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Farmed wild boars exposed to Toxoplasma gondii and Trichinella spp.
Veterinary Parasitology 187 (2012) 323–327
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Farmed wild boars exposed to Toxoplasma gondii and Trichinella spp. Pikka Jokelainen a,∗ , Anu Näreaho a , Outi Hälli b , Mari Heinonen b , Antti Sukura a a b
Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, FI-00014 University of Helsinki, Finland Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, FI-04920 Saarentaus, Finland
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 12 December 2011 Accepted 20 December 2011 Keywords: Toxoplasma gondii Trichinella spp. Zoonosis Serology Sus scrofa L. Wild boar Farmed game
a b s t r a c t The meat of wild boar (Sus scrofa L.) can be a source of human infections with zoonotic parasites Toxoplasma gondii and Trichinella spp. We screened 197 wild boar sera collected at slaughter from 25 Finnish farms in 2007–2008 for serological evidence of infections with these parasites. Using a commercial direct agglutination test at a serum dilution of 1:40, T. gondii-specific IgG antibodies were detected in 65 (33.0%) samples, on 14 (56.0%) farms. Females, animals older than 24 months, animals of small herds, and animals originating from south-western parts of Finland were more often T. gondii-seropositive than were males, younger animals, animals of larger herds, and animals originating from the north and east, respectively. Four (2.0%) of the sera, originating from three (12.0%) farms, tested Trichinella-seropositive with an in-house ELISA and a conservative cut-off for seropositivity. One farm had both T. gondii- and Trichinella-seropositive animals. Taken together, an infection source had been present on 16 (64.0%) farms, and 69 (35.0%) of the 197 farmed wild boars intended for human consumption had specific serological evidence of exposure to a zoonotic parasite. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The European wild boar (Sus scrofa L.) is a popular game animal species whose meat is also available for consumers as a farmed delicacy. Unfortunately, it can be a source of zoonotic parasites, such as Toxoplasma gondii and Trichinella spp., if enjoyed undercooked (EFSA, 2005, 2007). At the slaughterhouse level, meat intended for retail is monitored for Trichinella, but not for T. gondii infections. Although many epidemiological and parasitological studies of wild boar pertain to free-ranging animals (e.g. for T. gondii, reviewed by Fornazari et al., 2009), investigating the role of farmed wild boars as hosts for zoonotic parasites is also of both public health and veterinary importance. Contrary to expectations for free-ranging game animals, farmed animals could be at least partly protected from such infections. In Finland, approximately 100 free-ranging
∗ Corresponding author. Tel.: +358 9 191 57183; fax: +358 9 191 57194. E-mail address: pikka.jokelainen@helsinki.fi (P. Jokelainen). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.12.026
wild boars are shot by hunters every year, and 600 farmed wild boars are slaughtered for meat. This serological survey aimed to estimate the exposure of the farmed wild boar population to T. gondii and Trichinella spp. 2. Materials and methods 2.1. Farms and animals This survey was part of a nationwide epidemiological study for which a sampling frame was compiled from the official records of Finnish wild boar farmers. All 117 farms were contacted and 32 (45% of active farms) participated in the study. The farmers sampled their wild boars at slaughter and provided the background information on the animals and farms. For this survey, we selected serum samples from 2007 to 2008 to obtain a sufficient sample size, calculated based on expected seroprevalences of 2–15% for both parasites. Samples from 197 farmed wild boars, 1–43 per farm (median 4), were thus included in this survey (Table 1). The samples
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P. Jokelainen et al. / Veterinary Parasitology 187 (2012) 323–327
Table 1 Distribution of categorical variables of the 197 wild boars included in the survey, and Toxoplasma gondii and Trichinella seroprevalences by category. Variable
na
%
n T. gondii seropositive
% (95% CI)
n Trichinella seropositive
% (95% CI)
Age of the animal, months
0–12b 12–23b 24–
24 110 56
13 58 29
7 27 27
29 (14–49) 25 (17–33) 48 (35–61)
0 2 2
0 (0–12) 2 (0–6) 4 (1–11)
Gender of the animal
Male Female
120 74
62 38
29 34
24 (17–32) 46 (35–57)
2 2
2 (0–5) 3 (0–9)
Herd type
Slaughter Integrated
75 122
38 62
18 47
24 (15–35) 39 (30–47)
2 2
3 (0–9) 2 (0–5)
Number of animals in the herd
0–50 51–100 101–
97 25 75
49 13 38
39 6 20
40 (31–50) 24 (10–43) 27 (18–38)
2 0 2
2 (0–7) 0 (0–11) 3 (0–9)
Location of the farm
Southwest East North
16 138 33
9 74 18
9 40 12
56 (32–78) 29 (22–37) 36 (21–54)
0 3 1
0 (0–17) 2 (1–6) 3 (0–14)
Sampling year
2007 2008
48 149
24 76
9 56
19 (10–32) 38 (30–46)
1 3
2 (0–10) 2 (1–5)
65
33 (27–40)
4
2 (1–5)
Total a b
197
The location of three farms (ten animals), the age of seven animals from three farms, and the gender of three animals from two farms were unknown. In the simple logistic regression, age group was used as a dichotomous variable: these two groups were combined.
