Avian schistosomes are trematodes of the family Schistosomatidae that require two hosts to complete their life cycle: aquatic snails as intermediate hosts and waterbirds as definitive hosts. Free-swimming larval stages called cercariae, released from infected snails, can penetrate the skin of accidental hosts, including humans, causing cercarial dermatitis, commonly known as swimmer’s itch. This re-emerging waterborne disease has a worldwide distribution, and its public health relevance makes accurate detection and monitoring of these parasites in recreational waters a pressing concern.

Traditionally, identifying avian schistosomes has depended on the morphology of adult worms, which are difficult to obtain from bird hosts. Cercariae are much easier to collect from infected snails, but they are morphologically nearly indistinguishable from one another, so genetic typing is essential. The researchers’ earlier comprehensive work showed that only 25 of 85 described species have been molecularly studied, while 52 genetic lineages could not yet be linked to known species. Standard parasitological examination, which involves stimulating cercarial shedding and dissecting snails, is time-consuming, requires taxonomic expertise and often underestimates true diversity because infected snails are rare and early-stage infections within snail tissues are easily overlooked.

To overcome these limitations, the team designed a new pair of primers, ITS_F1670 and ITS_R2120, targeting the ITS1-5.8S ribosomal DNA region and specific to avian schistosomes. The primer database was built from 195 ITS sequences of avian schistosomes deposited in GenBank, spanning 13 genera including Trichobilharzia, Anserobilharzia, Bilharziella and Dendritobilharzia. In silico specificity testing with Primer-BLAST yielded 402 matches, of which 357, or 89 percent, were from the family Schistosomatidae, with the remaining cross-amplifications belonging to other digenean trematodes. The theoretical PCR products of target taxa ranged from 340 to 349 base pairs, distinguishable by length from the 357 to 402 base pair products of non-target organisms. A complementary primer pair targeting the mitochondrial cox1 barcoding gene was also designed but failed to amplify avian schistosome DNA from environmental water samples previously confirmed positive by ITS amplification, so it was not used further, a limitation the authors note given cox1’s superior resolution for closely related species complexes.

The study compared three complementary detection approaches across 37 waterbodies sampled for snails and 45 localities sampled for water. First, standard parasitological examination of 17,663 snails belonging to 26 species and 10 families involved cercarial shedding stimulated by artificial light, followed by dissection. Second, metabarcoding of snail hepatopancreas tissues from 445 seemingly uninfected individuals, selected from 1,035 snails of nine species across three families, sought pre-patent infections invisible to conventional methods. Third, metabarcoding of environmental water samples, concentrated either through a 20-micrometre plankton net from 30-litre volumes or through polypropylene membrane filters tested in the final phase of the study, targeted free-living parasite stages such as eggs, miracidia and cercariae, along with particle-associated DNA. All metabarcoding samples were amplified with barcoded primers and sequenced on an Illumina NovaSeq platform across three runs, yielding 7,380,836 raw reads that were quality-filtered, demultiplexed, checked for chimeras and clustered into OTUs at 97 percent sequence similarity, leaving 5,175,786 processed reads.

Standard examination detected avian schistosomes in only 11 of 37 localities, with 71 snails, or 0.4 percent of those examined, testing positive. Five species were identified this way: Bilharziella polonica, Trichobilharzia anseri, T. franki, T. regenti and T. szidati. Notably, T. anseri and T. regenti were found exclusively by this method, underscoring that conventional examination still captures taxa that the molecular approaches missed. Prevalence was higher in the Czech Republic, at 0.6 percent of 11,753 snails, than in Slovakia, at 0.08 percent of 5,910 snails. The method remains indispensable because it provides live parasitic material for morphological characterisation, molecular sequencing, experimental infections and unequivocal host-parasite associations that environmental samples cannot directly deliver.

