Prevalence of Parasitic Contamination of Raw Vegetables in Villages of Qazvin Province,Iran

Abstract

We conducted a study to determine the extent of parasitic contamination of raw vegetables distributed by wholesalers in villages of Qazvin Province, Iran. Vegetables collected for the study were leek, parsley, lettuce, coriander, radish, spring onion, tarragon, basil, spearmint, and cress. Samples of each vegetable were collected at monthly intervals for 6 months from May to October 2008, yielding a total of 654 samples. Samples of each vegetable collected at each monthly interval were selected randomly and allocated to be washed or to remain unwashed before being analyzed for parasitic contamination. Of the 654 samples collected over the study period, 218 samples were analyzed for parasitic contamination without washing and 436 were washed before being analyzed, with the wash procedure consisting of submersion in a solution of 200 ppm of calcium hypochlorite and rinsing in an automated vegetable washer for 10 minutes. The samples were analyzed according to the method used by the U.S. Food and Drug Administration for identifying parasitic contamination of raw fruits and vegetables, with specimens weighing 200 g each being prepared from the vegetable samples, followed by sonication of each specimen for 10 minutes in 1.5 L of detergent wash solution, collection and centrifugation of a 50-mL aliquot of the wash solution for 15 minutes at 1500 g, and examination of the resulting sediment for parasites by light microscopy. No parasitic contamination was found on any of the 436 washed samples of vegetables. Of the 218 unwashed samples of vegetables, 82 (37.6%) were found to be contaminated with parasites, of which 69 (31.6%) were contaminated with metazoa (helminth eggs in 9.6% and rhabditoid larvae in 22.0%), and 13 (6.0%) were contaminated with protozoa (Entamoeba coli cysts in 2.8%, Entamoeba histolytica/E. dispar cysts in 1.4%, and Giardia lamblia cysts in 1.8%). The difference in parasitic contamination of the washed and unwashed groups of vegetables was significant at p < 0.05. Helminth eggs detected in unwashed samples included those of nematodes (Ascaris lumbricoides [2.3%], Trichuris trichiura [0.9%], and Trichostrongylus spp. [2.8%]), trematodes (Dicrocoelium dendriticum [1.4%]), and cestodes (Taenia spp. [1.8%] and Hymenolepis nana [0.5%]). The eggs recovered in greatest number were those of nematodes (n = 13), followed by those of cestodes (n = 5) and trematodes (n = 3). No helminth eggs were found on unwashed samples of spearmint, tarragon, coriander, or radish. The percent parasitic contamination of unwashed samples was highest for leek (66.7%) and lowest for radish (20.7%). The highest percent parasitic contamination was with rhabditoid larvae (22.0%) and the lowest with H. nana eggs (0.5%). These findings emphasize the importance of properly washing and disinfecting raw vegetables before they are consumed.

Introduction

R aw vegetables are a major source of vitamins, minerals, and dietary fiber, and are considered an important food group for maintaining good human health (Su and Arab, 2006). The increased demand for foods, especially soft fruits and raw vegetables, and their global provision and rapid transport enhance both the likelihood of their surface contamination and the survival on them of the transmissive stages of pathogenic human parasites (Slifko et al., 2000). Trends in many countries toward eating more raw or slightly cooked vegetables, to preserve their taste and content of heat-labile nutrients, may further increase the likelihood of foodborne infection (Erdogrul and Sener, 2005).

Fruits and vegetables normally become potential sources of human infection through their contamination, during their growth, collection, transport, processing, and preparation, with the source of contamination commonly being animal feces or soil or water contaminated with such feces (Slifko et al., 2000). In developing countries in particular, the use of water contaminated with human and animal feces to irrigate fruit and vegetable crops has been reported as responsible for high rates of contamination with pathogenic parasites (Guilherme et al., 1999; Habbari et al., 2000; Takayanagui et al., 2000; Ulukanligil et al., 2001).

