International Journal of Hygiene and Environmental Health 218 (2015) 132–138
Contents lists available at ScienceDirect
International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh
Cryptosporidium spp. and Giardia duodenalis as pathogenic contaminants of water in Galicia, Spain: The need for safe drinking water José Antonio Castro-Hermida ∗ , Marta González-Warleta, Mercedes Mezo Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, Instituto Galego de Calidade Alimentaria-Xunta de Galicia, Carretera na), Espa˜ na AC-542 de Betanzos a Mesón do Vento, Km 7,5, CP 15318 Abegondo (A Coru˜
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 14 September 2014 Accepted 15 September 2014 Keywords: Cryptosporidium Giardia Drinking water Water quality Public health Surface and ground water
a b s t r a c t The objectives of this cross-sectional study were to detect the presence of Cryptosporidium spp. and Giardia duodenalis in drinking water treatments plants (DWTPs) in Galicia (NW Spain) and to identify which species and genotype of these pathogenic protozoans are present in the water. Samples of untreated water (surface or ground water sources) and of treated drinking water (in total, 254 samples) were collected from 127 DWTPs and analysed by an immunofluorescence antibody test (IFAT) and by PCR. Considering the untreated water samples, Cryptosporidium spp. were detected in 69 samples (54.3%) by IFAT, and DNA of this parasite was detected in 57 samples (44.8%) by PCR, whereas G. duodenalis was detected in 76 samples (59.8%) by IFAT and in 56 samples (44.0%) by PCR. Considering the treated drinking water samples, Cryptosporidium spp. was detected in 52 samples (40.9%) by IFAT, and the parasite DNA was detected in 51 samples (40.1%) by PCR, whereas G. duodenalis was detected in 58 samples (45.6%) by IFAT and in 43 samples (33.8%) by PCR. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0% in all samples analysed. Cryptosporidium andersoni, C. hominis, C. parvum and assemblages A-I, A-II, E of G. duodenalis were identified. The results indicate that Cryptosporidium spp. and G. duodenalis are widespread in the environment and that DWTPs are largely ineffective in reducing/inactivating these pathogens in drinking water destined for human and animal consumption in Galicia. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination. © 2014 Elsevier GmbH. All rights reserved.
Introduction Among the pathogens that contaminate water, Cryptosporidium spp. and Giardia duodenalis are known to be major aetiological agents of waterborne outbreaks of disease worldwide (MacKenzie et al., 1994; Savioli et al., 2006; Chalmers et al., 2010; Mason et al., 2010; Badursson and Karanis, 2011; Damiani et al., 2013; Burnet et al., 2014; Hlavsa et al., 2014; Kitajima et al., 2014; Puleston et al., 2014; Widerström et al., 2014). In fact, these parasites are the most common food and waterborne protozoans that affect humans and a wide range of animals, which can shed large numbers of the transmissive stages of these enteropathogens (i.e. oocysts and cysts, respectively) in their surroundings (Slifko et al., 2000; Thompson, 2000; Fayer, 2004; Smith et al., 2007; Santín et al., 2009; Smith and Nichols, 2010; Thompson et al., 2010; Ongerth, 2013; Ryan
∗ Corresponding author. Tel.: +34 881881840; fax: +34 881881800. E-mail address: [email protected] (J.A. Castro-Hermida). http://dx.doi.org/10.1016/j.ijheh.2014.09.001 1438-4639/© 2014 Elsevier GmbH. All rights reserved.
