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Am. J. Trop. Med. Hyg., 75(2), 2006, pp. 295-302
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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Right arrow Fascioliasis

PLANT-BORNE HUMAN CONTAMINATION BY FASCIOLIASIS

KEYHAN ASHRAFI, MARIA ADELA VALERO, JAFAR MASSOUD, ABDOLRASOUL SOBHANI, SHAHRAM SOLAYMANI-MOHAMMADI, PATRICIA CONDE, MESSAUD KHOUBBANE, MARIA DOLORES BARGUES, AND SANTIAGO MAS-COMA*
Department of Medical Microbiology, Faculty of Medicine, Gilan University of Medical Sciences, Gilan University Complex, Rasht, Iran; Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Valencia, Spain; Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran; Department of Pharmacology, Faculty of Medicine, Gilan University of Medical Sciences, Gilan University Complex, Rasht, Iran


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contamination by fasciolids takes place through ingestion of metacercariae attached to vegetables. Experimental studies were performed with plant-made foods suggesting a role in human contamination in Iran and on the usefulness of potassium permanganate as a preventive tool for killing metacercariae attached to vegetables used in salads in Egypt. In the foods assayed, although viability decreases with time, a high percentage of the metacercariae were still alive 2 and 4 weeks after preparation. Infection of laboratory animals proved that metacercariae kept their infectivity. The 5-minute tests of potassium permanganate effects showed that metacercarial viability was not affected even at the very high doses of 300, 600, and 1,200 mg/L. Careful, subsequent washing of leaves and vegetables with water is therefore needed after its application. A review on similar studies performed with metacercariae belonging to fasciolid and other trematode species affecting humans is included.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The need to implement control measures against important food-borne trematode infections has recently been emphasized.1 Among them, plantborne trematodiases have been included in the list considered by the Institute of Food Technologists’ Expert Panel on Food Safety and Nutrition.2 Fascioliasis and other food-borne trematodiases were added to the list of important helminthiases with a great impact on human development at the Third Global Meeting of the Partners for Parasite Control held in World Health Organization (WHO) Headquarters in Geneva in November 2004.

Fascioliasis is caused by digenean species of the genus Fasciola (Fasciolidae): F. hepatica in Europe, Asia, Africa, the Americas, and Oceania; F. gigantica in Asia and Africa. A different fasciolid species belonging to another genus, Fasciolopsis buski, causes fasciolopsiasis in Asia. These three species are transmitted by freshwater snail vectors. Rediae produce cercariae that emerge and swim in water until encystment on a substratum, mainly aquatic plants. Human contamination takes place through ingestion of infective metacercariae.3

Concerning fascioliasis, recent studies have shown that there are more contamination sources than the one traditionally noted, through free-living (= non-parasitic), encysted metacercariae attached to watercress.4 The most important human infection sources seem to be 1) ingestion of wild freshwater plants; 2) ingestion of cultivated freshwater plants; 3) ingestion of wild terrestrial plants; 4) ingestion of cultivated terrestrial plants; and 5) drinking of beverages made from local plants.5

Consequently, studies on the viability and infectivity of metacercariae and on strategies and agents potentially useful as control measures to impede human contamination by metacercariae-carrying vegetables and plant-made foods become crucial. Different studies have furnished a baseline on metacercarial viability and infectivity.6,7 Metacercarial infectivity is dependent on storage time, being lower when metacercariae are older. Moreover, metacercarial viability and infectivity did not show differences between isolates from different reservoir species.7,8

However, the number of studies on strategies and agents potentially useful as control measures to impede human contamination is surprisingly low. The purpose of this paper is to contribute to filling this gap by exposing results obtained in experimental studies performed with 1) local Iranian, plant-made foods served as an appetizer or a paste that have been suggested to play a role in human contamination in the northwestern endemic province of Gilan, at the Caspian Sea, where human fascioliasis is a public health problem, including outbreaks involving thousands of individuals1,9–11 and 2) on the potential usefulness of potassium permanganate, which has been suggested to be the most effective preventive tool for killing metacercariae attached to leaves and vegetables used in salads.12,13

