Parasitic Contamination of Fresh Vegetables in Open Air Markets of Aksum, Ethiopia

Background: Intestinal parasitic diseases are the foremost causes of illness in humans, responsible for signicant morbidity and mortality. Recently, there have been so many reports related to a foodborne illness linked to consuming fresh vegetables, especially in developing countries. The study was aimed to assess the parasitic contamination level of fresh vegetables collected from open air markets in Aksum town, Tigray region, Ethiopia. Method: A total of 384 samples belonging to six vegetable types were collected randomly from local markets in Aksum town. Samples were washed in saline solution followed by sedimentation for 24 hours.After that the supernatant was decanted leaving the sediment. The sediment was then transferred to a centrifuge tube and centrifuged at 3000 revolution per minute for 5minutes. Finally, the supernatant was decanted and the sediment was examined under light microscope for protozoan cyst, helminthes ova, and larvae Results: The overall prevalence of parasitic contamination was 57.3%. The Cabbage was the most frequently contaminated (79.7%) vegetable succeeded by Spinach (75%), Lettuce (73.4%), Potato (46.9%), Green pepper (37.5%), and tomato (31.2%). Among the parasites detected, Taenia spp. proglottides/egg was the most frequent contaminant with 41.1% prevalence rate followed by Enetrobius vermicularis egg (20.3%), Hookworm spp.(19.5%), Fasciola spp. egg (17.2%), Giardia lamblia cyst (18.5%), Entamoeba spp. cyst(15.9%), Strongyloides stercoralis larva (12.5%),Ascarislumbricoidese gg (9.4%), and Cryptosporidium spp.Chai-square analysis showed that the type of fertilizer used, educational status of the vendors, status of wash before display, and type of vegetable they produce was statistically and signicantly associated with the prevalent parasite forms detected (P < 0.001). Conclusion: This study identied a high prevalence of fresh vegetables contaminated with medically important parasites, thus


Background
Intestinal parasitic diseases are the foremost causes of illness in humans, responsible for signi cantmorbidity and mortality. It is estimated that 3.5 billion people are affected, and 350 million people are ill as a result of these infections, the majority being children 28,29 . Recently, there have been so many reports related to a foodborne illness linked to consuming fresh fruits and vegetables especially in developing countries 30 . These foodborne diseases which lead to serious health and economic problems are mainly caused by Protozoan and Helminthes parasites 1 , 7 . Vegetables are vehicles for the transmission of parasites if contaminated as a result of different associated factors related to planting, collecting, transportation, showcase chain, and improper treatment at domestic 3,4 . Untreated wastewater irrigation, the use of raw manure as fertilizer, improper on-harvest handling and hygienic conditions during preparation are factors that determine the level of contamination 2 .
Studies showed that the numbers of tourists who are visiting Aksum are increasing from time to time, and the number of Hotels and restaurants is increasing to meet the increasing ow of tourists 36 . It is a fact that fresh vegetables are a common diet in Ethiopia particularly in Aksum during the fasting period. It is also the commonly prepared food item in hotels and restaurants.
Hence, the rate of intestinal parasitic infection from the utilization of contaminated vegetables may increase particularly in case the vegetables are inappropriately washed. Thus monitoring and evaluating on the level of parasitic contamination is important to control intestinal parasitic infection in the area. However to our knowledge there is no published data on the status of parasitic contamination of vegetables in and around Aksum. Therefore, this study was conducted to assess the parasitic contamination status of vegetables and associated risk factors in Aksum town, Ethiopia.

Materials And Methods
Study area and period.
The study was carried out in Aksum town, in northern Ethiopia approximately 1010 km from Addis Ababa, Ethiopia's capital. In the town, the majority of populations (88.3%) are Orthodox Christian followers 35 . Since the area in and around Aksum town is suitable to grow fruits and vegetables many farmers are engage in producing a variety of vegetables .The vegetables are supplied from the surrounding villages directly by the farmers. Customers purchase the vegetables either from the farmers or retailers. This study was conducted from March 1 to March 23, 2019, which is the fasting period for the Orthodox Christian followers living in the town.

