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Background Many parasites (protozoa and helminths) can be transmitted through food and lead to infections with high morbidity, as well as disease outbreaks. Although the importance of foodborne parasites (FBP) is recognised by many sectors of the food industry, standardized analytical methods and validation procedures for testing food for FBP are lacking. Scope and approach:Current methods for detection of FBP, and their validation, are critically reviewed, focusing on priority FBP in Europe: the helminths Echinococcus multilocularis, Echinococcus granulosus, Taenia saginata, Trichinella spp., and Anisakidae, and the protozoa Toxoplasma gondii, Cryptosporidium spp., and Giardia duodenalis. Key findings and conclusions:Standard methods exist for detection of T. saginata in beef, and Trichinella spp. in meat (and are mandatory at meat inspection in Europe), Anisakidae in fish, and Cryptosporidium spp. and G. duodenalis in leafy green vegetables and berry fruits. For other FBP or foods, methods used in sample surveys have been described, but validation data are generally absent; limits of detection are not provided, ring trials have rarely been performed, and for most FBP quality control materials, proficiency schemes, and reference standards are lacking. The use of surrogate particles or organisms for method development or validation purposes needs to be carefully considered. Documented procedures for validation, such as ISO17468 and ISO16140-2:2016 that were established for bacteria, are mostly inappropriate for FBP. The development and application of standardized and validated detection methods would enhance understanding of the foodborne route of transmission, improve risk assessments, and help identify and verify critical control points.

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Herbivores can be accidental hosts for the zoonotic nematode parasites Trichinella spp., which are endemic at high prevalence in wildlife in northeastern Europe. Using direct and indirect detection methods for Trichinella spp., we investigated samples from 463 wild moose (Alces alces) harvested by hunters in Estonia in 2015. A total of 460 moose were tested directly by artificial digestion of diaphragm muscle, 463 moose were tested indirectly by enzyme-linked immunosorbent assay (ELISA), and 34 moose also by western blot. Positive-control reference sera were from other host species. Nematode larvae were found in six muscle samples; five of which were pooled samples. None of the larvae were identified as Trichinella spp., based on their morphology and molecular analyses. Twelve moose (2.6%) were positive by ELISA, but none were positive by the western blot test. Trichinella spp. infection was not detected, but ELISA results may suggest Trichinella spp. exposure in a small proportion of moose in Estonia.

In this study, we summarize Trichinella findings from four wild, free-ranging host species from Estonia during 2007&ndash, 2014. Trichinella spp. larvae were detected in 281 (0.9%, 95% confidence interval (CI) 0.8&ndash, 1.0) of 30,566 wild boars (Sus scrofa), 63 (14.7%, 95% CI 11.6&ndash, 18.3) of 429 brown bears (Ursus arctos), 59 (65.56%, 95% CI 55.3&ndash, 74.8) of 90 Eurasian lynxes (Lynx lynx), and three (60.0%, 95% CI 18.2&ndash, 92.7) of five badgers (Meles meles). All four European Trichinella species were detected: T. britovi in 0.7% of the wild boars, 7.2% of the brown bears, 45.6% of the lynxes, and 40.0% of the badgers, T. nativa in 0.1% of the wild boars, 5.8% of the brown bears, and 20.0% of the lynxes, T. pseudospiralis in 0.02% the wild boars, and T. spiralis in 0.03% of the wild boars and 4.4% of the lynxes. The results include the first description from Estonia of T. britovi in brown bear and badgers, T. pseudospiralis in wild boars, and T. spiralis in wild boars and lynxes. The results indicate high infection pressure in the sylvatic cycles across the years &ndash, illustrating continuous risk of spillover to domestic cycles and of transmission to humans.

Publisher's version (útgefin grein), The world is changing, and parasites adapt. The Nordic-Baltic region in northern Europe – including the Nordic countries Denmark, Finland, Iceland, Norway and Sweden, and the Baltic States Estonia, Latvia and Lithuania – is facing new parasitological challenges due to changes in populations of parasites and their hosts and the spread of new parasites to the region due to climate change. Some changes can also be ascribed to increased awareness and detection. In this paper, we review and discuss a convenience selection of ten timely examples of recent observations that exemplify trends and challenges from different fields of parasitology, with particular focus on climate change and potential changes in epidemiology of pathogens in northern Europe. The examples illustrate how addressing parasitological challenges often requires both intersectoral and international collaboration, and how using both historical baseline data and modern methodologies are needed., The authors thank Brian Lassen for the graphical abstract. The World Federation for Parasitologists is thanked for this Special Issue initiative.