Until a few years ago, the use of agents useful to wash off the metacercariae was neglected mainly because people living in poor, rural endemic areas were not expected to use them. However, today, fascioliasis is an emerging/re-emerging disease in many countries; infected patients are increasingly being diagnosed in large urban areas such as La Paz and El Alto (Bolivia), Alexandria, Damanhour and Cairo (Egypt), and Bandar Anzali and Rasht (Iran).5,10,11,14 In these cities, agents such as potassium permanganate are easily available and could thus be very useful for individual prevention if they are effective.

A short review on similar studies performed with metacercariae belonging to fasciolid and other trematode species affecting humans is also included.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiments with Iranian local foods. Gilan traditional foods assayed. In the Iranian province of Gilan, there are several very popular kinds of wild plants, such as species of Eryngium and Mentha, which are eaten raw, ground and mixed with walnuts, various spices, garlic, and fresh olives for the preparation of an appetizer called "zeitoon-parvardeh," or used in the preparation of a paste called "delar" along with a great quantity of salt. The very high quantity of salt used for delar preparation is at the base for the local name "green salt" also given to this specialty. The amount of salt and kinds of vegetables in delar differ somewhat depending on geographical zones and people’s habits (usually 30–40%). However, in some regions, the mean weight of salt is almost equal to that of vegetables used (50%). The aromatic vegetables used for these two traditional foods are usually sold throughout the year, mainly in the streets of all endemic areas in Gilan Province. Although the consumption of these plants, raw or elaborated in those appetizer and paste, has been suggested to be the main source of human infections, no study on the viability of liver fluke metacercariae in zeitoon-parvardeh and delar under typical preparation conditions has been performed thus far.

For zeitoon-parvardeh preparation (Figure 1AGo), plants of the species Eryngium coucasicum (locally known as "choochagh"), walnuts, and garlic were ground. Ground materials were thoroughly mixed with olives after the removal of olive stones. Spices and sour-pomegranate juice were added afterward. This foodstuff is served as an appetizer and may be eaten right after preparation. However, it cannot be stored for > 2 weeks because growth of fungi prevents its further consumption.


Figure 1
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  		    FIGURE 1. Traditional culinary specialties made from very popular kinds of aromatic wild plants, involved in human contamination by fasciolid liver flukes in the endemic province of Gilan, Iran. (A) Zeitoon-parvardeh, an appetizer dish, which is made by mixing the grounded local wild plants with other ingredients and fresh olives. (B) Delar, a traditional herbal paste, which may be stored for consumption over several months.

 
For delar preparation (Figure 1BGo), amounts of Mentha pulegium and Mentha piperita ("khlivash" and "bineh," respectively) were washed and ground thoroughly using an electrical grinder or an ancient instrument (locally named "namakyar"). After thorough grinding, salt (40%) was added to the raw plants and mixed well. Delar is edible after preparation, but the paste may be stored for consumption over several months. It is usually eaten with cucumber, prunes, yogurt, etc. The pH, measured with an electrical pH meter (Crison Micro pH 2000, Barcelona, Spain), was 5.0.

Liver fluke metacercariae. To determine whether the ingredients used in the appetizer or paste alter the viability of liver fluke metacercariae, the following two experiments, using snails of the species Lymnaea gedrosiana15 collected from water bodies around the locality of Bandar Anzali, were carried out. 1) Miracidia from fasciolid eggs collected in bile from gallbladders of cattle naturally infected with, the most prevalent fasciolid species,16 from the Bandar Anzali slaughterhouse, were used to infect 150 snails; metacercariae obtained from cercariae shed by the snails were used to orally infect mice. 2) Miracidia from eggs obtained from the uterus of F. gigantica adults recovered from livers of naturally infected cattle slaughtered in Rasht, capital of Gilan Province, were used to infect 120 snails; metacercariae obtained from cercariae shed by the snails were used to orally infect hamsters. Metacercariae were collected on a plastic sheet and lettuce and stored in drinking water at 4°C for 2 weeks before adding them to the prepared foods.

