Parasitology 2.

Dr. Fontanilla
January 6, 2014

PROTOZOA - FLAGELLATES
OUTLINE

Protozoa
Nucleus and Cytoplasm
Locomotor Organelles

Pseudopodia

Cilia

Flagella
Encystment
Feeding and Metabolism
Classification

Flagellates
Genus Trypanosoma

Trypanosoma brucei

Trypanosoma cruzi
Genus Leishmania

Visceral

Cutaneous

Mucocutaneous

PROTOZOA
Was once a phylum name
Actually refers to a number of phyla
Explains diversity and complexity of members
Currently used colloquially as a common noun
NUCLEUS AND CYTOPLASM
Protozoa consist of a single cell
Many species contain more than one nucleus during all or portions
of their life cycles.
Nucleus and cytoplasm
Like all cells, the bodies of protozoa are covered by plasma
membrane which may contain glycocalyx that may have
immunologic importance
Pellicular microtubules or fibrils may course beneath the plasma
membrane.

Examples:
o Kinetoplastid flagellates - microtubules underlie a
flexible membrane
o Trypanosome and trichomonas - adjoining membranes
have a fibrous connection between them such as
between the body and undulating membrane

Mitochondria

Examples:
o Amoeba - branched tubular cristae
o Flagellates - a single, large body
o Ciliates - arranged as elongated sausage-shaped
structures

Golgi apparatus (dictyosome)

Examples:
o Flagellates - large and/or multiple parabasal bodies in
association with kinetosomes, the basal bodies or
parabasal bodies

Microbodies

Peroxisomes contain oxidases and catalases.

Examples:
o Trichomonas spp - hydrogenosomes
o Kinetoplastida glycosomes which contain glycolytic
enzymes used in carbohydrate and fat synthesizing
pathway called glyoxylate cycle

Cytoplasmic matrix

Low density colloid

Can exist as fluid (sol state) or relatively solid (gel state)

Central zone of cytoplasm

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

o ENDOplasm
o SOL state
o Contains nucleus, mitochondrion, Golgi bodies

Peripheral zone of cytoplasm

o ECTOplasm
o GEL state
o More transparent than sol
o Maintains cell shape
o Base of flagella or cilia are embedded in the ectoplasm
Protozoa, like fungi, are described as eukaryotes.

Eukaryotes - the genetic material, DNA, is carried on welldefined chromosomes contained within a membrane-bound
nucleus

Nuclei - oval, discoid, or round that appear vesicular with

irregular distribution of chromatin
o Examples:
Ciliates
Micronucleus reproductive; undergo meiosis
prior to sexual reproduction (conjugation)
*some protozoan members reproduce by
asexual reproduction such as binary fission or
budding
Macronucleus dense, elongated, or
chainlike; considered as somatic; function in
cell metabolism and growth; does not undergo
meiosis

Nucleoplasm finely granular with aggregation of dense

chromatin

Endosomes nucleoli; do not disappear during mitosis

Nuclear envelope consists of two membranes

LOCOMOTOR ORGANELLES
Protozoa move by 3 basic types:
Pseudopods (amoeba)
Flagella (flagellates)*
Cilia (ciliates)*
*called undulopodia
Some amoebas possess both flagella and pseudopods transformation
from flagellated to amoeboid cell occurs in response to environmental
conditions and is a recognized life cycle event.

Figure 1. Euglenoid Flagellum; Paramecium Cilia, Amoeba Pseudopodia

PSEUDOPODIA
Temporary extensions of the cell membrane and are found in amoebas
as well as in a variety of cell types
Movement by means of pseudopodia is a complex form of
protoplasmic streaming involving protrusion of the cell, adhesion to
substrate, and subsequent contraction.
Pseudopodia may also be used for amoeboid feeding

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Figure 2. Mechanism of pseudopod feeding

An amoeba feeds on small organisms such as bacteria.

As an amoeba approaches food, pseudopodia form and eventually

surround the food.

The food becomes enclosed in a food vacuole.

Digestive enzymes break down the food, and the nutrients diffuse into
the cytoplasm.

