Prevalence of helminth eggs on raw vegetables used for salads

Esma Kozan
a
, Bahadir Gonenc
b
, Oguz Sarimehmetoglu
b
, Hasan Aycicek
c,
*
a
Department Helminthology, Veterinary Faculty of Afyon Kocatepe University, 03010 Afyon, Turkey
b
Department Helminthology, Veterinary Faculty of Ankara University, 06029 Diskapi, Ankara, Turkey
c
Department Food Hygiene and Technology, Gulhane Military Medical Academy, 06018 Etlik, Ankara, Turkey
Received 11 October 2003; received in revised form 23 February 2004; accepted 24 February 2004
Abstract
The presence of helminth eggs on raw vegetables, including lettuce, parsley, green onions, cucumbers, carrots, red cabbage,
tomatoes, rockets (Eruca sativa), and green-peppers from wholesalers in Ankara, Turkey was determined. A total of 203 unwashed
and 406 washed samples were assayed by light microscopy. Helminth eggs were detected in 12 (5.9%) of 203 unwashed samples and
not in any washed samples (p < 0:05).
Helminth eggs detected in unwashed samples included Taenia spp. (3.5%), Toxocara spp. (1.5%), and Ascaris lumbricoides (1.0%)
eggs. Taenia spp. eggs were recovered to be highest number (n 130), followed by Toxocara spp. eggs (n 21). Approximately 11%
of unwashed lettuce and parsley was contaminated compared with only 2.5% of carrot samples. No helminth eggs were detected in
red cabbage, rockets, tomatoes or green-peppers.
These results highlight the potential for transmission of helminth eggs by unwashed salad vegetables in Turkey and the
importance of properly washing/disinfecting raw vegetables before consumption.
2004 Elsevier Ltd. All rights reserved.
Keywords: Vegetables; Human pathogens; Helminth eggs; Washing; Disinfecting
1. Introduction
In recent years, studies conducted in countries with
widespread helminthic infections (Amahmid, Asmama,
& Bouhoum, 1999; da Silva, Marzochi, Camillo-Coura,
Messias Ade, & Marques, 1995; de Oliveira & Germano,
1992; Ogunba & Adedeji, 1986; Rude, Jackson, Bier,
Sawyer, & Risty, 1984) have shown that consumption of
raw vegetables and fruits without proper washing or
peeling represents an important potential for the spread
of related infections. The increased demand, global
sourcing and rapid transport of foods, especially soft
fruit and salad vegetables, enhance both the likelihood
of source contamination and survival of the transmissive
stages of parasites pathogenic to man (Slifko, Smith, &
Rose, 2000). In developing countries, especially,
uncontrolled use of water from sources contaminated
with human and animal faeces to irrigate such vegeta-
bles and fruits has been reported to be responsible for
their high rates of contamination with helminth eggs
(Guilherme et al., 1999; Takayanagui et al., 2000;
Ulukanligil, Seyrek, Aslan, Ozbilge, & Atay, 2001).
Considering the fact that a part of the vegetables grown
in these developing countries are exported to the
developed world, the threat that these contamina-
tions cause to other countries cannot be overlooked
(Robertson & Gjerde, 2001).
Failure to adhere to hygienic standards in the kitch-
ens of institutions like schools, hospitals, restaurants
and hotels that deal in catering services can lead to the
widespread and easy spread of helminthic infections like
A. lumbricoides, hookworms, Enterobius vermicularis,
Trichuris spp. Toxocara spp. and Trichostrongylidae to
humans as a result of consumption of improper washed
vegetables and fresh fruits used as salad ingredients
(Coelho, Oliveira, Milman, Karasawa, & Santos, 2001;
Mesquita, Serra, Bastos, & Uchoa, 1999; Takayanagui
et al., 2001; Vazquez Tsuji, Martinez Barbarosa, Tay-
Zavala, Ruiz Hernandez, & Perez-Torres, 1997).
The objective of this study was to determine the
prevalence of helminth eggs on raw salad vegetables
obtained from wholesalers.
*
Corresponding author. Fax: +90-312-3042150.
E-mail address: haycicek@gata.edu.tr (H. Aycicek).
0956-7135/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2004.02.005
Food Control 16 (2005) 239242
www.elsevier.com/locate/foodcont
2. Materials and methods
A total of 203 unwashed and 406 washed raw salad
vegetables, including lettuce, red cabbage, cucumbers,
green onions, carrots, tomatoes, parsley, rockets and
green-peppers, were obtained between January 2002 and
June 2003 for assay. The vegetables were collected from
wholesalers in the villages of Ankara during the summer
months and from villages of Antalya, Adana, Mersin,
and Hatay in the Mediterranean region during the
winter months. The total of 3035 samples were col-
lected monthly from ve wholesalers. Specic informa-
tion regarding water used to irrigate elds from which
the vegetables were grown could not be obtained. Half
of the samples analyzed before washing and half after
washing/disinfecting.
