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Introduction to Protozoa PDF Print E-mail
Sunday, 07 February 2010 00:00

Eric Villegas


One of the most important natural resources in the world is water. Tremendous amounts of resources and efforts have been invested in keeping our water clean and safe. But in spite of these efforts and advances in water treatment technologies, waterborne disease outbreaks still occur and continue to threaten drinking water quality and safety in developed and developing countries. In 1996, the World Health Organization reported that at least 25% of the developing world’s population does not have access to safe drinking water and over 66% lack adequate sanitation systems contributing to the continued occurrences of waterborne infectious diseases related illnesses worldwide ( WHO 1996). There are many protozoan parasites that have been documented to cause a significant proportion of the reported cases of waterborne disease with cryptosporidiosis and giardiasis being the leading two illnesses reported ( Karanis et al. 2007). This section will currently focus on Giardia intestinalis, Cryptosporidium spp., Naegleria fowleri, Toxoplasma gondii, and Cyclospora cayetanensis. Organisms like Entamoeba histolytica, Acanthamoeba, Balantidium coli, Sarcocystis spp., Isospora belli, and Blastocystis spp. may be added as more information (in regard to epidemiology, prevalence, detection methodology, etc.) becomes available. What makes these parasites so successful in causing waterborne disease outbreaks? What are the major issues concerning water quality and human health risks? Are current disinfection treatment practices effective at inactivating these parasites? What do we know about occurrences, and population diversity of these parasites in water? What steps are being taken to minimize exposure to these contaminants? This portal is intended to start addressing these key questions and provide the reader a landscape of the diversity, occurrences and human health risks of these groups of pathogens. It describes seminal papers that have pushed the field and will also provide the reader the current trajectory of researches conducted globally.

Typical symptoms arising from parasitic infections can vary from causing mild gastro intestinal distress and diarrhea (e.g., Giardia, Cryptosporidium and C. cayetanensis), flu-like symptoms, lethargy, splenomegaly, lymphadenopathy, to severe neurological disease and death (T. gondii and N. fowleri) in healthy individuals. There are only a few drugs available to treat these diseases but most are expensive not very efficacious. There are also no available vaccines making protozoan parasites one of the most important group of waterborne contaminants threatening human health. Although a substantial amount of research to advance environmental monitoring and clinical and molecular epidemiology of waterborne protozoan diseases have been conducted, significant knowledge gaps still exist.

What make these parasites so successful at causing disease are their inherent abilities to survive in the environment for years. Oocysts and cysts from this group of pathogens are ubiquitous. Many species are zoonotic and use humans and animals as reservoirs. Other key features that enable them to persist in drinking water systems include their relatively small size and their ability to survive various disinfection treatment conditions (e.g., chlorine). For example, Cryptosporidium oocysts are around 4 - 6 mm in size allowing them to easily pass through the filtration barrier system ( Fayer R 2008). These parasites along with T. gondii are also resistant to chlorine disinfection ( Betancourt and Rose 2004; Erickson and Ortega 2006; Wainwright et al. 2007). Both can survive chlorine levels well above concentrations in finished drinking water. Studies by Wainwright and others demonstrated that T. gondii oocysts exposed to 100mg/L are still infectious to mice ( Wainwright et al. 2007). More recently, T. gondii oocysts treated with 10% Clorox® bleach (equivalent to at least 6,000 mg/L free chlorine) for twenty four hours only resulted in a 1-log10 reduction in infectivity (Villegas, unpublished). Ozone is generally considered a more efficient disinfectant against protozoa in water systems than chlorine or chlorine dioxide. Other physical treatment processes that can affect survival of protozoan pathogens include freezing, heating, filtration, sedimentation, UV irradiation, high pressure, and ultrasound ( Erickson and Ortega 2006). UV disinfection has received much attention in the water industry and showed promise to be efficient at inactivating these pathogens ( Erickson and Ortega 2006). Further details about inactivation of protozoan pathogens in food, water, and environmental systems and their removal from drinking water systems can be found in reviews by Erickson and Ortega ( Erickson and Ortega 2006) and Betancourt and Rose ( Betancourt and Rose 2004).

