Parasitology Research

, Volume 108, Issue 4, pp 955–962

Parasitological and molecular study of the furcocercariae from Melanoides tuberculata as a probable agent of cercarial dermatitis


  • Mehdi Karamian
    • Department of Parasitology and MycologyShiraz University of Medical Sciences
  • Jitka A. Aldhoun
    • Department of ZoologyNatural History Museum
  • Sharif Maraghi
    • Department of Parasitology and Mycology, Tropical Diseases, Thalassemia and Haemoglobinopathy Research CenterJundishapur University of Medical Sciences
  • Gholamreza Hatam
    • Department of Parasitology and MycologyShiraz University of Medical Sciences
  • Babak Farhangmehr
    • Department of Parasitology and MycologyShiraz University of Medical Sciences
    • Department of Parasitology and MycologyShiraz University of Medical Sciences
Original Paper

DOI: 10.1007/s00436-010-2138-x

Cite this article as:
Karamian, M., Aldhoun, J.A., Maraghi, S. et al. Parasitol Res (2011) 108: 955. doi:10.1007/s00436-010-2138-x


Cercarial dermatitis is caused by animal schistosomes in many parts of the world including Iran. Various stages of the parasites have been studied in intermediate and definitive hosts in northern and southwestern Iran; however, no molecular investigation for species identification and classification of these agents has been carried out, so far. In the present study, more than 3,800 aquatic snails were collected from water sources of Khuzestan, southwest Iran. The snails were identified as Lymnaea gedrosiana, Radix auricularia, Melanoides tuberculata, Melanopsis sp. and Physa acuta. They were examined for schistosome cercariae. Two specimens of M. tuberculata were infected with ocellate furcocercariae belonging to the family Schistosomatidae. Molecular studies were carried on these schistosomatid samples. Both samples belong to an unknown schistosome species and genus in sister position to GigantobilharziaDendritobilharzia clade. They differ from other species in their ITS sequence region as well as in their intermediate host specificity—This is one of the first reports on schistosome cercariae from M. tuberculata and the first including molecular data. Due to adaptability and invasiveness of this snail species, this new schistosome species, as a potential causative agent of cercarial dermatitis in humans, needs to be studied further.


Cercarial dermatitis or swimmer's itch is an allergic response that often occurs in humans after repeated exposure to water infected with furcocercariae of animal schistosomes (Kolárová 2007; Rudolfová et al. 2007). These cercariae belong to different species, and a variety of aquatic snails are susceptible to be infected with them (Brant and Loker 2009a, b).

The cercariae of Trichobilharzia sp., as the main agent of cercarial dermatitis, find and recognize their bird hosts by different phases of host-finding behaviour (Feiler and Haas 1998a, b), but due to similar lipid composition of avian and human skin, they cannot differentiate humans and water birds and accidentally invade human skin (Haas and van de Roemer 1998). Furthermore, some of these avian schistosomes are more successful in dermal invasion of humans compared with human schistosomes (Haas and Haeberlein 2009). Clinical signs of the disease include maculopapular lesions usually accompanied by severe itching (Olivier 1949). Recent studies show that schistosomes can develop to a certain degree in non-specific hosts as well, and schitosomula may migrate to visceral parts of the human body. Infections of lungs and other organs have been observed in experimentally infected mice (Horák and Kolárová 2000; Chanová et al. 2007). Trichobilharzia regenti has been found in the central nervous system of mice, and it could cause nerve palsy (Horák et al. 1999). Due to this issue, as well as multiple epidemic occurrence and its economic effects on agricultural and recreational areas, cercarial dermatitis is classified as an important emerging disease today (Larsen et al. 2004; Rao et al. 2007).

In Iran, in the past, the main focus was on human schistosomiasis caused by Schistosoma haematobium. It was prevalent in Khuzestan province, southwest Iran (Ansari and Faghih 1953; Massoud et al. 1982); however, it was eradicated (Rokni 2008). Recently, cercarial dermatitis and its causative agents gain more attention. It has been reported in the northern part of Iran (Sahba and Malek 1979) and in Khuzestan (Farahnak and Essalat 2003). Adult worms of dermatitis-producing schistosomes have been isolated from bird hosts in northern Iran (Athari et al. 2006). A number of animal schistosomes, i.e. Orienthobilharzia, have been found in Fars province (Maleki et al. 1994).

