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Detection of microsporidia in drinking water, wastewaterand recreational rivers

Fernando Izquierdo a, Jose Antonio Castro Hermida b, Soledad Fenoy a, Mercedes Mezo b,Marta Gonzalez-Warleta b, Carmen del Aguila a,*aUniversidad San Pablo CEU, Laboratorio de Parasitologıa, Facultad de Farmacia, Urbanizacion Monteprıncipe, CP 28668 Boadilla del Monte,

Madrid, SpainbCentro de Investigaciones Agrarias de Mabegondo, Laboratorio de Parasitologıa, Instituto Galego de Calidade Alimentaria-Xunta de Galicia,

Carretera AC-542 de Betanzos a Meson do Vento, Km 7.5, CP 15318 Abegondo (A Coruna), Spain

a r t i c l e i n f o

Article history:

Received 26 January 2011

Received in revised form

22 June 2011

Accepted 22 June 2011

Available online 2 July 2011

Keywords:

Drinking water treatment

plant (DWTP)

Wastewater treatment

plant (WWTP)

Recreational river area (RRA)

IDEXX Filta-Max

Microsporidia

Encephalitozoon intestinalis

* Corresponding author. Tel.: þ34 91 372 47 9E-mail addresses: ferizqui@ceu.es (F. I

(S. Fenoy), mercedes.mezo@xunta.es (M. Me0043-1354/$ e see front matter ª 2011 Elsevdoi:10.1016/j.watres.2011.06.033

a b s t r a c t

Diarrhea is the main health problem caused by human-related microsporidia, and water-

borne transmission is one of the main risk factors for intestinal diseases. Recent studies

suggest the involvement of water in the epidemiology of human microsporidiosis.

However, studies related to the presence of microsporidia in different types of waters from

countries where human microsporidiosis has been described are still scarce. Thirty-eight

water samples from 8 drinking water treatment plants (DWTPs), 8 wastewater treatment

plants (WWTPs) and 6 recreational river areas (RRAs) from Galicia (NW Spain) have been

analyzed. One hundred liters of water from DWTPs and 50 L of water from WWTPs and

RRAs were filtered to recover parasites, using the IDEXX Filta-Max� system.

Microsporidian spores were identified by Weber’s stain and positive samples were

analyzed by PCR, using specific primers for Enterocytozoon bieneusi, Encephalitozoon intesti-

nalis, Encephalitozoon cuniculi, and Encephalitozoon hellem. Microsporidia spores were identi-

fied by staining protocols in eight samples (21.0%): 2 from DWTPs, 5 from WWTPs, and 1

from an RRA. In the RRA sample, the microsporidia were identified as E. intestinalis.

To the best of our knowledge, this is the first report of human-pathogenic microsporidia

in water samples from DWTPs, WWTPs and RRAs in Spain. These observations add further

evidence to support that new and appropriate control and regulations for drinking,

wastewater, and recreational waters should be established to avoid health risks from this

pathogen.

ª 2011 Elsevier Ltd. All rights reserved.

1. Introduction Giardia and Cryptosporidium, are frequently transmitted by

Waterborne transmission is one of the main risk factors for

intestinal diseases causing an important morbidity and

mortality worldwide. However, it is surprising that even

though known agents that produce intestinal disease, such as

6/84; fax: þ34 91 351 04 9zquierdo), jose.antonio.czo), marta.gonzalez@xunier Ltd. All rights reserved

water (Graczyk et al., 2007b), over 50% of waterborne infec-

tions are produced by unknown agents (Dowd et al., 1998).

This finding is of special interest if we bear in mind that, for

economic and environmental reasons, spreading sewage

sludge on agricultural lands has increased during recent years

6.astro.hermida.@xunta.es (J.A. Castro Hermida), sfenrod@ceu.esta.es (M. Gonzalez-Warleta), cagupue@ceu.es (C.del Aguila)..

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 8 3 7e4 8 4 34838

(Rimhanen-Finne et al., 2004). This might affect not only the

circulation of recognized pathogens such as Cryptosporidium

and Giardia, but also emerging pathogens, such as micro-

sporidia. Moreover, the results obtained in different studies

carried out to establish the quality of depuration end products

associated with the presence of parasites seem to be contra-

dictory (Straub et al., 1993; Wiandt et al., 2000; Caccio et al.,

2003; Graczyk et al., 2007a). The general impression is that

treatment to obtain sewage sludge end products has demon-

strated a high efficacy of pathogen removal. However, as

viable pathogens have been detected in these end products,

they could be considered a serious health threat (Graczyk

et al., 2007a).

