Introduction
Cystic echinococcosis (CE) is a severe parasitic disease affecting both humans and animals (Jenkins et al., Reference Jenkins, Simon, Bachand and Stephen2015; Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson and Jenkins2017; Kern et al., Reference Kern, Menezes da Silva, Akhan, Müllhaupt, Vizcaychipi, Budke and Vuitton2017). CE is caused by a complex of tapeworm species collectively referred to as Echinococcus granulosus sensu lato (s.l.). These include the species E. granulosus sensu stricto (s.s.; genotypes G1 and G3; Kinkar et al., Reference Kinkar, Laurimäe, Sharbatkhori, Mirhendi, Kia, Ponce-Gordo, Andresiuk, Simsek, Lavikainen, Irshadullah, Umhang, Oudni-M'rad, Acosta-Jamett, Rehbein and Saarma2017), E. equinus (G4), E. felidis, E. ortleppi (G5) and the E. canadensis cluster (G6–G8, G10) for which the number and name(s) of species are under dispute (e.g. Moks et al., Reference Moks, Jõgisalu, Valdmann and Saarma2008; Thompson, Reference Thompson2008; Saarma et al., Reference Saarma, Jõgisalu, Moks, Varcasia, Lavikainen, Oksanen, Simsek, Andresiuk, Denegri, González, Ferrer, Gárate, Rinaldi and Maravilla2009; Knapp et al., Reference Knapp, Nakao, Yanagida, Okamoto, Saarma, Lavikainen and Ito2011; Nakao et al., Reference Nakao, Lavikainen and Hoberg2015; Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017; Laurimäe et al., Reference Laurimäe, Kinkar, Moks, Romig, Omer, Casulli, Umhang, Bagrade, Irshadullah, Sharbatkhori, Mirhendi, Ponce-Gordo, Soriano, Varcasia, Rostami-Nejad, Andresiuk and Saarma2018a; Vuitton et al., Reference Vuitton, McManus, Rogan, Romig, Gottstein, Naidich, Tuxun, Wen and Menezes da Silva2020). The life cycle of these tapeworms involves canids as definitive hosts, harbouring the adult worm, and various herbi- and omnivores that act as intermediate hosts. In the latter, the larval stage develops predominantly in the liver or lungs in the form of fluid-filled cysts (Thompson, Reference Thompson2017). Several E. granulosus s.l. species are transmitted mostly between domesticated animals, with dogs acting as the main definitive and livestock ungulates as intermediate hosts, whereas others infect largely wildlife species, such as wolves and moose (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017). Humans are accidental aberrant hosts and do not generally contribute to the perpetuation of the life cycle of the parasite (Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson and Jenkins2017). A systematic review, based on European E. granulosus s.l. data published in 2000–2021, identified 599 genetically confirmed human echinococcal cysts, majority of which belonged to E. granulosus s.s. (76.8%), followed by genotypes G6/7 and G10 of the E. canadensis cluster (21.7%) (Casulli et al., Reference Casulli, Massolo, Saarma, Umhang, Santolamazza and Santoro2022). However, the number of human CE cases is likely an underestimation. A recent Europe-wide analysis has revealed that the disease is under-reported (Casulli et al., Reference Casulli, Abela-Ridder, Petrone, Fabiani, Bobić, Carmena, Šoba, Zerem, Gargaté, Kuzmanovska, Calomfirescu, Rainova, Sotiraki, Lungu, Dezsényi, Ortiz, Karamon, Maksimov, Oksanen, Millon, Sviben, Shkjezi, Gjoni, Akshija, Saarma, Torgerson, Snabel, Antolova, Muhovic, Besim, Chereau, García, Chappuis, Gloor, Stoeckle, Müllhaupt, Manno, Santoro and Santolamazza2023). The most abundant species – E. granulosus s.s. – is predominantly transmitted among domesticated animals (e.g. sheep and dogs) (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017).
