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Serological Evidence of Exposure to Globally Relevant Zoonotic Parasites in the Estonian Population

We investigated Estonian population and its selected subgroups for serological evidence of exposure to Ascaris lumbricoides, Echinococcus spp., Taenia solium, Toxocara canis, Toxoplasma gondii, and Trichinella spiralis. Serum samples from 999 adults representing general population, 248 children aged 14–18, 158 veterinarians, 375 animal caretakers, and 144 hunters were tested for specific immunoglobulin G antibodies against the selected parasites using commercial enzyme immunoassays (ELISA). Sera yielding positive or twice grey zone Echinococcus spp, T. solium, T. canis, and T. spiralis results were subjected to western blot (WB) analysis. In the general population, based on the ELISA results, the A. lumbricoides seroprevalence was 12.7%, Echinococcus spp. seroprevalence was 3.3%, T. solium seroprevalence was 0.7%, T. canis seroprevalence was 12.1%, T. gondii seroprevalence was 55.8%, and T. spiralis seroprevalence was 3.1%. Ascaris lumbricoides seroprevalences were higher in children and in animal caretakers than in the general population, and T. canis seroprevalence was higher in animal caretakers than in the general population. Compared with the general population, Echinococcus spp. seroprevalence was higher in children. By contrast, T. gondii seroprevalence was higher in animal caretakers, and lower in children, than in the general population. In the general population, the WB-confirmed Echinococcus spp. seroprevalence was 0.5%, T. solium cysticercosis seroprevalence was 0.0%, Toxocara spp. seroprevalence was 14.5%, and Trichinella spp. seroprevalence was 2.7%. WB-confirmed Toxocara spp. seroprevalence was higher in animal caretakers than in the general population. We found serological evidence of exposure to zoonotic parasites in all tested groups. This calls for higher awareness of zoonotic parasitic infections in Estonia.


Introduction
hunters gave written informed consent before the blood samples were taken by nurses. The sera were stored and analysed coded.
Those veterinarians, animal caretakers, and hunters who had provided contact information were informed of their serology results and given a short description of what seropositivity means. In addition, they were provided the contact information for designated research group members, to whom further questions could be addressed. Those with medical questions were guided to consult their own family physician.

Setting
Estonia is located in the north-eastern Europe and has a population of 1.3 million inhabitants [27]. Approximately 1000 veterinary practitioners are licenced to work in the country [28]. The number of persons working as animal caretakers is unknown. There are over 15000 hunters [29].

Samples
The general population samples (n = 999), from individuals 18 years and older, were obtained as a random sample stratified by county and gender from the serum bank of the Estonian Genome Center. The samples had been collected in 2004-2011. Sera from apparently healthy children aged 14-18 years (n = 248) were obtained from a serum bank that had been collected in 2003. The samples originated from different parts of the country.
Veterinarians (n = 158) were sampled at a local veterinary conference in October 2012. Animal caretakers (n = 375) included persons involved with dairy cattle (n = 193), beef cattle (n = 51), pigs (n = 68), and sheep and goats (n = 63). From those involved with dairy cattle, blood samples were collected in March-May 2013, whereas those involved with beef cattle were sampled in January 2014. Animal caretakers involved with pigs were sampled in September-November 2013, and those involved with sheep and goats were sampled in October-November 2013. Those involved with pigs were sampled during farm visits, whereas the collection of the other samples was arranged at local professional meetings and events.
Hunters (n = 144) were sampled during a national meeting of hunters in July 2013. General population samples and children samples were stored frozen at -20°C and thawed prior to the analyses. The samples from veterinarians, animal caretakers, and hunters were allowed to clot and then centrifuged, at the sampling site. The sera were separated within 24 hours. The sera were stored at +4°C for up to two days, during which the first analyses were performed, and then frozen at -20°C until thawed prior to further analyses.
The samples that tested positive with ELISA were considered seropositive. The samples that yielded a grey zone result were retested, and the second test result was considered the final ELISA result.
Samples that had tested positive or yielded a grey zone result twice with the Echinococcus spp., T. solium, T. canis and T. spiralis ELISA were further tested using ECHINOCOCCUS Western Blot IgG, CYSTICERCOSIS Western Blot IgG, TOXOCARA Western Blot IgG, and TRICHINELLA Western Blot IgG (LDBIO DIAGNOSTICS, Lyon, France), respectively. The positive controls provided in the kits were used in each analysis. The results were interpreted following the manufacturer's instructions.
The samples that tested positive with the western blot (WB) were considered confirmed seropositives. It is noteworthy that the T. solium ELISA is intended for detecting antibodies against T. solium antigens, both taeniasis and cysticercosis, while the corresponding WB is based on antigens from crude larval extract and intended for detecting cysticercosis caused by the larval stages of the parasite only.
The crude seroprevalences were calculated using the number of seropositives (ELISA result) as the numerator. The WB-confirmed seroprevalences were calculated using the number of confirmed seropositives (ELISA and WB in series) as the numerator. The true seroprevalences (Rogan-Gladen) were calculated using the number of seropositives (ELISA result) and taken into account the Sp and Se, with EpiTools [30]. If the Sp or Se was given as ">95%", the calculation was done using 95%. The denominator was the number of samples tested in each group.