On the other hand, it is important to understand that the

presence of human pathogens in surface water may suggest

the presence of living environmental reservoirs, such as

domestic and wild animals. Among the latter, aquatic birds

may play an important role in the transmission of different

pathogens (Slodkowicz-Kowalska et al., 2006).

Microsporidia are obligate intracellular eukaryote patho-

gens that may cause infection in both vertebrate and inver-

tebrate hosts. Diarrhea is the most frequent health problem

caused, mainly in immunocompromised people. The trans-

mission routes indicated are via airborne, person-to-person,

zoonotic, and waterborne means (Didier et al., 2004; Graczyk

et al., 2007c).

Waterborne transmission ofmicrosporidian spores has not

yet been appropriately addressed in epidemiological studies,

due to the small size of spores (1e4 m) (Mathis et al., 2005).

Their presence, associated with waterborne outbreaks and

also with recreational and river water, has rarely been docu-

mented (Sparfel et al., 1997; Dowd et al., 1998, 2003; Cotte et al.,

1999; Fournier et al., 2000; Thurston-Enriquez et al., 2002;

Coupe et al., 2006; Graczyk et al., 2007b, 2007c; Lucy et al.,

2008). On the other hand, the demonstration of waterborne

microsporidian spores of species known to infect humans,

proceeding from common waterfowl which have unlimited

access to surface waters, has only recently been documented

(Slodkowicz-Kowalska et al., 2006).

In spite of this, microsporidia are recognized category B

biodefense agents on the National Institutes of Health list, and

the transmission of microsporidian spores is seriously

considered by American agencies concerned with the quality

of drinking water (Nwachcuku and Gerba, 2004). These path-

ogens have been included in the Contaminant Candidate List

of the U.S. Environmental Protection Agency ((EPA), 1998)

because spore identification, removal, and inactivation in

drinking water are technologically challenging, and human

microsporidial infections are difficult to treat (Slodkowicz-

Kowalska et al., 2006; Graczyk et al., 2007b).

In Europe, the regulation related to the quality of sanitary

water for human consumption is adapted from Directive 98/

83/EEC (Communities, 1998), which specifies the need to

detect fecal bacterial indicators and also establishes a water

turbidity limit to determine the presence of Cryptosporidium or

other microorganisms and parasites, when considered

appropriate by authorities. However, microsporidia are not

specifically monitored.

The quality of bathing water and the use of sewage sludge

in agriculture are governed by Directives 76/160/EEC

(Community, 1976) and 86/278/EEC, respectively (Community,

1986). However, parasites are not covered by these directives,

so microsporidia are not routinely monitored.

Finally, the use of regenerated water has recently been

regulated in our country, (R.D 1620/2007). However, although

Giardia, Cryptosporidium, and helminth eggs are included, there

is no mention of the search for microsporidia. Considering

that this type of water is planned for use in urban, agricultural,

industrial, recreational, and environmental practices, this

may represent a sanitary risk for users.

The present work studies, for the first time, the presence of

microsporidia in different types of water in Spain.

2. Materials and methods

2.1. Water sampling

Thirty-eight water samples from 8 drinking water treatment

plants (DWTPs), 8 wastewater treatment plants (WWTPs) and

6 water samples from recreational river areas (RRAs) from

Galicia (NWSpain) were analyzed (Fig. 1). Thewater treatment

carried out in all the DWTPs included coagulation, floccula-

tion, and clarification through sedimentation, filtration, and

disinfection by chlorination. Neither UV treatment nor ozon-

ation was carried out in any of DWTPs included in the study.

The main processes in the selected WWTPs consisted of

a primary treatment (screening, storage and preconditioning)

and a secondary treatment (coagulation and flocculation,

sedimentation and decantation). A tertiary treatment (UV or

ozone) was not carried out. All water sampling areas were

located in areas with high livestock (cattle and sheep) activity,

predominantly cattle farming.

