Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.


Introduction
Waterborne diseases caused by protozoans have a worldwide distribution in developed and developing countries and cause epidemics and endemic diseases in hum ans (COTRUVO et al., 2004).Cryptosporidiosis and giardiasis are the most prevalent parasitic diseases that cause waterborne diarrhea in humans (LANE & LLOYD, 2002).Cryptosporidium spp.and Giardia spp.produce infectious forms (oocysts or cysts, respectively) that are transmitted by the feces of the infected host and are transmitted through direct contact with contaminated food or water (CACCIÒ et al., 2005;JEX et al., 2011).Molecular methods have identified different species and subtypes of these parasites (XIAO et al., 2001), many of which are infectious to humans (CHALMERS, 2011;FAYER, 2011).
In Brazil, the Ministry of Health Law 2914/11 outlines the procedures for the control and surveillance of water quality for human consumption and potability standards.This law determines that an annual geometric mean greater than or equal to 1,000 Escherichia coli/100 mL in raw water is necessary to monitor Giardia spp.cysts and Cryptosporidium spp. in different locations of the water catchment, with the goal of decreasing their occurrence (BRASIL, 2011).
There is a highest prevalence of waterborne protozoan infections in developing countries, due to their low hygiene standards, but there is no data that report the true occurrence of Cryptosporidium and Giardia in water supply sources.This lack of data leads to an underestimation of the real rate of waterbone parasite protozoan in developing countries (LIMA & STAMFORD, 2003;BALDURSSON & KARANIS, 2011).
Therefore, the purpose of this study was to investigate the occurrence of Cryptosporidium spp.and Giardia spp. in raw and treated water in a public water treatment system to perform a molecular characterization of isolates and to relate the results with physico-chemical and climate parameters.
There are two public water treatment systems (WTS) in Londrina; however, only one was chosen for this study.The chosen WTS performs the entire water treatment cycle (catchment, coagulation, flocculation, decantation, flotation, disinfection with chlorine, fluoridation, storage and distribution) and serves 32% of the city's population (SANEPAR, 2010).

Samples
Samples of 100 liters of treated water and 30 liters of raw water were collected every two weeks between September 2012 and September 2013.The samples were stored in plastic containers and transported to the State University of Londrina.

Water sample concentrations
The samples were analyzed using a membrane-filtration technique with cellulose acetate membranes that had a 47 mm diameter and 1.2 µm porosity (Millipore, Brazil).A vacuum pump system was used, and a filtration rate of 4 liters per minute was defined.The protocols followed those proposed by Dawson et al. (1993), Aldom & Chagla (1995) and Cantusio & Franco (2004).

Direct immunofluorescence test
We used 10 µL aliquots of each sample to perform the direct immunofluorescence test using the Merifluor Cryptosporidium/Giardia commercial kit (Meridian Bioscience, USA) according to the manufacturer's protocol, using positive and negative controls.Simultaneously, a confirmatory test was performed with inclusion of the fluorogenic vital stain DAPI (4´,6´-diamidine-2-phenylindole; Sigma Chemicals Co., USA) to reveal the morphological characters (CANTUSIO & FRANCO, 2004).
To read the immunofluorescence, we used a Nikon Eclipse epifluorescent microscope with a 450-490 nm excitation filter and a 520 nm emission filter.To read the DAPI, we used a 365-400 nm excitation filter and a 395 nm emission filter (REDLINGER et al., 2002).To identify oocysts and cysts, we used an immunofluorescence defined by a bright apple-green color, the absence of pores or appendices that had a compatible size and format: 8-12 µm in size and an oval shape for Giardia spp.and 3-8.5 µm in diameter, a spherical shape and the presence (not necessary) of sutures in the oocyst for Cryptosporidium spp.(REDLINGER et al., 2002).
After enumerating the cysts and oocysts present in the samples, a formula proposed by Cantusio & Franco (2004) was used to estimate the number of cysts and oocysts per liter.

Nested-PCR for Cryptosporidium spp. and Giardia spp.
All samples were subjected to DNA extraction using the NucleoSpin Tissue commercial kit (Macherey-Nagel, Germany), according to the manufacturer's protocol.
The PCR products were subjected to electrophoresis on agarose gel (Invitrogen, Brazil) 1.5% stained with SYBR  Safe (Invitrogen, Brazil) for 45' for Cryptosporidium spp.and 30' for Giardia spp..The products were visualized using ultraviolet light and photodocumented using the program LPix Image ST (Loccus Biotechnology, USA).
The positive bands were extracted from the gel and purified using the commercial kit QIAquick Gel Extraction.Sequencing was performed using an ABI3500 sequencer Genetic Analyzer (Applied Biosystems, USA), with the support of the BigDye Terminator v3.1 Cycle Sequencing commercial kit (Applied Biosystems, USA).Both forward and reverse strands were sequenced.The nucleotide sequences obtained were compared with the standard Cryptosporidium and Giardia sequences deposited in Genbank using the BLAST System (Basic Local Alignment and Search Tool) and by manual alignment using the BioEdit program (Biological Sequence Alignment Editor).The sequences obtained were deposited in Genebank under number KP334118 and KP334117 for Cryptosporidium and for Giardia under number KP334119 and KP334120.

Physico-chemical parameters
The turbidity was calculated using a HACH turbidimeter 2100Q, the pH was measured using a pH meter DM-Digimed 2P and the color was calculated using a HACH spectrophotometer DR/890 Colorimeter.

Climate parameters
Climate data (precipitation, relative humidity and temperature) were taken from the Instituto Agronômico do Paraná site (IAPAR, 2013).