Khuzestan province with tropical weather is suitable for the establishment of the life cycle of schistosomes, including the agents of cercarial dermatitis. Residents use the water resources for agriculture and swim in the ponds and rivers where many snails live. A few investigations regarding the causative agents of cercarial dermatitis have been carried out in Iran, and Lymnaea and Planorbis snails have been reported as intermediate hosts (Farahnak and Essalat 2003; Athari et al. 2006). Melanoides tuberculata has also been reported to be an intermediate host of a number of cercariae including Schistosomatidae (Farahnak et al. 2005). However, no molecular identification of these agents has been carried out so far. In the present study, efforts were made to find out the agents of cercarial dermatitis based on molecular analysis of selected DNA fragments. Different species of aquatic snails were studied as possible intermediate hosts.

Materials and methods

The study was conducted from October 2008 to February 2009 in the Khuzestan province, southwest of Iran. Different localities including Ahwaz, Shadegan, Dezful and Susangerd were investigated (Fig. 1). A total of 3,830 snails were collected from agriculture canals, rivers, swamps and drains.
Fig. 1

Map of the state of Khuzestan, Iran. Numbers correspond to locality data in Table 2

The snails were collected by hand and a handle paddle and transferred to Ahwaz Health Research Centre where preliminary works were carried out. In the laboratory, the snails were pooled according to species in bottles with de-chlorinated tap water. The snails were then transferred to the taxonomy laboratory at the Medical School of Shiraz University of Medical Sciences. They were divided to beakers and placed under artificial light for at least 2 h or overnight to induce shedding. Snails from positive batches were individually isolated for a second assay. Cercariae found were identified by systematic key references (Frandsen and Christensen 1984).

Ocellate furcocercariae (with pigmented eye spots) were first observed and photographed while alive and then preserved in 96% ethanol for molecular investigations or for staining with FAAL (formalin, azocarmine, alcohol and lactic acid; Sadjjadi and Massoud 1999) and Ehrlich's alum haematoxylin and eosin.

The furcocercariae stained with FAAL were photographed using normal light as well as phase contrast. They were also graphed with camera lucida. Morphological features of the stained furcocercariae were measured using micrometry.

Also, a number of collected snails were crushed against a glass plate and then examined for schistosome cercariae and/or sporosysts.

Snails infected with furcocercariae were preserved in individual containers using 96% ethanol.

Sequencing of ITS region of rDNA (containing a part of 28S and 18S rRNA genes, and the whole 5.8S rRNA gene, ITS1 and ITS2) was carried out for the identification of furcocercariae from infected snails. Sequencing of ITS2 was used for snail typing. Genomic DNA was extracted from alcohol-fixed samples (furcocercariae collected from individual snails or foot of an infected snail) using the Qiamp DNA Mini Kit (Qiagen) according to the manufacturer's instruction. For the cercariae, genomic DNA was amplified using its4Trem and its5Trem primers (Dvorák et al. 2002). For amplification of the snail DNA, C1a and ITS3 primers (Bargues et al. 2001) were used. Polymerase chain reaction (PCR) products were purified from the PCR mixture using SureClean (Bioline) or from agarose gel using MinElute Gel Extraction Kit (Qiagen). Purified PCR products were sequenced directly using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). For cercariae, internal primers its2Trem, its3Trem (Dvorák et al. 2002), ITS6-F and ITS6-R (Kolárová et al. 2006) were used in addition to the PCR primers.

The sequences were deposited in the GenBank database under the accession numbers HM803239 and HM163469 (Table 1).
Table 1

Isolates of schistosomes were found in snails used for phylogenetic analysis






Latitude; longitude

Avian schistosomatid

SMS1 accession number (HM163469)


M. tuberculata

Shadegan, Iran

30.6450° N; 48.6312° E

Avian schistosomatid

SMS2 accession number (HM803239)


M. tuberculata

Ahwaz, Iran

31.3257° N; 48.6143° E

Phylogenetic analyses were performed using the newly obtained sequences, and sequences were downloaded from GenBank (Table 2). Sequences of Diplostomum huronense and Diplostomum indistinctum (accession numbers AY123044 and AY123043) were used as an outgroup.
Table 2

