Protozoa: Structure, Classification, Growth, and Development

General Concepts


Protozoa


Protozoa are one-celled animals found worldwide in most habitats. Most species are free living, but all higher animals are infected with one or more species of protozoa. Infections range from asymptomatic to life threatening, depending on the species and strain of the parasite and the resistance of the host.
Structure


Protozoa are microscopic unicellular eukaryotes that have a relatively complex internal structure and carry out complex metabolic activities. Some protozoa have structures for propulsion or other types of movement.
Classification


On the basis of light and electron microscopic morphology, the protozoa are currently classified into six phyla. Most species causing human disease are members of the phyla Sacromastigophora and Apicomplexa.
Life Cycle Stages


The stages of parasitic protozoa that actively feed and multiply are frequently called trophozoites; in some protozoa, other terms are used for these stages. Cysts are stages with a protective membrane or thickened wall. Protozoan cysts that must survive outside the host usually have more resistant walls than cysts that form in tissues.
Reproduction


Binary fission, the most common form of reproduction, is asexual; multiple asexual division occurs in some forms. Both sexual and asexual reproduction occur in the Apicomplexa.
Nutrition


All parasitic protozoa require preformed organic substancesthat is, nutrition is holozoic as in higher animals.



INTRODUCTION


The Protozoa are considered to be a subkingdom of the kingdom Protista, although in the classical system they were placed in the kingdom Animalia. More than 50,000 species have been described, most of which are free-living organisms; protozoa are found in almost every possible habitat. The fossil record in the form of shells in sedimentary rocks shows that protozoa were present in the Pre-cambrian era. Anton van Leeuwenhoek was the first person to see protozoa, using microscopes he constructed with simple lenses. Between 1674 and 1716, he described, in addition to free-living protozoa, several parasitic species from animals, and Giardia lamblia from his own stools. Virtually all humans have protozoa living in or on their body at some time, and many persons are infected with one or more species throughout their life. Some species are considered commensals, i.e., normally not harmful, whereas others are pathogens and usually produce disease. Protozoan diseases range from very mild to life-threatening. Individuals whose defenses are able to control but not eliminate a parasitic infection become carriers and constitute a source of infection for others. In geographic areas of high prevalence, well-tolerated infections are often not treated to eradicate the parasite because eradication would lower the individual's immunity to the parasite and result in a high likelihood of reinfection.
Many protozoan infections that are inapparent or mild in normal individuals can be life-threatening in immunosuppressed patients, particularly patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that many healthy persons harbor low numbers of Pneumocystis carinii in their lungs. However, this parasite produces a frequently fatal pneumonia in immunosuppressed patients such as those with AIDS. Toxoplasma gondii, a very common protozoan parasite, usually causes a rather mild initial illness followed by a long-lasting latent infection. AIDS patients, however, can develop fatal toxoplasmic encephalitis. Cryptosporidium was described in the 19th century, but widespread human infection has only recently been recognized. Cryptosporidium is another protozoan that can produce serious complications in patients with AIDS. Microsporidiosis in humans was reported in only a few instances prior to the appearance of AIDS. It has now become a more common infection in AIDS patients. As more thorough studies of patients with AIDS are made, it is likely that other rare or unusual protozoan infections will be diagnosed.
Acanthamoeba species are free-living amebas that inhabit soil and water. Cyst stages can be airborne. Serious eye-threatening corneal ulcers due to Acanthamoeba species are being reported in individuals who use contact lenses. The parasites presumably are transmitted in contaminated lens-cleaning solution. Amebas of the genus Naegleria, which inhabit bodies of fresh water, are responsible for almost all cases of the usually fatal disease primary amebic meningoencephalitis. The amebas are thought to enter the body from water that is splashed onto the upper nasal tract during swimming or diving. Human infections of this type were predicted before they were recognized and reported, based on laboratory studies of Acanthamoeba infections in cell cultures and in animals.
The lack of effective vaccines, the paucity of reliable drugs, and other problems, including difficulties of vector control, prompted the World Health Organization to target six diseases for increased research and training. Three of these were protozoan infectionsmalaria, trypanosomiasis, and leishmaniasis. Although new information on these diseases has been gained, most of the problems with control persist.
Structure