One hundred liters of water from DWTPs and 50 L from

WWTPs and RRAs were collected. In all cases, water samples

were concentrated by the IDEXX Filta-Max� system for the

capture and recovery of Cryptosporidium sp and Giardia sp.,

following the 1623 method used by the United States Envi-

ronmental Protection Agency (USEPA) (U.S.E.P.A., 2005). Water

was collected using a portable water pump connected to

a foam filter module, following the manufacturer’s instruc-

tions and USEPA 1623 (U.S.E.P.A., 2005). Organisms were

recovered by elution in a final volume of 5 ml.

2.2. Staining methods

Thin smears from concentrated water samples were prepared

and stained, using Weber’s chromotrope-based stain (Weber

et al., 1992) to detect microsporidia. However, this method

cannot determine the species of microsporidia.

2.3. DNA extraction and purification

To determine the microsporidian species, DNA from unpre-

served concentrated water samples was extracted following

the methods described earlier (del Aguila et al., 1997a). DNA

was extracted by bead disruption of spores using the Fast-

DNA-Spin kit, according to the manufacturer’s instructions

(Bio 101, Carlsbad, Calif.). PCR inhibitors were removed using

the QIAquick PCR kit (QIAGEN, Chatsworth, CA).

Fig. 1 e Geographical location of the sampling points in relation to the 11 municipalities in Galicia (NW Spain), where the

following water samples were obtained: recreational river areas (RRAs; no. [ 6); influent and final effluent from drinking

water treatments plants (DWTPs; no. [ 8) and wastewater treatment plants (WWTPs; no. [ 8). * Locations where

microsporidia were detected.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 8 3 7e4 8 4 3 4839

2.4. PCR amplification

Microsporidial-small subunit rRNA (SSU-rRNA) coding regions

were amplified, using the following species-specific primers:

EBIEF1/EBIER1 for Enterocytozoon.bieneusi (Da Silva et al., 1996),

SINTF/SINTR for Encephalitozoon intestinalis (Da Silva et al.,

1997), EHELF/EHELR for Encephalitozoon hellem (Visvesvara

et al., 1994), and ECUNF/ECUNR for Encephalitozoon cuniculi

(De Groote et al., 1995). The PCR amplification was carried out

with the GenAmp kit (PerkineElmer Cetus, Norwalk, CT),

according to manufacturer’s procedures and the conditions

for the reaction described previously (Da Silva et al., 1997).

Purified samples were tested for the presence of PCR inhibi-

tors, as described previously (Da Silva et al., 1997). Amplifi-

cation products were analyzed by 2% agarose gel

electrophoresis and visualized by ethidium bromide staining

(Da Silva et al., 1997).

3. Results

Eight samples (21.0%) out of 38 water samples (2 from DWTPs,

5 fromWWTPs and 1 from an RRA) showed a variable number

of spores that stained pinkish red when theWeber’s stain was

used. The characteristic morphology ofmicrosporidian spores

with a clear vacuole-like polar end was observed; they were

ovoid and ranged from 0.9 to 1.6 mm (Fig. 2; Table 1). In only

one of the positive treatment plants, microsporidian spores

were detected solely in the final effluent. It was in the WWTP

of Municipality No.6 (Fig. 1, Table 2). On the other hand, in

Municipality No. 8, microsporidian spores were detected in

the influent water of both DWTPs andWWTPs studied (Fig. 1).

From all RRAs, only one case of microsporidial contamination

was detected by Trichrome stain (Municipality No. 7).

DNA amplification of positive samples in the staining

technique, with specific primers for the four most common

microsporidia infecting humans, allowed us to confirm the

presence of microsporidian species in the water sample from

an RRA (Municipality No. 7). Themicrosporidia were identified

as E. intestinalis, showing the diagnostic band of 528 bp in the

agarose gels. No positive samples for E. bieneusi, E. cuniculi, or E.

hellem were detected. No PCR inhibitors were detected (Fig. 2).

