In the north of Portugal, there are 5 major hydrographical basins forming the most important water resources of the country (Fig. 1). These hydrographical basins are named after the main rivers; (1) Minho, (2) Lima, (3) Cávado, (4) Ave, and (5) Douro (Fig. 2). Cávado and Ave rivers run entirely inside national (Portuguese) borders, while Minho, Lima, and Douro are international rivers, with sources in Spain.

Raw water samples were collected twice a year between January 2004 to December 2006 from 97 sources, including main rivers and respective affluents, from upriver to downriver. The volume of each sample ranged from 25 to 100 L. Samples were 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 the filter, passing the sample through a membrane, and centrifugation. A sample pellet (around 2 ml) was obtained and transferred to a Leighton tube for subsequent immunomagnetic separation (IMS).

The IMS procedure was performed according to the USEPA method 1623 [20]. Briefly, anti-Giar 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 1 hr at room temperature. Then, using 2 magnetic particle concentrators, beads were collected, washed, and transferred into a 1.5 µl tube. Fifty-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, Sydney, 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 Giardia spp. using the Filta-Max system and IMS procedures from water samples is, according to the manufacturer, 50 ± 13% and 41 ± 79%, respectively [21].

PCR analysis was performed in the samples with the highest density of infectious stages of both parasites detected by DFA. The criterion utilized was the detection of a minimum of 100 cysts/oocysts 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 microscope observation that the slide had no remaining cysts or oocysts. The tip of the cotton swab was cut and placed in a 1.5 ml tube for subsequent DNA extraction with a QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's instructions.

For determining the species of Cryptosporidium and Giardia present in the samples, PCR analyses were performed. A 2-step nested PCR was performed to amplify a portion of the small-subunit (SSU) ribosomal RNA gene of Cryptosporidium spp. [22]. For the molecular typing of Giardia spp., a semi-nested PCR was performed to amplify a portion of the β-giardin gene [23]. 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 PCR products of the successful reaction were purified by Wizard SV Gel and PCR Clean-up System (Promega, Madison, Wisconsin, USA) and sequenced in both strands by an external laboratory (EUROFINS MWG OPERON, Ebersberg, Germany). Chromatograms were examined with the software ChromasPro (http://www.technelysium.com.au/ChromasPro.html) and the sequences with the software ProSeq (http://www.biology.ed.ac.uk/research/institutes/evolution/software/filatov/proseq.htm). Sequences were compared with the GenBank database with the tool BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and deposited in the database Zoop-Net of the Med-Vet-Net network (http://www.medvetnet.org/cms/).

The number of validated raw water samples in this study was 283. Environmental stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, including 15.2% (43 of 283) positive for cysts of G. duodenalis, 9.5% (27 of 283) for oocysts of Cryptosporidium spp., and 48.1% (136 of 283) for both parasites. In Fig. 3, the percentages of positive and negative samples from the 5 hydrographical basins are shown individually. The Ave basin showed the highest percentage of positive samples; 90.2% of the samples were positive. Minho basin showed the lowest percentage of positive samples, even though this value was more than 64%. In all the 5 hydrographic basins, the co-presence of Cryptosporidium and Giardia counted for the majority of positive samples, with the exception of Minho basin in which Giardia positive samples were slightly more than Cryptosporidium and Giardia positive samples. In the cases where both parasites were present in the same sample, the number of G. duodenalis cysts always outnumbered Cryptosporidium spp. oocysts. We also found no correlation between the concentrations of both parasites, meaning that, when the concentration of Giardia cysts is high, Cryptosporidium oocysts are not necessarily high (data not shown). Furthermore, the range of the concentrations of G. duodenalis cysts was much higher than Cryptosporidium spp. oocysts (0.17-50,000 cysts per 10 L and 0.2-726.1 oocysts per 10 L, respectively). In positive water samples, no empty (i.e., without internal characteristics, or ghosts) or DAPI negative Cryptosporidium spp. oocysts or G. duodenalis cysts were found.

In all cases, it was possible to observe an increase of parasite load from upriver to downriver. The majority of water samples from the international rivers (Minho, Lima, and Douro) collected at the border with Spain was negative.

By PCR, it was unable to amplify DNA extracted from slides containing less than 100 oocysts of Cryptosporidium and less than 100 cysts of Giardia. Furthermore, positive amplifications over 3 replicates were never obtained when the number of cysts and oocysts was less than 1,000 per slide. With this criterion, of all the positive IMS samples, PCR amplification was performed over 80 samples.

Genetic characterization was successful in 8 samples for G. duodenalis and 20 samples for Cryptosporidium spp. In 59 samples, PCR amplification was not successful. A summary of the PCR results is shown in Table 1. Cryptosporidium andersoni was found in 16 samples, Cryptosporidium parvum in 2 samples, Cryptosporidium hominis in 1 sample, and Cryptosporidium muris in 1 sample. G. duodenalis assemblage A-II was found in 4 samples, assemblage B in 1 sample, and in the remaining 3 samples assemblages A, B, and E were found.