Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

ORIGINAL ARTICLE

Presence of Giardia cysts and Cryptosporidium oocystsin drinking water supplies in northern SpainD. Carmena1, X. Aguinagalde1, C. Zigorraga2, J.C. Fernandez-Crespo1 and J.A. Ocio1

1 Department of Health, Laboratory of Public Health of Alava, Basque Government, Vitoria-Gasteiz, Spain

2 Department of Health, Laboratory of Public Health of Guipuzcoa, Basque Government, San Sebastian, Spain

Introduction

Protozoan enteroparasites of the genera Cryptosporidium

(phylum Apicomplexa) and Giardia (subphylum Sarco-

mastigophora) have emerged over the past decades as

major waterborne pathogens. Both parasites are among

the major causative agents of gastroenteritis in humans

and can potentially shorten the life spans of immunocom-

promised individuals. These micro-organisms are trans-

mitted via the faecal–oral route, with the consumption of

contaminated drinking water and use of recreational

waterways significant avenues for acquisition of infection

in developed countries (Slifko et al. 2000). Cryptospori-

dium and Giardia are ubiquitous in the aquatic environ-

ment and their transmission stages (oocysts and cysts,

respectively) may remain viable for several months under

Keywords

Cryptosporidium, Giardia, drinking water,

public health, recreational water, water

quality.

Correspondence

D. Carmena, MRC Clinical Sciences Centre,

Faculty of Medicine, Imperial College,

Hammersmith Hospital Campus, Du Cane

Road, London W12 0NN, UK.

E-mail: [email protected]

2006/0916: received 28 June 2006, revised

23 August 2006 and accepted 6 September

2006

doi:10.1111/j.1365-2672.2006.03193.x

Abstract

Aims: To evaluate the prevalence of Cryptosporidium and Giardia in surface

water supplies from the province of Alava, northern Spain, and to investigate

possible associations among the presence of these pathogenic protozoa with

microbiological, physicochemical and atmospheric parameters.

Methods and results: A total of 284 samples of drinking and recreational water

supplies were analysed. Cryptosporidium oocysts were found in 63Æ5% of river

samples, 33Æ3% of reservoirs samples, 15Æ4% and 22Æ6% of raw water samples

from conventional and small water treatment facilities (respectively), 30Æ8% of

treated water from small treatment facilities, and 26Æ8% of tap water from

municipalities with chlorination treatment only. Giardia cysts were found in

92Æ3% of river samples, 55Æ5% of reservoirs samples, 26Æ9% and 45Æ2% of raw

water samples from conventional and small water treatment facilities (respect-

ively), 19Æ2% of treated water from small treatment facilities, and 26Æ8% of tap

water from municipalities with chlorination treatment only. The presence of

Cryptosporidium and Giardia had significant Pearson’s correlation coefficients

(P < 0Æ01) with the turbidity levels of the samples, and a number of significant

associations were also found with the count levels for total coliforms and

Escherichia coli. The samples were positive for Cryptosporidium significantly

(P < 0Æ05) more frequently during the autumn season than during the spring

and winter seasons. No significant differences were found in the seasonal pat-

tern of Giardia. A moderate association (r ¼ 0Æ52) was found between rainfall

and the presence of Cryptosporidium oocysts.

Conclusions: Cryptosporidium and Giardia are consistently found at elevated

concentrations in surface waters for human consumption from the province of

Alava, northern Spain.

Significance and Impact of the Study: Water treatments based on rapid filtra-

tion process and/or chlorination only are often unsatisfactory to provide safe

drinking water, a situation that represents an important public health problem

for the affected population because of the risk of waterborne outbreaks.

Journal of Applied Microbiology ISSN 1364-5072

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 619–629 619

a range of environmental conditions (Smith et al. 1995).

In addition, Cryptosporidium oocysts and Giardia cysts

are resistant to conventional disinfectants at the concen-

trations and exposure times commonly used, and their

infectious doses in humans have been estimated to be as

low as 30 oocysts for Cryptosporidium (DuPont et al.

1995) and 10 cysts for Giardia (Adam 2001). Taking

together, these data indicate that Cryptosporidium and

Giardia represent a significant threat to public health.

More than 160 waterborne outbreaks of cryptosporidi-

osis and giardiasis have been reported worldwide, with

most cases reported in the US and UK (Slifko et al. 2000;

Craun et al. 2002). This situation has become a major

concern for water utilities and sanitary authorities that

are responsible for providing safe drinking water supplies

for human consumption. In the US, the US Environmen-

tal Protection Agency (USEPA) initiated an effort in 1996

to collect data related to the biology and the epidemiol-

ogy of these protozoa, and to evaluate their risk to

human health. As a result, the USEPA has implemented

national drinking water regulations and developed inno-

vative technologies to improve the detection, monitoring

and surveillance of these micro-organisms in drinking

water (U.S. Environmental Protection Agency 2002). Sim-

ilar initiatives have been implemented in other countries

including England, Wales (Drinking Water Inspectorate

2003), Canada (Health Canada 2004) and New Zealand

(Ministry of Health of New Zealand 2005). The new

European Drinking Water Directive (Directive 98/83/CE)

establishes the goal that all the state members should pro-

vide drinking water supplies with the absence of patho-

genic organisms. However, for practical purposes, the

highly variable sensitivities of the methods available for

the detection of Cryptosporidium and Giardia and prob-

lems associated with the determination of the (oo)cysts

viability/infectivity make the establishment of maximum

acceptable concentrations very difficult (Messner and

Wolpert 2003). Concentrations of ‡3–30 (oo)cysts per

100 l in treated water have been proposed as an ‘action

level’, where the possibility of an outbreak may exist

(Haas and Rose 1995; Wallis et al. 1996).