Statistical analysis
To verify the correlation between the presence of Cryptosporidium spp.and Giardia spp. with water physico-chemical parameters and seasonality, the Pearson Correlation Coefficient (r) was calculated using the BioEstat 5.0 statistical package.The intensity correlation was defined as follows: weak (0 < r < 0.40), moderate (0.40 ≤ r <0.7) and strong (0.70 ≤ r ≤ 1.0) (FIGUEIREDO & SILVA, 2009).

Results
A total of 48 water samples, 24 raw and 24 treated, were analysed.Considering the results of the direct immunofluorescence, Cryptosporidium spp. was not detected in the raw or treated water samples.However, Giardia spp. was detected in 8.33% (2/24) of the raw water samples and was absent in the treated water samples.The Giardia spp.concentrations were 0.42 cysts per liter and 4.2 cysts per liter.

Discussion
This study identified the presence of C. parvum and G. duodenalis in the water supply source studied.We obtained a similar result to those of other studies that have demonstrated the occurrence of protozoans in water supply sources in different regions around the world (NISHI et al., 2009b;CANTUSIO et al., 2010;JÚLIO et al., 2012;MAHMOUDI et al., 2013).
Genetic sequencing showed the presence of C. parvum in positive raw water samples.C. parvum is considered zoonotic because it has a lower host specificity, and it is thus of considerable interest to public health (FAYER, 2004).In China, Xiao et al. (2013) found oocysts of this parasite in 86.4% of water catchment areas and Lee et al. (2014) reported its prevalence in 23.1% of Malaysian rivers.In Brazil, Araújo et al. (2011) described the occurrence of Different studies involving the molecular analysis and sequencing of positive samples for Cryptosporidium spp.have been conducted with the goal of better understanding the environmental epidemiology of this parasite.Many studies describe the occurrence of C. meleagridis, C. hominis, C. parvum and C. andersoni in water samples (ARAÚJO et al., 2011;LEE et al., 2013;RUECKER et al., 2013).
It is also possible to quantify the degree of water contamination using direct immunofluorescence.It was observed concentrations of Giardia spp.cysts of 0.42 cysts per liter and 4.2 cysts per liter in two samples of raw water.The infectious dose for humans is relatively low: 10 cysts can begin an infection (STEINER et al., 1997).
Genetic sequencing showed the presence of G. duodenalis in positive raw water samples.There are six known species of Giardia, but only G. duodenalis has a wide host range and is the only species of interest to public health (THOMPSON & MONIS, 2004), highlighting the importance of the findings of this study.Giardia spp.have a significant public health impact because, in addition to the prevalence and disease treatment costs, it can cause large outbreaks and affect the growth and cognitive development of children (FENG & XIAO, 2011).
Descriptions of the occurrence of Giardia spp. in water samples have been reported.It has been found in 92.3% of river water, 55.5% of water reservoirs and 45.2% and 26.9% of raw water from small and conventional water treatment companies, respectively, and in 19.2% of small water treatment systems and 26.8% of tap water from municipalities that performed only water chlorination (CARMENA et al., 2007).
Sequencing positive water samples for Giardia spp.has also been conducted to determine the species involved: G. duodenalis has been described in cisterns containing drinking water and in an outbreak of diarrhea in humans due to the consumption of contaminated drinking water (AHMED et al., 2010;CHEUN et al., 2013).
In recent years, interest has grown in the zoonotic transmission of C. parvum and G. duodenalis, especially in relation to their transmission caused by cattle and other livestock (COKLIN et al., 2007).This finding explains the occurrence of both protozoans in the sources examined because many dairy cattle reside along the Ribeirão Cafezal, until it arrives at the WTS analyzed.
This study found a moderate correlation between the Cryptosporidium spp.presence and both color and turbidity in raw water and between Giardia spp.presence and turbidity in raw water.Correlations between the parameters analyzed and the presence of Cryptosporidium spp.and Giardia spp.were also described by Ahmad et al. (1997), Cantusio & Franco (2004), Lopes (2009) and Nishi et al. (2009a).Low turbidity values are fundamental to obtaining better performance in the disinfection process inside water treatment systems (CERQUEIRA, 2008).
High concentrations of these two protozoa in raw water have been correlated with high concentrations or frequent detections in treated water.Thus, monitoring raw water should be an important tool for controlling and monitoring treated water quality (HELLER et al., 2004).Furthermore, Cryptosporidium and Giardia are refractory to the conventional treatments used in water treatment plants, which makes them even more important to public health (KARANIS et al., 2007).Thus, it is critically important to determine any sources of contamination and maintain water quality (FAYER, 2004;THOMPSON & MONIS, 2004;AUSTIN et al., 2012).
The United States Environmental Protection Agency (USEPA) proposed Method 1623 to detect Cryptosporidium and Giardia in water.It is internationally recognized for its elevated efficiency.Method 1623 includes concentration phases, immunomagnetic separation and immunofluorescence microscopy (USEPA, 2005).However, at the present moment, there is not a globally preferred method for detecting Cryptosporidium and Giardia in water (FREGONESI et al., 2012).
In conclusion, this is the first report of the occurrence of C. parvum and G. duodenalis in the water supply of Londrina, Paraná State, Brazil.The circulation of Cryptosporidium spp.and Giardia spp. in the study area enhances the necessity for water treatment companies to comply with Ministry of Health Law No. 2914/11 to ensure a safe water supply for the population and avoid possible future waterborne outbreaks and negative impacts on public health.

Table 1 .
Osaki et al. (2013)on Coefficient (r)between the physico-chemical and climate parameters for Cryptosporidium spp.andGiardia spp. in raw water from a public water treatment system in Londrina, Paraná, Brazil, September 2012 and September 2013.parasite in 30% of the water samples collected in state of São Paulo, whereasOsaki et al. (2013)found Cryptosporidium spp. in 2/4 of the water catchment systems in Curitiba, Paraná State.
*significant correlation.this