Salmonella enterica subspecies enterica serotype Choleraesuis is a swine adapted serovar. S. Choleraesuis variant Kunzendorf is responsible for the majority of outbreaks among pigs. S. Choleraesuis is rare in Europe, although there have been serious outbreaks in pigs including two outbreaks in Denmark in 1999-2000 and 2012-2013. Here, we elucidate the epidemiology, possible transmission routes and sources, and clonality of European S. Choleraesuis isolates including the Danish outbreak isolates. A total of 102 S. Choleraesuis isolates from different European countries and the United States, covering available isolates from the last two decades were selected for whole genome sequencing. We applied a temporally structured sequence analysis within a Bayesian framework to reconstruct a temporal and spatial phylogenetic tree. MLST type, resistance genes, plasmid replicons, and accessory genes were identified using bioinformatics tools. Fifty-eight isolates including 11 out of 12 strains from wild boars were pan-susceptible. The remaining isolates carried multiple resistance genes. Eleven different plasmid replicons in eight plasmids were determined among the isolates. Accessory genes were associated to the identified resistance genes and plasmids. The European S. Choleraesuis was estimated to have emerged in ∼ 1837 (95% credible interval, 1733-1983) with the mutation rate of 1.02 SNPs/genome/year. The isolates were clustered according to countries and neighbor countries. There were transmission events between strains from the United States and European countries. Wild boar and pig isolates were genetically linked suggesting cross-border transmission and transmission due to a wildlife reservoir. The phylogenetic tree shows that multiple introductions were responsible for the outbreak of 2012-2013 in Denmark, and suggests that poorly disinfected vehicles crossing the border into Denmark were potentially the source of the outbreak. Low levels of single nucleotide polymorphisms (SNPs) differences (0-4 SNPs) can be observed between clonal strains isolated from different organs of the same animal. Proper disinfection of livestock vehicles and improved quality control of livestock feed could help to prevent future spread of S. Choleraesuis or other more serious infectious diseases such as African swine fever (ASF) in the European pig production system.

Taenia solium and Taenia saginata are food-borne parasites of global importance. In eastern Europe only fragmented information is available on the epidemiology of these zoonotic parasites in humans and animal populations. In particular for T. solium, on-going transmission is suspected. The aim of this systematic review was to collect the available data and describe the current knowledge on the epidemiology of T. solium and T. saginata in eastern Europe.Literature published in international databases from 1990 to 2017 was systematically reviewed. Furthermore, local sources and unpublished data from national databases were retrieved from local eastern European experts. The study area included 22 countries.Researchers from 18 out of the 22 countries provided data from local and unpublished sources, while no contacts could be established with researchers from Belarus, Kosovo, Malta and Ukraine. Taeniosis and human cysticercosis cases were reported in 14 and 15 out of the 22 countries, respectively. Estonia, the Former Yugoslav Republic of Macedonia, Lithuania, Moldova, Poland, Romania, Serbia, and Slovakia reported cases of porcine cysticercosis. Croatia, Czech Republic, Estonia, Former Yugoslav Republic of Macedonia, Moldova, Poland, Romania, Serbia, Slovakia, and Ukraine reported bovine cysticercosis.There is indication that taeniosis and cysticercosis are present across eastern Europe but information on the occurrence of T. solium and T. saginata across the region remains incomplete. Available data are scarce and species identification is in most cases absent. Given the public health impact of T. solium and the potential economic and trade implications due to T. saginata, notification of taeniosis and human cysticercosis should be implemented and surveillance and notification systems in animals should be improved.

Table S3. Individual taeniosis cases identified in case reports in eastern Europe available from 1990 to 2017. Table S4. Aggregated taeniosis cases reported in documents (authorities’ reports, epidemiological bulletins, national registries and publications) identified in eastern Europe available from 1990 to 2017. Table S5. Taeniosis prevalence data reported in epidemiological studies published between 1990–2017. Table S6. Individual human cysticercosis cases identified in case reports in eastern Europe (1990–2017). Table S7. Aggregated human cysticercosis cases identified in case reports and publications in eastern Europe (1990–2017). Table S8. Porcine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990–2017). Table S9. Bovine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990–2017). (DOCX 207 kb)

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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.

