Genetic Diversity of an Avian Nasal Schistosome Causing Cercarial Dermatitis in the Black Sea-Mediterranean Migratory Route

Parasitology Research https://doi.org/10.1007/s00436-018-6087-0

ORIGINAL PAPER

Genetic diversity of an avian nasal schistosome causing cercarial dermatitis in the Black Sea-Mediterranean migratory route

Keyhan Ashrafi1,2 & Alireza Nouroosta3 & Meysam Sharifdini1 & Mohammad Reza Mahmoudi1 & Behnaz Rahmati1 & Sara V. Brant4

Received: 30 July 2018 /Accepted: 11 September 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract This study is part of an effort to document the diversity of avian schistosomes in ducks and snails in Northern Iran, a major flyway (Black Sea/Mediterranean) for migratory birds and where cercarial dermatitis (CD) is prevalent in rice growing areas. CD is an allergic skin reaction from schistosome trematodes that emerge from aquatic snails. Most CD cases are reported from recreational swimmers or aquaculture farmers. Much of the work on the epidemiology of CD has focused in recreational waters in the Americas and Europe, with fewer studies in aquaculture, particularly in Iran. The artificial environment at aquaculture sites support dense populations of snails that are hosts to schistosomes, as well as domestic ducks. Thus, are domestic ducks reservoir hosts of species of Trichobilharzia, one of the main etiological agents of CD in Northern Iran? This study focused on a survey of domestic ducks for the presence of the nasal schistosome, T. regenti, that has been reported widely in Europe. Trichobilharzia regenti were found in domestic ducks in the Guilan Province of Iran based on morphological and molecular analyses. The presence of this species in Northern Iran indicates that the domestic duck can serve as a reservoir host for this species and that one of the local snail species is likely the intermediate host. The continued study and surveillance of this species is important because it is a neuropathic schistosome that can use a diversity of bird definitive hosts and Radix snails that are widespread across Eurasia.

Keywords Cercarial dermatitis . Schistosome . Schistosomatidae . Trichobilharzia regenti . Iran

Introduction except Antarctica, in mammals or birds as definitive hosts and either freshwater or marine aquatic gastropods as intermediate Cercarial dermatitis (CD) is an allergic skin reaction at the hosts. Most cases of CD are reported from recreational swim- penetration site of larval schistosome trematodes (Digenea: mers, such as at beaches, or aquaculture farmers, such as rice Schistosomatidae) that emerge from aquatic snails in the sum- farms. Much of the work to understand the epidemiology of mer months in temperate latitudes, and likely year-round in CD has been focused in recreational waters in the Americas tropical latitudes. Schistosomes are found on all continents, and Europe (see references in review Horak et al. 2015), but fewer studies in aquaculture sites. Areas of aquaculture are Section Editor: Ramaswamy Kalyanasundaram where people and animals have prolonged contact with the water, particularly countries in Asia that have some of the * Sara V. Brant highest rice production (International Rice Research Institute [email protected] 2009). Rice producing countries in the Middle East have recently been taking notice of the prevalence of CD in their 1 Department of Medical Parasitology and Mycology, School of countries (e.g., Farahnak and Essalat 2003;Atharietal. Medicine, Guilan University of Medical Sciences, Rasht, Iran 2006; Al-Khuzaee 2009; Schuster et al. 2014; Omar et al. 2 Cellular and Molecular Research Center, School of Medicine, Guilan 2016). University of Medical Sciences, Rasht, Iran The last decade has seen an increase in reports 3 Student Research Committee, Guilan University of Medical documenting cases and the etiological agents of CD, particu- Sciences, Rasht, Iran larly in Iran (Farahnak and Essalat 2003; Athari et al. 2006; 4 Museum of Southwestern Biology, Division of Parasites, Department Karamian et al. 2011; Gohardehi et al. 2013; Maleki et al. of Biology, University of New Mexico, 1 University of New Mexico 2012; Mahdavi et al. 2013a, b; Imani-Baran et al. 2013; MSC03 2020, Albuquerque, New Mexico 87131, USA Parasitol Res

Ghobaditara et al. 2015; Fakhar et al. 2016; Yakhchali et al. Province includes 238,000 acre of rice fields and produce 2016). The artificial environment of, for example, a rice field, about 40% of rice product of the country. After rice harvesting supports populations of snails that are hosts to schistosomes, from early August to late September, the rice paddies, espe- as well as domestic ducks, which also host avian schisto- cially those located near the farmer’s houses, become appro- somes. Thus, an important question in the epidemiology of priate places for livestock grazing, domestic ducks breeding CD, particularly in aquaculture is, are domestic ducks reser- and snail intermediate hosts reproduction. The domestic ducks voir hosts of one or more species of Trichobilharzia,oneof constitute an important part of the diet of indigenous popula- the main etiological agents of CD (Horak et al. 2015)? Wild tion, so they are sold in weekly local markets. Late fall also ducks, geese, and swans are well known hosts of avian schis- coincides with the start of the rainy season in Guilan and when tosomes and because these hosts can migrate long distances, the rice fields receive large amounts of water. These paddies so too can their parasites, or can they? Can they migrate and then become a suitable environment to establish and sustain maintain local life cycles? Discovering the species of schisto- the snail intermediate hosts and ducks feeding, including the some and host use diversity and source populations is a foun- many water collections and small streams around Guilan vil- dational step to initiating a targeted control program (Horak lages, all of which facilitate the schistosome life cycle. See et al. 2015). This study is part of a larger effort to document Table 1 and Fig. 1 for the collection localities and locality the diversity of avian schistosomes in ducks and local snails in descriptions. Northern Iran where CD is prevalent in aquaculture areas. This study focused on a survey of domestic ducks in Guilan Parasite collection Province as reservoir hosts for the presence of the nasal schistosome that has been reported widely in Europe (Horak et al. Domestic ducks, Anas platyrhynchos domesticus,werepur- 1998; Picard and Jousson 2001; Dvorak et al. 2002; chased directly from villager’s houses surrounded by rice Rudolfova et al. 2007; Jouet et al. 2008, 2010a, 2010b; fields where the ducks were feeding (Fig. 1). Locality data Skirnisson et al. 2012;Christiansenetal.2016) and in some were determined by GPS (Table 1). The ducks were trans- localities in Northern Iran (Gohardehi et al. 2013;Malekietal. ferred to the parasitology laboratory at the Guilan University 2012; Mahdavi et al. 2013a, b;Fakharetal.2016). The con- of Medical Sciences and decapitated to examine for presence tinued study and surveillance of this species is important be- of nasal schistosomes. The following procedure was used for cause it is a neuropathogenic schistosome that can use a di- all duck heads: (a) two longitudinal incisions through the bird versity of bird definitive hosts and Radix snails that are wide- nostrils along the beak and then a transverse cut to connect the spread across Eurasia (Leontovyc et al. 2016). longitudinal incisions were made, (b) the nasal mucosa was cut open and surrounding vascular tissues were removed, (c) Materials and methods the mucosa and tissue were then transferred into two Petri dishes, one with water and the other with saline to obtain intact eggs and miracidia, and (d) the nasal mucosa were teased apart Study area gently to look for adult worms. Guilan Province has an area of 14,042 km2 (38°28′ 58″ N, 50° 35′ 59″ E) and is situated at the western shores of the Caspian Morphological and genetic analyses Sea. Guilan Province contains 16 districts located at different geographical zones, for example, coastal plain lowlands that For morphological studies, some of the eggs were transferred are below sea level in some regions, foothills, and forested onto a glass slide and coverslip and measured under a micro- mountainous areas. This province has a humid subtropical scope (Olympus BX50) equipped with digital camera climate with the heaviest rainfall in Iran reaching as high as (TrucChrome Metrics, China) and Nomarski Piece (U-DICT, 1900 mm in some regions. Mean rainfall in Guilan Province is Olympus, Japan). The length and width of the eggs were mea- about 1500 mm which is four to six times more than mean sured, and the data analyzed in SPSS Ver. 22 (minimum, max- annual rainfall in Iran. Humidity is very high because of the imum, average, and SD). The remaining eggs, miracidia, and marshy character of the coastal plains and can reach 90% in worm fragments, if any, were transferred to microtubes con- summer with temperature of over 26 °C. The coastal plain taining 96% alcohol for molecular studies. Some of the col- along the Caspian Sea is similar to that of Mazandaran (area lected samples were also kept in labeled microtubes in 90% also reports CD) that is mainly used for rice paddies. These alcohol in the Department of Parasitology and Mycology of areas have altitudes ranging from − 27 m below sea level (b.s. the Guilan University of Medical Sciences as a permanent l) to 300 m above sea level (a.s.l), where 60% of the land- museum voucher. It is critical for the evolutionary character- cover consists of wetlands, with numerous irrigation canals ization of organisms to have a permanent museum voucher and rice cultivation as the main agriculture activity. Guilan (Pleijel et al. 2008; Valkiunas et al. 2008; Hoberg et al. 2009). Parasitol Res

