1. Diagnosis of Endoparasitism
Fecal collection
Find life-cycle stages in feces
Eggs, oocysts, larvae, segments, adults
Collection techniques
At home—Store in clean, airtight container
At clinical—Gloved finger or fecal loop (turn glove inside out, tie, and label)
Detection of parasites involves the collection and microscopic examination of feces.
What are some of the precautions that should be taken during fecal examinations?
(Fecal samples should be handled with care
The laboratory area should be cleaned thoroughly after fecal examinations complete
Accurate and thorough records should be maintained)

2. Gross Examination of Feces
Characteristics that should be recorded and reported to veterinarian:
Consistency
Color
Blood
Mucus
Gross parasites
Soak dried tapeworm segment in saline 1-4 hours
Locate small mineral deposits (calcareous bodies) by crushing segment for microscopic examination
Figure 14-40: Mature segments of the most common tapeworms of dogs and cats. Left, Taenia spp. Right, Dipylidium caninum.
Figure 14-41: Chains and individually mature segments of tapeworms of horses, Anoplocephala (left), and cattle, Moniezia (right).

3. Microscopic Examination of Feces
Most reliable detection method
Requirements
Compound microscope
4×, 10×, and 40× objectives
Mechanical stage and micrometer helpful
Technique
Move slide systematically corner to corner
Eggs in air bubbles
Calibration
Fecal specimens should be examined routinely using the 10X objective.
The size of the various stages of many parasites is often important for correct identification.
The initial plane of focus should be that of air bubbles because most helminth eggs are found in this plane.
Calibration must be performed on every microscope to be used.

4. Examination of Direct Smears
Performed on a small amount of fresh feces
Microscopic examination
Used to rapidly estimate a parasite load
Used to detect motile protozoa in feces
Debris may interfere with seeing parasite eggs or protozoa
Observe contamination precautions
Keep accurate records
Because only a small amount of feces is used, any parasites present in the fecal sample may be missed.
This is a preliminary check and shouldn’t be the only examination of the feces.

5. Concentration Methods: Fecal Flotation
One method to concentrate parasitic material in feces
Uses a solution with a specific gravity higher than the specific gravity of parasite material
Feces will sink to bottom
Parasitic material will float to the surface
Salt, sugar, and zinc solutions with an S.G between and 1.250
Zinc sulfate solution: least amount of distortion; usually used in combination with other solutions
Sodium nitrate solution is most common solution used in veterinary hospitals; expensive; forms crystals and distorts eggs after 20 minutes.
Sugar solution least expensive; no distortion; easy to make, store, and clean up.
Zinc sulfate most common in diagnostic labs; least distortion of protozoal organisms.
Saturated sodium chloride is the least desirable solution.

6. Standard Fecal Flotation
Feces and flotation solution mixed in tube or vial
Coverslip or microscope slide set on top of tube
Let stand undisturbed so eggs can float to top and adhere to coverslip or slide
Remove coverslip and place on clean slide or remove slide and place a clean coverslip
Examine with microscope
This is the most common flotation technique used in veterinary hospitals.
Figure 14-45: Vial filled with flotation solution, showing appearance of meniscus.
Figure 14-46: Ovassay (left) and Ovatector (right) are two examples of commercial fecal flotation kits.

7. Centrifugal Fecal Flotation
Most efficient concentration method
Requires centrifuge and 15-mL tubes
Can batch samples
This method works on the same principle as the simple flotation method.
The eggs/parasitic material float to the top of the solution.
If testing multiple samples, number them to keep in order.

8. Fecal Sedimentation
A second method to concentrate parasitic material in feces
Used to detect heavy eggs that will not float in flotation solution
Used to detect eggs that will distort in flotation solution
Eggs settle to the bottom of the tube
Water is the usual solution
Fluke eggs are often detected by this method because of their large size.
Debris will also settle on the bottom of the tube and will interfere with examination for parasite eggs.

