The first records of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae), a causative agent of human sparganosis, in Latvian wildlife

Diphyllobothriid tapeworms of the genus Spirometra are causative agents of sparganosis, food-borne zoonotic parasitic disease. They have been recorded in broad spectrum of hosts, including humans, in all continents except Antarctica. Spirometra tapeworms have been intensively studied in several Asian countries; however, they have been rather neglected in Europe. The aim of this study was to provide a pilot screening of Spirometra spp. in Latvia, where data on sparganosis are not available. Tapeworms morphologically identified as diphyllobothriid species were isolated from grey wolves Canis lupus and Eurasian lynxes Lynx lynx from Latvia during the hunting periods 2013–2019. The parasites were subjected to molecular genotyping using sequences of the partial large (LSU rDNA; 615 bp) and small (SSU rDNA; 720 bp) subunits of the nuclear ribosomal RNA gene and complete (1566 bp) cytochrome c oxidase subunit I gene of the mitochondrial DNA (cox1 mtDNA). Analyses of both ribosomal subunits of 13 tapeworms revealed no intraspecific variation within the respective rDNA subunits. On the other hand, sequence analysis of mitochondrial cox1 revealed intraspecific polymorphism displayed by 12 cox1 haplotypes. Comparison of the current data with sequences of the corresponding DNA regions deposited in the GenBank revealed 99.3–99.5% (LSU rDNA), 99.2% (SSU rDNA) and 99.6–100% (cox1 mtDNA) identity of studied tapeworms with Spirometra erinaceieuropaei, which provided the first confirmation of this diphyllobothriid tapeworm in Latvia. Since S. erinaceieuropaei is probably prevalent in Latvian wildlife and may also occur in other potential host species, further studies are needed in order to acquire complex data on its geographic distribution and transmission in the natural environment of Latvia, as well as on the spectrum of its intermediate, paratenic, and definitive hosts.


Introduction
Sparganosis is a food-borne zoonosis caused by larval stages (plerocercoids) of diphyllobothriid tapeworms of the genus Spirometra (Liu et al. 2015). They have been recorded in humans, domesticated and wild animals in all continents except Antarctica (Scholz et al. 2019). Although several species have been recognized in the genus, the taxonomy of Spirometra tapeworms is not fully resolved and requires further clarification (Kuchta et al. 2020). Currently, Spirometra erinaceieuropaei (Rudolphi 1819) is the species most often identified in natural environments in Europe (Kondzior et al. 2018;Kołodziej-Sobocińska et al. 2019;Scholz et al. 2019;Kuchta et al. 2020); however, recent data also reported the first confirmation of S. mansoni in amphibians in Romania (Kuchta et al. 2020).
While most human cases have been reported from Asia, e.g. China, South Korea, and Thailand (Wiwanitkit 2005;Liu et al. 2015;Hong et al. 2016;Wang et al. 2018), some reports have also been published from Europe, e.g. Italy, Poland, France, Czech Republic, and Germany (Pampiglione et al. 2003;Czyżewska et al. 2019). Humans can be infected either by drinking water contaminated with infected copepods, or by ingestion of plerocercoids present in raw or undercooked meat of second intermediate or paratenic hosts. Adult Spirometra tapeworms are also able to mature in the human intestine and can cause a rare disease called spirometrosis, which usually does not result in clinical symptoms (Le et al. 2017). However, the most serious and lifethreatening form is proliferative sparganosis (Kikuchi and Maruyama 2020). In Asia, the majority of human cases were most probably connected with the consumption of amphibians and reptiles (Wiwanitkit 2005;Liu et al. 2015), which are not considered to be a source of infection in Europe due to different eating habits. However, wild boar Sus scrofa meat cannot be ruled out as a source of infection in Europe (Kołodziej-Sobocińska et al. 2016;Czyżewska et al. 2019), since it has been recorded as a paratenic host of S. erinaceieuropaei in Serbia, Belarus, Ukraine, and recently in Poland (Rukavina et al. 1957;Shimalov 2009;Nevolko and Litvinenko 2014;Kołodziej-Sobocińska et al. 2016).