2.1. Evaluation of the unwashed samples
The vegetable samples from wholesalers were brought
to a hospital kitchen. They were transported to the
laboratory for analysis in nylon bags before washing
process. Samples from the outer leaves of lettuce and red
cabbage, and the peeled skins of carrots, tomatoes, and
cucumbers were obtained, whereas parsley, green
onions, rockets, and green-peppers were evaluated in
tact. Portion of the samples (200 g) were weighed into
sterile nylon.
2.2. Evaluation of washed samples
The washing/disinfecting procedure of the vegetables
in the kitchen included:
1. The sorting stage: Before the washing procedure,
crushed and rotten leaves of the fruits and vegetables
were removed and not used.
2. First washing stage: The leaves of leafy vegeta-
bles were separated and washed with tap water.
Thus, mud and dust of these vegetables were re-
moved.
3. Chlorination stage: After the rst washing stage, the
vegetables were disinfected by submersion in a wash
solution containing 200 ppm active calcium hypo-
chloride for 30 min.
4. The rinsing stage: The chlorinated vegetables were
washed in an automated vegetablefruit washer
(Electrolux, Mod. LV198N-Italy) to rinse.
5. The slicingpeeling and weighing stage: Skins of
carrot, tomato, cucumber samples were peeled. After
peeled parts of the samples were weighed (200 g) into
nylon bags. Lettuce, red cabbage, parsley, green
onion, rocket, and green-pepper samples were sliced
and weighed (200 g) into nylon bags.
2.3. Detection of helminth eggs
The washed and unwashed samples that were brought
from the hospitals kitchen to the laboratory once every
week, were weighed in portions of 200 g. The weighed
samples were placed in a 1.5-l detergent solution (con-
taining 1% sodium dodecyl sulphate, and 0.1% Tween
80) and subjected for 10 min of sonication using a so-
nicator (Bandelin Sonorex, Mod. RK100H, Sonorex
Electronic, Germany) (Bier, 1991). Later 50 ml of the
liquid obtained from the sonication procedure were
transferred to centrifugation tubes and centrifuged for
15 min at 1500g (Bier, 1991). The sediment was exam-
ined at 10 magnication under a light microscope for
helminth egg content. The eggs were identied at 40
magnication. Thus, the helminth eggs were counted in
200 g each of samples. Eggs that were deformed were
excluded from the evaluation.
2.4. The statistical analysis
The data were analyzed by with Statsdirect (Statsdi-
rect Ltd., ver. 2, 2.0) statistical software. Helminth egg
contamination percentages of two samples were com-
pared by the Test for two proportions procedure. The
number of eggs of two vegetables was compared by
using Comparing two counts test at p < 0:05 (Kanji,
1999; Zar, 1996).
3. Results and discussion
Results of helminth egg contamination of unwashed
and washed samples are presented in Tables 1 and 2.
Helminth eggs were detected in 12 (5.9%) of 203 un-
washed. However, neither of washed samples have any
eggs (p < 0:05). The helminth eggs recovered from raw
unwashed samples were Taenia spp. (7/203; 3.5%),
Toxocara spp. (3/203; 1.5%), and Ascaris lumbricoides
(2/203; 1.0%). The recovery percentage of helminth eggs
in unwashed lettuce samples was highest rate (11.4%),
whereas, the carrot samples had the lowest infestation
rate (2.5%). No infestation was detected on red cabbage,
tomatoes, rockets, and green-peppers. Dierences
among the rates of positive samples were not statistically
signicant (p > 0:05).
The number of helminth eggs detected per 200 g of the
vegetables is presented in Table 2. The greatest number
of eggs after sample was Taenia spp. (n 130) followed
by Toxocara spp. (n 21). The highest helminth egg
count was found in parsley samples (n 107), whereas
cucumber samples (n 4) contained lowest (Table 2)
(p < 0:05). Eggs that were deformed (7, Taenia spp.; 2,
Toxocara spp.) were excluded from the evaluation.
240 E. Kozan et al. / Food Control 16 (2005) 239242
Studies on helminthological quality of vegetables in
Turkey are insucient. In previous studies by Ulukan-
ligil et al. (2001) in Sanliurfa (south-east Anatolian
region) in Turkey, A. lumbricoides and Taenia spp. were
detected, in 11.0% and 1.0%, respectively of unwashed
vegetables tested. In our study, the rate of A. lumbrico-
ides was found lower than those of Ulukanligil et al.
(2001), whereas the rate of Taenia spp. was higher. Choi
and Lee (1972) found Ascarid eggs in 49.0% of 147
lettuce samples that were collected from markets in
Taegu, Korea.