Most parasitic protozoa in humans are less than 50 µm in size. The smallest (mainly intracellular forms) are 1 to 10 µm long, but Balantidium coli may measure 150 µm. Protozoa are unicellular eukaryotes. As in all eukaryotes, the nucleus is enclosed in a membrane. In protozoa other than ciliates, the nucleus is vesicular, with scattered chromatin giving a diffuse appearance to the nucleus, all nuclei in the individual organism appear alike. One type of vesicular nucleus contains a more or less central body, called an endosome or karyosome. The endosome lacks DNA in the parasitic amebas and trypanosomes. In the phylum Apicomplexa, on the other hand, the vesicular nucleus has one or more nucleoli that contain DNA. The ciliates have both a micronucleus and macronucleus, which appear quite homogeneous in composition.
The organelles of protozoa have functions similar to the organs of higher animals. The plasma membrane enclosing the cytoplasm also covers the projecting locomotory structures such as pseudopodia, cilia, and flagella. The outer surface layer of some protozoa, termed a pellicle, is sufficiently rigid to maintain a distinctive shape, as in the trypanosomes and Giardia. However, these organisms can readily twist and bend when moving through their environment. In most protozoa the cytoplasm is differentiated into ectoplasm (the outer, transparent layer) and endoplasm (the inner layer containing organelles); the structure of the cytoplasm is most easily seen in species with projecting pseudopodia, such as the amebas. Some protozoa have a cytosome or cell "mouth" for ingesting fluids or solid particles. Contractile vacuoles for osmoregulation occur in some, such as Naegleria and Balantidium. Many protozoa have subpellicular microtubules; in the Apicomplexa, which have no external organelles for locomotion, these provide a means for slow movement. The trichomonads and trypanosomes have a distinctive undulating membrane between the body wall and a flagellum. Many other structures occur in parasitic protozoa, including the Golgi apparatus, mitochondria, lysosomes, food vacuoles, conoids in the Apicomplexa, and other specialized structures. Electron microscopy is essential to visualize the details of protozoal structure. From the point of view of functional and physiologic complexity, a protozoan is more like an animal than like a single cell. Figure 77-1 shows the structure of the bloodstream form of a trypanosome, as determined by electron microscopy.
FIGURE 77-1 Fine structure of a protozoan parasite, Typanosoma evansi, as revealed by transmission electron microcopy of thin sections. (Adapted from Vickerman K: Protozoology. Vol. 3 London School of Hygiene and Tropical Medicine, London, 1977, with permission.)
Classification


In 1985 the Society of Protozoologists published a taxonomic scheme that distributed the Protozoa into six phyla. Two of these phylathe Sarcomastigophora and the Apicomplexa--contain the most important species causing human disease. This scheme is based on morphology as revealed by light, electron, and scanning microscopy. Dientamoeba fragilis, for example, had been thought to be an ameba and placed in the family Entamoebidae. However, internal structures seen by electron microscopy showed that it is properly placed in the order Trichomonadida of flagellate protozoa. In some instances, organisms that appear identical under the microscope have been assigned different species names on the basis of such criteria as geographic distribution and clinical manifestations; a good example is the genus Leishmania, for which subspecies names are often used. Biochemical methods have been employed on strains and species to determine isoenzyme patterns or to identify relevant nucleotide sequences in RNA, DNA, or both. Extensive studies have been made on the kinetoplast, a unique mitochondrion found in the hemoflagellates and other members of the order Kinetoplastida. The DNA associated with this organelle is of great interest. Cloning is widely used in taxonomic studies, for example to study differences in virulence or disease manifestations in isolates of a single species obtained from different hosts or geographic regions. Antibodies (particularly monoclonal antibodies) to known species or to specific antigens from a species are being employed to identify unknown isolates. Eventually, molecular taxonomy may prove to be a more reliable basis than morphology for protozoan taxonomy, but the microscope is still the most practical tool for identifying a protozoan parasite. Table 77-1 lists the medically important protozoa.

Life Cycle Stages


During its life cycle, a protozoan generally passes through several stages that differ in structure and activity. Trophozoite (Greek for "animal that feeds") is a general term for the active, feeding, multiplying stage of most protozoa. In parasitic species this is the stage usually associated with pathogenesis. In the hemoflagellates the terms amastigote, promastigote, epimastigote, and trypomastigote designate trophozoite stages that differ in the absence or presence of a flagellum and in the position of the kinetoplast associated with the flagellum. A variety of terms are employed for stages in the Apicomplexa, such as tachyzoite and bradyzoite for Toxoplasma gondii. Other stages in the complex asexual and sexual life cycles seen in this phylum are the merozoite (the form resulting from fission of a multinucleate schizont) and sexual stages such as gametocytes and gametes. Some protozoa form cysts that contain one or more infective forms. Multiplication occurs in the cysts of some species so that excystation releases more than one organism. For example, when the trophozoite of Entamoeba histolytica first forms a cyst, it has a single nucleus. As the cyst matures nuclear division produces four nuclei and during excystation four uninucleate metacystic amebas appear. Similarly, a freshly encysted Giardia lamblia has the same number of internal structures (organelles) as the trophozoite. However, as the cyst matures the organelles double and two trophozoites are formed. Cysts passed in stools have a protective wall, enabling the parasite to survive in the outside environment for a period ranging from days to a year, depending on the species and environmental conditions. Cysts formed in tissues do not usually have a heavy protective wall and rely upon carnivorism for transmission. Oocysts are stages resulting from sexual reproduction in the Apicomplexa. Some apicomplexan oocysts are passed in the feces of the host, but the oocysts of Plasmodium, the agent of malaria, develop in the body cavity of the mosquito vector.
Reproduction


