Laboratory diagnosis

There are different laboratory diagnostics

tequinechens
Parasitology laboratory
Microbiology laboratory
Serology laboratory
Molecular laboratory
Pathology laboratory
Clinical pathology laboratory
 Parasitology laboratory
• Inroduction
• A parasite is a living organism that lives upon or
within another living organism, known as the host.
• Helminth parasites are found in cattle, sheep and
goats in all countries and regions of the world.
•Many of these parasites are commonly associated
with poor production and unthriftness and can
produce acute disease and even death.
  

Internal organs of ruminants showing predilection sites of helminth parasites :

 Classification of helminthes parasites of domestic animals

namatodes

• Haemonchus • Bunostomum(hookworms)

• Ostertagia • Strongyloides

• Trichostrongylus • Oesophagostomum

• Mecistocirrus • Chabertia ovina

• Cooperia • Trichuris (whipworms)

• Nematodirus • Dictyocaulus

• Protostrongylus • Parafilaria

• Muellerius • Onchocerca

• Toxocara • Setaria

• Stephanofilaria • Thelazia
 Cestodes:

• Monezia • Cysticercus bovis

• Avitellina • Cysticercus tenuicollis

• Thysaniezia • Coenurus cerebralis

• Stilesia • Hydatid cysts

 Trematodes:

• Fasciola • Paramphistomum

• Dicrocoelium • Schistosoma

Protozoa:

* Members of this family Eimeriidae are

referred to here as Coccidia
 Faecal Sample Examination
Purpose
• To demonstrate the presence of helminths and identify species or
helminth groups present.
• To provide a quick and simple but relatively insensitive method for
demonstrating helminth infection and identifying the eggs and larvae
present.
• The demonstration of eggs or larvae in faeces can indicate the presence
of parasitic infection and facilitate the diagnosis of parasitic disease.
• The eggs and larvae can be identified and quantified.  
• 
•Principles
• Eggs and larvae produced by adult helminths in the host animal are
often passed out in the faeces
•Equipment List:
• Wide mouthed screw capped bottles, minimum 30 ml size
• Plastic containers with lids, minimum 30 ml size
• Disposable plastic sleeve-gloves used for collecting samples
• Plastic bags
• Pen marker for labelling
• Cool box for transportation of samples
• Refrigerator in laboratory suitable for the storage of faecal samples
•Faecal sampling:
– 1. Faecal samples for parasitological examination should
preferably be collected from the rrrrrectum. Appropriate
disposable gloves should be worn.
– 2. Collection from large animals can be more easily
accomplished than from smaller animals.
– 3. Smaller animals such as lambs can be induced to
defecate by inserting a moistened finger gginto the
rectum and massaging until the external sphincter relaxes.
– Samples can be collected from the ground if the animal is seen
defecating.
• As soon as the faecal samples arrive at the
laboratory they should be stored in a
refrigerator at 4°C until they are processed.
•* If prolonged transport time to a laboratory is expected, the
following may help to prevent the eeggs 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) Adding 10% formalin to the faeces (5-20 ml, depending on the
volume of faeces).
•Samples can be kept in the refrigerator for up to 3 weeks without significant
changes in the egg counts and the morphology of eggs
• * This is to preserve parasite eggs.
 GROSS EXAMINATION OF FAECES

•Procedure:

•1. Examine faeces before mixing with formalin.

•2. Observe color, (clay color is evidence of bile obstruction; bloody evidence of
hemorrhage in jfthe colon or rectum, black or tarry is of hemorrhage in the
stomach or small intestine).
•3. Observe consistency of the faeces to know whether the animal has diarrhea or
constipation.
•4. Observe whether the faeces contains tape worm segments or some nematode
worms.
 Direct microscopy examination
•1. Faecal smear

•Principles:
• Helminth eggs and larvae can be identified in a thin smear of
faeces on a microscope slide.
•Equipment:
• Microscope slides
• Cover slips
• Saline solution (0.85%) or water
• Microscope
•Faecal smear:
•Procedure
1. Smear a small quantity of faeces on a clean microscope slide.
2. Mix with a few drops of water or physiological saline.
3. Place a cover slip over the smear.
4. Examine first using the low power (10x) and then the high dry
power (40x) to confirm
 Floatation method

