Figure 1. Scheme of samples process treatment. TOTAL SAMPLES ANALYZED Occurrence of Giardia in wastewater treatment plants in Spain 1Alonso J.L., 1Amorós I, 1Navarro M.D., 1Navarro J.A., and 2Bernácer I. 1Instituto de Hidrología y Medio Natural, Universidad Politécnica, Camino de Vera 14, 46022 Valencia, Spain 2Entidad de Saneamiento de Aguas Residuales, c/Alvaro de Bazán 10 Entlo, 46010 Valencia, Spain INTRODUCTION In many regions there is a shortage of fresh water resources that can be used for irrigation. Using reclaimed wastewater for irrigation provides a vital resource to enhance agricultural productivity. Among water-borne pathogens, protozoa of the genera Giardia are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. The objective of this study was to investigate the occurrence of Giardia in three wastewater treatment plants (WTPs) in the Comunidad Valenciana (Spain) where treated wastewater is reused for agricultural activities, using immunofluorescence (IFA) and polymerase chain reaction (PCR) techniques. Efficiencies of wastewater treatment processes to remove cysts were investigated. MATERIAL AND METHODS Castellón plant receives 40,000 cubic domestic wastewater per day and employed secondary treatment (activated sludge) and UV disinfection (wave length 254 nm, doses of 0.50-0.80 mJ per square millimeter), Pinedo 1 (Valencia) plant receives 144,000 cubic meters of domestic wastewater per day and Rincón de León (Alicante) plant receives 80,000 cubic meters of domestic wastewater per day. Monitoring was undertaken at two locations within the process train (raw sewage and final effluent). Samples were collected fortnightly throughout the 1-year study. Samples were concentrated by centrifugation at 2,000 X g for 15 min. Giardia cysts were further purified by immunomagnetic separation (IMS), using magnetic beads coated with an anti-Giardia monoclonal antibody (Dynabeads GC-Combo, Dynal). Presence of the parasites was assessed by immunofluorescence (EasyStain™ C+G FITC, IC-IFA with monoclonal antibody IgG1 from Biotechnology Frontiers) (figure 1). A common problem encountered with PCR-mediated amplification of DNA extracted from environmental samples is inhibition by humic-type materials that coextract with the DNA. In the present study, bovine serum albumin (BSA) and the high High Pure PCR Template Preparation Kit of Roche were used to overcome PCR inhibition by contaminants in environmental samples (figures 1 and 2). A single PCR using primers MM1/MM2 (Kuhn et al., 2000) was used to detect Giardia. To characterize the cysts at the molecular level, a PCR assay (primer sets G7/G759 and G376/G759) that target the beta-giardin gene was amplified, and the products were analysed by restriction according to the method of Caccio et al. (2002). For scanning electron microscopy, cysts samples after immunomagnetic separation were prepared by fixing in 0.1 M sodium phosphate buffer (PBS), pH 7.2, containing 2.5% glutaraldehyde and 2% osmium tetroxide. The samples were examined by using a JEOL JSM-5410 scanning electron microscopy at 20kV. For Giardia §  Fifteen freeze-thaw cycles, each cycle consisted of 1 min in liquid nitrogen followed by thawing at 65ºC. §  Add 1l of lysozime (100 mg/ml). §  Incubate at 37ºC for 1 h. §  Add 2 l of proteinase K (20 mg/ml). §  Incubate at 55ºC for 2 h. §  Inactivation of proteinase K by boiling 10 min at 95ºC.. §  Incubate the samples for 10 min in ice. §  Centrifuge at 13000 rpm for 10 min. §  Pipet all of the supernatant into a new 1,5 ml microcentrifuge tube. §  Store DNA at –20ºC. Figure 2.Scheme of the cyst DNA release by a freeze-thaw method. We used TES (50mM Tris-HCl pH 8.5, 1mM EDTA, 0.5% SDS) (Nichols et al., 2001) as extraction buffer. DNA extraction and PCR First step: DNA extraction by criofracture (see Figure 2 ) Centrifugation at 2,000 X g for 15 min Influent, 300ml; Effluent, 800ml Immunocapture with Kit Dynabeads.GC-Combo of Dynal Biotech Second