Exclusion of samples
The database was searched for double entries: one sample from general population and one sample from animal caretakers (persons involved with beef cattle) were excluded.

Statistical analyses
The statistical analyses were performed using the free software OpenEpi [31]. Confidence intervals (95% CI) were calculated using Mid-P exact test. Two-by-two tables were used to evaluate differences in crude seroprevalences between the groups. Bonferroni adjustment was used to reduce the likelihood of type 1 error, and differences with two-tailed p-values < 0.01 (Mid-P exact test) were considered statistically significant. When comparing a single result of ours to another published result, a difference with two-tailed p-value < 0.05 (Mid-P exact test) was considered statistically significant.

Ascaris lumbricoides
The A. lumbricoides seroprevalence was 12.7% in the general population. All groups showed evidence of exposure to A. lumbricoides ( Table 1). The seroprevalence was significantly higher in children and in animal caretakers than in the general population (p < 0.001).

Echinococcus spp.
The Echinococcus spp. seroprevalence was 3.3% and the WB-confirmed Echinococcus spp. seroprevalence was 0.5% in the general population. All groups showed evidence of exposure to Echinococcus spp. ( Table 2). The seroprevalence was higher in children (p < 0.001) than in the general population. WB-confirmed seropositives were detected in the general population and animal careretakers group.

General
Comparison with the general population:

Toxocara canis
The T. canis seroprevalence was 12.1% and the WB-confirmed Toxocara spp. seroprevalence was 14.5% in the general population. All groups showed evidence of exposure to T. canis ( Table 4). The seroprevalence was higher in animal caretakers (p < 0.001) than in the general population. The WB-confirmed Toxocara spp. seroprevalence was also higher in animal caretakers (p < 0.001) than in the general population. The

Toxoplasma gondii
The T. gondii seroprevalence was 55.8% in the general population. All groups showed evidence of exposure to T. gondii ( Table 5). The seroprevalence was higher in animal caretakers (p < 0.01), and lower in children (p < 0.001), than in the general population.  Trichinella spiralis

Discussion
We detected evidence of exposure to all of the zoonotic parasites tested. These zoonotic parasites present a threat to human health and life quality, animal health and welfare, food safety, the economy, and the environment [1,32,33]. The results of this study call for higher awareness of zoonotic parasitic infections in Estonia.
The A. lumbricoides seroprevalence in general population was lower than an estimate from the Netherlands (33.0%, p < 0.001) [34]. The A. lumbricoides seroprevalence estimate in children in Estonia was higher than estimates from Poland (15.0%, p < 0.001) [35] and from the Netherlands (7.2%, p < 0.001) [36]. In our study, the seroprevalence was higher in the children than in adults. This is in contrast to an increase in seroprevalence with age that was observed in the Netherlands [34]. One possible explanation could be that exposure to this parasite would have increased recently in Estonia, but there are no direct data to support that. The higher seroprevalence in animal caretakers might be due to contact with Ascaris eggs in the agricultural environment.
The Echinococcus spp. seroprevalence estimate in general population was higher than that observed in Austria (0.0%, p < 0.001) [37] and Greece (1.1%, p < 0.01) [38], but similar to that Table 6. Trichinella spiralis ELISA and Trichinella spp. western blot (WB) results including those that tested positive (POS) and those that tested positive or yielded a grey zone result twice (POS+GREY) in the general population, children, veterinarians, animal caretakers, and hunters in Estonia.

