Diagnosis of helminthic infections

Faecal sample

Parasite diseases are common in animals and as such effective prevention and control of diseases depends on early diagnosis of the disease status. Faecal sample examination for the presence of eggs or larvae is the routine aid to diagnosis of parasitic infections. Therefore the faecal samples must be properly collected in plastic/glass vials or bottles, preserved in 10 per cent formalin and despatched to the laboratory or processed scientifically for confirmatory diagnosis
1. Direct smear method: A few drops of water plus an equal amount of faeces are mixed on a clean glass slide. A cover slip is applied before examining under the microscope.
2. Concentration method:
A. Sedimentation method: Sedimentation is more reliable than direct smear examination in terms of number of organisms demonstrated and also due to the fact that much of the debris is removed by this technique.
Procedure
1. Mix about 2 g of faeces with 15 ml of water or 10 % formalin in a mortar with a pestle.
2. Strain it to remove the coarse particles. Transfer the filtrate into a test tube and centrifuge at 3000 rpm for 2 min.
3. Pour off the supernatant and transfer a drop of sediment on a clean glass slide for examination after applying a cover slip.
B. Floatation method: The basis of floatation is that when the faeces is suspended in a liquid with a specific gravity higher than that of eggs, the eggs will float up to the surface. Commonly employed solutions for floatation are saturated solution of sodium chloride, zinc sulphate or sugar.
Procedure
1. A small quantity of faecal sample is added to 10 ml of floatation solution and mixed well.
2. Then the suspension is transferred carefully into a test tube placed vertically and more floatation solution is added to fill the tube to the top without overflowing.
3. A cover slip is placed on top of the surface of liquid and the tube is left undisturbed for 15-30 min.
4. The cover slip is then removed vertically and inverted on a glass slide for microscopical examination.
The floatation of eggs in the floatation solution can be accelerated by centrifugation.

Skin scrapings

Skin scrapings are generally examined to detect mange mites and parasitic fungi. It is always advised to clip off the hairs in and around the area of lesions as closely as possible before attempting scraping. The periphery of the lesions should be selected to collect scrapings since these areas contain more number of parasites. Depending upon the type of parasites superficial or deep scrapings are taken. Superficial scrapings are taken using a sharp scalpel, the blade of which is held at an acute angle. For this the area is rather shaved than scraped, removing the outer epidermis together with the hair stumps. It will be some times more beneficial to moisten the skin with liquid paraffin so that scrapings will adhere to the scalpel. In instances when deep scrapings are to be obtained the area is shaved with the scalpel until blood oozes out. The pustular contents, if any, may also be taken for examination
It is always better to use stopper bottles or vials to collect the skin scraping since some of the mites are too active so that they escape if an envelope is employed for the purpose. It is preserved in 70% alcohol before despatch.

Processing of skin scrapings
1. Transfer the sample to a boiling tube containing about 5 ml of 10% aqueous potassium hydroxide or sodium hydroxide solution.
2. The contents are then gently boiled for about 5 min or until the hairs are digested, taking care to avoid vigorous boiling. Splashing of the contents also should be carefully avoided.
3. Once the larger particles are digested and the fluid becomes almost transparent, cool the tube and transfer the contents into a centrifuge tube. Centrifuge it at low speed (1000 rpm) for 5 min, pour off the supernatant and examine the sediment.

Blood
Blood is collected during the height of temperature for identification of extra-cellular parasites and intracellular haemoprotozoans. The proper site is clipped of hairs and cleaned with spirit or 70 % alcohol. Adopting aseptic precautions, blood is collected for serum preparations / hematological examination
Blood Smears:-
Animals: Collected from ear veins
Poultry: Collected from comb/ wattle
Whole blood:-
Cattle, horse, sheep and goat: Jugular vein
Pigs: Anterior vena cava, tip of the ear
Fowl: Wing vein
There are different methods of examination for detection of blood parasites. Hence the technique adopted for sample preparation also varies.
Wet film examination
1. Obtain a drop of blood from the ear vein /ear tip
2. Place a drop of blood on a clean glass slide
3. Apply a cover slip before examination.
Preparation of blood smears
There are thin smears and thick smears.
Thin smear:
1. Collect a drop of blood onto a clean glass slide
2. Place another slide at 30 0 angle, touch the blood and allow to spread along its edge
3. Push the second slide along the first with a smooth, rapid movement to form a thin film.
4. Air dry.