A brief historical review of CE in Iceland
CE ('sullaveiki’ in Icelandic) was likely introduced to Iceland during the settlement period in the 9th century and was considered endemic already in the 12th–13th centuries (mentioned in Icelandic sagas written in this period) (Skírnisson et al., Reference Skírnisson, Richter, Eydal, Akkuffo, Linder, Ljungström and Whalgren2003 and references therein; Kristjansdottir and Collins, Reference Kristjansdottir and Collins2011). In addition to these documents, there is also archaeological evidence of CE in humans in Iceland in the past. A cyst as large as 15 cm in diameter was found in a female skeleton, aged about 45 years, from a cemetery dated to the 18th century at Viðey in Southwest Iceland (Gestsdottir, Reference Gestsdottir2004). Interestingly, the most ‘prolific’ site has been Skriðuklaustur, a medieval Augustinian monastery in eastern Iceland that also functioned as a hospital in 1493–1554. In this region, Zoega (Reference Zoega2007) reported of a skeleton diagnosed with ‘hydatism’. Later on, 8 additional cases were recovered by Kristjansdottir and Collins (Reference Kristjansdottir and Collins2011), including a skeleton with large cysts of 17–20 cm in diameter. Echinococcus cysts in archaeological material have also been reported in other parts of Europe, such as Denmark (Weiss and Møller-Christensen, Reference Weiss and Møller-Christensen1971).
Reports dating back to the 19th century indicate that CE had devastating health consequences in Iceland during this time (Fig. S1), and according to a local medical doctor, nearly every fifth adult died of CE or its complications (Hjaltelin, Reference Hjaltelin1869). In the 19th century and in the first half of the 20th century, Iceland had the highest prevalence of human CE ever recorded anywhere (Dungal, Reference Dungal1957). For example, of 2272 autopsies carried out in Reykjavik (during 1932–1950), CE prevalence was documented to range between 15 and 22% among people born between 1841 and 1880. The infection rate in humans was likely high due to effective transmission of the parasite between sheep and dogs. Slaughtering occurred in farms and offal were often fed to dogs. In the mid-19th century, approximately 70 000 humans inhabited Iceland, alongside with 600 000–700 000 sheep and 15 000–20 000 dogs. Systematic data on the prevalence of CE in animals in Iceland are scarce and the information is scattered. Jón Finsen in his medical reports from 1858 considered ‘hydatids’ (i.e. Echinococcus cysts) so common in ‘kvíaám’ (ewes being milked without lambs) and cattle, that animals free of infection were an exception. Thus, almost all sheep and cattle were infected when slaughtered (Pálsson et al., Reference Pálsson, Sigurðsson and Henriksen1953). Also, Krabbe (Reference Krabbe1865) reported that Echinococcus was very common in sheep and cattle, and was found also in a pig. In 1863, 28% of dogs were reported to be infected (Krabbe, Reference Krabbe1865).
Eradication efforts of CE in Iceland
Measures to control CE were already formulated in the 1860s to reduce parasite transmission and included preventing dogs from accessing raw offal, restricting home slaughter of sheep, improved meat inspection and appropriate offal disposal and educating people about hygiene and the life cycle of the parasite. These measures were first applied in 1863, whereas in 1869 the first nationwide legislation to control CE was enforced to regulate the number of dogs and placing a tax on all dogs not required for work. The law also stipulated the instructions for appropriate destruction of cysts and infected offal (Beard, Reference Beard1973). For deworming, the kamala fruit (Mallotus philippinensis) extract was used in the beginning, although this was soon replaced with a more effective areca nut (Areca catechu) extract containing arecoline as a key ingredient. From 1924, a synthetic arecoline hydrobromide was used (reviewed in Craig et al., Reference Craig, Hegglin, Lightowlers, Torgerson and Wang2017). This drug caused diarrhoea and did not eliminate all cestodes from the organism. Eradication efforts of CE in Iceland were further strengthened by changes in sheep husbandry, favouring exporting and marketing wethers, decreasing thereby the rates of home slaughter. As a result, human CE cases were reduced significantly.
