Protozoa are unicellular eukaryotes, meaning that they have characteristic organelles -- a specialized cellular part (as a mitochondrion, lysosome, or ribosome) that’s analogous to an organ -- and are large (some are visible to the naked eye). Although they’re single-celled organisms, they’re by no means simple in structure. In fact, many diverse forms are observed among the more than 65,000 named species. Such morphological variability, evolved over hundreds of millions of years, has enabled protozoan adaptation to a wide variety of environments. Along with algae and other unicellular eukaryotes, protozoa are classified under the kingdom Protista.

Protozoa are found in nearly all terrestrial and aquatic environments and play a valuable role in ecological cycles. Many species are able to exist in extreme environments, from polar regions to hot springs and dry desert soils. Protozoa are known to aid in a number of natural processes such as contributing to the food chain, microbial population control, and environmental decomposition rates. Able to act as predator and prey, they may control bacterial biomass and in turn provide a major fraction of the primary food source for higher animals in surface waters. Sewage treatment plant filters may sustain protozoan populations as high as 5 × 10⁴ cells per milliliter (ml) of activated sludge where they aid in particulate removal and the reduction of bacteria via grazing and flocculation to provide a higher quality effluent.

Parasitic protozoa may either cause significant disease or actually help the invaded host. For example, protozoa found in the intestines of wood-eating termites digest cellulose material to acceptable nutrients for the insect, without which the decomposer would starve to death. In recent years, however, protozoan pathogens such as Giardia, Cryptosporidium and Microsporidia have emerged to become a leading concern with regard to safe drinking water and the human host.

Structural characteristics
Protozoa may be free-living, capable of growth and reproduction outside any host, or parasitic -- meaning they colonize host cell tissues. Some are opportunists, adapting either a free-living or parasitic existence as their environment dictates. The size of a protozoan varies from 2 microns (µm) to several centimeters, depending on the species. Many have locomotive structures and are mobile in aqueous environments.

All protozoa have the equipment necessary to complete essential functions of life, such as to obtain energy, metabolize and reproduce. Most protozoa obtain their energy by feeding on bacteria, algae or other protozoa. Many parasitic protozoa have an environmentally resistant stage as part of their life cycle. The resistant stage of Cryptosporidium is called an oocyst, which is similar to the cyst stage of Giardia. Cysts or oocysts are defined as a metabolically dormant, protective phase that allows the protozoa to survive adverse conditions such as aging, starvation, desiccation or other environmental stress.

Each Cryptosporidium oocyst is 4 µm in diameter and stores as many as four infective structures known as sporozoites. Each sporozoite has the ability to develop into an infective, intracellular form, known as a trophozoite, which eventually produces eight progeny organisms known as merozoites. The merozoites burst the host cell and invade neighboring cells, which may ultimately result in adverse health effects.

The primary reason for encystment of protozoa -- transforming into a hard-shelled cyst or oocyst -- is survival under adverse conditions during widespread dispersal throughout the ecosystem via air, water, birds, or passage through the intestinal tracts of these or other animals.

Parasitic protozoa
Of the more than 65,000 species of protozoa -- approximately 20 percent are parasitic -- meaning that they require a host for completion of their life cycle, which may or may not be a detriment to the host. Some species have commensal relationships with their host where one uses the other but neither is harmed, while others join in symbiotic relationships where both host and parasite benefit. Nearly all disease-causing protozoa, however, are tissue parasitic -- feeding on the tissues of host organisms -- causing mild to serious illness and possible death.

A variety of parasitic protozoa are of obvious public health concern, causing such diseases as malaria, sleeping sickness, Chagas disease, leishmaniasis, giardiasis and cryptosporidiosis, to name a few. They’re able to evade host immune responses and have adapted to long-term survival and continual reproduction in hosts, producing chronic illnesses. A number of protozoa are intestinal parasites of humans and domestic animals and may be present in the environment in an encysted form. These hearty cysts are known to survive conventional methods of disinfection and thus can be transmitted to their host via a water route.

Parasitic protozoa commonly have water-related routes of infection. Entamoeba histolytica is a parasitic amoeba, with a cyst stage, causing diarrhea and dysentery. Naegleria is a free-living amoeba, sometimes present in fresh water, capable of infecting nasal passages of humans and invading brain tissues, always resulting in fatality. Toxoplasma -- a highly nonspecific, invasive protozoa, thought to infect any mammal and all cell types within a given host -- causes blindness and serious illness or death in unborn fetuses. Plasmodium species are responsible for the mosquito-borne disease malaria, which affects human populations worldwide. Cryptosporidium is responsible for a number of epidemics including the largest U.S. waterborne outbreak to date -- Milwaukee in 1993.