The province of Alava (northern Spain) extends over

3037 km2, has winter/summer temperatures ranging from

)6�C to 16�C and 7�C to 38�C, respectively, and its

annual precipitation is in the range of 650–900 l m)2. It

has an estimated population of 294 360 people, of which

223 257 are living in the capital, Vitoria-Gasteiz. The

province bears a significant cattle farming activity whose

runoff may be a potential source of contamination in sur-

face waters. The Zadorra Reservoir (225 Hm3) is the main

water supply for human consumption, providing drinking

water to approx. 2 million people in Vitoria-Gasteiz and

the Gran Bilbao area of the adjacent province of Vizcaya.

Other smaller water bodies, as the Marono (2Æ2 Hm3) and

Arceniega (1Æ4 Hm3) reservoirs, supply drinking water to

a number of minor towns. These drinking water sources

are generally open to varied recreational uses, including

swimming recreation, especially during the summer

months. The treatment of the water is carried out in con-

ventional large and medium-size water plants and includes

coagulation, flocculation, clarification through sedimenta-

tion, filtration and disinfection processes. However, there

are also approx. 300 small communities that use surface

water supplies with minimal treatment, usually only chlor-

ination. Because Cryptosporidium oocysts and Giardia

cysts are resistant to this disinfectant, these communities

(estimated total population of approx. 50 000 people) are

potentially at risk of waterborne outbreaks of crypto-

sporidiosis and/or giardiasis. These facts, together with the

lack of previous data in the province of Alava, have

induced the Department of Health of the Basque Govern-

ment to carry out the present study with the aims of

(i) evaluating the prevalence of Cryptosporidium and Giar-

dia in surface water supplies; (ii) estimating the efficiency

of treatment plants in removing these parasites; and

(iii) determining the relationship with microbiological,

physicochemical and atmospheric parameters.

Materials and methods

Sample collection and filtration

A total of 284 samples of water supplies for human

consumption and/or recreation were analysed. Samples

were collected over a 30-month period between April

2000 and September 2002, and comprised natural surface

waters from rivers (n ¼ 52) and reservoirs (n ¼ 36), raw

(n ¼ 26) and treated (n ¼ 31) water from conventional

water treatment facilities (CWTF, facilities that include

coagulation, flocculation, sedimentation, filtration and

disinfection processes), raw (n ¼ 31) and treated (n ¼26) water from small water treatment facilities (SWTF,

facilities that include rapid filtration and/or disinfection

processes only), and tap water (n ¼ 82) from municipal-

ities with chlorination treatment only. Sampling was

mainly directed to those points where Cryptosporidium

and Giardia were suspected. Sampling points were tested

at an interval of approx. 2 months, although when

increased numbers of pathogens in waters were observed

surveillance was intensified to weekly intervals. Samples

were collected in the field by using a portable sampling

apparatus equipped with a peristaltic pump. A minimum

of 100 l of water was filtered through a polypropylene

MicroWynd D-PPPY filter (Cuno Europe, France) of

1-lm nominal pore size at a flow rate of 3–5 l min)1.

However, less volume of water was processed in some

Giardia and Cryptos. in surface waters D. Carmena et al.

620 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 619–629

ª 2006 The Authors

samples with high turbidity because of filter clogging.

Filters were returned to the laboratory in clean poly-

thene containers, typically within 2–5 h after filtration.

Upon receipt, the samples were stored refrigerated at

4�C until processed. Water sub samples were also inde-

pendently collected following the standard procedures

for subsequent physicochemical and microbiological

analysis.

Sample elution and concentration

Samples were processed within 24 h after filtration.

Briefly, filters were kept in the housing with the water that

remained after sampling and subjected to a crosscurrent

air flow for 15 min to unpack the fibres and facilitate the

removal of cysts/oocysts. Filters were then cut longitudi-

nally and the fibres divided into an outer, middle and

inner layer. Portions were washed separately three times

for 5 min in 1 l of PBS buffer pH 7Æ2 containing 0Æ1%

SDS, 0Æ1% Tween 80, and 0Æ01% Antifoam A (Sigma-Ald-

rich, Dorset, UK). In order to increase the cyst/oocyst

recoveries, fibre portions were sonicated in a water bath

for 5 min at 40 Hz between the second and the third

wash. The material that was washed from the fibres was

centrifuged at 1050 g for 10 min using 50-ml centrifuge

tubes in a swinging-bucket rotor, and the supernatant

carefully discarded by aspiration. The packed pellet vol-

ume was recorded, and the pellet resuspended in a suitable

volume of deionized water and sonicated in a water bath

for 10 min at 40 Hz to prevent cysts/oocysts aggregation.

