Detection of Parasitic Contamination of Commonly Consumed Vegetables Sold at Obollo-Afor Market, Nigeria

Background and Objective: Vegetables are plant edible components that are eaten whole or in pieces, raw or cooked as part of a main dish or salad. The parasitic fauna of ten different vegetables distributed at Obollo-Afor Market, Enugu State, Nigeria, were investigated. The objective was to determine the prevalence of parasitic contamination of different commonly consumed vegetables sold at Obollo-Afor Market. Materials and Methods: The samples were centrifuged after being cleaned with normal saline and left to stand for 10-12 hrs to allow for sedimentation and the sediments were then inspected under a microscope. Using a modified Zieehl-Nelsen approach, the oocysts of the parasite Cryptosporidium parvum were found in vegetables. A questionnaire was used to gather information about the vegetables. Results: The overall prevalence of parasites found in the vegetables was 44 (36.7%). Ascaris lumbricoides ova (18.3%) was the most prevalent parasite whereas Trichuris ova (0.8%) was the least. False cubeb leaf (60%) was the most contaminated vegetable while African spinach (10%) was the least. As 17 (41.5%) vegetable sellers used clean water for irrigation, 1 (2.4%) used wastewater, 11 (26.8%) used animal (human) faeces as manure, 3 (7.3%) used organic manure whereas 4 (9.8%) used inorganic manure. Conclusion: There is a moderate prevalence of parasitic infestation, probably due to handling procedures from production to sales.


INTRODUCTION
The relative nature of the host-parasite relationship varies and this made it possible to illustrate a list of parasite species that show an increasing degree of metabolic dependence on their hosts.Poulin and Morand 1 drew a hypothetical scale in which at one end of the scale is zero dependence, implying a free-living organism and on the other end is 100% or total parasitism.In between these two extremes lies a range of organisms that satisfy their metabolic requirements to a varying extent at the expense of the host.
Vegetables are planted edible components that are eaten whole or in pieces, raw or cooked as part of a main dish or salad 2 .A vegetable includes leaves, stems, roots, flowers, seeds, fruits, bulbs, tubers and fungi 3 .Vegetables are an essential part of a healthy human diet owing to their nutritional value 4 .To prevent chronic diseases like heart disease, cancer, diabetes and obesity as well as to prevent and treat several micronutrient deficiencies, the World Health Organization recommends consuming at least 400 g of fruits and vegetables daily, especially in less developed countries 5 .Regular consumption of vegetables helps to prevent the human body against a number of diseases by providing nutrients such as vitamins, minerals, proteins and fibers, it also plays a vital role in body weight regulation and related conditions such as diabetes and hypertension 6 .Ononogbu 7 , reported that vegetable fats and oils lower blood lipids thereby reducing the occurrence of disease associated with the damage of the coronary artery.Vegetable fats and oil serve as precursors of prostaglandins which are known to perform the role of vasoconstriction and vasodilation of the blood vessels.Vegetable fats and oil are known to serve as precursors of thromboxane which facilitate blood clotting in humans 7 .
However, some vegetables are consumed raw in other to maintain their natural taste and to prevent heat liable nutrients 8 .Vegetables are potential sources of transmission for intestinal parasites 6 .This means of parasitic contamination is a bigger issue in resource-constrained nations like Nigeria, where feces in undesignated areas by scavenging animals and people as well as the use of dirty water for vegetable irrigation are all common 9 .For instance, helminths that are spread by eating raw vegetables, such as Hymenolepis nana, Toxocara canis, Ascaris lumbricoides, Ancylostoma duodenale and Enterobius vermicularis, are well recorded among Nigerians 10 .The high levels of contamination with harmful parasites in many poor nations have been attributed to the use of untreated sewage to irrigate vegetables 11 .Due to aging, hunger, HIV infection and other underlying medical problems, many people are extremely vulnerable to parasite infections 12 .Fresh veggies have been associated with an increase in cases of foodborne illness that have been reported 8 .Research has shown that humans can become infected with parasites like Ascaris lumbricoides, Cryptosporidium species, Entamoeba histolytica, Enterobius vermicularis, Fasciola species, Gardia intestinalis, Trichuris trichiura, hookworm, Hymenolepis, Taenia and Toxocara species by eating tainted, raw, or improperly cleaned vegetables 13 .It has been known for at least 100 years that raw vegetables can spread sickness to people 14 .The usage of untreated manure, primarily composed of pig and poultry feces and other practices connected to vegetable contamination are widespread in the Obollo-Aforaxis [13][14][15] .
To prevent and control medically important parasitic diseases, assessing the sources of infectious agents and their level of contamination is crucial because these parasites are highly resilient and able to tolerate challenging circumstances 12 .There is little information on the dangers of parasite diseases linked to vegetable consumption in the research domain, therefore, a need to carry out this research work.This study is aimed at the detection of parasitic contamination of commonly consumed vegetables in Obollo-Afor Market, Udenu Local Government Area (LGA), Enugu State, Nigeria and the specific objectives include determining the prevalence of parasitic contamination of different commonly consumed vegetables sold at Obollo-Afor Market, the prevalence of parasite groups in different vegetables sold, the demographic characteristics of respondents using questionnaires and the major sources of parasitic contamination of vegetables in the study area in relation to the educational level of the respondents.