Knowledge about the geographic distribution of S. erinaceieuropaei in Europe has been scarce. Since 2000, the tapeworm has been recorded in various wildlife species in Belarus (Shimalov and Shimalov 2000, 2002a, b, 2003Anisimova 2004;Shimalov 2009), Serbia (Nevolko and Litvinenko 2014), Ukraine (Kornyushin et al. 2011), and most recently in Poland, where S. erinaceieuropaei was confirmed by molecular analyses for the first time in Europe (Kołodziej-Sobocińska et al. 2014Kondzior et al. 2018). It is highly probable that S. erinaceieuropaei is also present in other European countries but has been overlooked due to a number of factors. Firstly, some of the hosts are protected species, which can be subjected to detailed parasitological examination only under strict conditions and special permissions. Secondly, the specific location of plerocercoid larvae in subcutaneous tissues and/or muscles of intermediate or paratenic hosts means that the presence of this parasite may be overlooked during standard dissection and parasitological examination, if specific methodology focusing on detection of spargana is not utilized. Finally, taxonomic identification of larval Spirometra stages, including S. erinaceieuropaei, is complicated owing to the absence of reliable species-specific morphological markers (Kuchta et al. 2015(Kuchta et al. , 2020. Although identification of adult tapeworms based on morphological features is possible (Scioscia et al. 2014), DNA-based genotyping is often recommended in order to confirm the results (Badri et al. 2017;Kołodziej-Sobocińska et al. 2018;Kuchta et al. 2020). The aim of the present work was to acquire additional data on the occurrence of S. erinaceieuropaei in Europe, with particular focus on Latvia, where a pilot screening of this zoonotic tapeworm in definitive hosts was performed by molecular genotyping.

Materials and methods
The material in the current work originated from the helminthological collection at the Latvian State Forest Research Institute "Silava" in Salaspils, Latvia. In the 2013-2019 hunting seasons, grey wolves (July-March) and Eurasian lynxes (December-March) were hunted within the national legal framework according to the quota allocated by the Latvian Hunting Regulations. A total of 35 hosts, 11 Eurasian lynxes and 24 grey wolves of different ages (juveniles and adults) and sexes (males and females) were examined (Table 1). Figure 1 provides scheme of geographic location of examined Eurasian lynxes and grey wolves in Latvia; all hosts except for one originated from the eastern part of the country.
After complete helminthological dissection, all helminths were taxonomically identified using morphological criteria, preserved in 70% ethanol and deposited in the helminthological collection of the above mentioned institution. For the current work, 44 adult tapeworms were selected from the helminthological collection and subjected to molecular genotyping. The tapeworms were isolated from intestines of Eurasian lynxes and grey wolves immediately after dissection, and they were identified as "diphyllobothriid tapeworms." Taxonomical identification of diphyllobothriideans is difficult and requires properly fixed and processed material for an accurate morphological description which should be supplemented with molecular genotyping. Since the condition of tapeworms isolated from Eurasian lynxes and grey wolves did not allow reliable morphological description, the material was fixed in ethanol for further identification by molecular genotyping.
