HAV in fresh vegetables: a hidden health risk in district Mardan, Pakistan
© Khan et al.; licensee Springer. 2014
Received: 30 September 2014
Accepted: 6 November 2014
Published: 18 November 2014
Hepatitis A is an acute inflammation of the liver caused by the hepatitis A virus (HAV) in human. The path of entry of HAV to the bloodstream is the epithelium of the intestine. Liver inflammation occurs when HAV multiplies within the hepatocytes and Kupffer cells of the liver. HAV is mostly transmitted by contaminated water, fruits and vegetables. The purpose of the current study was to evaluate fresh vegetables raised on the fecal contaminated water for the detection of HAV by PCR method. Twenty nine samples were collected from 13 different locations of district Mardan and screened for the presence of HAV. Village Bajowro near Takht Bhai was the most contaminated site having HAV in all vegetables grown over there. Water samples collected from this area proved to be contaminated with HAV. It may be concluded that fecal contaminated water is unsafe for irrigation because of the health risk associated with such practices.
Viruses are the most familiar causes of food-borne infections in human population. They remain inert in or on contaminated food items which may transmit infection. Besides several groups of viruses responsible for the contamination of food items, HAV and gastroenteritis viruses are the major food-borne viral pathogens which infect via the gastrointestinal tract (Vaughan et al. 2013; Lee et al. 2013). Viruses responsible for infection via the gastrointestinal tract are expelled in vomit and may likewise be excreted in feces. Viral pathogen may contaminate foods either directly from infected people or through sewage pollution (Seymour and Appleton 2001). Untreated or inadequately treated sewage is one of the key factors contaminating food and water (Iritani et al. 2014). Fruits and vegetables that are consumed without cooking are particularly risky in spreading viral pathogens.
HAV may make its way to vegetables from sewage containing human feces (Bosch 1998; Rodríguez-Lázaro et al. 2012). HAV effectively resists detergents, acids (even pH 1), solvents and extreme temperatures (up to 60°C) (Wright 2013; Cappellozza et al. 2012). These characters make it suitable for survival in sewage of domestic origin. Its survival time may reach up to months even years in fresh and salt water (Sobsey et al. 1986).
Mardan is the second largest city of Khyber Pakhtunkhwa Pakistan, which occupies an area of 1,632 km2, located at 34°12'0 N 72°1'60E and an altitude of 283 metres (928 ft) (Khan et al. 2011). A project of sewage treatment plant (STP) with total cost of Rs. 51.89 million was started by the Mardan Development Authority with the collaboration of Asian Development Bank in 1980. The project was completed and its control was handed over to Tehsil Municipal Administration (TMA) on July 14, 1999 to manage its functioning (Dawn News 2012: Mardan sewage treatment project rendered useless From the Newspaper Updated Apr 18, 2012 12:08 am). However, the project never worked and the dream of cleaner Mardan did not come true. For the same reason, practice of dumping sewage is directly into canals and other freshwater bodies without any pre-treatment is continued. Water in the canals are predominantly contaminated with sewage which is used to irrigate agriculture fields. Situation become verse in areas where domestic sewage is directly used for the purpose or irrigation. Purpose of the current study was to investigate the presence of HAV in fresh vegetables grown on the field irrigated with fecal contaminated water in the target area.
Material and methods
Sequence of sense and antisense primers used in PCR and RT-PCR
Croci et al. 2002
During our survey, we found that domestic sewage was running directly to canals irrigating different areas of Mardan. In some places, such as Bajawro kalay and Guli Bagh, fields were irrigated directly with domestic sewage containing human feces. The areas were also best for raising vegetables among different locations of district Mardan. According to the local community, different vegetables including tomato, peppermint, coriander, radish, turnip, cabbage, potato, okra, chili, apple gourd and bottle gourd etc.
Location-wise list of vegetables along with results indicating the presence or absence of HAV in these vegetables determined by RT-PCR
L1. Sazodin near Rehman cotton mill Takht Bhai.
L2. Samar Bagh Takht Bhai
L3. Bajawro kalay near Tablighi Markaz Takht Bhai
L4. Chail, Takht Bhai
L5: Western Bypass near Research Form Malakand road Mardan
L6: Sharmakhano bridge and Lodhi Abad Gujargharai, Mardan
L7: Deuband colony Bypass road Muqam, Mardan
L8: Miangul kalay near, Bypass road, Mardan
L9: Bachakalay, Mardan
L10: Guli Bagh, Mardan
L11: Meervas kalay Charsadda road Mardan
L12: Tauheed colony near Charsadda chowk, Mardan
L13: Near Fazli Haq College Mardan
Contaminated water drains and field of Samar Bagh irrigated with feces loaded water supported the growth of fresh and healthy vegetables, Solanum melongeana, L. acutangula and Cucurbeta pepo. PCR results proved that the vegetables of the area were free of HAV and hence, may be recommended for human consumption.
