MICROSCOPIC EXAMINATION OF BLOOD FOR PROTOZOAN PARASITES

Most hemoprotozoan parasites are intracellular in erythrocytes or white blood cells and may cause anemia. A routine thin blood smear is therefore useful both for assessing erythrocyte abnormalities and for detecting the presence of parasites. Parasites are most likely to be detected in blood smears during acute infection. Once infections become chronic, immunologic or molecular diagnostic techniques are usually more sensitive as parasitemias can drop below detectable limits by light microscopy.

For microscopic examination of blood smears for hemoprotozoa, Giemsa stain is most effective, but Wright’s stain can also be used in most cases. Commercial stain kits used in many veterinary practices (an example is Dip Quick Stain, Jorgensen Laboratories, Loveland, CO, www.jorvet.com) will also stain hemoprotozoa when used as directed, but the stain will be of poorer quality. The following procedure can be used for Giemsa stain.

To prepare a thin blood smear, place a drop of blood on one end of a microscope slide and draw the blood into a thin film as shown in Figure 3.1.

MICROSCOPIC EXAMINATION OF BLOOD FOR NEMATODE PARASITES

Many species of parasitic worms enter the bloodstream of the host to reach certain organs, where they develop to maturity. These parasites usually stay in the blood only minutes or hours; thus, they are seldom seen in blood samples taken for diagnostic purposes. There are some filarial nematodes, however, whose larvae (e.g., microfilariae) are normally found in the peripheral blood. The microfilarial stage of these parasite species remains in the circulation until ingestion by the bloodsucking intermediate host. Microfilaria testing is often performed for detection of canine heartworm infection. The following discussion of techniques for microfilaria detection is directed specifically to Dirofilaria immitis testing. However, all species of microfilariae in the blood could be detected by the same microscopic techniques.

Although the techniques for microscopic detection of heartworm microfilariae are presented below, the ELISA antigen test for diagnosis of canine heartworm infection is a commonly used screening test. Antigen tests are significantly more sensitive than microfilaria tests because many heartworm infections are amicrofilaremic (occult infections). The absence of microfilariae may be due to low or single‐sex worm burdens or immune clearance of microfilariae. Moreover, some heartworm preventives are microfilaricidal and may render infected dogs amicrofilaremic after one or several months of administration.

The American Heartworm Society and the Companion Animal Parasite Council currently recommend annual testing using an antigen test and a microfilaria test to identify D. immitis infection in dogs. Dogs testing positive on an antigen test should always be tested for microfilariae to determine if microfilaricidal treatment is necessary. Antigen tests currently available in the United States are available in ELISA and immunochromatographic formats. Differences in sensitivity among these tests have been found experimentally and are particularly evident when only a few adult worms are present. False‐negative antigen results can occur, especially when immune complexes have formed, precluding detection. Pre‐treatment of the serum or plasma to disrupt immune complexes using heat or chemicals prior to performing the antigen test releases the antigen and allows detection. Because specialized equipment is required, sending a sample to a diagnostic laboratory is recommended when blocked antigen is suspected. Available tests are considered highly specific although false‐positive results have been reported both before and after treatment to reveal blocked antigen.

Diagnosis of heartworm infection in cats is more difficult than in dogs. Several of the antigen tests can be used in cats, but false‐negative results are common because of immune complex formation as well as the low worm burdens usually found in cats. Similarly, cats are rarely microfilaremic and may develop disease before the adult stage, detectable by antigen testing, is present. To improve the sensitivity of heartworm detection in cats, heartworm antibody tests have been developed. These tests can detect infection earlier than antigen tests but may only indicate exposure to the parasite rather than active infection. Care should be taken in interpreting a feline antibody test, and the results of that test alone should not be used to establish a diagnosis of heartworm infection. In a cat showing clinical signs consistent with heartworm infection, both antigen and antibody tests should be performed as part of the diagnostic workup.

For current recommendations of the American Heartworm Society and the Companion Animal Parasite Council relating to diagnosis and treatment of heartworm infection in dogs and cats, consult the websites of the two organizations: www.heartwormsociety.org and www.capcvet.org.

Tests for Canine Heartworm Microfilariae in Blood Samples

The following techniques can be used to detect microfilariae in blood samples. The canine heartworm, Dirofilaria immitis, is found throughout the world. In North America, dogs may also be infected with Acanthocheilonema (= Dipetalonema) reconditum or, rarely, with Dirofilaria striata, a parasite of wild felids in North and South America. In parts of Europe, Asia, and Africa, Dirofilaria repens and Acanthocheilonema dracunculoides parasitize dogs. When a microfilaria test is used for heartworm diagnosis, the microfilariae of other species must be differentiated from those of D. immitis.