originated from 25 farms with 6–150 animals (median 47) and a surface area of 1–65 ha (median 3). All the animals were used for human consumption after passing the official meat inspection that included examination for Trichinella using the digestion method (EU 2075/2005). The predilection site sampled for this examination was diaphragm, and five grams of the sample were digested. 2.2. Sampling The wild boars were stunned and bled on the farms prior to the transportation of their carcasses to the slaughterhouses. The farmers collected the blood samples during bleeding and sent them to the laboratory within three days. The sera were separated by centrifugation (10 min at 920 × g) and stored at −18 to −21 ◦ C until analyzed.
control sera were obtained earlier from wild boars with known infection levels of 0.07 larvae per gram of muscle tissue (lpg) (P1), 1.13 lpg (P2), and 1.65 lpg (P3), and two digestion-negative wild boars (N1 and N2). The controls and samples were diluted to 1:100. After subtracting the mean optical densities (OD) of the buffer-only wells from all the values, we corrected for plate-to-plate variation by using the mean of the ODs of the two positive control samples (P1 and P2) as the denominator for the sample ODs. To avoid false-positive results, the cut-off for seropositivity was set to the mean of the ODs of the negative controls (N1 and N2) plus three times their standard deviation. This chosen cut-off is conservative; the mean corrected OD of the positive control with the lowest infection level (P1) falls below it. 2.5. Statistical analyses
2.3. Toxoplasma gondii serology The sera were screened for T. gondii -specific IgG antibodies using a commercial direct agglutination test (Toxo-Screen DA; bioMérieux SA, Marcy-l’Étoile, France) according to the manufacturer’s instructions and alongside the controls provided. The sera were diluted to 1:40, and samples that tested positive at this dilution were defined as T. gondii-seropositive.
Cross-tabulations and test statistics (chi-square, Mid-P exact, t-test) served to evaluate associations prior to regression analyses with Stata 11.0 software (StataCorp, College Station, TX, USA). Both animal-level and herd-level risk factors were included in the analyses. P-values <0.05 were considered statistically significant. 3. Results
2.4. Trichinella spp. serology
3.1. Prevalences
Trichinella-specific antibody responses were determined with an in-house ELISA (Sukura et al., 2002), which was further optimized and adjusted for wild boar sera. To improve specificity, we used Trichinella spiralis excretorysecretory antigen (provided by Dr. Bien, Witold Stefanski Institute of Parasitology, Warsaw, Poland) to coat the plates. HRP-conjugated anti-pig-IgG (Serotec, Oxford, UK), diluted to 1:10,000, was the secondary antibody. The
T. gondii-specific IgG antibodies were detected in 65 (33.0%; 95% CI 26.7–39.8%) sera (Table 1). At least one T. gondii-seropositive animal was found on 15 (60.0%; 95% CI 40.2–77.6%) farms (Fig. 1). Based on the cut-off that we set, four (2.0%; 95% CI 0.6–4.8%) samples were interpreted as Trichinellaseropositive (Table 1). Three (12.0%; 95% CI 3.1–29.3%) farms had at least one Trichinella-seropositive animal. In
P. Jokelainen et al. / Veterinary Parasitology 187 (2012) 323–327
325
45 40 35 30 25 20 15 10 5 0 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
No. of posives
No. of negaves
Fig. 1. The number of Toxoplasma gondii -seropositive and -seronegative farmed wild boars from 25 farms.
