Hemoflagellates

Trypanosoma
Lecture 5

Prof. Dr. Ayhan R. Mahmood

General Characteristics of Hemoflagellates
 Hemoflagellates are live in the blood and tissues of human and other
vertebrate hosts, belong to the family Trypanosomatidae
 Most of the Trypanosomatids complete their life cycle in two hosts:
vertebrate host and a blood sucking arthropod as a biological vector
therefore, called as digenetic or heteroxenous parasites.
 Hemoflagellates are oval to elongated body, with a single nucleus which
usually lies near the center of the body, and a single flagellum arising and
passes forward over the cell surface to extend freely at the anterior end;
where the flagellum is adherent to the cell surface, its sheath is expanded and
forms the undulating membrane.
 Kinetoplast: It represents network of circular DNA (called kDNA) inside a large
mitochondrion that contains multiple copies of the mitochondrial genome.
 Axoneme: It extends from blepharoplast to the cell membrane. It represents the base of
flagellum. Based upon arrangement of flagellum, Hemoflagellates exist in two or more of
morphological stages: amastigote, promastigote, epimastigote and trypomastigote.
 Multiplication in both the vertebrate and invertebrate host is by binary fission. No sexual
cycle is known.
 There are two medical importance genera of Trypanosomatids; Leishmania and
Trypanosoma.

Classification of Hemoflagellates
Phylum : Sarcomastigophora
Subphylum : Mastigophora
Class : Kinetoplastidea
Order : Trypanosomatida
Family : Trypanosomatidae
Genus : Leishmania and Trypanosoma
Morphological forms of hemoflagellates
 Amastigote form: Round to oval in shape, lacks flagellum, 2-5 microns in
diameter, lives and reproduces by longitudinal binary fission in macrophage
of skin, mucosa, lymph node and reticuloendothelial cells of human infected
with Leishmania spp. and Trypanohsoma cruzi. The vesicular nucleus with
large central karyosome, and kinetoplast can be seen. The axoneme extends
from near the kinetoplast to the cell membrane. This form is a non-motile
intracellular stage.
Figure: Amastigotes in Heart tissue.
 Promastigote form: Lanceolate shaped; measuring 15-20 um, the large single
nucleus is located in or near the center, and the kinetoplast is at the base of
the flagellum anterior to nucleus. There is no undulating membrane. A single
free flagellum extends anteriorly from the axoneme. It is found in the mid
gut of insect vector. This is the infective stage of Leishmania.

Promastigotes
 Epimastigote form: Elongated, nucleus located in posterior end, kinetoplast is
more centrally located, usually is placed close to the nucleus. A single flagellum
arise anteriorly from the axoneme forming a shorter undulating membrane
than observed in trypomastigotes extending half of the body length, and
project from the anterior end as a free flagellum. Epimastigotes are less motile
than trypomastigotes. It is the form in which Trypanosoma brucei occur in
salivary gland of the vector tsetse fly and Trypanosoma cruzi in the midgut of
the vector reduviid bug. Note: This stage is lacking in Leishmania.

Epimastigotes: long slender form.

 Trypomastigote form: Elongated appears as C or U shape in stained blood
films, with central nucleus. Kinetoplast lies near the posterior end of the
parasite. Flagellum arises posteriorly and runs as long undulating membrane
extending entire body length. It is the infective stage of Trypanosoma found
in arthropod vector and peripheral blood of humans.

