Salmonella infection in children from the wastewater-spreading zone of Marrakesh city (Morocco)
Correspondence to: Professor Lahcen Hassani, Laboratoire de Microbiologie, Faculté des Sciences Semlalia, Université Cadi Ayyad, BP 2390, 40 000 Marrakech, Morocco (e-mail: firstname.lastname@example.org).
The available circumstantial evidence gained from epidemiological and microbiological investigations suggests that the use of untreated wastewater causes an excess of Salmonella infection among children living in El Azzouzia (the wastewater-spreading area of Marrakesh city, Morocco) compared with those from a control area that does not practice sewage irrigation (Sidi Moussa). The prevalence in the exposed group (32·56%) was significantly (P < 0·001) higher than for the control group (1·14%). Serogroups B and C were the most commonly isolated. Boys were at greater risk (37·61%) of contracting Salmonella infection than girls (26·66%). Age-specified rates showed that children of less than 10 years old were infected at a higher rate than older children in the area (exposed group), with 40·32% and 19·72% rates of infection, respectively. Crop irrigation with untreated wastewater caused a significantly higher rate of infection with Salmonella in the children of agricultural workers (39·33%) than in the children of non-agriculturists (24·58%).
Wastewater irrigation is practised in many countries around the world. The practice has special significance for developing countries with arid and semi-arid climates and limited water resources. In Morocco, the wastewater-spreading area of Marrakesh city (El Azzouzia) is the seat of intense agricultural activity where the surface irrigated is about 3000 ha. The crops are diverse and are destined for both farmers consumption and marketing. Land application of wastewater provides nutrients for crop growth as well as organic matter for soil conditioning, and it is often the most economic means of wastewater disposal. However, the problem with using untreated wastewater for irrigation is that it contains disease-causing pathogenic organisms (bacteria, viruses, protozoa and helminths). Wastewater may spread excreta-related infections which can be a danger to public health when crops are consumed raw ( Shuval et al. 1986b ; Mara & Cairncross 1989; Armon et al. 1994 ; Lerman et al. 1994 ). Despite the extensive worldwide practice of wastewater irrigation dating back many years, there have been few studies that have assessed the epidemiological evidence of quantifiable human health effects associated with wastewater irrigation ( Bertranou et al. 1983 ; Shuval et al. 1984 ; Lerman et al. 1994 ). The purpose of this research was to determine the prevalence of Salmonella by a copro-epidemiological study of children living in the wastewater-spreading field of Marrakesh (El Azzouzia) compared with children from a control community (Sidi Moussa) not practising wastewater irrigation, in order to evaluate the risk associated with wastewater re-use in the transmission of Salmonella.
Investigation of some epidemiological factors (sex, age, occupation of the parents) allowed an assessment to be made of which groups were at high risk of exposure among the studied population.
Materials and methods
An epidemiological prospective study was carried out among children in the wastewater disposal area of Marrakesh city (El Azzouzia) and in a control area (Sidi Moussa). This study was conducted from March 1997 to August 1997. During visits to both areas, children (boys and girls) aged between three and 15 years were randomly selected from families. A total of 740 in-patient children, including 390 children living in the wastewater irrigation zone and 350 control children, were examined ( Table 1). The two groups studied lived in a Marrakesh peri-urban area and had similar socio-economic and demographic characteristics.
Table 1. Distribution of children by age and sex
|Total/area||390|| ||350|| |
|Total number||740|| || || |
A complete report, including information on the residence from where the specimen was collected, age, sex, and family occupation, was undertaken.
Stool samples were collected in sterile glass bottles, brought to the laboratory and examined within 2 h. The isolation of Salmonella strains was carried out according to the methods of Baleux et al. (1988) . Three test-tubes, each containing 10 ml of single strength enrichment medium, were inoculated with 1 g of faecal sample. For enrichment, N-selenite cystine broth (Biokar) and tetrathionate broth (Diagnostics Pasteur) were simultaneously tested. All the enrichment tubes were incubated at 42 °C for 24 h ( Morinigo et al. 1986 ). Following incubation, sub-cultures were made from each tube of enrichment broth on plates of Salmonella–Shigella (S-S) agar (Biomérieux) and Hektoen enteric agar (Difco). The plates were incubated at 37 °C for 24 h, then two suspected Salmonella colonies from each plate were sub-cultured for purification on nutrient agar. Then, they were examined using Kligler iron agar (Biomérieux). The isolates that matched the biochemical profile of the genus Salmonella were later confirmed with the API Z System (Biomérieux, France); their serological characteristics were investigated by the slide agglutination test using commercial polyvalent O antisera (OMA, OMB and OMC) and monovalent antisera (O1,2, O4,5, O6,7,8 and O9) (Sanofi-Pasteur, France).
The statistical analysis was made using the chi-square test ( Scherrer 1984).
The prevalence of Salmonella infection was 32·56% (127/390) among children living in the wastewater-spreading field of Marrakesh city (El Azzouzia). Serogroups B and C were the most commonly isolated at infection rates of 9·8% and 15·6%, respectively. Other serogroups, including group A (1·79%) and group D (3·33%), were also isolated ( Table 2).
