Protocol for Parasite Egg Identification in Faecal Samples in Parasitology Unit

To diagnose gastro-intestinal parasites of ruminants, the parasites or their eggs/larvae must be recovered from the digestive tract of the animal or from faecal material. These should be subsequently identified and quantified. The following are the main tasks involved in this process:

· Collection of faecal samples

· Separation of eggs/larvae from faecal material, and their concentration
· Macroscopically examination of prepared specimens

· Preparation of faecal cultures

· Isolation and identification of larvae from cultures

Limitations of faecal examination in the diagnosis of gastro-intestinal parasitism.

(a) The demonstration of parasite eggs or larvae in the faeces provides positive evidence that an animal is infected but does not indicate the degree of an infection.

(b) The failure to demonstrate eggs or larvae does not necessarily mean that no parasites are present; they may be present in an immature stage or the test used may not be sufficiently sensitive.

Various factors can limit the accuracy and significance of a faecal egg count.

(a) There is a fairly regular fluctuation in faecal egg output.

(b) Eggs are not evenly distributed throughout the faeces.

(c) The quantity of faeces passed will affect the number of eggs per unit weight.

(d) The egg output is influenced by the season of the year (large infections may be acquired during rainy seasons).

 (e) An egg count often refers to the total number of eggs of a mixture of species, which differ widely both in their biotic potential and their pathogenicity.

(f) Eggs may not be detected due to low numbers of them or to a low test sensitivity.

Faecal samples for parasitological examination should be collected from the rectum of animal.

If rectal samples cannot be obtained, fresh Faecal samples may be collected from the pasture.

Several samples should be collected. Samples should be dispatched as soon as possible to a laboratory in suitable containers such as:

· screw cap bottles

· plastic containers with lids

· disposable plastic sleeves/gloves used for collecting the samples

· plastic bags

Each samples should be clearly labeled with animal identification ,date and place of collection.

Samples should be packed and dispatched in a cool box to avoid the eggs developing and hatching. If prolonged transport time to a laboratory is expected, the following may help to prevent the eggs developing and hatching.

(a) Filling the container to capacity or tightening the sleeve/glove as close to the faeces as possible. This is to exclude air from the container.

(b) Add 3% formal in to the faeces (5-20 ml, depending on the volume of faeces). This is to preserve parasite eggs. (N.B Formalin-fixed faeces cannot be used for faecal cultures.) When samples are received in the laboratory they should immediately be stored in the refrigerator (4 °C) until they are processed. Samples can be kept in the refrigerator for up to 3 weeks without significant changes in the egg counts and the morphology of eggs. SAMPLES SHOULD NEVER BE KEPT IN THE FREEZER.

Qualitative techniques for separating and concentrating eggs/larvae

1:Simple flotation method

2:Sedimentation technique (for trematode eggs)

1:Simple flotation method

This is simple technique for use in initial surveys. It can be used in conjunction with the McMaster technique to detect low numbers of eggs.

Equipment

· Two beakers or plastic containers

· A tea strainer or cheesecloth

· Measuring cylinder or other container graded by volume

· Fork, tongue blades or other type of stirring rod

· Test tube (dry)

· Microscope

· Microslides, coverslips

· Balance or teaspoon

· Flotation fluid

Procedure:

 (A) Put approximately 3 g of faeces (weigh or measure the faeces with a precalibrated teaspoon) into container 1

 (B) Pour 50ml of floatation fluid into container 1

 (C) Mix the contents thoroughly with a stirring device (tongue blade fork).

 (D) Pour the resultant faecal suspension through a tea strainer or a double-layer of cheesecloth into container 2.

(E) Leave the container to stand for 10 minutes.

(F) Fill the  test tube with Faecal suspension up to full

 (G) Place the test tube in a test tube stand or rack.

 (H) Cover the test tube by a cover slip on top

(I) Mount the cover slip on micro slide for microscopic examination for egg /larvae identi fication

This is a procedure to assess the presence of trematode infections.The procedure can be used to detect liver fluke (Fasciola) and Paramphistomum eggs.

Equipment

· Beakers or plastic containers

· A tea strainer or cheesecloth

· Measuring cylinder

· Stirring device (fork, tongue blade)

· Test tubes

· Test tube rack

· Methylene blue

· Microslide, coverslips

· Balance or teaspoon

· Microscope

 (A) Weigh or measure approximately 3 g of faeces into container 1

 (B) Pour 40-50 ml of tap water into container 1

 (C) Mix (stir) thoroughly with a stirring device (fork,tongue blade).

 (D) Filter the faecal suspension through a tea strainer or double layer of cheesecloth into container 2.

 (E) Pour the filtered material into a test tube.

(F) Allow to sediment for 5 minutes.

 (G)Remove (pipette, decant) the supernatant very carefully

 (H) Resuspend the sediment in 5 ml of water

(I) Allow to sediment for 5minutes.

 (J) Discard (pipette, decant) the supernatant very carefully.

 (K) Stain the sediment by adding one drop of methyline blue

 (L) Transfer the sediment to a micro slide. Cover with a cover slip and examine under micro scope.

Quantitative techniques for separating and concentrating eggs/larvae:

The simplest and most effective method for determining the number of eggs or oocysts per gram of faeces is the McMaster counting technique

This technique can be used to provide a quantitative estimate of egg output for nematodes, cestodes and coccidia. Its use to quantify levels of infection is limited by the factors governing egg excretion.