and Cacciò, 2013; Lalancette et al., 2014; Palos Ladeiro et al., 2014). Waterborne infections caused by these parasites are particularly important because (oo)cysts are highly stable in the environment and are largely resistant to disinfectants such as chlorine and chloramines (Fayer et al., 2000; Clancy and Hargy, 2008; Nichols, 2008; Plutzer et al., 2010). In the study region (Galicia, NW Spain) water bodies are generally used for a variety of recreational purposes, including swimming (especially during spring and summer), and as sources of drinking water. In previous studies, we have observed the presence of Cryptosporidium spp. and G. duodenalis in domestic (cattle and sheep) and wild animals (roe deer and wild boars) next to DWTP pumping areas (Castro-Hermida et al., 2011). We have also observed both parasites in animal slurry being applied to agricultural land in these areas (data not published). Moreover, Cryptosporidium spp. and G. duodenalis have been detected in the final effluents from wastewater treatment plants which are discharged directly into rivers (recreational areas, river beaches and DWTP pumping areas) and into marine estuaries where bivalve molluscs are cultured for
J.A. Castro-Hermida et al. / International Journal of Hygiene and Environmental Health 218 (2015) 132–138
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human consumption (Gómez-Couso et al., 2005; Castro-Hermida et al., 2008, 2010). Outbreaks of cryptosporidiosis and giardiosis in humans are therefore expected to occur in the region. All these data suggest a recurrent exposure of our population to these pathogens and therefore imply that cryptosporidiosis and giardiosis could be a potential threat to public health in Spain (Navarro-i-Martinez et al., 2011; Carmena et al., 2012). The overall aim of the present study was to provide an accurate estimate of the extent of contamination of drinking water by Cryptosporidium spp. and G. duodenalis in Galicia. Materials and methods Study area and sampling The study was carried out in Galicia (NW Spain). The region occupies an area of 29,575 km2 (between 43◦ 47 –41◦ 49 N and 6◦ 42 –9◦ 18 W). The climate of Galicia is temperate and rainy, with a dry period in summer, and is usually classified as Oceanic in the west and north, and Mediterranean in the southeast of the region. The target population consisted of all DWTPs of Galicia. The sampling frame contained DWTPs from 161 municipalities (51.1% of all municipalities in Galicia in 2011) who had conventional water treatment facilities, and a suitable drinking water distribution system infrastructure, i.e., including all water utility components (pipes, pumps, valves, storage tanks, reservoirs, meters, fittings, and other hydraulic appurtenances) for the distribution of finished or potable water by means of gravity storage feed or pumps though distribution pumping networks to customers or other users. The sample size was calculated using the sample size estimate of the Win Episcope 2.0 software. Based on an estimated prevalence of 50%, an allowable error of 4%, and a confidence level of 95% a sample of 127 DWTPs was required. Finally, a total of 130 DWTPs were randomly selected and 127 agreed to participate in the study (Fig. 1). In each DWTP, the information compiled included the main features of the drinking water treatment plant processes, estimation of the total population that is served by the plant and the type water source (surface or ground water). According these data, the DWTPs were grouped into two groups (Table 1). For the purposes of the study, one sample of untreated water (100 l) and another of treated drinking water (100 l) were collected at each of 127 DWTPs in the region during 2011–2012 (Fig. 1). Concentration of Cryptosporidium and Giardia (oo)cysts from water samples The 254 water samples obtained were filtered through FiltaMax filters (IDEXX Laboratories, Inc., Westbrook, ME, USA) with the aid of a motorized pump. The filters were transported to the laboratory in a cool box and processed within 24 h of collection. Samples were therefore taken immediately to the laboratory and processed with the Filta-Max Automatic System (IDEXX Laboratories, Inc., Westbrook, ME, USA). All samples were filtered at recommended flow rates, according to the manufacturer’s instructions. Elution of filters and final concentration of the sample were carried out with the same equipment, according to the manufacturer’s instructions. Briefly, this involved placing a membrane filter (3 m pore size; polysulphone) at the base of the sample concentrator, inserting the filter module into the apparatus, adding 600 ml of phosphate-buffered saline (PBS) containing 0.01% Tween 20 (PBST; Sigma-Aldrich, USA) to the reservoir and unscrewing the filter housing to allow expansion of the foam pads. The foam pads were washed by pumping the plunger and then transferring the PBST elution volume into the magnetic particle concentrator and filtering the entire volume to approximately 20 to 25 ml, under
Fig. 1. Geographical location of the sampling points in relation to the 127 municipalities in Galicia (NW, Spain) where samples of the untreated and treated water were obtained from drinking water treatment plants.