Table 1 Districts and coordinates of localities. The duck host number matches the numbers on the collection localities in Fig. 1

District Coordinates (latitude/longitude) Duck host # Infected Altitude

Astara 38° 25′ 11.3″ N/48° 51′ 55.5″ E1 Yes − 26 Astara 38° 26′ 10.0″ N/48° 51′ 44.9″ E2 No − 27 Astara 38° 26′ 08.7″ N/48° 51′ 46.1″ E3 No − 27 Astara 38° 26′ 01.9″ N/48° 51′ 45.1″ E4 No − 26 Talesh 37° 36′ 46.5″ N/49° 03′ 30.1″ E5 Yes 16 Talesh 37° 37′ 29.0″ N/49° 02′ 39.0″ E6 Yes 7 Talesh 37° 37′ 36.7″ N/49° 02′ 52.4″ E 7* Yes 4 Talesh 37° 36′ 46.4″ N/49° 02′ 23.6″ E8 Yes 23 Talesh 37° 38′ 07.3″ N/49° 02′ 50.7″ E9 Yes − 8 Talesh 37° 49′ 02.9″ N/48° 54′ 58.8″ E10 No 26 Talesh 37° 497′ 03. ″ N/48° 55′ 02.0″ E11No26 Talesh 37° 49′ 01.0″ N/48° 55′ 10.1″ E12No26 Talesh 37° 48′ 48.3″ N/48° 55′ 27.5″ E13No24 Talesh 37° 46′ 25.5″ N/48° 56′ 40.3″ E14No8 Rezvanshahr 37° 33′ 22.1″ N/49° 09′ 09.8″ E 15 Yes 0 Rezvanshahr 37° 33′ 08.6″ N/49° 08′ 54.9″ E 16 Yes 5 Rezvanshahr 37° 33′ 24.5″ N/49° 08′ 19.0″ E 17 Yes 7 Rezvanshahr 37° 32′ 47.1″ N/49° 09′ 57.0″ E18No2 Rezvanshahr 37° 32′ 36.7″ N/49° 10′ 00.3″ E19No − 1 Masal 37° 22′ 28.0″ N/49° 09′ 13.5″ E20Yes32 Masal 37° 22′ 23.2″ N/49° 09′ 11.4″ E21Yes33 Masal 37° 222′ 27. ″ N/49° 08′ 42.0″ E22No41 Masal 37° 22′ 26.6″ N/49° 08′ 41.0″ E23No41 Masal 37° 22′ 23.0″ N/49° 08′ 36.6″ E24No42 Masal 37° 22′ 17.6″ N/49° 08′ 31.2″ E25No45 Sowme’eh Sara 37° 16′ 44.2″ N/49° 22′ 46.7″ E26Yes − 5 Sowme’eh Sara 37° 16′ 35.3″ N/49° 22′ 21.0″ E27Yes − 4 Sowme’eh Sara 37° 16′ 35.3″ N/49° 22′ 20.0″ E 28* Yes − 4 Sowme’eh Sara 37° 16′ 57.0″ N/49° 22′ 28.3″ E29Yes − 8 Sowme’eh Sara 37° 17′ 15.3″ N/49° 22′ 26.5″ E30No − 7 Sowme ’eh Sara 37° 17′ 23.4″ N/49° 22′ 51.7″ E31 No − 8 Fouman 37° 12′ 44.9″ N/49° 18′ 19.9″ E32 Yes 33 Fouman 37° 12′ 55.2″ N/49° 18′ 22.8″ E33 Yes 31 Fouman 37° 13′ 03.1″ N/49° 19′ 36.0″ E34 Yes 27 Fouman 37° 13′ 01.7″ N/49° 19′ 10.4″ E 35* Yes 30 Fouman 37° 13′ 59.0″ N/49° 19′ 49.7″ E36 Yes 21 Fouman 37° 12′ 54.0″ N/49° 18′ 40.5″ E37 Yes 33 Fouman 37° 12′ 50.0″ N/49° 18′ 46.1″ E38 No 33 Anzali 37° 27′ 10.1″ N/49° 35′ 49.9″ E39 Yes − 27 Anzali 37° 25′ 22.8″ N/49° 26′ 11.8″ E40 Yes − 27 Anzali 37° 27′ 34.4″ N/49° 30′ 37.5″ E41 Yes − 25 Anzali 37° 27′ 32.3″ N/49° 30′ 38.7″ E42 No − 21 Shaft 37° 08′ 37.6″ N/49° 23′ 27.9″ E 43* Yes 59 Shaft 37° 08′ 39.9″ N/49° 23′ 31.9″ E44 No 59 Shaft 37° 08′ 38.2″ N/49° 23′ 07.0″ E45 No 61 Shaft 37° 08′ 29.9″ N/49° 22′ 53.1″ E46 No 65 Shaft 37° 08′ 57.6″ N/49° 23′ 09.8″ E47 No 59 Rasht 37° 10′ 50.5″ N/49° 41′ 10.4″ E48 Yes 26 Rasht 37° 10′ 43.8″ N/49° 41′ 16.8″ E 49* Yes 29 Rasht 37° 10′ 31.8″ N/49° 41′ 24.1″ E50 Yes 29 Rasht 37° 16′ 16.2″ N/49° 45′ 30.9″ E 51 Yes 1 Rasht 37° 16′ 21.5″ N/49° 45′ 43.8″ E52 No 1 Rasht 37° 16′ 40.0″ N/49° 45′ 40.1″ E53 No 1 Rasht 37° 16′ 46.3″ N/49° 45′ 45.4″ E54 No 0 Rasht 37° 16′ 30.2″ N/49° 45′ 09.0″ E55 No − 1 Rasht 37° 16′ 30.0″ N/49° 45′ 15.9″ E56 No 1 Rasht 37° 11′ 19.5″ N/49° 31′ 49.9″ E57 No 31 Rasht 37° 11′ 16.7″ N/49° 31′ 44.5″ E58 No 31 Rasht 37° 13′ 04.3″ N/49° 30′ 34.8″ E59 No 13 Roudbar 37° 01′ 08.2″ N/49° 36′ 46.2″ E60 No 87 Roudbar 37° 01′ 06.5″ N/49° 36′ 46.3″ E61 No 84 Roudbar 37° 00′ 59.4″ N/49° 36′ 29.6″ E62 No 88 Roudbar 37° 00′ 43.2″ N/49° 35′ 55.0″ E63 No 90 Siahkal 37° 09′ 35.1″ N/49° 52′ 58.3″ E64 No 36 Siahkal 37° 09′ 35.0″ N/49° 52′ 57.8″ E65 No 36 Parasitol Res