Momordica charantia and C. pepo were collected from fields of Chail, Takht Bhai. No band of DNA was detected in the PCR products using RNA extract from these vegetables, confirming the absence of HAV. Hence, the vegetables may be safe for human consumption.
People of Western bypass near Tobacco Research station, Malakand road Mardan are mostly illiterate, involved in agriculture by profession and green vegetables are grown all over the area. We sampled M. charantia and C. pepo from the area. The vegetables were free of HAV contamination as confirmed by the absence of PCR product on the gel.
C. pepo and L. acutangula, sampled from the fields in Sharmakhanon Bridge, Gujar Garhai. These vegetables were free from HAV and may be acceptable for human consumption. Similarly, L. acutangula and Abelmoschus esculentus grown in Deuband Muqam bypass road, Mardan were also free of HAV. No HAV RNA was detected in C. pepo and Capsicum annuum of Miangulkalay bypass barafkhana, Mardan.
Canal flowing through Bacha kalay, Mardan, located at a distance of 2.5 Km from Mardan city was also heavily polluted by human feces. However, vegetables grown in the area, C. annuum, Cucurbeta pepo and S. melongeana, were free of viral (HAV) contamination.
Guli bagh is an area located in the main city in close proximity to District Headquarter Hospital, Mardan. Canal passing through this area is heavily polluted with domestic sewage and hospital waste. Domestic sewage is also directly used for irrigating fields on which a number of different vegetables are grown. A. esculentus and L. acutangula were collected from this contaminated area. No HAV was confirmed by PCR.
The site of Sabzi Mandi Mirwas, Mardan, located at a distance of 1 Km from Mardan city, is moderately populated with 40 houses of the local population. Cucurbeta pepo and L. acutangula were collected from this area. No band corresponding to HAV RNA was detected on the gel indicating that the vegetables collected from this area were free from HAV contamination.
HAV contamination was not detected in the vegetables collected from Tauheed colony, Charsadda road, Mardan, which is located at a distance of 2 Km from Mardan city and are near Fazli Haq College, Mardan, about 3 Km from main city.
Vegetables from the most polluted area of district Mardan, where irrigation with fecal contaminated water is a common practice, were analyzed for HAV, a food born pathogen. Out of thirteen different locations, HAV was detected in the vegetables grown in Bajowro kalay only. We used RT-PCR based detection method for HAV detection. The method was previously used with success to determine the absorption of mammalian virus by vegetables (Seymour and Appleton 2001). Molecular biology techniques have been proposed for the sensitive and specific detection of some enteric viruses (Jean et al. 2001). The presence of HAV in three vegetables (C. pepo, M. piperita and L. acutangula) sampled from the area may be regarded as a hidden health risk to human population consuming such contaminated vegetables. The result of this study supported the idea that irrigation with fecal contaminated water may lead to HAV contamination of the crop. There are reports about the importance of HAV as a waterborne pathogen and the phenomenon is well recognized (Deboosere et al. 2012; Miura et al. 2013; Schultz and Myrmel 2013). Among the vegetables contaminated with HAV, M. peperita is consumed in its raw form and hence, may be a greater risk. Based on this postulation, other vegetables grown in such areas and consumed in raw form can be equally hazardous for humans. The initial survey has indicated the cultivation of tomato cucumber, onion, coriander, radish, carrot etc. on these fields which may magnify the intensity of the associated risk because of their consumption in raw form. Additionally, the HAV contaminated vegetables which are consumed in cooked form, may also contribute to the risk by communicating the virus to the HAV free vegetables and fruits consumed in raw form, through the use common kitchen utensils. Several HAV outbreaks due to consumption of berries and vegetables have been reported previously (Butot et al. 2007). Reports on contamination of vegetables from several parts of the world (Mukomolov et al. 2012; Hernández et al. 1997; Felix-Valenzuela et al. 2012) indicate the gravity of the situation.
It may be concluded that crop irrigated with sewage water may be screened not only for HAV, but also for other mammalian viruses, especially human pathogens of viral, bacterial, fungal and other origin. Based upon this study, the human consumption of vegetables grown on fields irrigated with fecal contaminated water is not recommended. Such ill practices need prompt attention of the government and civil society. Society mobilization against such practices is highly desired and the purpose may be achieved by educating people to understand this hidden health risk.
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