Measures to combat CE taken since 1860s in Iceland proved to be highly successful, resulting in the disease being virtually eliminated by 1920 (prevalence in the human population between 1881 and 1920 ranging from 0 to 3%; Dungal, Reference Dungal1957) and only 8 cases being reported in the 20th century (Skírnisson et al., Reference Skírnisson, Richter, Eydal, Akkuffo, Linder, Ljungström and Whalgren2003; Skírnisson, Reference Skírnisson2017). Four of these cases were diagnosed in 1984–1988 (Arinbjarnar, Reference Arinbjarnar1989; Fig. S2). These patients had likely acquired the infection in the first half or middle of the century. The last mortality case was in 1960, when a 23-year-old woman died of complications during surgery (Jónsson, Reference Jónsson1962). The same trend occurred in livestock animals. In 1924 there was a survey in slaughter houses, where 17 424 sheep were examined and 2164 had hydatids (12.4%), 763 in the liver, 15 in the lungs and 75 both in the liver and lungs. The prevalence decreased in the following decades. By the middle of the 20th century, CE in sheep and cattle had more or less disappeared. The last infected cattle was recorded in 1961 and sheep in 1979 (Pálsson et al., Reference Pálsson, Sigurðsson and Henriksen1953; Pálsson, Reference Pálsson1984; Skírnisson, Reference Skírnisson2017). The cestode survived regionally in dogs in the eastern part of the country until 1970s (Pálsson, Reference Pálsson1984; Skírnisson, Reference Skírnisson2017). It is worth noting that no anthelmintic effective against cestodes was used in Iceland until praziquantel was adopted in the 1970s. Although several factors contributed to the gradual eradication of CE, education on the disease and prevention, as well as decreased onsite slaughter of adult sheep coupled with restricting access of dogs to raw offal, played a major role (Beard, Reference Beard1973). Thus, the control programme to eliminate CE in Iceland was highly successful and has served as an example for other countries faced with similar challenges. In the middle of the 20th century, CE control programmes were initiated in other islands such as New Zealand, Tasmania, Cyprus and Falkland Islands (Malvines), eventually resulting in the eradication of the disease (Craig et al., Reference Craig, Hegglin, Lightowlers, Torgerson and Wang2017).
To date, no Echinococcus cyst material from Iceland has been molecularly characterized. To understand which species/genotypes of E. granulosus s.l. were present in Iceland before the eradication of CE, we aimed here to sequence the coding regions of the mitochondrial genome of Echinococcus isolates obtained from one of the last known infected sheep from 1977. We also explored their phylogenetic relations with other samples from Europe for which mitochondrial genome sequences are publicly available.
Material and methods
Biological material
Three Echinococcus cysts, 2 from the liver and 1 from the lungs, were collected in 1977 from an adult female sheep (ID number H3479/77; Óseyri, Stöðvarfjörður, Iceland; Fig. S3). The sheep was the second to last animal found to be infected in Iceland. The affected organs were collected by veterinarian Hákon Hanson during a routine inspection in the slaughterhouse of Breiðdalsvík and sent to the Keldur Institute where a local expert Halldór Vigfússon identified the isolates as ‘Sullaveiki’ (i.e. E. granulosus). After microscopical examination, he noted the following: ‘Huge numbers of protoscolices present with typical hooks, the hooks have the typical shape of those of Echinococcus’ (Fig. S4). Halldór Vigfússon was an experienced scientist who had worked on the identification of Echinococcus isolates for decades. In addition to the 3 cysts, more were found in the lungs and liver, both on the surface and deeper inside the organs (most appearing greyish), but were not collected. Calcification was not noticed in any of the cysts. After the examination, the collected samples were stored frozen (−20°C). In 2022, scrapings were taken from the inside of the 3 cysts and placed in 99% ethanol. It is worth noting that the samples had thawed up at least once prior to this.
