Laboratory Diagnosis of Parasitism

Diagnostic stages of parasites

• Faeces: parasite eggs, larvae, oocysts, cysts, trophozoites, cestodes
segments and adults
• Blood: Babesia, Theileria, Anaplasma, Trypanosoma, Dirofilaria immitis,
Plasmodium
• Sputum: lung parasite eggs (Dictyocaulus viviparus), Capillaria species in
dogs and cats
• Skin : used to diagnose external parasites such as : Mange (Sarcoptes,
Psorptes)
• Autopsy : from dead animals .
• Biopsy: from live animals
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Cestode: Anoplocephala perfoliata gravid segments
 A B

Nematode; Haemonchus contortus: A: adult B: eggs

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Feces
Water containing product produced by gastrointestinal tract (GIT)
• Water,
• Undigested feed materials (starch, granules, cellulose fibers, etc.)
• Bile pigments,
• Bile salts,
• Inorganic salts, enzymes
• Cells of intestinal mucosa (occasional red blood cells and leucocytes)
• Bacterial fermentation products & bacteria in large numbers.
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Indications
1. Diagnosis of some parasitic disease of GIT through detection of ova,
larvae or intact worms
2. Some bacterial diseases Johne’s disease, salmonellosis & Colibacillosis.
3. Some viral disease as rota, corona and BVD
4. Chemical examination for detection of blood or fats

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Collection and handling of fecal samples
Materials
➢ Glass rod or thermometer
➢ Glass slide
➢ Polythene bag
➢ Petri plates/dishes
➢ Containers (Plastic and glass containers)

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Collection and submission of samples
1. Fecal Samples:
❖ Fecal exams should be conducted for fresh fecal material.
❖ In large animals : feces should be collected directly from the rectum by
using disposable plastic glove.
❖ In small animals feces should be collected immediately after defecation.
❖ If material is collected from the ground, it should be from the top of freshly
passed deposit- Avoid deposit area in contact with the ground
❖ Minimum sample size is 5g. Preferably, submit a “golf ball” size sample in
plastic container (air tight and water tight)
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 Collection and submission of samples
❖ Feces should be placed in a sealed glass or plastic container, clearly marked
(Label) with the :
1. Time and date of collection.
2. Species of animal, sex, age
3. History of clinical disease.
4. Owner's name , and any other information relevant to the case.
❖ If collected feces can not be examined within a few hours, the sample should be
stored at 4°C until examined.
❖ Feces should not be frozen, because freezing can distort parasite eggs and
trophozoites .
❖ If feces inspected for the presence of protozoan trophozoites (e.g. Giardia,
Trichomonad) they should be examined immediately after collection
❖ When the material is to be sent to another laboratory it should be packaged in
cold packs, helminths eggs may also be preserved in equal volume of 10% formalin
or 70% isopropyl alcohol
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 Storage and Preservation of Faecal Specimens

Objective: to preserve morphology of eggs and prevent from hatching and

deterioration
• For parasitic examination, 10% formalin can be added as preservative in
faeces with the 3:1 ration, respectively
• Faecal samples for lungworm examination should be transferred in sealed
container without adding any preservative
• Diarrhoeaic samples should be examined within 30min of collection and
should be stored at room temperature
• Faeces can be mixed with preservative as PVA (polyvinyl alcohol), MIF
(Mertholiate Iodine Formalin), PAF (Phenol Alcohol Formaldehyde), SAF
(Sodium acetate acetic acid formaldehyde), Schaudins fixatives, formalin
etc. 11
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 Transportation of Faecal samples

1. The specimen should not be more than one tenth of the capacity of
container
2. The container should be tightly stoppered and the stopper is held in place
with adhesive tape or cord, to prevent leakage
3. The specimen should be properly labelled
4. Container should be properly wrapped to prevent breakage
5. Send special delivery so that the laboratory may receive the sample within
12-24 hours.

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Examination of the Fecal Samples
Several procedures commonly used to examine feces for internal parasites
1. Gross examination of feces
2. Microscopic examination of feces
1. Direct smear
2. Indirect smear/Concentration methods
1. Qualitative (Flotation, Sedimentation)
2. Quantitative (Stoll technique, McMaster technique, Baermann method)

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 Gross Examination of Feces
❖ Consistency : The condition of the feces that is : soft, watery (diarrheic) or
very hard soiled, this description will vary with the animal species, for
example, cattle feces are normally softer than those of horses or sheep
❖ Color : Unusual fecal colors
❖ Mucous : Mucous on the surface of fresh feces may be associated with
intestinal parasitism or some other metabolic diseases
❖ Blood : blood may indicate severe parasitemia
❖ Age of feces : If the feces appear old and dry, this should be noted in aged
sample, parasite eggs have embryonated or larvated, oocyst may have
sporulated or pseudoparasites may be present
❖ Gross parasites : Tapeworm segments, round worms, and larval
arthropods
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 Gross Examination of Feces

Species Color, Odor and Consistency

Equines
Dry Feed Yellowish brown firm regular balls
Grazing Dark green soft regular balls and of slightly unpleasant odour
Cattle & Buff
Dry Feed Dark brown firm flat cakes (balls in camel)
Grazing Dark green soft flat cakes (balls in camel) and of slightly unpleasant
odour
Sheep and Goat Dark green to black firm spherical pellets and of slightly unpleasant
odour
Dogs and Cats Firm elongated cylindrical masses of dark brown or light gray color
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and of offensive odour
Microscopic examination of feces

Materials for Direct Smear

• Physiological saline/Distilled water
• Glass slide
• Coverslip
• Wooden tongue depressor / applicator stick

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 Microscopic examination of feces

Procedure of direct smear:

1. Place small amount of feces directly on the microscope slide, by a
stick
2. Dilute this quantity with water or normal saline
3. Mix by using applicator stick.
4. Apply coverslip and examine under the microscope.