Sample purification

Cysts/oocysts in the samples were isolated from other par-

ticulate material by immunomagnetic separation using the

commercial kit Dynabeads GC-Combo (Dynal Biotech,

Bromborough, UK), as described in the method 1623

(U.S. Environmental Protection Agency 1999). Briefly, the

procedure involves adding magnetic beads labelled with

Cryptosporidium- and Giardia-specific monoclonal anti-

bodies to 10 ml of resuspended pellet and allowing

the antibody-antigen reactions to bind the (oo)cysts to

the beads. The sample is then magnetized, separating the

(oo)cyst-magnetic bead complex from the sample debris,

which is then discarded. The beads are then detached and

the (oo)cysts are added to a well slide for sample screen-

ing, allowed to air-dry completely, and fixed with acetone.

Sample staining and examination

The identification and enumeration of cysts/oocysts was

carried out by immunofluorescence assay using the

commercial kit Crypto/Giardia IF Test (Cellabs, Brookvale,

Australia), according to the manufacturer’s instructions.

Well slides were washed three times with abundant PBS

buffer pH 7Æ2, and after adding mounting medium, the

coverslip was sealed with nail polish. The slides were sys-

tematically examined by using epifluorescence microscope

(Zeiss Standard Lab 16; Carl Zeiss, Gottingen, Germany)

at 400· magnification, searching for brilliant apple-green

fluorescing round to oval objects. Magnification was

increased to 1000· for confirmation of presumptive sam-

ples and differential interference contrast microscopy was

used for identification of internal morphological features

such as number of sporozoites or nuclei, and presence of

axonema or median bodies. Estimations of the total

amount of cysts/oocysts were calculated considering the

volume of water filtered and the fraction of the pellet ana-

lysed. Positive and negative staining controls were rou-

tinely included.

Physicochemical analysis

Turbidity and free chlorine were measured in the laborat-

ory immediately after the arrival of the samples. Turbidity

was measured for each thoroughly stirred sample with a

Hach 2100N turbimeter (Hach, Loveland, CO, USA) and

the results were expressed in nephelometric turbidity

units (NTU). Free chlorine was measured in treated water

samples only by using the DPD chlorine test kit (La-

Motte, Chestertown, MD, USA) and expressed in milli-

gram per litre.

Analyses of Escherichia coli and total coliforms

Escherichia coli counts were determined by filtering

100-ml sample through a 0Æ45-lm pore size cellulose

filter (Millipore, Bedford, MA, USA). The filters were

incubated at 36�C for 24 h on the Chromocult� coliform

agar (Merck Biosciences, Nottingham, UK) and the dark

blue- to violet-coloured colonies were considered to be

E. coli. Total coliforms counts were determined by filter-

ing 100-ml samples through 0Æ45-lm pore size cellulose

filter (Millipore). The filters were incubated at 36�C for

24 h on the m-Endo total coliform agar (Millipore) and

the deep red with distinct green metallic sheen colonies

were counted as total coliforms.

Stock suspension preparation and enumeration

Human stools from patients infected with Giardia lamblia

were obtained from the Donostia Hospital, San Sebastian

(Spain). Stools from calves with cryptosporidiosis were

kindly provided by Dr Enrique Perez, Faculty of Veterin-

ary, University of Extremadura (Spain). Faecal samples

were filtered through a 0Æ5-mm sieve and concentrated by

D. Carmena et al. Giardia and Cryptos. in surface waters

ª 2006 The Authors


centrifugation at 1050 g for 10 min. Cysts/oocysts were

isolated by using percoll-sucrose gradient (specific gravity:

1Æ09–1Æ10) and centrifugation at 1050 g for 10 min.

Purified cysts/oocysts were stored at 4�C in PBS buffer

pH 7Æ2 complemented with 10 000 U ml)1 penicillin and

10 mg ml)1 streptomycin (Sigma-Aldrich) to prevent bac-

terial growth. Spiking suspensions containing 10 000–

15 000 cysts/oocysts were prepared in reagent water with

0Æ01% Tween 20. Stock and spiking suspensions were

enumerated by using both haemocytometer chamber and

well slide for immunofluorescence detection (U.S. Envi-

ronmental Protection Agency 1995). A total of 10 differ-

ent haemacytometer chambers were counted and averaged

for each cyst/oocyst suspension to achieve optimal count-

ing accuracy. Well slide counting was performed by ana-

lysing a 20-ll aliquot of each cyst/oocyst suspension, in

triplicate. Stock suspensions of cysts/oocysts were used for

no more than 12 weeks following the purification.

Initial precision and recovery of the method

In order to determine the initial recovery achieved using

the method, 12 independent spiking experiments were

carried out by filtering 50 l of distilled water as described

above. Spiking suspensions with 2500 cysts of Giardia

and 5000 oocysts of Cryptosporidium were sequentially

delivered into the inlet tube of the sampling housing by

injection using a syringe with a 21-gauge needle. This

procedure avoids the loss off cysts/oocysts that may

remain attached to the internal wall of the carboy. The

percent recovery (R) of the method was calculated by

using the following equation:

R ¼ N=T � 100

where N is the number of cysts/oocysts detected and T

the number of cysts/oocysts spiked. The average percent

recovery and the SD of the recoveries for Cryptosporidium

and Giardia were also calculated.