MATERIALS AND METHODS Study area:
The study was carried out during the rainy season from June to August, 2019.The vegetables were purchased from Obollo-Afor Market where other surrounding markets get their supply of vegetables.The administrative centre of Udenu LGA of Enugu State, Nigeria, is located in Obollo-Afor.It is the point at which the Igbo drive turns north.Amalla and Idoma in Benue State, Enugu-Ezike Northwest and the Umundu, Igugu and Imilike people in the South form its Northern, Eastern and Southern borders, respectively.Obollo-Afor is at a good location.Due to its advantageous position, prostitution is prevalent there as it serves as the entrance to both the Northern and Eastern States of Nigeria 16  Vegetable sample collection: On each day of sample collection, the vegetables were collected randomly from different vendors to obtain a qualitative estimation of the parasitic content in each vegetable.A 50 g sample was taken of each vegetable randomly purchased from different vegetable vendors.The screening of the vegetable samples was carried out in the University of Nigeria, Nsukka, Department of Zoology and Environmental Biology laboratory.
Processing the vegetable samples: The vegetable samples were subjected to parasitological examination according to Endale et al. 12 .A portion (50 g) of each vegetable was washed separately in 125 mL of normal saline (0.85% NaCl) for detaching the stages (ova, larvae, cysts and oocysts) of the parasites commonly assumed to be associated with contamination.According to Salavati et al. 17 the washing solution was fixed in formalin (95 mL of the solution against 5 mL of 4% formaldehyde) to preserve the different parasitic stages.The washing solution was allowed to stand on the bench for 10-12 hrs for proper sedimentation.The supernatant was discarded, 15 mL of the sediment was transferred to a centrifuge tube.The tube was centrifuged at 3000 rpm for 5 min in order to concentrate the parasite stages.After centrifugation, the supernatant was gently decanted without shaking.To disseminate the parasite stages, the silt was carefully stirred 12 .

Parasite detection:
As 100 µL of sediment was transferred to a clean glass slide, covered with cover glass and inspected using ×10 and ×40 objectives on a light microscope 12 .
Modified Zeihl-Neelsen staining approach: Clarke and Mclntyre 18 described a modified Zeihl-Neelsen staining method for identifying Cryptosporidium species oocysts.A thin smear was formed straight from the silt and left to air-dry.The slides were fixed in methanol for 5 min before being stained with carbon fusion for 30 min.The slides were then washed with tap water and decolonized with acid alcohol (1 mL concentration HCl in 99 mL of 96% ethanol) for 1-3 min.Finally, the slides were rinsed with tap water and allowed to air-dry.The slides were then examined under a light microscope (the manufacturer: Philip Harris, Location: China, serial No. 0006149) at ×100 magnification with immersion oil.
Statistical analysis: Data were analyzed using the Statistical Packages for Social Sciences (SPSS) version 23.0 (IBM Corporation, Armonk, USA).The prevalence of vegetable contamination by parasites was compared using Chi-square analysis.Frequency distribution was used to summarize questionnaire responses.Chi-square analysis was used to compare the frequency of responses relative to respondent characteristics, as questionnaire responses were not matched to contamination status.Only where responses to the questionnaire relate significantly to respondents' demographic characteristics was the result displayed in the result section.The level of significance was set at 95% probability (i.e., p<0.05).

