Collection of blood in goats at bunda,lilongwe; diagnostic techniques that determine blood cell parasites-thick and thin smears.

1. AUTHOR: SHAREEF NGUNGUNI
TITLE: IDENTIFICATION OF BLOOD PROTOZOA IN GOATS
AIM
To perform thin and thick blood smears in laboratory; and to identify blood protozoa in the
smears on microscope.
INTRODUCTION
Blood parasites include species of protozoa, rickettsia and nematodes. Protozoa are
unicellular, eukaryotic, micro-organisms and while most are free living, some are parasitic
causing significant stock and production losses in many parts of the world. Several
pathogenic protozoa are found in blood and tissues of animals (Alan, 1967) and include the
protozoal species of Babesia, Trypanosoma, Theileria, and Cytauxzoon. Reproductive
methods vary, depending on the species and include binary fission, schizogony, and or sexual
reproduction. Transmission is usually by biting and sucking insects, and ticks with the
exception of Toxoplasma gondii which is acquired by ingestion of infectious agents (Soulsby,
1968).
According to Johannes (1996), blood parasitic diseases have severely hindered development
of livestock production in many developing countries, particularly those in sub-Saharan
Africa. The protozoan diseases of veterinary importance are trypanosomosis, theileriosis and
babesiosis, of which trypanosomosis is the most widely distributed. Diagnoses of these
diseases can be through direct microscope examination of blood for the presence of the
parasites or by serologically to detect antibodies against the parasites.
Blood from goats can be withdrawn from venous like such as jugular veins and saphenous
veins, arterial blood vessels or heart chambers from goats for a wide range of scientific
research (Charles & Robinson, 2012). It is necessary when collecting blood from goats
should be least stressful and painful since stress will affect the outcome of study. The blood
was collected from jugular vein of the goats on the farm and when collecting blood from
jugular vein one person has to restrain the animal and monitor the animal; and another is
required to collect blood from the animal. Restraining the animal is important since it
enhances an easiest way to handle the animal without difficulties and the sample efficiently
collected. The neck region is shaved and blood is withdrawn slowly to avoid collapse of
blood vessels (Symth, 1994).If the attempt to collect blood fails, the needle is slowly
removed and the site is monitored for bleeding. If they is no bleed, one more attempt can be
made but make sure that further attempts should avoided to avoid bleeding.
According to Johannes (1996), in the laboratory, thick and thin smears of blood can be
prepared for detecting the presence of the parasites. Thin smear or film is a drop of blood that
is spread across a large area of the slide and allows to identify the species because the
parasite appearance is best preserved in this preparation while thick films allow to screen
larger volume of blood and more sensitive than thin films meaning that it highly detects the
presence of parasites. But microscopic diagnosis is difficult on thick stained blood smears
since the appearance of the parasites is much distorted.

2. METHODOLOGY
A. Materials
Light microscopes, Giemsa stains, immersion oil, EDTA tubes, gloves, syringe, needles,
slides, distilled water, ethanol, cooler box
B. procedures
Procedure for Blood collection.
The goats were restrained and the jugular vein was located on the neck. With an alcohol pad,
Blood collection site was disinfected. The plastic cover from the needle tip was removed and
the needle was inserted into the vein. With the needle in the vein, the EDTAtube was pushed
inside the needle holder and onto the bottom end of the needle. Then the EDTAtube was let
to be filled with blood until blood stops flowing near the top of the tube. The tubes were then
left in cooler box and the needle was removed from the vein and disposed of.
Procedure for thick Blood Film Staining Technique
A thick film was made by placing a large drop of blood (about 15mm in size) on the center of
microscopic slide. Without delay, the blood was spread with another slide clockwise to
achieve a thick smear covering an area of 15 x 15mm. This was allowed to air dry in a
horizontal position. Then a Giemsa stain (3% solution diluted with buffer for 30 minutes
staining) was applied on the thick film and allowed for 30 minutes. After this
time, the stain was washed off using distilled water and again allowed to air-dry. The dried
stained thick smear was finally viewed under a light microscope using 100x oil immersion
objective lens.
Procedure for thin Blood Film Staining Technique
A thin film was made by placing a small drop of blood on the centre of a microscopic slide.
The drop of blood was then spread with a slide held at an angle of 45 degree to obtain a thin
film with a smooth tail end. This was allowed to air dry in a horizontal position and then
fixed with absolute methanol for two minutes. After this, a Giemsa stain diluted with buffer
for 30 minutes staining was applied on the thin film and allowed to air dry for 30 minutes.
The stain was then washed off using distilled water and also air dried. The stained thin film
was viewed under a light microscope using 100x oil immersion objective lens.
RESULTS AND DISCUSSION
Examination of stained blood films showed the organisms to be within red cells. The parasite
showed to be in pairs, elongated and cigar shaped The protozoon that was found in the
erythrocytes is of Genus Babesia this is due to Babesia spp often occur in pairs of pyriform
bodies joined at their narrow posterior ends within erythrocytes (Charles & Robinson,
2012).According to Georgi, (1985) the various species of Babesia are grouped into the small
Babesia whose pyrilorm bodies are 1.0-2.0µm long and large Babesia which are 2.5-5.0µm
long. With Giemsa stains the cytoplasm appears blue and the nucleus red.

3. On electron microscope, Babesia appear to have at its blunt end an electron dense called
apical complex which assist in penetration of erythrocytes (Schimdt & Roberts, 1977).
The more prominent Babesia spp in goats are B.bovis and B.motasi; B bovis is smaller than
B.motasi (Alan, 1967).Since the observed Babesia spp was at least large enough and the
blood was from goats, it could be concluded that its B motasi.
On thin smear blood its where all the morphologies of the Babesia spp were observed
because in this smear blood cells are spread in a layer such that the thickness decreases
toward the feathered edge(Symth,1994). In thick blood smears, the appearance of the Babesia
spp were much distorted and could not be easily distinguished this is due to in this smear the
blood elements (including parasites if any) are more concentrated than in an equal area of
thin blood smear; this method only detects the presence of parasites and do not permit an
optimal review of parasite morphology (Johannes, 1996)
CONCLUSION
All in all, the goats in which the blood where corrected from have Babesia spp which
specifically could be B.motasi and they need to be treated. It was difficult to focus the
protozoa on thick blood smears due to its limitations. However, the aims of the experiment
were successfully achieved.
REFERENCES
Alan W.R., (1967).Introduction to Parasitology.Edward and Arnold(publishers)
Ltd.London,Uk.
Charles M.H and Robinson E.,(2012).Diagnostic Parasitology for Veterinary Technicians.4th
Edition.Linda Rudolph.Elsevier,inc.St Louis,US
Georgi J.R.(1985).Parasitology for Vetereninarians.4th
Edition. W.B.Saunders
Company.Canada.USA.
Johannes K.(1996).Parasitic Infections of Domestic Animals;a Diagnostic Mannual.1st
Edition. Springer Basel AG.Switzerland.
Schimdt D.G.,and Roberts S.R. (1977).Foundations of Parasitology.C.V Mosby Company.St
Louis,Missouri 63141-US.
Soulby E.J. (1968).Helmith,Arthropods and Protozoa of Domestic animals.6th
Edition.Baillire,Tindall and Cassel Ltd.london,Uk.
Symth J.D,(1994).An introduction to Animal parasitology.3rd
edition.Cambridge University
Press.London