Data about the presence of Cryptosporidium species and Giardia genotypes in water samples from Brazil are scarce. We investigate the occurrence of these pathogenic protozoa in raw water samples of Atibaia and Capivari Rivers which are the main water supplies of Campinas city (Southeast Brazil). These rivers show high degree of eutrophication. All samples were subjected to membrane filtration technique using 45°C heated elution solution followed by immunofluorescence and nested PCR. Recovery efficiencies were estimated using water aliquots spiked with Color Seed®. Average recovery efficiencies of spiked samples in Atibaia River were 18.0% ± 18.4 and 74.0% ± 22.1 for Cryptosporidium and Giardia, respectively. For Capivari River, recovery efficiencies were 29.7% ± 24.6 and 65.1% ± 33.0 for oocysts and cysts. Cryptosporidium oocysts were found in 42.8% samples from Atibaia River and in 85.7% samples from Capivari River, whereas Giardia cysts were present in 100% of samples in both rivers using immunofluorescence. Molecular analysis revealed the presence of C. hominis and C. parvum as well as G. duodenalis subgroup BIII in Atibaia River. All samples from Capivari River were PCR negative, probably due to inhibitors. These findings suggest anthroponotic and zoonotic contamination of Atibaia River. The occurrence of these pathogenic protozoa in both rivers highlight the potential risk for human and animal health. Close monitoring of water quality of these rivers is highly recommended.

Bueno Franco RM, Branco N, Trainotti Amaro BC, Neto RC, da Silva Fiuza VR (2016) Cryptosporidium Species and Giardia Genotypes Detected in Surface Water Supply of Campinas, Southeast Brazil, by Molecular Methods.. J Vet Med Res 3(3): 1053.

•    Pathogenic protozoa
•    Water supply
•    Nested PCR
•    Recovery

MF: Membrane Filtration Technique; IFA: Immunofluorescence Assay; DAPI: 4’, 6-diamidino-2- Phenylindole Fluorescent Stain; DIC: Differential Interference Contrast Microscopy; SD: Standard Deviation; BSA: Bovine Serum Albumin; DMSO: Dimethylsulfoxide; LaCTAD: High Performance Technology Center Laboratory in Life Sciences/Campinas State University, Brazil; UT: Turbidity Unit

Cryptosporidiosis is one of the most common food borne and waterborne diseases reported worldwide [1,2]. Self - limiting diarrhea is usually observed in immune competent people, whereas in young children, elderly and immune compromised people death can occur [3]. Cryptosporidium incidence is higher among children from developing countries [4]; in developed countries, adults are infected by ingestion of water or food contaminated with oocysts [5].

Thirty species and more than 40 genotypes are described in Cryptosporidium genus [6]. Recently, three new species were reported: Cryptosporidium rubeyi [7], C. proliferans [8] and C. avium [9]. However, not all species comprise risks to human health [6]. C. hominis and C. parvum are responsible for over 90% cases of infections in humans [10]. However, C. parvum has a wider variety of hosts, including animals and is considered the most important zoonotic agent of cryptosporidiosis [11].

The greatest waterborne cryptosporidiosis outbreak occurred in Milwaukee, Wisconsin, EUA, in 1993, where over 400,000 people were reportedly infected [12]. Filter effluents reached a turbidity of 2.7 UT in few days signalizing a failure of treatment process. The largest waterborne outbreak ever reported in Europe occurred in Sweden [13] affecting 27,000 individuals.

Giardia duodenalis is a parasite of human and other mammals. Six species are described in the Giardia genera according to morphological characteristics and hosts infected [14]. It is currently accepted that G. duodenalis is a complex of eight distinct genetic groups (designated A-H). These groups are morphologically identical but differ in genomic mutations [15,16]. Humans are mainly infected by genetic groups A and B, which are divided into five sub - groups (named AI-III and BIIIBIV) [17].

About 280 million people worldwide are estimated to acquire Giardiasis each year and an incidence of 1.2 million cases are annually reported in the United States [18]. Waterborne Giardiasis outbreaks in humans are generally caused by contamination of water sources intended for human consumption [19]. In addition to gastrointestinal disorders, this flagellate protozoan can cause extra intestinal symptoms [20].

While there are other Cryptosporidium and Giardia transmission routes to humans, water is considered as the main route due to the low infectious dose, chlorine tolerance and zoonotic potential. These factors reinforces the need of knowing which species and genotypes occur in water sources in order to evaluate the risks for human and animal health; then, proper control measures can be outlined [21].

Data about the presence of Cryptosporidium species and Giardia genotypes in water samples from Brazil are scarce. In Paraná state, C. parvum oocysts were detected in raw water of Ribeirão Cafezal watershed [22]; C. hominis and C. meleagridis oocysts were found in superficial water from São Paulo state [23].