GenBank sequences of bird schistosomes used for phylogenetic analysis


Accession number

Allobilharzia visceralis


A. visceralis W241


A. visceralis W246


A. visceralis W262


Austrobilharzia variglandis Pf


A. variglandis O4


A. variglandis O9


A. variglandis O10


A. variglandis O12


Bilharziella polonica Cz39


Dendritobilharzia pulverulenta Dp


D. pulverulenta W230


Gigantobilharzia huronensis W30


G. huronensis W53


Avian schistosome W214


Avian schistosome W217


Avian schistosomatid AvL


Avian schistosomatid AvDS


Avian schistosomatid AvN


Avian schistosomatid AvNd


Avian schistosomatid F4


Avian schistosomatid F5


Avian schistosomatid F6


Trichobilharzia sp. W205


Trichobilharzia sp. A W149


Trichobilharzia sp. A W192


Trichobilharzia sp. A W213


Trichobilharzia sp. C W173


Trichobilharzia sp. E W376


Trichobilharzia sp. 1 Is2


Trichobilharzia sp. 1 Is29


Trichobilharzia sp. 1 Is32


Trichobilharzia sp. 1 Is47


Trichobilharzia sp. 1 Is49


Trichobilharzia sp. 2 Is5


Trichobilharzia sp. 2 Is18


Trichobilharzia sp. 2 Is20


Trichobilharzia sp. 2 Is27


Trichobilharzia sp. 2 Is38


Trichobilharzia sp. 3 Pl7


Trichobilharzia sp. 3 Pl10


Trichobilharzia sp. 3 Is11


T. brantae W331


T. brantae W340


T. brantae W346


T. franki F3


T. franki FPC


T. franki Is23


T. franki Is25


T. franki M2


T. franki Ra1


T. franki Ra3


T. franki O14


T. franki V2


T. franki


T. physellae W171


T. physellae W230


T. physellae W234


T. physellae W263


T. physellae W249


T. physellae W256


T. querquedulae W135


T. querquedulae W137


T. querquedulae W156


T. querquedulae W158


T. querquedulae W183


T. querquedulae W190


T. querquedulae W203


T. querquedulae SDS


T. regenti AP


T. regenti Cz79


T. regenti Pl17


T. regenti Pl27


T. regenti


T. stagnicolae W164


T. stagnicolae W224


T. stagnicolae W229


T. stagnicolae W230


T. stagnicolae W240


T. stagnicolae DouglasLake


T. szidati F1


T. szidati Hrd


T. szidati Par


T. szidati Prib


T. szidati Vlk


T. szidati ToA


T. szidati ToE


T. szidati BlindSuckerLake


T. szidati FlatHeadLake


T. szidati Ls2


T. szidati Ls5


T. szidati Pl3


T. szidati Pl21


Orientobilharzia turkestanicum


Schistosoma bovis SbITSbt


S. edwardiense


S. haematobium ShITSmir


S. hippopotami


S. mansoni


S. mansoni JE25


S. mansoni JE27


S. rodhaini


Sequences were aligned in ClustalX 2.0, and the alignment was manually refined in Bioedit 7.0.9. The non-repetitive region containing part of ITS1 and whole 5.8S rDNA and ITS2 was used for tree construction.

Maximum likelihood analysis was done in the program Phyml 3.0 (Guindon and Gascuel 2003) using the GTR + I + Γ model with parameters estimated by the software. Maximum parsimony analyses were carried out in PAUP* v.4.0 (Swofford 2002) using the heuristic search method and TBR swapping. The starting tree was constructed by stepwise addition, and one tree was held at each step. Nodal support was assessed by bootstrapping (100 replicates for both ML and MP). The phylogenetic analyses using the Bayesian method was performed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). Four simultaneous Markov Monte Carlo chains (temperature 0.2) were run for 500,000 generations under covarion GTR + I + Γ substitution model and sampled every 100 generations. The first 2,000 trees were discarded as the ‘burnin’.