Table 2. Prevalence (%) of different Salmonella serogroups isolated from the studied populations
|Control group||–||–|| 0·28||–|| 0·85|| 1·14|
In the control area (Sidi Moussa), the prevalence of Salmonella infection was only 1·14% ( Table 2). The infection rate was significantly higher (χ2 = 125·92; P < 0·001) among children in the exposed group than in the control group.
In the wastewater-spreading zone of Marrakesh city, a high proportion of children showed diarrhoea symptoms. The prevalence was 35·38% (138/390) in the exposed group whereas for the control group, it was 14·85% (52/350) (χ2 = 40·84; P < 0·001).
In the exposed group, Salmonella infection was significantly higher in boys than in girls (χ2 = 5·30; P < 0·05), with 37·61% and 26·66% infection rates, respectively.
hildren aged between three and 10 years were shown to carry a significant excess (χ2 = 17·73; P < 0·001) of Salmonella (40·32%) compared with those aged between 11 and 15 years (19·72%) ( Table 3).
Table 3. Prevalence of Salmonella in the test group according to age of children
The occupation of the parents was a significant factor influencing the prevalence of Salmonella. For the children of agriculturists, the rate of Salmonella infection (39·33%) was significantly higher (χ2 = 9·60; P < 0·01) than for those from non-agriculturist families (24·58%).
Throughout this study, it became clear that re-use of raw wastewater leads to the contamination of children by Salmonella. The rate of infection with Salmonella was much higher among children in the exposed group than that found in the control group of the same age and socio-economic background. El Azzouzia is irrigated with untreated wastewater which contains high concentrations of pathogenic micro-organisms ( Boussaid et al. 1991 ; Hassani et al. 1992 ). The consumption of raw vegetables irrigated with this wastewater, and the direct contact with wastewater, may in part explain this excess of infection. During site visits to the area, farm workers were observed furrow-irrigating many vegetables, some of which were to be consumed raw, including lettuce, tomatoes, parsley, potatoes and cereals. Furthermore, it was observed that children played in the fields irrigated with raw wastewater.
A high incidence of Salmonella infection in children living in the wastewater-spreading area has previously been reported in other cities. In Mendoza (Argentina), the incidence of Salmonella was 23% in the exposed group compared with 4% in the control zone ( Bertranou et al. 1983 ). One of the most well known outbreaks associated with wastewater irrigation of vegetables was the outbreak of cholera in Jerusalem in 1970 ( Shuval et al. 1984 ). The disease almost totally disappeared when irrigation of vegetables and salad crops with wastewater was stopped. In Santiago (Chile), strong circumstantial evidence was found indicating that typhoid fever can be transmitted by vegetables and salad crops irrigated by raw sewage ( Shuval et al. 1986a ) . The incidence of typhoid in Santiago was much higher than in comparable cities in Chile that did not practice sewage irrigation.
The excessive diarrhoea infection in children from the wastewater-spreading area was similar to that observed by Bertranou et al. (1983) and Black et al. (1985) , who reported an excessive prevalence of diarrhoea in the exposed group compared with the control group. In Sidi Moussa, 14·85% of children had diarrhoea but from only 1·14% was Salmonella isolated. This could be explained by other bacterial or viral infections.
In El Azzouzia, boys were significantly more infected by Salmonella than girls because they accompanied and helped their parents in the fields while the girls usually stay at home. During visits to El Azzouzia, young children could sometimes be found playing in the fields irrigated with untreated wastewater, or in water polluted by sewage effluent. In Santiago (Chile), the incidence of typhoid fever was also higher in young children ( Black et al. 1985 ).
In the wastewater-spreading area of Marrakesh city (El Azzouzia), the children of agriculturists showed an excess of Salmonella infection. During some visits to the fields, it was observed that as the farmers’ houses usually abut wastewater-irrigated fields, there is ample opportunity for the children to come into direct contact with wastewater, and indeed were observed to do so. For instance, the children of agriculturists involved in controlling the flow of irrigation water used their hands, in addition to their tools, to make earth dams.
Salmonella is a natural inhabitant of the intestinal tract of domestic and wild animals and is shed by infected persons in faecal material. From this source, the bacteria contaminate the environment and can grow in water and food. The contaminated food can transmit pathogenic micro-organisms to agricultural workers and their families, and also to consumers if crops are consumed raw or brought raw into the home where they can contaminate other food ( Shuval et al. 1984 ; Mara & Cairncross 1989; Hedberg et al. 1994 ).
In developing countries, human salmonellosis continues to be a major disease. The use of wastewater in agriculture represents an important source of spread of the disease into the environment. In conclusion, this study provides evidence that the prevalence of Salmonella among children living in El Azzouzia, the municipal wastewater-spreading field of Marrakesh city (Morocco), is higher than in the control area not practising wastewater irrigation. The problem could be overcome if the wastewater was treated to remove all microbial pathogens and to meet the quality guideline for allowing unrestricted irrigation ( Mara & Cairncross 1989).
This work was financially supported by the European Union (Project no. SEM 03/204/017) and the society SOABIMEX (Group Yves Rocher, France). The authors are grateful to the Regional Services of the Moroccan Ministry of Public Health for technical assistance with this project (Delegation du MSP à la Wilaya de Marrakech, SIAAP Menara-Gueliz, and Dispensaire de Sidi Moussa). The also wish to thank Dr R. Serraj for critical reading and correction of the manuscript.