PROTOZOA

INTRODUCTION
• Unicellular, chemoheterotrophic, eucaryotic organisms of kingdom Protista (3-2000
mm).
• Protozoan means “first animal”.
• 20,000 species, only a few are pathogens. Most are free-living organisms that inhabit
water and soil. Some live in association with other organisms as parasites or symbionts.
• Reproduce asexually by fission, budding, or schizogony.
• Some exhibit sexual reproduction (e.g.: Paramecium).
• Trophozoite: Vegetative stage which feeds upon bacteria and particulate nutrients.
• Cyst: Some protozoa produce a protective capsule under adverse conditions (toxins,
scarce water, food, or oxygen).

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.

Fine structure of a protozoan parasite, Typanosoma evansi, as revealed by transmission electron microcopy of
thin sections.
Locomotion

a. Protozoa employ a variety of mechanisms of locomotion and may be

classified according to these mechanisms of locomotion:
i. trophozoite
ii. flagellates
iii. ciliates
iv. amoebas
v. sporozoans
b. Additionally there exist fungus-like protozoa.

Trophozoite: The trophozoite is the motile feeding stage of protozoa. Some protozoa
exhibit no other stage. Usually a sufficient moisture levels and food supplies are
necessary to maintain this stage of the protozoa life cycle.

Flagellates: The flagellates are protozoa which move by means of flagellar action.
Some flagellates have their flagella attached in a structure called an undulating
membrane. Interestingly, flagellates tend to have symbiotic relationships with
multicelled organisms.

Ciliates: The ciliates are protozoa which move by means of cilia action. Because of
the tremendous variety in ciliary arrangements and functions, ciliates are among the
most diverse and awesome cells in the biological world." The ciliates, interestingly,
represent a branch of the protozoa particularly close evolutionarily to the fungi,
plants, and animals. They are also among the more advanced of protozoa. Example:
The paramecia are ciliates

Amoebas: Amoebas are protozoa which move by employing pseudopodia, which are
membrane covered cytoplasmic extensions. Many amoebas employ their pseudopodia
also to engulf food. Amoebas live in moist terrestrial or aquatic environments.
Amoebas include among their members various protozoa which form calcium-based
or silicon-based shells (the foraminiferans and the radiolarians, respectively). Chalk
deposits such as the White Cliffs of Dover formed from the shells of numerous, long
dead foraminiferans.

Sporozoans : The sporozoans are parasitic spore formers which do not locomote
under their own power. Plasmodium spp., the cause of malaria, are sporozoans.
Microspora are an extremely ancient eucaryote lineage also classified as sporozoans.

Fungus-like protozoa

Exoenzymes and nutrient absorption:

i. Protozoa exist which occupy a fungus-like niche and employ the

very fungal nutrient acquiring strategy of secreting exoenzymes
and absorbing the solubilized nutrients.
ii. These include the following:
1. slime molds
2. cellular slime molds
3. water molds

Nutrition:

Varied nutrient sources:

Descriptions of protozoa nutrient acquisition types include:

1. holozoic

2. holophytic

3. saprozoic (or saprophytic)

Holozoic

a. Holozoic describes the obtainment of nutrients by

engulfing.
 b. Generally engulfers:
i. Free living protozoa tend to consume:
1. animal debris
2. plant debris
3. bacteria
4. or other protozoa
ii. The majority of protozoa are holozoic.
c. Adaptation to wet environments:
i. Engulfment is probably a good strategy
particularly in wet, well mixed
environments.
ii. Here exoenzymes would probably be
unsuitable due to the large diffusion factor.
iii. Engulfment results in intracellular (though
not cytoplasmic) digestion thus limiting the
volume to which digestive enzymes are
exposed.

Holophytic [photoautotrophic]: Holophytic describes the

obtainment of nutrients by the same method as algae,
cyanobacteria, and green plants (i.e., photoautotrophic). Some,
but only a minority of protozoa are holophytic

Saprozoic [saprophytic]: Saprozoic (or saprophytic) protozoa

absorb nutrients from the extracellular environment.

Classification

In 1985 the Society of Protozoologists published a taxonomic scheme that distributed the
Protozoa into six phyla. Two of these phyla the 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.
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 falciparum perhaps ten or fewer in
rare instances are 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 erythrocytes about 400 million parasites per milliliter of blood.