Metacercariae were divided into three groups: 1) added to zeitoon-parvardeh; 2) added to delar; and 3) kept in water as control. During the process of food preparation by a native person, metacercariae were added and mixed thoroughly. Finally, the foods were kept at 4°C until oral infection of mice and hamsters. For animal infection, metacercariae were collected, by means of a pipette, from a small amount of zeitoon-parvardeh and delar previously put in a Petri dish containing water.

Verification of metacercarial viability and infectivity. The viability and infectivity of metacercariae kept in zeitoon-parvardeh and delar was checked by microscopical analyses and animal infection assays performed in a laboratory of the Gilan University of Medical Sciences.

Microscopical analyses included the weekly checking of metacercarial viability according to the refractile appearance of secretory granules.17 Metacercarial cysts recovered from the foods were studied between a glass slide and coverslip under a light microscope. This method was used for metacercariae kept in zeitoon-parvardeh only for 2 weeks after food preparation and before contamination with fungi. For delar, the verification of metacercarial viability was continued throughout a longer period of 4 weeks after preparation, just until infection of mice, as this culinary speciality can be stored for use over several months.

For animal infection analyses, mice and hamsters were kept in an animal house under daily observation until killed. The study was approved by the institutional committee on animal care of Gilan University of Medical Sciences (Iran). Their liver, abdominal subcutaneous, peritoneal, and thoracic cavities were checked under a stereomicroscope for flukes.7 Animals were used in the above-mentioned experiments as follows. 1) A total of 30 female Swiss mice were divided into two identical groups (15 mice/group); each mouse was orally infected with 10 metacercariae; in group 1, metacercariae were kept in delar for 30 days, and in group 2, metacercariae were kept for the same time in water at 4°C as controls. All mice in the test and control groups were analyzed at 10 weeks post-infection (pi). 2) A total of 21 golden hamsters were divided into three groups (7 hamsters/group); each hamster was orally inoculated with 25 metacercariae. In group 3, metacercariae were kept in delar for 7 days, in group 4, metacercariae were in zeitoon-parvardeh for 7 days, and in group 5, metacercariae were kept in water at 4°C for 7 days as a control. All hamsters in the test and control groups were analyzed at 8 weeks pi.

Experiments with potassium permanganate. Liver fluke metacercariae. Metacercariae were obtained after cercarial shedding obtained in a laboratory of Valencia University from two naturally infected lymnaeid snails of the species Galba truncatula collected in the human hyperendemic area of the governorate of Behera, Nile Delta region, Egypt,18 and kept in drinking water in a Petri dish under conditions of a temperature of 20°C, photoperiod of 12-hour/12-hour light/ dark, and relative humidity of 90% in a climatic chamber Heraeus-Vötsch HPS-500 (Heraeus-Vötsch GmbH, Balingen, Germany). The classification of the metacercariae as belonging to F. hepatica was verified by obtaining the adult stage and eggs in laboratory Wistar rats (Iffa Credo, Barcelona, Spain) after experimental oral inoculation by means of a gastric tube. The animals were housed in Micro-Isolator boxes (Iffa Credo, Barcelona, Spain) and maintained in a pathogen-free room, electrically heated with a 12-hour/12-hour light/dark cycle (conditions in compliance with the European Agreement of Strasbourg, 18 March 1986). Food and water were provided ad libitum.7

