Detection of Cryptosporidium spp. oocysts and Giardia spp. cysts in surface water destined for public supply in the state of Goiás, Brazil

Detecção de oocistos de Cryptosporidium spp. e cistos de Giardia spp. em mananciais superficiais destinados ao abastecimento público do estado de Goiás, Brasil

ABSTRACT

The protozoa Cryptosporidium spp. and Giardia spp. are etiological agents responsible for the transmission of gastroenteritis, mainly due to the consumption of contaminated water. Their (oo)cysts are resistant to adverse environmental conditions, as well as to most conventional water treatment processes. Thus, the objective of this work was to evaluate the occurrence of (oo)cysts of these protozoans in surface water collected for human consumption in the state of Goiás, Brazil. Fifteen samples of raw water were collected to assess the occurrence of (oo)cysts of the protozoa using the Membrane Filtration method, in addition to turbidity and pH analyses. Recovery rates in tests with ultrapure water reached the USEPA (2012) criteria for Giardia (78.1% ± 0%) and for Cryptosporidium (60.6% ± 32.6%); however, recovery in raw water was lower due to turbidity. All samples (n = 15) were positive for Cryptosporidium spp. oocysts, with a maximum concentration of 250 oocysts/L, demonstrating that these protozoa are disseminated in the aquatic environment of the state of Goiás and pose a risk to public health. Due to the use of water sources for public consumption, it is recommended that public authorities and sanitation companies act to preserve and maintain water courses, carry out periodic monitoring of treatment plants that supply the Cerrado, Santana and São Manoel streams and improve existing treatment technologies. The results did not allow to infer whether animal load and grazing area promote an increase in contamination of the lotic aquatic systems.

Keywords:
Cryptosporidium; Giardia; hydrographic basin; protozoa; surface water; treatment water

RESUMO

Os protozoários Giardia spp. e Cryptosporidium spp. são agentes etiológicos responsáveis pela transmissão de gastroenterites, principalmente por causa do consumo de água contaminada. Seus (oo)cistos são resistentes às condições ambientais adversas, bem como à maioria dos processos convencionais de tratamento da água. Assim, o objetivo deste trabalho foi avaliar a ocorrência de (oo)cistos desses protozoários em águas superficiais captadas para consumo humano no estado de Goiás, Brasil. Quinze amostras de água bruta foram coletadas para avaliar a ocorrência de (oo)cistos dos protozoários pelo método de Filtração por Membrana, além da análise de turbidez e pH. As taxas de recuperação nos testes com água ultrapura atingiram os critérios da USEPA (2012) para Giardia (78.1 ± 0%) e para Cryptosporidium (60.6 ± 32.6%), entretanto, em água bruta, a recuperação foi inferior devido à turbidez. Todas as amostras (n = 15) foram positivas para oocistos de Cryptosporidium spp., com valor máximo de 250 oocistos/L, demonstrando que esses protozoários se disseminam no meio aquático do estado de Goiás e geram risco à saúde pública. Devido à utilização de mananciais para consumo público, recomenda-se que as autoridades públicas e as empresas de saneamento atuem na preservação e manutenção dos cursos d’água, realizem o monitoramento periódico das plantas de tratamento que realizam adução no Córrego Cerrado, Córrego Santana e Córrego São Manoel e aprimorem as tecnologias de tratamento existente. Os resultados não permitiram inferir se a carga de animais e a área de pastagem promove o aumento da contaminação dos sistemas aquáticos lóticos.

Palavras-chave:
Cryptosporidium; Giardia; bacia hidrográfica; protozoário; água superficial; tratamento de água

INTRODUCTION

Waterborne diseases are a relevant public health concern. Indeed, around 829,000 deaths from diarrhea worldwide each year (WHO, 2019) have been related to the use of water contaminated by microorganisms from untreated domestic sewage (CHENG et al., 2009) or surface runoff from livestock farms (IMRE et al., 2017).

Among the protozoa transmitted by water, the genera Cryptosporidium (4.0-6.0 μm in diameter) and Giardia (8-18 μm in length by 5-15 μm in width), stand out as being widely distributed in developed and developing countries (COTRUVO et al., 2004).

These protozoa have been responsible for numerous waterborne outbreaks worldwide (KARANIS; KOURENTI; SMITH, 2007), since the oral ingestion of at least 10 cysts or a single oocyst can cause infection in susceptible individuals (KARANIS, 2011).

The outbreaks caused by these etiological agents are associated with severe diarrhea, and have been well documented (Table 1).

Table 1 -
Number of waterborne outbreaks caused by protozoan parasitic agents worldwide.

Year	Giardia spp.	Cryptosporidium spp.	Key reference
previous century-2004	132 outbreaks of Giardia duodenalis	165 outbreaks of Cryptosporidium parvum	Karanis, Kourenti and Smith (2007)
2004-2010	70 outbreaks of Giardia lamblia	120 outbreaks of Cryptosporidium spp.	Baldursson and Karanis (2011)
2011-2016	142 outbreaks of Giardia spp.	239 outbreaks of Cryptosporidium spp.	Efstratiou, Ongerth and Karanis (2017b)

The occurrence of these outbreaks can be attributed to the high resistance of these organisms to the environment (KARANIS, 2011) and their high infectivity (THOMPSON, 2004).

Means of transmission begins with the ingestion of (oo)cysts via the fecal-oral route, through contact between people and between people and other animals; the ingestion of water contaminated by host feces (humans and other non-human animals), recreational activities, and contaminated surface water, in addition to eating contaminated food (RYAN; FAYER; XIAO, 2014; PLUTZER; KARANIS, 2016).

Several studies have reported the occurrence of these protozoa in surface water destined for public supply in Brazil (ALMEIDA et al., 2015; DIAS et al., 2008; FREITAS et al., 2015; SATO et al., 2013; TOLEDO et al., 2017), indicating a risk for infection by gastrointestinal diseases to consumers and highlighting the need for special attention from public authorities.

Temporal and seasonal variation in the occurrence of these protozoa in the environment and in water supply systems justifies the importance of their monitoring since the absence of fecal indicators, such as E. Coli, does not guarantee the absence of Cryptosporidium spp. and Giardia spp., and, thus, are not considered standard indicators (HORMAN et al., 2004).

Therefore, the objective of the present study was to analyze the occurrence of (oo)cysts of Cryptosporidium spp. and Giardia spp. in surface waters destined for public supply in the state of Goiás, Brazil.

METHODOLOGY

Investigated area

The study area encompasses 15 surface water sources for public water supply in the state of Goiás, as described in Table 2 and spatially distributed as shown in Figure 1. The state is located in the Center-West Region of Brazil and has a territorial extension of 340,106,492 km² bordering the states of Mato Grosso do Sul, Mato Grosso, Tocantins, Bahia, Minas Gerais and Distrito Federal.