general, higher ODs seemed to be clustered on some of the farms (Fig. 2). Indirect evidence that animals were exposed to zoonotic parasites was thus found for 16 (64.0%; 95% CI 44.1–80.8%) farms. One farm had both T. gondii- and Trichinellaseropositive animals. No animals showed evidence of a mixed infection with the two parasites. 3.2. Risk factors The seroprevalences according to the age and gender of the animals, the type and size of the herd, the location of the farm, and the sampling year appear in Table 1. Further risk factor analyses were performed for T. gondii data only, due to the small number of Trichinella-seropositive animals. The univariable analyses revealed several significant associations. Females were T. gondii-seropositive more often than were males. Animals older than 24 months were more likely to be T. gondii-seropositive than were younger
animals, and the results of the oldest animals were in line with this pattern: 19 (70%; 95% CI 51–85%) of 27 wild boars that were 30 months or older, and 14 (93%; 95% CI 71–100%) of the 15 oldest wild boars (all older than 40 months) tested seropositive. Animals that originated from small herds or south-western Finland more often tested seropositive than did animals from herds with more than 50 animals or from eastern and northern areas, respectively. The overall T. gondii seroprevalence was higher in 2008 than in the previous year. In the final simple logistic regression model for T. gondii seropositivity, including three dichotomous variables, the odds for seropositivity in the female wild boars was 2.5 (95% CI 1.3–4.8) times higher than in the male ones. Animals older than 24 months had 2.1 (95% CI 1.1–4.3) times higher odds for seropositivity than did the younger age group, and samples from the year 2008 more often tested positive than did those from 2007, with an odds ratio of 2.8 (95% CI 1.2–6.5). The combined effect of age group and
Fig. 2. Serological evidence of Trichinella spp. exposure of farmed wild boars from 25 farms, shown as the optical density of each sample, corrected for plate-to-plate variation. The cut-off value for seropositivity was 0.745, and the mean corrected optical densities of the positive controls P1 (0.07 larvae per gram tissue), P2 (1.13 lpg), and P3 (1.65 lpg), were 0.64, 1.36, and 1.34, respectively.
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gender was no different from the sum of their individual effects (no interaction) even though they were related to each other and to seropositivity (confounders). When accounting for herd effects by building a random effects logistic model for T. gondii seropositivity, females had 3.8 (95% CI 1.6–8.8) times higher odds to test seropositive than males, and samples from the year 2008 more often tested positive than did those from the previous year, with an odds ratio of 5.0 (95% CI 1.4–18.7). None of the herdlevel risk factors (location and surface area of the farm, and number of animals in the herd) were statistically significant, and the age of the animal was neither a significant variable nor a confounder.
4. Discussion The T. gondii seroprevalence in Finnish farmed wild boars (33%) was seven times higher than the seroprevalence in captive wild boars in Brazil (4.5%) despite our higher threshold for defining a positive sample (Fornazari et al., 2009). The method used in the Brazilian study is fundamentally the same as the direct agglutination test used in our study. The Trichinella seroprevalence in Finnish farmed wild boars (2%) was lower than the previous estimate (11%) (Sukura et al., 2001). However, that earlier study included animals from a highly exposed farm and used a slightly different methodology. In that study, as in ours, higher ODs were associated with some of the farms, thus indicating farm-level differences in exposure to Trichinella spp. Although all of the wild boars in our study had tested negative for Trichinella at meat inspection, four of them were interpreted as seropositive with the ELISA method despite the conservative cut-off. ELISA is more sensitive and can detect infection levels as low as 0.01 lpg (Gamble et al., 1983), whereas the actual detection threshold of the routine 1 g digestion method is 3–5 lpg (Gamble, 1996; Webster et al., 2006). The possibility of false negative digestion results cannot be excluded, although in wild boar meat inspection, the sensitivity of the method is higher due to the digestion of five grams of the sample taken from a predilection site instead of one gram of tissue. False seropositive results are unlikely with the conservative cut-off chosen for this study. Moreover, Trichinella nativa infection induces intensive antibody response in pigs but causes, at most, a low larval burden in their muscles (Murrell, 1985; Kapel et al., 1998). The prevalence of T. nativa in Finnish wildlife is high (Oivanen et al., 2002; Airas et al., 2010), and infections with this species could explain the detectable antibody levels in wild boars that had passed meat inspection. In a recent Dutch study, T. gondii seroprevalence by age proved inconsistent with the assumption of the lifelong persistence of antibodies in free-ranging wild boars: the oldest age group had a lower seroprevalence than expected (Opsteegh et al., 2011). In our study, all but one of the 15 oldest animals tested seropositive, and both the univariable analysis and the simple logistic regression model showed that older animals more often tested seropositive than did young ones. Surprisingly, however, age was a non-significant, non-confounding factor and was thus