Sample preparation, PCR and sequencing
Genomic DNA was extracted from parasite material. The samples were digested in ATL buffer with protease K, and the DNA was extracted using the Qiamp96 DNA Qiacube HT Kit (Qiagen, Germany) following manufacturer's instructions. Before polymerase chain reaction (PCR) amplification, the quality of the DNA was verified by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA). Three DNA samples, 1 from each cyst, were PCR amplified. PCR reactions were set up and amplification was performed as described in Laurimäe et al. (Reference Laurimäe, Kinkar, Romig, Omer, Casulli, Umhang, Gasser, Jabbar, Sharbatkhori, Mirhendi, Ponce-Gordo, Lazzarini, Soriano, Varcasia, Rostami Nejad, Andresiuk, Maravilla, Gonzalez, Dybicz, Gawor, Šarkunas, Šnabel, Kuzmina and Saarma2018b), involving mitogenome amplification with 13 primer pairs and yielding overlapping DNA fragments. PCR reaction was performed in a volume of 20 μL, with 1× BD Advantage 2 PCR buffer (BD Biosciences, Franklin Lakes, NJ, USA), 0.2 μM deoxyribonucleoside triphosphate (Thermo Fisher Scientific), 0.25 μM of each primer, 1 U of Advantage 2 Polymerase mix (BD Biosciences) and 10–100 ng of template DNA. PCR amplification was performed in a touchdown regime: 95°C for 1 min, followed by 10 cycles of 95°C for 20 s, 55°C for 45 s (annealing temperature reduced in each cycle by −0.5°C) and 68°C for 2 min; followed by 25 cycles of 95°C for 20 s, 50°C for 45 s, 68°C for 2 min; and finishing with 68°C for 3 min. Nine microlitres of the PCR products were examined on a 1.0% agarose gel and 10 μL were purified with 1 U shrimp alkaline phosphatase/1 U exonuclease I mix (both from Thermo Fisher Scientific) by incubating at 37°C for 30 min and then inactivating the enzymes at 80°C for 15 min. Sequencing was performed at the Core Facility at the Institute of Genomics (University of Tartu, Estonia), using the same set of primers as for the initial PCR. Both forward and reverse strands were sequenced.
Sequence assembly, quality control and alignment
Consensus sequences were assembled in Codon Code Aligner v.10.0.2. Chromatograms were checked by eye and mistakes were corrected. Sequence alignment was performed by using Clustal W (Thompson et al., Reference Thompson, Higgins and Gibson1994), integrated into BioEdit v.7.2.5 (Hall, Reference Hall1999).
Phylogenetic analysis
Mitochondrial genome sequences of other E. granulosus s.s. G1 isolates from Europe were retrieved from GenBank (Table 1) and aligned with the sequence of this study. To evaluate the phylogenetic position of the Icelandic G1 haplotype among the sequences retrieved from GenBank, a median-joining network was constructed, using Network v.10.1.00 (Fluxus Technology Ltd., Colchester, UK) software, with both indels and point mutations considered.
Table 1. A list of haplotypes and the GenBank accession numbers of the corresponding sequences. Abbreviations: ISL – Iceland, SPA – Spain, ITA – Italy, ROM – Romania, MOL – Moldova, ALB – Albania, FRA – France, GRE – Greece.
* Sequenced in this study. The other sequences are from Kinkar et al. (Reference Kinkar, Laurimäe, Acosta-Jamett, Andresiuk, Balkaya, Casulli, Gasser, van der Giessen, González, Haag, Zait, Irshadullah, Jabbar, Jenkins, Kia, Manfredi, Mirhendi, M'rad, Rostami-Nejad, Oudni-M'rad, Pierangeli, Ponce-Gordo, Rehbein, Sharbatkhori, Simsek, Soriano, Sprong, Šnábel, Umhang, Varcasia and Saarma2018b)
Results
Genotypic identity
All 3 DNA samples analysed from different cysts were identified as E. granulosus s.s. genotype G1 and were identical in their sequence. However, the DNA samples were not of equal quality, and a single isolate (from lung) that produced the best sequence quality was selected for further analysis. This sequence was of 8141 bp in length, and included the complete cox1, rrnL, rrnS, cytb, nad4L, atp6, nad2, nad1 and nad3 gene sequences, as well as partial nad5, cox2 and nad4 gene sequences. The gene sequences were concatenated and deposited to the GenBank database (accession number OQ269593).
