1. THE UNIVERSITY OF ZAMBIAINARY
SCHOOL OF VETERINARY MEDICINE
DEPARTMENT OF DISEASE CONTROL
NAME: MUSALO BRIAN
COMPT #: 10008047
COURSE: VMP 4300 - PROTOZOOLOGY
LAB #: ONE
TITLE: DIAGNOSIS OF TRYPANOSOMES
ATTN: MR. A CHOTA
LECTURER: DR. H. CHITAMBO
DUE DATE: 13 ND JUNE, 2014

2. TITLE: Diagnosis of trypanosomes
AIM: To diagnose and identify the type of trypanosome in the blood sample provided
INTRODUCTION
Trypanosomes are hemoflagellates and three species of the genus Trypanosoma are
responsible for disease in humans such as sleeping sickness. Trypanosomes 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. However, only a
few species are of importance as serious cause of morbidity and mortality in animals and
humans in tropical regions. 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
trypanosomes.
Transmission of trypanosomes can be categorically be divided into Salivaria and Stercolaria.
Salivaria trypanosomes (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 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 (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

3. Trypanosomiasis is the Tsetse fly, Glossina spp. 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
Wear gloves, Label pre-cleaned slides (preferably frosted-end) with name (or other identifier),
take drop of blood and place it on the glass slide and cover by a cover slip, mount on the
microscope for observation.
 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.

4. 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
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
The mode of transmission mentioned above, metacyclic transmission, requires to be
separated from mechanical transmission, a process in which trypanosomes survive, for a

5. 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.
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.
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, 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
parasites, Examination of aspirates from enlarged lymph glands for the parasites,
Examination of the CSF for the parasite, Detection of trypanosomal antibodies in the serum.

6. 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 thick 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.
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.

7.  http://www.google.co.zm/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web&cd=10&ved=0
CGgQFjAJ&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