statistically eliminated from the random effects logistic model that took into account heterogeneity among farms. A different age distribution (the mean age among the male wild boars was 19 months, whereas that among the females was 31 months) might be expected to explain why female wild boar tested T. gondii-seropositive more often than males did. However, further analyses indicated that the joint effect was unimportant. The simple logistic regression model for T. gondii seropositivity showed no interaction between the age group and gender of the animal, and gender was also a significant factor in the random effects regression model, which excluded age. Intriguingly, the T. gondii seroprevalence was highest in south-western Finland, whereas three of the four Trichinella-seropositive wild boars originated from the eastern parts of the country. In previous nationwide studies, the highest T. gondii seroprevalences in both wild and domestic animal hosts were also found in south-western Finland (Jokelainen et al., 2010, 2011). This finding suggests a higher level of environmental contamination with oocysts, likely due to the presence of more domestic cats in this area where the human population is concentrated. For both T. gondii and Trichinella spp., animals farmed outdoors are good hosts. A fenced-off area may be efficiently contaminated with T. gondii oocysts by felids and can be accessed by small free-ranging hosts: both parasites have many opportunities to infect the hosts farmed for consumption by yet other hosts. Further investigations of herd-level differences in exposure to these parasites would benefit from a specific questionnaire focusing on the presence of possible infection sources. Although processing the meat of infected wild boars can hamper the further transmission of both of these zoonotic parasites, prevention of the infections should be the primary goal. Of the recommended preventive measures for domestic pigs (van Knapen, 2000; Lehmann et al., 2003; Dubey, 2009; Gottstein et al., 2009), rodent control, offal hygiene, and securing a barrier against wildlife are the most important. In addition, sties, pastures, feed, and water should be kept free of feces of felids excreting T. gondii oocysts (Lehmann et al., 2003; Dubey, 2009). Prevention of these zoonotic parasite infections in wild boar farming is needed; currently in Finland, farmed wild boars commonly encounter T. gondii and sporadically Trichinella spp.
Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgements The authors thank the farmers for their contribution in sampling the animals, the personnel of the Quality Game Farming project for their assistance, and Antti Oksanen for commenting on the manuscript during its preparation. Financial support was provided by the Finnish Ministry of Agriculture and Forestry, the Association for Animal Disease Prevention (ETT), and the Mercedes Zachariassen Foundation.
P. Jokelainen et al. / Veterinary Parasitology 187 (2012) 323–327
References Airas, N., Saari, S., Mikkonen, T., Virtala, A.-M., Pellikka, J., Oksanen, A., Isomursu, M., Kilpelä, S.-S., Lim, C.W., Sukura, A., 2010. Sylvatic Trichinella spp. Infection in Finland. J. Parasitol. 96, 67–76. Dubey, J.P., 2009. Toxoplasmosis in pigs – The last 20 years. Vet. Parasitol. 164, 89–103. EFSA, 2005. Opinion of the Scientific Panel on Biological Hazards on “Risk assessment of a revised inspection of slaughter animals in areas with low prevalence of Trichinella”. EFSA J. 200, 1–41. EFSA, 2007. Scientific Opinion of the Panel on Biological Hazards on a request from EFSA on Surveillance and monitoring of Toxoplasma in humans, food and animals. EFSA J. 583, 1–64. Fornazari, F., Langoni, H., da Silva, R.C., Guazzelli, A., Ribeiro, M.G., Chiacchio, S.B., 2009. Toxoplasma gondii infection in wild boars (Sus scrofa) bred in Brazil. Vet. Parasitol. 164, 333–334. Gamble, H.R., 1996. Detection of trichinellosis in pigs by artificial digestion and enzyme immunoassay. J. Food Prot. 59, 295–298. Gamble, H.R., Anderson, W.R., Graham, C.E., Murrell, K.D., 1983. Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory-secretory antigen. Vet. Parasitol. 13, 349–361. Gottstein, B., Pozio, E., Nöckler, K., 2009. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin. Microbiol. Rev. 22, 127–145. Jokelainen, P., Isomursu, M., Näreaho, A., Oksanen, A., 2011. Natural Toxoplasma gondii infections in European brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization. J. Wildl. Dis. 47, 154–163. Jokelainen, P., Näreaho, A., Knaapi, S., Oksanen, A., Rikula, U., Sukura, A., 2010. Toxoplasma gondii in wild cervids and sheep in Finland: North–south gradient in seroprevalence. Vet. Parasitol. 171, 331–336.