9. Examining Feces for Protozoa
Trophozoites—one-cell, motile organisms that lack rigid wall of a cyst
Flotation not possible
Direct smear with saline and stain preferred
Stains aid visualization, not preservation
Trophozoites recognized by movements
All of the previously described procedures may be used to detect protozoal cysts. However, some protozoans do not form cysts and pass out of the host in the trophozoite form.
Since trophozoites lack the rigid wall of a cyst, making flotation without distortion or death of the trophozoite is impossible. Therefore, the direct smear technique is the preferred procedure for examination of a fecal sample for protozoal organisms.

10. Special Tests Acid-fast staining Diff-Quik stain Antigen tests
Cryptosporidium
Almost undetectable in flotation
Diff-Quik stain
Cystoisospora
Faster than conventional flotation
Essential in saving piglets by finding oocysts
Antigen tests
Giardia
For accurate results with any of these procedures, several samples should be examined.

11. Sample Collection at Necropsy
Postmortem examination
Two collection methods
Decanting method
Sieving method
Handling precautions
Examining postmortem is an important method in diagnosing many diseases, including parasitism.
With each method, the veterinary technician must separate the different parts of the digestive tract wand work with the contents of each individually.

12. Shipping Specimens Preserving and containing Labeling
Packaging and cover letter
Any parasitologic specimen shipped to a diagnostic laboratory should be preserved with alcohol or formalin, unless otherwise directed by laboratory personnel.

13. Detection of Endoparasites
Baermann technique
Examination of blood samples
Direct blood smear
Thin blood smear
Thick blood smear
Concentration techniques
Buffy coat method
Modified Knott’s technique
Filter techniques
There are many miscellaneous procedures used to detect endoparasites.

14. Baermann Technique Sample placed on net with warm water
Air bubbles released
Sample sits 12 to 24 hours
Warm water stimulates larvae to move and sink to bottom of funnel for collection
Drop of fluid from bottom of funnel removed and larvae examined microscopically
In this picture, a Baermann apparatus is used to recover larvae of roundworms from feces, soil, or animal tissues. This apparatus is most useful in recovering larvae of lungworms.

15. Detection of Blood Parasites
Direct blood smear
To detect microfilaria
Detects movement among the red blood cells
Mild infections can be missed
Microfilaria of D. immitis, D. reconditum spp. can be found on direct blood smears
The small sample size makes it easy to miss mild infections.
The direct smear is not a good diagnostic test.

16. Detection of Blood Parasites
Thin blood smear
Prepared and stained the same as for a differential
Microfilaria will be found along the feathered edge
Trypanosomes, protozoans, and rickettsiae may be found in or around blood cells
Thick blood smear
Uses more blood than thin smear
Microfilaria still seen but difficult to identify
Microfilaria will be stained.
Because of their large size, they will be pushed to the feathered edge when the smear is made.
Other blood parasites difficult to find on a thick smear because the cells are stacked so densely.

17. Detection of Blood Parasites: Concentration Techniques
Buffy coat method
Microfilaria found on the top of the buffy coat
Modified Knott’s technique
Allows for differentiation of microfilaria
With concentration techniques, a small volume of blood is used.

18. Buffy Coat Method Blood centrifuged in a microhematocrit tube
Separates into three layers:
Plasma
White blood cell layer (buffy coat)
Red blood cell layer
Microfilaria found on top of buffy coat
The buffy coat method does not allow for identification of the microfilaria.
Figure 14-48: Buffy coat in a hematocrit tube lies between the plasma above and packed red blood cells below. A plug of clay prevents the blood from escaping the tube during centrifugation.

19. Modified Knott’s Technique
Concentrates, “relaxes,” and stains microfilariae
Lyses RBCs to make microfilariae more visible
Examine as many as possible; mixed infections can occur
Differentiation
Measure length and width
Learn general characteristics
Cranial (A) and caudal (B) ends of a Dipetalonema reconditum microfilaria.

20. Filter Techniques Most common method used to detect microfilaria
Commercial kits available
RBCs are lysed and blood is forced through filter
Microfilaria are trapped by the filter
Filter is examined microscopically
If microfilariae are present, it is best to perform other diagnostic procedures for identification purposes.

21. QUESTIONS?