Table 1 (continued)

District Coordinates (latitude/longitude) Duck host # Infected Altitude

Siahkal 37° 09′ 50.3″ N/49° 52′ 60.0″ E66 No 32 Siahkal 37° 09′ 51.6″ N/49° 52′ 17.5″ E67 No 33 Astaneh 37° 15′ 46.8″ N/49° 53′ 28.4″ E68 No − 7 Astaneh 37° 15′ 46.2″ N/49° 53′ 30.3″ E69 No − 8 Astaneh 37° 16′ 22.6″ N/49° 54′ 30.1″ E70 No − 9 Astaneh 37° 16′ 22.7″ N/49° 54′ 40.3″ E71 No − 10 Astaneh 37° 16′ 22.7″ N/49° 54′ 40.6″ E72 No − 9 Astaneh 37° 16′ 40.1″ N/49° 55′ 38.3″ E73 No − 8 Lahijan 37° 13′ 10.2″ N/49° 59′ 47.6″ E74 No − 11 Lahijan 37° 13′ 07.1″ N/49° 59′ 45.3″ E75 No − 12 Lahijan 37° 13′ 06.4″ N/49° 59′ 03.4″ E76 No − 8 Lahijan 37° 13′ 20.0″ N/49° 58′ 21.1″ E77 No − 10 Lahijan 37° 13′ 14.8″ N/49° 58′ 51.5″ E78 No − 10 Lahijan 37° 12′ 22.6″ N/50° 02′ 21.2″ E79 No − 9 Lahijan 37° 16′ 03.0″ N/50° 07′ 17.6″ E 80* Yes − 19 Lahijan 37° 16′ 02.4″ N/50° 07′ 15.4″ E81 No − 20 Langroud 37° 11′ 55.4″ N/50° 10′ 03.9″ E82 No − 24 Langroud 37° 12′ 12.7″ N/50° 10′ 30.0″ E83 No − 26 Langroud 37° 12′ 13.4″ N/50° 11′ 16.6″ E84 No − 25 Langroud 37° 11′ 11.8″ N/50° 12′ 44.1″ E85 No − 23 Langroud 37° 11′ 11.9″ N/50° 12′ 44.5″ E86 No − 23 Amlash 37° 05′ 39.9″ N/50° 11′ 52.9″ E87 No 19 Amlash 37° 05′ 40.1″ N/50° 11′ 53.9″ E88 No 19 Amlash 37° 03′ 37.8″ N/50° 16′ 10.9″ E 89* Yes 25 Amlash 37° 03′ 37.9″ N/50° 16′ 10.5″ E90 No 25 Roudsar 37° 11′ 29.2″ N/50° 10′ 04.1″ E91 No − 23 Roudsar 37° 08′ 19.8″ N/50° 16′ 37.8″ E92 No − 22 Roudsar 37° 08′ 28.3″ N/50° 16′ 41.6″ E93 No − 21 Roudsar 37° 08′ 34.8″ N/50° 16′ 47.2″ E94 No − 20 Roudsar 37° 08′ 20.5″ N/50° 16′ 33.2″ E95 No − 22 Roudsar 37° 02′ 08.2″ N/50° 20′ 15.4″ E96 No 38 Roudsar 37° 02′ 09.0″ N/50° 20′ 17.4″ E97 No 38

*Samples used for molecular studies

The genomic DNA was extracted from 96% ethanol pre- denaturationat95°Cfor6min,30cyclesof95°Cfor45s, served worm fragments using a commercial kit (High Pure 55 °C for 60 s, and 70 °C for 1 min, followed by a final extension PCR Template Preparation Kit; Roche, Mannheim, Germany) at 72 °C for 6 min. These PCR products were submitted to according to the manufacturer’s recommended protocol. Bioneer Company (Korea) and sequenced in both directions Primers BD1 (5′-GTCGTAACAAGG TTTCCGTA-3′) using the same PCR primers. (Bowles and McManus 1993 MBP) and 4S (5′-TCTA GATGCGTTCGAARTGTCGATG-3′) (Bowles et al. 1995) Reconstruction of evolutionary relationships were used to amplify the internal transcribed spacer 1 region (ITS1) nuclear rDNA. For a partial cytochrome oxidase 1 The phylogenetic relationship of the schistosomes found in (cox1) region of the mitochondrial DNA the primers used were this study were reconstructed using a partial gene region of Cox1_SchistoF (5′-TCTTTRGATCATAAGCG-3′)and cox1 and ITS1. Sequences were aligned by eye in Se-Al v Cox1_SchistoR (5′-TAATGCATMGGAAAAAAACA3′) 2.0a11 (tree.bio.ed.ac.uk). Phylogenetic analyses of the cox1 (Lockyer et al. 2003). The PCR reactions were performed in a and ITS datasets were performed using Bayesian Inference in 30-μl reaction mixture containing 15 μl of PCR mix including MrBayes (Huelsenbeck and Ronquist 2001) with default 1.25 U Taq DNA polymerase, 200 μMofdNTPsand1.5mM priors for the ITS1 gene region (Nst = 6, rates = gamma, MgCl2 (2 × Master Mix RED Ampliqon, Denmark), 10 pmol of ngammacat = 4) and cox1 (parameters unlinked, each partition each primer and 3 μl of DNA sample. The thermal PCR profiles by codon had its own set of parameters; Nst = 6 rates = for cox1geneincludedaninitialdenaturationstepat94°Cfor invgamma). The partitions by codon evolved under different 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, rates (preset applyto = (all) ratepr = variable). Model selection annealingat52°Cfor30sandextensionat72°Cfor120s, was estimated using ModelTest (Posada and Crandall 1998). followed by a final extension step at 72 °C for 7 min. The PCR Four chains were run simultaneously for 2 × 105 generations, conditions of the ITS1 gene amplification consisted of initial the first 2000 trees discarded as burnin. The remaining trees Parasitol Res

