Abstract
Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.
INTRODUCTION
Cyclospora cayetanensis and several species of microsporidia are recognized as emerging human pathogens (1, 2). They have transmission stages that can be highly resistant to external environmental conditions and to many physical and chemical disinfection methods routinely used as bactericides in drinking water plants, swimming pools, and irrigation systems. These exogenous transmission stages are microscopic in size and of low specific gravity, facilitating their easy dissemination in freshwater or seawater. Also animals, both vertebrates and invertebrates, have been reported as reservoirs for microsporidia, which contribute to the dissemination of the parasites by environmental contamination with stool and/or urine from these reservoirs (1).
C. cayetanensis and microsporidia have been found in drinking water, wastewater, and recreational water (3–5), and they have been associated with waterborne outbreaks worldwide (6, 7). Because of their waterborne transmission potential, both Cyclospora and microsporidia (Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae) have been included in the last two drinking water contaminant candidate lists (contaminant candidate list 2 [CCL2] and CCL3) of the U.S. Environmental Protection Agency (EPA) (8, 9).
In Europe, regulation related to the quality of sanitary water for human consumption has been adapted from Directive 98/83/EEC, which specifies the need to detect fecal bacteria indicators and also establishes a water turbidity limit to determine the presence of Cryptosporidium and/or other microorganisms and parasites when this is considered appropriate by the authorities. However, microsporidia and Cyclospora are not specifically monitored. Likewise, current environmental regulation of bathing water quality (Directive 2006/7/EC of the European Parliament and of the Council, concerning the management of bathing water quality; and Real Decreto 1341/2007 of the Spanish Government, concerning the management of bathing water quality) and of the use of sewage sludge (Directive 86/278/EEC of the Council Directive on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture) and regenerated water (Real Decreto 1620/2007 of the Spanish Government, concerning the management of regenerated water) does not involve a search for these parasites.
So far, in Spain there has been only one study on the presence of microsporidia in water samples (10), and in relation to C. cayetanensis there are no up-to-date published data on environmental samples. Similarly, to date, data regarding the prevalence of these parasites in the Spanish population are scarce. For this reason and to contribute to the understanding of the epidemiology and risk factors associated with them, we investigated their presence in waters obtained from drinking water and wastewater treatment plants (DWTPs and WWTPs, respectively) and locations of influence (LI) of such plants during a 1-year survey.
MATERIALS AND METHODS
Sample collection.
A year-long longitudinal study from spring 2008 to winter 2009 was designed to evaluate and characterize the presence of microsporidia and Cyclospora spp. in different kinds of water. Four river basins (RB) were selected for this study. All of them had livestock activity, mainly bovines for breeding or milk production. The 17 sampling points chosen are shown in Fig. 1. Water treatment in DWTPs includes preoxidation, precloration, coagulation, flocculation, decantation, and disinfection (ozone and chloramines). Wastewater procedures in all the WWTPs evaluated in our study were based on physicochemical and biological treatments with activated sludge.
Fig 1.
Geographical location of the studied sampling points, including four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) located on four river basins (RB): Guadalix/Jarama (1), Manzanares (2), Lozoya (3), and Guadarrama (4). The template map was republished from reference 11 with kind permission from Springer Science+Business Media B.V.
Sampling was done as described by Magnet et al. (11). Each season of the year, sampling was done in duplicate. A total of 223 water samples were obtained. For DWTPs, up to 100 liters of water was collected from each site (at the point of entry, raw water, and at the end of the process, finished water). For WWTPs (both raw and treated water) and LI, up to 50 liters of water was collected. In all cases water samples were concentrated using an IDEXX Filta Max system as per the manufacturer's instructions (12). A total of 5 ml was finally eluted from each concentrated sample. Three-hundred microliters was used for DNA extraction, and 50 μl was used for microscopy. Samples for molecular analysis were kept at −80°C.
Staining methods.
All water samples were stained with Weber's chromotrope stain to investigate microsporidia and with Kinyoun stain to search for structures morphologically compatible with Cyclospora spp. Microscopic analysis was done at a magnification of ×1,000. Viewing patterns for microsporidia included spores with a bright pinkish-red stain and either a clear vacuole-like polar end or a belt-like stripe in the middle of the spore. For Cyclospora, structures ranging from 8 to 10 μm with variable morphology—round, collapsed, or distorted on one side—and variable staining (colorless to deep purple) were evaluated.
DNA extraction and purification.
DNA was obtained from 300 μl of the 223 concentrated water samples by bead disruption of spores using a Fast-DNA-Spin soil kit following the protocol described by da Silva et al. (13). PCR inhibitors were removed using a QIAquick PCR kit (Qiagen, Chatsworth, CA) following the manufacturer's instructions. Extracted DNA was stored at 4°C until PCR amplification.