Sample collection and parasitological Examination
To conduct the study a random sample of 384, 64 from 6 distinct vegetable types namely; Lettuce (Lactuca sativa), Cabbage (Brassica Oleracea), Spinach (Spinacia oleracea), Pepper (Capsicum annum), Potato (Solanumtuberosum) and Tomato (Lycopersicon esculentum) was purchased from the multiple vendors in Aksum markets. Just one vegetable from a single vendor was collected. Each vegetable sample was placed in an isolated plastic sack and labeled with a code and date of collection. Samples were transported to the Biology laboratory of Aksum University for parasitological analysis. The samples were prepared for examination within three hours of collection time.Each vegetable sample was cuted into pieces and weighted to 200 grams.
The chopped samples were washed separately using 1LNormal saline to detach the parasite stages (Ova, cyst, and larvae) of helminths and protozoa. After overnight sedimentation of the washing solution, 15 ml of the sediment was then transferred to a centrifuge tube using a sieve to remove undesirable matters. To concentrate the parasite stages, the tube was centrifuged at 3000 rpm for ve minutes (using Labtech, Model.No.AC 2305 centrifuge). After centrifugation, the top water was decanted carefully leaving about ve milliliters of the sediment. The sediment was then agitated gently to redistribute the parasite stages.
Finally, Lugol (Iodine) stained slides were prepared from each sample sediment and examined under a light microscope for protozoa cyst, helminths ova, and larvae using × 10 and × 40 objectives 15,7 . Besides, Modi ed ZiehlNeelson staining technique was employed to identify Oocysts of Cryptosporidium, Isospora beli and Cyclospora spp. The parasite stages were identi ed based on morphological details as described by Souls 32 .
To identify the factors associated with vegetable contamination, vendors were asked to answer questions about the possible source of contamination for vegetables including; their level of education, the fertilizer type used, and the status of the wash of the vegetables before display and the type of vegetable they produced to identify the factors related to the prevalence of parasites.