Fruits and vegetables eaten raw and without proper washing or peeling have especially been demonstrated to be vehicles for the transmission of a range of parasites (Rude et al., 1984; Ogunba and Adedeji, 1986; de Oliveira and Germano, 1992; da Silva et al., 1995). Among parasitic bodies associated with vegetable- or fruit-borne outbreaks as opposed to individual cases of infection are cysts and oocysts of species of the protozoan genera Giardia, Entamoeba, Toxoplasma, and Isospora, and eggs and larvae of species of the helminthic genera Hymenolepis, Taenia, Fasciola, Trichuris, Trichostrongylus, Strongyloides, and Hookworm (Choi and Lee, 1972; Choi et al., 1982; Raisanen et al., 1985; Mintz et al., 1993; Coelho et al., 2001; Erdogrul and Sener, 2005).

In terms of sewage management and food contamination, protozoans and helminths are the parasites of primary concern for public health. An important characteristic of these organisms is their development through the stage of a cyst or ova that facilitates their survival. More than 40 million persons worldwide are infected with protozoan or helminthic parasites, and more than 10% of the world's population is at risk of such infection (Abdussalam et al., 1995).

In Iran, studies have reported intestinal parasites, including Giardia lamblia, Ascaris lumbricoides, Hymenolepis nana, and Taenia spp., as contaminants of raw fruits and vegetables in different parts of the country (Sarkari, 1996; Sayyed Tabai and Sadjjadi, 1998; Akhlaghi and Oormazdi, 2000; Zohour and Molazadeh, 2001; Daryani, 2002; Gharavi et al., 2002; Daryani and Ettehad, 2005), and different rates of parasitic contamination of these raw foods, ranging from 1.94% to 68.3%, have been found in different regions of Iran (Hamzavi, 1997; Sayyed Tabai and Sadjjadi, 1998; Sahebani et al., 1999; Akhlaghi et al., 2001; Davami et al., 2001). The differences observed in these studies are likely to have been related to the level of development of sanitation and public hygiene in these areas (Daryani et al., 2008).

Because eating of raw or only slightly cooked vegetables is customary in Iran, and little study has been done of parasitic contamination of vegetables in the province of Qazvin, Iran, we conducted a study of such contamination of raw vegetables obtained from wholesalers in villages within the province, and of its epidemiologic implications.

Materials and Methods

Between May and October 2008, we obtained from 15 wholesalers in the villages of Qazvin Province a total of 38 samples per month of vegetables that were grown in these villages under natural conditions, yielding a total of 654 vegetable samples collected over this 6-month period. The vegetables sampled, and the total number of samples of each vegetable collected over the 6-month study period were leeks (Allium porrum), n = 90; parsley (Petroselinum crispum), n = 63; lettuce (Lactuca sativa), n = 69; coriander (Coriandrum sativum), n = 72; radish (Raphanus sativus), n = 87; spring onions (Allium cepa L.), n = 66; tarragon (Artemisia dracunculus), n = 57; basil (Ocimum basilicum), n = 45; spearmint (Mentha viridis), n = 54; and cress (Lepidium sativum), n = 51. The vegetable samples were divided into two groups consisting of 218 samples and 436 samples, respectively, of which the samples in the latter group were washed before being analyzed for parasitic contamination and those in the former group were analyzed directly without washing.

Sample collection and treatment

Portions weighing 200 g of each of the raw vegetable samples collected each month from wholesalers were placed in sterile nylon bags at the time of collection and transported to the laboratory for analysis. Samples of leek, parsley, coriander, spring onions, tarragon, basil, spearmint, and cress were evaluated as the intact vegetables, whereas those of lettuce consisted of the outer leaves, and those of radish consisted of the peeled skins.

