The aim of this study was to elucidate the species diversity of larva trematodes in the Mazandaran Province, Northern Iran. In this cross-sectional study, a total of 3,266 lymnaeid snails from 3 species were collected from different parts of streams, swamps, rice fields and rivers in the central areas of Mazandaran Province (Sari, Neka, Qaemshahr and Savad-Koh cities), during April to September, 2008. The samples were tested by crushing and emerging methods. From the total of examined snails, 119 (3.6%) were found to be infected with the lymnaeid snails. Lymnaea gedrosiana were found to be infected with the Furcocercariae of Diplostomidae, Clinostomidae, Echinostomatidae and also cercariae of the Plagiorchiidae. The latest infection was found to be in L. palustris. The Mazandaran Province with its temperate climate is a suitable place for living of snails, particularly lymnaeidae, that could have a significant role as an intermediate host of diseases.
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Pond snails (Lymnaeidae) are fresh water gastrepoda. Their preferred habitats are stagnant in the slow streaming water with heavy vegetation. These snails are hermaphroditic.
Three species of lymnaeids were reported throughout the Mazandaran Province: L. (stagnicola) palustris, a second intermediate host of F. hepatica; Galba (Lymnaea) truncatula, the main intermediate host of F. hepatica and Radix (Lymnaea) gedrosiana, a member of the auricularia complex transmitting F. gigantica (Moghaddam et al., 2004a). Athari et al. (2006) reported that infection rate of animal schistosoma in L. gedrosiana and L. palustris in North of Iran was 0.17 and 0.06%. Furthermore, infection rate with other Furcocercariae in these snails was 0.27 and 0.2%, respectively (Athari et al., 2006). In the Khouzestan Province, South Western of Iran, from 2000 L. gedrosiana snails collected, 48 (2.1%) were infected with animal Schistsoma (Farahnak and Essalat, 2003).
The role of lymnaeid snails as the first intermediate host of animal schistsoma has been shown throughout the world (Ferte et al., 2005).
The susceptibility of L. gedrosiana to F. hepatica under laboratory conditions has been reported in Iran. Lymnaeid snails associated with larvae stages of Fasciolid sp. have been reported from Guilan Province (Ashrafi et al., 2004) and Mazandaran Province (Moghaddam et al., 2004b). In Guilan Province, from the total of 4,830 different snails studied, only seven (0.35%) of L. gedrosiana were found to be infected with larvae stages of Fasciola sp. (Ashrafi et al., 2004).
The most important and widespread intermediate host of F. hepatica in Europe, Asia, Africa and North America was found to be L. truncatula (Soliman, 2008) while, in Iran, L. gedrosiana was reported to be a proper intermediate host for F. gigantica (Ashrafi et al., 2004) and cercarial dermatitis (Athari et al., 2006; Farahnak and Essalat, 2003).
The various studies in many countries indicated that pond snails (Lymnaeidae) were infected with larvae of Fasciolid sp. (De kock et al., 2003; Pointier et al., 2006; Bargues et al., 2007; Soliman, 2008; Barragan-Saenz et al., 2009) and trematodes such as Paramphistomum (Dryffuss et al., 2004), Holostom (Klockars et al., 1928), Trichobilharzia, Diplostomidae, Plagiorchiidae, Echinostomatidae, Monostom, Tetracotyle Metacercariae, Schistosomatidae, Azygiidae, Notocotyidae and Strigeidae (Faltynkova and Haas, 2006). In contrast, Bin Dajem (2009) in his study revealed none trematode immature stages in different snails under study.The lack of comprehensive studies on these snails, especially in relation to rate of infection, in the Mazandaran Province, was the major factor for the decision to conduct the present study. The aim of this study was to evaluate the occurance of trematodea infections in Lymnollect and recognize of lymnaeid snails using the crushing and emerging methods.