Genomic DNA was isolated from 20 mg of tissue of 44 diphyllobothriidean tapeworms using the QIAamp® DNA Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions, diluted in deionized water and stored at − 20°C. Molecular genotyping was performed by Polymerase Chain Reaction (PCR) amplification of a partial fragment of  the nuclear large subunit of the ribosomal RNA gene (LSU rDNA) and a partial fragment of the nuclear small subunit of the ribosomal RNA gene (SSU rDNA). LSU rDNA was amplified using the LSU-5 (5′-TAGGTCGACCCGCT G A A Y T T A A G C A -3 ′ ) a n d 1 5 0 0 R ( 5 ′ -G C T A TCCTGAGGGAAACTTCG-3′) primers originally described by Olson et al. (2003). Universal primers WormA (5′-GCGA ATGGCTCATTAAATCAG-3′) and WormB (5′-CTTG TTACGACTTTTACTTCC-3′) (Littlewood and Olson 2001) were used for amplification of SSU rDNA. In addition, the complete cytochrome c oxidase subunit I of the mitochondrial DNA (cox1 mtDNA) was amplified using the Diphyllo-Cox1-F (5′-TAGACTAAGTGTTTTCAAAACACTA-3′) and Diphyllo-Cox1-R (5′-ATAGCATGATGCAAAAGG-3′) primers described by Yanagida et al. (2010). The PCR amplification conditions were 5 min at 94°C as an initial step, then 30 cycles of 1 min at 94°C, 1 min at 55°C, 2 min at 72°C, and finally 10 min at 72°C. The PCR products were visualized on a 1.5% agarose gel and purified using exonuclease I and shrimp alkaline phosphatase (Werle et al. 1994). Sequencing was performed by the automatic genetic analyser Applied Biosystems 3130xl (Applied Biosystems, Foster City, California, USA) and the BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems). Sequencing of LSU rDNA, SSU rDNA, and cox1 mtDNA was performed from both directions using following PCR primers and internal primers. The 5′end of LSU rDNA amplification product was sequenced with LSU-5 primer (primer used for PCR amplification) and two internal primers, 300F (5′-CAAGTACCGTGAGGGAAAGTTG-3′) and 400R (5′-GCAGCTTGACTACACCCG-3′) (Olson et al. 2003). Similarly, the 5′end of SSU rDNA was sequenced with PCR primer WormA and two internal primers 600F (5′-GGTG CCAGCAGCCGCG-3′) and 600R (5′-ACCGCGGC TGCTGGCACC-3′) (Littlewood and Olson 2001). The complete sequence of cox1 mtDNA was obtained with two PCR amplification primers Diphyllo-Cox1-F and Diphyllo-Cox1-R (Yanagida et al. 2010) and two internal primers, DCox1-R2 (5′-AAACACCGGCTCACGTAAAG-3′) and Cox1-R3 (5′-CGCAAATGCCGAATAAAGAG-3′ ) (Koł odziej-Sobocińska et al. 2019). Contiguous sequences were assembled and inspected for errors using the Geneious software Fig. 1 Map of collection sites of diphyllobothriid tapeworms isolated from Eurasian lynxes (green circle) and grey wolves (blue circle) in Latvia with specification of hosts in which Spirometra erinaceieuropaei was molecularly confirmed (full circles). Details of individual hosts are provided in Table 1; the numbering of hosts follows the numbers indicated in the column "number of host." The map was created using Photoshop CS4, version 11.0 (version 10.0.5,Biomatters,Auckland,New Zealand). Obtained sequences were compared with sequences deposited in GenBank using nucleotide BLAST search.

Results and discussion
Of the 44 diphyllobothriid tapeworms analysed in the current work, PCR products of partial LSU rDNA, partial SSU rDNA and complete cox1 were successfully amplified in 13 specimens. PCR amplifications of both ribosomal subunits and mitochondrial cox1 were repeatedly carried out under different reaction conditions; however, 31 samples did not yield any amplicons. A possible explanation may be that hunted animals are often provided to the parasitological laboratories at an advanced level of decomposition of the content of their gastro-intestinal tracts. A prolonged period between animal necropsy and parasite fixation may consequently result in a lower level of effectiveness of molecular analyses due to DNA degradation.