Undulipodia
Slender, whip-like structures
Composed of a central axoneme and an outer sheath that is a
continuation of the cell membrane
The axoneme consists of nine peripheral and one central pair of
microtubules.
The axoneme arises from a kinetosome (basal body) which is similar to
centrioles of other eukaryotic cells.
The flagellum may also be bent back along and loosely attached to the
lateral cell surface, forming a fin-like undulating membrane (an
adaptation in a viscous environment).
A dark staining body, the kinetoplast found near the kinetosome; disc
made of DNA circles (kDNA)
May be directed anteriorly, posteriorly or laterally regardless of
direction of movement
Heterkonts are flagellates with two or more flagella with differeing
structures

CILIA

Figure 5. Flagellum, kinetosome, and associated organelles are called the

mastigont or mastigont system.

Figure 3. Schematic representation of a ciliate

Structurally similar to flagella

With a kinetosome and an axoneme composed of two central and nine
peripheral microtubules
Appear to beat regularly, with a back-and-forth stroke in a twodimensional plane
FLAGELLA

ENCYSTMENT
Many protozoa can secrete a resistant covering and enter a resting
stage cyst.
Conditions favoring encystment involve some adverse environmental
events such as food deficiency, desiccation, increased tonicity,
decreased oxygen concentration or pH, or temperature change.
During encystment, a cyst wall is secreted and starch or glycogen is
incorporated in the cyst as energy source
During excystation, there is return to a favorable environment usually
associated with absorption of water from environment, cell swelling,
activation of lytic enzymes and of normal physiologic pathways
In coccidians, the cystic form is an oocyst which is formed after gamete
union and in which multiple fission occurs (sporogony) with cytokinesis
to produce sporozoites.

Figure 4. Cilia and Flagella Structure

Figure 6. Different flagellate morphologies

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Edited: AFV

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They are called hemoflagellates - laboratory culture media usually

must contain blood.
In the past (in manual), stages were named after the genera they most
resembled. (e.g. Leptomonad for a stage resembling species of genus
Leptomonas)
The nomenclature used today refers to kinetoplast and nucleus
position.
STAGES
Various species pass through amastigote, promastigote, epimastigote,
and/or trypomastigote stages

Figure 7. Different amoebae morphologies

FEEDING AND METABOLISM

Protozoa lacking chloroplasts are all heterotrophic. They get energy
from complex carbohydrates and nitrogen from amino acids.
Mouth parts:

Amoeba temporary pseudopod

Ciliates permanent cytostome

Excretion of indigestible material
Ciliates cytopyge
CLASSIFICATION
Protozoa have been divided traditionally on the basis of their means of
locomotion, although this character is no longer believed to represent
genuine relationships.
Flagellates (e.g. Giardia lamblia)
Amoeboids (e.g. Entamoeba histolytica)
Sporozoans (e.g. Plasmodium)

Apicomplexa

Microsporidia
Ciliates (e.g. Balantidium coli)

FLAGELLATES

Presence of several, long, thread-like extensions of ectoplasm called

flagella during their trophozoite stage

Flagella arises from axoneme which is associated with a kinetoplast

constituting the neuromotor apparatus

Free-living or parasitic

Flagellates of importance to man:

Flagellates of the blood and tissues (to be discussed here)

Hemoflagellates

Requires a blood-sucking insect to complete life cycle

Four basic morphologic forms differing in position of

kinetoplast, presence/absence of undulating membrane
Flagellates of the digestive tract and genitals ( Trans 2.2)
Phylum Euglenozoa
Class Kinetoplasta
Order Trypanosomatida
Genera: Trypanosoma
Leishmania
Leptomonas (no medical importance)
Crithidia (in insects)

TRYPANOSOMA
All trypanosomes are heteroxenous.
During one stage of their lives, they live in the blood and/or fixed
tissues of all vertebrate classes. During other stages, they live in
the intestines of bloodsucking invertebrates.

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Figure 8. Stages of Trypanosoma

Amastigote - basal body anterior of nucleus, with a short, essentially

non-functional flagellum
Promastigote - basal body anterior of nucleus, with a long, detached
flagellum
Epimastigote - basal body anterior of nucleus, with a long flagellum
attached along the cell body
Trypomastigote - basal body posterior of nucleus, with a long flagellum
attached along the cell body
These names are derived from the Greek mastig, meaning whip,
referring to the trypanosome's whip-like flagellum.

Figure 9. Trypanosoma VS Leishmania morphologies

Edited: AFV

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The mammalian stages of T. brucei exist primarily in the bloodstream.