PCR amplification.
Amplification reactions were performed using a GeneAmp kit (PerkinElmer, MA) in 25 μl of reaction mixture with 0.2 mM deoxynucleoside triphosphates, 0.2 μM each primer, and buffer with 1× MgCl2 and 1.25 U of Taq polymerase. A Gene Amp PCR system 9700 thermocycler (PerkinElmer, MA) was used. PCR products were analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide and examined under UV light. The sizes of the amplicons were compared with a standard 100-bp DNA ladder. For every PCR, both positive and negative controls were included.
Microsporidian phylum and species amplification.
PCR was performed by using different diagnostic primer pairs. Generic microsporidian primer pairs MicR1 and MicF1 were used to confirm the presence of microsporidia (4); microsporidian small-subunit rRNA coding regions (SSU-rRNA) were amplified using the amplification protocol described for each species and the following species-specific primers: EBIEF1/EBIER1 for Enterocytozoon bieneusi (14), SINTF/SINTR for Encephalitozoon intestinalis (15), ECUNF/ECUNR for Encephalitozoon cuniculi (16), EHELF/EHELR for Encephalitozoon hellem (17), NALGf2/NALGR1 for A. algerae (18), and NCORF1/NCORR1 for V. corneae (19).
Enterocytozoon bieneusi genotyping.
Enterocytozoon bieneusi genotyping was performed by sequence analysis of the internal transcribed spacer (ITS) region of rDNA. For this purpose, a fragment of 536 bp containing the 243 bp of the ITS was amplified using the two primers described by Galván et al. (20). PCR amplifications were performed using the following cycling conditions: 35 cycles of denaturing at 94°C for 30 s, alignment at 55°C for 30 s, and extension at 72°C for 90 s.
Encephalitozoon cuniculi genotyping.
E. cuniculi genotyping was performed by sequence analysis of a fragment of the polar tube protein (PTP) gene. A 363-bp fragment of the PTP was amplified from E. cuniculi DNA by PCR using the primers 5′-GCAGTTCCAGGCTACTAC-3′ and 5′-AGGAACTCCGGATGTTCC-3′ and following the protocol described by Xiao et al. (21).
Cyclospora.
A nested PCR described by Relman et al. (22) which amplifies the coding region of the rRNA small subunit (SSU-rRNA) was used to confirm the presence of Cyclospora spp. For primary PCR, primers CYCF1 and CYCR2 were used. For secondary PCR, a fragment of 308 bp was amplified from 10 μl of the primary PCR product with primers CYCF3 and CYCR4. Positive samples with this PCR were sequenced to determine Cyclospora species.
DNA sequence analysis.
PCR products were purified with a QIAquick PCR kit (Qiagen, Chatsworth, CA) and sequenced on both ends through the sequencing service of Macrogen Laboratories (South Korea). The resulting sequences were edited and aligned with the Bioedit Sequence Alignment Editor, version 7.0.5.3 (23).
Statistical analysis.
Statistical analysis was performed using PASW 18 (IBM SPSS) software for Windows. Differences in microsporidian and Cyclospora sp. prevalence rates between seasons were evaluated using nonparametric Cochran and McNemar tests, and P values of <0.05 were considered statistically significant.
RESULTS
Prevalence of microsporidia and Cyclospora spp. in water samples.
A total of 223 samples were collected over a 1-year period from 17 sampling points that included four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins (RB) (Fig. 1). Microsporidia and/or Cyclospora spp. were detected by PCR in 52% (117 out of 223) of the water samples, with 49% (109 out of 223) positive for microsporidia, 9% (20 out of 223) positive for Cyclospora spp., and 5.4% (12 out of 223) positive for both parasites.
In reference to the presence of microsporidia in water samples analyzed by PCR, the highest percentages of positive samples were found in WWTPs and LI, with an annual prevalence rates of 61 and 50%, respectively (Table 1). In WWTPs it was common to find the parasite in both raw and finished water, with 64.3% (36/56) and 57.1% (32/56), respectively, of positive samples (Fig. 2 and Table 2). Microsporidian prevalence in DWTPs was lower (27%) (Table 1), and this parasite was more prevalent in raw water (35.5%; 11/31) than in finished water (18.8%; 6/32) (Fig. 2 and Table 2). Distribution of results by season revealed that although microsporidia were present throughout the year of the study, they have a higher prevalence in spring (64%) and summer (55%), with WWTPs showing the highest number of positive samples (22 and 20, respectively) (Table 1).
Table 1.