K
10 mm

Figure: Trypomastigotes, Trypanosoma brucei sp. In

blood smear stained with Giemsa .
 A B
Figures A and B: Trypanosoma brucei sp. in thin blood smears stained with Giemsa .
TRYPANOSOMA
Genus Trypanosoma
Trypanosomes are hemoflagellates that most species reside in peripheral blood and tissues
of their host. In these hosts, they appear in the trypomastigote form and divide by
longitudinal binary fission. One notable exception is T. cruzi, which has become adapted to
intracellular life in the amastigote form and does not multiply in the trypomastigote form.
Trypanosomes belong to the family Trypanosomatidae, which includes a number of genera,
only two of which parasitize humans:
1. Trypanosoma cruzi: It is the causative agent of South American trypanosomiasis (also
called as Chagas’ disease) in human, which occurs in Central and South America, and
parts of the United States, and transmitted by insect vector reduviid bugs (Genus
Triatoma).
2. Trypanosoma brucei: It causes African trypanosomiasis, also called sleeping sickness, a
disease found in Africa transmitted by tsetse fly, which belongs to genus Glossina. It has
three important subspecies out of which only two of them infect humans:
- Trypanosma brucei rhodesiense: It is the causative agent of East African sleeping
sickness.
- Trypanosma brucei gambiense: It is the causative agent of West African sleeping
sickness.
- Trypanosoma brucei brucei: It causes “nagana”, a disease affecting cattle in Africa.
Trypanosoma cruzi
Trypanosoma cruzi is a parasite that lives in the blood and reticuloendothelial tissues of
humans and many domestic and wild mammalian reservoir hosts, including dogs, cats, bats,
raccoons, foxes, squirrels, monkeys and pigs. It is the causative agent of South American
trypanosomiasis or Chagas’ disease.
Epidemiology: Chagas’ disease is mainly found in South and Central American countries; Brazil,
Argentina, Venezuela, etc. it is estimated that 8 million people are chronically infected with T.
cruzi and 14,000 deaths occur due to the illness every year.
Habitat:
 In humans, T. cruzi exists in two forms: amastigote and trypomastigote form. Amastigotes
are intracellular parasite found in reticuloendothelial cells, spleen, liver, lymph node, bone
marrow, and myocardium. They are also found in cells of epidermis and striated muscles.
Trypomastigotes are extracellular and found in peripheral blood.
 In insect vector (reduviid bug), it exists: trypomastigote form, and epimastigote form.
Kissing bug Triatomine
Vector of Trypanosoma cruzi

Triatoma infestans
Life cycle
Infective form: Metacyclic trypomastigotes forms are the infective forms found in feces of
reduviid bugs.
When reduviid bugs feeding on the blood of infected human, the reduviids obtain the
parasite either as free flagellates in the trypomastigote stage, or as intracellular amastigotes
within host macrophages. With the blood meal, the parasites pass first into the midgut of
the insect where development transforms the flagellates into epimastigotes. The latter
migrate to the hindgut where they are further transformed into infective metacyclic
trypomastigotes and are excreted in the bug’s feces. The insect cycle takes about 10–15
days. While infected bugs feed, they defecate, voiding feces containing infective
trypomastigotes. The Human infection occurs when infective trypomastigotes in
contaminated feces enter; through the skin punctures made at the biting site by the biting
bugs, through skin abrasions, or through penetration the mucous membranes of the eye and
mouth that are rubbed with contaminated fingers. Entrance of the infective trypomastigotes
initiates an acute local inflammation. The parasite circulates in the blood for a time and then
invade tissue cells. During the early stages of infection, the trypomastigotes are abundant
circulates in the blood and then invade tissue cells; but they do not undergo multiplication
there. They eventually invade, and are engulfed by reticuloendothelial cells of the liver,
spleen, and lymphatics, glial cells, and myocardial and skeletal muscles. Other tissues
infected include nervous, gonadal, bone marrow and placenta. Within the various host cells,
the trypanosomes rapidly transform into amastigotes that repeatedly multiply by binary
fission producing numerous individuals.
The parasites then transform into motile slender C shaped non-multiplying trypomastigote
stages, and are released when the destroyed host cells rupture. The released
trypomastigotes are infective to other host cells, as well as to the insect vectors, and may re-
invade the host cells and transformed into amastigote form, then revert to the
trypomastigote form and again invade the blood.
Figures A-C: Trypanosoma cruzi morphological forms. (A) The replicating epimastigote form in culture (Giemsa stain). (B)
Trypomastigote in a peripheral blood smear from a patient with acute Chagas disease (Giemsa stain). (C) amastigotes within
a cardiac myocyte in a patient with chronic Chagas disease (hematoxylin and eosin stain). Courtesy of the Division of Parasitic
Diseases and Malaria, U.S. Centers for Disease Control and Prevention.
Mode of transmission
 Transmission of T. cruzi infection to human and other reservoir hosts takes
place when abraded skin, mucus membranes, conjunctiva, become
contaminated with reduviid bug’s faeces of the bug containing infective
form of the parasite (metacyclic trypomastigotes).
 In endemic areas transmission may occur from infected donors during
blood transfusions, can also be transmitted by the organ transplantation.
 The parasites also may be transmitted from mother to fetus through the
placenta.
Pathogenesis and Clinical feature
Average incubation period is between 1-2 weeks. American trypanosomiasis or Chagas’
disease caused by T. cruzi can be acute or chronic type:
1. Acute Chagas’ disease: Acute phase occurs after infection and may last for 1–4 months.
Chagas’ disease appears in an acute stage primarily in young children. Frequently, early
symptoms of the acute form appear as inflammatory swellings or nodules (Chagoma) at the
sites of the insect bite. Inoculation of the parasite in conjunctiva causes unilateral, painless
oedema of the eyelid and conjunctiva, and swelling of the pre-auricular lymph nodes known
as Romana’s sign. Usually within 4–8 weeks, patient either recovers spontaneously or
develops chronic T. cruzi infection.