In contrast, most of the mammalian developmental stages of T. cruzi
and Leishmania spp. reside within the cytoplasm of a wide range of
host cells or the phagolysosome of host macrophages respectively.
Proliferative and non-proliferative (boxed) stages and the locations of
the flagellum (blue) and kinetoplast (red) relative to the nucleus (grey)
are indicated. T. cruzi and Leishmania amastigotes may undergo
periods of proliferative and non-proliferative growth.
CLASSIFICATION
Trypanosomes are divided into two broad groups based on the
characteristics of their development in the insect hosts.
Section Salivaria - the species develops in the anterior portion of
the digestive tract of the insect host
Section Stercoraria - the species develops in the vectors hindgut
SECTION SALIVARIA TRYPANOSOMA BRUCEI
The genus includes about 30 species and subspecies.
Most of these are not associated with transmission of sleeping sickness,
although many transmit animal trypanosomiasis to game and domestic
livestock.
Members of the genus Glossina are divided conveniently into three
groups which are often given subgeneric status. These divisions are:
The fusca group (subgenus Austenina)
The palpalis group (subgenus Nemorhina)
The morsitans group (subgenus Glossina s.s.)
VECTORS - tsetse flies (Dipteran: Glossinidae; Genus: Glossina)
Tsetse flies are now restricted to continental Africa. About half of
Africa is infested, some 10.4 million square kilometres in all.
Glossina species are large (6-15mm), narrow-bodied flies,
brownish or greyish in color, with a stout proboscis that projects
well forward in front of the head.

Figure 10. Tsetse Fly

3 subspecies that are morphologically indistinguishable:

Trypanosoma brucei brucei
Trypanosoma brucei gambiense
Trypanosoma brucei rhodesiense

Reservoirs
Found mostly in domestic pigs, cattle, and dogs
There is evidence that antelopes in certain areas may also carry
the parasite.
Man-fly-man transmission is hence, more common in West and Central
Africa.
Asymptomatic persons can carry the parasites in their blood for long
periods and could be continuously infective for the vectors.
TRYPANOSOMA BRUCEI RHODESIENSE
ACUTE form of sleeping sickness
Found in east central and central Africa
Vectors
G. morsitans, G. pallidipes, G. swynnertoni - inhabit the open
savannah and pupate in dry friable earth
Reservoirs
Wild game mammals (bushbuck, hartebeest, lion, hyena) as well
as cattle
The more virulent of the two, is thus, maintained in the most
resistant reservoirs, resulting in continuous selection of aggressive
strains.
LIFE CYCLE of Trypanosoma brucei gambiense and Trypanosoma brucei
rhodesiense
During a blood meal on the mammalian host, an infected tsetse fly
(genus Glossina) injects metacyclic trypomastigotes into skin tissue.
The parasites enter the lymphatic system and pass into the
bloodstream inside the host. They transform into bloodstream
trypomastigotes which are carried to other sites throughout the body,
reach other blood fluids (e.g. lymph, spinal fluid), and continue the
replication by binary fission.
The entire life cycle of African Trypanosomes is represented by
extracellular stages. The tsetse fly becomes infected with bloodstream
trypomastigotes when taking a blood meal on an infected mammalian
host.
In the flys midgut, the parasites transform into procyclic
trypomastigotes, multiply by binary fission, leave the midgut, and
transform into epimastigotes.
The epimastigotes reach the flys salivary glands and continue
multiplication by binary fission.
The cycle in the fly takes approximately 3 weeks. Humans are the main
reservoir for Trypanosoma brucei gambiense but this species can also
be found in animals. Wild game animals are the main reservoir of
Trypanosoma brucei rhodesiense.
IMPORTANT

Infective stage to man - metacyclic trypomastigote

Mode of transmission - inoculation of metacyclic trypomastigote

from a bite of a tsetse fly

Diagnostic stage - trypomastigote in blood or lymph

TRYPANOSOMA BRUCEI BRUCEI

A bloodstream parasite of native antelopes and other African ruminant
Produces a disease called nagana
Humans are not susceptible.
TRYPANOSOMA BRUCEI GAMBIENSE
The etiologic agent of African sleeping sickness
CHRONIC form of sleeping sickness
Found in west central and central Africa
Vectors
G. palpalis and G. tachinoides - riverine flies
Breed in shady, moist areas along rivers