Potassium permanganate application. Potassium permanganate is a water-soluble solid, very dark purple, crystalline product, used chiefly as an oxidizing agent, disinfectant, and in medicine as an astringent and antiseptic. A total of 87 metacercariae encysted on lettuce were collected. The metacercariae were stored in drinking water in total darkness at 4°C until required. Only metacercariae of the same age of 15–30 days were used for the test. The effect of potassium permanganate (KMnO4) on metacercariae was tested for a period of 5 minutes at laboratory temperature (22°C) by submerging metacercariae in different water concentrations of 300, 600, and 1,200 mg/L. The experiment was carried out twice, including controls (metacercariae not treated with potassium permanganate) also in duplicate (Table 3Go). After exposure, metacercarial cysts were washed with modified Earle saline medium.19


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TABLE 3
Test results of potassium permanganate effects on the viability of F. hepatica metacercariae
 
Verification of metacercarial viability. The viability of metacercariae was analyzed by their in vitro excystation capability.20 Metacercariae were placed in a Petri dish with natural water, and the outer cyst walls were removed by gently pressing with a dissecting needle under a stereomicroscope. Once the outer wall was removed, metacercariae were transferred to a test tube with 10 mL of modified Earle saline medium19,21 containing 39 mmol/L NaHCO3, 103 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L NaH2PO4, 0.8 mmol/L MgSO4, 1.8 mmol/L CaCl2, and 11 mmol/L glucose. Metacercariae were activated by incubation for 1 hour at 38°C in this medium in a shaking water bath (100 strokes/min) under a constantly refreshed 60% CO2/40% N2 gas phase. During this experimental phase, a pH of 6.3 was maintained. The incubation was continued for another 30 minutes in aerobiosis, during which the gas mixture was removed and air was used to restore the initial pH of 7.2. After this activation phase, the total volume was transferred to a Petri dish for observation and counting of the activated metacercarial number. Metacercariae were considered activated when presenting movements. Afterward, the medium was returned to the crystal test tube. To stimulate the excystment, taurocholic acid (9 g/L) was added to the medium, and metacercariae were incubated in aerobiosis for another 6 hours at the same temperature (+38°C) and shaking speed (100 strokes/min). After incubation, the total volume of the medium with metacercariae was transferred to a Petri dish and examined under a black-bottomed stereomicroscope. The number of empty cyst walls, free mobile juvenile flukes, and unexcysted metacercariae were surveyed, and the percentage of excystation was calculated as the number of empty cyst walls (verified by observation of mobile free juvenile flukes) from the total number of initially incubated metacercariae. All excystment assays were made in duplicate, and data are expressed as the mean of the two observations.

Statistical analyses. The n/tn% (n = number of metacercariae found alive or empty cyst walls; tn = total number of metacercariae microscopically checked), with its 95% confidence interval (95% CI; EPIINFO), was used for the statistical analysis of the viability of metacercariae. Because the size of the samples was small (results obtained were so clear that no additional assays were needed), it was considered that significant differences (P < 0.05) in n/tn% were present when there was no overlapping in the values of 95% CIs.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Metacercariae kept in traditional foods. Results of microscope analyses to check metacercarial viability are shown in Table 1Go. In both zeitoon-parvardeh and delar, although viability seems to decrease with time, a total of 66.6% and 57.8% of the metacercariae were still alive 2 weeks after food preparation, respectively. The statistical comparison of the percentages of living metacercariae in relation to the total number of metacercariae checked in experiment A does not show any significant difference between viability of metacercariae kept in zeitoon-parvardeh, delar, and water (= control) at days 1, 7, and 14.


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TABLE 1
Viability of F. gigantica metacercariae kept in traditional Iranian foods (zeitoon-parvardeh and delar) and water (= control), checked by microscopical analysis according to the refractile appearance of secretory granules and movements of juveniles within cysts
 
In delar, metacercarial viability is lower than in water in the third and fourth weeks, differences being statistically significant. However, although delar seems to decrease the viability of metacercariae with time, there is still a relatively high proportion of metacercariae surviving at days 21 and 28 (60.8% and 47.0%, respectively).