The most infamous of all the protozoa is Cryptosporidium. In 1993, a massive cryptosporidiosis outbreak infecting over 400,000 people was reported ( Mac Kenzie et al. 1994). Today, Cryptosporidium along with Giardia remain the two leading causes of waterborne protozoan illnesses worldwide ( Karanis et al. 2007). Extensive source tracking and occurrence data is readily available for these two organisms since these pathogens are regulated and/or monitored in the drinking water systems in the United States, United Kingdom, and other countries around the world ( Hunter and Thompson 2005; Xiao and Fayer 2008; Xiao and Ryan 2008). Waterborne outbreaks of cryptosporidiosis continue to rise in the US with an increase in outbreaks related to treated recreational waters ( Yoder and Beach 2009). The increase may be due to improved case reporting, detection methodologies, and standardizations used by health laboratories. Little is still known about the occurrences of C. cayetanensis and T. gondii, primarily due to the lack of standardized detection methods for these organisms in drinking water, but both have been implicated to have caused several waterborne disease outbreaks ( Bahia-Oliveira et al. 2003; Erickson and Ortega 2006; Jones and Dubey 2010). N. fowleri occurrence data is even scarcer, predominantly in the south or in warmer regions of the US ( Yoder et al. 2009).

The gold standard for detecting environmental protozoa is US Method 1622 and US Method 1623 which are used to monitor for the presence of Giardia and/or Cryptosporidium in source waters ( USEPA 2005a; USEPA 2005b). These methods consist of three main parts: 1) filtration of at least 10L of water, 2) (oo)cyst concentration using antibody based immunomagnetic separation procedure and a 3) detection component using immunofluorescence and differential interference contrast microscopy to enumerate total (oo)cysts from the sample. While these approaches are useful in monitoring for total numbers of oocysts in a sample, they do not have the ability to determine the species/genotype of the (oo)cysts. They also cannot assess infectivity/viability of the organism. A significant amount of efforts have been focused on developing a “protozoan detection toolbox” that will allow for the detection of protozoan contaminants in environmental water matrices. A partial list of the tools that have been made to date is briefly described below. A more exhaustive description of the advantages and limitations of these assays are described within each the protozoan section.

For species identification and genotyping tools for Cryptosporidium oocysts, PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), Randomly Amplified Polymorphic DNA (RAPD), multi-locus sequence typing, Single Stranded Conformational Polymorphism (SSCP), and sequencing of the SSUrRNA or the gp60 genes have all been used successfully, albeit at varying degrees ( Bouzid et al. 2008; Ferguson et al. 2006; Morgan et al. 1996). Similar approaches have also been developed for Giardia ( Bouzid et al. 2008; Caccio and Ryan 2008; Xiao and Fayer 2008), T. gondii ( Ajzenberg et al. 2005; Dumetre and Darde 2003; Miller et al. 2004; Schwab and McDevitt 2003), and N. fowleri ( Schuster and Visvesvara 2004). Other advances in molecular detection technologies that have been applied for water quality monitoring include fluorescence in-situ hybridization (FISH) (16, 24), real-time quantitative PCR (qPCR) ( Varma et al. 2003; Yang et al. 2009), Loop-mediated isothermal amplification LAMP ( Karanis and Ongerth 2009; Karanis et al. 2007; Plutzer and Karanis 2009; Sotiriadou and Karanis 2008; Zhang et al. 2009) and nucleic acid sequence based amplification (NASBA) ( Baeumner et al. 2001). More recently, multiplex PCRs and microarray technologies have been utilized to develop a multi-pathogen detection assay, which have the potential to simultaneously detect several dozen waterborne pathogens in a reaction ( Haque et al. 2007; Straub et al. 2002; Wang et al. 2004). Tools to determine viability/infectivity of the organism have also been developed. The techniques range from traditional in vitro cell culture assays ( Arrowood MJ 2008; Arrowood MJ 2002; Dumetre et al. 2008; Villegas submitted), integrated cell culture/PCR detection techniques ( Di Giovanni and LeChevallier 2005; Rochelle et al. 1996), targeting messenger RNA ( Jenkins et al. 2003; Widmer et al. 1999; Villegas submitted), to using photocatalytic nucleic acid intercalating dyes in conjunction with PCR to determine viability ( Brescia et al. 2009). With all these assays in our protozoan detection toolbox, no single assay has been standardized and no consensus on which assays are best suited for environmental monitoring has been established. The community must also be cognizant that our research should be question driven instead of technology or tool driven. There is still no one method or “silver bullet” that can simultaneously address the critical questions of: how much, what is it, is it alive and does it cause disease, nor will it be available in the near future.

Each section in this website describes the state-of-the science for each protozoan listed in hopes of providing critical information to the community about the importance of these pathogens in our water and provides the state-of-the science and future directions for each pathogen.


Last Updated on Tuesday, 17 August 2010 07:50


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