Fig. 1 Map of Guilan Province, Iran. Districts are named and the number negative for T. regenti infected ducks. The numbers in the of ducks examined for each district listed. The black circles indicate districts circleds refer to the samples that were included in the molecular positive for T. regenti infected ducks and gray circles indicate districts phylogenetic analysis were used to calculate a 50% majority-rule consensus tree Trichobilharzia regenti was found in 31/97 (32% prevalence) with posterior probabilities. Outgroups used for the cox1were ducks from 11 of the 16 localities (Table 1). The adult worm T. mergi, T. szidati,andT. stagnicolae (Brant and Loker 2009; morphological and egg measurements were within the range Fakhar et al. 2016). Because of the emerging increase in ge- of measurements presented in other studies reporting netic diversity within nasal species of Trichobilharzia found T. regenti (Fig. 2; Tables 3 and 4). recently (Jouet et al. 2010a, b; Fakhar et al. 2016), we used the The phylogenetic analysis of the cox1 (816 bp) datasets most variable nuclear region of the ITS, ITS1. However, to place the samples from this study within specimens de- align ITS1 with species other than the nasal species was not scribed genetically as T. regenti (Fig. 3),andtheydidnot possible, thus an ingroup analysis was performed. The new group with the previously reported clade of sequences generated by this study were deposited in GenBank Trichobilharzia cf. regenti (Jouet et al. 2010b;Fakhar (accession numbers MH198311–MH198319). See Table 2 for et al. 2016). The same pattern of relationships was support- the list of specimens with locality, references, and GenBank ed with the ITS1 (1032 bp) dataset (Fig. 4). Interestingly, accession numbers. This study was approved by ethics com- the previous genetic studies of nasal schistosomes from mittee of Guilan University of Medical Sciences (IR.GUMS. Iran included worms only from Spatula clypeata REC.1395.337). (formally Anas clypeata, Gonzales et al. 2009), while this study focused on domestic ducks, A. p. domesticus.Both duck species are dense and common inhabitants of rice Results fields and surrounding areas, compared to the other duck species that migrate through the area. Based on previous From our collections of 97 domestic ducks (Anas molecular studies thus far, nasal Trichobilharzia from platyrhynchos domesticus) from 16 localities, ducks other than S. clypeata typically have grouped with Table 2 Samplesusedinthisstudy

Avian schistosome species Snail host Avian host Country of Original GenBank GenBank Museum catalog number* Reference origin identifier ITS cox1

Trichobilharzia regenti Radix balthica France EAN9 HM439499 No record of museum voucher Jouet et al. 2008, 2010b Trichobilharzia regenti Mergus merganser France HAR1 HM439501 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Cygnus olor France CYA18 HM439500 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Anas platyrhynchos France BERS58 HM439502 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Anas platyrhynchos France CAN5 HM439494 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Mergus merganser France HAR11 HM439498 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Radix peregra France EAN9 HM439495 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Radix peregra France EAN78 HM439496 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Cygnus olor France CYA18 HM439497 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Anas platyrhynchos Iceland AC122 HM439503 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Anas platyrhynchos Iceland AC125 HM439504 No record of museum voucher Jouet et al. 2010b Trichobilharzia regenti Anas platyrhynchos Czech Republic AP GU233740 No record of museum voucher Trichobilharzia regenti Anas platyrhynchos Czech Republic Cz31 EF094538 No record of museum voucher Rudolfova et al. 2007 Trichobilharzia regenti Anas platyrhynchos Czech Republic Cz79 EF094540 No record of museum voucher Rudolfova et al. 2007 Trichobilharzia regenti Anas platyrhynchos Poland Pl20 EF094535 No record of museum voucher Rudolfova et al. 2007 Trichobilharzia regenti Aythya fuligula Poland Pl17 EF094534 No record of museum voucher Rudolfova et al. 2007 Trichobilharzia regenti Anas platyrhynchos Switzerland ad1_1400 AJ312049 No record of museum voucher Picard and Jousson 2001 Trichobilharzia regenti Radix balthica Switzerland ov2_1400 AJ312048 No record of museum voucher Picard and Jousson 2001 Trichobilharzia regenti Radix balthica Switzerland ov1_1400 AJ312047 No record of museum voucher Picard and Jousson 2001 Trichobilharzia regenti Radix peregra Denmark DK KP271015 ZMUC-TRE-10–12* Christiansen et al. 2016 Trichobilharzia regenti Anas platyrhynchos Iran NS7 MH198311 MH198315 Currently at university This study domesticus collection Trichobilharzia regenti Anas platyrhynchos Iran NS10 MH198316 Currently at university This study domesticus collection Trichobilharzia regenti Anas platyrhynchos Iran NS11 MH198312 MH198317 Currently at university This study domesticus collection Trichobilharzia regenti Anas platyrhynchos Iran NS17 MH198313 MH198318 Currently at university This study domesticus collection Trichobilharzia regenti Anas platyrhynchos Iran NS29 MH198314 MH198319 Currently at university This study domesticus collection Trichobilharzia regenti Anas platyrhynchos Iran NL MH410290 MH410294 Currently at university This study domesticus collection Trichobilharzia cf. regenti Anas clypeata France JIT11 HM439505 No record of museum voucher Jouet et al. 2010b Trichobilharzia cf. regenti Anas clypeata France JIT10 HM439493 No record of museum voucher Jouet et al. 2010b Trichobilharzia cf. regenti Anas clypeata Iran T39 KJ583237 KR108325 No record of museum voucher Fakhar et al. 2016 Trichobilharzia cf. regenti Anas platyrhynchos Iran T44 KJ583238 KR108326 No record of museum voucher Fakhar et al. 2016 Trichobilharzia cf. regenti Anas clypeata Iran 2IRF AB594831 No record of museum voucher Maleki et al. 2012 Trichobilharzia cf. regenti Anas clypeata Iran 15IRF AB594834 No record of museum voucher Maleki et al. 2012 Res Parasitol Trichobilharzia cf. regenti Anas clypeata Iran 9IRF AB594833 No record of museum voucher Maleki et al. 2012 Trichobilharzia cf. regenti Anas clypeata Iran 4IRF AB594832 No record of museum voucher Maleki et al. 2012 Trichobilharzia cf. regenti Anas clypeata Iran Tr1IRF AB594830 No record of museum voucher Maleki et al. 2012 aaio Res Parasitol Table 2 (continued)