Results of experimental animal infection tests to check metacercarial infectivity are shown in Table 2Go. Among the 15 mice experimentally exposed to metacercariae from delar and dissected at 10 weeks pi, 2 mice (13.3%) were infected with one fluke each and another 2 showed pathologic effects, although no fluke was recovered. Among the 15 mice infected with control metacercariae (from water), 3 mice (20.0%) were infected by one, one, and three worms each. The percentage of fluke recovery was lower with metacercariae from delar than with those from water (1.3 and 3.3, respectively). Flukes were recovered from different body parts (Table 2Go), and one mouse in the control group showed liver pathology but no fluke.


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TABLE 2
Viability of F. gigantica metacercariae kept in traditional Iranian foods (zeitoon-parvardeh and delar) and water (= control), checked by experimental oral infection in laboratory animals
 
The results of the experimental infection of hamsters showed infectivity percentages of 42.8%, 71.4%, and 57.1% with metacercariae kept in delar, zeitoon-parvardeh, and water (= control), respectively. Percentages of flukes recovered were 2.3%, 3.4%, and 4.6% with the same metacercarial groups, respectively. In all infected hamsters, mild to mostly well-patent pathology on the surface and in the parenchyma of the liver was present. There was severe pathology in one hamster of the delar group and another of the control water group, both in the liver surface and parenchyma, although no flukes were recovered.

Metacercariae treated with potassium permanganate. Results of the tests of potassium permanganate effects on the viability of the metacercariae are shown in Table 3Go. The results obtained show that metacercarial viability is not affected by potassium permanganate, even at the very high doses of 300, 600, and 1,200 mg/L. The analyses of n/tn% of control metacercariae in relation to metacercariae treated with the different concentrations showed no significant difference (P < 0.005) between them in any group.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Effects of food handling and control measures against trematode metacercariae. Despite human infection by food-borne trematodes being very common in numerous countries,1,3,4 the number of studies on food handling effects (elaboration and conservation conditions) and agents potentially useful as control measures to impede human contamination is relatively small. A summarized review of studies performed from these points of view on metacercariae belonging to fasciolid and other trematode species affecting humans is included in Table 4Go.


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TABLE 4
Effect of different conditions on the viability and infectivity of metacercariae of some foodborne trematodes
 
The effect of acidity and salt has been analyzed only in trematode species whose metacercariae encyst in a second intermediate host (i.e., fish). In Opisthorchis species and the heterophyid Haplorchis taichui, the viability of metacercariae is affected by salt, acidity, and fermentation in a period ranging from 3 to 24 hours.22–24 Clonorchis sinensis is the only species whose metacercarial viability does not seem to be affected, at least in a 30% salt concentration throughout a period of 5–7 days.25

Concerning human fascioliasis, studies carried out in human hyperendemic areas have shown that many raw-eaten, cultured, or wild-grown plant species are involved in human contamination, for example, in Bolivia26 and Egypt.12,27 Human contamination by fascioliasis through ingestion of metacercariae included in local beverages is also known in other human endemic areas, as Cape Verde.1,5 However, this study seems to be the first to analyze the effects of traditional food preparation and conservation on the metacercariae of liver fluke species of the genus Fasciola.

This is, nevertheless, not the first study of this kind on free-living metacercariae encysted in the external environment (i.e., non-parasitic in an intermediate host). Several analyses were carried out on metacercariae of the Giant Asian intestinal fluke F. buski a long time ago (Table 4Go).3 Metacercariae of F. buski were killed by 1.0% hydrochloric acid in 18 days, by 2% acetic acid in 9 days, by 3.0% acetic acid in 6 days, by 5% salt solution in 3 hours, by soybean sauce in 30 minutes, by 10% cane sugar in 3 days,28 by direct solar radiation in 20–30 minutes,29 by desiccation in 19 hours at 27°C, and by boiling water in 1–2 minutes.30

Articles on the efficacy of different agents on Fasciola metacercariae are restricted to only two studies in Egypt.12,27 These studies concluded that the fasciolid metacercarial detaching capacity of running water was very low compared with the high capacity of citric acid (10 mL/L), commercial vinegar (120 mL/L), liquid soap (12 mL/L), potassium permanganate (24 mg/L),12 and sodium dichloroisocyanurate (1 mg/L).27 The latter two are, moreover, considered the only ones able to kill the metacercariae.12,27 The commercial availability of potassium permanganate makes it the agent of choice.