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Kapel, C.M.O., Webster, P., Lind, P., Pozio, E., Henriksen, S.-A., Murrell, K.D., Nansen, P., 1998. Trichinella spiralis, T. britovi, and T. nativa: infectivity, larval distribution in muscle, and antibody response after experimental infection of pigs. Parasitol. Res. 84, 264–271. van Knapen, F., 2000. Control of trichinellosis by inspection and farm management practices. Vet. Parasitol. 93, 385–392. Lehmann, T., Graham, D.H., Dahl, E., Sreekumar, C., Launer, F., Corn, J.L., Gamble, H.R., Dubey, J.P., 2003. Transmission dynamics of Toxoplasma gondii on a pig farm. Infect. Genet. Evol. 3, 135–141. Murrell, K.D., 1985. Strategies for the control of human trichinosis transmitted by pork. Food Technol. 39 (65–68), 110–111. Oivanen, L., Kapel, C.M.O., Pozio, E., La Rosa, G., Mikkonen, T., Sukura, A., 2002. Associations between Trichinella species and host species in Finland. J. Parasitol. 88, 84–88. Opsteegh, M., Swart, A., Fonville, M., Dekkers, L., van der Giessen, J., 2011. Age-related Toxoplasma gondii seroprevalence in Dutch wild boar inconsistent with lifelong persistence of antibodies. PLoS One 6, e16240, doi:10.1371/journal.pone.0016240. Sukura, A., Näreaho, A., Mikkonen, T., Niemi, M., Oivanen, L., 2002. Trichinella nativa and T. spiralis induce distinguishable histopathologic and humoral responses in the raccoon dog (Nyctereutes procyonoides). Vet. Pathol. 39, 257–265. Sukura, A., Näreaho, A., Veijalainen, P., Oivanen, L., 2001. Trichinellosis in farmed wild boar: meat inspection findings and seroprevalence. Parasite 8, 243–245. Webster, P., Maddox-Hyttel, C., Nöckler, K., Malakauskas, A., van der Giessen, J., Pozio, E., Boireau, P., Kapel, C.M.O., 2006. Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation. Euro Surveill. 11, 50–55.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Farmed wild boars exposed to Toxoplasma gondii and Trichinella spp. Pikka Jokelainen a,∗ , Anu Näreaho a , Outi Hälli b , Mari Heinonen b , Antti Sukura a a b
Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, FI-00014 University of Helsinki, Finland Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, FI-04920 Saarentaus, Finland
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 12 December 2011 Accepted 20 December 2011 Keywords: Toxoplasma gondii Trichinella spp. Zoonosis Serology Sus scrofa L. Wild boar Farmed game
a b s t r a c t The meat of wild boar (Sus scrofa L.) can be a source of human infections with zoonotic parasites Toxoplasma gondii and Trichinella spp. We screened 197 wild boar sera collected at slaughter from 25 Finnish farms in 2007–2008 for serological evidence of infections with these parasites. Using a commercial direct agglutination test at a serum dilution of 1:40, T. gondii-specific IgG antibodies were detected in 65 (33.0%) samples, on 14 (56.0%) farms. Females, animals older than 24 months, animals of small herds, and animals originating from south-western parts of Finland were more often T. gondii-seropositive than were males, younger animals, animals of larger herds, and animals originating from the north and east, respectively. Four (2.0%) of the sera, originating from three (12.0%) farms, tested Trichinella-seropositive with an in-house ELISA and a conservative cut-off for seropositivity. One farm had both T. gondii- and Trichinella-seropositive animals. Taken together, an infection source had been present on 16 (64.0%) farms, and 69 (35.0%) of the 197 farmed wild boars intended for human consumption had specific serological evidence of exposure to a zoonotic parasite. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The European wild boar (Sus scrofa L.) is a popular game animal species whose meat is also available for consumers as a farmed delicacy. Unfortunately, it can be a source of zoonotic parasites, such as Toxoplasma gondii and Trichinella spp., if enjoyed undercooked (EFSA, 2005, 2007). At the slaughterhouse level, meat intended for retail is monitored for Trichinella, but not for T. gondii infections. Although many epidemiological and parasitological studies of wild boar pertain to free-ranging animals (e.g. for T. gondii, reviewed by Fornazari et al., 2009), investigating the role of farmed wild boars as hosts for zoonotic parasites is also of both public health and veterinary importance. Contrary to expectations for free-ranging game animals, farmed animals could be at least partly protected from such infections. In Finland, approximately 100 free-ranging