Avian schistosome species Snail host Avian host Country of Original GenBank GenBank Museum catalog number* Reference origin identifier ITS cox1

Trichobilharzia cf. regenti Anas platyrhynchos Poland Pl27 EF094537 No record of museum voucher Rudolfova et al. 2007 Trichobilharzia cf. regenti Anas clypeata Poland Pl14 EF094533 No record of museum voucher Rudolfova et al. 2007 Trichobilharzia franki Radix auricularia Czech Republic FJ174530 No record of museum voucher Brant and Loker 2009 Trichobilharzia sp. A Anas americana USA W192 FJ174471 MSB:Para:18609 Brant and Loker 2009 Trichobilharzia sp. A Anas americana USA W182 FJ174525 MSB:Para:18574 Brant and Loker 2009 Trichobilharzia sp. B Anas americana USA W205 FJ174528 MSB:Para:18638 Ebbs et al. 2016 Trichobilharzia sp. C Lophodytes cucullatus USA W173 FJ174529 MSB:Para:18562 Brant and Loker 2009 Trichobilharzia physellae Physa gyrina USA W263 FJ174523 MSB:Para:178 Brant and Loker 2009 Trichobilharzia physellae Aythya affinis USA W193 FJ174518 MSB:Para:18610 Brant and Loker 2009 Trichobilharzia Spatula discors USA W137 FJ174498 MSB:Para:19497 Brant and Loker 2009 querquedulae Trichobilharzia Spatula discors USA E45 FJ174510 MSB:Para:24778 Brant and Loker 2009 querquedulae Trichobilharzia Spatula cyanoptera USA W180 FJ174505 MSB:Para:18573 Brant and Loker 2009 querquedulae Trichobilharzia Spatula clypeata USA W203 FJ174508 MSB:Para:18636 Brant and Loker 2009 querquedulae Trichobilharzia mergi Radix ampla Belarus NVRau24 JQ681539 No record of museum voucher Chrisanfova et al. 2009 Trichobilharzia mergi Radix ampla Belarus NRau5 JQ681535 No record of museum voucher Chrisanfova et al. 2009 Trichobilharzia sp. Radix luteola Nepal W515 KF672863 MSB:Para:18709 Devkota et al. 2014 Trichobilharzia sp. E Stagnicola sp. Canada W332 FJ174483 MSB:Para:18614 Brant and Loker 2009 Trichobilharzia sp. D Anas acuta Canada W344 FJ174487 MSB:Para:18625 Brant and Loker 2009 Trichobilharzia sp. E Stagnicola sp. Canada W376 FJ174485 MSB:Para:27676 Brant and Loker 2009 Trichobilharzia szidati Stagnicola elrodi USA FHL FJ174495 MSB:Para:27677 Brant and Loker 2009 Trichobilharzia cf. szidati Lymnaea stagnalis USA BSL FJ174496 MSB:Para:27678 Brant and Loker 2009 Trichobilharzia stagnicolae Mergus merganser USA W230 FJ174493 MSB:Para:18652 Brant and Loker 2009 Trichobilharzia stagnicolae Stagnicola USA DL FJ174489 MSB:Para:24775 Brant and Loker 2009 emarginata Trichobilharzia stagnicolae Stagnicola sp. USA TMont FJ174488 MSB:Para:27679 Brant and Loker 2009 Trichobilharzia stagnicolae Stagnicola USA W229 FJ174491 MSB:Para:179 Brant and Loker 2009 emarginata

*The Natural History Museum of Denmark, Copenhagen Parasitol Res

samples available for ITS1 than there were for cox1, highlight- ing the importance of using multiple gene regions for genetic comparisons. Some clades in the ITS1 tree were grouped as A and B (they do not represent monophyletic groups) to better summarize the variation within and between the different in- dividual specimens of T. regenti across the migration route of their bird hosts. But in the cox1 tree, most of the samples were from group A, or the sample did not have ITS1 sequence and thus we could not assign a group as in the ITS1 tree. Second, the intraspecific genetic variation of cox1 within T. regenti was higher compared to other species of Trichobilharzia,but still within the intraspecific range of variation (Table 5). The average intraspecific uncorrected p-distance for T. querquedulae, a species found in North and South America, South Africa, and New Zealand (Ebbs et al. 2016), was 1.1% and it was 0.53% for T. regenti only across Eurasia. Particularly for schistosomes, 5% for cox1 is a reasonable estimate as a cut off for species. Third, while it is still not clear what is T. cf. regenti, is it the same or a distinct species, the genetic variation estimated is within a 5% cut off for species. Since these are gene trees, not species trees, and the need for more information on the biology of this clade, it cannot be determined if T. regenti and T. cf. regenti are conspecifics. Fig. 2 Representative variation in egg morphology of T. regenti from the nasal cavity of the duck Discussion T. regenti, rather than the clade of T. cf. regenti (Jouet et al. 2010b;Fakharetal.2016). The results of this work show that Trichobilharzia regenti,a We used uncorrected p-distances as both a proxy for spe- species of avian schistosome that lives in the nasal cavities of cies differentiation and to highlight the genetic diversity in its anatid hosts, is not just restricted to Europe, but is likely a samples collected thus far (Table 5). First, there appears to parasite of Eurasia. The geographic distribution of T. regenti is be more genetic variation in ITS1 than cox1. This result is facilitated by the availability and migration of the duck hosts ambiguous since there were many more GenBank accession and the ability of this schistosome to use several species of Radix, a genus that is found commonly in the Eastern Table 3 Mean size (micrometer) of male Trichobilharzia regenti from Hemisphere. The survey of domestic ducks in rice fields to Anas platyrhynchos collected in Iceland and Iran understand the epidemiology and etiological agents of CD Skirnisson et al. 2012 This study showed that the life cycle of T. regenti can be completed in Northern Iran. While the snail host species is not yet known, Width at esophagus (middle region) 87 ± 12 79 domestic ducks do not migrate, and thus for them to become Maximum width (at acetabulum) 108 ± 11 94 infected, there must be a local population of snails that can Width at VSE region 87 ± 11 74 host T. regenti. In Europe, this is most commonly Radix Oral sucker length 61 ± 5 56 balthica (= peregra,=ovata); although, a laboratory main- Oral sucker width 50 ± 4 45 tained T. regenti is cycled through R. lagotis where we have Acetabulum length 50 ± 5 46 learned much about the biology of this species (e.g., Acetabulum width 56 ± 4 49 Rimnacova et al. 2017). Domestic ducks can also act as res- Acetabulum to anterior end 634 ± 53 622 ervoirs of this parasite in these rice fields, likely increasing the Acetabulum to VSE 74 ± 27 59 prevalence of outbreaks and densities of the schistosome. Acetabulum to gynaecophoric canal 756 ± 100 745 Different species of Lymnaeidae including Galba truncatula, Seminal vesicle length 632 ± 76 627 Lymnaea shiraziensis, Stagnicola palustris,andRadix Gynaecophoric canal, length 506 ± 55 474 auricularia have been reported from Guilan Province and Gynaecophoric canal, width 152 ± 20 127 are potential hosts (Ashrafi and Mas-Coma 2014; Ashrafi Longest fragment 15.4 mm 4.7 mm et al. 2015). Except G. truncatula restricted to the highlands of the province, the other three species are distributed in the Parasitol Res

