2. THE UNIVERSITY OF ZAMBIA
SCHOOL OF VETERINARY MEDICINE
DEPARTMENT OF DISEASE CONTROL
NAME: MWAMBA CHANDA
COMPT #: 10057633
COURSE: VMP 4300 - PROTOZOOLOGY
LAB #: ONE
TITLE: DIAGNOSIS OF TRYPANOSOMES
ATTN: MR. A CHOTA
LECTURER: DR. H. CHITAMBO
DUE DATE: 13 ND JUNE, 2014

3. DATE: 16/06/2014
TITLE: Diagnosis of trypanosomes
AIM: To diagnose and identify the type of trypanosome in the blood sample provided
INTRODUCTION
Trypanosomes have been around for more than 300 million years. They are microscopic
unicellular protozoa that are ubiqitous parasites of insects, plants, birds, bats, fish, amphibians
and mammals. Because they have been around for so long, they and their natural hosts have
evolved together to ensure their mutual survival. Few species of trypanosomes are pathogenic.
Trypanosomes, and other parasites, mainly cause disease when they spread to new hosts, like
humans and their domestic animals, especially recent imports into endemic areas of species
that diverged since continents separated.
The scanning electron micrograph of a single trypanosome shows how the flagellum, which is
responsible for movement, emerges from the posterior end, then adheres to the body of the
cell, causing the characteristic appearance of the moving cells. In contrast to what one normally
expects, the direction of motion is towards the end of the flagellum.
Trypanosoma are responsible for disease in humans such as sleeping sickness they occur in the
blood of the majority of vertebrate animals. The life cycle involves intermediate host, which
usually is an insect. Many species of trypanosomes can live in harmony with their hosts
producing no pathogenic effect, but the best known species are those that are pathogenic to
their definitive hosts. The genus Trypanosoma contains a large number of parasitic species
which infect both domestic/wild animals and humans. Members of this genus are found in the
blood stream and tissues of vertebrates throughout the world. The disease caused by the
pathogenic species is called trypanosomiasis. The life cycle of trypanosomes involve an
intermediate host which usually is an insect (Urquhart, 2007).
Trypanosomes that are pathogenic to livestock in Africa include; Trypanosoma vivax,
Trypansoma brucei and Trypanosoma congolense. These are referred to as African Animal
trypanosomes (AAF) but some can also affect human beings.
Transmission of trypanosomes can be categorically be divided into Salivaria and Stercolaria.
Salivaria trypanosomes include Trypansoma brucei rhodesiense & Trypansoma brucei
gambiense, Trypanosoma vivax, Trypanosoma congolense, Trypanosoma equiperdum are all
transmitted cyclically by glossina (Glossina morsitans most spread) in much of sub-Saharan

4. Africa, they are found in the proboscis of the insect vector and infection is therefore
inoculative. This is the causative agent of African trypanosomiasis and is zoonotic.
Stercolarian trypanosomes include Trypanosoma cruzi, Trypanosoma theileri, Trypanosoma
melophagium occupy the posterior portion of the gut of the insect vector (Triatoma bugs -
Triatoma infestans) and therefore are passed out in the feces and infection is therefore
contaminative (www.phsource.us/US/PARA/Chapter_11.htm).
The life cycle of trypanosomes involves transmission from one vertebrate to another is carried
out by blood-sucking invertebrates, usually an insect. The vector for African Trypanosomiasis is
the Tsetse fly, Glossina species. which cause the diseases Trypanosoma brucei gambiense and
Trypanosoma brucei rhodesiense.
Metacyclic (infective) trypomastigotes are inoculated through the skin when a tsetse fly takes a
blood meal. The parasites develop into long slender trypomastigotes which multiply at the site
of inoculation where ulceration occurs. The trypanosomes continue to develop and then may
invade the lymphatic tissues, the heart, various organs and in later stages, the central nervous
system. Trypomastigotes are taken up by the tsetse fly (male and female) during a blood meal.
The parasites develop in the midgut of the fly where they multiply. 2-3 weeks later the
trypomastigotes move to the salivary glands transforming from epimastigotes into metacyclic
(infective) trypomastigotes. The tsetse fly remains infective for life i.e. about three months.
MATERIALS
 Light microscope
 Slides
 Cover slips
 Blood sample
 Centrifuge
 Giemsa stain
 Alcohol
 Gloves
 Immersion oil
PROCEDURE
Wet smear
The slides were pre-cleaned and the drop of blood was placed on the slide and cover slip, and
then dried followed by microscopic examination.

5. Thin smear and thick smear (both were made on one slide)
Take a drop of blood and place it on slide. Bring a clean spreader slide, hold at a 45° angle, toward
the drop of blood on the specimen slide. Wait until the blood spreads along the entire width of
the spreader slide. While holding the spreader slide at the same angle, push it forward rapidly
and smoothly.
For thick smear, place a drop of blood on the glass slide. Using the corner of a clean slide, spread
the drop of blood in a circle the size of a dime (diameter 1-2 cm). Do not make the smear too
thick or it will fall off the slide. (Should be able to read newsprint through it.)
Giemsa staining blood smears.
After preparing thin OR thick blood smear (in this case both are prepared on one slide) hemolyze
the thick blood smear by putting it in distilled water, then after that the rest process are same as
thin smear. Fix dried smears with methanol for 3 min then flood slide for 30 min with 10 % Giemsa
stain. Rinse slides in Giemsa buffer or running water and dry the slides. Examine slides with a high
power microscope (at x 1000 magnification) with immersion oil.
Buffy coat
Using a capillary tube, get blood from the test tube up to three quarters or full the capillary tube.
Then seal one part using sealant and centrifuge the tube at 3000rpm for 15 minutes. After
centrifugation cut the capillary tube just above the buffy coat and drop the buffy coat alongside
some plasma onto a slide and cover using coverslip then observe on microscope.
DATA COLLECTION
Parasitological test Observation
Wet smear  The parasite (trypanosome) was observed
but with less distinctive structure
differentiation and therefore need for
other tests to identify the trypanosome.
Thin smear  Observed a lot of different length of
trypanosome, mostly the overall length
was 3-4 red blood cells to one
trypanosome
 The undulating membrane was visible