· Beakers or plastic containers

· Balance

· A tea strainer or cheesecloth

· Measuring cylinder

· Stirring device (fork, tongue depressor)

· Pasteur pipettes and (rubber) teats

· Flotation fluid

· McMaster counting chamber

· Microscope

 (a) Weigh 4 g of faeces and place into container 1.

 (b)Add 56 ml of  a floatation fluid.

 (c) Mix ( stir) the contents thoroughly with a stirring device (fork,tongue blade).

 (d) Filter the faecal suspension through a tea strainer or a double layer of cheesecloth into container 2.

 (e) While stirring the filtrate in container 2, take a sub sample with pasture pipette.

 (f) Fill the both slides of the McMaster counting chamber with the sub samples

 (g) Allow the counting chamber to stand for 5 minutes .

 (h) Examine the sub-sample of the filtrate under a microscope at a10x 10 magnification.

(i) Count all eggs and coccidia oocytes within the engraved area of both chambers.

 (j) The number of eggs per gram of faeces can be cal culated as followe:add the egg count of the two chambers togather

Multiply the total by 50. This gives the e.p.g. of faeces. (Example: 12 eggs seen in chamber 1 and 15 eggs seen in chamber 2 = (12 + 15) x 50 = 1350 e.p.g.)

The prepared samples on microslides from the simple test tube flotation method, the simple flotation method and the sedimentation method are examined under a microscope at the magnifications listed below.

Annex:

MAGNIFICATION LEVELS FOR EXAMINING PREPARED SAMPLES

Magnification

Parasites

10 x 10

Nematode and cestode eggs

10 x 40

Coccidia oocysts

10 x 4

Trematode eggs

Guideline to the interpretation of faecal egg counts in animals:

FAECAL EGG COUNTS IN ANIMALS

Parasite

Degree of infection (eggs per gram of faeces)

Light

Moderate

Heavy

CATTLE

Mixed infection

50-200

200-800

800+

Pure Haemonchus infection

200

200-600

600+

Pure Trichostrongylus infection

50-100

100-400

400+

Pure Cooperia infection

200-300

300-2500

2500+

SHEEP

Mixed infection

50-800

800-1200

1200+

Mixed infection with Haemonchus absent

300-800

800-1000

1000+

Pure Haemonchus

100-2000

2000-7000

7000+

Pure Trichostrongylus

100-500

500-2000

2000+

Pure Nematodirus

50-100

100-600

600+

Pure Oesophagostomum

100-800

800-1600

1600+

If possible guidelines for the interpretation of faecal egg counts should be established for each area/country/region according to different climatic zones, as the composition and pathogenicity of parasite populations may differ from area to area.

Formulations for flotation fluids and other reagents for use in diagnostic tests.

FLOTATION FLUIDS

The preparation of three different flotation fluids is described below. Any one of them can be used, depending on the availability of reagents. However, the salt/sugar solution (3) gives the best results due to its high specific gravity.

Good-quality inexpensive salt and/or sugar that gives a clear solution should be used for the preparation of flotation fluids. For convenience, a stock supply can be prepared (preferably in a clear container so the amount of salt/sugar not in solution can be seen). The solution should be stirred thoroughly before use to ensure that it is saturated.

(1) Saturated salt solution

Sodium chloride (kitchen salt)

400 g

Water

1000 ml

Specific gravity: 1.200

(2) Saturated sugar solution

Sugar

Q.S.

Water

1000 ml

Specific gravity: 1.120-1.200

Add sugar until saturation, indicated by the presence of sugar crystals at the bottom of the container after stirring for 15 minutes. Stir well before use.

(3) Salt/sugar solution

Sodium chloride (kitchen salt)

400 g

Water

1000 ml

Sugar

500 g

Specific gravity:

1.280

Dissolve the salt in water (saturated solution). Add the sugar to the saturated salt solution. Stir until the sugar is dissolved.

OTHER REAGENTS FOR USE IN DIAGNOSTIC TESTS

(1) Physiological saline solution (0.9%).

Sodium chloride (kitchen salt)

9 g

Distilled water

1000 ml

Dissolve the salt in water

(2) Aqueous iodine solution.

Iodine re-sublimed crystals

10 g

Potassium iodide

50 g

Water

1000 ml

Dissolve the potassium iodide in the water.

Then add and dissolve the iodine crystals.

(3) Formalin 3% solution.

Commercial formalin (40% formaldehyde)

3 parts

Water

97 parts

NOTE. The commercially available 40% formaldehyde solution is regarded as 100% formalin.

(4) Sodium thiosulphate.

Sodium thiosulphate crystals

124.1 g

Water

1000 ml

Dissolve the crystals in water.

Reference:

The epidemiology, diagnosis and control of helminth parasites of ruminants: A Handbook Jørgen Hansen, DVM, PhD Animal Production and Health Division Food and Agriculture Organization Rome, Italy  Brian Perry, BVM&S, DTVM, MSc, DVM&S, MRCVS International Laboratory for Research on Animal Diseases Nairobi, Kenya ILRAD 1994  Published by the International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya Printed by the International Livestock Centre for Africa Addis Ababa, Ethiopia ISBN 92-9055-703-1 .Chapter. 3. Techniques for parasite assays and identification in faecal samples,3.1-3.8