Table 4 Measurements of Trichobilharzia regenti eggs from Anas platyrhynchos and Spatula clypeata in various localities

Definitive host Egg length Egg width L/W Country Reference

Anas platyrhynchos 316 ± 29 (260–397, n = 60) 72 ± 9 (56–90, n = 60) 4.4 Iceland Skirnisson et al. 2008 Anas platyrhynchos 300 ± 15 (n = 30) 68 ± 7 (n = 30) 4.4 Iran Fakhar et al. 2016 Anas platyrhynchos 290 ± 21 (n = 15) 68 ± 9 (n = 15) 4.3 France Jouet et al. 2010 Anas platyrhynchos 317 ± 27 (260–382, n = 56) 76 ± 7 (52–93, n = 56) 4.2 Iran This study Spatula clypeata 270 ± 10 (n = 30) 63 ± 4 (n = 30) 4.3 Iran Fakhar et al. 2016 Spatula clypeata 259 ± 17 (n = 10) 62 ± 6 (n = 10) 4.2 France Jouet et al. 2010 lowlands of Guilan Province where rice cultivation is the main to the (a) permanent and still water bodies, warmer tempera- agricultural activity. There are some studies with evidence of ture and lowlands (Bargues et al. 2001;Mas-Comaetal.2009) R. auricularia (= L. gedrosiana) as an intermediate host of and b) habitat present in the lowlands of Guilan at the coastal Trichobilharzia spp. in Iran (Farahnak and Essalat 2003; regions of Caspian Sea to include an altitude between − 27 and Athari et al. 2006; Gohardehi et al. 2013). It appears that 300 m a.s.l. Radix auricularia has been reported to transmit R. auricularia likely plays a major role in transmission of F. gigantica in the same area where T. regenti is prevalent avian schistosomes in the country. This snail is well adapted (Ashrafi et al. 2015).

Fig. 3 Phylogenetic tree based on Bayesian analysis of cox1. The gray shaded clade includes the nasal specimens of Trichobilharzia and the taxa analyzed for this study are in bold. All taxa are listed with their corresponding GenBank accession number (see Fig. 1 and Table 2 for additional information) and include taxa from Iran. Nodal support is indicated at the branching nodes as Bayesian posterior probabilities Parasitol Res

Fig. 4 Phylogenetic tree of nasal Trichobilharzia based on Bayesian analysis of ITS1. The taxa analyzed for this study are in bold and taxa with asterisks (*) include taxa from Iran. All taxa are listed with their corresponding GenBank accession number (see Fig. 1 and Table 2 for additional information). Nodal support is indicated at the branching nodes as Bayesian posterior probabilities

Table 5 Pairwise uncorrected p-distances among taxa in the ITS1 cox1 trees T. regenti–T.cf.regenti 2.1–4.0% (avg. 2.9–3.8% (avg. 3.2%) 3.16%) T. regenti A+B 1.0–2.8% (avg. 1.9%) 0.2–1.1% (avg. 0.53%) T. regenti group A 0.1–1.5% (avg. 0.8%) – T. regenti group B 0–1.5% (avg. 0.8%) – T.cf.regenti 0–1.8% (avg. 1%) 0.7–1.1% (avg. 0.9%) Trichobilharzia querquedulae*0.2%1.2% Trichobilharzia physellae 0.6% 0.9% Trichobilhazia franki 0.1% 0.4% T. querquedulae–Trichobilharzia Brazil 1.4% 9.8% KJ855995/KJ855997 T. physellae–Trichobilharzia Brazil KJ855995/KJ855997 1.6% 6.6% T. querquedulae–T. franki 1.6% 7.7% T. querquedulae–T. physellae 1.2% 9.5% T. physellae–T. franki 1.4% 8.1%