Phylogeographic relations of the Icelandic isolate with other G1 isolates in Europe
The median-joining phylogenetic analysis, based on an alignment of 31 sequences (8141 bp), produced a star-like network, where the Icelandic G1 haplotype was separated from the central putative haplotype (a hypothetical common ancestor of all analysed isolates) by 2 mutations and did not specifically belong to any haplogroup. None of the haplotypes is highly diverged, the closest to the central haplotype is 2 mutational steps apart (the one from Iceland and an isolate from Spain (SPA11). The other haplotypes are 3–11 mutational steps apart from the putative common ancestor. In the network, several groups can be identified, comprising of isolates from: (1) Italy, (2) Italy and Spain, (3) Spain; (4) Romania and Moldova, (5) Albania, Romania and Moldova.
Discussion
It is likely that the genotype identified in this study (G1) was one of the representative genotypes circulating among humans and livestock in Iceland prior to the eradication of CE, and possibly contributed significantly to the high rates of infection in humans. Although other genotypes may have been present in Iceland that are also known to infect humans, their impact was likely lower, as historic records in Iceland indicate that CE was mainly recorded from sheep, and E. granulosus s.s. (i.e. the former ‘sheep strain’; G1) is known to cause the majority of human CE infections in Europe (Casulli et al., Reference Casulli, Abela-Ridder, Petrone, Fabiani, Bobić, Carmena, Šoba, Zerem, Gargaté, Kuzmanovska, Calomfirescu, Rainova, Sotiraki, Lungu, Dezsényi, Ortiz, Karamon, Maksimov, Oksanen, Millon, Sviben, Shkjezi, Gjoni, Akshija, Saarma, Torgerson, Snabel, Antolova, Muhovic, Besim, Chereau, García, Chappuis, Gloor, Stoeckle, Müllhaupt, Manno, Santoro and Santolamazza2023), as well as globally (Alvarez Rojas et al., Reference Alvarez Rojas, Romig and Lightowlers2014). As sporadic CE infections were also recorded from cattle and pigs (Pálsson, Reference Pálsson1984), we cannot exclude the presence of E. ortleppi (G5) and/or E. granulosus s.l. genotype G7 in Iceland, though E. granulosus s.s. is known to infect these hosts as well (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017). Although other potential E. granulosus s.l. hosts – reindeer and horse – were also present before eradication, CE has never been reported from these animals in Iceland (Skírnisson, Reference Skírnisson2017).
The current study highlights that mitochondrial DNA from Echinococcus cyst material that has been stored for several decades at −20°C can be successfully sequenced, despite at least 1 freeze/thaw cycle. Mitochondrial DNA integrity appeared to be relatively high, as the primer pairs used in the current study were designed to amplify approximately 1000–1500 bp. This effort yielded a total sequence length of 8141 bp, which is a relatively large portion of the complete mitochondrial genome of G1 (17 675 bp; Kinkar et al., Reference Kinkar, Korhonen, Cai, Gauci, Lightowlers, Saarma, Jenkins, Li, Li, Young and Gasser2019). There are also other reports of DNA analysis from long-term preserved samples of Echinococcus. For example, DNA sequencing was successfully performed (and viability demonstrated) for metacestodes of Echinococcus multilocularis, cryopreserved for 35 years (Laurimäe et al., Reference Laurimäe, Kronenberg, Alvarez Rojas, Ramp, Eckert and Deplazes2020).
