**2. Materials and methods**

This is a descriptive observational study approved by the Human and Animal Research Ethics Committee at Hospital das Clínicas of Universidade Federal de Goiás.

#### **2.1 Spatial and temporal sample delimitation**

A total of 72 samples were collected on a monthly basis for one year (February 2006 to January 2007), from one point in the center of each of the following bodies of water: Meia Ponte river, João Leite river, Vaca Brava Park lake, Bosque dos Buritis lake.

#### **Meia Ponte river**

In this river two sites were selected for sampling: the first, 1 km after the emission of wastewater treated by the municipal wastewater treatment plant of Goiânia, located at 16°37'40.94"S latitude and 49°16'13.41"W longitude (MP1), and the second, located at 16°38'22.39"S latitude and 49°15'50.68"W longitude (MP2) (Figure 1).

Fig. 1. Photograph of Meia Ponte river at the time of sampling during the rainy season, showing the high volume of water and its coloring (Santos et al., 2008).

#### **João Leite river**

344 Environmental Monitoring

the Midwestern Region of Brazil, focusing on *Cryptosporidium* sp., *Cyclospora cayetanensis,* 

This is a descriptive observational study approved by the Human and Animal Research

A total of 72 samples were collected on a monthly basis for one year (February 2006 to January 2007), from one point in the center of each of the following bodies of water: Meia Ponte river, João Leite river, Vaca Brava Park lake, Bosque dos Buritis

In this river two sites were selected for sampling: the first, 1 km after the emission of wastewater treated by the municipal wastewater treatment plant of Goiânia, located at 16°37'40.94"S latitude and 49°16'13.41"W longitude (MP1), and the second, located at

Fig. 1. Photograph of Meia Ponte river at the time of sampling during the rainy season,

showing the high volume of water and its coloring (Santos et al., 2008).

Ethics Committee at Hospital das Clínicas of Universidade Federal de Goiás.

16°38'22.39"S latitude and 49°15'50.68"W longitude (MP2) (Figure 1).

*Isopora belli* and Microsporidia*.*

**2. Materials and methods** 

lake.

**Meia Ponte river** 

**2.1 Spatial and temporal sample delimitation** 

In this river two sites were selected for sampling: one located at 16°37'40.18"S latitude and 49°14'26.08"W longitude (JL1) (Figure 2), when this body of water reaches Goiânia, and the other located at 16°19'37.52"S latitude and 49°13'24.53"W longitude (JL2), before Goiânia. Figure 3 shows hydrographic map with the four sampling points in the rivers under study: João Leite (JL1 and JL2) and Meia Ponte (MP1 and MP2).

Fig. 2. João Leite river upstream of Goiania, after interbreeding Jurubatuba stream with the Posse stream, municipality of Goianapolis (Santos et al., 2008).

#### **Vaca Brava Park lake**

This park encompasses an area of approximately 72.7 thousand m2, distributed among green areas, walking and jogging tracks, sports courts, playground, and exercise facilities. The site selected for sampling is located at 16°42'31.18"S latitude and 49°16'15.67"W longitude (VB) (Figure 4).

#### **Bosque dos Buritis lake**

Bosque dos Buritis is an urban park encompassing an area of approximately 125 m2 with three artificial lakes supplied by Buriti stream. The site selected for sampling is located at 16°40'58.51"S latitude and 49°15'38.35"W longitude (BB) (Figure 5)

(Santos et al., 2008).

eutrophication) (Santos et al., 2008).

Opportunistic Protozoa in Rivers and Lakes: Relevance to Public Health in the Neotropics 347

Fig. 4. Photography of Vaca Brava lake, demonstrating the puopulsion system of water

Fig. 5. Bosque dos Buritis lake, where we observe the dark Green water (an indicator of

Fig. 3. Hydrographic map showing the four sampling points in the rivers under study: João Leite (JL1 and JL2) and Meia Ponte (MP1 and MP2).

Fig. 3. Hydrographic map showing the four sampling points in the rivers under study: João

Leite (JL1 and JL2) and Meia Ponte (MP1 and MP2).

Fig. 4. Photography of Vaca Brava lake, demonstrating the puopulsion system of water (Santos et al., 2008).

