**1. Introduction**

340 Environmental Monitoring

Unwin AR, Wills G. Eyeballing time series. *Proceedings of the 1988 ASA Statistical Computing* 

Ware C. *Information Visualization: Perception for Design* (2nd edn). Morgan Kaufmann

*Section* 1988; pp 263−268.

Publishers, 2004.

Water is a natural resource of vital importance to living beings, but due to anthropic action several microorganisms are disseminated into aquatic environments. In developing countries, over one billion people do not have access to clean, properly treated water and approximately three billion people do not have access to adequate sanitary facilities (Kraszewski et al., 2001) This scenery is probably a consequence of the increased environmental degradation, depletion of water resources, and constant contamination of bodies of water with wastewater and industrial effluents (Pedro & Germano, 2001), causing microorganisms from soil, faeces, decomposing organic matter, and other pollutant sources to spread into water.

Goiania, the capital of the state of Goiás, located in the Midwestern Region of Brazil, has ca. 1.221.654 inhabitants and is considered a regional metropolis, among the major Brazilian cities that receive a large number of migrants (Alves & Chaveiro 2007). As a result, the city faces problems of disorderly and unsustainable urban growth with a consequent increase in superficial waste, which is a continuous source of contamination of water courses.

The current sources of public water supply for the city of Goiania, the Meia Ponte river basin and its tributary river João Leite, are constantly submitted to degradation processes due to anthropic action, such as agriculture, intensive livestock production, and urbanization. And although all the water supplies of Goiânia come from this basin (52% from the Joa˜o Leite River and 48% from the Meia Ponte River), this municipality is its largest polluter (Silva et al., 2010).

Among the microorganisms that contaminate the aquatic environment, special attention should be given to opportunistic protozoa, such as Coccidea (*Cryptosporidium parvum*, *Isospora belli*, *Sarcocystis* sp., and *Cyclospora* sp.) and Microsporidia that infect the

professional activities.

1996).

these pathogens.

(Thurston-enriquez et al., 2002).

*Cryptosporidium* sp. oocysts in water environments.

States (Clancy et al., 2003; Franco, 2004).

Opportunistic Protozoa in Rivers and Lakes: Relevance to Public Health in the Neotropics 343

Human health is likely to be affected either directly by drinking water contaminated with biological agents such as bacteria, viruses, and parasites, indirectly by consuming food or drinks prepared with contaminated water, or accidentally during recreational or

A massive waterborne outbreak of cryptosporidiosis occurred in 1993, in Milwaukee, Wisconsin, in the United States. Approximately 403.000 people experienced illness, 4.400 of them were hospitalized, and 100 deaths were registered (Corso et al., 2003). In 1996, the United States American Environmental Protection Agency (U.S. EPA) started a program to identify, standardize, and validate new methods for the detection of *Giardia* sp. cysts and

From 1984 to 2000, 76 outbreaks of waterborne *Cryptosporidium* sp. have been associated with in countries like USA, England, Northern Ireland, Canada, Japan, Italy, New Zealand and Australia, affecting about 481.026 people, of these 59.2% were related to drinking water and 40.7% to the recreational use of water (Fayer et al., 2000; Fricker et al. 1998; Glaberman et al., 20; Howe et al., 2002). The most frequent causes of contamination are due to operational failures of treatment systems and water contact with sewage or faecal accident in the case of recreational waters In the U.S., factors such as deterioration in raw water quality and decrease the effectiveness of the process of coagulation and filtration of one of the local water supply companies showed an increase in turbidity of treated water and inadequate removal of *Cryptosporidium* sp. (Kramer et al.,

Programs to monitor these pathogens in water have been spontaneously carried out in some countries such as the United States and the United Kingdom (Clancy et al., 1999). Since this, methods 1622 and 1623 (USEPA, 1999) have been used as reference procedures in the United

In Brazil, the concern about water quality prompted the Health Ministry to issue one Decree - Ordinance 518 (Brasil, 2004) - establishing procedures and responsibilities regarding the control and surveillance of water quality for human consumption and pattern of potability, and other measures. Nowadays, in Brazil, routine monitoring of protozoa is not performed in bodies of water used for the abstraction of water intended for human consumption. Nonetheless, the Brazilian Health Ministry recommends the inclusion of methods for the detection of *Giardia* sp. cysts and *Cryptosporidium* sp. oocysts aiming to reach a standard in which the water supplied to the population must be free of

It should be emphasized that the detection of cysts and oocysts in superficial water is a crucial component to control these pathogens. However, the current methods present high variability of recovery efficiency of *Cryptosporidium* sp. oocysts and *Giardia* sp. cysts (Hsu et al., 2001), leading to the need of aggregating other types of methodology to guarantee that water potability achieves a higher degree of reliability. Due to lack of specific techniques for detection of Microsporidia and Coccidea in water and food, the analysis has been carried out by adaptations of methods used for clinical testing

The goal of this study was to optimize and use parasitological and molecular techniques in the analysis and seasonal monitoring of opportunistic protozoa in water from fluvial systems for human usage in the municipality of Goiânia, the capital of the state of Goiás, in

gastrointestinal tract, are considered emergents (Gomes et al., 2002), and also *Giardia* sp., which causes diarrhea episodes (States et al., 1997), can be spread through water.