Although one of the main goals here was to identify the species/genotype of the Icelandic E. granulosus s.l. isolate, for which analysis of short sequences is sufficient (e.g. the nad5 and nad2 gene fragments; Kinkar et al., Reference Kinkar, Laurimäe, Acosta-Jamett, Andresiuk, Balkaya, Casulli, Gasser, González, Haag, Zait, Irshadullah, Jabbar, Jenkins, Manfredi, Mirhendi, M'rad, Rostami-Nejad, Oudni-M'rad, Pierangeli, Ponce-Gordo, Rehbein, Sharbatkhori, Kia, Simsek, Soriano, Sprong, Šnábel, Umhang, Varcasia and Saarma2018a; Laurimäe et al., Reference Laurimäe, Kinkar, Romig, Umhang, Casulli, Omer, Sharbatkhori, Mirhendi, Ponce-Gordo, Lazzarini, Soriano, Varcasia, Rostami-Nejad, Andresiuk, Maravilla, Gonzalez, Dybicz, Gawor, Šarkunas, Šnabel, Kuzmina, Kia and Saarma2019), we also aimed to explore its genetic relations to other G1 isolates from Europe for which sequences are available. As for this purpose, sequencing a significantly larger portion of mtDNA is recommended (e.g. Kinkar et al., Reference Kinkar, Laurimäe, Simsek, Balkaya, Casulli, Manfredi, Ponce-Gordo, Varcasia, Lavikainen, Gonzále, Rehbein, Giessen, Sprong and Saarma2016, Reference Kinkar, Laurimäe, Acosta-Jamett, Andresiuk, Balkaya, Casulli, Gasser, van der Giessen, González, Haag, Zait, Irshadullah, Jabbar, Jenkins, Kia, Manfredi, Mirhendi, M'rad, Rostami-Nejad, Oudni-M'rad, Pierangeli, Ponce-Gordo, Rehbein, Sharbatkhori, Simsek, Soriano, Sprong, Šnábel, Umhang, Varcasia and Saarma2018b, Reference Kinkar, Laurimäe, Balkaya, Casulli, Zait, Irshadullah, Sharbatkhori, Mirhendi, Rostami-Nejad, Ponce-Gordo, Rehbein, Kia, Simsek, Šnábel, Umhang, Varcasia and Saarma2018c; Laurimäe et al., Reference Laurimäe, Kinkar, Romig, Omer, Casulli, Umhang, Gasser, Jabbar, Sharbatkhori, Mirhendi, Ponce-Gordo, Lazzarini, Soriano, Varcasia, Rostami Nejad, Andresiuk, Maravilla, Gonzalez, Dybicz, Gawor, Šarkunas, Šnabel, Kuzmina and Saarma2018b; Ohiolei et al., Reference Ohiolei, Li, Yan, Fu and Jia2020); we aimed here to sequence 11 682 bp of mtDNA as performed for G1 and G3 isolates (Kinkar et al., Reference Kinkar, Laurimäe, Acosta-Jamett, Andresiuk, Balkaya, Casulli, Gasser, van der Giessen, González, Haag, Zait, Irshadullah, Jabbar, Jenkins, Kia, Manfredi, Mirhendi, M'rad, Rostami-Nejad, Oudni-M'rad, Pierangeli, Ponce-Gordo, Rehbein, Sharbatkhori, Simsek, Soriano, Sprong, Šnábel, Umhang, Varcasia and Saarma2018b, Reference Kinkar, Laurimäe, Balkaya, Casulli, Zait, Irshadullah, Sharbatkhori, Mirhendi, Rostami-Nejad, Ponce-Gordo, Rehbein, Kia, Simsek, Šnábel, Umhang, Varcasia and Saarma2018c). However, the isolate from Iceland did yield somewhat shorter high-quality sequence (8141 bp).