Table 2 -
Coordinates of the sampling points.

Municipality	Superficial Water Source	Coordinates
Adelândia	São Manoel Stream	16°24.387'S	50°11.267'W
Anicuns	Boa Esperança Stream	16°27.722'S	49°58.185'W
Barro Alto	Barro Alto Stream	14°57.647'S	48°55.452'W
Ceres	Das Almas River	15°18.679'S	49°35.373'W
Crixás	Forquilha Stream	14°33.388'S	49°58.334'W
Goianésia	Anda Só Stream	15°19.073'S	49°6.469'W
Jaraguá	Pari River	15°44.630'S	49°21.565'W
Morro Agudo de Goiás	Olho D’água Stream	15°18.746'S	50°3.300'W
Nova Glória	Jatobá Stream	15°8.806'S	49°34.110'W
Quirinópolis	Das Pedras Stream	18°27.084'S	50°28.643'W
Rubiataba	Novo River	15°10.416'S	49°48.112'W
Sanclerlândia	Cerrado Stream	16°12.942'S	50°18.640'W
São Luiz dos Montes Belos	Santana Stream	16°32.253'S	50°21.247'W
Uruanã	Uru River	15°30.638'S	49°40.742'W
Vianópolis	Dirceu Stream	16°44.418'S	48°31.022'W

Figure 1 -
Location of sampling points of water supply sources in the state of Goiás.

Water sources were selected according to the occurrence of Escherichia Coli in raw water, with an annual geometric average greater than 1,000 most probable number (MPN)/100 mL, as established by Annex XX of Consolidation Ordinance No. 5 (BRASIL, 2017), in at least four years between 2009 and 2018. Monitoring raw water quality using only indicators (E. Coli) is not sufficient to assess the occurrence of specific pathogens (HORMAN et al., 2004), and so the occurrence of pathogens in water courses must be assessed.

Sampling campaign

Parasitological analysis

The collections were made at the entrance of the treatment plants in August 2019, which is considered the dry period (May to September) in regions with a humid tropical climate, according to the classification of Köppen (1948). A sample of twenty liters of raw water was collected from each water source and stored in a polyethylene container that was previously cleaned with Tween 80 (0.1%).

The Membrane Filtration (MF) method was chosen due to the ease of implementation and the possibility of preserving (oo)cysts for future infectivity assays (MACIEL; SABOGAL-PAZ, 2016). Furthermore, the method has greater recovery efficiency than the cartridge filtration method (HSU et al., 2001), and reduced cost of implantation and analysis than standard methods recommended by USEPA (2012).

This reduced cost is due to the fact that most water treatment stations (WTS) already have the necessary equipment to perform the technique, which makes its implementation feasible in developing countries, and due to its wide use in research carried out in different regions of the world to evaluate the occurrence of these protozoa in water supply sources (BAUTISTA et al., 2018; NISHI et al., 2009; ALMEIDA et al., 2015).

Prior to quantifying (oo)cysts of the protozoa in raw water, the analytical quality control of the method employed was tested in triplicate to verify its reliability and sensitivity regarding the recovery of (oo)cysts in ultrapure and in natura raw waters from the Santana stream, with turbidity of 8.20 Nephelometric Turbidity Unit (NTU). The samples were contaminated with 10 µL of protozoan inoculum from Merifluor® Kit (Meridian Bioscience Diagnostics, Cincinnati, USA), with the average concentration of protozoans being quantified in two 10 µL aliquots of the inoculum.

Recovery efficiency of (oo)cysts was determined according to Equation 1 and correlated with criteria established by USEPA (2012) [mean ± standard deviation (SD)]: 27 - 100 ± 39% for cysts and 38 - 100 ± 37% for oocysts in ultrapure water, and 8 - 100 ± 97% for cysts and 32 - 100 ± 46% for oocysts in raw water. Recovery rates above 100% were disregarded from the results, following Franco et al. (2012). The results were discussed in terms of descriptive statistics.

$R=\frac{N}{T}\times 100$ (1)

Where:

• R =   recovery efficiency (%);
• N =   number of recovered cysts and (oo)cysts;
• T =   number of inoculated cysts and (oo)cysts;

To evaluate the contamination of the method, 1.0 L of ultrapure water was analyzed in duplicate, with subsequent concentration of in natura raw water, whose results should indicate the absence of cysts/oocysts (CANTUSIO NETO, 2008).

Membrane Filtration was performed by the following steps:

• concentration of (oo)cysts of protozoa through filtration of 2.0 L of each sample through a mixed cellulose esters membrane (Millipore®), with a diameter of 47 mm and nominal porosity of 3.0 µm;

• elution to recover the (oo)cysts by scraping and washing the material retained on the membrane with elution solution Tween 80 (0.1%);

• centrifugation and concentration of the material resulting from the elution by double centrifugation process for 15 minutes with a rotational force of 1,500 × g, as recommended by USEPA (2012);

• slide preparation using the direct Immunofluorescence Assay (FA) technique with Merifluor® Kit, according to the manufacturer’s guidelines for the simultaneous detection of oocysts and cysts, with aliquots of 10 μL for each slide well and three aliquots for each sample;

• reading of slides using a Zeiss Axio Imager M2 microscope for epifluorescence with 400X magnification, 450-490 nm excitation filter, and 520 nm barrier filter for detection and enumeration of (oo)cysts.

Fluorescence defined by bright apple-green- color was adopted to identify (oo)cysts: absence of pores or appendages; 8-18 μm in length and 5-15 μm in width, and oval shape for cysts of Giardia spp.; size of 4-6 μm in diameter, spherical shape and presence (not mandatory) of suture for Cryptosporidium spp. (CANTUSIO NETO, 2008; USEPA, 2012). The results were expressed as (oo)cysts/L according to Equation 2 described by Cantusio Neto and Franco (2004).

$CP=\frac{Numberof\left(\mathrm{o}\mathrm{o}\right)\mathrm{c}\mathrm{y}\mathrm{s}\mathrm{t}\mathrm{s}\mathrm{}\mathrm{s}\mathrm{e}\mathrm{e}\mathrm{n}}{Volumeofanalyzedsediment\left(\mu L\right)}\times \frac{Volumeofobtainedsediment\left(\mu L\right)}{Volumeoffilteredsample\left(L\right)}$ (2)

Where:

• CP =   concentration of protozoa [(oo)cysts/L].

Limit of Detection (LD) consists of the minimum number of recoverable organisms in an analyzed sample volume (ONGERTH, 2013), which varies according to the volume of the processed sample, which depends on the physical-chemical characteristics of the water, the final volume of the pellet, and the volume of aliquots. Calculation of LD follows Equation 3 (CANTUSIO NETO, 2008).