•. Purpose: For demonstrating the majority of Nematoda

eggs, Coccidia oocysts, and some of gCestoda eggs:
•Common Floatation Solutions
•A- Saturated sodium chloride solution
•B- Zinc sulphate solution
•C.Magnesium sulphate: Mgso4 specific gravity 1.2
•D. Sodium Nitrate: NaNO3 specific gravity 1.18
•E. Sugar Saturated: sugar solution specific gravity 1.27
•I. Simple Floatation Technique
•Procedure:
Take 3gm of well-mixed faecal specimen.
Mix the specimen with one of the floatation fluid (saturated salt solution or
sugar solution, etc) of 30-50ml
If the specimen is very coarse, it is advisable to strain through a sieve to remove
the large faecal particles.
Fill the test tubes with prepared suspension until a convex meniscus is formed
to the top.
Place microscope cover slip on the meniscus making sure no air bubbles are
present.
Allow standing for 10 – 15 minutes.
Remove the coverslip vertically, invert and place the preparation on a
microscopic slide.
Examine the preparation under the microscope starting from the lower power
lens & record any protozoan cysts, eggs, or gross parasites seen.
 Sedimentation methods

Purpose: For the detection of Trematoda,

Acanthocephala and some of Cestoda eggs.
•Procedure (Method 1):
 3gms of faecal sample is mixed/ suspended in 30-50ml of H 2O
 The mixture is poured into the test tube.
 Allow it to sediment by standing for 5 minutes.
 Remove the supernatant very carefully using pipette
 Resuspend the sediment with the same quantity of clean water.
 Allow it to sediment by standing for 5 minutes.
 Remove the supernatant very carefully using pipette.
 Stain the sediment by adding one drop of Methylene Blue.
 Transfer the sediment to a microscopic slide & cover with cover
slip.
 Examine under the lower magnification of microscope.
 The result is that if the eggs stain yellow it is Fasciola eggs where
as Paramphistomoum eggs takes & stains blue colour or remain
colorless.
 Centrifugation Technique (Method 2):
•Procedure
 Take 5g of faecal sample & mix with water.
 Pour some of the mixture into centrifuging tube.
 Centrifuge the tube at 1500-2000 RPM for 2 minutes.
 Pour & discard the supernatant.
 Repeat the process until the supernatant becomes very
clear.
 Take some of the sediment & put on to a microscopic
slide put cover slip.
 Examine under low power magnification of the
microscope.
 Mc MASTER EGG COUNTING TECHNIQUE

 Is a standard quantitative technique used to count the number of eggs or larvae

per gram of faeces. -i.e. indicate the number of eggs, cysts or larvae present in
each gram of faeces.
 Is important to have accurate information with regard to the severity of infection.
 Flotation fluids are used to separate eggs from faecal material in a counting
chamber (MC Master) with two compartments.
 It can provide a quantitative estimation of egg output for nematodes, Cestodes &
Coccidia.
•Procedure
 Weigh 3 gram of faeces or if faeces are diarrheic take 3 teaspoonfuls.
 Break up thoroughly in 42 ml of tape water in a plastic container.
 Pour the mixture through a fine mesh sieve aperture 250 μm.
 Collect the filtrate, agitate and fill into 15ml flat-bottomed test tube.
 Then centrifuge at 2000 rpm for 2 minutes.
 Pour off supernatant, agitate the sediment very well and fill the test tube to the previous level
with the desired flotation solution.
 Then invert the test tube 6 times so as to have homogenous distribution of eggs.
 And then immediately remove fluid with Pasteur pipette to fill both chambers of Mc Master
slide without interruption to avoid formation of bubbles in the compartments.
 After 5 minutes eggs float & all eggs in the ruled area of both chambers of the Mc Master are
counted.
 Examine one chamber and multiply number of eggs or larvae under one etched area by 100 or
two chambers and multiply by 50 to arrive at the number of eggs per gram of faeces (epg).
• 
•Mathematics of Mc Master Chamber is that:
• 
• 3gram of faeces dissolved-----------42 ml H2O
• Total volume ----------------42+3= 45ml
• Therefore:
• 1g-------- (3g=45ml) ------45/3=15ml
•The volume under (1g) faeces etched area is 0.15ml
•Therefore the number of eggs is multiplied by 100.
•If two chambers are examined, multiply by 50.
Techniques of Larvae recovery from fresh & cultured faecal
samples

A number of methods can be used to examine larvae of helminths.