Thick smears:
1. Collect medium sized drop of blood onto the centre of glass slide
2. Mix using a tooth picks
3. Dry the smear
4. Place the slide in water until the colour disappears. This is called laking.
5. Dry the smear.

Staining of blood smears by Giemsa’s stain
1. Fix the smear in methanol for 2-3 min
2. After drying, flood the smear with freshly diluted Giemsa stain (1 part Giemsa stock solution and 9 parts tap water) and allow act for 30 to 60 min
3. Pour off the stain, wash in tap water and dry before examination
Two or more slides should be prepared. The dried smears or stained smears are separated with broomsticks/match sticks/any applicator sticks in between, wrapped in paper and packed well in cotton and despatched.

Lymph node biopsy:
After properly holding the lymph node, a sterilized needle is pricked into it. Sterile PBS /normal saline solution (0.5-1 ml is ideal) is injected into the gland, rubbed and contents aspirated back in the syringe. The contents are then smeared on glass slides and air dried before despatch.
Nasal discharge
1. Collect the nasal discharge with a scoop and transfer it to a glass slide.
2. Add a few drops of 5% Potassium/Sodium hydroxide.
3. Wait for few minutes
4. Apply a cover slip and keep ready for examination

Copious nasal discharges/sputum can also be collected into plastic bottles/vials along with 10% formalin. If the discharge is too scanty, use a scoop to collect the material. Moist cotton swabs are not advised, as they are liable to get dry, thereby losing the parasite. Granulomatous growths may be cut and fixed in 10 % formalin for despatch to the laboratory.


Egg per gram or EPG

To obtain accurate information with regard to the severity of an infection, egg counting methods have been devised in order to determine the number of eggs per gram of faeces. It is useful in determination of the efficacy of anthelmintic medicines.
Routine procedure
Weigh one gram of faeces in a test tube .Add to this 15 ml of water. The tube is corked firmly and shaken vigorously till the faeces is completely emulsified. Allow the emulsion to settle till all the froth has disappeared. Pipette out 0.15 ml of the emulsion after lightly shaking it and spread the pipetted fluid evenly on a clean grease free slide .Count all the eggs in that sample beginning from one end of the slide .The number obtained multiplied by 100 gives the number of eggs per gram. The average of three counts should be taken. Multiplication of this number by the total weight of the faeces passed during the day gives the total output of eggs. This count should be made on three consecutive days and the average is to be taken for correct assessment.

Stoll’s method
Three grams of faeces is weighed in to a test tube, which is graduated to 45 ml. The tube is then filled to the 45 ml mark with 1/10 N caustic soda solution and 10 to 12 glass beads are added to and is shaken to give a homogenous suspension of the faecal material. Then 0.15 ml of this suspension is drawn out using a calibrated pipette. The total number of eggs in 0.15 ml of the sample is counted and this number is multiplied by 100 to obtain eggs per gram of the sample.
Interpretation of EPG
The following can be taken as a guide though clinical signs and circumstances also should be considered during interpretation.
1. Nematodes: In cattle 300- 600 epg indicates the need of treatment. In lambs 2000-6000 epg indicates severe infection and a value of 1000 or more indicates the necessity for treatment. In equines, 500 epg suggests mild infection, 800-1000 indicates moderate infection and 1500- 2000 indicates severe infection.
Trematodes : the presence of 100 -200 eggs of Fasciola hepatica per gram of faeces of cattle, or 300- 600 epg in sheep indicates pathogenicity

Dr.R.Radhika