Quality control of the method

In order to determine the inter-assay variation of the

method for the recovery of Cryptosporidium oocysts and

Giardia cysts, a spiking experiment was carried out every

15 field samples processed, as previously described. The

acceptance criterion for Cryptosporidium and Giardia

recoveries during routine use of the method was defined

as their initial mean recovery ±1Æ5 SD.

Meteorological data

Rainfall data recorded at representative weather stations

were utilized to study the extent to which heavy rains

influence the concentration of Cryptosporidium oocysts

and Giardia cysts in water for human consumption. The

obtained precipitation data were used to calculate weekly

sums and monthly arithmetic means.

Statistical analysis

Pearson’s correlation coefficient and nonparametric

Spearman’s rho were calculated to evaluate how physico-

chemical, microbiological and atmospheric parameters

are related with cysts/oocysts rates. Spearman’s rho test

was chosen because it is less sensitive to extreme values

than the standard Pearson’s correlation coefficient.

Chi-squared test was used to estimate possible significant

differences between the seasonal prevalence of Crypto-

sporidium and Giardia and rainfall. Values of P < 0Æ05

were considered statistically significant. All the analyses

were performed with the Statistical Package for Social

Sciences 12Æ0 for Windows (SPSS Inc., Chicago, IL, USA)

software.

Results

Precision of the method

The initial average percent recovery of the method

(n ¼ 12) was 16Æ7% (SD: 7Æ7) for Cryptosporidium

oocysts and 41Æ5% (SD: 10Æ6) for Giardia cysts. The

acceptable recovery intervals (accuracy) ranged from

5Æ2% to 28Æ2% for Cryptosporidium oocysts and from

25Æ5% to 57Æ5% for Giardia cysts. Inter-assay variation

during routine use of the method was investigated by

assaying a total of 18 independent spiking experiments

carried through out the course of the study (Fig. 1).

Only 3/18 (16Æ7%) of the Crypstosporidium and Giardia

seeded samples failed to fall inside the acceptable range

for (oo)cysts recovery.

Prevalence of Cryptosporidium and Giardia in water

samples

Over the course of the 30-month study period, a total of

284 water samples were analysed for the detection of

Cryptosporidium oocysts and Giardia cysts. Both protozoa

were frequently found in all water sources, except those

from CWTF (Table 1). Natural surface water from rivers

and reservoirs showed the highest rates of protozoa

occurrence, with concentrations that reached 1767

Cryptosporidium oocysts and >25 000 Giardia cysts per

100 l. CWTF achieved at least 3-log (99Æ9%) (oo)cyst

removal, and no protozoa were detected in the finished

water. However, SWTF only achieved 0Æ53 log10 Crypto-

sporidium oocysts and 1Æ62 log10 Giardia cysts removals,



ª 2006 The Authors

respectively. In addition, differences in the parasitological

quality of the raw water were also noted, having the

samples from SWTF higher (oo)cysts concentrations than

the equivalent samples from the CWTF. Worryingly, an

important proportion of the SWTF finished water and

tap water samples analysed was found positive for the

presence of both Cryptosporidium and Giardia (ranging

from 26% to 31% and from 19% to 27%, respectively).

The detected concentrations were always <8 (oo)cysts per

100 l.

Physicochemical parameters

Physicochemical and microbiological data of the water

samples studied are shown in Table 2. Turbidity values

varied between 0Æ3 and 181 NTU, with river and reservoir

water samples recording the highest levels (mean: 15Æ3and 10Æ7 NTU, respectively). CWTF achieved an average

fivefold reduction of turbidity levels (mean: 0Æ4; SD: 0Æ2).

In contrast, a much lower performance of 2Æ7-fold reduc-

tion rate was accomplished by SWTF (mean: 3Æ1; SD:

9Æ4). In order to evaluate the efficiency of the disinfection

process, free chlorine levels were measured in finished

and tap water. The tested samples showed values ranging

from 0Æ1 to 1Æ08 mg ml)1 (mean: 0Æ31–0Æ40; SD: 0Æ22–

0Æ42).

Microbiological parameters

Occurrence of E. coli and total coliforms was determined

as these micro-organisms are traditional faecal indicators.

The presence of E. coli was detected in all the categories

of samples, except those from CWTF-treated water

(Table 2). As expected, the highest counts were obtained

in natural surface and raw water samples (means ranging

from 4Æ6 to >100 CFU 100 ml)1). Although absence

of E. coli was recorded in CWTF-finished water, low lev-

els of this indicator were frequently observed in SWTF-

treated water samples (mean: 17Æ0 CFU 100 ml)1; SD:

30Æ9). The presence of E. coli was also sporadically detec-

ted in tap water samples (mean: 0Æ04 CFU 100 ml)1; SD:

0Æ24). Total coliforms counts reveal the same pattern

obtained for E. coli., although with slightly higher rates.