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Edited: AFV

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Figure 12.
(Left) Winterbottoms sign - swollen nodes at base of skull
(Right) Kerandels sign - delayed sensation of pain after the release of
pressure of the hands
3.
Figure 11. Domestic and Wild Cycles of Gambian and Rhodesian types of
African Sleeping Sickness
(A) In West Africa, riverine tsetse flies (palpalis group) living in the bush
transmit the Gambian forms to humans (man-fly cycle) and sometimes to
domestic animals, particularly pigs.
(B) In East Africa, tsetse flies (morsitans group) of the open savannah
transmit the Rhodesian form to various mammals, mainly antelopes, and to
humans. The Gambian cycle can result in an epidemic.

PATHOGENESIS of Trypanosoma brucei gambiense and Trypanosoma

brucei rhodesiense
The clinical features of Gambian and Rhodesian disease are the same;
however, they vary in severity and duration.
Rhodesian disease progresses more rapidly and the symptoms are
often more pronounced.
The symptoms of the two diseases are also more pronounced in
Caucasians than in the local African population.
Classically, the progression of African trypanosomiasis can be divided
into three stages:
1. The bite reaction (chancre)

A non-pustular, painful, itchy chancre forms 1-3 weeks after

the bite and lasts for 1-2 weeks. It leaves no scar.
2. Parasitemia (blood and lymphoid tissues)

Parasitemia is more prominent during the acute stage than

during the recurrence episodes.

Parasitemia and lymph node invasion is marked by attacks of

fever which starts 2-3 weeks after the bite and is
accompanied by malaise, lassitude, insomnia, headache,
lymphadenopathy, and edema.

Febrile episodes may last few months as in Rhodesian disease

or several years as in Gambian disease.

Painful sensitivity of palms and ulnar region to pressure

(Kerandel's sign) may develop in some Caucasians.

Very characteristic of Gambian disease is visible enlargement

of the glands of the posterior cervical region (Winterbottom's
sign).
o Swollen nodes at the base of the skull
o Lymph nodes become swollen and congested especially
in the neck, groin, and legs
o Named after a British officer who recognized the sign
among slaves bound for the Caribbean Market
o Symptoms are more marked in newcomers than in
people native to the area

CNS stage

Marked by changes in character and personality

Lack of interest and disinclination to work

Avoidance of acquaintances

Morose and melancholic attitude alternating with exaltation

Mental retardation and lethargy

Low and tremulous speech

Tremors of tongue and limbs

Slow and shuffling gait

Altered reflexes
Males become impotent.
There is a slow progressive involvement of cardiac tissue.
The later stages are characterized by drowsiness and uncontrollable
urge to sleep.
The terminal stage is marked by wasting and emaciation.
Death results from coma, malnutrition, intercurrent infection or cardiac
failure, or a severe fall.
Some differences of T b rhodesiense from T b gambiense
Rarely invades the nervous system
Rapid weight loss and heart involvement
No somnambulism or other protracted nervous disorders
Patients die before CNS disorders develop
Death within a few months of infection
Causes a more rapid course toward death

Figure 13. Comparison of West and East African Sleeping Sickness

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Edited: AFV

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IMMUNOLOGY
African trypanosomes express a glycosylphosphatidyl inositol (GPI) anchored variable surface glycoprotein (VSG) as a protective coat.
During infection, large amounts of VSG molecules are released
into the circulation.
Their interaction with various cells of the immune system
underlies the severe infection-associated pathology.
Recent results have shown that anti-GPI vaccination can prevent
the occurrence of this pathology.
The two properties of the VSG coat that allow immune evasion are:
Shielding - the dense nature of the VSG coat prevents the immune
system of the mammalian host from accessing the plasma
membrane or any other invariant surface epitopes (such as ion
channels, transporters, receptors, etc.) of the parasite
Periodic antigenic variation - the VSG coat undergoes frequent
genetic modification, 'switching', allowing variants expressing a
new VSG coat to escape the specific immune response raised
against the previous coat

Figure 14. VSG, the major surface component of Trypanosomes, is also

released in host fluids. VSG induce resistance to complement lysis, escape to
specific immune response, persistent cytokine production, autoantibody
synthesis by molecule mimicry with host tissues.

