In the north of Portugal there are 5 major hydrographical basins forming the most important water resources of the country. These hydrographical basins are named after the main rivers; Minho, Lima, Cávado, Ave, and Douro (Figs. 1, 2). Cávado and Ave rivers run entirely inside national (Portuguese) borders, while Minho, Lima, and Douro are international rivers, with sources in Spain. Global features of the area, based on data provided by CCDRN, are as follows: The resident population in 2006 is 3,744,341 inhabitants; animal husbandry is an important economic activity; there are poorly developed sanitation infrastructures with wastewater plants discharging into these hydrographical basins; 64% of resident population has access to sanitation and 84% has access to treated drinking water; the region has the highest population density of the country, reaching 176 inhabitants per km², there are a high number of recreational areas, river beaches as well as pumping areas for drinking water plants.

Drinking water samples were collected twice a year from January 2004 to December 2006 in 43 sampling points in the drinking water treatment plants, from upriver to downriver, from all the 5 hydrographical basins (Fig. 2). The volume of each sample ranged from 80 to 100 L. Samples were collected locally, filtered through Filta-Max filters (IDEXX Laboratories, Inc., Westbrook, Maine, USA) with a pump on the inlet side of the filter according to the recommendation of the manufacturer. Intact filters were kept in refrigerated containers and transported immediately to the laboratory. The filter was taken from the container and processed with the aid of a Filta-Max Manual Wash Station (IDEXX Laboratories, Inc.) for further elution and concentration process which consisted of decompression of the filter, passing the sample through a membrane, and centrifugation. A sample pellet (around 2 µl) was obtained and transferred to a Leighton tube for subsequent immunomagnetic separation (IMS).

The IMS procedure was performed according the US EPA method 1623 (USEPA, 2001). Briefly, anti-Giardia and anti-Cryptosporidium magnetic beads were mixed with SL Buffer A and SL Buffer B in each Leighton tube containing the sample concentrate (Dynabeads GC-Combo, Invitrogen Dynal, A. S., Oslo, Norway) and incubated for 1 hr at room temperature. Using 2 magnetic particle concentrators, beads were collected, washed, and transferred into a 1.5 µl tube. Then, 50 µl of 0.1 N HCl were added to each sample to dissociate beads from the target organisms, the beads were rejected and the suspension was transferred to the wells of the slides containing 5 µl of 1.0 N NaOH. The samples were air dried overnight and stained with FITC-conjugated anti-Cryptosporidium spp. and anti-Giardia spp. monoclonal antibodies, according the manufacturer's instructions (Crypto/Giardia Cells, Cellabs, Australia). The excess of FITC-MAb was removed by adding 100 µl of PBS to each well, leaving the slides for 5 min, and aspirating the excess of PBS. A 50-µl aliquot of 4'-6'-diamino-2-phenylindole (DAPI) solution (0.4 µg/ml in PBS) was introduced into each well. The slides were left at room temperature for 15 min, and excess DAPI solution removed by washing the slides in PBS. Slides were examined by epifluorescence microscope. Giardia cysts and Cryptosporidium oocysts were indentified and counted based on their shape and size using a Nikon Optiphot fluorescence microscope (Nikon Corporation, Tokyo, Japan). The number of cysts and oocysts per each well was recorded and concentrations extrapolated per 10 L of sample. Positive and negative controls were performed as indicated by the manufacturer and recommended in the Method 1623.

The mean recovery percentages of oocysts of Cryptosporidium spp. and cysts of G. duodenalis using Filta-Max system and IMS procedures from water samples were, according to the manufacturer, 50 ± 13% and 41 ± 79%, respectively [23].

PCR analysis was performed in the samples with the highest density of infectious stages of both parasites detected by the direct fluorescence assay (DFA). The criterion utilized was the detection of a minimum of 100 cyst or oocyst stage of any parasite in the total sample volume. In this context, the genetic characterization was executed in 80 samples. The cover slip was separated from the slide, and with the aid of cotton swab soaked with 100 µl of distillated water, the surface of the slide was scraped in order to collect the sample. It was confirmed, under microscopic observations, that the slide had no remaining cysts or oocysts. The tip of the cotton swab was cut and placed in a 1.5 µl tube for subsequent DNA extraction with a QIAamp DNA Mini Kit (QIAGEN GmbH, Berlin, Germany), according to the manufacturer's instructions.

For determining the species of Cryptosporidium and Giardia present in the samples, a PCR analysis was performed. A 2-step nested PCR was performed to amplify a portion of the small-subunit (SSU) ribosomal RNA gene of Cryptosporidium [24]. For the molecular typing of G. duodenalis, a semi-nested PCR was performed to amplify a portion of the β-giardin gene [25]. For all PCR reactions, negative and positive controls were prepared, with sterile water and reference DNA, respectively. The PCR products were analyzed in agarose gel (1.4%) stained with ethidium bromide under UV light. Images were captured with a gel documentation system (GelDoc2000, BioRad, Hercules, California, USA).

The number of validated drinking water samples in this study was 167. In Fig. 3, the percentage and concentration of isolates from the 5 hydrographical basins are shown. Negative results, for both protozoa, were obtained in 124 out of 167 drinking water samples. Infectious stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples. Among them, 8.4% (14 out of 167) of the samples were with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium, and 7.2% (12 out of 167) with both parasites. In positive water samples, no empty (i.e., without internal characteristics, or ghosts) or DAPI negative oocysts of Cryptosporidium spp. or cysts of G. duodenalis were found. Furthermore, the mean concentrations of G. duodenalis cysts were much higher than those of the Cryptosporidium spp. oocysts (0.1-108.3 cysts per 10 L and 0.1-12.7 oocysts per 10 L, respectively). A number of cysts and oocysts greater than to the minimal infectious dose (83-123 oocysts for Cryptosporidium and 19-50 cysts for Giardia) were detected in 2 samples. Except a third sample, where the amount of cysts of Giardia was 12.6 cysts per 10 L, none of the samples had more than 5 cysts or oocysts per 10 L, showing a low level of contamination.

It was not possible by PCR to amplify DNA extracted from slides containing less than 50 oocysts of Cryptosporidium and 50 cysts of Giardia. Furthermore, positive amplifications over 3 replicates were never obtained with the number of cysts and oocysts smaller than 1,000 per slide. With this criterion, of all the positive IMS samples, PCR amplification was performed only over 4 samples. In these samples, it was not able to obtain amplification. In the correspondent raw water samples at the entrance of the drinking water plant, Cryptosporidium andersoni, Cryptosporidium parvum, Cryptosporidium hominis, and Cryptowsporidium muris have been detected (personal communication). Also, G. duodenalis assemblage A-II (in 4 samples), assemblage B (1 sample), and assemblages A, B, and E together (in 3 samples) was found in the same raw water (personal communication). It is expected that at the end of the treatment process the same species and genotypes will be found.