vacuum. The process was repeated with a second volume (600 ml) of PBST, and the resulting concentrate was combined with the first concentrate and then filtered under vacuum to produce a final volume of around 20 ml. This was then transferred to a 50 ml centrifuge tube. The filter membrane was transferred to a small sealable plastic bag, and 8–10 ml of PBST was added; the membrane was kneaded manually, and the spent wash volume was pooled with the primary eluate. The membrane washing procedure was repeated twice. The sample was resuspended to 50 ml with PBST and centrifuged at 1500 × g for 5 min. The supernatant was aspirated to 10 ml, and the resuspended pellet was transferred to a Leighton tube and subjected to Immunomagnetic Separation technology (IMS). The IMS procedure was performed as described in US Environmental Protection agency method 1623, for the isolation and detection of Cryptosporidium and Giardia (USEPA, 2001, 2002). Briefly, each 10 ml sample concentrate was added to a Leighton tube containing 1 ml of 10 × SL buffer A and 1 ml of 10× SL buffer B (Dynabeads® GC-Combo, Invitrogen Dynal, A.S., Oslo, Norway). One hundred microlitres of Cryptosporidium and Giardia IMS beads were added to each tube, and samples were incubated for 1 h at room temperature with constant rotation. The Leighton tubes were then placed in a magnetic particle concentrator and rocked gently for 2 min through an angle of 90◦ . The supernatant was decanted, the tubes were removed from the magnetic particle concentrator, and 1 ml of 1× SL buffer A was added to each. The tubes were rocked gently to resuspend the bead(oo)cyst complexes. The suspension was transferred, with a Pasteur pipette, to 1.5 ml polypropylene tubes, which were then placed in
134
J.A. Castro-Hermida et al. / International Journal of Hygiene and Environmental Health 218 (2015) 132–138
Table 1 General characteristics of the conventional drinking water treatment plants (CDWTP). CDWTP (n)
Treatment
Population served
Water source
Large and medium-size (n = 100)
Coagulation, flocculation, sedimentation, filtration and disinfection by chlorination Rapid filtration and disinfection processes by chlorination
2000–110,000
Surface water (rivers or streams)
Contents lists available at ScienceDirect
International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh
Cryptosporidium spp. and Giardia duodenalis as pathogenic contaminants of water in Galicia, Spain: The need for safe drinking water José Antonio Castro-Hermida ∗ , Marta González-Warleta, Mercedes Mezo Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, Instituto Galego de Calidade Alimentaria-Xunta de Galicia, Carretera na), Espa˜ na AC-542 de Betanzos a Mesón do Vento, Km 7,5, CP 15318 Abegondo (A Coru˜
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 14 September 2014 Accepted 15 September 2014 Keywords: Cryptosporidium Giardia Drinking water Water quality Public health Surface and ground water
a b s t r a c t The objectives of this cross-sectional study were to detect the presence of Cryptosporidium spp. and Giardia duodenalis in drinking water treatments plants (DWTPs) in Galicia (NW Spain) and to identify which species and genotype of these pathogenic protozoans are present in the water. Samples of untreated water (surface or ground water sources) and of treated drinking water (in total, 254 samples) were collected from 127 DWTPs and analysed by an immunofluorescence antibody test (IFAT) and by PCR. Considering the untreated water samples, Cryptosporidium spp. were detected in 69 samples (54.3%) by IFAT, and DNA of this parasite was detected in 57 samples (44.8%) by PCR, whereas G. duodenalis was detected in 76 samples (59.8%) by IFAT and in 56 samples (44.0%) by PCR. Considering the treated drinking water samples, Cryptosporidium spp. was detected in 52 samples (40.9%) by IFAT, and the parasite DNA was detected in 51 samples (40.1%) by PCR, whereas G. duodenalis was detected in 58 samples (45.6%) by IFAT and in 43 samples (33.8%) by PCR. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0% in all samples analysed. Cryptosporidium andersoni, C. hominis, C. parvum and assemblages A-I, A-II, E of G. duodenalis were identified. The results indicate that Cryptosporidium spp. and G. duodenalis are widespread in the environment and that DWTPs are largely ineffective in reducing/inactivating these pathogens in drinking water destined for human and animal consumption in Galicia. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination. © 2014 Elsevier GmbH. All rights reserved.