Reproduction in the Protozoa may be asexual, as in the amebas and flagellates that infect humans, or both asexual and sexual, as in the Apicomplexa of medical importance. The most common type of asexual multiplication is binary fission, in which the organelles are duplicated and the protozoan then divides into two complete organisms. Division is longitudinal in the flagellates and transverse in the ciliates; amebas have no apparent anterior-posterior axis. Endodyogeny is a form of asexual division seen in Toxoplasma and some related organisms. Two daughter cells form within the parent cell, which then ruptures, releasing the smaller progeny which grow to full size before repeating the process. In schizogony, a common form of asexual division in the Apicomplexa, the nucleus divides a number of times, and then the cytoplasm divides into smaller uninucleate merozoites. In Plasmodium, Toxoplasma, and other apicomplexans, the sexual cycle involves the production of gametes (gamogony), fertilization to form the zygote, encystation of the zygote to form an oocyst, and the formation of infective sporozoites (sporogony) within the oocyst.
Some protozoa have complex life cycles requiring two different host species; others require only a single host to complete the life cycle. A single infective protozoan entering a susceptible host has the potential to produce an immense population. However, reproduction is limited by events such as death of the host or by the host's defense mechanisms, which may either eliminate the parasite or balance parasite reproduction to yield a chronic infection. For example, malaria can result when only a few sporozoites of Plasmodium falciparumperhaps ten or fewer in rare instancesare introduced by a feeding Anopheles mosquito into a person with no immunity. Repeated cycles of schizogony in the bloodstream can result in the infection of 10 percent or more of the erythrocytesabout 400 million parasites per milliliter of blood.
Nutrition


The nutrition of all protozoa is holozoic; that is, they require organic materials, which may be particulate or in solution. Amebas engulf particulate food or droplets through a sort of temporary mouth, perform digestion and absorption in a food vacuole, and eject the waste substances. Many protozoa have a permanent mouth, the cytosome or micropore, through which ingested food passes to become enclosed in food vacuoles. Pinocytosis is a method of ingesting nutrient materials whereby fluid is drawn through small, temporary openings in the body wall. The ingested material becomes enclosed within a membrane to form a food vacuole.
Protozoa have metabolic pathways similar to those of higher animals and require the same types of organic and inorganic compounds. In recent years, significant advances have been made in devising chemically defined media for the in vitro cultivation of parasitic protozoa. The resulting organisms are free of various substances that are present in organisms grown in complex media or isolated from a host and which can interfere with immunologic or biochemical studies. Research on the metabolism of parasites is of immediate interest because pathways that are essential for the parasite but not the host are potential targets for antiprotozoal compounds that would block that pathway but be safe for humans. Many antiprotozoal drugs were used empirically long before their mechanism of action was known. The sulfa drugs, which block folate synthesis in malaria parasites, are one example.
The rapid multiplication rate of many parasites increases the chances for mutation; hence, changes in virulence, drug susceptibility, and other characteristics may take place. Chloroquine resistance in Plasmodium falciparum and arsenic resistance in Trypanosoma rhodesiense are two examples.
Competition for nutrients is not usually an important factor in pathogenesis because the amounts utilized by parasitic protozoa are relatively small. Some parasites that inhabit the small intestine can significantly interfere with digestion and absorption and affect the nutritional status of the host; Giardia and Cryptosporidium are examples. The destruction of the host's cells and tissues as a result of the parasites' metabolic activities increases the host's nutritional needs. This may be a major factor in the outcome of an infection in a malnourished individual. Finally, extracellular or intracellular parasites that destroy cells while feeding can lead to organ dysfunction and serious or life-threatening consequences.
REFERENCES


Englund PT, Sher A (eds): The Biology of Parasitism. A Molecular and Immunological Approach. Alan R. Liss, New York, 1988
Goldsmith R, Heyneman D (eds): Tropical Medicine and Parasitology. Appleton and Lange, East Norwalk, CT, 1989
Lee JJ, Hutner SH, Bovee EC (eds): An Illustrated Guide to the Protozoa. Society of Protozoologists, Lawrence, KS, 1985
Kotler DP, Orenstein JM: Prevalence of Intestinal Microsporidiosis in HIV-infected individuals referred for gastrointestinal evaluation. J Gastroenterol 89: 1998, 1994
Neva FA, Brown H: Basic Clinical Parasitology, 6th edition, Appleton & Lange, Norwalk, CT, 1994