$LD=\frac{1}{Volumeanalyzedsediment\left(\mu L\right)}\times \frac{Volumeofobtainedsediment\left(\mu L\right)}{Volumeoffilteredsampl\left(L\right)}$ (3)

Analysis of physical parameters

The variables turbidity and pH were analyzed according to Standard Methods (APHA; AWWA; WEF, 2012), since they are part of the routine operational control of WTS.

Pasture area and animal density

Occupation data for the percentage of pasture area were obtained using ArcGIS software in conjunction with MapBiomas - Collection.3.0 database (PROJETO MAPBIOMAS, 2017), for the year 2017. Cattle load, which corresponds to the quotient between the number of cattle and the area of pasture in hectares, and the load of swine and horses as a function of the total area of the basin, was obtained from the Censo Agropecuário 2017 (IBGE, 2017).

RESULTS AND DISCUSSION

Validation of the membrane filtration method

Results regarding the performance of the MF method with samples contaminated with Giardia spp. cysts and Cryptosporidium spp. oocysts are provided in Table 3 for ultrapure water, and Table 4 for in natura raw water samples from the Santana stream. The absence of contamination in the analytical process is noteworthy, as no (oo)cysts were detected in the duplicated test.

Table 3 -
Initial Precision and recovery of Giardia spp. and Cryptosporidium spp. inoculated in ultrapure water, containing the inoculated (oo)cysts, recovery efficiency, mean, SD and CV.

Samples	Cysts of Giardia spp.			Oocysts of Cryptosporidium spp.
	Number of inoculated cysts	Number of recovered cysts	Recovery efficiency (%)	Number of inoculated oocysts	Number of recovered oocysts	Recovery efficiency (%)
1	32.0	25.0	78.1	268.0	325.0	121.3
2	32.0	25.0	78.1	268.0	250.0	93.3
3	32.0	50.0	156.3	268.0	75.0	28.0
		Mean	78.1		Mean	60.6
		SD	0.0		SD	32.6
		CV	0.0		CV	53.8

• SD: standard deviation; CV: coefficient of variation.

Table 4 -
Continued precision and recovery of Giardia spp. and Cryptosporidium spp. inoculated in raw water in natura of Santana Stream with turbidity of 8.20 NTU, containing the inoculated (oo)cysts, recovery efficiency, mean, SD and CV.

Samples	Cysts of Giardia spp.			Oocysts of Cryptosporidium spp.
	Number of inoculated cysts	Number of recovered cysts	Recovery efficiency (%)	Number of inoculated oocysts	Number of recovered oocysts	Recovery efficiency (%)
1	32.0	0.0	0.0	268.0	16.5	6.2
2	32.0	83.5	260.9	268.0	66.5	24.8
3	32.0	33.5	104.7	268.0	16.5	6.2
		Mean	-		Mean	12.4
		SD	-		SD	8.8
		CV	-		CV	71.1

• SD: standard deviation; CV: coefficient of variation.

The individual results in Table 3 show anomalous recovery efficiency values, with 156.3 and 121.3% for Giardia spp. cysts and Cryptosporidium spp. oocysts, respectively, which may be explained by possible agglomeration of organisms in the aliquot. Table 4 shows atypical results of 260.9 and 104.7% for Giardia spp. cysts, which may be explained by possible agglomeration of organisms in the aliquot due to the turbidity of samples, which requires more of membranes to filter samples and a greater final pellet volume.

Agglomerations can be caused by age, origin of organisms, storage time of the inoculum suspension (CANALE, 2014), and presence of algae, yeasts and debris that self-fluoresce and can affect the detection of (oo)cysts under the microscope by generating false positives (USEPA, 2012).

The average recovery rates in ultrapure water were 78.1 ± 0% for Giardia spp. cysts and 60.6 ± 32.6% for Cryptosporidium spp. oocysts (Table 3), as recommended by USEPA (2012).

Recovery in raw water was lower than that recommended by USEPA (2012), with values of 12.4 ± 8.8% for Cryptosporidium spp. oocysts (Table 4), due to the turbidity of the samples which, according to Efstratiou, Ongerth and Karanis (2017a), affects the detection performance for protozoa toward underreporting.

Particle is one of the limitations faced by the MF method in recovering (oo)cysts (FRANCO; BRANCO; LEAL, 2012; CANTUSIO NETO, 2008), since, under conditions of greater turbidity, a large number of membranes is required for filtration, which generates a greater volume of sediment destined for slide preparation. Using the MF method, Bautista et al. (2018) reported that the lowest means for detected (oo)cysts/L were in samples with turbidities of 554 and 800 NTU, during the rainy season, in Rímac stream of Peru.

Recovery outside the range recommended by USEPA (2012) for raw water was reported by Canale (2014) and Maciel (2006). The latter associated the method in question with immunomagnetic separation (IMS) and obtained mean recoveries of 31.5% cysts and 5.7% oocysts. Efstratiou, Ongerth and Karanis (2017a) reported that, in general, Giardia spp. cysts achieve greater recovery than Cryptosporidium spp. oocysts.

To improve performance in recovery efficiency with raw water samples, a more voluminous aliquot can be added to each slide well, once that according to Maciel (2006), the variability of tests may be associated with low aliquot representativeness(10 µL) in relation to the final sediment volume (250-1,000 µL), mainly for techniques that do not use IMS.

In addition, a rotation energy greater than 1,500 × g (15 minutes) could be adopted, as verified by Clancy et al. (2000), who achieved best recovery performance for Cryptosporidium spp. with a gravitational force of 2,170 × g and a centrifugation time of 15 minutes.

Prevalence of (oo)cysts in surface water sources

Data on the occurrence of E. Coli in raw water (frequency and range), the percentage of pasture area and animal load in the catchment hydrographic basins are presented together, with the results of the physical and parasitological parameters, in Table 5. As it turns out, in the present scenario, (oo)cysts are distributed in the aquatic environment of the state of Goiás, suggesting a potential risk to the population’s public health.

Table 5 -
Percentage pasture area; densities of cattle, swine and horses; incidence of E. Coli from 2009 to 2018 (frequency and amplitude); and results of physical and parasitological parameters, as well as the LD.