Introduction Among the pathogens that contaminate water, Cryptosporidium spp. and Giardia duodenalis are known to be major aetiological agents of waterborne outbreaks of disease worldwide (MacKenzie et al., 1994; Savioli et al., 2006; Chalmers et al., 2010; Mason et al., 2010; Badursson and Karanis, 2011; Damiani et al., 2013; Burnet et al., 2014; Hlavsa et al., 2014; Kitajima et al., 2014; Puleston et al., 2014; Widerström et al., 2014). In fact, these parasites are the most common food and waterborne protozoans that affect humans and a wide range of animals, which can shed large numbers of the transmissive stages of these enteropathogens (i.e. oocysts and cysts, respectively) in their surroundings (Slifko et al., 2000; Thompson, 2000; Fayer, 2004; Smith et al., 2007; Santín et al., 2009; Smith and Nichols, 2010; Thompson et al., 2010; Ongerth, 2013; Ryan
∗ Corresponding author. Tel.: +34 881881840; fax: +34 881881800. E-mail address: [email protected] (J.A. Castro-Hermida). http://dx.doi.org/10.1016/j.ijheh.2014.09.001 1438-4639/© 2014 Elsevier GmbH. All rights reserved.
and Cacciò, 2013; Lalancette et al., 2014; Palos Ladeiro et al., 2014). Waterborne infections caused by these parasites are particularly important because (oo)cysts are highly stable in the environment and are largely resistant to disinfectants such as chlorine and chloramines (Fayer et al., 2000; Clancy and Hargy, 2008; Nichols, 2008; Plutzer et al., 2010). In the study region (Galicia, NW Spain) water bodies are generally used for a variety of recreational purposes, including swimming (especially during spring and summer), and as sources of drinking water. In previous studies, we have observed the presence of Cryptosporidium spp. and G. duodenalis in domestic (cattle and sheep) and wild animals (roe deer and wild boars) next to DWTP pumping areas (Castro-Hermida et al., 2011). We have also observed both parasites in animal slurry being applied to agricultural land in these areas (data not published). Moreover, Cryptosporidium spp. and G. duodenalis have been detected in the final effluents from wastewater treatment plants which are discharged directly into rivers (recreational areas, river beaches and DWTP pumping areas) and into marine estuaries where bivalve molluscs are cultured for
J.A. Castro-Hermida et al. / International Journal of Hygiene and Environmental Health 218 (2015) 132–138
133
human consumption (Gómez-Couso et al., 2005; Castro-Hermida et al., 2008, 2010). Outbreaks of cryptosporidiosis and giardiosis in humans are therefore expected to occur in the region. All these data suggest a recurrent exposure of our population to these pathogens and therefore imply that cryptosporidiosis and giardiosis could be a potential threat to public health in Spain (Navarro-i-Martinez et al., 2011; Carmena et al., 2012). The overall aim of the present study was to provide an accurate estimate of the extent of contamination of drinking water by Cryptosporidium spp. and G. duodenalis in Galicia. Materials and methods Study area and sampling The study was carried out in Galicia (NW Spain). The region occupies an area of 29,575 km2 (between 43◦ 47 –41◦ 49 N and 6◦ 42 –9◦ 18 W). The climate of Galicia is temperate and rainy, with a dry period in summer, and is usually classified as Oceanic in the west and north, and Mediterranean in the southeast of the region. The target population consisted of all DWTPs of Galicia. The sampling frame contained DWTPs from 161 municipalities (51.1% of all municipalities in Galicia in 2011) who had conventional water treatment facilities, and a suitable drinking water distribution system infrastructure, i.e., including all water utility components (pipes, pumps, valves, storage tanks, reservoirs, meters, fittings, and other hydraulic appurtenances) for the distribution of finished or potable water by means of gravity storage feed or pumps though distribution pumping networks to customers or other users. The sample size was calculated using the sample size estimate of the Win Episcope 2.0 software. Based on an estimated prevalence of 50%, an allowable error of 4%, and a confidence level of 95% a sample of 127 DWTPs was required. Finally, a total of 130 DWTPs were randomly selected and 127 agreed