∗ Corresponding author. Tel.: +358 9 191 57183; fax: +358 9 191 57194. E-mail address: pikka.jokelainen@helsinki.fi (P. Jokelainen). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.12.026
wild boars are shot by hunters every year, and 600 farmed wild boars are slaughtered for meat. This serological survey aimed to estimate the exposure of the farmed wild boar population to T. gondii and Trichinella spp. 2. Materials and methods 2.1. Farms and animals This survey was part of a nationwide epidemiological study for which a sampling frame was compiled from the official records of Finnish wild boar farmers. All 117 farms were contacted and 32 (45% of active farms) participated in the study. The farmers sampled their wild boars at slaughter and provided the background information on the animals and farms. For this survey, we selected serum samples from 2007 to 2008 to obtain a sufficient sample size, calculated based on expected seroprevalences of 2–15% for both parasites. Samples from 197 farmed wild boars, 1–43 per farm (median 4), were thus included in this survey (Table 1). The samples
324
P. Jokelainen et al. / Veterinary Parasitology 187 (2012) 323–327
Table 1 Distribution of categorical variables of the 197 wild boars included in the survey, and Toxoplasma gondii and Trichinella seroprevalences by category. Variable
na
%
n T. gondii seropositive
% (95% CI)
n Trichinella seropositive
% (95% CI)
Age of the animal, months
0–12b 12–23b 24–
24 110 56
13 58 29
7 27 27
29 (14–49) 25 (17–33) 48 (35–61)
0 2 2
0 (0–12) 2 (0–6) 4 (1–11)
Gender of the animal
Male Female
120 74
62 38
29 34
24 (17–32) 46 (35–57)
2 2
2 (0–5) 3 (0–9)
Herd type
Slaughter Integrated
75 122
38 62
18 47
24 (15–35) 39 (30–47)
2 2
3 (0–9) 2 (0–5)
Number of animals in the herd
0–50 51–100 101–
97 25 75
49 13 38
39 6 20
40 (31–50) 24 (10–43) 27 (18–38)
2 0 2
2 (0–7) 0 (0–11) 3 (0–9)
Location of the farm
Southwest East North
16 138 33
9 74 18
9 40 12
56 (32–78) 29 (22–37) 36 (21–54)
0 3 1
0 (0–17) 2 (1–6) 3 (0–14)
Sampling year
2007 2008
48 149
24 76
9 56
19 (10–32) 38 (30–46)
1 3
2 (0–10) 2 (1–5)
65
33 (27–40)
4
2 (1–5)
Total a b
197
The location of three farms (ten animals), the age of seven animals from three farms, and the gender of three animals from two farms were unknown. In the simple logistic regression, age group was used as a dichotomous variable: these two groups were combined.
originated from 25 farms with 6–150 animals (median 47) and a surface area of 1–65 ha (median 3). All the animals were used for human consumption after passing the official meat inspection that included examination for Trichinella using the digestion method (EU 2075/2005). The predilection site sampled for this examination was diaphragm, and five grams of the sample were digested. 2.2. Sampling The wild boars were stunned and bled on the farms prior to the transportation of their carcasses to the slaughterhouses. The farmers collected the blood samples during bleeding and sent them to the laboratory within three days. The sera were separated by centrifugation (10 min at 920 × g) and stored at −18 to −21 ◦ C until analyzed.
control sera were obtained earlier from wild boars with known infection levels of 0.07 larvae per gram of muscle tissue (lpg) (P1), 1.13 lpg (P2), and 1.65 lpg (P3), and two digestion-negative wild boars (N1 and N2). The controls and samples were diluted to 1:100. After subtracting the mean optical densities (OD) of the buffer-only wells from all the values, we corrected for plate-to-plate variation by using the mean of the ODs of the two positive control samples (P1 and P2) as the denominator for the sample ODs. To avoid false-positive results, the cut-off for seropositivity was set to the mean of the ODs of the negative controls (N1 and N2) plus three times their standard deviation. This chosen cut-off is conservative; the mean corrected OD of the positive control with the lowest infection level (P1) falls below it. 2.5. Statistical analyses
2.3. Toxoplasma gondii serology The sera were screened for T. gondii -specific IgG antibodies using a commercial direct agglutination test (Toxo-Screen DA; bioMérieux SA, Marcy-l’Étoile, France) according to the manufacturer’s instructions and alongside the controls provided. The sera were diluted to 1:40, and samples that tested positive at this dilution were defined as T. gondii-seropositive.
