The mouse bile duct tapeworm, Hymenolepis microstoma in free-living small mammals in Slovakia: occurrence and genetic analysis

Among tapeworms belonging to the genus Hymenolepis, three species are medically relevant and are commonly found worldwide in sites where murid rodents occur: Hymenolepis nana (Siebold, 1852), Hymenolepis diminuta (Rudolphi, 1819), and Hymenolepis microstoma (Dujardin, 1845) Blanchard, 1891 (Cunningham & Olson, 2010). Hymenolepis (= Rodentolepis) microstoma, the mouse bile duct tapeworm, has a wide variety of reported definitive hosts of rodent genera as mice (Hopkins et al., 1977), rats (Goodall, 1972), hamsters (Bogh et al., 1986), voles (Litchford, 1963), and gerbils (Schmidt, 1986). A zoonotic potential of this tapeworm was indicated by a single report in which mixed infections of H. nana and H. microstoma were identified in four humans from a remote region of Western Australia (Macnish et al., 2003). H. microstoma is recognized as requiring an appropriate tenebrionid grain beetle or other insect as intermediate host (originated mostly from Tribolium and Tenebrio genera), in which the eggs develop into cysticercoids after ingestion (Bogh et al., 1986; Smyth & Wakelin, 1994). Nevertheless, the feasibility of a direct life cycle in immunodeficient mice has also been demonstrated for the mouse bile duct tapeworm (Andreassen et al., 2004).

H. microstoma has attracted considerable attention since it was first described by Dujardin in 1845. In 1891, Blanchard relocated the species to the genus Hymenolepis and provided a more detailed description of the species. Since 1950s, H. microstoma is being commonly used as an experimental model. Due to its easy maintenance in the laboratory in mice host and short life cycle, H. microstoma has been established as a preferable model to both H. nana and H. diminuta, the maintenance of which is more costly (Clark, 2013). Moreover, the mouse bile duct tapeworm is one of the four tapeworm species having its complete genome assembled and characterized (Cunningham & Olson, 2010; Tsai et al., 2013). Although adult H. microstoma specimens may be differentiated based on morphological characteristics and predilection site (Baer & Tenora, 1970; Czaplinski & Vaucher, 1994), in some cases these characteristics are difficult to use, especially when dead animals are sampled (e.g. in national parks) and the carcasses (along with their intestinal parasites) are in some stage of decay. Furthermore, a variety of other hymenolepidid species, morphologically similar to H. microstoma, can potentially infest free-living small mammals (Riebold et al., 2019). Thus, the use of molecular tools, especially in cases when identification via morphology is ambiguous, is essential for identifying and classifying species (Peniche et al., 2017). The study was designed to survey the occurrence of H. microstoma in free-living small mammals in selected areas of Slovakia and to conduct a retrospective analysis of epidemiological data published in Slovakia. In addition, the genetic typing at species level of collected isolates was carried out.

The research related to animals has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

Although H. microstoma was not recorded to date in any study of intestinal parasites of small mammals conducted in the territory of Slovakia, the present research documented its occurrence in the country. The parasite was recorded in two animals, in the common shrew (Sorex araneus) and in the European hamster (Cricetus cricetus), in two geographically distinct sites and ecosystems of Slovakia.

To our knowledge, these are also the first documented reports H. microstoma in Central Europe, considering that no data about its occurrence are available in the literature including records deposited in the Host-Parasite database of the Natural History Museum (Gibson et al., 2005). As the parasite was not included in the checklist ´Synopsis of Cestodes in Slovakia´, encompassing records from a number of local and broader surveys in Slovakia during 1955 – 1996 (Hanzelová & Ryšavý, 1996), it is likely that the mouse bile duct tapeworm was introduced into the Slovak territory over the last two decades.

The parasite was found close to Tatranské Matliare village in the vicinity of the protected area of the Tatra National Park in a common shrew (Sorex araneus). As far as we are aware, the present study also provides the first record of H. microstoma infection in S. araneus, which is one of the most common and abundant woodland micromammals in different types of forests (Mitchell-Jones et al., 1999; Rychlik, 2000), whose presence has also been confirmed in 90.3 % of the area of Slovakia (Krištofík et al., 2012).

Two H. microstoma-specific DNA profiles (assigned as SK-CS and SK-EH) were identified among 10 hymenolepidid isolates examined with pmy and ITS-1 markers. In the partial pmy gene, the shrew isolate (SK-CS) has formed the subgroup with H. microstoma, thus differentiating this couple from the close H. nana with whom one of two main clades in the NJ tree inferred from Hymenolepis-deposited sequences was formed. The percentage nucleotide difference between the SK-CS isolate and the H. microstoma sequence obtained from a Portuguese mouse isolate (Macnish, 2001) was 4.86 %, which is a relatively high value, comparable to the interspecific nucleotide difference (4.21 %) between H. diminuta and H. citelli in the screened pmy region. Nevertheless, given that we have found no record of the H. microstoma grouping with any other species when H. nana was present as the closest relative in phylograms inferred from nuclear and/or mitochondrial data, and the strong genetic cohesiveness between the H. microstoma reference and our sample (their subclade received the bootstrap value of 84 %), the classification of the studied isolate as belonging to H. microstoma is highly plausible.