*Ebbs et al. 2016 Parasitol Res

Considering the species of Trichobilharzia that have been The occurrence of CD is high in aquatic areas where peo- reported thus far in Iran, there are two possibly three species. ple, snails, and birds or mammals all come into prolonged There were six studies (to include this one) that examined contact with water. Recent years have seen more effort to A. platyrhynchos and four that examined S. clypeata that survey and understand the transmission dynamics in aquacul- could be either T. regenti or T. cf. regenti (Athari et al. 2006; ture sites in Asia (e.g., Lui et al. 1980;Oshimaetal.1992; Gohardehi et al. 2013; Maleki et al. 2012; Mahdavi et al. Kullavanijaya and Wongwaisayawan 1993;Narainetal. 2013a, b; Fakhar et al. 2016). Of those, only Fakhar et al. 1994; Joshi and Phil 2002;Devkotaetal.2014, 2015; (2016), Maleki et al. (2012) and this study have genetic con- Jauhari and Nongthombam 2014). Domestic ducks harbor firmation of lineages. Other results found the following: (a) T. regenti in rice fields in Northern Iran and likely act as Maleki et al. (2012)examinedonlyS. clypeata and found T. reservoir hosts, contributing to the high prevalence of CD in cf. regenti, (b) Fakhar et al. (2016) examined both these areas. In this study, only nasal schistosomes were stud- A. platyrhynchos and S. clypeata, but found only T. cf. regenti, ied, but efforts need to be made to identify the other species of and (c) while this study found only T. regenti in A. p. schistosomes that reside in wild and domestic ducks, provide domesticus. Visceral species of schistosomes were also found sequence data for cercariae from snail hosts, as well as genet- in some of the duck hosts, but those have yet to be genetically ically determine the snail host. With these data, we will learn and morphologically characterized. Species of more about the specific epidemiology of CD in this area, and Trichobilharzia have been reported in snails from Iran, can focus on which species during which part of the season is Lymnaea gedrosiana, Stagnicola palustris,andLymnaea the most prevalent, and does it change over the season, or by stagnalis; however, none were characterized genetically ducks or snail species? These are the data that can be used to (Atharietal.2006; Gohardehi et al. 2013). In Europe, initiate appropriate control programs. L. stagnalis is the host for T. szidati. Yakhchali et al. (2016) genetically characterized cercariae from Radix auricularia Acknowledgements This work was performed as a MSc thesis at Guilan and reported two species that were similar to T. franki and to University of Medical Sciences (GUMS), Rasht, Iran, with collaboration of the University of New Mexico, USA. T. szidati, both species that are common in European ducks. Thus far, we do not know what species of snail the nasal Funding The project was funded by the Deputy of Research of GUMS schistosome uses in Iran. (No. 95110217). The variation in T. regenti and T. cf. regenti might be explained by the following non-mutually exclusive hypotheses: Compliance with ethical standards (1) All known samples from nasal tissues are T. regenti and the observed genetic variation can be explained by ecological sep- Conflict of interest The authors declare that they have no conflict of aration of host species (Spatula clypeata versus other anatids). interest. Snail hosts are critical here too (Ebbs et al. 2016), but have not Animal welfare All procedures performed in studies involving animals been found yet to host T. cf. regenti; (2) seasonality and host were in accordance with the ethical standards of the institution or practice can influence morphological and molecular variation at which the studies were conducted. This study was approved by ethics (Bayssade-Dufour et al. 2006; Jouet et al. 2010a; Stillson committee of Guilan University of Medical Sciences and Platt 2007) and may explain some of the differences; (3) (IR.GUMS.REC.1395.337). as proposed by Jouet et al. (2010a), the egg morphology of T. cf. regenti look like a species reported from Africa (Radix natalensis?). Thus, it might be proposed that the snail host References for T. cf. regenti is found in the African continent, rather than found in Eurasia, where species of nasal Trichobilharzia have Al-Khuzaee JHR (2009) Study about Swimmer’s itch disease (Al-Sharaa) been reported, but not from S. clypeata (Fain 1955, 1956). in AL Najaf-AL-Ashraf. Mag Al-Kufa Univ Biol 1:100–107 Thus, T. regenti transmission may be restricted to Eurasia. It Ashrafi K, Mas-Coma S (2014) Fasciola gigantica transmission in the is only with increased sampling across more species in more zoonotic fascioliasis endemic lowlands of Guilan, Iran: experimen- localities along the migratory flyway that we can understand if tal assessment. Vet Parasitol 205:96–106 T. cf. regenti represents a new species or encompasses intra- Ashrafi K, Valero MA, Peixoto RV, Artigas P, Panova M, Mas-Coma S (2015) Distribution of Fasciola hepatica and F. gigantica in the specific variation across geography and host-use. Ebbs et al. endemic area of Guilan, Iran: relationships between zonal overlap (2016) reported T. querquedulae across the northern and and phenotypic traits. Infect Genet Evol 31:95–109 southern hemispheres, but the genetic variation (cox1 avg. Athari A, Gohardehi S, Rostami-Jalilian M (2006) Determination of de- 1.2%) was not as much as it is between T. regenti and T. cf. finitive and intermediate hosts of cercarial dermatitis producing agents in northern Iran. Arch Iran Med 9:11–15 regenti (cox1avg.3.2%;Table5), but without more samples, it Bargues MD, Vigo M, Horak P, Dvorak J, Patzner RA, Pointier JP, is hard to know if that translates to different species nor how Jackiewicz M, Meier-Brook C, Mas-Coma S (2001) European much time since T. querquedulae has been so widespread. Lymnaeidae (Mollusca: Gastropoda), intermediate hosts of Parasitol Res