MATERIALS AND METHODS
This study was conducted on 3,266 lymnaeid snails in the central area of Mazandaran Province (Sari, Neka, Qaemshahr, Svad-koh cities) from April to September 2008, by a wooden handle paddle with 1.5 m long and net size of 30x30 cm. Snails were collected from agriculture canals, various streams, swamps, rice fields and rivers. Samples then transferred to Sari Health Research Center. After identification of species, each 1 to 2 snails were placed in a Petri-dish containing dechlorinated water and then, they were placed against light for 3 to 5 h or over night at room temperature. The snails were examined for the presence of cercariae by shedding and crushing methods. If no cercariae shedding were observed, snails were pressed and crushed between two square pieces of 15x15 glasses and studied in order to find cercariae, sporocysts and redia. Collected cercariae were observed carefully and mixed in 90% ethanol and 10% formalin and cleared in lactophenol and/or stained with azocarmin and neutral red. After measuring and drawing different parts of cercariae, the genus of cercariae were identified by a systematic key reference (Frandsen and Christensen, 1984; Christian, 2003).
RESULTS AND DISCUSSION
All of furcocercariae and Echinostom cercariae were found in L. gedrosiana in Sari, center of Mazandaran Province. Plagiorchiidae cercariae were common in all localities (Table 1).
In this study, Sari with 92 (9%) positive cases, showed the most infection, followed by, Savad-koh with 15 (2%) positive cases, Neka with 9 (1.2%) positive cases and Qaemshahr with 3 (0.4%) positive cases (Table 2). Cercariae with 4 families of digenean parasites were recorded from L. gedrosiana in which infection rate of this snail was 4.6%. L. Palustris was infected only by plagiorchiidae cercariae and its infection rate was 1.7%. Athari et al. (2006) in North of Iran showed less prevalence of trematode infection in these snails. This difference may be due to difference in study area. L. truncatula showed no infection in the above cities (Table 2). Ashrafi et al. (2004) also in a study on Fasciola in Guilan Province didnt report any cases of Fasciola cercaria. Prevalence of digenean larva, including Diplostomidae, was extremely high in L. gedrosiana from Sari (Table 1).
Lymnaeid snails from Mazandaran Province of Northern Iran
Snails occurrence from four geographical area in mazandaran province
1Cercariae species were as follows: Diplostomidae cercariae (n = 84), 2Plagiorchiidae cercariae (n = 32), Echinostomatidae cercariae (n = 1), 3Clinostomidae (n = 2), 4One case of Plagiorchiidae sporocyst was obtained
The prevalence of infected snails in central areas of Mazandaran Province
Kinds of detected cercariae (a) Echinostomatidae, (b) Clinostomidae, (c) Diplostomidae and (d) Plagiorchiidae
Trematodes are a diverse group of endoparasite requiring molluscan and vertebrate as intermediate and definitive host in their life cycle. Intramolluscan trematode parasitism is frequently associated with the alteration of a host's growth, fecundity or survival (Sorensen and Minchella, 1998) and snails susceptibility to trematodes is highly specific (Kalbe et al., 1997). The distribution of freshwater mollusks varies with physical, chemical and biological characteristics (Sharpe, 2002). The DNA sequence and isoenzyme studies have been done for the detection of larva stages trematodes and distinction of snails (Ashrafi et al., 2004; Mas-coma et al., 1999).
This study provides to estimate larva trematode parasites among lymnaeid snails and their zoonotic importance in animal or on human health. The characteristics of Mazandaran Province includes high humidity, rain fall, temperature 20-30°C (April to September), agricultural tradition, mainly rice, numerous stagnant water collections and irrigation canals rich in aquatic vegetation. The presence of all necessary conditions above suggest an appropriate condition for snails and all of the examinations performed verify the role of lymnaeid snails as intermediate host for larvae trematodes.
Most prevalence of snails in these regions is May to September. Moreover, in this study, most infection rates of snails were found in August to September, in Seyedmahale area of Sari.
Sporocyst of Plagiorchiidae
In the present study, Plagiorchiidae, Diplostomidae, Clinostomidae and Echinostomatidae families cercariae were found by shedding and crushing methods.
In this study, 0.97% of snails L. gedrosiana and L. palustris were infected with species of Plagiorchiidae (Fig. 3). The first intermediate hosts for Plagiorchiidae are freshwater snails; the second intermediate ones are fish, arthropods, mollusks (snails) and definitive hosts are vertebrates including human (Ghobadi and Farahnak, 2004; Fried et al., 2004; Farahnak et al., 2005). Plagiorchiids are parasite of intestine, gallbladder, bileduct or cloaca of vertebrates (Fried et al., 2004). The only genus that infects human is Plagiorchis. P. muris has been reported in natural infections in human (Hong et al., 1996).