Campinas is the third most populous city in the state of São Paulo. This city is supplied by Atibaia and Capivari Rivers which belong to the basin of Piracicaba, Capivari and Jundiaí Rivers. The region where this basin is located is one of the most industrialized of São Paulo state [24]. Consequently, there is a high eutrophication degree in these rivers.

This study aimed to investigate the occurrence of Cryptosporidium species and Giardia genotypes in raw water samples from Atibaia and Capivari Rivers at Campinas city through immunofluorescence and molecular methods (nestedPCR), after membrane filtration (MF).

All water samples 20 cm in depth were collected at the intake pipe of the Atibaia and Capivari Water Treatment Plants of Campinas city in new polystyrene bottles previously rinsed with Tween 80 0,1% solution. Samples were immediately transported to the laboratory in iceboxes at 4°C. Raw water samples were processed by the membrane filtration technique according Franco et al., (2001) [25] with the following modifications: organisms were eluted from filter through rinsing and scrapings of membrane using Tween 80 (0.1%) at 45°C and concentrated by 1500 x g/15 min. Cysts and oocysts were purified by immune magnetic separation using CGCombo Dynal® kit in accordance to USEPA Method 1623.1 [26] with dissociation by heat and use of 50µL of beads for initial incubation step.

Immunofluorescence assay (IFA) was performed following Cellabs® kit (Cellabs Pty. Ltd., Australia) instructions with minor modifications. Briefly, DAPI stain was applied on slide wells prior to monoclonal antibodies. Samples were microscopically examined at 200x and 400x magnification, using epifluorescence and DIC microscopy. Only organisms compliant with USEPA Method 1623.1 were considered, with internal morphological characteristics assessed by DIC microscopy.

In order to evaluate the water turbidity influence on MF technique using heated elution solution, replicates of water samples were also collected. In these samples Color Seed® (Biotechnology Frontiers, Australia) were seeded at a level of 100 ± 1 SD per water sample (1L). Further analyses were performed according to the methods previously described. Estimate number of organisms/L and recovery efficiency were calculated according Franco et al., 2012 [27]. Results were compared to acceptance criteria of USEPA Method 1623.1.

DNA extractions were performed using the DNeasy Blood & Tissue® kit (Qiagen, Hilden, Germany) or PowerSoil® kit (MoBio) for Cryptosporidium, and ZR Fungal/Bacterial DNA MiniPrep® kit for Giardia according to the availability of these kits in our laboratory. All reactions followed the manufacturer’s instructions, using 300µL of the pellet obtained for each eluted water sample.

An 830 bp fragment of the 18S rRNA gene of Cryptosporidium was amplified by nested PCR using primers described by Xiao et al., 1999 [28] (Table 1). The mixture of first PCR reaction contained 1X PCR buffer, 3 mM MgCl2 , 0.2 mM of each dNTP, 2.5µL of BSA (bovine serum albumin - 0.1 g/10 mL), 2.5 U Taq, and 1 mM of each primer in a final reaction volume of 50µL [29]. The first PCR samples were subjected to initial denaturation at 94°C for 3min, 35 cycles of 94°C for 45s, 59°C for 45s and 78°C for 1 min. The final extension was at 72°C for 7 minutes. The second amplification reaction mix was identical to the first except for the MgCl2 concentration (1.5 mM). Forty cycles of 94°C for 30s, 58°C for 90s and 72°C for 2 minutes, after initial denaturation at 94°C for 3 minutes and final extension at 72°C for 7 minutes [29].

For Giardia DNA amplification, genetic markers SSU-rRNA and beta-giardin were addressed using the protocols described by Appelbee et al., (2003) [30] and Hopkins et al., (1997) [31], respectively. Primer sets of first and second reactions are described in Table (1). For primary amplification of SSU-rRNA genetic marker, a mix was prepared containing 1X PCR buffer; 1.5 mM MgCl2 ; 0.2 mM of each dNTP; 2 U Taq; 2.5 µL of dimethyl sulfoxide (DMSO) and 0.5 µM of each primer in a final volume of 50 µl. First PCR conditions were: initial denaturation of 96°C (2 min.), 35 cycles of 96°C for 45s, 58°C for 30s, 72°C for 45s, and final extension at 72°C for 4 min. For the second amplification the PCR mixture and cycling conditions were identical to the first [29].