In Estonia, northeastern Europe, antibodies against Toxoplasma gondii are common in many host species, including wildlife, domestic animals, and humans. Our nationwide study aimed to estimate T. gondii seroprevalence and its geographical distribution, and to evaluate plausible risk factors for seropositivity in free-ranging moose (Alces alces) hunted for human consumption in 2015. We screened sera or plasma from 463 moose for presence of anti-T. gondii immunoglobulin G antibodies with a commercial direct agglutination test, using a cut-off titer 40 for seropositivity. Altogether 111 moose tested seropositive, yielding a seroprevalence estimate of 23.97%. Seropositive moose were detected in all the 13 counties where the samples originated from. Based on a multivariable logistic regression model, 'being female', 'being adult', and 'being from the northern part of the country' were significant risk factors, with odds ratios of 2.91, 3.07, and 3.11, respectively, and there was interaction between the variables 'being female' and 'being from the northern part of the country'. A substantial proportion of the moose investigated had been exposed to T. gondii. Presence of the parasite in edible tissues of the moose was not shown in the present study, but moose hunted in Estonia should be considered a potential T. gondii infection source to other hosts, including humans. Seropositivity indicates previous exposure, and because the seroprevalence was higher in adults than in calves and because moose are herbivores, the exposure was likely exposure to and ingestion of T. gondii oocysts. The results can thus be interpreted to indicate that the environment in Estonia was widely contaminated with T. gondii oocysts, in particular in the northern part of the country. Investigation of samples from a free-ranging herbivorous host that is hunted in large numbers appears useful in revealing environmental distribution patterns of T. gondii.

In this work the prevalence and serovar diversity of Salmonella in various food products including non-thermally processed food and ready-to-eat (RTE) food in Estonia in 2008–2012 are summarized. The findings demonstrate that the overall prevalence of Salmonella in these food categories was low. A total of 260 (0.54%) of 47,927 food samples were found to be positive for Salmonella, the overall prevalence in non-thermally processed food was 0.81% (256/31,576) and in RTE products only 0.02% (4/16,351). Salmonella was most often isolated from raw eggs and products thereof (2.17%, 5/230), followed by raw meat products (0.95%, 207/21,723), RTE mayonnaises (0.90%, 2/221) and raw meat (0.89%, 38/4252). In the raw meat category, Salmonella was most frequently isolated from turkey meat (6.96%, 11 positive samples out of 158), broiler chicken meat (4.00%, 7/175) and from layer hen meat (2.22%, 11/496). Salmonella was isolated in lesser extent from meat preparations (1.91%, 82/4292), minced meat and mechanically separated meat products (0.97%, 100/10,344) and from raw sausages (0.35%, 25/7087). Altogether 24 different serovars were identified among the 260 Salmonella positive samples. Salmonella Typhimurium was the most frequent serovar (26.90% of the positive samples) and it was isolated most commonly in raw food products. The next most frequent serovars were Salmonella Derby (17.50%), Salmonella Enteritidis (8.37%) and Salmonella Newport (7.57%). The only serovars isolated from the Salmonella positive RTE food samples were Salmonella Infantis (two isolates) and S. Enteritidis (two isolates).

Trichinella spp. are relevant zoonotic pathogens in Estonia. The aim of this nationwide cross-sectional study was to estimate the seroprevalence of Trichinella spp. in domestic pigs (Sus scrofa domestica) and hunted wild boars (Sus scrofa). Serum samples from 374 pigs, originating from 14 farms, and meat juice samples from 470 wild boars were tested for immunoglobulin G antibodies against Trichinella excretory/secretory antigens using a commercial enzyme-linked immunosorbent assay (ELISA). Antibodies against Trichinella were not detected in the domestic pigs, indicating effective parasite control strategies in the farms. By contrast, 42.1% of the wild boars tested positive, indicating substantial infection pressure in the sylvatic cycle. Further analysis of a subset of the wild boar samples, using another ELISA and Western blot, yielded a confirmed seroprevalence estimate of 17.4%. A substantial proportion of wild boars in Estonia had evidence of exposure to Trichinella spp. and may have carried infective larvae. Undercooked Estonian wild boar meat is a potential source of Trichinella spp. infections to humans and other hosts.

