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

Authors

  • 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

Abstract

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.

Introduction

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.
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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

Determination

Isolate

Stage

Host

Locality

Latitude; longitude

Avian schistosomatid

SMS1 accession number (HM163469)

Cercariae

M. tuberculata

Shadegan, Iran

30.6450° N; 48.6312° E

Avian schistosomatid

SMS2 accession number (HM803239)

Cercariae

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

Isolate

Accession number

Allobilharzia visceralis

DQ067561

A. visceralis W241

EF071989

A. visceralis W246

EF071990

A. visceralis W262

EF071991

Austrobilharzia variglandis Pf

AY713963

A. variglandis O4

FJ469818

A. variglandis O9

FJ469822

A. variglandis O10

FJ469815

A. variglandis O12

FJ469813

Bilharziella polonica Cz39

EF094539

Dendritobilharzia pulverulenta Dp

AY713962

D. pulverulenta W230

EF071988

Gigantobilharzia huronensis W30

EF071986

G. huronensis W53

EF071987

Avian schistosome W214

GQ920622

Avian schistosome W217

GQ920621

Avian schistosomatid AvL

FJ786030

Avian schistosomatid AvDS

FJ786029

Avian schistosomatid AvN

FJ786028

Avian schistosomatid AvNd

FJ786027

Avian schistosomatid F4

FJ609412

Avian schistosomatid F5

FJ609413

Avian schistosomatid F6

FJ609414

Trichobilharzia sp. W205

FJ174571

Trichobilharzia sp. A W149

FJ174574

Trichobilharzia sp. A W192

FJ174572

Trichobilharzia sp. A W213

FJ174570

Trichobilharzia sp. C W173

FJ174576

Trichobilharzia sp. E W376

FJ174537

Trichobilharzia sp. 1 Is2

FJ469784

Trichobilharzia sp. 1 Is29

FJ469786

Trichobilharzia sp. 1 Is32

FJ469788

Trichobilharzia sp. 1 Is47

FJ469790

Trichobilharzia sp. 1 Is49

FJ469791

Trichobilharzia sp. 2 Is5

FJ469792

Trichobilharzia sp. 2 Is18

FJ469807

Trichobilharzia sp. 2 Is20

FJ469794

Trichobilharzia sp. 2 Is27

FJ469796

Trichobilharzia sp. 2 Is38

FJ469799

Trichobilharzia sp. 3 Pl7

EF094531

Trichobilharzia sp. 3 Pl10

EF094532

Trichobilharzia sp. 3 Is11

FJ469804

T. brantae W331

FJ174532

T. brantae W340

FJ174533

T. brantae W346

FJ174534

T. franki F3

FJ609411

T. franki FPC

FJ469820

T. franki Is23

FJ469805

T. franki Is25

FJ469809

T. franki M2

FJ469821

T. franki Ra1

AY713969

T. franki Ra3

AY713966

T. franki O14

FJ469811

T. franki V2

FJ469812

T. franki

AF356845

T. physellae W171

FJ174564

T. physellae W230

FJ174567

T. physellae W234

FJ174569

T. physellae W263

FJ174562

T. physellae W249

FJ174565

T. physellae W256

FJ174575

T. querquedulae W135

FJ174557

T. querquedulae W137

FJ174558

T. querquedulae W156

FJ174554

T. querquedulae W158

FJ174549

T. querquedulae W183

FJ174560

T. querquedulae W190

FJ174550

T. querquedulae W203

FJ174552

T. querquedulae SDS

FJ174548

T. regenti AP

GU233740

T. regenti Cz79

EF094540

T. regenti Pl17

EF094534

T. regenti Pl27

EF094537

T. regenti

AF263829

T. stagnicolae W164

FJ174540

T. stagnicolae W224

FJ174542

T. stagnicolae W229

FJ174543

T. stagnicolae W230

FJ174545

T. stagnicolae W240

FJ174544

T. stagnicolae DouglasLake

FJ174546

T. szidati F1

FJ609409

T. szidati Hrd

GU233735

T. szidati Par

GU233736

T. szidati Prib

GU233737

T. szidati Vlk

GU233739

T. szidati ToA

AY713970

T. szidati ToE

AY713971

T. szidati BlindSuckerLake

FJ174538

T. szidati FlatHeadLake

FJ174539

T. szidati Ls2

AY713961

T. szidati Ls5

AY713967

T. szidati Pl3

EF094530

T. szidati Pl21

EF094536

Orientobilharzia turkestanicum

HM803240

Schistosoma bovis SbITSbt

FJ588862

S. edwardiense

AY197344

S. haematobium ShITSmir

FJ588861

S. hippopotami

AY197343

S. mansoni

AF531314

S. mansoni JE25

AY446080

S. mansoni JE27

AY446081

S. rodhaini

AF531312

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’.

Results

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

Snails

No. examined

No. infected

Locality

Latitude; longitude

Melanoides tuberculata

232

1. Dezful, Iran

32.4172° N; 48.3570° E

Melanoides tuberculata

57

3. Susangerd, Iran

31.5492° N; 48.1633° E

Melanoides tuberculata

933

1

4. Ahwaz, Iran

31.3257° N; 48.6143° E

Melanoides tuberculata

1073

1

7. Shadegan, Iran

30.6450° N; 48.6312° E

Radix auricularia

4

1. Dezful, Iran

32.4172° N; 48.3570° E

Radix auricularia

30

6. Ahwaz, Iran

30.7176° N; 48.6760° E

Lymnaea gedrosiana

106

1. Dezful, Iran

32.4172° N; 48.3570° E

Lymnaea gedrosiana

30

2. Susangerd, Iran

31.5772° N; 48.1135° E

Physa acuta

330

1. Dezful, Iran

32.4172° N; 48.3570° E

Physa acuta

140

2. Susangerd, Iran

31.5772° N; 48.1135° E

Melanopsis spp.

470

3. Susangerd, Iran

31.5492° N; 48.1633° E

Melanopsis spp.

425

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.
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Fig. 2

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

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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

Dimensions

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)

Acetabulum

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

0.46

Ratio, body/furcae

1.33

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.
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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.

Discussion

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.

Acknowledgements

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