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its

resistance to a lytic factor present in human serum.
Host immune response
Ig (host immune system greatly stimulated)
complement lysis of RBC anemia
DIAGNOSIS
Demonstration of parasite in blood, bone marrow, CSF card
agglutination test (CATT) to detect antibodies in whole blood or serum

EPIDEMIOLOGY AND PREVENTION

Transmission of trypanosomiasis involves four interacting organisms:
The human host
The insect vector
The pathogenic parasite
The domestic and wild animal reservoirs
Glossina are efficient vectors and are responsible for linking these
organisms. Any reduction of their numbers should lead to significantly
reduced transmission and hence, contribute to elimination and the
sustainability of control efforts.
Current vector control interventions involve the use of insecticides
either through:
Sequential aerosol spraying technique (SAT)
Ground spraying
Insecticide-treated targets or insecticide-treated animals live
baits
Use of traps
Sterile insect technique (SIT)
Bush clearing (tsetse fly habitat destruction) or elimination of wild
animals (tsetse reservoir hosts) have been discarded for ecological and
environmental concerns.
Odor baited traps and screen impregnated with insecticide and
appropriate attractive colors have been used in many countries to
effectively suppress tsetse fly population by 99%.
These artificial bait methods are cheaper than ground and aerial
spraying but communities and governments cannot deploy them
on sustainable bases, as they are labor and management
intensive.
The Sterile Insect Technique (SIT) is another approach to reduce tsetse
fly populations.
Females mate only once in their lifetime thus, any mating with a
sterile male will prevent females from giving birth to any offspring.
SIT consists in rearing a large numbers of laboratory male tsetse
flies which are irradiated and subsequently released in the wild to
compete with wild (naturally occurring) males so that females
inseminated by them produce no offspring.
SECTION STERCORARIA TRYPANOSOMA CRUZI
Depending on its host environment, the organism occurs in three
different forms. The trypanosomal (trypomastigote) form, found in
mammalian blood, is 15 to 20 microns long and morphologically similar
to African trypanosomes.
The crithidial (epimastigote) form is found in the insect intestine.
The leishmanial (amastigote) form, found intracellularly or in
pseudocysts in mammalian viscera (particularly in myocardium and
brain), is round or oval in shape, measures 2-4 microns, and lacks a
prominent flagellum.

Figure 15. Diagnosis by demonstration of parasite in blood sample

TREATMENT
Arsenicals eye damage; trypanosomes become tolerant
Other drugs
Suramin
Pentamidine
Berenil
Difluoromethylornithine (DFMO) brain infections

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Edited: AFV

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LIFE CYCLE
The organism is transmitted to mammalian host by many species of
kissing (riduvid) bug.
Transmission takes place during the feeding of the bug which normally
bites in the facial area (hence the name, kissing bug) and has the habit
of defecating during feeding.
The metacyclic trypomastigotes, contained in the fecal material, gain
access to the mammalian tissue through the wound which is often
rubbed by the individual that is bitten.
Subsequently, they enter various cells, including macrophages, where
they differentiate into amastigotes and multiply by binary fission.

Figure 16. Trypanosoma cruzi

American trypanosomiasis (Chagas Disease)

Widespread in the American continent chiefly among small wild
mammals (enzootic sylvatic cycle)
Human Chagas Disease - bio-ecological and socioeconomic factors
leave rural poor populations of South and Central America in
contact with the sylvatic cycle, where the parasite is transmitted
by natural vectors of the infection
From the Public Health standpoint, the importance of Chagas
Disease remains correlated to so called "domestic cycle, not only
because millions of human beings are involved but also because all
the available control measures are directed against it.
The most important mechanism of transmission of T. cruzi to
humans and other mammals is the feces of infected triatomines.
The vectors of Chagas Disease are insects of the order Hemiptera,
family Reduviidae, subfamily Triatominae.
These are species that colonize poorer quality rural houses, where
colonies of hundreds of individuals (or even thousands) can be
found.
The Triatominae (commonly known as kissing bugs) are defined as
subfamily of Reduviidae (commonly known as assassin bugs) that
suck vertebrate blood (strictly hematophagous) and are mainly
restricted to the New World
Within the subfamily, genera Triatoma, Rhodnius, and
Panstrongylus contain species of bugs that are especially
important vectors of Trypanosoma cruzi, the agent of Chagas
Disease in humans.