Data analysis
The data were analyzed through descriptive prevalence and count statistics. The onesample proportion test was also used to assess the signi cance of vegetable contamination prevalence. To identify the factors related to the prevalence of parasites in vegetables, a Chai-square analysis was used. This is achieved using the statistical software of SPSS version 20 and 95% con dence.
Result From a total of 384 samples screened for parasites,220(57.3%) had at least one and at most seven parasites detected ( Table 1).
Out of 384, 71 vegetable samples were contaminated by only one parasite and 52 vegetable samples were contaminated by two parasites. However, there were only six vegetable samples with seven parasite contamination. The highest prevalence of parasites was detected in cabbage (Brassica oleracea) (79.7%) succeeded by spinach (Spinacia oleracea) (75%), and lettuce (Lactuca sativa) (73.4%). Tomato (Solanum tuberosum) with 31.2% prevalence is the least contaminated vegetable. The onesample proportion test on the contaminated vegetables indicated that the prevalence is statistically signi cant with all Pvalues less than 5%.
As shown in Table 2, the average prevalence of parasites contamination in the variety of vegetable samples was 57.3%. Nine different kinds of parasite forms were detected from the vegetables examined. Its prevalence, however, showed a considerable variation. Particularly, the most frequently observed was Taenia spp. proglottides/egg (41.1%) followed by Enetrobius vermicularis egg (20.3%) and Hookworm spp. egg (19.5%). Besides, Fasciola Spp.egg, Giardia lamblia cyst, and Entamoeba spp.cyst showed considerable decrement namely, 17.2%, 18.5%, and 15.9% respectively. However, Strongyloides stercoralis larva, and Ascaris lumbricoides egg was the least frequently detected parasitic forms with 12.5% and 9.1% prevalence rates, respectively.
The distribution of the parasites on fresh vegetables ( Table 2) also showed that Taenia spp. was the most prevalent of all parasites in all vegetable samples with a prevalence rate of 60.9% in Spinach, 56.2 in Cabbage, 45.3% in Lettuce, 37.5% in Potato, 26.6% in Green pepper, and 20.3% in Tomato.Next to Taenia saginata the second frequently detected parasite in Lettuce was, Hookworm spp. (31.25%) followed by G.lamblia(29.7%).
In Cabbage, G. lamblia, and Hookworm spp. egg/larvae were detected as second and third most prevalent parasites with 26.6% and 25% prevalence rate respectively. In spinach E. vermicularis egg and Fasciola spp.was the second (28.1%) and third frequently detected parasites. On the other hand, G. lamblia cyst was the second prevalent parasite (17.2%) in Green pepper samples succeeded by E. vermicularis, Hook worm spp.which was observed in 12.5% each.
In tomato samples G .lambilia cyst and Cryptosporidium spp.Oocyst.were recorded as the second most prevalent parasite stagess with a 10.9% prevalence each followed by E. histolytica, Hook worm spp., and E.vermicularis which were detected 6% each. The onesample proportion test result showed that all of the parasite detection proportions were found to be statistically signi cant with Pvalues less than 5% ( Table 2).
The chaisquare analysis showed that the type of fertilizers used, the educational status of the vendors, the type of vegetable they produce, and the status of wash before display was found statistically and signi cantly associated with the prevalent parasite contamination (P < 0.001)( Table 3).   The average prevalence is higher compared to some studies reported in Ethiopia 9, 10 , where they reported, 54.4% and 32.41% prevalence rate respectively but lowers than 2 which reported 57.8%. Moreover, compared to the study from Sudan 20 , Libya 21 , Iran 13 , and Jordan 23 , the prevalence rate is highest. But it is lower than the comparable study reported from Ghana 16 , Nigeria 23 , Kenya 33 and Iran 33 that showed prevalence rates of 57.5%, 76%, 75.9%, 79%, and Dahmar city, Yemen's highest rates (100%) 26 .
The geographic position and type and number of samples examined, detection methods, different techniques employed in the laboratory, water types used for irrigation, post-harvesting methods and the type of water used for the wash of vegetables may be attributed to this variation 8,21,20 .
Cabbage (79.7%), followed by spinach (75%) and lettuce (73.4%) were among the most commonly contaminated vegetables of the vegetables examined (Table 1). This difference in contamination rates between the vegetables could be due to shape and sizedifferences 34 . In particular, Cabbage and Spinach have larger and uneven surfaces which make the parasites attach more easily to the surface. This study is consistent with 15 reports that leafy vegetables are contaminated highly in Hanoi, Vietnam in comparison to root and fruit vegetables. Beside, Spinach's rough surface and leaf fold can retain dirt which cannot be removed easily 15 .
The smooth surface of green pepper and tomato, by contrast, can reduce the parasite attachment rate 34  Strongyloides stercoralis larvae were the seventh detected parasite with a prevalence rate of 12.5% ( Table 2).The nding of this study is similar to the previous report from Jimma, Ethiopia (21.9%) 2 . The low prevalence rate was reported in Awash, Ethiopia (6.2%) 10 , and Kogi state, Nigeria (6.06%) 3 . The Highest prevalence rate was reported in two markets in Ghana (36.4%) 16 . Ascaris lumbricoides egg was identi ed as the eighth detected parasite stage (9.1%)contaminating fresh vegetables in this study ( The presen study showed that the type of fertilizer used,the status of wash before display, the typeof vegetable they produce, and the educational status of the vendors was statistically and signi cantly associated with the contamination level of the vegetables (P < 0.001)( Table 3). This nding is consistent with previous reports by 9, 24 who revealed a high prevalence rate of parasites detected from raw manure fertilized vegetables in Mekelle city, Ethiopia, and Amman, Jordan respectively. This result is also in agreement with 34 who showed the high prevalence of vegetables contamination in Tercha town, southern Ethiopia was associated with the type of vegetables they produce, status of wash, and education status of vendors. In this study, the high prevalence of contamination was detected in washed vegetables than unwashed ones. This result is contrary to 7 who reported a high prevalence rate of parasites detected from vegetable samples unwashed before display in Arbaminch, Ethiopia. The high prevalence of washed vegetables in the present study may be due to the use of wastewater to wash the vegetables.

Conclusion
The highest levels of fresh vegetables contaminated with intestinal parasite stages were identi ed. The most frequently detected parasite forms were, in particular, Taeniasaginataproglottides /eggs and Enterobiusvermiculariseggs. The Cabbage was among the most contaminated vegetables screened for parasites. The high incidence of parasite contamination was statistically and signi cantly related to the type of fertilizer used; status of wash before display, vendor education status and vegetable variety they produce. In terms of transmission of the intestinal parasites to men, contaminated fresh vegetables can play an important role in Aksum markets. Therefore it is highly recommended, for instance, to avoid the use of raw manure as fertilizer, and standard washing before using raw vegetables, to reduce the risk of intestinal parasitic infections from contaminated vegetables.

Ethics approval
Ethical clearance was obtained from Aksum Universi thical review board.The purpose of the study was explained to the vendors of vegetables prior to data collection, and verbal consent was obtained from every study participant.