Figure: Acute Chagas Figure: Real image

disease in a young child. showing romana’s sign
Romaña's sign is present. in the eyelid.
2. Chronic Chagas’ disease: The chronic form is found in adults and older children and
becomes apparent years or even decades after the initial infection. Myocardial and
neurological dysfunctions represent more severe manifestations of the chronic form of the
disease. In chronic phase, T. cruzi produces inflammatory response, cellular destruction and
fibrosis of muscles and nerves, which can present with cardiac myopathy, megaoesophagus
and megacolon.

Figure: Patient with chagasic megacolon undergoing

xenodiagnosis. Note pillbox, which contains the reduviid bugs,
Figure: Megacolon in chronic Chagas’ disease.
attached to the right forearm.
Laboratory Diagnosis

 Microscopic examination: used for detection of the trypomastigotes in thick and thin
Giemsa-stained peripheral blood films. Histopathology Biopsy specimens may reveal
amastigotes of T. cruzi.
 Culture: Blood is inoculated in NNN medium, incubated at 25 °C, and observed for
epimastigote forms for up to 30 days before they are considered negative. Culture is more
sensitive than smear microscopy.
 Animal inoculation: The blood or CSF of the patient is inoculated intraperitoneally into
mice or rats. Trypomastigotes can be demonstrated in the blood of mice within 10 days,
and detection of amastigotes in cardiac tissues occurs after two months of inoculation.
 Serological tests: are used for the detection of antibodies such as complement fixation,
immunofluorescence, IHA (indirect hemagglutination test), and ELISA tests. Chronic
Chagas’ disease is diagnosed by the detection of specific IgG antibodies against T. cruzi
antigens. IgM antibodies are diagnostic for congenital infection. T. cruzi specific antigens
from the serum and urine of the infected patients are detected, which are very useful for
diagnosing acute infection and congenital transmission.
 Xenodiagnosis: by allowing clean, uninfected laboratory breed reduviid bugs to feed on the
suspected patients and examining the hindgut two weeks later for epimastigotes.
 Molecular Methods: PCR is available that detects T. cruzi specific kinetoplasts or nuclear
DNA in blood. It is more sensitive than microscopy and serology for the diagnosis of chronic
disease. It can detect as low as one trypomastigote per 20 mL of blood. It is also useful in
monitoring the response to treatment and for the diagnosis of congenital infections.