Cross-tabulations and test statistics (chi-square, Mid-P exact, t-test) served to evaluate associations prior to regression analyses with Stata 11.0 software (StataCorp, College Station, TX, USA). Both animal-level and herd-level risk factors were included in the analyses. P-values <0.05 were considered statistically significant. 3. Results
2.4. Trichinella spp. serology
3.1. Prevalences
Trichinella-specific antibody responses were determined with an in-house ELISA (Sukura et al., 2002), which was further optimized and adjusted for wild boar sera. To improve specificity, we used Trichinella spiralis excretorysecretory antigen (provided by Dr. Bien, Witold Stefanski Institute of Parasitology, Warsaw, Poland) to coat the plates. HRP-conjugated anti-pig-IgG (Serotec, Oxford, UK), diluted to 1:10,000, was the secondary antibody. The
T. gondii-specific IgG antibodies were detected in 65 (33.0%; 95% CI 26.7–39.8%) sera (Table 1). At least one T. gondii-seropositive animal was found on 15 (60.0%; 95% CI 40.2–77.6%) farms (Fig. 1). Based on the cut-off that we set, four (2.0%; 95% CI 0.6–4.8%) samples were interpreted as Trichinellaseropositive (Table 1). Three (12.0%; 95% CI 3.1–29.3%) farms had at least one Trichinella-seropositive animal. In
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45 40 35 30 25 20 15 10 5 0 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
No. of posives
No. of negaves
Fig. 1. The number of Toxoplasma gondii -seropositive and -seronegative farmed wild boars from 25 farms.
general, higher ODs seemed to be clustered on some of the farms (Fig. 2). Indirect evidence that animals were exposed to zoonotic parasites was thus found for 16 (64.0%; 95% CI 44.1–80.8%) farms. One farm had both T. gondii- and Trichinellaseropositive animals. No animals showed evidence of a mixed infection with the two parasites. 3.2. Risk factors The seroprevalences according to the age and gender of the animals, the type and size of the herd, the location of the farm, and the sampling year appear in Table 1. Further risk factor analyses were performed for T. gondii data only, due to the small number of Trichinella-seropositive animals. The univariable analyses revealed several significant associations. Females were T. gondii-seropositive more often than were males. Animals older than 24 months were more likely to be T. gondii-seropositive than were younger
animals, and the results of the oldest animals were in line with this pattern: 19 (70%; 95% CI 51–85%) of 27 wild boars that were 30 months or older, and 14 (93%; 95% CI 71–100%) of the 15 oldest wild boars (all older than 40 months) tested seropositive. Animals that originated from small herds or south-western Finland more often tested seropositive than did animals from herds with more than 50 animals or from eastern and northern areas, respectively. The overall T. gondii seroprevalence was higher in 2008 than in the previous year. In the final simple logistic regression model for T. gondii seropositivity, including three dichotomous variables, the odds for seropositivity in the female wild boars was 2.5 (95% CI 1.3–4.8) times higher than in the male ones. Animals older than 24 months had 2.1 (95% CI 1.1–4.3) times higher odds for seropositivity than did the younger age group, and samples from the year 2008 more often tested positive than did those from 2007, with an odds ratio of 2.8 (95% CI 1.2–6.5). The combined effect of age group and
Fig. 2. Serological evidence of Trichinella spp. exposure of farmed wild boars from 25 farms, shown as the optical density of each sample, corrected for plate-to-plate variation. The cut-off value for seropositivity was 0.745, and the mean corrected optical densities of the positive controls P1 (0.07 larvae per gram tissue), P2 (1.13 lpg), and P3 (1.65 lpg), were 0.64, 1.36, and 1.34, respectively.
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gender was no different from the sum of their individual effects (no interaction) even though they were related to each other and to seropositivity (confounders). When accounting for herd effects by building a random effects logistic model for T. gondii seropositivity, females had 3.8 (95% CI 1.6–8.8) times higher odds to test seropositive than males, and samples from the year 2008 more often tested positive than did those from the previous year, with an odds ratio of 5.0 (95% CI 1.4–18.7). None of the herdlevel risk factors (location and surface area of the farm, and number of animals in the herd) were statistically significant, and the age of the animal was neither a significant variable nor a confounder.