The life cycle of a plasmodial slime mold

 The life cycle of cellular slime moulds

Disease caused by protozoa

GIARDIASIS

Giardia lamblia (also known as G. duodenalis) is a protozoan parasite that colonizes the upper
portions of the small intestine. In fact, it was probably the first symbiotic protozoan ever
observed. Typically Giardia is non-invasive and often results in asymptomatic infections.
Symptomatic giardiasis is characterized by acute or chronic diarrhea and/or other gastro-intestinal
manifestations.

Metronidazole, although not licensed in the United States for giardiasis, effectively clears
the parasite (cure rates approximately 85%) and is the drug of choice. The recommended dosage
is 750 mg three times per day for five days. For children 15 mg/kg/d in three doses is
recommended. Other effective drugs include: quinacrine, tinidazole, furazolidone, and
paramomycin. Tinidazole is effective as a single two gram dose; paramomycin is not absorbed
and may be useful during pregnancy.
 Health promotion and education aimed at improving personal hygiene, and emphasizing
hand washing, sanitation and food handling, are effective control activities for the reduction of
person-to-person transmission. Boiling or iodine treatment kills Giardia cysts, but standard
chlorination does not. There are no safe or effective chemoprophylatic drugs for giardiasis.

TRICHOMONIASIS

Trichomonas vaginalis was first described from purulent vaginal discharges in 1836 and by the
early part of the twentieth century was recognized as an etiological agent of vaginitis.
Trichomoniasis is believed to be the most common non-viral sexually transmitted disease.
Despite the frequency of trichomoniasis it has in the past been considered more of a nuisance
parasite rather than a major pathogen. However it is now recognized a factor in promoting HIV
infection, causing low-weight and premature births, and predisposing women to substantial
discomfort and stress. T. vaginalis infection typically elicits a local cellular immune response
with inflammation of the vaginal epithelium and cervix in women and the urethra of men. In
addition, T. vaginalis can cause punctate hemorrhages on the vaginal walls and cervix.

Diagnosis is confirmed by the demonstration of trophozoites in vaginal, urethral,

prostatic secretions, or urine sediment. Antibody and DNA-based tests with high sensitivity and
specificity are being developed. Metronidazole and other nitroimidazoles, such as tinidazole, are
highly effective against trichomoniasis.

DIENTAMOEBIASIS

Dientamoebiasis is a medical condition caused by infection with Dientamoeba fragilis.

Dientamoeba fragilis is a single celled parasite that infects the lower gastrointestinal tract
of humans. Dientamoeba fragilis include abdominal pain (69%) and diarrhea (61%).
Additional symptoms include weight loss, fatigue, nausea and vomiting, fever, uritcaria
(skin rash), Pruritis (itchiness), and biliary infection

Successful treatment of the infection with Iodoquinol, Doxycycline, Metronidazole,

Paromomycin, and Secnidazole have been reported
BALANTIDOSIS

B. coli usually lives as a non-pathogenic commensal in the large intestine and produces no
symptoms. Mild or chronic infections are characterized by intermittent diarrhea and constipation,
weight loss, and abdominal pain. On rare occasions the trophozoites will invade the intestinal
epithelium and produce ulceration. Clinically this results in an acute diarrhea with mucus and
blood (ie, dysentery). Rare extra-intestinal infections involving lungs, vagina, ureter and urinary
bladder and intestinal perforations leading to peritonitis have been reported.

The treatment of choice is tetracycline given at 500 mg four times per day for 10 days.
Iodoquinol is the recommended alternate drug. Metronidazole has not produced consistent results.
Preventive measures are the same as other diseases transmitted by the fecal-oral route. In
addition, pig sewerage should be kept away from supplies of drinking water and food.

AMEBIASIS

E. histolytica is considered pathogenic and the disease it causes is called amebiasis or amebic
dysentery. E. histolytica exhibits a typical fecal-oral life cycle consisting of infectious cysts
passed in the feces and trophozoites which replicate within the large intestine. Contaminated food
and water are probably the primary sources of infection. However, E. histolytica is rarely the
cause of travelers' diarrhea and is usually associated with a long-term stay in an endemic area.
Upon ingestion the cysts pass through the stomach and excyst in the lower portion of the small
intestine.
Amebiasis Treatment

Drugs Uses

Iodoquinol, Paromomycin, or Luminal agents to treat asymptomatic cases and as a follow up

Diloxanide furoate treatment after a nitroimidazole.
Treatment of nondysenteric colitis, dysentery, and extra-intestinal
Metronidazole or Tinidazole
infections.