General
Children observed in Poland (3.2%) [39] and Spain (3.4%) [40]. The seroprevalence in veterinarians was similar to that in veterinary surgeons in Turkey (2.2%) [41]. The Echinococcus spp. seroprevalence in hunters was lower than both E. granulosus seroprevalence (10.7%, p < 0.001) and E. multilocularis seroprevalence (5.4%, p < 0.05) in hunters in Austria [7], while none of the seropositives in either of the studies tested positive with WB. The data available on Echinococcus spp. infections in animal hosts originates mainly from research projects, but E. multilocularis, E. canadensis (G8 and G10) of E. granulosus (G1) have been diagnosed in animal hosts in Estonia recently [14]. We found no comparable data on T. solium seroprevalence, while the parasite is considered relevant in Europe [42,43]. Cysticercosis has been reported from Estonian pigs [44].
The T. gondii seroprevalences were worryingly high when compared with recent results from other European countries, where the seroprevalence has decreased [53,54]. The burden caused by T. gondii infections is high [1,2,32] and the parasite merits attention. Toxoplasma gondii seroprevalence typically increases with age, indicating acquired infections. The high T. gondii seroprevalence in children in Estonia suggests that the infection pressure is substantial, while different age distribution might partly explain some of the differences noted between other groups. Contact with contaminated environment on farms [55] may partly explain the higher seroprevalence in farm workers. High T. gondii seroprevalence in domestic cats in Estonia [15] indicates that the environment has been contaminated with oocysts, which is supported by results from wild and domestic animals [12,16].
The T. spiralis seroprevalence in the general population was similar to an estimate in forest workers in Poland (6.0%) [39], and the WB-confirmed Trichinella spp. seroprevalence was similar to an estimate in hunting communities in Greenland (3.3%) [56]. Trichinella spp. merit higher awareness as relevant zoonotic parasites in Estonia, in animal hosts particularly in the sylvatic cycle [17].
The sample sizes were adequate for estimating and comparing the seroprevalences. The general population samples were a good representation of the Estonian population. The children group only included samples from youngsters aged over 14 years; thus the seroprevalences in younger children remain unknown. The convenience samples from children, veterinarians, animal caretakers, and hunters may be limited by geographical representativeness; those interested in research activities and further professional education may be overrepresented.
Serology is an indirect detection method. For detecting chronic T. gondii infections, serology is widely used, whereas for other parasites, serology results should be interpreted with more caution. Detecting antibodies provides evidence of exposure and can indicate infection [57] and has been used in follow-up of patients. For example, patients with alveolar echinococcosis appear to maintain ELISA-seropositivity despite the intensity of the WB bands may fade during the follow-up, while a curative resection results in seronegativity in some patients in a few years [58].
We chose to investigate the presence of IgG antibodies because they are commonly longlasting and suitable for epidemiological studies. However, investigating only one class of antibodies is a limitation of the study.
The assays used are based on purified antigens, but some potential cross-reactions are listed by the manufacturer. Of the 499 T. canis positive samples, 130 (26.1%) tested positive for antibodies against A. lumbricoides and ten (2.0%) tested positive for antibodies against Echinococcus spp. with the corresponding ELISA assays. A majority (99.1%) of T. canis results tested positive with the corresponding WB. One sample that had tested positive for antibodies against T. canis and for antibodies against Echinococcus spp. tested positive for both with the corresponding WB assays.
Overall, it appeared to be a good decision to include also samples that yielded a grey zone ELISA result twice to be tested with WB. Several samples yielded a grey zone result twice with ELISA but tested positive with WB (Tables 2, 4 and 6).
The methods were evaluated to be suitable for an epidemiological study, although communicating an individual result required explaining the main limitations of the methods used. Informing the veterinarians, animal caretakers, and hunters of their results was evaluated to be an ethically reasoned choice.
These results provide baseline data, which can inform public health decision makers and suggest where further research and prevention efforts should be targeted. It is obvious that the zoonotic parasites circulating in Estonia reach also humans, but the locally relevant risk factors for encountering the parasites are currently largely unknown.

Conclusions
People living in Estonia had evidence of having been exposed to several zoonotic parasites, which calls for evaluation of need of prevention strategies and higher awareness. Antibodies against zoonotic parasites appeared to be formed already in childhood, indicating considerable infection pressure. The results suggest that zoonotic parasitic infections are underdiagnosed or underreported in Estonia.