In various studies (Amahmid et al., 1999; Ogunba &
Adedeji, 1986; Robertson & Gjerde, 2001; Takayanagui
et al., 2001), dierent rates of helminthic infections were
associated with vegetables and fruits in various coun-
tries. The dierences observed in these studies were re-
lated to the level of development of the various countries
as well as the dierences within same country (Robert-
son & Gjerde, 2001; Takayanagui et al., 2001). The
water used in irrigating these elds especially was
implicated as a major route of direct contamination of
the fruits and vegetables grown on them by parasites
contained in these waters. In addition, in areas where
the hygienic conditions of the canalization system were
not appropriate, the spread of human-borne helminthic
infections especially was apparent (Bergstrom &
Langeland, 1981; Guilherme et al., 1999; Takayanagui
et al., 2000; Ulukanligil et al., 2001).
In Turkey, hydatidosis infection is widespread among
humans and animals (Tinar & Coskun, 1991; Unat,
1991). In this study, some of the eggs thought to be
Taenia spp. were suspected to be Echinococcus granulo-
sus that are indistinguishable from Taenia spp. and are
transmitted through waters contaminated with faeces of
dogs. The high rate of Taenia spp. contamination of
unwashed vegetables indicate that unwashed raw vege-
tables are of major importance in the epidemiology of
helminthic infections from Taenia spp.
Eggs thought to be those of A. lumbricoides are very
dicult to distinguish from A. suum. However, swine
production, with which A. suum is associated, is ex-
tremely limited and pork consumption rarely occasion
in Turkey. Therefore, it was suggested that the eggs are
most likely those of A. lumbricoides. This infection, seen
especially in children and which leads to growth retar-
dation, has been reported to be of high prevalence in
Turkey (Yasarol, 1984). Presence of A. lumbricoides and
Taenia spp. in the samples indicates that the canaliza-
tion system as well as the toilet habits in the areas where
these products are grown are not up to the required
standards.
Almost all studies performed on this topic have fo-
cused on the rate of parasitic infections carried by veg-
etables and fruits cultivated in the dierent regions of
various countries for marketing (Amahmid et al., 1999;
Ogunba & Adedeji, 1986; Robertson & Gjerde, 2001;
Table 1
Results of the analysis of unwashed vegetables
Sample (n) Taenia spp. Toxocara spp. A. lumbricoides Total
No. (%) No. (%) No. (%) No. (%)
Lettuce (n 35) 0 (0.00) 2 (5.71) 2 (5.71) 4 (11.42)
Parsley (n 44) 4 (9.09) 1 (2.27) 0 (0.00) 5 (11.36)
Green onion (n 15) 1 (6.66) 0 (0.00) 0 (0.00) 1 (6.66)
Cucumber (n 16) 1 (6.25) 0 (0.00) 0 (0.00) 1 (6.25)
Carrots (n 40) 1 (2.50) 0 (0.00) 0 (0.00) 1 (2.50)
Red cabbage (n 14) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)
Tomato (n 15) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)
Rocket (n 8) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)
Green-pepper (n 16) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)
Total (n 203) 7 (3.45) 3 (1.48) 2 (0.98) 12 (5.91)
Table 2
Determined egg counts on infected samples
Sample Number of samples infected Egg counts in 200 g of sample
Taenia spp. Toxocara spp. A. lumbricoides
Lettuce (n 35) 4 0 6, 12 3, 5
Parsley (n 44) 5 8, 12, 16, 68 3 0
Green onion (n 15) 1 7 0 0
Cucumber (n 16) 1 4 0 0
Carrot (n 40) 1 15 0 0
Total 12 130 21 8
E. Kozan et al. / Food Control 16 (2005) 239242 241
Takayanagui et al., 2000) and evaluation of the various
methods available for obtaining the eggs and larvae of
these parasites from vegetables and fruits (Bier, 1991;
Robertson & Gjerde, 2000; Rude, Peeler, & Risty, 1987).
Besides, studies on the nature of the washing process of
potentially contaminated vegetables and fruits from re-
gions with high rates of parasitic infections and the level
of change of this potential after the washing procedure
are especially important. In present study, absence of
any helminth eggs indicates that washing/disinfecting
procedures are proper in the establishment.
Vegetables from the Mediterranean region are ex-
ported to European and Asian countries. Thus, the
study is also important with regard to this point of view.
The results indicate that irrigation methods of vegeta-
bles elds and protection of these areas against to stray
animals are very poor. Besides, detected helminth eggs
are evidence for human-borne and animal-borne con-
taminations. It is certain that irrigation process of these
vegetables elds has been performed using contaminated
water sources. Besides, it suggests that cats and dogs (or
wild canidae) have entered into the elds freely. These
poor conditions should be improved urgently. There-
fore, legal preventive precautions should be forced for
natural water sources and agricultural environments.
Frankly, food safety principles from eld to table for
vegetables in Turkey need reformation.
Consequently, the data emphasized that if washing
and disinfecting procedures of the raw eaten salad veg-
etables are neglected, they can be important vehicles of
transmission human pathogen helminths.
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