Prevalence of microsporidia in water samples from different treatment plants and locations of influence during a year-long longitudinal study: comparison between results obtained by a modified trichrome stain and PCR
| Sampling point (n)a | Prevalence of microsporidia by time period and detection method (no. of positive samples [%])b |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Spring |
Summer |
Autumn |
Winterc |
Year |
||||||
| MTS | PCR | MTS | PCR | MTS | PCR | MTS | PCR | MTS | PCR | |
| DWTP (16) | 3 | 6 | 4 | 4 | 3 | 6 | 2 | 1 | 12 (19) | 17 (27) |
| WWTP (28) | 7 | 22 | 13 | 20 | 4 | 12 | 3 | 14 | 27 (24) | 68 (61) |
| LI (12) | 2 | 8 | 4 | 7 | 4 | 4 | 1 | 5 | 11 (23) | 24 (50) |
| Total (no. of positive samples/total no. of samples [%]) | 12/56 (21) | 36/56 (64) | 21/56 (37) | 31/56 (55) | 11/56 (20) | 22/56 (39) | 6/55 (11) | 20/55 (36) | 50/223 (22) | 109/223 (49) |
n, number of sampling points; DWTP, drinking water treatment plant; WWTP: wastewater treatment plant; LI: location of influence.
MTS, modified trichrome stain.
One sample from a DWTP in this season could not be obtained.
Fig 2.
Distribution of results obtained by PCR for microsporidia in raw and finished waters from DWTPs and WWTPs during a year-long longitudinal study. The percentage of positive samples is shown on the y axis. RW, raw water; FW, finished water.
Table 2.
Microsporidian presence detected by PCR in water samples according to season and fluvial basins
| Fluvial basin (region no.) | Sampling pointa | Microsporidian detection by sampling time and no. |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Spring |
Summer |
Autumn |
Winter |
||||||
| 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | ||
| Guadalix/Jarama (1) | DWTP 1-R | − | + | − | − | − | + | − | − |
| DWTP 1-F | − | − | − | − | − | + | − | − | |
| WWTP 1.1-R | + | + | − | + | − | + | + | − | |
| WWTP 1.1-F | + | + | + | + | − | − | − | + | |
| WWTP 1.2-R | + | + | + | − | + | + | − | + | |
| WWTP 1.2-F | + | + | + | − | − | + | − | + | |
| LI 1.1 | + | + | − | + | − | − | − | + | |
| LI 1.2 | + | + | − | + | + | − | − | - | |
| LI 1.3 | + | + | − | + | − | − | − | + | |
| Manzanares (2) | DWTP 2-R | − | − | − | + | − | − | − | + |
| DWTP 2-F | − | − | − | − | − | − | − | - | |
| WWTP 2-R | − | + | + | + | − | + | − | + | |
| WWTP 2-F | − | + | + | + | − | + | − | + | |
| LI 2 | − | + | + | − | + | − | − | + | |
| Lozoya (3) | DWTP 3-R | + | + | + | + | + | − | NDb | − |
| DWTP 3-F | + | + | − | − | + | − | − | − | |
| WWTP 3-R | + | + | + | + | + | − | − | + | |
| WWTP 3-F | + | − | + | + | − | + | − | + | |
| LI 3 | − | + | − | + | − | − | − | + | |
| Guadarrama (4) | DWTP 4-R | + | − | − | + | − | − | − | − |
| DWTP 4-F | − | − | − | − | + | + | − | − | |
| WWTP 4.1-R | − | + | + | + | − | − | − | + | |
| WWTP 4.1-F | − | + | − | − | − | − | − | + | |
| WWTP 4.2-R | − | + | + | − | + | − | − | + | |
| WWTP 4.2-F | + | + | + | − | + | + | − | + | |
| WWTP 4.3-R | + | + | + | + | + | − | − | + | |
| WWTP 4.3-F | + | + | + | − | − | − | − | + | |
| LI 4 | + | − | + | + | + | + | − | + | |
DWTP, drinking water treatment plant; WWTP, wastewater treatment plant; LI, location of influence; R, raw water; F, finished water.
ND, not done. One sample from a DWTP in this season could not be obtained.
Two of the four DWTPs (DWTP 3 and DWTP 4) were positive for microsporidia in spring, summer, and autumn (Table 2). Both raw and finished water were positive at DWTP 3 in spring and autumn, and in summer microsporidia were found only in raw water. DWTP 4 was positive for microsporidia only in raw water in spring and summer, but in autumn the parasite was detected only in finished water. The other plants (DWTP 1 and DWTP 2) had positive samples in two of the four seasons (Table 2). With regard to LI, spring and summer were the seasons with the highest number of positive samples (8 and 7, respectively), followed by winter (5) and autumn (4) (Tables 1 and 2). For all the treatment plants and LI evaluated, there was no association between the presence of microsporidia and the river basins studied (Table 2). Weber's chromotrope stain showed a lower sensitivity (22%) than PCR (49%) in the detection of this parasite (Table 1).
