Surveillance studies from around the world reported widely varying contamination rates for the three protozoan in leafy greens, attributable to differing sampling strategies, geographic location, sanitation and detection methodologies.
Parasitic (oo) cysts have usually been monitored in food matrices following three steps: elution, concentration and detection [22]. The difference in properties of the various food matrices could make it difficult to remove and detect protozoan (oo) cysts [2]. Despite the establishment of a standard ISO [23] for the microscopic detection of Cryptosporidium spp. and G. duodenalis in leafy greens and berry fruits, there are still other protozoan parasites like T. gondii that need development of standardized methods. In addition, this standard is based on immunomagnetic separation technique followed by immunofluorescent assay for detection that are expensive, time consuming and require a microscopy expertise, making this method difficult to use for routine checks by food processors. Therefore, there have been a multitude of described methods worldwide for parasitic detection in vegetable matrices with different recovery efficiencies and detection levels [22]. This contributes to the under diagnosis of protozoan parasites in food.
In our study, we used a rapid molecular method that involved elution with 100 ml of 0.01% Tween 80 / PBS pH 7.2 in filter stomacher bags, under horizontal shaking at 37°C. It allowed the detachment of (oo) cysts from vegetable leaves due to the capacity of Tween 80, as a nonionic surfactant, to enter the interface between the vegetable surfaces and the parasites to ease adsorption at the interface and to minimize the interfacial tension, and consequently reduce the attractive interactions between the microorganisms and vegetables surfaces [36]. Consistent with this, 0.01% Tween 80 / PBS elution buffer has already been successfully used to elute parasites from leafy green vegetables [37–39]. In addition, the filter stomacher bags allowed removing large particles (above 250 µm) that could interfere particularly at the DNA extraction phase. The duration of elution was sufficient to ensure (oo) cysts recovery with minimum formation of debris and matrix that could affect the process of elution. The recovery of the parasitic forms could have been improved by a purification step using the immunomagnetic separation “IMS” as recommended in the standardized method ISO [23] for the detection of G. duodenalis and C. parvum. However, the IMS is time consuming, more expensive and is not suitable for the detection of T. gondii since the only described monoclonal antibodies for T. gondii oocyst walls led to a low recovery rate ranging from 0.2 to 35% and a high value of LOD (33 oocysts/g of basil and raspberries) [35]. DNA extraction is mentioned to be affected by the technique that is used to prepare the DNA template, with superiority of some techniques over others [40]. Vegetables constituents (e.g., polysaccharides, polyphenols, pectin and xylan) may be co-extracted with the targeted parasite DNA and thereafter inhibit the PCR by cross-linking with nucleic acids and modifying their chemical properties [41]. Herein, the DNA extraction was performed using a kit based on mechanical disruption of the sample that has been successfully used to extract parasitic DNA in food matrices [29, 40, 42] as well as other matrices like soil [28] and cat feces [43]. The overall method led to LOD95 ranging between 0.04 and 4 parasites/g that are suitable with occurrence studies except for C. parvum in lettuce (LOD95 = 40 oocysts/g).
Compared to T. gondii and G. duodenalis, the detection of C. parvum is less sensitive in our study; this could be related to the use of a single copy target gene. Shapiro et al. [44] recently designed a multiplex system also based on the 18S ribosomal RNA that should be tested on our samples in the future. Despite the low limits of detection of oocysts, we were not able to observe a linear response (r² > 0.98) between the Cq and the number of parasites spiked on leafy greens, probably due to the presence of inhibitors in vegetable samples. Further adjustments could include inhibition control and efforts have still to be made to overcome inhibition problems and to succeed in quantifying the detected parasites.
This study achieved its goal of determining the occurrence of T. gondii, G. duodenalis and C. parvum in leafy greens marketed in Marrakech, over the period April 2018 and October 2019. It is known that leafy greens can be exposed to parasitic contamination, given the nature of their foliage and the structure of their surface, for instance lettuce has broad and irregular leaves, while coriander and parsley have flat leaves and dense foliage providing a large contamination surfaces and favoring parasitic attachment. Indeed, we have detected a relatively high proportion of contaminated leafy greens (32%), similarly to the finding of our recent study undertaken in 2017, in Marrakech [20]. However, studies from some other more populated and largest countries of North Africa have indicated different levels of contamination in various leafy greens (e.g., 35.6% in Alexandria, Egypt [17]; 2.2% in Tripoli, Libya [14]). In more developed countries, the proportion of contaminated vegetables with parasites tends to be lower [e.g., less than 1% in Canada [2]; 6 % in Norway [13]].
Recently, it has become evident that ingestion of oocysts in fresh produce is an under recognized transmission route of contamination. A recent source attribution meta-analysis has highlighted the involvement of vegetables in sporadic toxoplasmosis [45]. The present study revealed a high rate of T. gondii (29.6%) in leafy greens. The detection of this parasite has been reported elsewhere such as in Czech Republic 9.6% (28/292) [46] and Portugal and Spain 42.9% (14/35) [47], using molecular methods. In North Africa, only two studies have been conducted to investigate the presence of this parasite in fresh vegetables: the study performed in Egypt [15] has revealed using microscopy a contamination rate of 5.6 % (19/212), while our previous study [20] showed an overall rate of 21% (18/86), using the same qPCR.
In our study, G. duodenalis was detected in 2.6% of leafy greens, this was in agreement with the contamination rates reported in other studies, using microscopy, as Libya with a rate of 2.2% (12/54) [14] and Egypt with a rate of 4% (2/49) [19]. In contrast, our present finding was lower than those reported recently in Morocco with 7% (6/86) [20], Egypt with 9% (47/530) [18] and India 5% (13/284) [3]. Previously, Bouhoum and Amahmid [21] have evaluated the presence of Giardia cysts in crops irrigated with treated and untreated wastewater, in Marrakech: this study revealed the presence of G. duodenalis in 20.3% of the 9 analyzed samples of coriander, while it was not detected in crops irrigated with treated wastewater. This may confirm that the use of raw wastewater for irrigation contributes to parasitic contamination.
Cryptosporidium spp. was not detected in any of the analyzed samples, while the rates observed in leafy greens in other studies were considerably higher: 20% (99/494) [16] and 4.6% (4/86) [20]. The difference between our previous [20] and present results could be due to two reasons: i) the oocysts could be present in low quantities that were considerably under the limit of detection of this described protocol, and/or ii) the observed Cryptosporidium spp. oocysts may not belong to C. parvum, C. hominis or C. meleagridis that are targeted by the qPCR used in the study.
The data presented here on the occurrence of T. gondii, G. duodenalis and C. parvum in leafy greens is a crucial step in identifying potential sources of parasitic infection and potential exposition of consumers in Marrakech. A limitation of molecular assays for the detection of protozoan (oo) cysts in produce is the inability to distinguish between living and dead organisms. Therefore, a positive result does not necessarily mean that there is a risk for consumers. However, populations of (oo) cysts often consist of viable and non-viable organisms in different proportions, and as very low dose of (oo) cysts are necessary to lead to human infection, any findings should be considered as an indicator of exposure.