Amplification of a beta - giardin gene fragment was performed using 12.5 µL PCR Master Mix (Promega), 0.6 mM of each primer, 2 µL of BSA (0.1/10 mL) and 4.5 µL of nuclease - free water. Three micro liters of sample DNA were used in each reaction. The following amplification conditions were performed: initial denaturation step for 5 minutes at 94°C, followed by 40 cycles, each consisting of: 30s at 94°C, 30s at 65°C and 60s at 72°C, and final extension of 7 minutes at 72°C. In the nested reaction, an internal fragment of 511 bp was amplified [32,33]. The mixture was prepared with 13.5 µL PCR Master Mix (Promega), 0.6 mM of each primer, and 4.5 µL of nuclease - free water and 4 µL of sample DNA. Initial denaturation was performed at 98°C for 15 minutes followed by 40 cycles of 30 seconds at 95°C, 55°C for 30s, and 60s at 72 °C. The final extension was done at 72°C for 7 min.

For all samples, IFA-DAPI-DIC visualizations were performed first, followed by DNA extractions for the PCR assays. PCRs for each sample were repeated in triplicate in order to reduce false positive or negative results. All gels were stained with GelRed® (Biotium, Hayward, USA) for visualization.

All PCR positive samples were purified using ExoSAP-IT® and sequenced in LaCTAD Center of Campinas State University with same primers used in the second PCR reaction. The sequences of each strand were aligned and examined with Lasergene software (DNASTAR), and submitted to the Basic Local Alignment Search Tool (BLAST) analysis to identify similarities with the GenBank sequences.

Seven water samples from each river were collected between September 2014 and June 2015. Water turbidity from Atibaia River was higher (average: 46.1 UT, SD: 34.3, minimum value: 17.1, maximum value: 110) than Capivari River (average: 34.4 UT, SD: 19.7, minimum value: 16.8, maximum value: 74.0) during this research. Overall recovery of Cryptosporidium oocysts and Giardia cysts from Atibaia River averaged 18.0% ± 18.4 (SD) and 74.0% ± 22.1 (SD) respectively (Table 2). From Capivari River, overall oocysts and cysts recovery averaged 29.7% ± 24.6 (SD) and 65.1% ± 33.0 (SD), respectively (Table 2). Cryptosporidium recovery efficiency was lower than Giardia in both rivers. MF technique was less affected by turbidity considering oocyst recovery in Capivari river. A decline in the ability of this technique to detect Cryptosporidium was observed with the increase of turbidity of water samples from Atibaia River.

Cryptosporidium oocysts were present in 42.8% samples from Atibaia River and in 85.7% samples from Capivari River. Cryptosporidium oocysts were found in fewer numbers than Giardia cysts in Atibaia River (Table 2). Cryptosporidium averaged 2.4 ± 3.5 (SD) oocysts/L and was not detected in four months of this study in Atibaia River. In Capivari River, Cryptosporidium averaged 5.2 ± 5.6 (SD) oocysts/L, and was not detected in one month (Table 2).

PCR amplification of genomic DNA extracted from water samples of Atibaia River produced a band of the expected size of 830 bp for Cryptosporidium in three samples (3A, 4A, 5A) (Figure 1). These samples were sequenced but one positive sample could not be identified due to insufficient sequence quality (sample 3A). Sample 4A was identified as C. parvum and matched the HCTX8 isolate (97.9% of homology) described by Xiao et al., 1999 [28]. Sample 5A was identified as C. hominis and had 88.0% homology with C. hominis isolate by Araújo et al., 2013, in São Paulo city [23]. No DNA amplification was achieved from Capivari River samples.

Giardia cysts were present in 100% of samples in both water sources. A higher number of cysts was found in Atibaia River (average: 65.1 ± 28.7 (SD) cysts / L) than in Capivari River (average: 27.7 ± 36.1cysts / L) (Table 2).

Only one sample (A3) was PCR positive for Giardia in beta giardin analysis (Figure 2). This sample had 99.8% homology with G. duodenalis sub - group BIII and matched the HC07 isolate described by Durigan et al., 2014 [16] with a different base at position 494 (a cytosine instead of thymine). All samples from Capivari River were PCR negative for Cryptosporidium and Giardia.

Table 1: Primers sets used for PCR reactions targeting genetic markers 18S - rRNA (Cryptosporidium), SSU-rRNA and beta - giardin (Giardia) to analyze water samples from Atibaia and Capivari Rivers, Campinas city, Southeast Brazil.

Table 2: Number of Cryptosporidium oocysts/L and Giardia cysts/L detected in raw water samples of Atibaia and Capivari Rivers (Campinas, Southeast Brazil) and recovery efficiencies of membrane filtration technique (followed by direct immunofluorescence assay using monoclonal antibodies) of seeded samples using ColorSeed®.

Concerns about water contamination by pathogenic protozoa has considerably increased during the past two decades in Brazil given the occurrence of two Cyclospora cayetanensis and one large toxoplasmosis waterborne outbreaks which affected 950 and 496 people, respectively [34,35].