Staining characteristics can be used in discriminating among species, but are not usually performed in veterinary practices. Measurement of total length, width, and the shape of the head can also aid microfilaria identification (Table 3.2). Sizes should be determined with an eyepiece micrometer (see Chapter 1 for micrometer calibration procedure). The standard measurements of microfilariae in Table 3.2 were determined with formalin‐fixed specimens; use of other fixatives or lysing solutions may alter the size of the organisms. Similarly, storage of microfilariae in blood samples for more than 3 days may cause D. immitis microfilariae to shrink in length to the size of A. reconditum.

Wet Mount

The wet mount is the simplest and most rapid of the procedures for microfilariae detection. It is not a very sensitive technique but can be used in conjunction with an adult heartworm antigen test to determine if microfilariae are present or to evaluate the pattern of movement of microfilariae when attempting to differentiate between Dirofilaria and Acanthocheilonema

1. Place one drop of anticoagulated venous blood onto a clean microscope slide and coverslip.

   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   

   	Dirofilaria immitis	Dirofilaria repens	Dirofilaria striata	Acanthocheilonema reconditum	Acanthocheilonema dracunculoides
   Length (μm)	295–325	268–360	360–385	250–288	189–230
   Width (μm)	5–7.5	5–8	5–6	4.5–5.5	5–6
   Head	Tapered	Blunt	Tapered	Blunt	Blunt
   Tail	Straight	Variable— straight or hooked	Curved	Variable—hooked (30%) or curved	Sharp and extended
   Body shape	Straight		S‐shaped	Curved
   Motion (live)	Stationary		Stationary	Progressive
   Relative number	Few to many		Few	Few
   Location of adult	Pulmonary arteries, right heart	Subcutaneous intramuscular tissues	Subcutaneous intramuscular tissues	Subcutaneous tissues	Peritoneum
   Geographic location	Worldwide	Europe, Africa, Asia	North and South America	Africa, Europe, North America	Africa, Europe, India

   
   
2. Examine the coverslip area under low magnification (10×) of the microscope. Look for undulating movements of larvae, which may retain motility for as long as 24 hours.

Hematocrit Test

This technique is only slightly more sensitive than the wet mount:

1. Draw fresh whole blood into a microhematocrit tube.
   
2. Spin for 3 minutes in a hematocrit tube centrifuge.
   
3. Examine the plasma portion of the separated blood, while still in the tube, under low magnification (10×). Moving microfilariae will be present in the plasma above the buffy coat (Fig. 3.2).

The wet mount and microhematocrit techniques may not detect infections with only small numbers of microfilariae. Therefore, if a microfilariae test is used as a screening procedure for heartworm infection, one of the following concentration techniques should be used.

Modified Knott’s Test

The modified Knott’s technique is the preferred concentration method for the detection and identification of microfilariae in blood:

1. Draw a sample of blood into a syringe containing anticoagulant such as EDTA or heparin.
   
2. Mix 1 mL of the blood with 9 mL of a 2% formalin solution. If not well mixed, the red cells will not be thoroughly lysed by the hypotonic formalin solution, making the test much more difficult to read. Microfilariae, but not red cells, will be fixed by 2% formalin. If 10% formalin is used (the concentration used for fixation of tissues), red cells will also be fixed and not lysed.
   
3. Centrifuge the mixture at 1200 rpm for 5 minutes (or as for fecal flotation procedures) and discard the supernatant.
   
4. Add one drop of 0.1% methylene blue to the sediment, mix well, and transfer the entire stained sediment to a microscope slide using a Pasteur pipette.
   
5. Examine using the 10× microscope objective. Microfilariae will be fixed in an extended position with nuclei stained blue.

An alternative procedure using the same amount of blood is the filter test, which traps microfilariae on a filter that is examined with the microscope. This technique can be performed more quickly than the modified Knott’s test, but microfilariae are not easily measured for identification. Materials for performing the filter test were sold as a kit (Difil‐Test^®), which is no longer available. Components of the test can be purchased individually if desired.

Filter Test

1. Mix 1 mL of blood with 9 mL lysing solution (2% formalin) in a syringe.
   
2. Attach the syringe to a filter holder containing a transparent 25 mm filter with a 5‐μm pore size and empty the syringe.
   
3. Refill syringe with water and pass it through the filter to wash away remaining small debris.
   
4. Refill syringe with air, reattach to the filter apparatus, and express.
   
5. Unscrew the filter assembly, remove the filter with forceps, and place the filter on a microscope slide.
   
6. Add one drop of 0.1% methylene blue, coverslip, and examine at 10×.