The following snails were identified: M. tuberculata, Radix auricularia, Lymnaea gedrosiana, Physa acuta and Melanopsis sp. Their distribution and collection sites are shown in Table 3 and Fig. 1. The species of M. tuberculata infected with avian schistosomatid was also confirmed by sequencing of the ITS2 rDNA of this snail. It was deposited in GeneBank with the accession number HQ199839.
Table 3

List of the snails screened and collection locality


No. examined

No. infected


Latitude; longitude

Melanoides tuberculata


1. Dezful, Iran

32.4172° N; 48.3570° E

Melanoides tuberculata


3. Susangerd, Iran

31.5492° N; 48.1633° E

Melanoides tuberculata



4. Ahwaz, Iran

31.3257° N; 48.6143° E

Melanoides tuberculata



7. Shadegan, Iran

30.6450° N; 48.6312° E

Radix auricularia


1. Dezful, Iran

32.4172° N; 48.3570° E

Radix auricularia


6. Ahwaz, Iran

30.7176° N; 48.6760° E

Lymnaea gedrosiana


1. Dezful, Iran

32.4172° N; 48.3570° E

Lymnaea gedrosiana


2. Susangerd, Iran

31.5772° N; 48.1135° E

Physa acuta


1. Dezful, Iran

32.4172° N; 48.3570° E

Physa acuta


2. Susangerd, Iran

31.5772° N; 48.1135° E

Melanopsis spp.


3. Susangerd, Iran

31.5492° N; 48.1633° E

Melanopsis spp.


5. Ahwaz, Iran

30.9344° N; 48.3044° E

Two (0.09%) out of 2,294 M. tuberculata examined were infected with ocellate furcocercariae from the family Schistosomatidae (Figs. 2 and 3). Dimensions of the cercariae are listed in Table 4. No specimen of the other examined snail species was infected by ocellate furcocercariae.
Fig. 2

Avian schistosomatid shed from Melanoides tuberculata using phase contrast microscopy, ×10
Fig. 3

Cercaria from Melanoides tuberculata. a General view. b Detail of cercarial body. AO anterior organ, FC flame cell, ES eye spot, VS ventral sucker, PG penetration gland. Scales in micrometres

Table 4

Measurements of cercariae from ethanol-preserved specimens


Min–max (mean) in μm

Body length

124–134 (130)

Body width

50–65 (58)

Tail stem length

260–320 (284)

Tail stem width

32–38 (35)

Furca length

96–101 (98)

Furca width

19–24 (22)


16–21 (18) × 18–24 (20)

Eye spots

10–13 (12) × 10–14 (13)

Anterior end–acetabular

70–85 (78)

Anterior end–eye

63–72 (67)

Frontal organ length

45–54 (50)

Frontal organ width

30–36 (34)

Ratio, body/tail


Ratio, body/furcae


The phylogenetic tree made using the newly obtained sequences and the sequences downloaded from GenBank are shown in Fig. 4. In this figure, our isolates (sms1 and sms2) from M. tuberculata were in sister position to GigantobilharziaDendritobilharzia clade.
Fig. 4

The phylogenetic tree of schistosome isolates based on partial sequence of the ITS region constructed by maximum likelihood method. Node support is indicated by maximum parsimony and maximum likelihood bootstrap values and Bayesian posterior probabilities, respectively. The dash indicates no significant node support. The sequences of D. huronense and D. indistinctum were used as outgroup

All described avian schistosomes, and only some of the mammalian ones have eyespots (Snyder and Loker 2000). Therefore, we can suppose that the ocellate furcocercariae from M. tuberculata may belong to avian schistosomes.


Khuzestan province in southwest Iran with its warm/hot weather, humidity and suitable habitats for intermediate snail hosts of schistosomes was an endemic area of schistosomiasis in the past (Ansari and Faghih 1953). With successful schistosomiasis control programmes that started nearly five decades ago (Massoud et al. 1982), no cases of human schistosomiasis have been reported in Iran in recent years (Rokni 2008). However, animal schistosomes remain active in this area. Several cases of cercarial dermatitis have been reported annually, and different developmental stages of these parasites have been isolated from their intermediate and definitive hosts (Farahnak and Essalat 2003). The most common intermediate hosts of animal schistosomes in Iran are supposed to be members of the genus Lymnaea (Radix)—only one report (Farahnak et al. 2005) mentions isolation of schistosome furcocercariae from M. tuberculata (family Thiaridae). However, it seems clear that climate change and global warming will result in changes in aquatic environments (Mas-Coma et al. 2009). As a consequence, species composition of snail populations will also change. Similarly to other countries, Iran, particularly in the southern regions, has been influenced by global warming. In Khuzestan province with tropical climate, severe rainfall reduction has been observed in recent years. Drying of water resources due to rainfall decrease in this region had a significant effect on the schistosome life cycle and survival of the intermediate snail host (Fenwick et al. 2006). In the present study, the number of Lymnaea snails, as the main intermediate host of the animal schistosomes, seems to have decreased in the study area. On the other hand, M. tuberculata, with better resistance to environmental changes, has survived and is abundant in irrigation canals. M. tuberculata is an oriental species belonging to the family Thiaridae and has the capacity to colonize many types of habitats (Pointier and McCullough 1989). This snail is considered to be invasive after its introduction into new territories, and it has become established in numerous countries outside its native range (Pointier 2001; Rocha-Miranda and Martins-Silva 2006; Derraik 2008). Also, M. tuberculata has proved to be a compatible intermediate host for several trematode species of medical and veterinary importance (Dung et al. 2010; Mukaratirwa et al. 2004; Bogéa et al. 2005; Lun et al. 2005). In general, reports on snails of the group Prosobranchia serving as intermediate hosts for bird schistosomes are rare (Aldhoun et al. 2009). Since there was no documented report on infection of Melanoides sp. by schistosomes previously, they were not considered as reservoir hosts of schistosome parasites. On the contrary, Melanoides proved to be an effective competitor against Biomphalaria glabrata (Giovanelli et al. 2002) and Biomphalaria pfeifferi (Gashaw et al. 2008), intermediate hosts of Schistosoma mansoni, and was considered as an effective biological agent for control of human schistosomiasis.