6. Thick smear  Was not well prepared, didn’t observe
anything
Buffy coat Observed moving colliding trypanosomes,
they were quite a lot of them.
The trypanosome looked colorless
The undulating were seen
DISCUSSION
Morphology; the parasite is an elongated cell with single nucleus which usually lies near the
centre of the cell. Each cell bears a single flagellum which appears to arise from a small granule
- the kinetoplast. The kinetoplast is a specialized part of the mitochondria and contains DNA.
The length and position of the trypanosome’s flagellum is variable. In trypanosomes from the
blood of a host the flagellum originates near the posterior end of the cell and passes forward
over the cell surface, its sheath is expanded and forms a wavy flange called an undulating
membrane.
The mode of transmission mentioned above, metacyclic transmission, requires to be separated
from mechanical transmission, a process in which trypanosomes survive, for a short time, on
and about mouth parts of an insect and are inoculated into a new host when the vector bites
again, without undergoing any developmental cycle. Metacyclic transmission requires a lapse of
time to allow the trypanosomes to reach an infective stage by a particular developmental
sequence in the vector, usually a period of several days. .
Development is characterized by the occurrence of three types of blood forms (polymorphic),
these are: Slender forms: long and thin, about 29μm long, free flagellum, Stumpy forms: thick
and short, average length 18μm, typically no free flagellum, but a short one may be present,
Intermediate forms: about 23μm long with a moderately thick body and a free flagellum of
medium length.
Clinical Disease of trypanosomes, the early stages of African trypanosomiasis may be
asymptomatic and there is a low grade parasitiaemia. This period may last for several weeks to
several months. The disease may terminate untreated at this stage or go on to invade the
lymph glands. Invasion of the lymph glands is usually accompanied by a high irregular fever
with shivering, sweating and an increased pulse rate. The lymph glands near the bite often
become swollen, in T. b. gambiense the glands at the back of the neck and T. b. rhodesiense
usually the glands under the jaw are affected (Winterbottom's sign). As the disease progresses,

7. edema of the eyelids, face and sleeplessness are features along with increasing lethargy and
listlessness.
Trypanosomes may invade the central nervous system giving symptoms of
meningoencephalitis, confusion, apathy, excessive sleeping and incontinence. At this stage, the
cerebrospinal fluid (CSF) usually contains mononuclear cells and a few trypanosomes may be
detected. If untreated, character changes, mental deterioration and coma develops, finally
resulting in death. Such signs are more commonly seen with gambiense than in rhodesiense in
which patients often die before these symptoms develop fully.
Laboratory Diagnosis of African trypanosomiasis is by: Examination of blood for the parasi tes,
Examination of aspirates from enlarged lymph glands for the parasites, Examination of the CSF
for the parasite, Detection of trypanosomal antibodies in the serum.
Blood smears are taken and a thick and thin blood smears will let doctors know the percentage
of red blood cells that are infected (parasite density) and what type of parasites are present. A
thick blood smear is a drop of blood on a glass slide. Thick blood smears are most useful for
detecting the presence of parasites, because they examine a larger sample of blood. (Often
there are few parasites in the blood at the time the test is done). A thin blood smear is a drop
of blood that is spread across a large area of the slide. Thin blood smears helps doctors discover
what species of trypanosome is causing the infection. Quantitative buffy coat (QBC) is a
laboratory test to detect infection with malaria or other blood parasites. The blood is taken in a
QBC capillary tube which is centrifuged. This test is more sensitive than the conventional thi ck
smear and in > 90% of cases the species of parasite can also be identified
(http://en.wikipedia.org/wiki/Buffy_coat).
Treatment of trypanosomiasis is done into two distinctive stages namely;
Stage I:
Pentamidine: 7-10 injections for T. b. gambiense infection. Side effects include: Painful
injections with risk of hypotension and shock, pancreatic, renal or hepatic dysfunction; bone
marrow suppression and polyneuropathy. Suramin – multiple doses on varying days for T.b.
rhodesiense infection. Side effect include: renal impairment, peripheral neuropathy and bone
marrow suppression.
Stage II:
Melarsoprol (arsenical compound) – slow IV injection. Side effects include: encephalopathy.
Eflornithine – infusion for 2 weeks every 6 hours. Drug is expensive and more effective against
T. b. gambiense.

8. Prevention is said to be better than cure and it can be achieved through Control in the
reservoirs like livestock and wildebeest, Remove scrub (where tsetse flies reproduce), DDT,
Education and Public awareness.
CONCLUSION
The trypanosome that was viewed under the microscope was identified as Trypanosoma brucei
REFERENCES
 Urquhart, G.M, Armour, J, Duncune, J.L, Dunn. J.L and Jenning, F.W (2007). Veterinary
parasitology .pp191-200. 2nd edition. Blackwell publishing.
 http://www.google.co.zm/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web&cd=10&ved=0C
GgQFjAJ&url=http%3A%2F%2Focw.usu.ac.id%2Fcourse%2Fdownload%2F1110000141-
tropical-medicine%2Ftmd175_slide_trypanosoma_leishmania.pdf&ei=ZLueU-G2CKmw7AapiIHABQ&
usg=AFQjCNEO_sC_zQWIFvhdxvXnPMv2pyzgTQ
 http://en.wikipedia.org/wiki/Buffy_coat