After analysis of the nuclear ribosomal ITS region, the hamster isolate (SK-EH) originated from an urban ecosystem in south-east Slovakia clustered with H. microstoma specimens from the two continents (Europe, Australia). Interestingly, SK-EH appeared to be the most distinctive sample among H. microstoma examined so far in ITS-1, differing in 1.4 – 1.9 % of nucleotide bases from the remaining isolates that markedly exceeded the value of the average intercontinental base difference measured between isolates from southern Europe and Australia (0.14 %). The difference was partially due to the two indel polymorphisms associated with two and five nucleotides, respectively, the latter of which accounted for the absence of arginine in the protein composition. It has been found that especially during starvation, flatworm parasites including H. diminuta develop enzymes to synthesize arginine and urea, offering an advantage for osmoregulation and serving as effective nutrients in the fasting state (Campbell, 1963; Mohamed et al., 2005). Nevertheless, some studies have suggested that arginine is not necessarily an essential amino acid for a variety of invertebrates including flatworms if ornithine can be synthesized (Campbell & Speeg, 1968; Tielens & van Hellemond, 2005).

With regard to the three most important Hymenolepis spp., phylogenetic analysis of nuclear targets herein employed corroborated that representatives of H. nana and H. microstoma form a strongly supported clade with respect to H. diminuta, which may have diverged from the common ancestral line earlier. Similar results with respect to the systematic position of these species were also obtained by other mitochondrial (cox1) and nuclear (ITS2, 18S) evidences (e.g. Okamoto et al., 1997; Macnish et al., 2002; Olson et al., 2003; Sharma et al., 2016). Besides, studies on immunobiological characteristics and cysticercoid development from eggs suggested that H. microstoma is more similar to H. nana than to H. diminuta (Voge, 1964; Ito et al., 1988).

In previous surveys, H. microstoma was genetically identified in two consecutive studies by Macnish et al. (2002, 2003) in mice (Mus musculus) from Portugal, Australia and the United Kingdom, and in humans from Australia. In the Canary Islands (Spain), Foronda et al. (2011) detected H. microstoma in 10.8 % of the examined house mice (Mus musculus) and in 0.3 % of black rats (Rattus rattus), but none of 13 brown rats (Rattus norvegicus) was positive. In twelve cestodes collected from house mice (Mus musculus) in Lima (Peru), morphologically and genetically (cox1 mt gene) categorized as H. microstoma, lower genetic variability between specimens from Peru and Spain compared to Japan indicated that the parasite was introduced into Peru from a European country (Gomez-Puerta et al., 2018). A close phylogenetic match to H. microstoma was also determined in the tapeworm derived from captive dormouse (Muscardinus avellanarius) in the United Kingdom (Peniche et al., 2017).

The dwarf tapeworm H. nana is the only cestode recognized as not requiring an intermediate host to develop into maturity under natural conditions (Dovč et al., 2017). The potential capability of the direct life cycle for H. microstoma (as another common feature with H. nana) has been discussed in the literature in the past (Skrjabin & Kalantarian, 1942; Macnish et al., 2003), and later evidenced in immunocompromised mice by Andreassen et al. (2004) who showed that oncospheres penetrated the intestinal tissues and developed into cysticercoids and subsequently into the adult stage in the bile duct and duodenum when infected with parasite eggs. It is therefore presumed that the development of patent infections of H. microstoma in humans could more likely occur in immunocompromised individuals. Malnourishment, type II diabetes, alcoholism, cancer, HIV-infection, immunosuppressive therapy, etc. (e.g. Kontogiorgi et al., 2013; Kumari et al., 2014) are factors contributing to depression of the immune system and may enhance the development of infections with parasites such as H. microstoma in humans.

To date, the majority of published surveys in free-living and domestic small mammals reported the occurrence of hymenolepidid tapeworms based on coprological examination (Jarošová et al., 2020). Due to striking morphological similarity of the eggs of H. microstoma and H. nana that are difficult to distinguish by morphology alone (Fitzgerald et al., 1970; Macnish, 2001), it is possible that some infections of H. microstoma in animals and humans may have been previously misdiagnosed as H. nana (Whary et al., 2015; Riebold et al., 2019). In addition, infection in humans may have been underdiagnosed due to sporadic or reduced egg shedding that was documented in H. nana, H. diminuta and H. citelli in response to factors such as the quality and quantity of the host diet (Kennedy, 1983; Schantz, 1996) and can occur also in H. microstoma.

A recently indicated zoonotic potential of H. microstoma (Macnish et al., 2003) along with a possibility of its direct transmission between animals and/or humans without the need for intermediate hosts pose a public health concern in contaminated areas of Slovakia. In the present study, the use of molecular techniques has enabled identification of the mouse bile duct tapeworm circulating in the two animal populations of Slovakia and will greatly facilitate a more thorough understanding of the epidemiological situation of H. microstoma and related tapeworms in various ecosystems of the country in the future.