An antigen detection kit (Trichin-L), based on latex agglutination and developed by the Bio-Rad company was validated at five European laboratories. The validation parameters included specificity, sensitivity, robustness and reproducibility. Specificity was evaluated by testing parasite antigens from five non-Trichinella parasites in addition to the Trichinella genus. To evaluate sensitivity, 10 pork samples spiked with 1, 3, 6 or 15 Trichinella larvae were tested in each laboratory. To evaluate the robustness of the test, the solubilized antigens were maintained at room temperature and tested at different times. Reproducibility was assessed in each laboratory using 40, 100g minced pork samples, each spiked with Trichinella spiralis. The use of larval homogenates obtained from the Trichin-L kit as a template for parasite identification at the species level by a multiplex PCR, was also evaluated. The results showed a high specificity and sensitivity where solubilized antigens maintained their stability and reactivity for up to three days. Reproducibility was high, as similar results were obtained in the five laboratories. The larval homogenates obtained using the Trichin-L kit were successfully used in multiplex PCRs to identify Trichinella species.

The investigation of antimicrobial resistance of pathogens isolated from animals was carried out during 2005-2009 by scientists of Estonian University of Life Sciences and was financed by Estonian Ministry of Agriculture. The aim of the investigation was to get an overview of the resistance situation in Estonia and according to the results work out national guidelines for prudent use of antibiotics in veterinary medicine. The investigation includes clinical mastitis pathogens, E.coli and Salmonella isolated from clinically ill swine and bovine, Salmonella isolated from food products of animal origin and indicator bacteriae from clinically healthy animals. Antimicrobial resistance of several pathogens shows tendency to increase. Wide and irrational use of different antibiotic treatments could be the reason for that. It is necessary to continue the investigation in order to follow trends of the resistance and to work out the appropriate measures to minimise the development of antimicrobial resistance in veterinary field., EE; et; birgit.aasmae@emu.ee

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Background Many parasites (protozoa and helminths) can be transmitted through food and lead to infections with high morbidity, as well as disease outbreaks. Although the importance of foodborne parasites (FBP) is recognised by many sectors of the food industry, standardized analytical methods and validation procedures for testing food for FBP are lacking. Scope and approach:Current methods for detection of FBP, and their validation, are critically reviewed, focusing on priority FBP in Europe: the helminths Echinococcus multilocularis, Echinococcus granulosus, Taenia saginata, Trichinella spp., and Anisakidae, and the protozoa Toxoplasma gondii, Cryptosporidium spp., and Giardia duodenalis. Key findings and conclusions:Standard methods exist for detection of T. saginata in beef, and Trichinella spp. in meat (and are mandatory at meat inspection in Europe), Anisakidae in fish, and Cryptosporidium spp. and G. duodenalis in leafy green vegetables and berry fruits. For other FBP or foods, methods used in sample surveys have been described, but validation data are generally absent; limits of detection are not provided, ring trials have rarely been performed, and for most FBP quality control materials, proficiency schemes, and reference standards are lacking. The use of surrogate particles or organisms for method development or validation purposes needs to be carefully considered. Documented procedures for validation, such as ISO17468 and ISO16140-2:2016 that were established for bacteria, are mostly inappropriate for FBP. The development and application of standardized and validated detection methods would enhance understanding of the foodborne route of transmission, improve risk assessments, and help identify and verify critical control points.