Prevention
 Prevention depends on the application of insecticide to control the vector bugs.
 Personal protection using insect repellant and mosquito net.
 Improvement in rural housing and environment to eliminate breeding places of bugs.
Treatment:
Nitrofurans, pyrimethamine, primaquine and nitroimidazole have been used with some
success. Surgery is indicated in cases showing megaesophagus and megacolon.
Trypanosoma brucei
Trypanosoma brucei causes African trypanosomiasis, transmitted by tsetse fly, which belongs
to genus Glossina. It has three important subspecies out of which only two of them common
trypanosomes infecting human beings are:
1. Trypanosma brucei rhodesiense: It is the causative agent of Rhodesian or East African
sleeping sickness.
2. Trypanosma brucei gambiense: It is the causative agent of Gambian or West African
sleeping sickness.
3. Trypanosoma brucei brucei: It causes “nagana”, a disease affecting cattle in Africa.
Geographical distribution: Trypanosoma gambiense is found mainly in the Western and
Central parts of the equatorial Africa and Trypanosoma rhodesiense occurs in Eastern and
Central Africa. Transmission: - by bite of tsetse fly. Organisms are in the salivary glands of the
fly.
Habitat: Trypanosomes live in human and other vertebrate hosts, they multiply in blood early
in disease; later from the blood, they invade regional lymph nodes and finally CNS.
Life cycle
 Trypanosoma brucei passes its life cycle in two hosts:
- The vector for both T. brucei species is the tsetse fly (genus Glossina); Glossina palpalis
serves as the vector for T. brucei gambiense whereas Glossina morsitans is the vector for T.
brucei rhodesiense.
 Humans are the reservoir for Trypanosoma brucei gambiense, whereas Trypanosoma
brucei rhodesiense has reservoirs in both domestic animals (especially cattle) and wild
animals.
 Infective form: The metacyclic trypomastigote forms are found in salivary gland of tsetse
fly.
 Mode of transmission: By the bite of tsetse fly, trypomastigote forms are transmitted to the
punctured wound from the saliva of the tsetse fly.
Human infection occurs when an infected tsetse fly takes a blood meal and injects the
infective metacyclic trypomastigotes contained in the saliva into the skin. At the site of
inoculation, the injected metacyclic trypomastigotes initiate inflammation, and transform into
long slender trypomastigote forms, and are carried to other sites through the blood stream.
Trypomastigotes multiply by binary fission in various body fluids (blood, lymph, and spinal
fluid). They transform into an intermediate stage and then into a non-dividing short stumpy
form without a free flagellum. The blood stream forms may be slender, intermediate, or
stumpy. Subsequently, the parasites invade the blood stream, resulting in parasitemia, and
migrate to various organs, including the CNS.
Trypomastigotes in the blood are ingested by tsetse fly; the short stumpy forms are the
infective form, which initiates the infection of the tsetse flies (Glossina spp.). They multiply in
the midgut of the tsetse fly and then migrate to the salivary glands, where they transform into
epimastigotes and then into metacyclic trypomastigotes, which are the infective forms of
humans. It takes around 3 weeks from the time of the blood meal until the fly becomes
infected.
Figure: Trypanosoma gambiense, in blood smear. Figure: Trypanosoma brucei, in a human blood smear.

Figure: Glossina sp. (tsetse fly), the vector of

African trypanosomiasis.
Pathogenesis and Clinical Features
 Trypanosoma brucei gambiense causes African trypanosomiasis (West African sleeping
sickness). The illness is chronic and can persist for many years. The incubation period is
about 1–2 weeks.
 A painless chancre appears on the skin at the site of the bite by the tsetse fly, followed by
fever, chills, rash, anemia, and weight loss. There are high levels of immunoglobulins, mainly
IgM.
 In Trypanosma gambiense patients, the lymph glands, mainly in the cervical and sub-
occipital regions, become enlarged (Winterbottoms sign).
 Invasion of the CNS occurs after several months later and is marked by an increasing
headache, convulsions, daytime sleepiness, and finally coma. Death generally occurs by
intercurrent infection.
 In Trypanosma rhodesiense infection runs a more rapid and fatal course than does
Trypanosma gambiense. The CNS involvement occurs within a few months.
Figure: Trypanosomal chancre. Panel A shows the trypanosomal chancre on the back of the patient at
presentation. The chancre had a diameter 5 cm with ulcerating and elevated edges. Panel B shows the
swollen chancre at higher magnification