Municipality	Superficial Water Source	Pasture area (%)	Cattle density (number/hectare)	Swine density (number/hectare)	Horse density (number/hectare)	Incidence of E. Coli ≥ 1000 MPN/100 mL	E. Coli (minimum - maximum)	Turbidity (NTU)	pH	Giardia (cysts/L)	Cryptosporidium (oocysts/L)	LD ((oo)cysts/L)
Adelândia	São Manoel Stream	71.60	1.78	0.066	0.022	6	211 - 3,890	6.80	7.89	66.7	208.3	8.3
Anicuns	Boa Esperança Stream	66.83	2.06	0.059	0.015	4	8 - 2,295	6.86	7.94	100.0	175.0	8.3
Barro Alto	Barro Alto Stream	40.90	0.77	0.020	0.009	7	974 - 4,685	7.47	7.71	< LD	58.3	8.3
Ceres	Das Almas River	52.23	1.36	0.105	0.033	5	157 - 6,432	4.84	7.84	58.3	66.7	8.3
Crixás	Forquilha Stream	34.73	0.96	0.020	0.011	6	48 - 2,727	2.11	7.8	16.7	62.5	4.2
Goianésia	Anda Só Stream	51.83	1.42	0.065	0.013	6	284 - 5,291	11.9	7.65	16.7	108.3	8.3
Jaraguá	Pari River	61.18	1.32	0.055	0.018	6	282 - 6,429	7.38	8.01	< LD	8.3	8.3
Morro Agudo de Goiás	Olho D’água Stream	67.05	1.57	0.038	0.024	8	630 - 4,770	16.3	7.43	116.7	250.0	16.7
Nova Glória	Jatobá Stream	24.16	1.88	0.053	0.012	5	473 - 9,289	2.44	7.27	8.3	16.7	4.2
Quirinópolis	Das Pedras Stream	45.59	1.38	0.018	0.008	5	39 - 2,003	5.98	7.85	116.7	133.3	8.3
Rubiataba	Novo River	61.11	1.57	0.052	0.019	5	498 - 6,125	4.99	7.8	50.0	66.7	8.3
Sanclerlândia	Cerrado Stream	79.33	1.63	0.050	0.022	7	337 - 10,089	7.19	7.88	58.3	216.7	8.3
São Luís de Montes Belos	Santana Stream	78.46	1.73	0.083	0.027	5	366 - 10,669	12.4	7.83	66.7	66.7	8.3
Uruana	Uru River	50.07	1.58	0.073	0.025	5	187 - 4,351	2.64	7.74	79.2	54.2	4.2
Vianópolis	Dirceu Stream	35.95	1.91	0.033	0.017	4	Absence - 4,133	5.93	7.74	58.3	108.3	8.3

• LD: limit of detection; MPN: most probable number; NTU: Nephelometric turbidity unit.

According to Annex XX of Consolidation Ordinance No. 5 (BRASIL, 2017), the E. Coli microbiological parameter is an indicator of (oo)cysts of Cryptosporidium spp. and Giardia spp. in water resources; however, Grott et al. (2016) and Bastos et al. (2004) failed to find a significant correlation between the prevalence of these organisms when analyzing water from surface sources in the states of Santa Catarina and Minas Gerais, respectively.

According to Horman et al. (2004), the lack of statistical correlation can be partly associated with the survival time of each species in the aquatic environment, since Cryptosporidium spp. oocysts can survive for more than six months while cysts can survive for a maximum of two months (KARANIS; KOURENTI; SMITH, 2007).

Table 5 shows that all samples were positive for Cryptosporidium spp. oocysts and 86,66% (13/15) for Giardia spp. cysts. Detention rates have not been compared with other investigations (IMRE et al., 2017) as, according to Hachich et al. (2004), lotic environments have a higher incidence of protozoa than lotic aquatic systems. Lopes et al. (2017) found maximum concentrations of 0.8 cysts/L and 0.4 oocysts/L in the Vargem das Flores reservoir, a lentic environment intended for supplying Belo Horizonte, Minas Gerais. The lower occurrence of protozoa is associated with removal factors, including sedimentation and inactivation by temperature, ultraviolet radiation (UV) and predation (BROOKES et al., 2004).

In the Olho D’água stream, the maximum concentrations of Cryptosporidium spp. (250 oocysts/L) and Giardia spp. (116.7 cysts/L) were detected, along with eight occurrences of the E. Coli indicator above 1,000 MPN/100 mL, due to the predominance of pasture area (67.05%) and cattle load (1.57 per hectare). This source presents intense agricultural activities with a pig load in the range of 0.038 per hectare, the main host of the species C. scrofarum and C. suis, and a horse density of 0.024 per hectare, the host of C. erinacei (RYAN; FAYER; XIAO, 2014).

These concentrations were notably higher than the maximum concentration of 75 cysts/L and 19.2 oocysts/L reported for raw water from Corumbataí river in Piracicaba, São Paulo (CANALE, 2014) and the maximum concentration of 35.8 cysts/L in raw water from Atibaia river in Campinas, São Paulo (CANTUSIO NETO, 2008). Since these studies did not provide data on land use and occupation, it appears that the higher prevalence of protozoa in the basins studied here is related to characteristics of land use, especially pasture area, as the presence of cattle feces in the vicinity of the water courses promotes water contamination, especially in the rainy season and when the pasture is located in the floodplain (GRACZYK et al., 2000).

A high occurrence of Giardia spp. (116.7 cysts/L) was also detected in the Pedras stream, with 45.59% of pasture area, five occurrences of E. Coli above the recommended level, and a cattle load of 1.38.

The predominance of pastures may indicate the extent of the source of contamination upstream of the catchment point (ADAMSKA, 2015). However, molecular characterization of the organisms is needed to confirm this relationship, with a focus on species infectious of humans, such as C. parvum and hominis(EFSTRATIOU; ONGERTH; KARANIS, 2017a).

These results corroborate the study by Almeida et al. (2015), which identified the presence of Cryptosporidium parvum and Giardia duodenalis in Ribeirão Cafezal in Londrina Paraná, and associated this finding with the existence of dairy cattle in the region, and that of Farizawati et al. (2005), which verified the contribution of cattle farms along the watercourse for contamination by protozoa, with high concentrations in the range of 1.5 to 3.9 × 10⁵ (oo)cysts/L.

The risk associated with C. parvum, isolated from cattle, horses, and humans (ROSE; HUFFMAN; GENNACCARO, 2002), in diarrheal children hospitalized in Goiânia, state of Goiás, has been associated with environmental factors such as contact with surface water in the last 30 days and distance between residence and a watercourse (PEREIRA et al., 2002).

The water sources Barro Alto stream and Pari river had Giardia spp. cyst concentrations below the LD and Cryptosporidium spp. at 58.3 and 8.3 oocysts/L, respectively. It should be noted that the hydrographic basin of the Pari river, with greater pasture area (61.18%) than that of Barro Alto stream (40.90%), six occurrences of E. Coli and high densities of cattle (1.32), swine (0.055) and horses (0.018) per hectare, had the lowest concentration of Cryptosporidium spp. of the studied surface water sources, although still with a risk to human health, since the infectious dose corresponds to at least one oocyst (KARANIS, 2011; STEINER; THIELMAN; GUERRANT, 1997).