Cyclospora spp. had an annual prevalence of 9% by PCR (Table 3). The distribution of results by season showed that spring had the highest prevalence of positive samples with 23% (13/56), followed by autumn with 7% (4/56), summer with 4% (2/56), and winter with 2% (1/55) (Table 3). WWTPs showed the highest percentage of positive samples, with an annual prevalence of 13% (Table 3), with the parasite being more frequent in raw water (16.1%; 9/56) than finished water (10.7%; 6/56) (Fig. 3). Only in spring was Cyclospora prevalence higher in WWTPs in finished water, with 35.7% (5/14), than raw water, with 21.4% (3/14) (Fig. 3). Both DWTPs and LI had positive samples only in the spring, with annual prevalences of 6% and 2%, respectively (Table 3). In DWTPs the parasite was detected in both raw water (37.5%; 3/8) and finished water (12.5%; 1/8) (Fig. 3 and Table 3). PCR showed a higher sensitivity (9%) in the detection of this coccidian than Kinyoun stain (4%) (Table 3).
Table 3.
Prevalence of Cyclospora spp. in water samples from different treatment plants and locations of influence during a year-long longitudinal study: comparison between results obtained by Kinyoun stain and PCR
| Sampling point (n)a | Prevalence of microsporidia by time period and detection method (no. of positive samples [%])b |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Spring |
Summer |
Autumn |
Winterc |
Year |
||||||
| KS | PCR | KS | PCR | KS | PCR | KS | PCR | KS | PCR | |
| DWTP (16) | 0 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 (0) | 4 (6) |
| WWTP (28) | 5 | 8 | 0 | 2 | 2 | 4 | 2 | 1 | 9 (8) | 15 (13) |
| LI (12) | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 (2) | 1 (2) |
| Total (no. of positive samples/total no. of samples [%]) | 6/56 (11) | 13/56 (23) | 0/56 (0) | 2/56 (4) | 2/56 (4) | 4/56 (7) | 2/55 (4) | 1/55 (2) | 10/223 (4) | 20/223 (9) |
DWTP, drinking water treatment plant; WWTP, wastewater treatment plant; LI, location of influence; n, number of sampling points.
KS, Kinyoun stain.
One sample from a DWTP in this season could not be obtained.
Fig 3.
Distribution of results obtained by PCR for Cyclospora spp. in raw and finished waters from DWTPs and WWTPs during a year-long longitudinal study. The percentage of positive samples is shown on the y axis. RW, raw water; FW, finished water.
C. cayetanensis was confirmed by sequence analysis of the PCR product in 17 of 20 samples that were positive (Table 4). Unfortunately, in three samples the quantity of DNA amplified was not suitable for sequencing. There was an association between the presence of the parasite and the Guadarrama River basin in spring since all samples that were positive belonged to this watershed (Table 4). All sampling points belonging to the Guadalix/Jarama basin were negative for the parasite (Table 4).
Table 4.
Presence of Cyclospora spp. detected by PCR in water samples according to season and fluvial basins
| Fluvial basin (region no.) | Sampling pointa |
Cyclospora detection by sampling time and no. |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Spring |
Summer |
Autumn |
Winter |
||||||
| 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | ||
| Guadalix/Jarama (1) | DWTP 1-R | − | − | − | − | − | − | − | − |
| DWTP 1-F | − | − | − | − | − | − | − | − | |
| WWTP 1.1-R | − | − | − | − | − | − | − | − | |
| WWTP 1.1-F | − | − | − | − | − | − | − | − | |
| WWTP 1.2-R | − | − | − | − | − | − | − | − | |
| WWTP 1.2-F | − | − | − | − | − | − | − | − | |
| LI 1.1 | − | − | − | − | − | − | − | − | |
| LI 1.2 | − | − | − | − | − | − | − | − | |
| LI 1.3 | − | − | − | − | − | − | − | − | |
| Manzanares (2) | DWTP 2-R | − | + | − | − | − | − | − | − |
| DWTP 2-F | − | − | − | − | − | − | − | − | |
| WWTP 2-R | − | − | − | − | +c | − | − | − | |
| WWTP 2-F | + | − | − | − | − | − | − | − | |
| LI 2 | − | − | − | − | − | − | − | − | |
| Lozoya (3) | DWTP 3-R | − | − | − | − | − | − | NDb | − |
| DWTP 3-F | − | − | − | − | − | − | − | − | |
| WWTP 3-R | − | − | − | +c | +c | − | − | − | |
| WWTP 3-F | − | − | − | + | − | − | − | − | |
| LI 3 | − | − | − | − | − | − | − | − | |
| Guadarrama (4) | DWTP 4-R | + | + | − | − | − | − | − | − |
| DWTP 4-F | − | + | − | − | − | − | − | − | |
| WWTP 4.1-R | − | − | − | − | − | − | − | − | |
| WWTP 4.1-F | − | − | − | − | − | − | − | − | |
| WWTP 4.2-R | + | − | − | − | + | + | − | + | |
| WWTP 4.2-F | + | + | − | − | − | − | − | − | |
| WWTP 4.3-R | + | + | − | − | − | − | − | − | |
| WWTP 4.3-F | + | + | − | − | − | − | − | − | |
| LI 4 | + | − | − | − | − | − | − | − | |
DWTP, drinking water treatment plant; WWTP, wastewater treatment plant; LI, location of influence; R, raw water; F, finished water.