Wash procedure

Washing and disinfection of the 436 vegetable samples in the group of vegetables that were subjected to this treatment before analysis was done in the laboratory according the method of Kozan et al. (2005). Crushed and rotten leaves of these vegetables were removed before washing and were not used in the analysis. The intact leaves of leafy vegetables were separated and washed with tap water to remove mud and dust, and these leaves and the whole samples of the other vegetables examined in the group of washed vegetables were then disinfected by submersion for 30 minutes in a wash solution containing 200 ppm of active calcium hypochlorite and then rinsed for 10 minutes in an automated vegetable washer (Electrolux, Mod. LV198N-Italy). The washed samples were then sliced and weighed (200 g) on automatic balance into nylon bags for storage.

The 218 samples of vegetables in the unwashed group were submitted directly to analysis without washing.

Detection of helminth eggs, rhabditoid larvae, and protozoan cysts

Specimens of vegetables from both the washed and unwashed groups of samples were selected for analysis randomly at the time of their monthly arrival in the laboratory, and were analyzed with the method used by the U.S. Food and Drug Administration for recovery of parasites from fruits and vegetables (Bier, 1991). In accordance with this method, a weighed 200-g specimen of each vegetable sample was immersed in 1.5 L of a detergent solution containing 1% (w/v) sodium dodecyl sulfate and 0.1% (v/v) polysorbate (Tween) 80 (ICI Americas, Inc.) and sonicated for 10 minutes (Bandelin Sonorex RK100H; Sonorex Electronic). Aliquots of 50 mL of the resulting liquid were then transferred to centrifuge tubes and centrifuged for 15 minutes at 1500 g. The sediment from centrifugation was examined at magnifications of 10 × and 40 × under a light microscope (Carl Zeiss; NT6V/10w stab, 467085) for helminth eggs, rhabditoid larvae, and protozoan cysts, and the number of each type of parasitic contaminant in each sample was counted and recorded. Deformed eggs were excluded from analysis.

Statistical analysis

The data were analyzed with the Statsdirect software package version 2.2.0 (Statsdirect Ltd.). The percent contamination with helminth eggs, rhabditoid larvae, and protozoan cysts of the washed and unwashed samples was compared using the Student's t-test for two proportions. The numbers of helminth eggs, rhabditoid larvae, and protozoan cysts in the washed and unwashed vegetable samples were compared using χ ²-test, with the level of significance set at p < 0.05 (Kanji, 1999).

Results

The numbers of helminth eggs detected in each 200-g specimen of each of the unwashed samples of vegetables examined in the study are shown in Table 1. Helminth eggs were found in 21 (9.63%) of the total of 218 unwashed vegetable samples and none of the washed samples, a difference that was significant at p < 0.05. The eggs recovered from the unwashed samples were identified as those of H. nana, Taenia spp., Trichuris trichiura, A. lumbricoides, Trichostrongylus spp., and Dicrocoelium dendriticum. The highest percent contamination by helminth eggs in unwashed samples of the vegetables examined in the study was observed in samples of leek and parsley (23.33% and 19.4%, respectively) and the lowest percent contamination in samples of basil (6.66%). No contamination was observed of samples of spearmint, tarragon, coriander, or radish. The differences in percent contamination with helminth eggs among the washed samples of the vegetables that were contamined with these eggs were not statistically significant (p > 0.05).

Table 1.

Number and Percent of Helminth Eggs in Unwashed Vegetable Samples

Sample size	Hymenolepis nana, n (%)	Taenia spp., n (%)	Trichuris trichiura, n (%)	Ascaris lumbricoides, n (%)	Trichostrongylus spp., n (%)	Dicrocoelium spp., n (%)	Total, n (%)
Leek (n = 30)	1 (3.33)	0 (0.00)	0 (0.00)	3 (10.00)	3 (10.00)	0 (0.00)	7 (23.33)
Spring onion (n = 22)	0 (0.00)	0 (0.00)	0 (0.00)	2 (9.09)	1 (4.54)	0 (0.00)	3 (13.63)
Basil (n = 15)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	1 (6.66)	1 (6.66)
Parsley (n = 21)	0 (0.00)	2 (9.52)	1 (4.76)	0 (0.00)	0 (0.00)	1 (4.76)	4 (19.04)
Lettuce (n = 23)	0 (0.00)	2 (8.69)	1 (4.35)	0 (0.00)	0 (0.00)	0 (0.00)	3 (13.04)
Cress (n = 17)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	2 (11.76)	1 (5.88)	3 (17.64)
Spearmint (n = 18)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Tarragon (n = 19)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Coriander (n = 24)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Radish (n = 29)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Total (n = 218)	1 (0.46)	4 (1.83)	2 (0.92)	5 (2.29)	6 (2.75)	3 (1.37)	21 (9.63)