RESULTS
A summary of the distribution of the prevalence of parasitic contamination in the vegetable types is presented in Table 1.A total of 120 vegetable samples consisting of 10 types of vegetables commonly consumed were examined for parasitic contamination.Overall, 44 (36.7%) of the 120 samples were contaminated with parasites.The prevalence of parasite contamination ranged from 10.0-60.0%,though the difference in parasite contamination between the vegetable types was not significant statistically (χ 2 = 9.187, p = 0.420).False cubed leaf was the most contaminated vegetable (60%) while the least contaminated vegetable in this study was African spinach (10%).
The distribution of helminth contamination by vegetable types is also shown in Table 2. Parasitic groups isolated were four nematodes (Ascaris ova, hookworm, Strongyloides larva and Trichuris ova), one cestode (Taenia spp.) and two protozoa (Entamoeba histolytica cyst and Cryptosporidium parvum oocyst).Nematodes were the most prevalent while cestode was the least.Among the nematodes, Ascaris ova 22 (18.3%)predominated while Trichuris ova 1 (0.8%) was the least.Taenia species 2 (1.7%) was the only cestode examined.Entamoeba histolytica 9 (7.5%) predominated among the protozoan while Cryptosporidium parvum oocyst 3 (2.5%) was the least.Generally, there was no significant difference in the prevalence of each parasite type between vegetable types (p>0.05).
Level of education was another important determinant of irrigation pattern.All the farmers who had non-formal education practised clean water irrigation farming 5 (100%).This contrasts strongly with those that had primary 6 (40.0%), secondary 5 (27.8%) and tertiary education 1 (33.3%) who practised clean water irrigation while 1 (33.3)tertiary respondents practiced wastewater irrigation (Table 5a).The difference in irrigation pattern between those who had non-formal education compared to other levels of education was observed significantly (χ 2 = 21.624,p = 0.001).Those vegetable farmers who had non-formal education always used either animal feces 4 (80.0%) or other organic manure 1 (20.0%).Those that had formal education sometimes used manure ranging from animal feces to organic and inorganic types.This was responsible for the significant difference observed (χ 2 = 18.353, p = 0.031) (Table 5b).