Figure 18. The amastigotes differentiate into non-replicating

trypomastigotes and the cells rupture to release them into the bloodstream.
Additional host cells, of a variety of types, can become infected and the
trypomastigotes once again form amastigotes inside these cells.

Figure 17. Kissing Bug (Rhodnius prolixus) Feeding on a Human

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Figure 19. Entry of metacyclic trypanomastigota via break on skin

Uninfected insect vectors acquire the organism when they feed on
infected animals or people containing trypomastigotes circulating in
their blood.
Inside the alimentary tract of the insect vector, the trypomastigotes
differentiate to form epimastigotes and divide longitudinally in the
midgut and hindgut of the insect where they develop into infective
metacyclic trypomastigotes.
Transmission may also occur from man to man by blood transfusion
and by the transplacental route.
IMPORTANT

Infective stage to man: metacycylic trypomastigote

Mode of transmission: entry of metacyclic trypomastigote from a

break on the skin

Diagnostic stage: trypomastigote in blood, CSF, fixed tissues, or

lymph

Edited: AFV

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SYMPTOMS
Chagas Disease can be divided into three stages:
The primary lesion

The primary lesion, chagoma, appearing at the site of

infection and within a few hours of a bite, consists of a
slightly raised, flat, non-purulent erythematous plaque
surrounded by a variable area of hard edema.

It is usually found on the face, eyelids, cheek, lips or the

conjunctiva, but may occur on the abdomen or limbs.

When the primary chagoma is on the face, there is an

enlargement of the pre- and post- auricular and the
submaxillary glands on the side of the bite.

Infection in the eyelid, resulting in a unilateral conjunctivitis

and orbital edema (Romaa's sign), is the most common
finding.
The acute stage

The acute stage appears 7-14 days after infection.

It is characterized by restlessness, sleeplessness, malaise,

increasing exhaustion, chills, fever, and bone and muscle
pains.

Other manifestations of the acute phase are cervical, axillary

and iliac adenitis, hepatomegaly, erythematous rash, and
acute myocarditis. There is a general edematous reaction
associated with lymphadenopathy.

Diffuse myocarditis, sometimes accompanied by serious

pericarditis and endocarditis, is very frequent during the
initial stage of the disease.

In children, Chagas Disease may cause meningo-encephalitis

and coma. Death occurs in 5-10 percent of infants.

Hematologic examination reveals lymphocytosis and

parasitemia.
The chronic stage

The acute stage is usually not recognized and often resolves

with little or no immediate damage and the infected host
remains an asymptomatic carrier. An unknown proportion
(guessed at 10-20%) of victims develop a chronic disease.
They alternate between asymptomatic remission periods and
relapses characterized by symptoms seen in the acute phase.

Cardiac arrhythmia is common.

The chronic disease results in an abnormal function of the

hollow organs, particularly the heart, esophagus, and colon.
The cardiac changes include myocardial insufficiency,
cardiomegaly, disturbances of atrio-ventricular conduction,
and the Adams-Stoke Syndrome. Disturbances of peristalsis
lead to megaesophagus and megacolon.
PATHOLOGY AND IMMUNOLOGY
The pathological effects of acute phase Chagas Disease largely result
from direct damage to infected cells.
In later stages, the destruction of the autonomic nerve ganglions may
be of significance. Immune mechanisms, both cell mediated and
humoral involving reaction to the organism and to autologous tissues,
have been implicated in pathogenesis.
T. cruzi stimulates both humoral and cell mediated immune responses.
The infection causes severe depression of both cell mediated and
humoral immune responses.
Antibody has been shown to lyse the organism, but rarely causes
eradication of the organism, perhaps due to its intracellular
localization.
Cell mediated immunity may be of significant value. While normal
macrophages are targeted by the organism for growth, activated
macrophages can kill the organism.
Unlike T. brucei, T. cruzi does not alter its antigenic coat.

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Antibodies directed against heart and muscle cells have also been
detected in infected patients leading to the supposition that there is an
element of autoimmune reaction in the pathogenesis of Chagas
Disease.
Immunosuppression may be due to induction of suppressor T-cells
and/or overstimulation of macrophages.
DIAGNOSIS
Demonstration of the causal agent is the diagnostic procedure in acute
Chagas Disease. It almost always yields positive results and can be
achieved by:
Microscopic examination of fresh anticoagulated blood or its buffy
coat for motile parasites and of thin and thick blood smears
stained with Giemsa, for visualization of parasites.

