# Protozoa as the \"Underdogs\" for Microbiological Quality Evaluation of Fresh Vegetables
## 3 Results and Discussion
monocytogenes_ was detected, but at levels below 4 \(\times\) 10 CFU/g (Table 2), all the sampled products were considered safe based on the microbiological criteria set by the European Commission Regulation (EC) No. 2073/2005 for \"Pre-cut fruit and vegetables (ready-to-eat)\", i.e., the absence of _Salmonella_ spp. and _L. monocytogenes_ in 25 g samples and counts of _E. coli_ not exceeding 1000 CFU/g. This is in agreement with previous studies, which demonstrated that the prevalence of these pathogens in this type of product is generally low [65, 66, 67]. Although the sample that tested positive for _L. monocytogenes_ was unwashed lettuce, \"Even if salad leaves and herbs are labelled with an instruction to wash before consumption, they are considered ready-to-eat. Although washing can reduce microbial contamination on the plant's surface, it is not effective to eliminate microorganisms of concern or reduce them to an acceptable level because some pathogens can become internalised within the plant's tissue.\" [68]. The other microbiological parameters are recognized as hygiene and spoilage indicator micro-organisms in RTE foods. Most of the parameter results fell within the Satisfactory-Questionable categories according to the values recommended by the Portuguese National Health Institute [69]. Yeasts and molds were exceptions, with some products being classified as \"Not Satisfactory\" (yeast and mold counts exceeding, respectively, 10\({}^{6}\) CFU/g and 10\({}^{3}\) CFU/g) (Supplementary Table S1). The knowledge gained in this field during the course of SafeConsume WP2 highlighted the need to include protozoa in the microbiological criteria for foodstuffs, as required by EU Law No. 1441/2007. This is particularly the case of _T. gondii_ and the enteric pathogens _G. duodenalis_, _Cryptosporidium_ spp., and _C.acetylanensis_, which are the most common protozoa associated with foodborne illness outbreaks. The absence of fecal contamination in vegetables and the guarantee of food safety for consumers can definitely no longer be defined only by the analysis of detection limits for bacteria, i.e., _E. coli_, _L. monocytogenes_, and _Salmonella_ spp. However, microbial risk assessment evaluation and definition of strategies for the management and control of these parasites requires total collaboration between academia and industry, and investments by major companies and governments. These efforts are required to support and foster epidemiological and surveillance studies, and for the development of new technologies and \"golden standard procedures\" for protozoan removal (or recovery) and detection along the food chain. Monitoring of irrigation and processing water and the efficiency of wastewater treatment plants should be regularly performed. In addition, analysis of soil and manure used as fertilizer should be frequently undertaken, and animal access to crops and personal hygiene regulations applied to food handlers should be controlled. For instance, in addition to the use of sodium hypochlorite as a disinfectant, improved, safer methods, such as UV treatment, treatment with chlorine dioxide or ozone, membrane filtration, and a multi-barrier approach could be used as treatments for (oo)cyst inactivation and removal [33, 37, 70]. Furthermore, improvement in tools used for genotyping is necessary for the rapid and reliable detection of protozoans of concern for human health and to clarify their transmission patterns. The development of accurate methods for determining the viability and infectivity of (oo)cysts would also be invaluable in surveillance and outbreak studies, and for estimating risk to consumers. Finally, controlling foodborne (and waterborne) diseases should always be accomplished from a collaborative One Health perspective, involving a multidisciplinary team of public health and food experts, epidemiologists, veterinarians, environmental scientists, microbiologists (bacteriologists, virologists, and parasitologists), farmers, industry managers, lawmakers, and media communication specialists.