The numbers of protozoan cysts detected in each 200-g sample of unwashed vegetables are shown in Table 2. Protozoan cysts were found in 13 (5.96%) of the total of 218 unwashed samples and in none of the washed samples (p < 0.05). The cysts were identified as those of Entamoeba coli, Entamoeba histolytica/E. dispar, and G. lamblia. The highest percent detection of protozoan cysts was in leek (16.66%) and the lowest percent detection of protozoan cysts was in samples of tarragon (5.26%). No protozoan cysts were observed on parsley, lettuce, or radish. The differences in percent contamination with protozoan cysts among the unwashed samples of vegetables that were positive for such contamination were not statistically significant (p > 0.05).

Table 2.

Number and Percent of Protozoan Cysts in Unwashed Vegetable Samples

Sample size	Entamoeba coli, n (%)	Entamoeba histolytica, n (%)	Giardia lamblia, n (%)	Total, n (%)
Leek (n = 30)	0 (0.00)	2 (6.66)	3 (10.00)	5 (16.66)
Spring onion (n = 22)	2 (9.09)	0 (0.00)	0 (0.00)	2 (9.09)
Basil (n = 15)	0 (0.00)	0 (0.00)	1 (6.66)	1 (6.66)
Parsley (n = 21)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Lettuce (n = 23)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Cress (n = 17)	1 (5.88)	0 (0.00)	0 (0.00)	1 (5.88)
Spearmint (n = 18)	0 (0.00)	1 (5.55)	0 (0.00)	1 (5.55)
Tarragon (n = 19)	1 (5.26)	0 (0.00)	0 (0.00)	1 (5.26)
Coriander (n = 24)	2 (8.33)	0 (0.00)	0 (0.00)	2 (8.33)
Radish (n = 29)	0 (0.00)	0 (0.00)	0 (0.00)	0 (0.00)
Total (n = 218)	6 (2.75)	3 (1.37)	4 (1.83)	13 (5.96)

No contamination with rhabditoid larvae was found on any of the washed samples of vegetables examined in the study, but rhabditoid larvare were detected in 48 (22.01%) of the 218 unwashed samples (p < 0.05) (Table 3). The highest percent contamination with rhabditoid larvae was found in unwashed samples of basil (33.33%) and the lowest percent contamination in samples of cress and coriander (11.76% and 12.50%, respectively).

Table 3.

Number and Percent of Rhabditoid Larvae in Unwashed Vegetable Samples

Sample size	Rhabditoid larvae, n (%)
Leek (n = 30)	8 (26.66)
Spring onion (n = 22)	5 (22.72)
Basil (n = 15)	5 (33.33)
Parsley (n = 21)	4 (19.04)
Lettuce (n = 23)	5 (21.73)
Cress (n = 17)	2 (11.76)
Spearmint (n = 18)	4 (22.22)
Tarragon (n = 19)	4 (21.05)
Coriander (n = 24)	3 (12.50)
Radish (n = 29)	6 (20.68)
Total (n = 218)	48 (21.01)

Table 4 shows the numbers of samples and percent contamination of samples in the unwashed group of vegetables according to the pathogenicity and nonpathogenicicity of the contaminating parasites. Leek was the vegetable most commonly contaminated with pathogenic parasites, which were found in 66.66% of its samples. The numbers of parasites detected in each 200-g specimen of each of the washed samples of vegetables examined in the study are not shown in the tables because none of the parasites were observed on washed samples.