The methods are recommended for the diagnosis of lungworms such as
Dictyocaulus, Protosrongylus & Muellerius
Also can be applied for the diagnosis of other helminths.
• MODIFIED BERMAN Method
 Is a simple adaptation of the Baermann apparatus technique.
 Faeces is enclosed in gauze in a glass filled with water and leave
overnight.
 The larvae will leave the faeces, migrate through the gauze and settle at
the bottom of the glass.
 After siphoning off the supernatant, the sediment is examined under the
low power of the microscope.
•BAERMAN TECHNIQUE
 It consists of a glass funnel held in a retort stand. A rubber tube attached to the
bottom of the funnel is constricted by a clip.
 A sieve (aperture 250 microns) is placed in the wide neck of funnel, which has been
partially filled with water, and double layer of gauze is placed on the top of the sieve.
 The main advantage of Baerman technique is recovering larvae of lungworms or
infective larvae of other helminths from cultured faeces.
•Procedure:
o Take 10 gram of fresh or cultured faeces.
o Wrap the faeces using gauze and place into the Baerman apparatus funnel to which a
sieve is placed.
o Then cover the faeces using lukewarm H2O (40-45oc)
o Allow standing overnight.
o The clipper on one end of the rubber tube is then released and the liquid collected in
a test tube.
o Centrifuge the collected material for 2 min. at 200rpm. Discard the supernatant and
examine the sediment for moving larvae.
 Examination of ectoparasite
•Sample collection
 A deep skin scrapping should be made around the periphery of the skin
lesion-using scalpel blade.
•Direct microscopic examination
 Cut the hair to short and make scrapping of the skin until some blood
appears, also from a new growing skin invaded by the mites.
 Put the scrapings in a tube unpreserved. If you are near to your clinic, you
can put the scrapings directly on the slide and mix it with distilled water
(tape water) and put cover slip and see under 10-x magnification.

•Examination of collected skin scrapings under stereomicroscopic can reveal

motile mange mites.
•II). Sedimentation technique
 Fresh or preserved skin scrapping material is placed in a test tube
 About 10 ml of 10% KOH is added to the test tube
 The test tube is placed in a beaker of hot water
 Avoid too boiling and control the heat, as bubbling would break up of mange mites
 Hairs and scabs soon begin to disintegrate by the action of hot KOH and liquid in the test tube
consequently becomes thicker
 The liquid in the test tube is allowed to sediment (either by leaving to stand for 15 minutes or
pouring the liquid into a centrifuge tube)
 The supernatant fluid is decanted and smears prepared from the deposit of sediments on a slide
covered with cover slip.
 Examine the slides under microscope using 10X objective.
 examination of protozoa parasite
• Protozoan parasites can be examined in blood,
body fluids (discharges), lymph, intestinal
contents (faeces) & muscle samples.
• Collection of Blood Samples
 Blood samples can be collected from animals for the
purpose of hematological examination, from
different sites:
 Jugular vein (in horses, cattle, sheep, goat, donkey, camel)
 Cephalic vein (in dogs and cats)
 Anterior vena cava (Cranial vena cava) in pigs
 Marginal ear vein (in pigs, small dogs and cats)
• Edge of the ear vein (in any animals
 Techniques of Preparation of blood for
Microscopic Examination
•There are two methods of blood film
Preparation for Microscopic examination viz:
A.Wet blood film
B.Blood Smears
 te
• 1.Wet blood smear
 Wet films are mostly used to diagnose extracellular blood parasites such as
Trpanosomes.
• Procedures
 A drop of blood is placed at the center of aclean slide
 Apply a clean cover slip
 Examine under the microscope (X40)
•  
• NB. The parasite can be seen the active movements(wriggling in the field or
crossing fast across the field) Trypanosomes.This technique can also be
used to diagnosis Trychomonosis in animals after making the wet smear
from vaginal secretions or prepual washings.
•2. Thin Blood Smear
•Procedure:
 Take a drop of blood on a grease free clean slide
 Spread the blood on a slide using a cover-slip or a clean slide at an
angle of 45%
 Dry it quickly and fix with methyl alcohol for 2 min.
 Stain with Giemsa diluted 1:10 in neutral phosphate buffer for 30
min. in a coplein jar or upside down on a staining plate.
 Wash with phosphate buffer at pH 6.8-7.2
 Allow it to dry by standing upright on the rack.
 Examine under the microscope(x100)
•Thick blood smear
• Procedure

 Take a small drop of blood on a clean grease free slide

 Spread with an applicator stick, needle or corner of another slide to a size
of about 2cm in diameter in such away that you can just read a script
through it
 Air dry quickly so that it is protected from flies
 Dehemoglobinize by gently running distilled water on the smear or by
immersing the smear in distilled water for 5 to 10 minutes
 Fix with methyl alcohol for 2 minutes
 Stain with Giemsa diluted in buffered distilled water 1:10 for 30 minutes
 Wash with buffered distilled water till it assumes abluish purple color
 Examine under the microscope ( X40 and X100)