Table 1 Descriptive statistics of the concentration of Cryptosporidium oocysts and Giardia cysts in the different water samples analysed

Rivers Reservoirs

Conventional

water

treatment

facilities

(CWTF) raw�

CWTF

treated�

Small water

treatment

facilities

(SWTF) raw� SWTF treated� Tap water§

Cryptosporidium Giardia Crypto. Giardia Crypto. Giardia Crypto. Giardia Crypto. Giardia Crypto. Giardia Crypto. Giardia

No. samples 52 52 36 36 26 26 31 31 31 31 26 26 82 82

% positive samples 63Æ5 92Æ3 33Æ3 55Æ5 15Æ4 26Æ9 0Æ0 0Æ0 22Æ6 45Æ2 30Æ8 19Æ2 26Æ8 26Æ8

Range (n 100 l)1) 0–1767 0->25 000 0–380 0–1320 0–276 0–50 0Æ0 0Æ0 0–1325 0–2997 0–184 0–25 0–61 0–62

Mean (n 100 l)1) 113Æ2 6522Æ8 17Æ6 63Æ3 17Æ2 4Æ6 0Æ0 0Æ0 26Æ4 53Æ9 7Æ8 1Æ3 2Æ3 2Æ0

SD 279Æ5 9452Æ7 31Æ7 110Æ8 26Æ7 5Æ0 0Æ0 0Æ0 58Æ2 94Æ8 13Æ6 2Æ2 3Æ6 2Æ7

�Conventional water treatment facilities that include coagulation, flocculation, sedimentation, filtration and disinfection processes.

�Small water treatment facilities that include rapid filtration and/or disinfection processes only.

§Tap water with chlorination treatment only.

Cryptosporidium

0

5

10

15

20

25

30

35

40

1

No. assay

Ooc

yst r

ecov

ery

(%)

Cys

t rec

over

y (%

)

Giardia

0

10

20

30

40

50

60

70

80

No. assay

1817 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2

1 18171615141312111098765432

Figure 1 Inter-assay variation (n ¼ 18) for the detection of Cryptos-

poridium oocysts (mean: 11Æ1; SD: 6Æ8) and Giardia cysts (mean: 32Æ3;

SD ¼ 11Æ0) during routine use of the method.


ª 2006 The Authors


Correlation between physicochemical parameters and

protozoa

The statistical analysis of the association between physico-

chemical and microbiological parameters and the occur-

rence of Cryptosporidium oocysts and Giardia cysts is

summarized in Tables 3 and 4. A strong linear correlation

(P < 0Æ01) between the presence of Cryptosporidium

oocysts and the turbidity of the water was found in all

the sample categories, except those from rivers. Similar

results were obtained for Giardia cysts in samples from

reservoirs, SWTF-treated water, and tap water. No signifi-

cant correlations (P > 0Æ05) were found between the inci-

dence of these protozoa and the levels of free chlorine in

drinking water samples.

Correlation between microbiological parameters and

protozoa

A significant correlation between Cryptosporidium oocysts

and E. coli was observed in raw (P < 0Æ05) and treated

water samples (P < 0Æ01) from SWTF. This parasite was

also found to be highly correlated with the presence of total

coliforms (P < 0Æ05) in all the categories of samples, except

those from rivers, SWTF-treated waters, and tap waters.

Finally, a number of significant associations between the

Table 3 Correlation between the number of Cryptosporidium oocysts detected and the physicochemical and microbiological parameters of the

water samples analysed

Turbidity Free-chlorine E. coli Total coliforms

Pearson

correlation

Spearman’s

rho

Pearson

correlation

Spearman’s

rho

Pearson

correlation

Spearman’s

rho

Pearson

correlation

Spearman’s

rho

r P r P r P r P r P r P r P r P

Rivers 0Æ001 0Æ092)0Æ075 0Æ596 – – – – 0Æ093 0Æ513 0Æ222 0Æ114 0Æ082 0Æ564 0Æ226 0Æ108

Reservoirs 0Æ575** 0Æ000 0Æ251 0Æ140 – – – – 0Æ290 0Æ086 0Æ233 0Æ171 0Æ209 0Æ221 0Æ362* 0Æ030

Conventional water treatment

facilities (CWTF) raw�

0Æ721** 0Æ000 0Æ532** 0Æ005 – – – – 0Æ040 0Æ984 0Æ157 0Æ444 0Æ367 0Æ065 0Æ435* 0Æ026

CWTF treated� – – – – – – – – – – – – – – – –

Small water treatment

facilities (SWTF) raw�

0Æ906** 0Æ000 0Æ171 0Æ365 – – – – 0Æ330 0Æ075 0Æ415* 0Æ022 0Æ228 0Æ227 0Æ395* 0Æ031

SWTF treated� 0Æ932** 0Æ000 0Æ297 0Æ140)0Æ381 0Æ527 )0Æ395 0Æ510 0Æ544** 0Æ004 0Æ338 0Æ091 0Æ404* 0Æ041 0Æ261 0Æ197

Tap water§ 0Æ316** 0Æ004 0Æ214 0Æ053 0Æ103 0Æ409 0Æ196 0Æ112)0Æ075 0Æ503)0Æ094 0Æ399)0Æ055 0Æ626 0Æ030 0Æ740




*Correlation significant at the 0Æ05 level; **Correlation significant at the 0Æ01 level.