Table 4.

Nonpathogenic and Pathogenic Parasitic Contamination of Unwashed Vegetable Samples

Sample size	Nonpathogenic parasites, n (%)	Pathogenic parasites, n (%)
Leek (n = 30)	10 (33.34)	20 (66.66)
Spring onion (n = 22)	12 (54.55)	10 (45.45)
Basil (n = 15)	8 (53.34)	7 (46.66)
Parsley (n = 21)	13 (61.91)	8 (38.09)
Lettuce (n = 23)	13 (56.53)	10 (43.47)
Cress (n = 17)	11 (64.71)	6 (35.29)
Spearmint (n = 18)	13 (72.23)	5 (27.77)
Tarragon (n = 19)	14 (73.69)	5 (26.31)
Coriander (n = 24)	19 (79.17)	5 (20.83)
Radish (n = 29)	23 (79.32)	6 (20.68)
Total (n = 218)	136 (62.38)	82 (37.61)

Discussion

Vegetables, particularly those eaten raw and without washing, have been shown to be vehicles for the transmission of a range of human parasites (Mintz et al., 1993). Several studies of such parasitic contamination conducted in Iran have reported high prevalence rates of parasites such as A. lumbricoides, Taenia spp., G. lamblia, and E. coli in unwashed raw vegetables (Sarkari, 1996; Sayyed Tabai and Sadjjadi, 1998; Sahebani et al., 1999; Akhlaghi and Oormazdi, 2000; Davami et al., 2000; Zohour and Molazadeh, 2001; Gharavi et al., 2002; Nematian et al., 2004; Sayyari et al., 2005; Arani et al., 2008; Daryani et al., 2008; Nasiri et al., 2009).

In our evaluation of the level of contamination by helminth eggs, rhabditoid larvae, and protozoan cysts of unwashed and washed raw vegetable samples, we found the highest percent contamination of unwashed vegetable samples by pathogenic parasites (66.7%) in unwashed samples of leek and the lowest percent contamination (20.7%) in samples of radish (see Table 4). In earlier studies, lettuce (Turkey) (Kozan et al., 2005) and spinach (Iran) (Daryani et al., 2008) were the vegetables most commonly contaminated with parasites. In our study in Qazvin Province, 1.3% of unwashed raw vegetable samples were contaminated with eggs of A. lumbricoides, whose environmental prevalence elsewhere in Iran has been reported as 90% in Hamadan (Sayyed Tabai and Sadjjadi, 1998), 6% in Yasouj (Sarkari, 1996), 2% in Ardabil (Daryani et al., 2008), and 1% each in Jiruft (Zohour and Molazadeh, 2001) and Tehran (Gharavi et al., 2002).

In previous studies, A. lumbricoides was detected in 11.0% and 1.0%, respectively, of unwashed vegetable samples in Sanliurfa (south-east Anatolian region) (Ulukanligil et al., 2001) and Ankara (Turkey) (Kozan et al., 2005). Choi and Lee (1972) found ascarid eggs in 49.0% of 147 lettuce samples collected from markets in Taegu, Korea. The presence of A. lumbricoides in these samples indicates that the canalization for drainage system and toilet habits in the areas where these vegetables were grown had been below the required United Nations standards.

We also found contamination with T. trichiura and Trichostrongylus eggs, respectively, of 1.3% and 2% of the unwashed vegetable samples that we examined. Gharavi et al. (2002) found T. trichiura eggs in 1% and Trichostrongylus eggs in 15% of 429 samples collected from farms and stores in Tehran, Iran.