Concentrations of 16.7 oocysts/L and 8,3 cysts/L were detected at the Jatobá stream, which had the smallest pasture area (24.16%), five loads of E. Coli higher than that established by the standard and low densities of cattle (1.88 per hectare). These results indicate that the characteristics of land occupation positively influenced the parasitological quality of raw water (DIAS et al., 2008), due to the smaller area of pasture that was reflected in a smaller number of cattle in the region, since studies in other countries reported that a bovine is capable of releasing 50 to 3.9 × 10⁵ (oo)cysts/g of feces (FARIZAWATI et al., 2005).

Among the basins studied, the highest cattle load (2.06) was observed for Boa Esperança stream, with an occurrence of 100 and 175 oocysts/L. It is noteworthy that this contamination is intensified in developing countries and rural areas due to untreated human and animal waste (HELLER et al., 2004).

The results did not allow to infer whether the animal load in the hydrographic basin generated increased concentrations of protozoa. According to Bagley et al. (1998), water sources that receive contributions of cattle feces and sewage discharge as a result of land use activities have 10 to 100 times higher concentrations of (oo)cysts. However, in the present study, the Barro Alto stream basin, which had the lowest cattle load (0.77), had an occurrence of 58.3 oocysts/L. This effect may be related to soil management, reducing the carrying of (oo)cysts (CHUAH et al., 2016), and the presence of uninfected cattle in the catchment basins with lower prevalence of protozoa, which depends on the water source given to the animals and their age, since young animals are more frequently contaminated due to having less immunity (DIAS et al., 2008).

The presence of protozoa in supply sources (Figure 1) in the state of Goiás represents a potential risk to consumer health if the water is not treated properly. It is recommended that sanitation authorities and companies dedicate themselves to the implementation of new treatment techniques, the improvement of existing techniques and the continuous monitoring of water courses (FREITAS et al., 2010).

Sanitation companies must analyze the (oo)cysts in fresh water as established in Annex XX of Consolidation Ordinance No. 5 (BRASIL, 2017), since the monitoring of raw water is an essential tool to control and monitor treated water (HELLER et al., 2004), in addition to preventing outbreaks of gastrointestinal diseases, negative impacts on public health and ensuring public health security, as the water resource is destined for different uses (LEE et al., 2013).

In addition, seasonal monitoring aims to characterize the aquatic environment, since bacteria in the coliform group are not good indicators of the prevalence of protozoa (HORMAN et al., 2004) and due to the underestimation of risk of infection caused by punctual samples (HELLER et al., 2006), as performed in the present study.

The range of variation in the physical parameters of turbidity and pH were 2.11-16.30 NTU and 7.27-8.01, respectively. This range of turbidity values is in concordance with the drought period, as found by Vasco et al. (2011), who reported that the increased flow of Poxim river in the state of Sergipe, due to the rainy period, directly interferes with turbidity.

Evaluating existing studies on the theme revealed that the present study is the first to report the occurrence of Giardia spp. and Cryptosporidium spp. in water sources intended for public water supply in the state of Goiás. Therefore, there is a need for basic sanitation companies to establish routines for monitoring these protozoa, in order to prevent outbreaks of gastrointestinal diseases, as well as urgent action for the preservation and maintenance of the respective water courses.

CONCLUSION

From the results obtained in the present study, it can be concluded that:

• The precision and initial recovery tests reached USEPA (2012) criteria for Giardia (78.1 ± 0%) and Cryptosporidium (60.6 ± 32.6%);

• The precision and continuous recovery tests with in natura raw water did not present any sample in accordance with the international standard. This was due to the turbidity of samples, which requires more of membranes to filter samples and a greater final pellet volume;

• The Olho D’água stream located in Morro Agudo de Goiás, had maximum concentrations of 250 oocysts/L for Cryptosporidium spp. and 116.67 cysts/L for Giardia spp. These values can be associated with the predominance of livestock activities in the hydrographic basin. However, the punctual sampling campaign did not allow to infer whether the animal load in the hydrographic basin generates increased concentrations of protozoa;

• The Jabotá stream, with a low predominance of pasture area, presented less contamination of the watercourse by protozoa Cryptosporidium spp. and Giardia spp.;

• Cysts and oocysts were detected in the state’s aquatic environment at concentrations ranging from < LD to 116.67 cysts/L and between 8.33 and 250 oocysts/L, which represents a potential risk to consumer health considering that the intake of at least 10 cysts of Giardia spp. and one oocysts of Cryptosporidium spp. can initiate the infectious process. Therefore, periodic monitoring of water sources by service delegates is recommended in order to improve existing technologies of treatment plants to ensure sanitary security;

• It is recommended that treatment plants that obtain water from Cerrado, Santana, and São Manoel streams monitor the occurrence of protozoa, as the predominance of pasture indicates a risk for cryptosporidiosis outbreaks.

