# Protozoa as the \"Underdogs\" for Microbiological Quality Evaluation of Fresh Vegetables
## 2 Materials and Methods  
Five bulk and 11 packaged and ready-to-eat (RTE) vegetables were collected from local producers in Portugal and Spain, or provided by retail sellers, between September 2018 and June 2019 (Table 1). The fresh produce comprised lettuce (_Lactuca sativa_), watercress (_Nasturtium officinal_), coriander (_Coriandrum sativum_), and parsley (_Petroselinum crispum_). Ready-to-eat mixed salads included different varieties of lettuce, arguula (_Eruca vesicaria sativa_), endive (_Cichorium endivia_), chicory (_Cichorium intybus_), carrot (_Daucus carota sativus_), red cabbage (_Brassica oleracea_ var. _capitata f. rubra_), and lamb's lettuce (_Valerianella locusta_). The concentration and recovery of _G. duodenalis_ and _Cryptosporidium_ spp. (oo)c cysts from the vegetable samples were performed by Filtration/Immunagnetic Separation (IMS)/Fluorescence Assay (FA) (Method 1623.1: _Cryptosporidium_ and _Giardia_ in water; US EPA 816-R-12-001-Jan 2012). Method 1623.1 is a validated and approved method used for surface water analysis [43]. This method was previously fully established in our laboratory, initially to gain knowledge regarding the contamination of water with _Cryptosporidium_ spp. and _Giardia_ spp. in the northern region of Portugal [44; 46]. We also previously designed a long-term program aiming to identify the sources of surface water and environmental \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sample** & **Product** & **Origin** & **Collection Date** & **Product** \\
**Number** & & & & **Presentation** \\ \hline
1 & Watercress & Portugal & 27 September 2018 & bulk \\
2 & Coriander & Portugal & 27 September 2018 & bulk \\
3 & Parsley & Portugal & 27 September 2018 & bulk \\
4 & Watercress & Portugal & 2 October 2018 & RTE \\
5 & Mixed salad & Portugal & 2 October 2018 & RTE \\
6 & Coriander & Portugal & 2 October 2018 & packaged \\
7 & Parsley & Portugal & 2 October 2018 & packaged \\
8 & Watercress & Portugal & 12 October 2018 & RTE \\
9 & Mixed salad & Portugal & 12 October 2018 & RTE \\
10 & Coriander & Portugal & 12 October 2018 & packaged \\
11 & Parsley & Portugal & 12 October 2018 & packaged \\
12 & Mixed salad & Portugal & 4 February 2019 & RTE \\
13 & Mixed salad & Spain & 17 June 2019 & RTE \\
14 & Mixed salad & Spain & 17 June 2019 & RTE \\
15 & Arugula & Spain & 17 June 2019 & RTE \\
16 & Lettuce & Portugal & 18 June 2019 & bulk \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Origin, collection date, and product presentation of the vegetable samples. contamination, working with the water-supply industry, and to provide cattle and human fecal screening and molecular characterization [47; 48; 49]. Briefly, vegetable samples weighing between 350 and 900 g each were manually washed in large volumes of distilled water (between 10 and 80 L) (Table 1). A 1 \(\upmu\)m Filta-Max(r) filter (IDEXX, Westbrook, ME, USA) applied to a peristaltic pump at a pressure of three bar was used for washing water filtration. Elution was performed in a Filta-Max(r) manual wash station and concentrated into 3 mL of phosphate-buffered solution (PBS) with 0.01% of Tween 20 (Merck KGaA, Darmstadt, Germany), after centrifugation at 1000\(\times\)\(g\) for 10 min at room temperature. Magnetic beads conjugated with specific antibodies (Dynabeads(tm) GC-Combo; Thermo Fisher Scientific, Waltham, MA, USA) were added to the concentrate, and potential magnetized _Cryptosporidium_ oocysts and _Giardia_ cysts were recovered from the extraneous material using a magnet. Fluorescein isothiocyanate (FITC) conjugated anti-_Cryptosporidium_ spp. and anti-_Giardia_ spp. monoclonal antibodies were used according to the instructions of the manufacturer, Aqua-Glo(tm)G/C (Waterborne, Inc., New Orleans, LA, USA), on slides containing the potential samples' parasites, and observed by epifluorescence microscopy at 200\(\times\) magnification. The total number of (oo)c cysts per slide/sample was screened by two different microscopists to cross-check the results.