Figure 20. The most reliable method for differentiating the trypomastigotes
(motile blood stage forms) of T. brucei and T. cruzi is by the size of the
kinetoplast (see arrows). T. brucei has a relatively small kinetoplast, while
T. cruzi has a larger kinetoplast. T. cruzi trypomastigotes also commonly
form a "C" shape, although this is a less reliable feature. Finally, T. cruzi may
also be found as a non-motile amastigote form in various tissues, while T.
brucei is only found in the trypomastigote form in humans.

Isolation of the agent:

Inoculation in culture with specialized media (e.g. NNN, LIT)
Inoculation into mice
Xenodiagnosis where uninfected triatomine bugs are fed on the
patient's blood and their gut contents examined for parasites 4
weeks later

TREATMENT
The most effective drugs kill only extracellular protozoa.
Nifurtimox and benznidazole
Somewhat effective in curing acute infections
Require long treatment duration
With significant side effects
Patients remain seropositive even after disappearance of parasites
in the blood.
LEISHMANIA
Like trypanosomes heteroxenous
Part of life cycle in sandflies promastigote
Remainder of life cycle in vertebrate tissues
Only amastigotes are found
Known as Leishmania-Donovan (L-D) bodies
Mammals most commonly affected - humans, dogs, and rodents

VECTORS: SANDFLIES
Family Psychodidae
Subfamily Phlebotominae
Genera: Phlebotomus (old world)
Sergentomyia (old world)
Lutzomyia (new world)
Bromptomyia (new world)

Edited: AFV

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ETIOLOGY
Several species of Leishmania are pathogenic for man:
L. donovani causes visceral leishmaniasis (Kala-azar, black disease,
dumdum fever)
L. tropica (L. t. major, L. t. minor, and L. ethiopica) cause
cutaneous leishmaniasis (oriental sore, Delhi ulcer, Aleppo, Delhi
or Baghdad boil)
L. braziliensis (also L. mexicana and L. peruviana) are etiologic
agents of mucocutaneous leishmaniasis (espundia, Uta, chiclero
ulcer)
VISCERAL LEISHMANIASIS
Caused by Leishmania donovani
Discovered by William Leishman (1900) in spleen smears of a soldier
who died of a fever in Dum-Dum, India
Disease known as Dum-Dum fever or kala-azar
Life Cycle
Female sand fly takes a blood meal from mammals and transfer
amastigotes in its mid gut
Amastigotes transform into procyclic promastigotes
Procyclic promastigotes change into metacyclic promastigotes by
simple division
Promastigotes migrate to the pharyngeal valve and are transferred
to mammals through blood meal as metacyclic promastigotes
Metacyclic promastigotes actively invade macrophages,
granulocyctes or are phagocytosed
Promastigotes transform into amastigotes and multiply by simple
division in the macrophages
Amastigotes leave infected cells to infect new macrophages or
transferred to vector via blood meal
Pathogenesis
Clinically, L. donovani infections may range from asymptomatic to
progressive to fully developed kala-azar
Incubation period 2-4 months
Begins slowly with low grade fever and malaise
Followed by progressive wasting and anemia, protrusion of the
abdomen from enlarged liver and spleen
Finally death in 2 -3 years
Treatment
Antimony compounds applied to lesions or injected intravenously
or intramuscularly
The immediate cause of death is often invasion of secondary
pathogen.

Figure 21. Amastigotes in macrophage (?? Unlabeled in slide)

CUTANEOUS LEISHMANIASIS
Caused by: Leishmania tropica and Leishmania major
Found in west central Africa, the Middle East, and Asia Minor into India
The 2 species are found in different localities and have different
reservoir and intermediate hosts.