Table 2 Descriptive statistics of other physicochemical and microbiological parameters of the water samples analysed

Turbidity (NTU) Free-chlorine (mg l)1)

Escherichia coli

(CFU 100 ml)1)

Total coliforms

(CFU 100 ml)1)

Range Mean SD Range Mean SD

% of +

samples Range Mean SD

% of +

samples Range Mean SD

Rivers 0Æ3–181 15Æ3 32Æ6 – – – 98Æ1 0->100 >100 – 100 2->100 >100 –

Reservoirs 0Æ3–100 10Æ7 25Æ4 – – – 69Æ4 0->100 29Æ1 39Æ8 94Æ4 0->100 67Æ5 37Æ2

Conventional water

treatment facilities (CWTF) raw�

0Æ7–7Æ8 2Æ0 1Æ8 – – – 34Æ6 0->100 4Æ6 19Æ5 80Æ8 0->100 30Æ3 39Æ7

CWTF treated� 0Æ3–1Æ0 0Æ4 0Æ2 0Æ1–1Æ08 0Æ40 0Æ23 0Æ0 0 0Æ0 0Æ0 3Æ2 0–4 0Æ12 0Æ71



0Æ4–95 8Æ5 17Æ5 – – – 74Æ2 0->100 37Æ9 41Æ4 74Æ2 0->100 57Æ2 45Æ5

SWTF treated� 0Æ3–49 3Æ1 9Æ4 0Æ1–1Æ03 0Æ39 0Æ42 46Æ1 0->100 17Æ0 30Æ9 50Æ0 0->100 25Æ1 37Æ3

Tap water§ 0Æ3–13Æ4 1Æ7 2Æ0 0Æ1–0Æ75 0Æ31 0Æ22 3Æ6 0->100 0Æ04 0Æ24 4Æ9 0->100 0Æ89 7Æ78






ª 2006 The Authors

occurrence of Giardia cysts and the E. coli/total coliform

counts were detected in samples from rivers, reservoirs,

CWTF raw water and SWTF-treated water.

Seasonality of prevalence

The distribution of results by season (Fig. 2) revealed that

autumn had a significantly higher incidence of Cryptos-

poridium oocyst positive samples than spring (v2 ¼ 2Æ92,

P < 0Æ1) and winter (v2 ¼ 3Æ85, P < 0Æ05). No significant

differences were detected in the seasonal pattern of Giar-

dia cysts.

Influence of rainfall on the occurrence of

Cryptosporidium and Giardia

Mean monthly rainfall values were calculated and plotted

against the percentage of monthly positive samples for

Cryptosporidium and Giardia (Fig. 3). On the basis of

Pearson’s correlation coefficient, moderate associations

were observed between the rainfall and the presence

of Cryptosporidium oocysts (r ¼ 0Æ52). The occurrence of

Giardia cysts was weakly correlated with the rainfall data

(r ¼ 0Æ34). Interestingly, peak prevalences for both proto-

zoa were achieved in October (autumn period), in coinci-

dence with the highest rate of rainfall. A second Giardia

peak was observed in April (spring period), also associ-

ated with elevated rainfall levels.

0

10

20

30

40

50

60

70

Spring

Pos

itive

sam

ples

(%

)

*

n = 65 n = 91 n = 62 n = 66 WinterAutumnSummer

Figure 2 Seasonality of Cryptosporidium ( ) and Giardia ( ) over a

30-month survey. Significant differences (*) on the percentage of

Cryptosporidium positive samples were found between autumn and

spring (P < 0Æ1) and between autumn and winter (P < 0Æ05).

0

10

20

30

40

50

60

70

80Ja

n.

Feb

.

Mar

ch

Apr

il

May

June

July

Aug

.

Sep

t.

Oct

.

Nov

.

Dec

.

Pos

itive

sam

ples

(%

)

0

100

200

300

400

500

600

700

800

900

1000

Rai

nfal

l (m

m m

onth

–1)

Figure 3 Prevalence of Cryptosporidium ( ) and Giardia ( ) over a

30-month survey, comparatively analysed with rainfall (s).

Table 4 Correlation between the number of Giardia cysts detected and the physicochemical and microbiological parameters of the water samples

analysed

Turbidity Free-chlorine E. coli Total coliforms

Pearson

correlation

Spearman’s

rho

Pearson

correlation

Spearman’s

rho

Pearson

correlation

Spearman’s rho Pearson

correlation

Spearman’s

rho

r P r P r P r P r P r P r P r P

Rivers )0Æ161 0Æ255 0Æ26 0Æ857 – – – – 0Æ161 0Æ255 0Æ382** 0Æ05 0Æ139 0Æ325 0Æ304* 0Æ029

Reservoirs 0Æ452** 0Æ060 0Æ172 0Æ315 – – – – 0Æ313 0Æ063 0Æ386* 0Æ020 0Æ344* 0Æ040 0Æ499** 0Æ002

Conventional water

treatment

facilities (CWTF) raw�

0Æ316 0Æ116 0Æ150 0Æ465 – – – – 0Æ256 0Æ207 0Æ635** 0Æ000 0Æ040 0Æ661 0Æ247 0Æ223

CWTF treated� – – – – – – – – – – – – – – – –



0Æ229 0Æ224 0Æ335 0Æ070 – – – – 0Æ275 0Æ142 0Æ194 0Æ305 0Æ189 0Æ317 0Æ307 0Æ099

SWTF treated� 0Æ848** 0Æ000 0Æ393* 0Æ047 )0Æ305 0Æ498 )0Æ388 0Æ452 0Æ539** 0Æ005 0Æ120 0Æ559 0Æ451* 0Æ035 0Æ090 0Æ660

Tap water§ 0Æ194 0Æ082 0Æ325** 0Æ003 0Æ059 0Æ635 0Æ078 0Æ532 )0Æ057 0Æ610 )0Æ094 0Æ400 )0Æ038 0Æ731 0Æ056 0Æ618

�Water treatment facilities that include coagulation, flocculation, sedimentation, filtration and disinfection processes.