Sequence analyses of 615 bp LSU rDNA and 720 bp SSU rDNA of 13 tapeworms revealed identical sequence structure within the respective ribosomal subunit with no intraspecific variation. Comparison of the obtained LSU rDNA sequences with sequences deposited in GenBank revealed 99.5% p a i r w i s e s e q u e n c e i d e n t i t y w i t h L S U r D N A o f S. erinaceieuropaei (GenBank accession number KY552835) isolated from dog Canis familiaris from Australia and 99.3% with S. erinaceieuropaei (KY552836) from yellow-spotted keelback snake Xenochrophis flavipunctatus from Vietnam (Waeschenbach et al. 2017). Similarly, comparison of the 720 bp fragment of partial SSU rDNA with sequences deposited in GenBank revealed 99.2% i d e n t i t y w i t h r e s p e c t i v e S S U r D N A r e g i o n o f S. erinaceieuropaei (KY552801) isolated from dog from Australia (Waeschenbach et al. 2017).
Sequences of LSU rDNA, SSU rDNA and cox1 of all 13 individuals were deposited in GenBank, EMBL and DDBJ databases under accession numbers summarized in Table 1. To conclude, all 13 diphyllobothriidean tapeworms obtained from three Eurasian lynxes and ten grey wolves were molecularly identified as S. erinaceieuropaei, providing the first record of this species in Latvia.
Carnivores are important definitive hosts of a wide range of protozoan and metazoan parasites and can play a significant role in the maintenance of different zoonoses (Craig and Craig 2005;Han et al. 2016). Eurasian lynx and grey wolf populations are distributed throughout the entire territory of Latvia. Both populations have increased since the beginning of the 2000s and numbers are estimated within 400-600 for Eurasian lynx and 300-500 for grey wolf (Ozoliņš et al. 2017a, b).
Since taxonomic identification of diphyllobothriidean tapeworms based only on morphology is difficult (Kuchta et al. 2015(Kuchta et al. , 2020 and the previously published occurrence of D. latus in a grey wolf in Latvia (Bagrade et al. 2009) was not confirmed by molecular methods, further studies focused on detailed morphological descriptions supplemented with molecular genotyping of diphyllobothriids are highly recommended for their more accurate taxonomic identification in Latvian wildlife. Dibothriocephalus latus is evidently circulating in the natural environment in Latvia because its larval stages, plerocercoids, were detected during ichthyoparasitological examinations in several fish species (Kirjušina and Vismanis 2007). In addition, human cases of diphyllobothriosis (up to 10 cases over 2010-2014) have been recorded in patients from Latvia by the Centre for Disease Prevention and Control (https://spkc. gov.lv). However, it is evident that more data on theoccurrence, prevalence, geographic distribution, and a spectrum of intermediate and definitive hosts of D. latus in Latvia are required for the up-to-date knowledge on circulation of this most frequent causative agent of diphyllobothriosis in Europe.
It is not possible to conclude if our findings represent a recent introduction of S. erinaceieuropaei to Latvia, or if this tapeworm has been present in the Latvian environment for a prolonged period. Since sparganosis has been confirmed in neighbouring Belarus (Shimalov and Shimalov 2000, 2002a, b, 2003Shimalov 2009) and geographically proximal Poland (Kołodziej-Sobocińska et al. 2014 and Finland (Lavikainen et al. 2013;Kuchta et al. 2020), it seems highly probable that S. erinaceieuropaei had been present in Latvia for a longer time.
The current study provided initial data on spirometrosis in Latvia, but reveals several perspectives for future studies. Investigating the role of particular intermediate and paratenic hosts of S. erinaceieuropaei is highly important for understanding its life cycle and dispersal in the natural environment in Latvia. In addition, the potential zoonotic risk of human sparganosis connected with wild boar meat consumption or drinking contaminated water should be examined from a medical health perspective.
Authors' contributions All authors contributed to the study conception and design. Guna Bagrade provided helminthological dissection of hosts, collection of parasitic material and its initial morphological identification. Molecular analyses were performed by Ivica Králová-Hromadová, Eva Bazsalovicsová, and Alžbeta Radačovská. Marta Kołodziej-Sobocińska, Ivica Králová-Hromadová, and Eva Bazsalovicsová wrote the first draft of manuscript; all authors read and approved the final manuscript. Guna Bagrade and Eva Bazsalovicsová prepared Table 1

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.