The present study is the first to have identified schistosome cercariae from M. tuberculata by molecular methods. The ability of M. tuberculata to spread to different parts of the world, colony production by a single snail using parthenogenetic reproduction, their adaptation by a wide range of water conditions even brackish water bodies (Pointier and McCullough 1989) and good adaptation to anthropomorphic habitats (Facon et al. 2003) could enable them to play an important role in the spread of schistosome infection in different parts of the world. Schistosome cercariae types of human and animal from Planorbidea have been isolated (Athari et al. 2006). Infected M. tuberculata snails kept under laboratory conditions had been shedding cercariae for nearly 2 months. Number of cercariae released per day by the snails, approximately 20 mm big, were several hundreds. Therefore, with regard to infinite cercarial production capacity of schistosomes (Mas-Coma et al. 2009), it can be supposed that the infected Melanoides snails have a high capacity of spreading schistosomes in their natural habitats even if the prevalence of infected snails is low. Low prevalence of infected snails seems to be normal in areas where cercarial dermatitis occurs in humans (Loy and Haas 2001), and M. tuberculata could be very efficient vector under these conditions.

In the phylogenetic tree made using ITS sequences, the schistosome samples isolated from M. tuberculata form a separate clade in sister position to the Gigantobilharzia-Dendritobilharzia clade. Their ITS region sequence does not correspond with any schistosome sequence deposited in GenBank. In addition, these isolates are unique with regard to their intermediate host, which is a prosobranch snail Melanoides tuberculata. There have been rare reports on prosobranch snails serving as intermediate hosts for schistosomes (e.g. Pomacea paludosa (Leedom and Short 1981), Semisulcospira libertina (Ito 1960), Littoridina australis (Szidat 1958), Littorina planaxis (Penner 1950) and Valvata macrostoma (Aldhoun et al. 2009); however, so far, there has been no finding of schistosome cercariae from M. tuberculata. Our study of two isolates of ocellate furcocercariae found in M. tuberculata leads to conclusion that based on their position in the phylogenetic tree, morphology and intermediate host they belong to a new bird schistosome species and genus. Further investigations are necessary to find other stages of life cycle of this parasite and its final host.

In conclusion, considering the range of dispersal of M. tuberculata and their better adaptability to warm weather in comparison to other snails (such as Lymnea), they could be a potential source of cercarial dermatitis for human in many parts of the world where Melanoides is prevalent. Although the life cycle and final host are not known, based on their position in phylogenetic tree, morphology and intermediate host, the newly found cercariae from M. tuberculata probably belong to a new species and genus of bird schistosomes. Life cycle, infectivity of this parasite for humans and the prevalence of infection in Melanoides in different areas with this parasite need to be further investigated.


The authors would like to thank Dr. Mowlavi from the Department of Parasitology and Mycology, Tehran University of Medical Sciences, Tehran, Iran, for his kind help; Dr. B. Vazirian-Zadeh and Dr. M. Rahdar from Ahwaz University of Medical Sciences Mrs S. Kazemian and all the people from the Health Center of Khuzestan province. This study has been supported by the Shiraz University of Medical Sciences Grant No. 4312.

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© Springer-Verlag 2011