*Correlation significant at the 0Æ05 level; **Correlation significant at the 0Æ01 level.


ª 2006 The Authors


Discussion

During the past 15 years, an increasing number of water-

borne outbreaks caused by Cryptosporidium and Giardia

have been documented worldwide (Lisle and Rose 1995;

Slifko et al. 2000; Craun et al. 2002; Fricker et al. 2002),

showing a trend in which protozoa and viruses are

replacing bacterial pathogens as agents of primary con-

cern in waterborne disease (Briancesco and Bonadonna

2005). Because of this situation, detection of Cryptospori-

dium oocysts and/or Giardia cysts in surface water,

especially in reservoirs for drinking water supply, is of

great public health importance. In order to provide safe

drinking water, drinking water systems must optimize five

key elements: source water protection, adequate treat-

ment, secure distribution, appropriate monitoring and

appropriate response to adverse monitoring results (Huck

and Coffey 2004).

In Spain, the prevalence of Cryptosporidium and

Giardia has been well documented in humans (Rodrı-

guez-Hernandez et al. 1996), livestock (Quilez et al.

1996; Castro-Hermida et al. 2002) and molluscs (Freire-

Santos et al. 2000; Gomez-Couso et al. 2004, 2005).

However, very few epidemiological surveys have been

conducted on the occurrence of these protozoa in sur-

face water for human consumption (Rodrıguez-Hernan-

dez et al. 1994; Montemayor et al. 2005) and no

significant waterborne outbreaks of cryptosporidiosis/

giardiasis have been reported. In addition, although the

European Community environmental legislation states

that water intended for human consumption should

not contain pathogenic organisms (Directive 98/83/CE),

in Spain there are no specific regulations relating to

Cryptosporidium and Giardia tolerable limits in drinking

water.

In the present study, the initial recovery results

(Cryptosporidium, 16Æ7%; Giardia, 41Æ5%) obtained during

the optimization of the analytical system were satisfactory,

meeting the acceptance criteria proposed by the method

1623 (U.S. Environmental Protection Agency 1999). Reli-

able values were also achieved in the inter-assay precision

tests, assuring the reproducibility of the method. These

facts demonstrate that the system performance is suitable

for analytical purposes.

Data obtained in this long-term survey show that

Cryptosporidium and Giardia (oo)cysts were consistently

detected in surface, raw and finished water samples from

the province of Alava (northern Spain), showing their

ubiquitous distribution. In most cases Giardia prevalence

was higher than that of Cryptosporidium, corroborating

the tendency observed in other countries (Horman et al.

2004; Rimhanen-Finne et al. 2004; Briancesco and Bona-

donna 2005).

Water treatment facilities play a key role in the process

to provide safe drinking water for human consumption.

Despite their small size, Cryptosporidium oocysts and Giar-

dia cysts can be effectively removed from water supplies

by conventional particle separation processes. However,

small number of these protozoa can be found in finished

water even in the absence of treatment problems (States

et al. 1997). In our study, CWTF achieved three orders of

magnitude removal for (oo)cysts, and no protozoa were

found in the treated water. These data are consistent with

those obtained in other investigations at pilot- and full-

scale conventional water treatment plants (reviewed by

Betancourt and Rose 2004). In addition, significant corre-

lations between water turbidity level and presence of Cryp-

tosporidium oocysts have been documented in previous

reports (Falabi et al. 2002; Hsu and Yeh 2003; Hsu 2003).

The observation that consistent removal rates of (oo)cysts

are achieved when the treatment facilities produce water

of consistently low turbidity (£0Æ3 NTU) has suggested

that turbidity is a useful in-plant measure of the degree of

(oo)cyst removal (Nieminsky et al. 1995; Hsu and Yeh

2003). In the present study, CWTF achieved fivefold tur-

bidity removals, with an average plant effluent turbidity of

0Æ4 NTU. We also found a strong association between

turbidity and presence of Cryptosporidium oocysts and

Giardia cysts in most of the categories of water analysed.

These findings strongly support the use of turbidity

removal as a reliable indicator of the effectiveness of

removal of (oo)cysts in water treatment plants. Finally, no

E. coli counts were detected in finished water from CWTF

and total coliforms were only detected in one sample at

very low concentration (mean: 0Æ12 CFU 100 ml)1). Over-

all, these results reveal that the CWTF system components

involved in the removal of water particles and disinfection

were intact and operating correctly, assuring the produc-

tion of safe drinking water.

With regard to SWTF, removals of 0Æ53 log10 for

Cryptosporidium oocysts and of 1Æ62 log10 for Giardia

cysts were obtained. However, both protozoa were found

in 30Æ8% and 19Æ2% of the finished water samples, respect-

ively (means: 7Æ8 oocysts/100 l, and 1Æ3 cysts/100 l). SWTF

also achieved 2Æ7-fold turbidity removal, producing an

average effluent turbidity of 3Æ1 NTU. As expected, these

data show that SWTF performance is inferior in compar-

ison with CWTF, demonstrating that water treatments

based on rapid filtration process and/or disinfection only

are clearly insufficient for removing protozoa and redu-

cing turbidity levels to acceptable limits. In addition, E.

coli and total coliforms counts were also found in finished

water from SWTF, indicating inadequacy of the disinfec-

tion procedures in some of the SWTF.

