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)

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).

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.

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.

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 1.200 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

QUESTIONS?