ND, not done. One sample from a DWTP in this season could not be obtained.
Species not identified.
Significant differences in microsporidian and Cyclospora sp. prevalence rates between the seasons evaluated could not be found.
Species identification and molecular characterization of microsporidia.
Microsporidian species identification was performed by amplification of the small subunit rRNA (SSU-rDNA) coding regions (Table 5). Encephalitozoon intestinalis was the most commonly detected species, with an annual prevalence of 26.6% in positive samples, followed by Enterocytozoon bieneusi (16.5%), Encephalitozoon cuniculi (11.9%), and A. algerae (1.8%) (Table 5). Enterocytozoon bieneusi, the more frequent microsporidia in humans, was detected in three of the four seasons and was most common in spring (27.7%), followed by winter (25%) and summer (9.7%). Encephalitozoon intestinalis was common in spring (58.3%) and winter (40%), and there were positive samples for E. cuniculi only in winter (65%). A. algerae was found in one sample from spring and another from autumn. Undetermined species represented 59.6% (65 out of 109) of samples. A sequence analysis of the phylum PCR product of 32 of these samples was done; however, we identified microsporidian species in only 3 samples. Sequences with similarities of 93% and 91% to Orthosomella operophterae (GenBank accession number AJ302316) were found in DWTP 1 and WWTP 1, respectively. In LI 1.2 we found a sequence with 88% identity to Pleistophora sp. (GenBank accession number D85500).
Table 5.
Seasonal distribution of microsporidian species in water samples from the central area of Spain by PCR
| Microsporidian species | Prevalence by time period (no. of positive samples [%])a |
||||
|---|---|---|---|---|---|
| Spring (n = 36) | Summer (n = 31) | Autumn (n = 22) | Winter (n = 20) | Year (n = 109) | |
| Encephalitozoon intestinalis | 21 (58.3) | 8 (40) | 29 (26.6) | ||
| Enterocytozoon bieneusi | 10 (27.7) | 3 (9.7) | 5 (25) | 18 (16.5) | |
| Encephalitozoon cuniculi | 13 (65) | 13 (11.9) | |||
| A. algerae | 1 (2.8) | 1 (4.5) | 2 (1.8) | ||
| Undetermined | 15 (41.6) | 28 (90.3) | 21 (95.5) | 1 (5) | 65 (59.6) |
n, total number of microsporidian-positive samples.
Enterocytozoon bieneusi genotyping was performed by sequence analysis of the ITS region of rDNA. A total of 18 samples were studied, but the genotype was determined in only 13 of the samples (Table 6). Genotype D-like (GenBank accession no. DQ836345) was the most frequent, being present in 8 of the 13 samples genotyped and in three of the four seasons evaluated. We also identified genotype C (GenBank accession number AF101199) (4/13) and genotype D (GenBank accession number AF023145) (1/13) (Table 6).
Table 6.
Identification of Enterocytozoon bieneusi genotypes in water samples from the central area of Spain
| Season | Sampling pointa | Haplotype | Group no. | Synonym | Hosts | Geographic distribution | References |
|---|---|---|---|---|---|---|---|
| Spring | WWTP 1.2-R | 1 | 1 | D | Humans, pets, and wild animals | Africa, Asia, America, Europe | 24, 25, 26, 27 |
| WWTP 1.2-F | 10 | 1 | C | Humans | Europe | 28, 29, 26, 30, 31, 27 | |
| LI 1.3 | 70 | 1 | D-like | Cats | South America | 26, 27, 32 | |
| WWTP 2-F | 10 | 1 | C | Humans | Europe | 28, 29, 26, 30, 31, 27 | |
| WWTP 4.2-F | 10 | 1 | C | Humans | Europe | 28, 29, 26, 30, 31, 27 | |
| WWTP 4.3-R | 10 | 1 | C | Humans | Europe | 28, 29, 26, 30, 31, 27 | |
| WWTP 4.3-F | 70 | 1 | D-like | Cats | South America | 26, 27, 32 | |
| LI 4 | 70 | 1 | D-like | Cats | South America | 26, 27, 32 | |
| Summer | WWTP 1.2-F | 70 | 1 | D-like | Cats | South America | 26, 27, 32 |
| DWTP 3-R | 70 | 1 | D-like | Cats | South America | 26, 27, 32 | |
| WWTP 4.3-R | 70 | 1 | D-like | Cats | South America | 26, 27, 32 | |
| Winter | WWTP 1.2-R | 70 | 1 | D-like | Cats | South America | 26, 27, 32 |
| WWTP 1.2-F | 70 | 1 | D-like | Cats | South America | 26, 27, 32 |
DWTP, drinking water treatment plant; WWTP, wastewater treatment plant; LI, location of influence; R, raw water; F, finished water.