The magnitude of enteric protozoan to public health should be emphasized because of their high prevalence, cosmopolitan distribution, and deleterious effects on the individuals' nutritional status and immune system. Although children are the most susceptible individuals to these pathogens, they also affect people from other age groups (Geldreich, 1996), mainly in subtropical and tropical areas.

According to Fayer et al. (2000) the *Cryptosporidium* is a protozoan parasite of vertebrates that causes diarrhea in humans in Different Geographical Regions of the world. Through molecular techniques, it is accepted that the *C. parvum* comprises at least two genotypes: 1 or H - only infectious for humans (anthroponotic), 2 or C - infecting cattle, men and various animals, confirming the zoonotic potential initially attributed to protozoa (Kosek et al. 2001).

Among the various water-borne pathogens (viruses, bacteria, fungi and parasites) are noted protozoa *Giardia duodenalis* (synonym *Giardia lamblia* and *Giardia intestinalis*) Thompson (2000) and *Cryptosporidium* sp., which cause gastroenteritis in humans and animals. These infectious agents are derived mainly from infected people and other warm-blooded animals, which undoubtedly pollute water (Gomes et al., 2002), highlighting some that are considered emerging, such as coccidia, *Cryptosporidium parvum, Isospora belli, Sarcocystis* sp., *Cyclospora* sp. and *Microsporidia* sp. *(*Garcia-Zapata et al., 2003).

For many years, *C. parvum* was considered the only emerging agent of opportunistic human infection. Recently, using molecular techniques was possible to prove that other animals and other genotypes also affect humans, such as *C. felis* (Caccio et al., 2002), *C. Muris* (Katsumata et al., 2001) or *C. meleagridis* (Pedraza-dias et al., 2000), thus showing that other species may also have an impact on public health, especially for people with immune system changes, such as patients infected with the AIDS (Acquired Immunodeficiency Syndrome), transplant recipients or patients undergoing chemotherapy, diabetics, elderly and very young children (Fayer et al., 2000). In developing countries, over one billion people do not have access to clean, properly treated water and approximately three billion people do not have access to adequate sanitary facilities (Kraszewski, 2001). This scenery is probably a consequence of the increased environmental degradation, depletion of water resources, and constant contamination of bodies of water with wastewater and industrial effluents (Pedro & Germano, 2001), causing microorganisms from soil, faeces, decomposing organic matter, and other pollutant sources to spread into water.

The magnitude of enteric protozoan to public health should be emphasized because of their high prevalence, cosmopolitan distribution, and deleterious effects on the individuals' nutritional status and immune system. Although children are the most susceptible individuals to these pathogens, they also affect people from other age groups (Geldreich, 1996), mainly in subtropical and tropical areas.

Criptosporidiosis is an important parasitic disease that can become a public health problem (Cimerman et al., 2000). The main modes of *Cryptosporidium* sp. transmission are frequently associated to contaminated water, which could be either treated or non-treated superficial water, treated water contaminated along the distribution systems, or inappropriate treated water, usually using only a simple chlorination method (Solo-gabriele & Neumeister, 1996).

gastrointestinal tract, are considered emergents (Gomes et al., 2002), and also *Giardia* sp.,

The magnitude of enteric protozoan to public health should be emphasized because of their high prevalence, cosmopolitan distribution, and deleterious effects on the individuals' nutritional status and immune system. Although children are the most susceptible individuals to these pathogens, they also affect people from other age groups (Geldreich,

According to Fayer et al. (2000) the *Cryptosporidium* is a protozoan parasite of vertebrates that causes diarrhea in humans in Different Geographical Regions of the world. Through molecular techniques, it is accepted that the *C. parvum* comprises at least two genotypes: 1 or H - only infectious for humans (anthroponotic), 2 or C - infecting cattle, men and various animals, confirming the zoonotic potential initially attributed to protozoa (Kosek

Among the various water-borne pathogens (viruses, bacteria, fungi and parasites) are noted protozoa *Giardia duodenalis* (synonym *Giardia lamblia* and *Giardia intestinalis*) Thompson (2000) and *Cryptosporidium* sp., which cause gastroenteritis in humans and animals. These infectious agents are derived mainly from infected people and other warm-blooded animals, which undoubtedly pollute water (Gomes et al., 2002), highlighting some that are considered emerging, such as coccidia, *Cryptosporidium parvum, Isospora belli, Sarcocystis* sp.,

For many years, *C. parvum* was considered the only emerging agent of opportunistic human infection. Recently, using molecular techniques was possible to prove that other animals and other genotypes also affect humans, such as *C. felis* (Caccio et al., 2002), *C. Muris* (Katsumata et al., 2001) or *C. meleagridis* (Pedraza-dias et al., 2000), thus showing that other species may also have an impact on public health, especially for people with immune system changes, such as patients infected with the AIDS (Acquired Immunodeficiency Syndrome), transplant recipients or patients undergoing chemotherapy, diabetics, elderly and very young children (Fayer et al., 2000). In developing countries, over one billion people do not have access to clean, properly treated water and approximately three billion people do not have access to adequate sanitary facilities (Kraszewski, 2001). This scenery is probably a consequence of the increased environmental degradation, depletion of water resources, and constant contamination of bodies of water with wastewater and industrial effluents (Pedro & Germano, 2001), causing microorganisms from soil, faeces, decomposing organic matter, and other