REFERENCES

• ADAMSKA, M. (2015) Molecular characterization of Cryptosporidium and Giardia occurring in natural water bodies in Poland. Parasitology Research, v. 114, n. 2, p. 687-692. http://doi.org/10.1007/s00436-014-4234-9
  » https://doi.org/http://doi.org/10.1007/s00436-014-4234-9
• ALMEIDA, J.C.; MARTINS, F.D.C.; FERREIRA NETO, J.M.; SANTOS, M.M.D.; GARCIA, J.L.; NAVARRO, I.T.; KURODA, E.K.; FREIRE, R.L. (2015) Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil. Revista Brasileira de Parasitologia Veterinária, v. 24, n. 3, p. 303-308. http://doi.org/10.1590/S1984-29612015051
  » https://doi.org/http://doi.org/10.1590/S1984-29612015051
• AMERICAN PUBLIC HEALTH ASSOCIATION (APHA); AMERICAN WATER WORKS ASSOCIATION (AWWA); WATER ENVIRONMENT FEDERATION (WEF). (2012) Standard Methods for the examination of water and wastewater 22^nd ed. Washington, D.C.: American Water Works Association, Water Environmental Federation.
• BAGLEY, S.T.; AUER, M.T.; STERN, D.A.; BABIERA, M.J. (1998) Sources and fate of Giardia cysts and Cryptosporidium oocysts in surface waters. Lake and Reservoir Management, v. 14, n. 2-3, p. 379-392. http://doi.org/10.1080/07438149809354345
  » https://doi.org/http://doi.org/10.1080/07438149809354345
• BALDURSSON, S.; KARANIS, P. (2011) Waterborne transmission of protozoan parasites: review of worldwide outbreaks - an update 2004-2010. Water Research, v. 45, n. 20, p. 6603-6614. https://doi.org/10.1016/j.watres.2011.10.013
  » https://doi.org/https://doi.org/10.1016/j.watres.2011.10.013
• BASTOS, R.K.X.; HELLER, L.; VIEIRA, M.B.M.; BRITO, L.A.; BEVILACQUA, P.D.; NASCIMENTO, L.E. (2004) Giardia cysts and Cryptosporidium oocysts dynamics in Southeast Brazil. Occurrence in surface water and removal in water treatment processes. Water Science and Technology, v. 4, n. 2, p. 15-22. http://doi.org/10.2166/ws.2004.0022
  » https://doi.org/http://doi.org/10.2166/ws.2004.0022
• BAUTISTA, M.; BONATTI, T.R.; FIUZA, V.R.D.S.; TERASHIMA, A.; CANALES-RAMOS, M.; JOSÉ, J.; FRANCO, R.M.B. (2018) Occurrence and molecular characterization of Giardia duodenalis cysts and Cryptosporidium oocysts in raw water samples from the Rímac River, Peru. Environmental Science and Pollution Research, v. 25, n. 12, p. 11454-11467. http://doi.org/10.1007/s11356-018-1423-6
  » https://doi.org/http://doi.org/10.1007/s11356-018-1423-6
• BRASIL. (2017) Ministério da Saúde. Portaria de Consolidação nº 5, de 28 de setembro de 2017 Brasília: Ministério da Saúde.
• BROOKES, J.D.; ANTENUCCI, J.; HIPSEY, M.; BURCH, M.D.; ASHBOLT, N.J.; FERGUSON, C. (2004) Fate and transport of pathogens in lakes and reservoirs. Environment International, v. 30, n. 5, p. 741-759. https://doi.org/10.1016/j.envint.2003.11.006
  » https://doi.org/https://doi.org/10.1016/j.envint.2003.11.006
• CANALE, I. (2014) Caracterização microbiológica, parasitológica e físico-química da água de lavagem de filtros recirculada em ETA de ciclo completo Dissertation (Mastering) - Faculdade de Tecnologia, Universidade Estadual de Campinas, Campinas.
• CANTUSIO NETO, R. (2008) Estudo dos métodos de floculação em carbonato de cálcio e adaptação das técnicas de filtração em membrana e separação imunomagnética para detecção de Cryptosporidium e Giardia em amostras hídricas Thesis (Doctorate) - Instituto de Biologia, Universidade Estadual de Campinas, Campinas.
• CANTUSIO NETO, R.; FRANCO, R.M.B. (2004) Ocorrência de oocistos de Cryptosporidium spp. e cistos de Giardia spp. em diferentes pontos do processo de tratamento de água, em Campinas, São Paulo, Brasil. Higiene Alimentar, v. 18, n. 118, p. 52-59.
• CHENG, H.W.A.; LUCY, F.E.; GRACZYK, T.K.; BROADERS, M.A.; TAMANG, L.; CONNOLLY, M. (2009) Fate of Cryptosporidium parvum and Cryptosporidium hominis oocysts and Giardia duodenalis cysts during secondary wastewater treatments. Parasitology Research, v. 105, n. 3, p. 689. http://doi.org/10.1007/s00436-009-1440-y
  » https://doi.org/http://doi.org/10.1007/s00436-009-1440-y
• CHUAH, C.J.; MUKHAIDIN, N.; CHOY, S.H.; SMITH, G.J.; MENDENHALL, I.H.; LIM, Y.A.; ZIEGLER, A.D. (2016) Prevalence of Cryptosporidium and Giardia in the water resources of the Kuang River catchment, Northern Thailand. Science of the Total Environment, v. 562, p. 701-713. http://doi.org/10.1016/j.scitotenv.2016.03.247
  » https://doi.org/http://doi.org/10.1016/j.scitotenv.2016.03.247
• CLANCY, J.L.; BUKHARI, Z.; MCCUIN, R.M.; HARGY, T.M. (2000) New Approaches for Isolation of Cryptosporidium and Giardia. United States: American Water Works Association.
• COTRUVO, J.; DUFOUR, A.; REES, G.; BARTRAM, J.; CARR, R.; CLIVER, D.O. (2004) Waterborne zoonoses: identification, causes, and control. Geneva: World Health Organization.
• DIAS, G.M.F.; BEVILACQUA, P.D.; BASTOS, R.K.X.; OLIVEIRA, A.A.; CAMPOS, G.M.M. (2008) Giardia spp. e Cryptosporidium spp. em agua de manancial superficial de abastecimento contaminada por dejetos humano e animal. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, v. 60, n. 6, p. 1291-1300. https://doi.org/10.1590/S0102-09352008000600001
  » https://doi.org/https://doi.org/10.1590/S0102-09352008000600001
• EFSTRATIOU, A.; ONGERTH, J.E.; KARANIS, P. (2017a) Evolution of monitoring for Giardia and Cryptosporidium in water. Water Research, v. 123, p. 96-112. http://doi.org/10.1016/j.watres.2017.06.042
  » https://doi.org/http://doi.org/10.1016/j.watres.2017.06.042
• EFSTRATIOU, A.; ONGERTH, J.E.; KARANIS, P. (2017b) Waterborne transmission of protozoan parasites: Review of worldwide outbreaks: An update 2011-2016. Water Research, v. 114, p. 14-22. https://doi.org/10.1016/j.watres.2017.01.036
  » https://doi.org/https://doi.org/10.1016/j.watres.2017.01.036