4. Discussion The T. gondii seroprevalence in Finnish farmed wild boars (33%) was seven times higher than the seroprevalence in captive wild boars in Brazil (4.5%) despite our higher threshold for defining a positive sample (Fornazari et al., 2009). The method used in the Brazilian study is fundamentally the same as the direct agglutination test used in our study. The Trichinella seroprevalence in Finnish farmed wild boars (2%) was lower than the previous estimate (11%) (Sukura et al., 2001). However, that earlier study included animals from a highly exposed farm and used a slightly different methodology. In that study, as in ours, higher ODs were associated with some of the farms, thus indicating farm-level differences in exposure to Trichinella spp. Although all of the wild boars in our study had tested negative for Trichinella at meat inspection, four of them were interpreted as seropositive with the ELISA method despite the conservative cut-off. ELISA is more sensitive and can detect infection levels as low as 0.01 lpg (Gamble et al., 1983), whereas the actual detection threshold of the routine 1 g digestion method is 3–5 lpg (Gamble, 1996; Webster et al., 2006). The possibility of false negative digestion results cannot be excluded, although in wild boar meat inspection, the sensitivity of the method is higher due to the digestion of five grams of the sample taken from a predilection site instead of one gram of tissue. False seropositive results are unlikely with the conservative cut-off chosen for this study. Moreover, Trichinella nativa infection induces intensive antibody response in pigs but causes, at most, a low larval burden in their muscles (Murrell, 1985; Kapel et al., 1998). The prevalence of T. nativa in Finnish wildlife is high (Oivanen et al., 2002; Airas et al., 2010), and infections with this species could explain the detectable antibody levels in wild boars that had passed meat inspection. In a recent Dutch study, T. gondii seroprevalence by age proved inconsistent with the assumption of the lifelong persistence of antibodies in free-ranging wild boars: the oldest age group had a lower seroprevalence than expected (Opsteegh et al., 2011). In our study, all but one of the 15 oldest animals tested seropositive, and both the univariable analysis and the simple logistic regression model showed that older animals more often tested seropositive than did young ones. Surprisingly, however, age was a non-significant, non-confounding factor and was thus
statistically eliminated from the random effects logistic model that took into account heterogeneity among farms. A different age distribution (the mean age among the male wild boars was 19 months, whereas that among the females was 31 months) might be expected to explain why female wild boar tested T. gondii-seropositive more often than males did. However, further analyses indicated that the joint effect was unimportant. The simple logistic regression model for T. gondii seropositivity showed no interaction between the age group and gender of the animal, and gender was also a significant factor in the random effects regression model, which excluded age. Intriguingly, the T. gondii seroprevalence was highest in south-western Finland, whereas three of the four Trichinella-seropositive wild boars originated from the eastern parts of the country. In previous nationwide studies, the highest T. gondii seroprevalences in both wild and domestic animal hosts were also found in south-western Finland (Jokelainen et al., 2010, 2011). This finding suggests a higher level of environmental contamination with oocysts, likely due to the presence of more domestic cats in this area where the human population is concentrated. For both T. gondii and Trichinella spp., animals farmed outdoors are good hosts. A fenced-off area may be efficiently contaminated with T. gondii oocysts by felids and can be accessed by small free-ranging hosts: both parasites have many opportunities to infect the hosts farmed for consumption by yet other hosts. Further investigations of herd-level differences in exposure to these parasites would benefit from a specific questionnaire focusing on the presence of possible infection sources. Although processing the meat of infected wild boars can hamper the further transmission of both of these zoonotic parasites, prevention of the infections should be the primary goal. Of the recommended preventive measures for domestic pigs (van Knapen, 2000; Lehmann et al., 2003; Dubey, 2009; Gottstein et al., 2009), rodent control, offal hygiene, and securing a barrier against wildlife are the most important. In addition, sties, pastures, feed, and water should be kept free of feces of felids excreting T. gondii oocysts (Lehmann et al., 2003; Dubey, 2009). Prevention of these zoonotic parasite infections in wild boar farming is needed; currently in Finland, farmed wild boars commonly encounter T. gondii and sporadically Trichinella spp.
Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgements The authors thank the farmers for their contribution in sampling the animals, the personnel of the Quality Game Farming project for their assistance, and Antti Oksanen for commenting on the manuscript during its preparation. Financial support was provided by the Finnish Ministry of Agriculture and Forestry, the Association for Animal Disease Prevention (ETT), and the Mercedes Zachariassen Foundation.
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