Human infections with P. philippiniensis, P. javensis and P. harinasutai have been reported in Philippines, Indonesia and Thailand (Kumar, 1999).
Plagiorchiidae have been reported in snails Bellamya (Viviparous) bengalensis (Ghobadi and Farahnak, 2004) and Mellanoides tuberculata (Farahnak et al., 2005) from the Khouzestan Province, in South Western Iran. In the study conducted in Germany, this trematode has been reported from L. palustris and Radix auricolaria (Faltynkova and Haas, 2006).
In this study, 0.1% of snails L. gedrosiana were infected with Echinostomatidae Cercariae. These Cercariae may infect fish, molluscs (Snails, clums) and amphibians (tadpoles, frogs) (Fried et al., 2004; Farahnak et al., 2005).
Adult Echinostomatidae infect a variety of mammals (including humans) and aquatic birds (Anonymous, 1995). Two separate life cycles were demonstrated in endemic areas, i.e., the human cycle and the Sylvatic cycle (Bundy et al., 1991). In the endemic areas, the disease occurs focally and is associated with common socio-cultural practices (Carney, 1991). In light to moderate infections, anemia, headache, gastric pain and lose stools have been reported. Heavy infections are associated with eosinophilia, abdominal pain, profuse watery diarrhea, anemia, edema and anorexia (Anonymous, 1995; Chattopayday et al., 1990). Farahnak et al. (2005) reported Echinostomatidae cercariae in the Melanoides tuberculata from Khouzestan Province, South Western Iran (Farahnak et al., 2005). This trematode has been reported in L. palustris and Radix auricularia from Germany (Faltynkova and Haas, 2006).
In this study, 8.2% of snails L. gedrosiana were infected by Diplostomidae cercariae. In the life cycle of the Diplostomidae, fish, snails, amphibians and reptiles are sources of metacercariae and piscivorous birds and mammals are considered as final hosts (Fried et al., 2004; Cribb et al., 2003).
It should be noted that cercariae of all diplostomatids are fork-tailed (Cribb et al., 2003). Diplostomidae cercariae encyst in a variety of organs, including inside of the eye ball (Machado et al., 2005). Infections with Neodiplostomum soulensis have been reported in humans. Examination of stool samples from military soldiers in Korea revealed that the prevalence of infection was 0.4% (Huh et al., 1994). Infections with F. seolensis are predominantly reported from Korea (Hong and Shoop, 1994; Chai and Lee, 2002; Chai and Shih, 2002).
In this study, 0.2% of snails L. gedrosiana were infected with clinostomidae cercariae. Definitive hosts of Clinostomidae are piscivorous birds, including, herons, pelicans, cormorants and darters, while the second intermediate hosts are fish. It should be noted that C.complanatum develops through species of lymnaea (Cribb et al., 2003). Human infection by Clinostomum is considered rare and is known to occur as a result of eating raw fresh-water fish such as carp, cyprinus carpio and crucian carp and carassius ouratus (Chung et al., 1995).
In the literature reviewed, 44 species of Clinostomum were recorded; from those, 19 species were recorded in Asia, Africa and America, which were identified as synonyms of C. complanatum (Chung et al., 1995).
The first human case of Clinostomum pharyngitis was reported in Taegu, Korea (Chung et al., 1995). The first human infection with C. complanatum had been reported from Japan, all additional case records have also been reported in Japan (Muller, 2002).
In addition to trematodes mentioned in this study, infection of L. palustris to larvae of Azygiidae (Faltynkova and Haas, 2006) and infection of Radix (Lymnaea) auricularia to trematodes such as Strigeidae, Schistosomatidae, Sanguinicolidae and Notocotylidae (Faltynkova and Haas, 2006), Trichobilharzia ocellata (Zbikowska, 2004) and T. franki (Ferte et al., 2005) have been demonstrated in the literature.
The suitable humid climate in Mazandaran Province, Northern Iran and presence of naturally infected snails, coupled with other conditions in this geographical area, verify the existence of zoonotic diseases which are transferable by snails.
This study was supported by the grant No. 87-24 from Deputy of Research, Mazandaran University of Medical Sciences, Sari, Iran. We wish to thank Dr. Athari and Dr. Mansorian for the review of snail samples and also to Mr. Gohar-Dehi, for his assistance with this project.
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