4. Discussion

Microsporidia, are emerging pathogens related to diarrhea in

both immunocompetent and immunosuppressed patients,

which in the last few years, have been recognized as water

contaminants, but their small size makes their detection

difficult. Using Weber’s stain, microsporidia spores were

detected in 8 water samples (21.0%): 2 samples from DWTPs, 5

Fig. 2 e A: Microsporidia spores stained with modified trichrome stain proceeding from RRA (Municipality No 7). B: PCR

amplification from RRA (Municipality No 7) using specific primers for E. intestinalis, M: 100 pb DNA ladder. Lane 1: DNA

extracted. Lane 2: DNA 1/10 dilution. Lane 3: DNA 1/100 dilution. Lane 4: positive control. Lane 5: negative control. RRA:

Recreational River Area.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 8 3 7e4 8 4 34840

from WWTPs and 1 from an RRA. The species E. intestinalis

could be identified by PCR methods only in the sample

proceeding from the RRA. It is necessary to point out that,

although the PCR technique is the most sensitive method for

species identification, the main problem involved is the

appearance of false-negative results, due to a low parasite

DNA concentration, and the presence of PCR inhibitors (Da

Silva et al., 1997). In the case of microsporidia, the presence

of extruded spores (non viable sporeswith noDNA) in samples

may be one additional reason for a low parasitic DNA

concentration, possibly influenced by treatments of DWTPs

andWWTPs. Finally, it is important to consider that the water

samples that tested positive with the staining methods may

not necessarily amplify with the specific primers used, due to

the presence of microsporidia other than the species studied.

To date, no agreement in the methods used to concentrate

microsporidia from water samples has been reached (Sparfel

et al., 1997; Fournier et al., 2000; Thurston-Enriquez et al.,

2002; Li et al., 2003; Hoffman et al., 2007; Kwakye-Nuako et al.,

2007). To our knowledge, in only one previous study, IDEXX

Filta-Max� was used for the concentration step, although the

system was considered unsuitable for detecting micro-

sporidia, based on the scarce recovery percentage (Stine et al.,

2005). However, the level of detection obtained in our study

(20.1% of samples) would be sufficient to include it among

techniques useful in detecting microsporidia. Studies in

Table 1 e Results obtained by Trichrome stain and PCRfrom drinking water treatment plants (DWTPs),wastewater treatment plants (WWTPs) and recreationalriver areas (RRAs) in the municipalities included in thestudy. No: number of samples analyzed.

Type of Water (No.) Positive Samples (%)

Trichrome stain PCR

DWTP (16) 2 (12.5%) e

WWTP (16) 5 (31.2%) e

RRA (6) 1 (16.6%) E. intestinalis

Total (38) 8 (21.0%) E. intestinalis

different types of water have shown the presence of micro-

sporidia such as E. bieneusi, E. intestinalis, E. hellem, Vittaforma

corneae, and Pleistophora, affecting humans (Avery and

Undeen, 1987; Dowd et al., 1998, 2003; Fournier et al., 2000;

Thurston-Enriquez et al., 2002; Graczyk et al., 2007a, 2007b;

Lucy et al., 2008). To the best of our knowledge, our results are

the first report of human-pathogenic microsporidia in water

samples from Spain. It is important to bear inmind that in our

countrymicrosporidia have been related to human diarrhea in

HIV positive (1.2e13.9%) and negative patients (5.1e17.02%)

(Subirats et al., 1996 del Aguila et al., 1997b; Gainzarain et al.,

1998; Lopez-Velez et al., 1999; Lores et al., 1999, 2002a, 2002b;

Abreu-Acosta et al., 2005); and 5.4% of blood-donors showed

seropositivity for Encephalitozoon sp. (del Aguila et al., 2001).

Additionally, human-related microsporidia have been identi-

fied in a high percentage (20.9%) in pigeons from urban parks

(Haro et al., 2005), reinforcing the convenience of studies to

discern the implication of waterborne transmission in the

epidemiology of these parasites.

A low contamination by microsporidia in DWTPs (only two

cases) was detected, compared with that shown in WWTPs (5

cases). The contamination detected in the DWTPs was only

found in the influent water but not in the final effluent.

Although the number of DWTPs positive for microsporidia

was low, the absence of this parasite in the final effluent in all

cases would suggest that the treatment used effected its

removal. To date, there are no similar studies on DWTPs,

although E. intestinalis have been demonstrated in drinking

waters (Dowd et al., 2003).

In one of the positive samples from WWTPs, micro-

sporidian spores were detected in the final effluent. Previous

studies have shown the presence of human microsporidia in

a tertiary effluent (Dowd et al., 1998) or in sewage sludge end

products orwetland outfalls (Graczyk et al., 2007a), whichmay

be explained because they are potentially resistant to disin-

fection (Dowd et al., 1998) or because these parasites would be

propagated by dogs, livestock and visiting wildlife (Graczyk

et al., 2009a).