Treatment of severe disease such as necrotic colitis, perforation of

Dehydroemetine or Emetine
intestinal wall, rupture of liver abscess.

COCCIDIOSIS

Coccidiosis is a disease of fowl caused by a microscopic animal or protozoa and is characterized

by diarrhea, unthriftiness and variable levels of mortality. In spite of much research to advance
the control and treatment of this disease, it remains the most costly disease of the poultry
industry.

Coccidiosis is transmitted by direct or indirect contact with droppings of infected birds. When a
bird ingests coccidia, the organisms invade the lining of the intestine and produce tissue damage
as the undergo reproduction. Within a week after infection, the coccidia shed immature
descendants that are referred to as oocysts. The oocysts shed in the droppings are not capable of
infecting another bird unless they pass through a maturation process (sporulation) in the litter.
This sporulation occurs within a one to three day period if the litter is warm and damp but can
take much longer if the conditions are cool and dry. After sporulation the coccidia are infective if
consumed by a new host bird.

Coccidiosis usually occurs in growing birds and young adults. It is seldom seen in birds under
three weeks or in mature birds. Signs of an outbreak include birds that are pale, droopy, tend to
huddle, consume less feed and water, have diarrhea, and may become emaciated and dehydrated.
Laying hens will experience a reduction in rate of egg production.
Cecal coccidiosis may produce bloody droppings and anemia that is often followed by death.
Intestinal coccidiosis is not as acute and is more chronic in nature. It produces less mortality than
the cecal form.

Cecal coccidiosis may be confused with blackhead and salmonellosis due to their similar lesions.
Intestinal coccidiosis may be confused with hemorrhagic anemia syndrome and other enteric
diseases. Definite diagnosis is made from the microscopic examination of scrapings of the
digestive tract and identification of the coccidia organisms. Since it is common for healthy birds
to possess some coccidia, consideration of flock history and lesions must be considered before
making diagnosis and treatment recommendations.

BLACKHEAD (Histomoniasis, Enterohepatitis)

Blackhead is an acute or chronic protozoan disease of fowl, primarily affecting the cecae and
liver. Blackhead is caused by a protozoan parasite called Histomonas meleagridis. The disease is
present wherever poultry are raised. Blackhead is one of the critical diseases of growing turkeys
and game birds. It may cause stunted growth, poor feed utilization and death. It is of lesser
economic importance in chickens since they are more resistant, but the incidence in chickens
apparently is increasing.

Lesions of uncomplicated blackhead are confined to the cecae and liver, thus the reason for the
synonymous term, enterohepatitis. The cecae are ballooned and walls may be thickened, necrotic
and ulcerated. Caseous (cheesy) cores within the cecae may be blood tinged. Peritonitis may be
present if ulcers have perforated the ceca walls. Livers are swollen and display circular depressed
areas of necrosis about one-half inch in diameter. Smaller lesions coalesce to form larger ones.
Lesions are yellowish to yellow-green and extend deeply into the underlying liver tissue. Healing
lesions may resemble those seen in visceral leukosis.

Good management practices can do much to control the blackhead problem. Do not keep birds of
different species on the same premises. Do not range turkeys on ground previously used by
chickens unless several years have elapsed. Rotate ranges periodically if possible. Cecal worm
control is necessary to reduce blackhead incidence. Wire or slatted floors reduce exposure.

Good management is the only effective method of preventing this disease since many of the
effective drugs used in past years are no longer available commercially. Drugs that reduce the
presence of cecal worms, and thus reduce the infection rate, are available but do not have an
effect on the Histomonas organism. Refer to the cecal worm section for recommended control
practices.

HEXAMITIASIS (Infectious Catarrhal Enteritis)

Hexamitiasis is an acute infectious disease of turkeys, quail, ducks, chukar partridges and
pigeons. Heavy losses have been reported in one outbreak in ring-necked pheasants. Chickens
apparently are not affected.