Metabarcoding of snail tissues proved a valuable complement, detecting parasite DNA in snails that had tested negative by both cercarial shedding and dissection. Of 83 tissue samples, 28 from five localities were positive, generating 1,197,717 quality-filtered reads that clustered into 31 OTUs, of which 210,363 reads, or 17.56 percent, fell into 15 OTUs assigned to avian schistosomes. Five OTUs matched known taxa, including B. polonica, Bilharziella sp. I, Anserobilharzia sp. I, T. franki and T. szidati, while ten could not be reliably assigned to any known species. The approach also revealed unexpected host associations, such as detections of T. franki and T. szidati in the planorbid snail Planorbarius corneus and Anserobilharzia sp. I in Lymnaea stagnalis and Stagnicola corvus, which the authors interpret cautiously, noting these could reflect contamination, transient cercarial contact or externally adhered DNA rather than true infections, and require confirmation by further work.

Water metabarcoding emerged as the most productive single technique. Of 368 water samples processed, 24 were positive, retaining 2,919,332 reads that clustered into 52 OTUs, with 81.10 percent of reads assigned to avian schistosomes. Twenty-two unique OTUs belonged to the target group, but only two were identical to previously known lineages, and three more were tentatively assigned based on their geographic origin in Central Europe. The remaining 17 OTUs represented unexplored diversity, including eleven nested within the genus Trichobilharzia, five forming a sister lineage to ‘T.’ filiformis and one grouping with Gigantobilharzia huronensis. Trichobilharzia franki was the most widespread species overall, recorded in 19 localities across both countries, followed by T. szidati in nine. The study also reports the first genetically confirmed record of T. szidati in Slovakia, the second records of Anserobilharzia sp. I and Schistosomatidae gen. sp. XVIII in the Czech Republic and the second report of Bilharziella sp. I in Europe after a Polish record.

The low detection frequency in water, 6.6 percent of samples, mirrors the low snail infection prevalence and was corroborated by a laboratory sensitivity assay in which only samples containing 50 cercariae of T. franki per litre of tap water produced positive results, yielding 6,506 reads of which 98.6 percent were assigned to T. franki, while samples with 5 or 10 cercariae did not. The authors attribute the low detection rate to the sparse release of cercariae into water, the heterogeneous spatial and temporal distribution of parasite DNA and probable loss of extracellular eDNA through the relatively large pore size of the plankton net. They also acknowledge the absence of technical replication during DNA extraction and PCR, which future studies should incorporate to better distinguish true biological signals from stochastic amplification artefacts. Crucially, non-detection in an eDNA sample does not necessarily indicate true absence of the parasites.

No single method recovered the complete diversity of avian schistosomes at any locality, and the complementary strengths of the three approaches were evident in taxa found exclusively by one technique: T. anseri and T. regenti only by standard examination, Schistosomatidae gen. sp. XVIII solely in water samples and Bilharziella sp. I exclusively in snail tissues. Vlkovský in the Czech Republic showed the highest diversity, with 15 species and genetic lineages detected. The researchers conclude that integrating water metabarcoding, snail-tissue metabarcoding and standard parasitological examination provides a robust framework for ecological monitoring, biodiversity assessment and public health risk evaluation. For rapid, large-scale surveillance of parasite presence, water metabarcoding is the most efficient option, though it cannot identify the snail host involved in transmission. The widespread occurrence of T. franki and T. szidati indicates a substantial risk of cercarial dermatitis in Central European recreational waterbodies and highlights the need for proactive ecological monitoring and public health awareness.

Subject of Research: Detection and diversity of avian schistosomes in Central European freshwater ecosystems using standard parasitology and eDNA metabarcoding

Article Title: A multi-source approach for the detection and diversity assessment of avian schistosomes ( Trematoda , Schistosomatidae ) in freshwater ecosystems

Article References: Kibet, C. J., Barčák, D., Lisnerová, M., Fiala, I., Orosová, M., Oros, M., Soldánová, M., & Kuchta, R. (2026). A multi-source approach for the detection and diversity assessment of avian schistosomes (Trematoda, Schistosomatidae) in freshwater ecosystems. Metabarcoding and Metagenomics, 10, Article e197780. https://doi.org/10.3897/mbmg.10.197780

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.197780

Keywords: avian schistosomes, swimmer's itch, environmental DNA, metabarcoding, Trematoda, Schistosomatidae, Trichobilharzia, freshwater ecosystems, snail hosts, cercarial dermatitis, Czech Republic, Slovakia