The Icelandic sample sequenced in the present study was genetically relatively similar to the other more ‘contemporary’ G1 samples (Fig. 1). It is likely that the ancestral genetic variant of G1 was introduced together with one of the past populations of sheep to Europe. Previously, Chessa et al. (Reference Chessa, Pereira, Arnaud, Amorim, Goyache, Mainland, Kao, Pemberton, Beraldi, Stear, Alberti, Pittau, Iannuzzi, Banabazi, Kazwala, Zhang, Arranz, Ali, Wang, Uzun, Dione, Olsaker, Holm, Saarma, Ahmad, Marzanov, Eythorsdottir, Holland, Ajmone-Marsan, Bruford, Kantanen, Spencer and Palmarini2009) have demonstrated, using genome integrated retroviral sequences (retrotypes), that sheep were dispersed over Europe by 2 different migration waves. During the first wave, sheep with more primitive characteristics (e.g. the Mouflon, Orkney, Soay and the Nordic short-tailed sheep) were confined to the periphery of northern Europe. The second wave involved sheep with improved production traits and these animals have shaped the majority of present-day breeds. Sheep from Iceland are part of the Nordic short-tailed sheep (Aðalsteinsson, Reference Aðalsteinsson1981), displaying retrotypes similar to those of Faroe Island and Fennoscandian populations (Chessa et al., Reference Chessa, Pereira, Arnaud, Amorim, Goyache, Mainland, Kao, Pemberton, Beraldi, Stear, Alberti, Pittau, Iannuzzi, Banabazi, Kazwala, Zhang, Arranz, Ali, Wang, Uzun, Dione, Olsaker, Holm, Saarma, Ahmad, Marzanov, Eythorsdottir, Holland, Ajmone-Marsan, Bruford, Kantanen, Spencer and Palmarini2009).
Figure 1. Median-joining network based on 8141 bp of mitochondrial genome sequences of Echinococcus granulosus sensu stricto G1 isolate from Iceland and across Europe. The numbers on the lines represent the number of nucleotide differences (mutational steps). Red dots are median vectors (haplotypes not sampled or extinct). The G1 haplotype from Iceland (ISL1) is in orange, surrounded by a dashed circle. Other haplotypes and their countries of origin are designated as 3 letter abbreviations (ALB, Albania; FRA, France; GRE, Greece; ITA, Italy; MOL, Moldova; ROM, Romania; SPA, Spain).
Control efforts of CE in Iceland and elsewhere
As a measure to prevent the reintroduction of E. granulosus s.l., the import of dogs to Iceland was restricted during 1909–1989. The ban was lifted in 1989, with the requirements of an enforced period of quarantine and of specific medical control and anticestodal treatment. At present, in 2023, each dog must be treated twice abroad with approved veterinary medicinal product(s) that has been registered for tapeworms; first at 3–4 weeks, then again 5–10 days prior to the import to Iceland. After arrival in Iceland, at the onset of a 2-week long stay in quarantine, a fecal sample must be examined for the presence of helminth parasites. Three days before leaving the quarantine station, and being handed over to the owner, the dog is treated for the third time with an approved anticestodal drug (Reglugerd ANR 201/2020). Further measures to prevent the re-emergence of CE in Iceland include a yearly, mandatory treatment of all dogs in Iceland with an approved anticestodal drug. Also, by slaughtering ruminants in abattoirs, hydatids are systematically searched for by veterinarians.
Although CE has been diagnosed in humans in other countries in the circumpolar region (e.g. Fennoscandia, Canada, Alaska and northern Russia), these continental regions represent somewhat different ‘settings’ in terms of CE transmission. To date, only island-based CE control programmes, such as in Iceland, have been successful in the eradication of CE transmission, whereas control in a continental setting has proved to be much more challenging (Craig et al., Reference Craig, Hegglin, Lightowlers, Torgerson and Wang2017). The predominant genotypes and species in continental circumpolar regions are E. canadensis genotypes G8 and G10, although surveillance data on CE is incomplete throughout the territories (Davidson et al., Reference Davidson, Lavikainen, Konyaev, Schurer, Miller, Oksanen, Skírnisson and Jenkins2016). In these areas, a synanthropic cycle involving semi-domesticated reindeer and dogs has caused hyperendemicity of CE in several arctic regions in Eurasia, including Fennoscandia and Alaska (Rausch, Reference Rausch2003). In both areas, the synanthropic cycle of CE has been largely eliminated owing to a major change in reindeer husbandry, namely by making herding dogs largely redundant by replacing them with snowmobiles (Rausch, Reference Rausch2003; Oksanen and Lavikainen, Reference Oksanen and Lavikainen2015).