Among our other findings were contamination by Taenia spp. of 1.3% of our unwashed raw vegetable samples, although some of the eggs thought to belong to Taenia spp. were suspected of belonging to Echinococcus granulosus, whose eggs are transmitted through water contaminated with dog feces and are indistinguishable from those of Taenia spp. Although the eggs of T. saginata are excreted only in human feces and are not infectious to humans (Kozan et al., 2005; Daryani et al., 2008), the eggs of Echinococcus spp. excreted by dogs are infectious to humans, and the eating of raw vegetables contaminated with these eggs may present an increased risk of infection with hydatid cysts.

Daryani et al. (2006), in a study of sheep and cattle slaughtered in an industrial abattoir in Ardabil, Iran, found that 74% and 38%, respectively, were infected with hydatid cysts as a result of eating fodder contaminated with Echinococcus spp. (Daryani et al., 2008). The high rate of contamination of water, soil, and vegetables with Taenia spp. in Iran indicates that unwashed raw vegetables are of major epidemiologic importance in infections caused by these species of helminths.

We also found that 1.3% of the unwashed raw vegetable samples in our study were contaminated with cysts of G. lamblia. The environmental prevalence of this parasite elsewhere in Iran was found to be 17% in Kargan village in Ardabil (Daryani, 2002), 10% in Ahwaz (Akhlaghi and Oormazdi, 2000), 11% in Yasouj (Sarkari, 1996), 5% in Hamadan (Sayyed Tabai and Sadjjadi, 1998), 14% in Jiruft (Zohour and Molazadeh, 2001), and 6.5% in Tehran (Gharavi et al., 2002). In another study, G. lamblia was found in 7% of imported vegetables and 8.9% of locally cultivated vegetables in Ardabil (Daryani et al., 2008), and intestinal parasitic infection with G. lamblia was found in 14% and 11.5%, respectively, of primary school children in Ardabil (Daryani and Ettehad, 2005) and Tehran (Nematian et al., 2004).

Among our other findings were that 2.66% and 1.33% of our unwashed vegetable samples were contaminated with E. coli and E. histolytica/E. dispar, respectively. Although these parasites are nonpathogenic to humans, they are considered indicators of local and regional health. Because these parasitic species infest only the human intestine, their presence on these vegetables indicates contamination of the vegetables with human fecal matter through sewage, and the probability of other intestinal pathogenic and nonpathogenic parasites on such vegetables (Gharavi et al., 2002; Daryani et al., 2008).

Rhabditoid larvae were present on 21.3% of the unwashed vegetable samples in our study. High levels of contamination of unwashed vegetables with rhabditoid parasites had previously been found in the Iranian cites of Yazd (Dehghani-Firoozabady et al., 2003) and Boushehr (Sahebani et al., 1999).

These high levels of contamination with rhabditoid larvae indicate that unwashed raw vegetables are of major importance in the epidemiology of helminthic infections caused by Strongyloides stercoralis and other nematodes.

We could not obtain specific information about the water used to irrigate the fields in which our vegetable samples were grown, but the water used in irrigating the fields in which parasite-contaminated vegetables were grown has been implicated as a source of such contamination (Craun, 1990; Daryani et al., 2008). In earlier studies, the spread of human-borne helminthic infections was especially noted in areas with unhygienic waste-drainage systems (Bergstrom and Langeland, 1981; Guilherme et al., 1999; Takayanagui et al., 2000; Ulukanligil et al., 2001). In some parts of Iran, such helminthic infection is also partly explained by a custom of eating raw vegetables cultivated in gardens and fertilized with untreated human fecal waste (“nightsoil”).

Conclusions

Our findings in the present study emphasize the important role of raw vegetables in the transmission of human intestinal parasites to new human hosts, and the need to improve the sanitary conditions in many areas where these vegetables are grown. The use of nightsoil as a fertilizer on farms may be made safe by its chemical disinfection or other types of sterilizing treatment. Our findings and those of other studies also suggest the need for legally enforced precautions to ensure the purity of both natural water sources and agricultural milieux.

Footnotes

Disclosure Statement

No competing financial interests exist.

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