to participate in the study (Fig. 1). In each DWTP, the information compiled included the main features of the drinking water treatment plant processes, estimation of the total population that is served by the plant and the type water source (surface or ground water). According these data, the DWTPs were grouped into two groups (Table 1). For the purposes of the study, one sample of untreated water (100 l) and another of treated drinking water (100 l) were collected at each of 127 DWTPs in the region during 2011–2012 (Fig. 1). Concentration of Cryptosporidium and Giardia (oo)cysts from water samples The 254 water samples obtained were filtered through FiltaMax filters (IDEXX Laboratories, Inc., Westbrook, ME, USA) with the aid of a motorized pump. The filters were transported to the laboratory in a cool box and processed within 24 h of collection. Samples were therefore taken immediately to the laboratory and processed with the Filta-Max Automatic System (IDEXX Laboratories, Inc., Westbrook, ME, USA). All samples were filtered at recommended flow rates, according to the manufacturer’s instructions. Elution of filters and final concentration of the sample were carried out with the same equipment, according to the manufacturer’s instructions. Briefly, this involved placing a membrane filter (3 m pore size; polysulphone) at the base of the sample concentrator, inserting the filter module into the apparatus, adding 600 ml of phosphate-buffered saline (PBS) containing 0.01% Tween 20 (PBST; Sigma-Aldrich, USA) to the reservoir and unscrewing the filter housing to allow expansion of the foam pads. The foam pads were washed by pumping the plunger and then transferring the PBST elution volume into the magnetic particle concentrator and filtering the entire volume to approximately 20 to 25 ml, under
Fig. 1. Geographical location of the sampling points in relation to the 127 municipalities in Galicia (NW, Spain) where samples of the untreated and treated water were obtained from drinking water treatment plants.
vacuum. The process was repeated with a second volume (600 ml) of PBST, and the resulting concentrate was combined with the first concentrate and then filtered under vacuum to produce a final volume of around 20 ml. This was then transferred to a 50 ml centrifuge tube. The filter membrane was transferred to a small sealable plastic bag, and 8–10 ml of PBST was added; the membrane was kneaded manually, and the spent wash volume was pooled with the primary eluate. The membrane washing procedure was repeated twice. The sample was resuspended to 50 ml with PBST and centrifuged at 1500 × g for 5 min. The supernatant was aspirated to 10 ml, and the resuspended pellet was transferred to a Leighton tube and subjected to Immunomagnetic Separation technology (IMS). The IMS procedure was performed as described in US Environmental Protection agency method 1623, for the isolation and detection of Cryptosporidium and Giardia (USEPA, 2001, 2002). Briefly, each 10 ml sample concentrate was added to a Leighton tube containing 1 ml of 10 × SL buffer A and 1 ml of 10× SL buffer B (Dynabeads® GC-Combo, Invitrogen Dynal, A.S., Oslo, Norway). One hundred microlitres of Cryptosporidium and Giardia IMS beads were added to each tube, and samples were incubated for 1 h at room temperature with constant rotation. The Leighton tubes were then placed in a magnetic particle concentrator and rocked gently for 2 min through an angle of 90◦ . The supernatant was decanted, the tubes were removed from the magnetic particle concentrator, and 1 ml of 1× SL buffer A was added to each. The tubes were rocked gently to resuspend the bead(oo)cyst complexes. The suspension was transferred, with a Pasteur pipette, to 1.5 ml polypropylene tubes, which were then placed in
134
J.A. Castro-Hermida et al. / International Journal of Hygiene and Environmental Health 218 (2015) 132–138
Table 1 General characteristics of the conventional drinking water treatment plants (CDWTP). CDWTP (n)
Treatment
Population served
Water source
Large and medium-size (n = 100)
Coagulation, flocculation, sedimentation, filtration and disinfection by chlorination Rapid filtration and disinfection processes by chlorination
2000–110,000
Surface water (rivers or streams)