Eradication or significant reduction of CE is more difficult to accomplish in large continental areas than in islands. However, recent signs of progress in the reduction of CE in continental territories are also evident. For example, in a highly endemic region of Campania in southern Italy, a multidisciplinary One Health approach to combat CE has been implemented since 2010. This involved a complex of procedures and tools, such as surveillance, diagnosis, anthelminthic treatment and public education, which has resulted in significant reduction of CE in livestock (Cringoli et al., Reference Cringoli, Pepe, Bosco, Maurelli, Baldi, Ciaramella, Musella, Buonanno, Capuano, Corrado, Ianniello, Alves, Sarnelli and Rinaldi2021).
Conclusion
The current study represents the first molecular characterization for one of the last CE cases recorded in Iceland, revealing E. granulosus s.s. genotype G1 as the causative agent. We conclude that it is likely that E. granulosus s.s. G1 was one of the representative genotypes circulating among humans and livestock in Iceland prior to the eradication of CE almost half a century ago, and may partly explain why such a large proportion of humans were infected in Iceland prior to the eradication of CE. Thus, decades-old samples hold significant potential to uncover genetic identities of parasites and other pathogens in the past (see also e.g. Flammer et al., Reference Flammer, Dellicour, Preston, Rieger, Warren, Tan, Nicholson, Přichystalová, Bleicher, Wahl, Faria, Pybus, Pollard and Smith2018; Guellil et al., Reference Guellil, van Dorp, Inskip, Dittmar, Saag, Tambets, Hui, Rose, D'Atanasio, Kriiska, Varul, Koekkelkoren, Goldina, Cessford, Solnik, Metspalu, Krause, Herbig, Robb, Houldcroft and Scheib2022). In the future, it would be interesting to explore the Icelandic isolate on a whole-genome scale, facilitated by the availability of a reference genome for genotype G1 (Korhonen et al., Reference Korhonen, Kinkar, Young, Cai, Lightowlers, Gauci, Jabbar, Chang, Wang, Hofmann, Koehler, Li, Li, Wang, Yin, Yang, Jenkins, Saarma, Laurimäe, Rostami-Nejad, Irshadullah, Mirhendi, Sharbatkhori, Ponce-Gordo, Simsek, Casulli, Zait, Atoyan, de la Rue, Romig, Wassermann, Aghayan, Hasmik, Yang and Gasser2022).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182023000355.
Data availability
The 8141 bp mtDNA sequence of the E. granulosus s.s. G1 isolate is deposited in GenBank under accession number OQ269593. The supplementary material for this article (Figs S1–S4) can be found at https://www.cambridge.org/core/journals/parasitology.
Acknowledgements
The authors would like to thank staff at the core facility at the Institute of Genomics, University of Tartu, for sequencing the DNA samples.
Author's contribution
U. S. conceived and designed the study, performed laboratory analyses and initial data analysis and wrote the original draft of the manuscript. K. S. conceived the study, provided samples and amended the manuscript. T. S. B. performed laboratory analysis and amended the manuscript. T. L. and L. K. performed data analysis and amended the manuscript.
Financial support
This work was supported by funding from the Estonian Ministry of Education and Research (grant PRG1209), and by funding from the European Union's Horizon 2020 Research and Innovation programme under grant agreement No. 773830: One Health European Joint Programme (MEME project; https://onehealthejp.eu/jrp-meme/).
Conflict of interest
None.
Ethical standards
No ethics permissions were required for the current study as all the biological material was donated to the University of Tartu (Estonia) and no biological material from animals was collected specifically for this study.
Open access
