# Protozoa as the \"Underdogs\" for Microbiological Quality Evaluation of Fresh Vegetables  
[PERSON]  
1 Associate Laboratory for Animal and Veterinary Sciences (AL4 AnimalsJ), Pracq do Coronel Pacheco 42, 4050-083 Porto, Portugal; [EMAIL_ADDRESS] (C.S.M.J. ; [EMAIL_ADDRESS] (S.S.);[EMAIL_ADDRESS] (A.C.); [EMAIL_ADDRESS] (J.M.C.d.C.) [PERSON]  
1 Associate Laboratory for Animal and Veterinary Sciences (AL4 AnimalsJ), Pracq do Coronel Pacheco 42, 4050-083 Porto, Portugal; [EMAIL_ADDRESS] (C.S.M.J. ; [EMAIL_ADDRESS] (S.S.);[EMAIL_ADDRESS] (A.C.); [EMAIL_ADDRESS] (J.M.C.d.C.) [PERSON]  
1 Associate Laboratory for Animal and Veterinary Sciences (AL4 AnimalsJ), Pracq do Coronel Pacheco 42, 4050-083 Porto, Portugal; [EMAIL_ADDRESS] (C.S.M.J. ; [EMAIL_ADDRESS] (S.S.);[EMAIL_ADDRESS] (A.C.); [EMAIL_ADDRESS] (J.M.C.d.C.) [PERSON]  
4 Cobel Avent  
seeds and an outbreak of listeriosis due to the consumption of frozen vegetables contaminated with _Listeria monocytogenes_[7, 8]. In addition to bacteria, several protozoan parasites can also contaminate fresh produce. _Giardia duodenalis_, _Cryptosporidium_ spp., _Toxoplasma gondii_, and _Cyclospora cayetanensis_ have been associated with several global outbreaks of foodborne illnesses related to the consumption of contaminated fresh vegetables and fruits, and are the most important emerging parasitic protozoans that commonly infect humans and animals [9, 10, 11, 12, 13, 14]. Despite guidelines and regulations that support the need for tracking and monitoring protozoa in fresh food products, their routine exclusion from detection and surveillance systems can be ascribed to the limited number of studies investigating parasite contamination of vegetables, the absence of consensual and accurate methodologies for their identification, and the wide acceptance among the expert authorities that the bacteriological aspects of the evaluation are more relevant for food safety [14]. Consequently, the monitoring of the microbial quality of fresh products in the industrial environment has mainly focused on bacterial indicators, and EU and national laws highly regulate the presence and microbiological limits of, in particular, the bacteria _E. coli_, _L. monocytogenes_, and _Salmonella_ spp. [15, 16, 17, 18]. In contrast, protozoan pathogens are rarely tested in fresh produce for food safety purposes. They are frequently neglected because they do not cause an immediate deleterious clinical response, but rather a chronic and long one. In addition, the associated diseases are also more common and prevalent in poor countries or communities. However, the harmful effects of these pathogens on humans in developed countries, especially on immunocompromised individuals, children, and pregnant women, and particularly their recent impacts, have increased the focus and attention of authorities [19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41]. _Giardia duodenalis_ is the causative agent of giardiasis, resulting in acute watery diarrhea in both humans and animals. Eight major genotypes of _G. duodenalis_ (assemblages) have been identified to date (A-H); however, assemblages A and B (and to a lesser extent assemblage E) are considered to have zoonotic interest due to their risk of infecting humans [24, 25]. _Cryptosporidium_ spp. are widespread protozoan parasites that infect humans and animals. Human cryptosporidiosis is the second commonest cause of diarrhea in children after rotavirus and can be caused either by the zoonotic _Cryptosporidium parvum_ or anthroponotic _Cryptosporidium hominis_[9, 26]. _Toxoplasma gondii_ is an intracellular coccidian protozoan, for which domestic and wild fields are the only known definitive hosts responsible for oocyst dissemination in the environment. Toxoplasma is usually asymptomatic in immunocompetent individuals, but may cause severe infections in immunocompromised individuals, fetuses during pregnancy, and newborns [27, 28].