Sanitation is still far from being available to everyone in Brazil whereas only 48.6% of the Brazilian population is served by sewage collection, and just 39% of sewage is treated (www. tratabrasil.org.br) [36]. Microbial load is a major concern regarding contamination of water supplies. Sato et al., (2013) [37] found high risks (> 1/10.000) for both Cryptosporidium and Giardia parasites in four metropolitan areas in São Paulo state, emphasizing the need for concerted efforts to maintain higher water quality in catchment points [38].

In order to determine the public health threat associated with two main water sources in Campinas, we applied molecular methods to the parasitological monitoring of Cryptosporidium and Giardia. Parallel processing of natural and spiked samples allowedus to evaluate the effect of water turbidity on performance of chosen method to analyze the occurrence of these protozoan parasites. This approach was important because water turbidity impacts negatively both membrane filtration method as well as PCR.

High turbidity of water matrix is the major factor for poor oocysts recovery from samples concentrated after filtration [39]. The recovery efficiency achieved for Cryptosporidium in water samples from Atibaia and Capivari Rivers did not meet the acceptance criteria established in USEPA Method 1623.1 (32%). According to Ongerth (2013) [40], the recovery of protozoa (especially Cryptosporidium ) is inversely proportional to the sample turbidity. The recovery of Giardia is less affected by the matrix effect due to the larger size of the cysts and lower hydrophobic behavior at neutral pH [41] as required by IMS purification. However, samples turbidity has also a negative effect on IMS [42]. The impact of water quality on recovery is still relatively poorly understood [43]. Several groups of constituents may be implicated with the matrix effect such as iron, manganese, and other metals as pointed by Rosen et al., (2014) [43], which found that cations and metals were strongly and negatively correlated with protozoan recoveries.

Furthermore, water sampling in this study coincides with a prolonged drought (“seca”) which affected São Paulo state during 2014/2015 years [44], impacting negatively the flow of these rivers and the dilution of contaminants discharged in these water bodies. In this scenario, it is not surprising that only three samples were IFA and PCR - positives for Cryptosporidium regarding samples collected in Atibaia River, and one for Giardia by PCR. From Capivari River, all samples IFA - positive by microscopy could not be amplified by nested PCR.

Pollution industrial loads, sand and clay extractions, and intense agricultural activity are registered in the banks of these rivers and its water may contain large amounts of silt, decaying organic material, humic acids and metals which are PCR inhibitors. Such inhibitors probably were not efficiently removed by the DNA extraction kits used in this study. Even the use of additives such as DMSO for Giardia and BSA for Cryptosporidium in our PCR reactions in order to reduce inhibitory effects and increase amplification efficiency, were not sufficient [45].

Both pathogenic protozoa were detected in Atibaia and Capivari Rivers. These findings corroborates with previous studies, where oocysts were found in minor concentrations than Giardia cysts [37,46,47].

Besides the three Cryptosporidium species reported in human (C. hominis, C. parvum, and C. meleagridis), the following species were described in animal fecal samples in Brazil: C. bovis, C. canis, C. felis, C. baileyi, C. galli, C. meleagridis, [48], C. scrofarum [49], C. andersoni [50-52], C. varanii, C. serpentis [53]; C. ryanae [54], C. tyzzeri, C. muris [55]; C. ubiquitum, C. xiaoi [56]. However, only C. parvum, C. hominis and C. meleagridis were described in water samples from São Paulo city [23]. C. parvum was recently found in Paraná state [22].

The present finding of C. hominis and C. parvum in Atibaia River is very important given the multiple uses of waters from this river. In an event of treatment failure, Cryptosporidium is a primary threat to public health [40]. As observed in this research, more than one Cryptosporidium species are usually reported. This river is also used for crop irrigation which representing a considerable impact, as the Metropolitan Region Campinas has 17 municipalities with 808 small agricultural farms [57]. Regarding Giardia, the presence of sub - group BIII in Atibaia River suggests the risk of Zoonotic transmission [16]. It should be noted that genetic group B shows a higher prevalence (50%) than genetic group A (37%) [58] worldwide. By studying the risks of Giardias is infections in most populous regions of São Paulo state, Razzolini et al., (2016) [38] found that the annual risk for Giardia exceeded the risks for Cryptosporidium by at least 1 Log.

Source tracking of fecal contamination through molecular methods is of primary relevance for detection of associated health risks.

The occurrence of C. hominis, C. parvum, and the genetic group BIII of G. duodenalis in Atibaia raw water samples highlights possible events of anthroponotic and zoonotic contaminations in this watercourse.

Financial support to this research was provided by the State of São Paulo Research Foundation/FAPESP (process number 2012/50522-0). B.C. T. Amaro received a grant of graduate student (FAPESP process number 2014/17997-0). V. R. S. Fiuza received a grant of Post - doctoral researcher (FAPESP process number 2014/21602-1).

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