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Microscopic examination of feces

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 Direct Smear
Advantages
• Rapid
• minimal equipment needed
• Need few mg of faeces
• Cost effective
• Detect eggs and larvae of all parasites
• Allows to see eggs or larvae undistorted
Disadvantages
• Can not detect eggs if mild infestation
• incorrectly assumed to be free of parasite
• lot of fecal debris on the slide
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• Needs 3 successive negative results to ensure negative 19
 Indirect Smear/Concentration Method
Advantages
• More accurate than direct method
• No chance of false negative results
• Can detect even mild infections
• Smear is free from debris
Disadvantages
• More time consuming
• Requires more equipment
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 CONCENTRATION METHODS FOR FECAL
EXAMINATION

Qualitative
1. Flotation Method
• Simple Flotation
• Centrifugal Flotation
2. Sedimentation Method
• Simple Sedimentation
• Centrifugal Sedimentation

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 CONCENTRATION METHODS FOR FECAL
EXAMINATION

Fecal Flotation
Indications
• Nematodes
• Cestodes
• Protozoal Cysts
Principle
• Based on differences in specific gravity of parasite eggs and larvae and that
of fecal debris
• Specific gravity refers to the weight of an object (for example: parasite eggs)
compared with the weight of an equal volume of pure water
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 Fecal Flotation
• Most parasite eggs have a specific gravity between 1.1 and 1.2, whereas tap
water is only slightly higher than 1, therefore, parasite eggs are too heavy to
float in tap water
• To make the eggs float, a liquid with a higher specific gravity than the eggs
must be used, such liquid are called flotation solution
• concentrated sugar or salts solution
• Flotation solution usually have specific gravity between 1.2 and 1.25

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 Flotation Solutions

➢Saturated salt/sugar solution

➢Sodium nitrate solution (NaNO3 solution)
➢Zinc sulfate solution (ZnSO4 solution)
➢Magnesium sulfate solution (MgSO4 solution)

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 MICROSCOPIC EXAMINATION OF FECES

Materials for Flotation

• Test tube or cylinder
• Flotation solution
• Glass slide and coverslip
• Wooden tongue depressor / applicator stick
• Muslin cloth / cheese cloth / tea strainer / fine sieve
• Beakers

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 PROCEDURE OF FLOTATION

1. Put about 1 gm. of the fecal sample in 100 ml glass beaker.

2. Add 15-30 ml of flotation solution
3. Mix the feces solution with solution
4. Strain the solution through a fine sieve (tea strainer) to remove the layer objects
5. The filtrate is then mixed with 4-5 mL of saturated salt solution
6. Pour the mixture in to (10 ml) test tube and fill the tube to the top.
7. Place a glass cover slip gently on the top of the fluid and allow the cover slip to
remain for 10 to 20 minutes.
8. Remove the cover slip carefully and immediately place it on the microscope
slide.
9. Examine the area of the slide under the cover slip with the microscope.
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Low power (10x) for helminth stages and at high power (40x) for protozoal 26stages
 PROCEDURE OF FLOTATION

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 Procedure of flotation

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 Ascaris “double wall –serrated outer
9/6/2024 membrane –concentric embryonic cell” 29
 Sedimentation Concentration Test

Indications:
Trematodes eggs as Paramphistomum & liver fluke (Fasciola)
Priniciple:
Specific gravity of water is 1 and SG of trematode is 1.3-1.5 (Eggs will
sink)
N.B. Flotation solutions used
• Zinc sulfate solution (ZnSO4 solution) and Zinc chloride(ZnCl).
Procedure
1. Place 5 gm of the fecal sample in 250 ml glass beaker
2. Add 30-40 ml of tap water or normal saline
3. Mix the water with the feces.
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 Procedure of Sedimentation:

4. Fill the beaker with tap water and strain suspension through cheesecloth
or graded sieves into a conical shaped flask.
5. Let settle it for 20-30 minutes
6. Pour off the liquid in the top of the tube without disturbing the sediment
at the bottom.
7. Using the Pasteur pipette, transfer a small amount of the top layer of the
sediment to a microscope slide.
8. Apply a cover slip to the drop and examine the slide microscopically.
If, the drop is too much thick, dilute it with a drop of water, Lugol’s iodine
solution may be used for dilution and for proper identification of
protozoan cysts.
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 Procedure of Sedimentation

Equipment Mixing Straining

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 Procedure of Sedimentation

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 Panel 1. Dogs (A-F) and Cats (G-I)
parasites.
A. Giardia intestinalis trophozoites
(100×).
B. Toxocara sp. Eggs (100×).
C. Dipylidium caninum egg packet
(40×).
D. Cystoisospora spp. (100×).
E. Giardia intestinalis cysts (100×).
F. Ancylostomideo egg (100×).
G. Toxocara sp. egg (40×).
H. Cystoisospora spp. (100×).
I. Giardia intestinalis trophozoites.
(100×).
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