon dog had a mixed infection. These two counties could thus be interesting for further studies.

We estimated that in 2011–2012, the Trichinella spp. biomass was more than 15 times higher in raccoon dogs and almost two times higher in red foxes than in 1992–2000 (based on mean larval burden), and almost 20 times higher in raccoon dogs and almost five times higher in red foxes than in 2000–2002 (based on median larval burden) (Table 4). Using the increased hunting bag in the calculation as an indication of increased population size or as an indication of biomass removed from the circulation by hunting, the role of these hosts as reservoirs was clearly illustrated (Table 4). The widespread distribution of Trichinella infections in Estonian wildlife underlines that there is a high infection pressure within the eastern European sylvatic cycles. Moreover, the results of this study indicate that there is an increase in the infection pressure. Trichinella spp. thrive in Estonia, and there is a continuous risk of spill-over to domestic animals and humans.

Conclusions

In Estonia, the proportion of both raccoon dogs and red foxes that hosted Trichinella were higher than ten years earlier. In addition, the larval burdens had also increased in these hosts, and an increased biomass of Trichinella larvae was circulating in sylvatic cycles. Trichinella nativa and T. britovi were found in both host species. There is a substantial and increasing Trichinella infection pressure to the food chains and humans.

Abbreviations

CI: 

confidence interval

nd: 

no data

OR: 

odds ratio

ROC curve: 

receiver operating characteristic (ROC) curve

Tb: 

Trichinella britovi

Tn: 

Trichinella nativa

Tp: 

Trichinella pseudospiralis

Ts: 

Trichinella spiralis

Declarations

Acknowledgements

We thank the hunters and Estonian Hunters Society as well as veterinarians, Veterinary and Food Laboratory, Veterinary and Food Board, Estonian Environment Agency, and European Union Reference Laboratory for their contributions to this study.

Funding

The work was partly supported by project funding M14143VLVP from the Strategic Development Fund of the Estonian University of Life Sciences and by project funding 8P160014VLVP from Base Financing of Estonian University of Life Sciences.

Availability of data and materials

All data used and analyzed during the current study are included in this article.

Authors’ contributions

AK presented the idea and designed the study. EN designed and organized the sampling, and KP was responsible for the sampling at necropsy. LH performed the artificial digestion analyses, and AV performed the molecular analyses. AK, PJ and BL analyzed the data and drafted the manuscript. All authors contributed to the writing, read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Authors’ Affiliations

(1)
Veterinary and Food Laboratory, Tartu, Estonia
(2)
Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Tartu, Estonia
(3)
Veterinary and Food Board, Tallinn, Estonia
(4)
University of Helsinki, Helsinki, Finland
(5)
Statens Serum Institut, Copenhagen, Denmark
(6)
Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg C, Denmark

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