Hexamitiasis is a problem in every commercial turkey-producing area. It may be a major problem

in localized areas during a particular year, followed by one or more years in which incidence is
low.

Hexamitiasis is caused by a one-celled parasite of the genus Hexamita. Hexamita meleagridis is

the cause in turkeys; in pigeons it is Hexamita columbae. This disease is found primarily in young
birds, and outbreaks seldom occur in poults past ten or eleven weeks. Losses are most severe in
birds three to five weeks old. Apparently, resistance develops rapidly with increasing age,
regardless of previous exposure.

The primary infection source is droppings from carrier birds. About a third of recovered birds
become carriers. Most outbreaks result from a buildup of organisms through several broods of
poults, making exposure of the following brood overwhelming. Indirect transmission may result
from fecal material carried from one location to another on shoes or equipment. Free-flying birds
also may be carriers.

Primary symptoms are listlessness and foamy or watery diarrhea with rapid weight loss due to the
dehydrating effect. Birds often huddle together near the heat source and cry or "chirp" constantly
as though in pain. Convulsions due to lowered blood sugar levels shortly precede death. Affected
birds suffer losses in weight and survivors remain stunted.

Dehydration and emaciation are the principal gross lesions. Muscles are dark and dry. The
intestine usually appears to have lost muscle-tone. Intestinal contents are usually thin and watery,
or may contain mucus.

Diagnosis depends upon history, symptoms and microscopic examination of intestinal

contents. A definite diagnosis cannot be made unless typical flagellated organisms can be
detected in intestinal contents of the duodenum. Most flagellate organisms in the cecae
are not disease producers.

Chagas' disease [Trypanosoma cruzi]

a. Arthropod vector:
i. Chagas' disease shows a step up in complexity from that of
toxoplamosis because the Chagas' disease parasite, Trypanosoma
cruzi, is spread via an arthropod vector.
ii. Trypanosoma cruzi parasitizes numerous mammalian hosts
including dogs, cats, opossums, armadillos, and foxes. These
presumably serve as the reservoir for T. cruzi from the point of
view of humanity.
iii. An insect vector parochially referred to as the "kissing bug" picks
up the protozoa when acquiring a blood meal from an infected
animal.
iv. T. cruzi replicates in this arthropod's intestinal lumen and
accumulates in its feces.
b. Infection:
i. Upon acquiring a blood meal the bug simultaneously deposits
feces on or near the bite.
ii. Scratching of the bite by the mammalian host contributes to the
entry of the parasite.
c. Disease:
i. Within mammalian hosts, T. cruzi initially has a white blood cell
and muscle tropism.
ii. Tropism can broaden over time ultimately leading to extensive
systemic infection and somtimes death.
iii. There is no cure or effective treatment for Chagas' disease and it is
endemic to South and Central America.

Malaria [Plasmodium spp.]

d. Highly complex life history:
i. Malaria means "bad air" in Italian (based upon the disproven
hypotheses that malaria is caused by bad air).
ii. Malaria is a step or two up in complexity from that exhibited by
Trypanosoma cruzi.
iii. Particularly, the malaria parasites (four species all of the
Plasmodium genus) have (or undergo):
1. a definitive host
2. an intermediate host.
3. an arthropod vector.
4. multiple tissue tropisms which they explore sequentially
5. distinct cellular phases depending on current tropism
6. intracellular replication
e. Globally important:
i. Malaria is humanity's dominant protozoan disease.
ii. Malaria kills two million people each year while infecting
hundreds of millions.
iii. The Plasmodium species together have a range which overlaps that
of one-third of humanity.
iv. Historically malaria has had a worldwide distribution. However, as
a consequence of mosquito control measures, malaria today is
restricted to the tropics. Note that this is a significant achievement
stemming from the germ theory of disease.
v. As a wonderfully perverse example of natural selection and
adaptation in action (i.e., evolution), cases of malaria are
apparently on the rise resulting on the one hand from Anopheles
mosquitoes developing resistance to mosquito control measures
(insecticides) and on the other hand Plasmodium spp. developing
resistance to anti-malarial chemotherapeutics.
f. Four species:
i. Four species of Plasmodium are implicated in human disease:
 1. P. malariae
2. P. vivax
3. P. falciparum
4. P. ovale
ii. They all are spread primarily by mosquito vectors, all of the genus
Anopheles.