While in the system
When present in water, Cryptosporidium oocysts are ingested by the host where body temperature and bile salts aid in the breakdown of the oocyst wall, resulting in excystation, the release of sporozoites. The protozoa may colonize the gut, be released into the blood stream, and eventually invade organ tissues or the lymphatic system. Infection in healthy hosts is usually self-limiting, albeit after a case of profuse, watery diarrhea lasting for days, but for the immunocompromised, protozoan infections can prove persistent and often fatal. Currently, there are no available drugs effective at controlling or preventing the disease.

Domestic animals are also at risk of serious illness and death from protozoan infections. For example, Histomonas has a 50-100 percent kill rate in infected turkeys and Trichomonas causes early abortion in 50-100 percent of infected cows.

Control of parasitic protozoa has proved problematic since they tend to be relatively small in size, are resistant to conventional water disinfectants, and have a wide range of transmission routes including water, food, vectors (mosquitoes, ticks, flies and fleas), and direct person-to-person contact. In addition, a variety of mammals (rodents, dogs, monkeys, cats, apes, humans, cows and many more), reptiles, and birds serve as reservoir hosts, aiding in the spread of parasitic protozoan populations.

In finished water
Cryptosporidium is common and widespread in ambient waters where they can persist for months. Outbreaks have occurred following state-of-the art municipal treatment, particularly in immunocompromised populations. Monitoring for Cryptosporidium directly is expensive and difficult and bacterial indicators often fail to predict the presence of protozoan pathogens.

In more than 25 studies, Cryptosporidium has been found in between 5.6 and 87.1 percent of source waters (i.e., surface, spring, and groundwater samples not impacted by domestic and/or agricultural waste) at a concentration of 0.0003 to 4.74 oocysts per liter (L).¹ Oocysts have also been reported in 60.2 percent of surface waters tested in the United States and Canada.² Recently of 199 groundwater samples surveyed, 5 percent of vertical wells, 20 percent of springs, 50 percent of infiltration galleries, and 45 percent of horizontal wells tested positive for Cryptosporidium oocysts, calling for a re-evaluation of the notion that groundwater is inherently free of protozoan parasites.³

Cryptosporidium is now regulated by the federal government as a primary drinking water contaminant. The USEPA’s Interim Enhanced Surface Water Treatment Rule is directly focused on the control of Cryptosporidium by setting a maximum contaminant level goal of zero for water utilities using surface water or groundwater under the direct influence of surface water and serving >10,000 people. For systems filtering water, the rule requires a minimum 2 log (99 percent) removal efficiency of Cryptosporidium. While this rule is expected to lower the rate of epidemic disease and reduce the likelihood of the occurrence of outbreaks, deficiencies in water treatment systems are often cited as a major reason for outbreaks. History has shown that even the best of systems can be overtaxed by a high density of oocysts entering the source waters over a short time period. To make matters worse, fewer than 30 oocysts may cause disease in humans.

Conclusion
Municipal water treatment alone has proven inadequate for protection of highly susceptible individuals with respect to protozoan pathogens. There is great need for application of effective disinfection (i.e., ultraviolet and ozone) and further development of alternative methods for inactivation and removal of Cryptosporidium at municipalities. Specific point-of-use filtration devices can offer additional protection at the tap, particularly those with an absolute pore size of 1 µm or smaller, certified by ANSI/NSF Standard 53 for cyst removal, or utilizing reverse osmosis. For maximum protection, immunocompromised individuals are advised by the USEPA and the Centers for Disease Control and Prevention to boil their drinking water for 1-2 minutes prior to use.

References
1. Lisle, J.T., and J.B. Rose, “Cryptosporidium contamination of water in the USA and UK: a mini-review,” Journal of Water SRT-Aqua, 44:3:103-117, 1995.

2. LeChevallier, M.W., and W.D. Norton, “Giardia and Cryptosporidium in raw and finished water,” Journal of the American Water Works Association (AWWA) , 87:9:54-68, 1995.

3. Hancock, C.M., et al., “Crypto and Giardia in U.S. groundwater,” Journal AWWA, 90:3:58-61, 1998.

About the author
Dr. Kelly A. Reynolds is a research scientist at the University of Arizona with a focus on development of rapid methods for detecting human pathogenic viruses in drinking water. She holds a master of science degree in public health (MSPH) from the University of South Florida and doctorate in microbiology from the University of Arizona. Reynolds has also been a member of the WC&P Technical Review Committee since 1997.