Metacercariae kept in traditional foods. Microscopical analyses (Table 1Go) showed that, during the 2 weeks between food preparation and consumption, the viability of liver fluke metacercariae kept in both zeitoon-parvardeh and delar is unaffected or only slightly decreased. During the third and fourth weeks in delar, around one half of the metacercariae kept their viability, indicating a high infection potential is still present despite having been in the food for a long time.

Experimental animal infection tests proved that metacercariae might keep their infectivity in both traditional Iranian foods (Table 2Go). Experiments performed with mice showed that the number of both infected animals and flukes recovered was higher in the control group (metacercariae kept in water) than in the delar group, suggesting that the high salt concentration used for delar preparation may alter the infectivity of metacercariae present. However, the host microhabitats other than liver where flukes were found (subcutaneous tissues of the abdominal region and the peritoneal cavity) indicate that the mouse is not a suitable host for F. gigantica, in agreement with previous studies,4 and that results obtained in mice should only be taken as suggestive.

Hamsters are, nevertheless, more appropriate hosts for F. gigantica,4 and consequently, results obtained with this animal species offer more conclusive data. The number of hamsters infected by metacercariae kept in zeitoon-parvardeh was higher than that infected by metacercariae from delar. The percentage of worms recovered when using metacercariae from zeitoon-parvardeh was higher than that from delar, and the fluke number recovery when infecting with metacercariae kept in both foods was lower than that in water. All this suggests 1) a negative impact of the two foods on metacercarial infectivity and 2) a greater negative impact of delar than zeitoon-parvardeh.

When comparing results of microscopical analyses of metacercarial viability and results of experimental infection tests with mice and hamsters about metacercarial infectivity, there is an agreement in indicating a decrease of both viability and infectivity with time. Although it is already known that metacercarial age decisively influences both aspects in this way,7 the results here obtained show that the two traditional foods analyzed seem to accelerate the decreasing processes of viability and infectivity of metacercariae. Moreover, data obtained indicate that delar has a greater negative impact than zeitoon-parvardeh on both metacercarial viability and infectivity, most probably because of the high salt concentration of delar.

In spite of this, results clearly prove that most metacercariae keep their viability and infectivity during the first two weeks after food preparation in both traditional specialties. Moreover, about half of the metacercariae keep their viability and infectivity in delar (salt = 40%; pH = 5) until at least four weeks. Thus, the present study shows the possible way of human contamination following the consumption of these two traditional food specialties of Gilan province, when prepared with raw vegetables presenting attached metacercariae. Results obtained indicate that the traditional methods of preparing these two local foods with fresh wild-grown plants offers little protection against contamination. The extended tradition of eating these foods in the Gilan Province, including a population of 2,000,000 people, may be a major source for human contamination with liver flukes.1,9

In Iran, human infection with fasciolids is mainly reported in this littoral region of the Caspian Sea, despite fascioliasis being prevalent in livestock throughout the country.31,32 Only sporadic cases have been reported from other areas of the country.33,34 Some of these sporadic cases may be linked to travelers who visit Gilan and sometimes take these foods as gifts.

Metacercariae treated with potassium permanganate. Even a low concentration of 24 mg/L of KMnO4 was considered the most useful culinary tool to detach and kill metacercariae attached to various leaves and vegetables used for salads, in a short period of 5–10 minutes, without softening the leaves or changing their color, according to results obtained relatively recently.12,13

Unfortunately, results obtained in this study prove that potassium permanganate is not effective in killing metacercariae, even when applying doses much higher than those initially recommended (Table 3Go). Thus, potassium permanganate may be considered as a tool useful only for detaching metacercariae, similar to commercial vinegar (at a concentration of 120 mL/L), citric acid (10 mL/L), or liquid soap (12 mL/L). A 97–100% efficacy to detach metacercariae had been recognized for all these products.12 The detaching capacity of potassium permanganate, by dissolving the external metacercarial attaching cement, was also observed in this study. Consequently, if it is used for such a purpose, careful, subsequent washing of leaves and vegetables with water is needed, as in the case of the above-mentioned products.