In reference to Encephalitozoon cuniculi genotyping, 13 samples were studied by sequence analysis of a 363-bp fragment of the PTP gene. Only three samples were successfully genotyped. Genotype I (rabbit strain) (GenBank accession number AF310677) was detected in one WWTP (WWTP 4.1-F) and in a river basin location (LI 2), and genotype III (dog strain) (GenBank accession number AF310679) was identified in one WWTP (WWTP 4.2-R).
DISCUSSION
Emerging waterborne pathogens include bacteria such as Campylobacter jejuni, Mycobacterium spp., Legionella pneumophila, and Pseudomonas aeruginosa, several viruses such as calicivirus and hepatitis E virus, and parasites such as Cryptosporidium spp., C. cayetanensis, and microsporidia (33). Although these organisms have been described as human infection agents for many years, it is only recently that they have been identified as waterborne pathogens. Development of detection methods, including molecular methods and immunological and immunomagnetic separation techniques, has improved the efficient search for these parasites, highlighting their presence in environmental samples (33).
In our study, apart from PCR, we used modified trichrome and Kinyoun stains to detect microsporidia and Cyclospora spp., respectively, in water samples, and although both stains had lower sensitivity than PCR, it is important to emphasize their usefulness as an additional diagnostic tool for confirmation of these organisms. With the modified trichrome stain, we detected microsporidian spores in several PCR-negative samples (data not shown). These results could be explained either by PCR inhibitors in environmental samples, which can be concentrated by the previous processes of filtration of water samples, or by the presence of spores belonging to microsporidia that were not detected with the phylum PCR used in our study, which could be possible given the fact that there are more than 150 genera described (1).
Microsporidian prevalence ranges between 5 to 50% in patients with AIDS and concomitant chronic diarrhea (1); these percentages vary depending on the geographic area studied and the diagnostic method used. In Spain, microsporidia have been described in patients with HIV/AIDS (34–36), in HIV-negative patients, including travelers (37), elderly people (38), and organ transplant recipients (20), and in the immunocompetent population (39), with Enterocytozoon bieneusi being the most frequent species identified. All these data confirm the cosmopolitan distribution of these parasites, which are considered ubiquitous organisms widely distributed in nature, and therefore they should have several transmission modes and infection sources for humans (1). Some epidemiological studies have identified water as a risk factor in the acquisition of microsporidiosis. Hutin et al. (40) determined that the use of public pools was a risk factor associated with microsporidian infection in AIDS patients, linking the water as a possible source of infection. In a retrospective study of an outbreak of intestinal microsporidiosis in Lyon, France, water was involved as the possible transmission vehicle (6), and use of hot tubs or spas and occupational contact with water were considered risk factors for acquiring intestinal microsporidiosis in a cohort of HIV-positive people (41). Additionally, several species of microsporidia that infect humans have been documented in water samples. Encephalitozoon intestinalis, Enterocytozoon bieneusi, A. algerae, Nosema spp., Pleistophora spp., and V. corneae spores have been identified in ground, surface, and recreational waters (42).
This study presents the first data of microsporidia in surface water in the central area of Spain during a year. There is only one report done by Izquierdo et al. (10) on the presence of microsporidia in several DWTPs, WWTPs, and recreational river areas (RRAs) from Galicia (Spain). Using staining protocols, they found 21% samples of positive. Parasites were present in the influent water but not in the final effluent of DWTPs, and in one WWTP microsporidia were found in the final effluent. Encephalitozoon intestinalis was identified by PCR in one sample from RRAs.
In our study, microsporidian presence was analyzed in each of the four seasons of the year, and both raw and finished waters from DWTPs and WWTPs were studied in order to evaluate the elimination methods of these organisms in these plants. Microsporidian data showed a tendency to rise in spring and summer, with a higher prevalence of these parasites in both raw and finished waters from WWTPs. It is important to highlight that human-pathogenic species were found with high prevalence in these seasons and that they were present in both raw and finished waters from WWTPs and also in influent locations. Other studies have also confirmed the presence of microsporidia in influent and effluent products of WWTPs, confirming their potential role as contaminants (3, 43, 44). With regard to seasonal variation, our results are similar to those obtained by Cheng et al. (43), who evaluated the presence of human-virulent microsporidia in wastewater treatment plants and found a seasonal increase of these parasites in April and July, both in inflowing wastewaters and in wastewater processing end products. Other authors who have studied microsporidian presence over a year-long period in water samples did not find a seasonal variation (45, 46). However, this could be possible because of the limited number of samples analyzed and also the kind of water evaluated, which included only surface water from a river (45) and water from swimming pools (46) and recreational lakes (45).