DISCUSSION
A total of 44 (36.7%) out of the 120 samples were contaminated with parasites.The prevalence of contamination of commonly consumed vegetables was not statistically significant.False cubed leaf was the most contaminated vegetable (60%) while African spinach (10%) was the least contaminated vegetable in this study.This study revealed that there was a considerably moderate level of parasitic contamination of vegetables in the Obollo-Afor Market.This is in line with the previous reports from Alexandria, Egypt and Naknon Si Thammarat Province, Southern Thailand 19 where the contamination rates were 31.7 and 35.1%, respectively.However, the present findings contrasted with the reports from Nsukka ecological zone, Nigeria 15 , Tripoli, Libya 20 , Metro Manila, Philipines 21 and Southern Iran 22 where the contamination rates were 51.8, 58, 51.6 and 43.7%, respectively.A lower contamination rate of 3.5% was observed in North-East Nigeria in a study conducted by Adamu et al. 23 whereas, Kozan et al. 24 , reported a 0% prevalence of helminthic eggs in washed vegetables in Turkey.Also, Saudi Arabia 25 and Bahir Dar City, Northwest Ethiopia 26 reported lower contamination rates of 16.2 and 39.1%, respectively.Vegetables are important means of parasitic contamination, especially intestinal parasites, which represent an important source of food-borne outbreaks in developing countries 27 .
In the present study, 10 different vegetable types were screened for different stages of parasites.False cubeb leaf and bushbuck were mostly contaminated (60.0 and 50.0%), respectively.This could be attributed to the creepy nature of these vegetables.They seem to be more vulnerable to parasitic contamination because they creep on soil that may contain contaminated night soil and other contaminated animal dung.On the other hand, African spinach and bitter leaves had the lowest contamination rates of 10 and 20%, respectively.This could be attributed to the fact that they grow taller and therefore, less vulnerable to parasitic contamination.
There are major potential determinants of parasitic contamination revealed from the questionnaire responses.Firstly, the type of water used for irrigation in town, where Obollo town practised only (31.3%) clean water irrigation and (68.8%) unclean water irrigation, Enugu-Ezike practised (50%) clean water and (50%) unclean water for irrigation purposes.The second likely determinant of parasitic contamination of vegetables in these areas could be attributed to the type of manure used.The majority of vegetable farmers used human/animal feces as manure.These types of manure are potential carriers of different stages of parasites.Thirdly, the display patterns of vegetable in the market matters a lot.Those vegetables displayed on the ground have higher tendencies to be contaminated because of dust and dirt from surface runoff.Also, the majority of the vegetable sellers washed the vegetables before display even though some were washed with dirty water.Washing the vegetables before display reduces the chances of parasitic transmission to humans.
Ascaris lumbricoides ova happened to be the most prevalent parasite in this study (18.3%).This is in consonant with research conducted in Mannuthy, India 28 where the Ascaris species was the only parasite isolated from the positive samples (2.7%).In another study carried out in West Bengal, India, the Ascaris species was the most predominant parasitic ova observed 29 .Ascaris species which is a roundworm affects about a quarter of the world's population 30 , It is one of the resistant intestinal infections that is frequently employed as a parasitological indicator 31 and is more common in developing countries 30 .The possession of a thick outer covering (cuticle) made Ascaris species more resistant to adverse environmental conditions and provides them with a long span in the soil 32 .
Therefore, the vegetables should not be eaten raw and/or must be properly washed before consumption.This study is applicable to vegetable handlers or vendors (sellers and consumers), they should always put on hand gloves to avoid being contaminated.

CONCLUSION
There is a moderate prevalence of parasitic contamination of vegetables sold at Obollo-Afor Market, probably due to handling procedures from production to sales.Ascaris lumbricoides was the most prevalent parasite.Vendors that washed their vegetables were almost two times more than those that didn't wash.Information gathered from this research should be notified to the general public of the tremendous increase in the rate of parasitic contamination of vegetables.A policy should be made on how best to manage vegetables from farm to table.The usual home practice of washing vegetables with saline water should be maintained.

SIGNIFICANCE STATEMENT
The purpose of this research was to determine the prevalence of parasites in commonly consumed vegetables sold at Obollo-Afor Market, Nigeria.It revealed vegetables sold at the market were contaminated with parasites of public health importance.The overall prevalence of parasites in the vegetables was 36.7% and Ascaris lumbricoides being the most predominated parasite.False cubeb leaf (60%) was the most contaminated vegetable whereas African spinach (10%) was the least.There was no significant difference in the prevalence of each parasite type between vegetable types.This study should be furthered by the use of molecular characterization of parasites of vegetables to confirm identification.

Table 2 :
Prevalence of parasite groups in different vegetables sold by retailers at Obollo-Afor Market

Table 3 :
Demographic characteristics of respondents summarized by frequency

Table 4 :
Summary of questionnaire responses of vegetable sellers at Obollo-Afor Market

Table 5 :
Classification of irrigation and manure type by town and level of education of vegetable sellers at Obollo-Afor Market (a) Irrigation type Irrigation type