• FARIZAWATI, S.; LIM, Y.A.L.; AHMAD, R.A.; FATIMAH, C.T.N.I.; SITI-NOR, Y. (2005) Contribution of cattle farms towards river contamination with Giardia cysts and Cryptosporidium oocysts in Sungai Langat Basin. Tropical Biomedicine, v. 22, n. 2, p. 89-98.
• FRANCO, R.M.B.; BRANCO, N.; LEAL, D.A.G. (2012) Parasitologia ambiental: métodos de concentração e detecção de Cryptosporidium spp. e Giardia spp. em amostras de água. Revista de Patologia Tropical, v. 41, n. 2, p. 119-135. http://doi.org/10.5216/rpt.v41i2.19320
  » https://doi.org/http://doi.org/10.5216/rpt.v41i2.19320
• FRANCO, R.M.B.; HACHICH, E.M.; ZANOLLI SATO, M.I.; NAVEIRA, R.M.L.; SILVA, E.D.C.; CAMPOS, M.M.D.C.; CANTUSIO NETO, R.; CERQUEIRA, D.A.; BRANCO, N.; LEAL, D.A.G. (2012) Performance evaluation of different methodologies for detection of Cryptosporidium spp. and Giardia spp. in water for human consumption to meet the demands of the Environmental Health Surveillance in Brazil. Epidemiologia e Serviços de Saúde, v. 21, n. 2, p. 233-242. http://doi.org/10.5123/S1679-49742012000200006
  » https://doi.org/http://doi.org/10.5123/S1679-49742012000200006
• FREITAS, A.G.; BASTOS, R.K.X.; BEVILACQUA, P.D.; PÁDUA, V.L.; PIMENTA, J.F.P.; ANDRADE, R.C. (2010) Recirculação de Água de Lavagem de Filtros e Perigos Associados a Protozoários. Engenharia Sanitária e Ambiental, v. 15 n. 1, p. 37-46. https://doi.org/10.1590/S1413-41522010000100005
  » https://doi.org/https://doi.org/10.1590/S1413-41522010000100005
• FREITAS, D.A.D.; PAIVA, A.L.R.D.; CARVALHO FILHO, J.A.A.D.; CABRAL, J.J.D.S.P.; ROCHA, F.J.S. (2015) Occurrence of Cryptosporidium spp., Giardia spp. and other pathogenic intestinal parasites in the Beberibe River in the State of Pernambuco, Brazil. Revista da Sociedade Brasileira de Medicina Tropical, v. 48, n. 2, p. 220-223. http://doi.org/10.1590/0037-8682-0174-2014
  » https://doi.org/http://doi.org/10.1590/0037-8682-0174-2014
• GRACZYK, T.K.; EVANS, B.M.; SHIFF, C.J.; KARREMAN, H.J.; PATZ, J.A. (2000) Environmental and geographical factors contributing to watershed contamination with Cryptosporidium parvum oocysts. Environmental Research, v. 82, n. 3, p. 263-271. https://doi.org/10.1006/enrs.1999.4022
  » https://doi.org/https://doi.org/10.1006/enrs.1999.4022
• GROTT, S.C.; HARTMANN, B.; SILVA FILHO, H.H.; FRANCO, R.M.B.; GOULART, J.A.G. (2016) Detecção de cistos de Giardia spp. e oocistos de Cryptosporidium spp. na água bruta das estações de tratamento no município de Blumenau, SC, Brasil. Ambiente e Água, v. 11, n. 3, p. 689-701. https://doi.org/10.4136/ambi-agua.1853
  » https://doi.org/https://doi.org/10.4136/ambi-agua.1853
• HACHICH, E.M.; SATO, M.I.Z.; GALVANI, A.T.; MENEGON, J.R.N.; MUCCI, J.L.N. (2004) Giardia and Cryptosporidium in source waters of Sao Paulo state, Brazil. Water Science and Technology, v. 50, n. 1, p. 239-245.
• HELLER, L.; BASTOS, R.K.X.; VIEIRA, M.B.C.M.; BEVILACQUA, P.D.; BRITO, L.L.A.; MOTA, S.M.M.; OLIVEIRA, A.A.; MACHADO, P.M.; SALVADOR, D.P.; CARDOSO, A.B. (2004) Oocistos de Cryptosporidium e cistos de Giardia: circulação no ambiente e riscos à saúde humana. Epidemiologia e Serviços de Saúde, v. 13, n. 2, p. 79-92. http://doi.org/10.5123/S1679-49742004000200002
  » https://doi.org/http://doi.org/10.5123/S1679-49742004000200002
• HELLER, L.; VIEIRA, M.B.C.M.; BRITO, L.L.A.D.; SALVADOR, D.P. (2006) Desempenho da filtração lenta em areia submetida a cargas de pico de oocistos de Cryptosporidium sp., bactérias e sólidos: uma avaliação em instalação piloto. Engenharia Sanitaria e Ambiental, v. 11, n. 1, p. 27-38. https://doi.org/10.1590/S1413-41522006000100005
  » https://doi.org/https://doi.org/10.1590/S1413-41522006000100005
• HORMAN, A.; RIMHANEN-FINNE, R.; MAUNULA, L.; VON BONSDORFF, C.H.; TORVELA, N.; HEIKINHEIMO, A.; HANNINEN, M.-L. (2004) Campylobacter spp., Giardia spp., Cryptosporidium spp., Noroviruses, and Indicator Organisms in Surface Water in Southwestern Finland, 2000-2001. Applied and Environmental Microbiology, v. 70, n. 1, p. 87-95. http://doi.org/10.1128/aem.70.1.87-95.2004
  » https://doi.org/http://doi.org/10.1128/aem.70.1.87-95.2004
• HSU, B.M.; HUANG, C.; HSU, Y.F.; JIANG, G.Y.; HSU, C.-I.I. (2001) Evaluation of two concentration methods for detecting Giardia and Cryptosporidium in water. Water Research, v. 35, n. 2, p. 419-424.
• IMRE, K.; SALA, C.; MORAR, A.; ILIE, M.S.; PLUTZER, J.; IMRE, M.; HORA, F.S.; BADEA, C.; HERBEI, M.V.; DĂRĂBUȘ, G. (2017) Giardia duodenalis and Cryptosporidium spp. as contaminant protozoa of the main rivers of western Romania: genetic characterization and public health potential of the isolates. Environmental Science and Pollution Research, v. 24, n. 22, p. 18672-18679. https://doi.org/10.1007/s11356-017-9543-y
  » https://doi.org/https://doi.org/10.1007/s11356-017-9543-y
• INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA (IBGE). (2017) Mapas por município do Censo Agro IBGE. Available at: <Available at: https://mapasinterativos.ibge.gov.br/agrocompara/ >. Accessed on: Apr. 24, 2019.
  » https://mapasinterativos.ibge.gov.br/agrocompara/
• KARANIS, P. (2011) Giardia and Cryptosporidium: Occurrence in Water Supplies. Encyclopedia of Environmental Health, p. 946-954. http://doi.org/10.1016/b978-0-444-52272-6.00565-1
  » https://doi.org/http://doi.org/10.1016/b978-0-444-52272-6.00565-1
• KARANIS, P.; KOURENTI, C.; SMITH, H. (2007) Waterborne transmission of protozoan parasites: A worldwide review of outbreaks and lessons learnt. Journal of Water and Health, v. 5, n. 1, p. 1-38. http://doi.org/10.2166/wh.2006.002
  » https://doi.org/http://doi.org/10.2166/wh.2006.002
• KÖPPEN, W. (1948) Climatologia: con un estudio de los climas de la tierra. Mexico: Fondo de Cultura Economica. 478 p.