Encephalitozoon intestinalis and Enterocytozoon bieneusi were the most frequent microsporidian species found in our study. The high prevalence can be explained by the fact that these are the two most prevalent species, both in humans and in a wide variety of hosts, including wild and domestic mammals (47) and birds (47–49). These animals could act as reservoirs, facilitating environmental contamination, a suggestion which has been supported in several studies (49, 50). Similar to findings in human samples, Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most frequent microsporidian species described in water samples (3, 51). In our study in addition to these two species, we also detected A. algerae and Encephalitozoon cuniculi spores in the water samples analyzed; we note that so far there are no data available on the presence of E. cuniculi in environmental samples. E. cuniculi showed a clear seasonal tendency as it was found in WWTPs and three LI only during winter. In the case of A. algerae, there is only one study on its presence in ditch water in Florida (1). Water contamination with both species is relevant because although they are most frequently associated with infections in other mammals, in the case of Encephalitozoon cuniculi (1), and in insects, in the case of A. algerae (52), they have also been reported as humans pathogens. E. cuniculi has been associated with respiratory, ocular, urinary tract, liver, central nervous system, and disseminated infections, among others (1), while A. algerae can produce ocular and muscle infections (1, 52).
The high percentage of samples containing undetermined species is not surprising since it is common to find in the environment, specifically water, the presence of other microsporidian genera that are not associated with disease in humans but which can be associated with fish and numerous invertebrates, including insects (1). Using the sequence analysis of positive phylum PCR products, we were able to establish homologies of less than 95% only with microsporidia which infect insects. O. operophterae and Pleistophora sp. were identified, with only the latter being associated with human infections although less frequently than other microsporidia (53). Species of this genus have also been found in ditch water, irrigation water used for crops, and surface water (53).
Molecular characterization of Enterocytozoon bieneusi did not exhibit a high diversity since only three genotypes were found in the 13 samples analyzed (D-like, D, and C genotypes). These genotypes are included in group 1, according to the classification based on the phylogenetic analysis of the ITS region of the rRNA gene (26, 27, 54). This group is the most diverse of the five that have been documented and includes several genotypes isolated from humans (both HIV positive and negative), as well as from domestic and wild animals (27, 54). The D-like genotype was the most frequent in the environmental samples analyzed. Until now this genotype has been found only in South America (32), and there are no data about its presence in Europe. It is important to point out that although the D-like genotype has until now been described only in cats, we should not underestimate the importance of this genotype as an agent of human infection, taking into account that there is only one study on the molecular characterization of E. bieneusi in two transplant patients in Spain (20).
Genotype D has a wide geographical distribution (26, 27, 54). It has been found in many animals, including pigs, cattle, foxes, monkeys, and dogs (54); it is common in HIV-positive patients (54), but it has also been found in seronegative patients (54). In Spain, this genotype has been found in two non-HIV-infected renal transplant recipients with intestinal symptomatology (20). Genotype C has, until now, been described in humans only in Europe (29), particularly in the transplant recipient population, but in Spain there are no data about its presence in either clinical or environmental samples.
Regarding Encephalitozoon cuniculi, genotypes I (rabbit strain) and III (dog strain) were found in three samples from WWTPs and LI; they have been well described both in humans and animals in Europe (35). As with Enterocytozoon bieneusi, there is little information about the strain variability of E. cuniculi in Spain. In fact, to our knowledge, there has only been one study carried out by del Aguila et al. (35), who identified E. cuniculi type strain III (dog strain) in the urine and sputum samples from a Spanish AIDS patient based on the sequence analysis of the ITS region of the rRNA gene. Taken together, the Enterocytozoon bieneusi and Encephalitozoon cuniculi genotype results suggest that both humans and animals could act as contamination sources for environmental samples, supporting the zoonotic potential of both species. Moreover, our data could contribute to the knowledge of the molecular epidemiology of these microsporidia, particularly in tracing the sources of infection and therefore helping to formulate preventive and control strategies.
C. cayetanensis has been found in patients with diarrhea worldwide (2). In Europe, cases of C. cayetanensis infection have been reported in Spain, Italy, Greece, Germany, United Kingdom, Switzerland, Sweden, France, and The Netherlands, among others, most of them associated with travel to areas of endemicity (2), although there have been a few reports of individuals without a travel history (2). Outbreaks of cyclosporiasis have also been described in Germany, Turkey, Sweden, and Spain (2), and these were usually associated either with consumption of food from developing countries or travel to areas of endemicity.