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Figure 22. Sandflies of the Genus Phlebotomus - Intermediate Hosts and

Vectors

Life Cycle
When a fly takes blood meal-containing amastigotes, parasites
multiply in the midgut and then move to the pharynx.
Sandfly saliva contains low molecular weight compound and
peptides that serve as vasodilators and facilitate infection.
Leishmaniasis is transmitted by the bite of infected female
phlebotomine sandflies.
The sandflies inject the infective stage (i.e. promastigotes) from
their proboscis during blood meals.
Promastigotes that reach the puncture wound are phagocytized by
macrophages and other types of mononuclear phagocytic cells.
Progmastigotes transform in these cells into the tissue stage of the
parasite (i.e., amastigotes), which multiply by simple division and
proceed to infect other mononuclear phagocytic cells.
Parasite, host, and other factors affect whether the infection
becomes symptomatic and whether cutaneous or visceral
leishmaniasis results. Sandflies become infected by ingesting
infected cells during blood meals.
In sandflies, amastigotes transform into promastigotes, develop in
the gut (in the hindgut for leishmanial organisms in the Viannia
subgenus; in the midgut for organisms in the Leishmania
subgenus), and migrate to the proboscis.

Figure 23. In cutaneous leishmaniasis, the organism (L. tropica) multiplies

locally, producing a papule 1-2 weeks (or as long as 1-2 months) after the
bite. The papule gradually grows to form a relatively painless ulcer. The
center of the ulcer encrusts while satellite papules develop at the periphery.
The ulcer heals in 2-10 months, even if untreated but leaves a disfiguring
scar. The disease may disseminate in the case of depressed immune function.
The sores can change in size and appearance over time. They often end up
looking somewhat like a volcano, with a raised edge and central crater. A

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PARASITOLOGY 2.1
scab covers some sores. The sores can be painless or painful. Some people
have swollen glands near the sores (for example, in the armpit if the sores
are on the arm or hand).

Diagnosis
Scrapings from the side of an ulcer smeared on a slide and stained
with Wrights or Giemsa stain will show parasites in endothelial
cells and monocytes, even if they cannot be found in circulating
blood.
Amastigotes of Leishmania are spherical to ovoid and measure 15m long by 1-2m wide. They possess a large nucleus, a
prominent kinetoplast, and a short axoneme, the last of which is
rarely visible by light microscopy. The organisms reside in
macrophages of the host and can be found throughout the body.

MUCOCUTANEOUS LEISHMANIASIS
Caused by Leishmania braziliensis
Produces a disease known as espundia, uta, mucocutaneous
leishmaniasis
Found in vast area between central Mexico and northern Argentina
Life cycle and Pathogenesis
Similar to L. tropica except that the promastigotes reproduce in
the hindgut of the sandfly

Figure 25. Mucocutaneous Leishmaniasis

Figure 24. Parasites in endothelial cells and monocytes

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Symptoms
Initial symptoms of mucocutaneous leishmaniasis are the same as
those of cutaneous leishmaniasis except that in this disease, the
organism can metastasize and the lesions can spread to mucoid
(oral, pharyngeal, and nasal) tissues and lead to their destruction
and hence, sever deformity.

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PARASITOLOGY 2.1
APPENDIX

Figure 1. Tsetse Fly Stages (Trypanosoma brucei)

Figure 2. Triatomine Bug Stages (Trypanosoma cruzi)

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

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PARASITOLOGY 2.1

Figure 3. Visceral Leishmaniasis

Figure 4. Sandfly Stages (Cutaneous Leishmaniasis)

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

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PARASITOLOGY 2.1
SUMMARY

CHARACTERISTIC STAGES OF SPECIES OF LEISHMANIA AND TRYPANOSOMA IN MAN AND IN THE INSECT HOST (FROM PARA MANUAL)
Stages of Parasite
Parasite
Leishmania
Leptomonad
Crithidial
Trypanosomal
L. tropica
In macrophages of skin & In midgut, proboscis of Absent
Absent
subcutaneous tissues
sandfly
L. braziliensis
Same as L. tropica
Same as L. tropica
Absent
Absent
L. donovani
In macrophages of liver, Same as L. tropica
Absent
Absent
spleen, bone marrow and
lymph nodes
T. rhodesiense
Absent
Absent
In salivary glands of tsetse fly In proboscis of tsetse fly; in
bloodstream and lymph
node
T. gambiense
Absent
Absent
Same as T. rhodesiense
Same as T. rhodesiense
T. cruzi
In macrophages of skin, Transitional stage only
In midgut of triatomid bug
In hindgut, feces of bug; in
lymph node, liver, spleen,
blood stream during acute
brain etc.
attacks

Group # 36 | Villanueva, M., Violanta, Yadao, Yau, Yee

Edited: AFV

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