step: DNA extraction with commercial kit (High Pure PCR Template Preparation Kit of Roche) Single PCR and nested PCR (PCR products were digested with HaeIII and HhaI) Immunofluorescence with the EasyStain™ C+G FITC GC-Combo from Biotechnology frontiers Figure 1. Scheme of samples process treatment. RESULTS AND DISCUSSION Of the 132 water concentrates analysed for the presence of Giardia lamblia cysts by IFA, 112 (84,9%) were positive, of which 65 (58,0 %) were raw and 47 were final water concentrates. The estimated mean number of cysts for Castellón WTP ranged from 745/L (influent) to 7/L (final effluent) (figure 3). The mean number of cysts for Pinedo 1 WTP were 402/L (influent) and 16/L (final effluent) (figure 4). The mean number of cysts for Rincón de León WTP were 445/L (influent) and 12/L (final effluent) (figure 5). The number of cysts in influent was found to vary from WTP to WTP and from month to month. Scanning electron micrographs from cyst samples obtained after immunocapture are shown in Figure 6. High-magnification SEM of individual cysts showed morphological alterations of the cyst wall. LeChevallier et al. (1991) reported in raw water samples that 12.8% of the 618 Giardia spp. observed contained viable type morphologies. These results suggest that the majority of cysts observed in the samples were not viable. Of the 120 water concentrates analysed for the presence of Giardia lamblia cysts by PCR between january and december 2003 (figure 7), 86 (72,4%) were positive, of which 57 (66,3 %) were raw and 29 were final water concentrates. To characterize the cysts at the molecular level, the beta-giardin gene was PCR amplified and the products were analysed by restriction. The PCR RFLP analysis revealed assemblage genotype A1 of G. lamblia in the raw water of Castellón WTP (figure 8). Figure 3. Giardia cysts concentration in Castellón WTP Figure 4. Giardia cysts concentration in Pinedo WTP Figure 5. Giardia cysts concentration in Rincón de León WTP The overall removal efficiency of cyst in the treatment plants ranged from 94,0 (Pinedo 1) to 98,8% (Castellón) (figure 9). Evaluation of wastewater treatment showed a 2 log cyst removal when the sand filtration and UV treatment was included after activated sludge process and sedimentation (98.8% versus 94.0). The results of Linden et al. (2002) indicate no phenotypic evidence of either light or dark repair of UV-damaged DNA in G. lamblia cysts irradiated with 16 or 40 mJ per square centimeter of LP UV. Thus, it appears that UV disinfection dose of 50 mJ per square centimeter provide adequate disinfection and also protect against DNA repair mechanisms and reactivation of G. lamblia cysts. The presence of cysts in final effluents without UV disinfection represents a potential risk for reuse programs. Figure 6. Scanning lectron micrographs of cysts after immunomagnetic separation of WTP samples. Scale bars = 5 µm (left, X10,000), 5 µm (middle, X10,000) and 20 µm (right, X2,000) Figure 9. Giardia removal efficiency G7/G759 fragments: CASTELLÓN (WWTP1) PINEDO (WTP2) RINCÓN DE LEÓN (WTP3) TOTAL SAMPLES ANALYZED 41 38 POSITIVE RESULTS 27 30 29 % 65’9 79,0 70’7 G376/G759 fragments: 1 E3 WTP 3 E2 WTP 2 E1 WTP 1 C+ GIARDIA DNA, CONTROL POSITIVE C- REACTION WITHOUT DNA, NEGATIVE CONTROL HaeIII 202 201 150 126 74 202 176 24 150 117 84 202 186 15 150 102 24 74 Genotype A Genotype B Genotype E 193 17 70 104 210 70 70 35 Genotype A1 Genotype A2/A3 HhaI 202/201 bp 150 bp 193 bp 126 bp 104 bp 74 bp 70 bp MARC E3 E2 E1 C+ C- MWM G7/G759 C+ MWM HaeIII HaeIII 2 17 bp MWM G376/G759 C+ MWM HhaI HhaI Figure 7.- PCR results for Giardia lamblia. 2.- Example of a enviromental samples analyzed for Giardia lamblia. Figure 8. Example of genotyping Giardia lamblia, that confirm genotype A1 in the raw water (influent) of Castellón WTP. Acknowledgements: Work supported by funding from the Entidad de Saneamiento de Aguas Residuales de la Generalitat Valenciana (Valencia, Spain)