All evidence suggests that trematode metacercariae encysting in the outer environment, as those of fasciolids, are more resistant than those encysting in intermediate hosts (Table 4Go). In the case of Fasciola species, studies on the potential effectivity of different products and methods to kill metacercariae attached to leaves and vegetables are needed. Although appropriate studies should still be performed, metacercariae of Fasciola species may perhaps be more resistant than metacercariae of F. buski.28–30

Received January 30, 2006. Accepted for publication April 16, 2006.

Acknowledgments: The support by Dr. L. Savioli and Dr. A. Montresor (PVC/CPE, WHO, Geneva) and Dr. Z. Hallaj (CDC, WHO-EMRO, Cairo) is greatly acknowledged for help in facilitating the international collaboration necessary for the research activities developed. The technical assistance by the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, and the help in obtaining fluke eggs in slaughterhouses by the Central Veterinary Office of Gilan Province are also acknowledged. We also thank Dr. Latif Salehi of Gilan University for assistance in verifying the scientific names of local plants, Mohammad Reza Hadiani of Bandar Anzali Health Center, and Miss Roohnavaz, Fariba Hadiani, and Behnaz Rahmati for assistance in this study.

Financial support: The study on Iranian culinary specialties was supported by funding from Gilan University of Medical Sciences in Rasht, Deputy of Research, Contract 5263. Spanish collaboration was funded by Project BOS2002-01978 of the Spanish Ministry of Science and Technology, Madrid, Project of the Fondo de Investigación Sanitaria (FIS) PI030545 of the Spanish Ministry of Health, Madrid, the Red de Investigación de Centros de Enfermedades Tropicales—RICET (Project C03/04 of the Programme of Redes Temáticas de Investigación Cooperativa) of FIS, Spanish Ministry of Health, Madrid, and Project 03/113 of Conselleria de Empresa, Universidad y Ciencia, Valencia, Spain. Six-month stay of the first author in the Valencia Department was funded by Gilan University of Medical Sciences and Iranian Ministry of Health.

* Address correspondence to Santiago Mas-Coma, Departamento de Parasitologia, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain. E-mail: S.Mas.Coma{at}uv.es Back

Authors’ addresses: Keyhan Ashrafi, Department of Medical Microbiology, Faculty of Medicine, Gilan University of Medical Sciences, Gilan University Complex, Rasht-Tehran Road km 7, PO Box 3363, Rasht, Gilan Province, Iran, Telephone: 98-131-5550596, Fax: 98-131-6690007, E-mail: k_fashi{at}yahoo.com. Maria Adela Valero, Patricia Conde, Messaud Khoubbane, Maria Dolores Bargues, and Santiago Mas-Coma, Departamento de Parasitologia, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain, Telephone: 34-96-3544298, Fax: 34-96-3544769, E-mail: S.Mas.Coma{at}uv.es. Jafar Massoud and Shahram Solaymani-Mohammadi, Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Enghelab Street, PO Box 6446-14155, Tehran, Iran, Telephone: 98-21-88951392, Fax 98-21-66462267, E-mail: massoud{at}yahoo.com. Abdolrasoul Sobhani, Department of Pharmacology, Faculty of Medicine, Gilan University of Medical Sciences, Gilan University Complex, Rasht-Tehran Road km 7, PO Box 3477, Rasht, Gilan Province, Iran, Telephone: 98-131-7723823, Fax: 98-21-66462267, E-mail: s_solaymani{at}yahoo.com.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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