Only one of the microsporidia-positive samples from RRAs

could be confirmed as E. intestinalis by PCR analysis. Previous

PCR studies have shown the presence of human-pathogenic

Table 2 e Results obtained by Trichrome stain and PCR ofmicrosporidia in the influent and effluent samples from drinkingwater treatment plants (DWTPs) and wastewater treatment plants (WWTPs) in the municipalities included in the study.No.: number of samples analyzed.

Plant (No.) Influent Effluent

Positive samples (%) Trichrome stain PCR Positive samples (%) Trichrome stain PCR

DWTP (8) 2 (25.0%) 2 0 0 0 0

WWTP (8) 4 (50.0%) 4 0 1 (12.5%) 1 0

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 4 8 3 7e4 8 4 3 4841

microsporidia, such as E. bieneusi and E. intestinalis, in surface

and recreational waters (Sparfel et al., 1997; Dowd et al., 1998,

2003; Fournier et al., 2000; Coupe et al., 2006; Graczyk et al.,

2007c; Lucy et al., 2008). The pathways of microsporidia

infections, modes, or routes of transmission, and the knowl-

edge of the epidemiology are still uncertain, although recent

studies point to a zoonotic origin (Didier et al., 2004; Haro et al.,

2005). The fact that wildlife that inhabits or visits rivers or

wetland systems can significantly contribute to human-

pathogenic microsporidia was previously suggested (Graczyk

et al., 2007c, 2009b). The presence of E. intestinalis in an RRA

reinforces this idea, since wild animals, including waterfowl,

have unlimited access to surface waters of the area under

study (Slodkowicz-Kowalska et al., 2006; Graczyk et al., 2009b).

It is notable that Municipalities No. 6, 7, and 8 have a very high

livestock density, mainly of bovine origin, with a density of

cattle population double that of the human population in

those areas. Although the livestock have no access close to

river water manure is frequently washed away from these

areas along well-defined drainage paths during rainfall

events, and the cows typically have free access to nearby

streams. In this scenario, both livestock manure and grazing

cattlemay contribute to contamination of the rivers. Although

there are no data on microsporidial infection of bovines in

Spain percentages between 13% and 15% have been described

in United States and Korea (Santin et al., 2004; Lee, 2007).

In our study, we could not establish the viability of

microsporidian spores detected. However, in previous studies,

the viability of microsporidia spores after water treatments

has been demonstrated. In a study on water proceeding from

sewage sludge end products, Graczyk et al. (2007a) observed

that most spores identified were potentially viable using

fluorescent in situ hybridization method (FISH). This method

indicated that the viability of microsporidia should be

considered even though the parasitic load is low, taking into

account their long-term environmental survival, and their

serious implications in human health (Weber et al., 1994).

Therefore, studies to discern the possible source-tracking of

contamination and the viability of these parasites after

treatments are necessary, as the water obtained in WWTPs

could be discharged into a river or be used for urban, agri-

cultural, industrial, recreational, or environmental practices

and might contribute to the contamination of the environ-

ment with the consequent risk to human health. In addition,

we must bear in mind that the ID50 for microsporidia in

humans is still unknown. However, previous reports indicated

that in animals the minimal infectious dose is very low

(Graczyk et al., 2010).

Our results are the first report on human-related micro-

sporidia in different kind of water from Spain, andwarn of the

possibility that exposure to recreational waters could play

a role in the epidemiology of human microsporidiosis.

Nevertheless, more complete epidemiology studies are

needed to understand the origin and the contribution of

microsporidia water contamination to human diarrhea.

5. Conclusions

This study shows the presence of human-related micro-

sporidia in water samples, highlighting the potential role of

water in microsporidiosis epidemiology. The difficulties

observed for themicrosporidia species determination in these

kinds of samples have made us aware of the need for the

development and standardization of good laboratorymethods

for an easier and more accurate detection of microsporidia in

water samples. This is a necessary first step that would

contribute to the development of a monitoring programme to

carry out source-tracking, risk assessment and linked epide-

miology studies to better understand these pathogens.

Acknowledgments

We are grateful to L. Hamalainen for help in the preparation of

themanuscript. This work was supported by the Ministerio de

Ciencia e Innovacion, within the Programa Nacional de

Recursos y Tecnologıas Agroalimentarias (RTA2010-00003-00-

00) and by grants from the Fundacion San Pablo-CEU 03/08.

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