The magnitude of enteric protozoan to public health should be emphasized because of their high prevalence, cosmopolitan distribution, and deleterious effects on the individuals' nutritional status and immune system. Although children are the most susceptible individuals to these pathogens, they also affect people from other age groups (Geldreich,

Criptosporidiosis is an important parasitic disease that can become a public health problem (Cimerman et al., 2000). The main modes of *Cryptosporidium* sp. transmission are frequently associated to contaminated water, which could be either treated or non-treated superficial water, treated water contaminated along the distribution systems, or inappropriate treated water, usually using only a simple chlorination method (Solo-gabriele & Neumeister, 1996).

which causes diarrhea episodes (States et al., 1997), can be spread through water.

1996), mainly in subtropical and tropical areas.

*Cyclospora* sp. and *Microsporidia* sp. *(*Garcia-Zapata et al., 2003).

pollutant sources to spread into water.

1996), mainly in subtropical and tropical areas.

et al. 2001).

Human health is likely to be affected either directly by drinking water contaminated with biological agents such as bacteria, viruses, and parasites, indirectly by consuming food or drinks prepared with contaminated water, or accidentally during recreational or professional activities.

A massive waterborne outbreak of cryptosporidiosis occurred in 1993, in Milwaukee, Wisconsin, in the United States. Approximately 403.000 people experienced illness, 4.400 of them were hospitalized, and 100 deaths were registered (Corso et al., 2003). In 1996, the United States American Environmental Protection Agency (U.S. EPA) started a program to identify, standardize, and validate new methods for the detection of *Giardia* sp. cysts and *Cryptosporidium* sp. oocysts in water environments.

From 1984 to 2000, 76 outbreaks of waterborne *Cryptosporidium* sp. have been associated with in countries like USA, England, Northern Ireland, Canada, Japan, Italy, New Zealand and Australia, affecting about 481.026 people, of these 59.2% were related to drinking water and 40.7% to the recreational use of water (Fayer et al., 2000; Fricker et al. 1998; Glaberman et al., 20; Howe et al., 2002). The most frequent causes of contamination are due to operational failures of treatment systems and water contact with sewage or faecal accident in the case of recreational waters In the U.S., factors such as deterioration in raw water quality and decrease the effectiveness of the process of coagulation and filtration of one of the local water supply companies showed an increase in turbidity of treated water and inadequate removal of *Cryptosporidium* sp. (Kramer et al., 1996).

Programs to monitor these pathogens in water have been spontaneously carried out in some countries such as the United States and the United Kingdom (Clancy et al., 1999). Since this, methods 1622 and 1623 (USEPA, 1999) have been used as reference procedures in the United States (Clancy et al., 2003; Franco, 2004).

In Brazil, the concern about water quality prompted the Health Ministry to issue one Decree - Ordinance 518 (Brasil, 2004) - establishing procedures and responsibilities regarding the control and surveillance of water quality for human consumption and pattern of potability, and other measures. Nowadays, in Brazil, routine monitoring of protozoa is not performed in bodies of water used for the abstraction of water intended for human consumption. Nonetheless, the Brazilian Health Ministry recommends the inclusion of methods for the detection of *Giardia* sp. cysts and *Cryptosporidium* sp. oocysts aiming to reach a standard in which the water supplied to the population must be free of these pathogens.

It should be emphasized that the detection of cysts and oocysts in superficial water is a crucial component to control these pathogens. However, the current methods present high variability of recovery efficiency of *Cryptosporidium* sp. oocysts and *Giardia* sp. cysts (Hsu et al., 2001), leading to the need of aggregating other types of methodology to guarantee that water potability achieves a higher degree of reliability. Due to lack of specific techniques for detection of Microsporidia and Coccidea in water and food, the analysis has been carried out by adaptations of methods used for clinical testing (Thurston-enriquez et al., 2002).

The goal of this study was to optimize and use parasitological and molecular techniques in the analysis and seasonal monitoring of opportunistic protozoa in water from fluvial systems for human usage in the municipality of Goiânia, the capital of the state of Goiás, in

**João Leite river** 

Opportunistic Protozoa in Rivers and Lakes: Relevance to Public Health in the Neotropics 345

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:

Fig. 2. João Leite river upstream of Goiania, after interbreeding Jurubatuba stream with the

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)

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

Posse stream, municipality of Goianapolis (Santos et al., 2008).

16°40'58.51"S latitude and 49°15'38.35"W longitude (BB) (Figure 5)

**Vaca Brava Park lake** 

**Bosque dos Buritis lake** 

(Figure 4).

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

the Midwestern Region of Brazil, focusing on *Cryptosporidium* sp., *Cyclospora cayetanensis, Isopora belli* and Microsporidia*.*