Escherichia coli and total coliforms are used as indicator

organisms worldwide for faecal contamination and



ª 2006 The Authors

microbial water hygiene (Edberg et al. 2000). In this

investigation, counts of these micro-organisms ranging

between 4Æ6 and >100 CFU 100 ml)1 were found in an

elevated proportion of the surface water samples analysed.

It is important to take into consideration that the

Zadorra Reservoir system bears a considerable cattle

farming activity, with up to 7000 cows, 8500 pigs, 25000

sheep and 700 horses. This picture suggests that livestock

faecal products are probably the main source of environ-

mental and water contamination. Some reports have

investigated the possible association between the preval-

ence of Cryptosporidium and Giardia and the presence of

other micro-organisms, with unclear results (Payment

and Franco 1993; Horman et al. 2004; Rimhanen-Finne

et al. 2004; Briancesco and Bonadonna 2005). We found

a number of significant correlations between the presence

of Cryptosporidium and Giardia and samples being posit-

ive for E. coli and/or total coliforms. These findings

indicate that micro-organism counts may be used as

predictors for the presence of these protozoan parasites.

The analyses of the tap water samples from municipal-

ities with chlorination treatment only show relevant

data. The presence of E. coli and total coliforms

was sporadically recorded at very low levels (average

E. coli, 0Æ04 CFU 100 ml)1; average total coliforms,

0Æ89 CFU 100 ml)1), demonstrating an acceptable per-

formance of the disinfection treatment. However, Cryptos-

poridium and Giardia were detected in the 26Æ8% of the

samples, at average concentrations of 2–2Æ3 (oo)cysts/

100 l. These data indicate that some of the tap water sam-

ples analysed contained (oo)cysts at concentrations in the

range considered as ‘action level’ by Haas and Rose

(1995) and Wallis et al. (1996). Although the viability

and the genotype of the (oo)cysts have not been assessed

in this study, this situation represents a potential risk for

waterborne infection for an estimated 50 000 population

in the province of Alava.

This 30-month survey has also shown that Cryptos-

poridium and Giardia (oo)cysts are present in surface

water throughout the year, with the highest frequency of

occurrence during the autumn. A second peak for

Cryptosporidium was recorded during the summer,

whereas Giardia prevalence was more homogeneously dis-

tributed during the rest of the year. The seasonality of

these parasite protozoa has been investigated in few stud-

ies, with diverse results. High frequencies of samples pos-

itive for Cryptosporidium and Giardia in environmental

water have been linked to activities associated with agri-

cultural practices and cattle farming such as calving,

lambing and muck spreading (Kemp et al. 1995; Ong

et al. 1996; Casemore et al. 1997). Additionally, runoff

from precipitation has been proposed as a mechanism for

entry of these organisms into surface waters (Bodley-Tick-

ell et al. 2002) and previous studies have reported moder-

ate correlations between rainfall and the presence of

Cryptosporidium and Giardia (Atherholt et al. 1998; Thur-

man et al. 1998; Bodley-Tickell et al. 2002). However,

unclear seasonality or association with rainfall has been

found in some other investigations (Carrington and Mil-

ler 1993; Robertson and Gjerde 2001). In the present

study, we found a reasonable correlation (r ¼ 0Æ52)

between the rainfall and the presence of Cryptosporidium,

with oocysts peak concentration in October coinciding

with the annual highest rate of rainfall. A weak associ-

ation was found for Giardia (r ¼ 0Æ34), but again cysts

peak concentrations were reached in October with a sec-

ond lesser peak in April. These data suggest that monitor-

ing of Cryptosporidium and Giardia must be intensified

during autumn rainfall months, the period of highest pre-

valence of these protozoa in surface water.

To address this situation, the Department of Health of

the Basque Government, in co-ordination with the local

authorities, has initiated a number of actions directed to

assure the quality of the most vulnerable drinking water

supplies in the province of Alava. These include protec-

tion of watersheds susceptible to be contaminated by

human or domestic animal faecal waste, construction of

new compact water treatment water facilities and

improvement of the disinfection procedures by imple-

mentation of UV light irradiation-based systems where

the construction of compact water treatment facilities was

not feasible. In addition, the sensitivity of the detection

assay has been enhanced by adoption of the Envirochek

capsule filters (Pall Gelman Sciences, Ann Arbor, MI,

USA) for routine use, as recommended in the Method

1623 (U.S. Environmental Protection Agency 1999). In

our hands, these capsules achieved a preliminary initial

average percent recovery (n ¼ 4) of 33Æ4% (SD: 2Æ2) for

Cryptosporidium oocysts, and 48Æ2% (SD: 3Æ9) for Giardia

cysts (Carmena et al., unpubl. data).

Acknowledgements

The authors are grateful to Phil Hobson (MRC Clinical

Sciences Centre, Imperial College London) for his advice

with the English language editing. This work was finan-

cially supported by a grant from the Health Department

of the Basque Government, Spain. David Carmena was a

recipient of a fellowship from the Health Department of

the Basque Government, Spain.

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Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain

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