Fig. 5. Bosque dos Buritis lake, where we observe the dark Green water (an indicator of eutrophication) (Santos et al., 2008).

*Cryptosporidium* sp. and

*Cryptosporidium* sp.

parasitological methods

*C. parvum* 

*C. parvum C. hominis* 

7 min.

7 min.

Opportunistic Protozoa in Rivers and Lakes: Relevance to Public Health in the Neotropics 349

Table 1. Primers selected to be used in confirmation/specification of protozoa detected by

PCR using primers AWA995f/AWA1206R and 14 μL DNA amplified by primers XIAF/XIAR was performed in a final volume of 25 μL with the following reagents: 2.5 μL buffer 10X, 1.5 mM Mg, 200 μM dNTP (dATP, dCTP, dTTP, and dGTP), 0.5 μM of each primer, and 1.25 U Taq DNA polymerase. The reaction conditions were an initial denaturation step for 7 min followed by another denaturation step of 40 cycles of 94°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 3 min, and final extension at 72°C for

PCR using primers LAX469F/LAX869R Laxer, (1991) and 14 μL DNA amplified by primers XIAF/XIAR was performed in a final volume of 25 μL with the following reagents: 2.5 μL buffer 10X, 2.0 mM Mg, 200 μM dNTP (dATP, dCTP, dTTP, and dGTP), 0.5 μM of each primer, and 1.25 U Taq DNA polymerase. The reaction conditions were an initial denaturation step for 7 min followed by another denaturation step of 40 cycles of 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for

The PCR products were separated by electrophoresis on 8% acrylamide gels stained with silver nitrate and on 1.5% agarose gels stained with ethidium bromide. Samples presenting

One aliquot of each sample concentrate was tested employing the MERIFLUOR® direct immunofluorescence assay kit using homologous monoclonal antibodies for the detection of *Cryptosporidium* sp. and *Giardia* sp. Each sample was analyzed in duplicate; however, due to a shortage of reagents, this technique was applied to 50% (36/72) of the samples taken at

The results obtained in this study were digitalized in spreadsheets using the software Microsoft Office Excel 2007. Statistical analyses were performed using the chi-squared test and the logistic regression analysis. Statistical significance level was set at p < 0.05 using the

Among the 72 samples processed, 8.33% (6/72) were positive for the protozoa researched. Using the MERIFLUOR® direct immunofluorescence assay kit, we found six positive

random and the positive samples detected by parasitological methods.

Statistical Package for the Social Sciences (SPSS) version 10.0.

5'-TTCTAGAGCTAATACATCCG-3' 5'-CCCATTTCCTTGAA ACAGGA-3'

5'-TAGAGATTGGAGGTTGTTCCT-3' 5'-CTCCACCACTA AGAACGGCC-3'

5'-CCGAGTTTGATCCAAAAAGTTACGA-3' 5'-TAGCTCCTCATATGCCTTATTGAGTA-3'

**Microorganism Primer Sequence** 

XIAF XIAR

AWA995F AWA1206R

LAX469F LAX869R

211-bp and 451-bp bands were considered positive.

**2.5 Direct immunofluorescence assay kit** 

**2.6 Statistical analyses** 

**3. Results** 

#### **2.2 Sample concentration**

Each sample was taken in a clean 10-L polyethylene container from one point in the center of the bodies of water approximately 20 cm under the surface and sent within 2 h to the Laboratório de Genética Molecular e Citogenética (Genetics and Molecular Diagnostic Laboratory) of the Universidade Federal de Goiás, and concentrated according to Silva et al. (2010).

Briefly, water samples were pre-filtered in a vacuum filter with qualitative paper filter, a process also called clarification, aiming to remove excessive amounts of organic matter, such as algae, plants, and other organisms, and immediately submitted to microfiltration using a positively nylon membrane with 0.45µm porosity with 47 mm of diameter (Hybond TM-N+, Amersham Pharmacia). The material adsorbed to the membrane was eluted by 5 ml of TE buffer (10 mM Tris-HCl, pH 8.0; 1mM EDTA) and 0.02% Tween-20, aliquoted and stored at - 20°C.