trematodiases, based on nuclear ribosomal DNA ITS-2 sequences. Lymnaea gedrosiana (Pulmunata: Lymnaeidae) in northwest Iran. Infect Genet Evol 1(2):85–107 Iran J Parasitol 8:423–429 Bayssade-Dufour C, Jouet D, Rudolfova J, Horak P, Ferte H (2006) International Rice Research Institute (2009) Annual Report, 40pp. http:// Seasonal morphological variations in bird schistosomes. Parasite books.irri.org/AR2009_content.pdf. Accessed 3 Oct 2018 13:205–214 Jauhari RK, Nongthombam PD (2014) Occurrence of a snail borne dis- Bowles J, McManus DP (1993) Rapid discrimination of Echinococcus ease, cercarial dermatitis (swimmer’s itch) in Doon Valley species and strains using a PCR-based RFLP method. Mol Biochem (Uttarakhand) India. Iran J Public Health 43:162–167 Parasitol 57:231–239 Joshi SK, Phil M (2002) Rice field work and the occupational hazards. Bowles J, Blair D, McManus DP (1995) A molecular phylogeny of the Occup Med 4:111–114 human schistosomes. Mol Phyl Evol 4:130–109 Jouet D, Ferte H, Depaquit J, Rudolfova J, Latour P, Zanella D, Brant SV, Loker ES (2009) Molecular systematics of the avian schisto- Kaltenbach ML, Leger N (2008) Trichobilharzia spp. in natural some genus Trichobilharzia (Trematoda: Schistosomatidae) in conditions in Annecy Lake, France. Parasitol Res 103:51–58 North America. J Parasitol 95:941–963 Jouet D, Skirnisson K, Kolarova L, Ferte H (2010a) Molecular diversity Christiansen AO, Olsen A, Buchmann K, Kania PW, Nejsum P, of Trichobilharzia franki in two intermediate hosts (Radix Vennervald BJ (2016) Molecular diversity of avian schistosomes auricularia and Radix peregra): a complex of species. Infect in Danish freshwater snails. Parasitol Res 115:1027–1037 Genet Evol 10:1218–1227 Devkota R, Brant SV, Thapa S, Loker ES (2014) Two avian schistosome Jouet D, Skirnisson K, Kolarova L, Ferte H (2010b) Final hosts and cercariae from Nepal, including a Macrobilharzia-like species from variability of Trichobilharzia regenti under natural conditions. Indoplanorbis exustus. Parasitol Int 63:374–380 Parasitol Res 107:923–930 Devkota R, Brant SV,Loker ES (2015) The Schistosoma indicum species Karamian M, Aldhoun JA, Maraghi S, Hatam G, Farhangmehr B, group in Nepal: presence of a new lineage of schistosome and use of Sadjjadi SM (2011) Parasitological and molecular study of the the Indoplanorbis exustus species complex of snail hosts. Int J furcocercariae from Melanoides tuberculate as a probably agent of Parasitol 45:857–870 cercarial dermatitis. Parasitol Res 108:955–962 Dvorak J, Vanacova S, Hampl V, Flegr J, Horak P (2002) Comparison of Kullavanijaya P, Wongwaisayawan H (1993) Outbreak of cercarial der- European Trichobilharzia species based on ITS1 and ITS2 se- matitis in Thailand. Int J Dermatol 32:113–115 quences. Parasitol 124:307–313 Leontovyc R, Young ND, Korhonen PK, Hall RS, Tan P, Mikes L, Kasny Ebbs ET, Loker ES, Davis NE, Flores V, Veleizan A, Brant SV (2016) M, Horak P, Gasser RB (2016) Comparative transcriptomic explo- Schistosomes with wings: how host phylogeny and ecology shap the ration reveals unique molecular adaptations of neuropathogenic global distribution of Trichobilharzia querquedulae Trichobilharzia to invade and parasitize its avian definitive host. (Schistosomatidae). Int J Parasitol 46:669–677 PLoS Negl Trop Dis 10:e0004406 Fain A (1955) Une nouvelle bilharziose des oiseaux: la trichobilharziose Lockyer AE1, Olson PD, Ostergaard P, Rollinson D, Johnston DA, nasale. Remarque sur l’importance des schistosomesd’oiseaux en Attwood SW, Southgate VR, Horak P, Snyder SD, Le TH, pathologiehumaine. Note préliminaire. Ann Soc Belge Med Trop Agatsuma T, McManus DP, Carmichael AC, Naem S, Littlewood 35:323–327 DT (2003) The phylogeny of the Schistosomatidae based on three Fain A (1956) Les schistosomes d’Oiseaux du genre Trichobilharzia genes with emphasis on the interrelationships of Schistosoma Skrjabin et Zakharow, 1920 au Ruanda-Urundi. Rev Zool Weinland, 1858. Parasitology 126:203–24 Botanique Africaines 54:147–178 Lui Z, Chen M, Kong H, Li L (1980) A survey of the aetiological agent of Fakhar M, Ghobaditara M, Brant SV, Karamian M, Gohardehi S, Bastani paddy field dermatitis with studies on the life cycle of R (2016) Phylogenetic analysis of nasal avian schistosomes Trichobilharzia jianensis. Acta Zool Sin 26:153–159 (Trichobilharzia) from aquatic birds in Mazandaran Province, north- Mahdavi SA, Farahnak A, Mobedi I, Molaei Rad MB, Azadeh H (2013a) ern Iran. Parasitol Int 65:151–158 Survey of migratory birds (Anatidae: Anas platyrhynchos) for schis- Farahnak A, Essalat M (2003) A study on cercarial dermatitis in tosome parasites from Mazandaran Province, northern Iran. Iran J Khuzestan province, southwestern Iran. BMC Public Health 3:35 Parasitol 8:333–336 Ghobaditara M, Fakhar M, Sharif M (2015) An overview on the present Mahdavi SA, Farahnak A, Mousavi SJ, Mobedi I, Rezaeian M, situation of cercarial dermatitis: a neglected zoonotic disease in Iran Golmohamadi T, Azadeh H, Gohardehi S (2013b) Prevalence of and the world. J Mazandaran Univ Med Sci 24:446–460 schistosome induced cercarial dermatitis in northern Iran. Asian Gohardehi S, Fakhar M, Madjidaei M (2013) Avian schistosomes and Pac J Trop Dis 3:37–40 human cercarial dermatitis in a wildlife refuge in Mazandaran Maleki SH, Athari A, Haghighi A, Taghipour N, Gohardehi SH, Tabaei Province, northern Iran. Zoonoses Public Health 60:442–447 SS (2012) Species identification of birds nasal Trichobilharzia in Gonzalez J, Düttmann H, Wink M (2009) Phylogenetic relationships Sari, north of Iran. Iran J Parasitol 7:82–85 based on two mitochondrial genes and hybridization patterns in Mas-Coma S, Valero MA, Bargues MD (2009) Fasciola, lymnaeids and Anatidae. J Zool 279:310–318 human fascioliasis, with a global overview on disease transmission, Hoberg EP, Pilitt PA, Galbreath KE (2009) Why museums matter: a tale epidemiology, evolutionary genetics, molecular epidemiology and of pinworms (Oxyuroidea: Heteroxynematidae) among pikas control. Adv Parasitol 69:41–146 (Ochotona princeps and O. collaris) in the American west. J Narain K, Mahanta J, Dutta R, Dutta P (1994) Paddy field dermatitis in Parasitol 95:490–501 Assam: a cercarial dermatitis. J Communicable Dis 26:26–30 Horak P, Kolarova L, Dvorak J (1998) Trichobilharzia regenti n. sp. Omar HM, Omer OH, Al-Dhubaibi MS (2016) Gigantobilharzia, possi- (Schistosomatidae, Bilharziellinae), a new nasal schistosome from ble cause of cercarial dermatitis: case report. Int J Health Sci 10: Europe. Parasite 5:349–357 147–150 Horak P, Mikes L, Lichtenbergova L, Skala V, Soldanova M, Brant SV Oshima T, Kitaguchi T, Saito K, Kanayama A (1992) Studies on the (2015) Avian schistosomes and outbreaks of cercarial dermatitis. epidemiology of avian schistosome dermatitis caused by the cercar- Clin Microbiol Rev 28:165–190 iae of Gigantobilharzia sturniae Tanabe, 1951 an attempt to prevent Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of paddy dermatitis using Cartap Padan. Jpn J Parasitol 41:194–201 phylogenetic trees. Bioinform Appl Note 17:754–755 Picard D, Jousson O (2001) Genetic variability among cercariae of the Imani-Baran A, Yakhchali M, Malekzadeh-Viayeh R, Farahnak A (2013) Schistosomatidae (Trematoda: Digenea) causing swimmer’s itch in Seasonal and geographical distribution of cercarial infection in Europe. Parasite 8:237–242 Parasitol Res

Pleijel F, Jondelius U, Norlinder E, Nygren A, Oxelman B, Schander C, Skirnisson K, Kolarova L, Horak P, Ferte H, Jouet D (2012) Sundberg P,Thollesson M (2008) Phylogenies without roots? A plea Morphological features of the nasal blood fluke Trichobilharzia for the use of vouchers in molecular phylogenetic studies. Mol regenti (Schistosomatidae, Digenea) from naturally infected hosts. Phylogenet Evol 48:369–371 Parasitol Res 110:1881–1892 Posada D, Crandall KA (1998) Modeltest: testing the model of DNA Stillson LL, Platt TR (2007) The crowding effect and morphometric substitution. Bioinformatics 14:817–818 variability in Echinostoma caproni (Digenea: Echinostomatidae) Rimnacova J, Mikes L, Turjanicova L, Bulantova J, Horak P (2017) from ICR Mice. J Parasitol 93:242–246 Changes in surface glycosylation and glycocalyx shedding in Valkiunas G, Atkinson CT, Bensch S, Sehgal RNM, Ricklefs RE (2008) Trichobilharzia regenti (Schistosomatidae) during the transforma- Parasite misidentifications in GenBank: how to minimize their num- tion of cercaria to schistosomulum. PLoS One 12:e0173217 ber? Trends Parasitol 24:247–248 Rudolfova J, Littlewood DTJ, Sitko J, Horak P (2007) Bird schistosomes Yakhchali M, Hosseinpanahi A, Malekzadeh-Viayeh R (2016) Molecular – of wildfowl in the Czech Republic and Poland. Folia Parasit 54:88 93 evidence of Trichobilharzia species (Digenea: Schistosomatidae) in ’ Schuster RK, Aldhoun JA, O Donovan D (2014) Gigantobilharzia the snails of Lymnaea auricularia from Urmia suburb, Northwest melanoidis n. sp. (Trematoda: Schistosomatidae) from Melanoides Iran. Iran J Parasitol 11:296–302 tuberculate (Gastropoda: Thiaridae) in the United Arab Emirates. Parasitol Res 113:959–972