Regarding water transmission of Cyclospora, several studies have been conducted in countries of South America and Africa, and they have identified water as an infection source for this parasite (4, 5, 7, 55). Oocysts of the parasite have also been reported in drinking water in Guatemala, Haiti, Ghana, Vietnam, and Egypt, among others (2).
In our study, distribution of positive samples for C. cayetanensis showed that the highest percentage belonged to WWTPs. These findings are consistent with the results obtained from studies in Nepal and Peru (55, 56), where Cyclospora spp. were detected in both drinking water and wastewater. Several authors have found that water from wastewater plants used either for irrigation or for processing vegetables was contaminated with Cyclospora oocysts (57, 58). Different studies have confirmed the seasonality of Cyclospora spp. in both clinical and environmental samples. Results vary according to the region studied (5, 55, 57, 59, 60). In clinical samples, seasonal variation has been documented in the humid and rainy seasons from November to May in Indonesia, from May to September in Nepal, and from May to August in Guatemala (55, 61, 62). However, although rainfall could be influencing the presence of the parasite, studies in Peru have detected a marked seasonality in a coastal area near Lima, which is a desert region (56). Unfortunately in Spain there are no longitudinal studies that establish seasonality for Cyclospora spp. in clinical samples. As previously mentioned, data come from sporadic cases or outbreaks (63).
In relation to environmental samples, only a few studies have detected Cyclospora and evaluated its seasonal behavior. As far as we know, this is the first description of the presence of C. cayetanensis over the course of a year in several drinking water treatment plants (both in raw and finished waters). Although there are some studies on the presence of this coccidian throughout a year (64, 65), they have included only treated potable water from tanks (65) and finished piped water (64). Cyclospora seasonality varied according to the region studied, with an increase in its presence in the pre-rainy season in Hanoi (57) and the rainy season in Cambodia (58). In our work, we show a seasonal tendency of Cyclospora during spring (April to June), with 23% of samples positive. However, a limitation in the interpretation of these results is the lack of data about cyclosporiasis cases during the period in which the research was done. Therefore, we could not correlate the infection pattern with C. cayetanensis and the seasonal variation described in this paper. Additionally, it may be important to determine the Cyclospora species in environmental samples and to investigate these protozoa in animals other than humans, which could act as possible reservoirs. Cyclospora-like oocysts resembling C. cayetanensis oocysts have been detected in fecal samples from several animals, including dogs, chickens, ducks, and primates (66–68). In Spain, Cordon et al. (66) studied fecal samples from birds of Almuñecar (Granada) and found 4.5% of samples positive for Cyclospora sp.; their results showed that this coccidian was present in not only humans but also animals, suggesting a possible role of the latter in the transmission of the parasite.
It is important that both microsporidia and Cyclospora were less frequent in finished water than in raw water in DWTPs. In WWTPs, both influent and effluent treated water showed similar frequencies of microsporidia and Cyclospora sp., which may suggest that both parasites can survive the treatments used in these plants. This could be explained by the thick outer walls of the microsporidian spore and Cyclospora oocyst that give them a high resistance to disinfection (1, 2), and in the case of microsporidia, their small size allows them to escape filtration systems commonly used in the treatment of drinking water and wastewater (69). However, it should be noted that the viability of these parasites was not determined in this study.
Conclusions.
To our knowledge this is the first report of a year-long study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence in Spain are presented. Results clearly suggest seasonality for Cyclospora during spring and for microsporidia in spring and summer. We describe for the first time the presence of A. algerae and Encephalitozoon cuniculi in water samples, with a marked seasonality for the latter, which was present only in winter. Enterocytozoon bieneusi and Encephalitozoon cuniculi genotyping results may suggest that both humans and animals are possible water contamination sources, confirming the zoonotic potential of these parasites. It is important to highlight that finished waters from DWTPs showed a lower prevalence of both parasites. Both WWTPs and LI had higher frequencies of microsporidia and Cyclospora spp. Molecular characterization of Enterocytozoon bieneusi showed that the D-like genotype was the most common. It is important that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites, which are not included in these regulations, were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters could be proposed to avoid health risks from these pathogens.
ACKNOWLEDGMENTS
We are indebted to Sergio Llorens for his valuable technical assistance and to Brian Crilly for helpful revision of the manuscript. We also thank Santiago Diaz-Parreño for his statistical advice.
This work was funded by grant PI061593 from the Instituto de Salud Carlos III (FISS). A.L.G. was supported in Spain by an overseas fellowship from Colciencias (Universidad de Antioquia, Colombia). A.M. was supported by the Ministerio de Educación y Ciencia, Spain (FPU grant AP2009-0415).
Footnotes
Published ahead of print 2 November 2012
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