• LEE, Y.T.; TSAIHONG, J.C.; TSENG, Y.C.; TSAI, C.M.; PENG, S.Y. (2013) Development of PCR-RFLP method to distinguish between Cryptosporidium parvum and C. hominis in Taiwan water samples. Southeast Asian Journal of Tropical Medicine Public Health, v. 44, n. 1, p. 13-18.
• LOPES, A.M.M.B.; GOMES, L.N.L.; MARTINS, F.D.C.; CERQUEIRA, D.A.; MOTA FILHO, C.R.; SPERLING, E.V.; PÁDUA, V.L.D. (2017) Dinâmica de protozoários patogênicos e cianobactérias em um reservatório de abastecimento público de água no sudeste do Brasil. Engenharia Sanitária e Ambiental, v. 22, n. 1, p. 25-43. https://doi.org/10.1590/s1413-41522016143529
  » https://doi.org/https://doi.org/10.1590/s1413-41522016143529
• MACIEL, P.M.F. (2006) Remoção de Giardia spp. e Cryptosporidium spp. em águas de abastecimento com turbidez elevada utilizando cloreto de polialumínio: estudo em escala de bancada e desafios analíticos Dissertation (Mastering) - Instituto de Biologia, Universidade Estadual de Campinas, Campinas.
• MACIEL, P.M.F.; SABOGAL-PAZ, L.P. (2016) Removal of Giardia spp. and Cryptosporidium spp. from water supply with high turbidity: analytical challenges and perspectives. Journal of Water and Health, v. 14, n. 3, p. 369-378. https://doi.org/10.2166/wh.2015.227
  » https://doi.org/https://doi.org/10.2166/wh.2015.227
• NISHI, L.; BAESSO, M.L.; SANTANA, R.G.; FREGADOLLI, P.; FALAVIGNA, D.L.M.; FALAVIGNA‐GUILHERME, A.L. (2009) Investigation of Cryptosporidium spp. and Giardia spp. in a Public Water‐Treatment System. Zoonoses and Public Health, v. 56, n. 5, p. 221-228. https://doi.org/10.1111/j.1863-2378.2008.01189.x
  » https://doi.org/https://doi.org/10.1111/j.1863-2378.2008.01189.x
• ONGERTH, J.E. (2013) The concentration of Cryptosporidium and Giardia in water - The role and importance of recovery efficiency. Water Research, v. 47, n. 1, p. 2479-2488. http://doi.org/10.1016/j.watres.2013.02.015
  » https://doi.org/http://doi.org/10.1016/j.watres.2013.02.015
• PEREIRA, M.D.G.C.; ATWILL, E.R.; BARBOSA, A.P.; SILVA, S.A.E.; GARCÍA-ZAPATA, M.T.A. (2002) Intra-familial and extra-familial risk factors associated with Cryptosporidium parvum infection among children hospitalized for diarrhea in Goiânia, Goiás, Brazil. The American Journal of Tropical Medicine and Hygiene, v. 66, n. 6, p. 787-793. http://doi.org/10.4269/ajtmh.2002.66.787
  » https://doi.org/http://doi.org/10.4269/ajtmh.2002.66.787
• PLUTZER, J.; KARANIS, P. (2016) Neglected waterborne parasitic protozoa and their detection in water. Water Research, v. 101, p. 318-332. http://doi.org/10.1016/j.watres.2016.05.085
  » https://doi.org/http://doi.org/10.1016/j.watres.2016.05.085
• PROJETO MAPBIOMAS. (2017) Coleção 3.0 da Série Anual de Mapas de Cobertura e Uso de Solo do Brasil Available at: <Available at: https://storage.googleapis.com/mapbiomas/legenda/Download_codigos_da_legenda_3.0_rev1.pdf >. Accessed on: Nov. 30, 2018.
  » https://storage.googleapis.com/mapbiomas/legenda/Download_codigos_da_legenda_3.0_rev1.pdf
• ROSE, J.B.; HUFFMAN, D.E.; GENNACCARO, A. (2002) Risk and control of waterborne cryptosporidiosis. FEMS Microbiology Reviews, v. 26, n. 2, p. 113-123. https://doi.org/10.1111/j.1574-6976.2002.tb00604.x
  » https://doi.org/https://doi.org/10.1111/j.1574-6976.2002.tb00604.x
• RYAN, U.N.A.; FAYER, R.; XIAO, L. (2014) Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology, v. 141, n. 13, p. 1667-1685. http://doi.org/10.1017/s0031182014001085
  » https://doi.org/http://doi.org/10.1017/s0031182014001085
• SATO, M.I.Z.; GALVANI, A.T.; PADULA, J.A.; NARDOCCI, A.C.; SOUZA LAURETTO, M.; RAZZOLINI, M.T.P.; HACHICH, E.M. (2013) Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil. Science of The Total Environment, v. 442, p. 389-396. http://doi.org/10.1016/j.scitotenv.2012.09.077
  » https://doi.org/http://doi.org/10.1016/j.scitotenv.2012.09.077
• STEINER, T.S.; THIELMAN, N.M.; GUERRANT, R.L. (1997) Protozoal agents: what are the dangers for the public water supply? Annual Review of Medicine, v. 48, n. 1, p. 329-340. http://doi.org/10.1146/annurev.med.48.1.329
  » https://doi.org/http://doi.org/10.1146/annurev.med.48.1.329
• THOMPSON, R.C.A. (2004) The zoonotic significance and molecular epidemiology of Giardia and Giardiasis. Veterinary Parasitology, v. 126, n. 1-2, p. 15-35. http://doi.org/10.1016/j.vetpar.2004.09.008
  » https://doi.org/http://doi.org/10.1016/j.vetpar.2004.09.008
• TOLEDO, D.S.R.; MARTINS, F.D.C.; FERREIRA, F.P.; ALMEIDA, J.C.; OGAWA, L.; SANTOS, M.M.; SANTOS, M.M.; PINHEIRO, F.A.; NAVARRO, I.T.; FREIRE, R.L. (2017) Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms. PloS One, v. 12, n. 4, e0175311. http://doi.org/10.1371/journal.pone.0175311
  » https://doi.org/http://doi.org/10.1371/journal.pone.0175311
• UNITED STATES ENVIROMENTAL PROTECTION AGENCY (USEPA). (2012) Method 1623.1 Cruptosporidium an Giardia in Water by Filtration/IMS/FA Office of Water (MS-140) EPA 816 -R-12-001.
• VASCO, A.N.; BRITTO, F.B.; PEREIRA, A.P.S.; MÉLLO JÚNIOR, A.V.M.; GARCIA, C.A.B.; NOGUEIRA, L.C. (2011) Avaliação espacial e temporal da qualidade da água na sub-bacia do rio Poxim, Sergipe, Brasil. Revista Ambiente e Água, Taubaté, v. 6, n. 1, p. 118-130. http://doi.org/10.4136/ambi-agua.178
  » https://doi.org/http://doi.org/10.4136/ambi-agua.178
• WORLD HEALTH ORGANIZATION (WHO). Home - Newsroom - Fact sheets - Detail -Drinking-water Geneva: WHO, 2019. Available at: <Available at: https://www.who.int/news-room/fact-sheets/detail/drinking-water >. Accessed on: Oct. 24, 2020.
  » https://www.who.int/news-room/fact-sheets/detail/drinking-water

• 1
  Reg. ABES: 20200312

Publication Dates

• Publication in this collection
  02 Dec 2020
• Date of issue
  Sep-Oct 2020

History

• Received
  31 Aug 2020
• Accepted
  14 Sept 2020