Background The goal of this study was to estimate the distribution of udder pathogens and their antibiotic resistance in Estonia during the years 2007-2009. Methods The bacteriological findings reported in this study originate from quarter milk samples collected from cows on Estonian dairy farms that had clinical or subclinical mastitis. The samples were submitted by local veterinarians to the Estonian Veterinary and Food Laboratory during 2007-2009. Milk samples were examined by conventional bacteriology. In vitro antimicrobial susceptibility testing was performed with the disc diffusion test. Logistic regression with a random herd effect to control for clustering was used for statistical analysis. Results During the study period, 3058 clinical mastitis samples from 190 farms and 5146 subclinical mastitis samples from 274 farms were investigated. Positive results were found in 57% of the samples (4680 out of 8204), and the proportion did not differ according to year (p > 0.05). The proportion of bacteriologically negative samples was 22.3% and that of mixed growth was 20.6%. Streptococcus uberis (Str. uberis) was the bacterium isolated most frequently (18.4%) from cases of clinical mastitis, followed by Escherichia coli (E. coli) (15.9%) and Streptococcus agalactiae (Str. agalactiae) (11.9%). The bacteria that caused subclinical mastitis were mainly Staphylococcus aureus (S. aureus) (20%) and coagulase-negative staphylococci (CNS) (15.4%). The probability of isolating S. aureus from milk samples was significantly higher on farms that had fewer than 30 cows, when compared with farms that had more than 100 cows (p < 0.005). A significantly higher risk of Str. agalactiae infection was found on farms with more than 600 cows (p = 0.034) compared with smaller farms. The proportion of S. aureus and CNS isolates that were resistant to penicillin was 61.4% and 38.5%, respectively. Among the E. coli isolates, ampicillin, streptomycin and tetracycline resistance were observed in 24.3%, 15.6% and 13.5%, respectively. Conclusions This study showed that the main pathogens associated with clinical mastitis were Str. uberis and E. coli. Subclinical mastitis was caused mainly by S. aureus and CNS. The number of S. aureus and Str. agalactiae isolates depended on herd size. Antimicrobial resistance was highly prevalent, especially penicillin resistance in S. aureus and CNS.

Graduation Thesis in Veterinary Medicine Annually around 6000 moose (Alces alces) are hunted in Estonia for human consumption. Moose can host Toxoplasma gondii and their meat can be a source of infection for humans and other hosts. The main aims of this thesis were to estimate the seroprevalence of T. gondii in moose hunted in Estonia and to evaluate different risk factors (age, sex, and geographical location) for seropositivity. Altogether 1004 sampling packages, which included questionnaires, were sent to hunters, who collected 463 blood samples post-mortem from hunted moose. Samples arrived to the laboratory by mail and sera were separated, divided into aliquots and stored frozen until analyzes. A commercial direct agglutination test was used to screen the sera, diluted 1:40, for T. gondii spesific IgG antibodies. We detected serological evidence of exposure to T. gondii in moose in Estonia: 111 moose tested seropositive. The seroprevalence estimate was 23.97%. Female moose had a seroprevalence of 27.53% and male moose 19.63%. Adults had a seroprevalence of 28.80% and calves 12.59%. Seropositive moose were found from all 12 counties where the samples originated. Moose were commonly exposed to T. gondii and the infections appeared to be acquired. Igal aastal kütitakse Eestis inimtoiduks ligikaudu 6000 põtra (Alces alces). Püsisoojaste loomadena on põdrad üheks võimalikuks Toxoplasma gondii peremeheks ja reservuaariks. Lõputöö eesmärgiks oli määrata T. gondii seroloogiline levimus Eestis kütitud põtradel ning hinnata selle erinevaid riskifaktoreid (vanus, sugu ja piirkond). Koostati küsimustik mis lisati välja prindituna selleks uuringuks komplekteeritud proovivõtuvahendite pakendisse. Vabatahtlikkudele jahiseltsidele saadeti kokku 1004 proovivõtuvahendite pakendit. Selleks uuringuks kogusid vabatahtlikud jahimehed kütitud põtradelt surma järgselt 463 vereproovi. Proovid toodi või saadeti posti teel laborisse, kus seerum eraldati, jaotati tuubidesse ja säilitati külmikus kuni analüüsimiseni. Analüüsimeetodina kasutati otsest agglutinatsiooni testi, millega uuriti T. gondii spetsiifilisi IgG antikehasid 1:40 lahjendatud seerumist. Uuringu tulemused tõendavad T. gondii infektsioonide esinemist Eestis kütitud põtradel: 111 põtra olid positiivsed. Tuvastasime T. gondii 23,97 % seroloogilise levimuse Eestis kütitud põtradel. Emastel põtradel oli seroloogiline levimus 27,53 %, isastel 19,63 %. Täiskasvanud loomadel oli seroloogiline levimus 28,80 %, vasikatel 12,59 %. Kõigis 12 uuritud maakonnas leiti seropositiivseid põtru. Põdrad puutuvad kokku sageli T. gondii oosüstidega ja infektsioonid tunduvad olevat omandatud.