An update on three important protozoan parasitic infections in cats: cryptosporidiosis, giardiasis, and tritrichomoniasis - Veterinary Medicine
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An update on three important protozoan parasitic infections in cats: cryptosporidiosis, giardiasis, and tritrichomoniasis
Our knowledge of these protozoan parasites in cats continues to expand. Here's what you need to know about diagnosing these infections and treating affected patients.


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DIAGNOSIS

Because there are so many infectious causes of diarrhea in cats, the American Association of Feline Practitioners (AAFP) Zoonoses Guidelines committee recommends that a fecal wet mount, fecal cytology, fecal flotation, and Cryptosporidium screening procedure be performed as the minimum diagnostic plan in all cats with diarrhea.41 Here are the common methods used to diagnose Cryptosporidium species, Giardia species, and T. foetus infections in cats.

Cryptosporidium species

In cats, few Cryptosporidium species oocysts per gram of feces are shed. The small numbers of oocysts combined with the small size of oocysts result in failure to diagnose the infection with microscopic examination of feces after routine fecal flotation. Staining a thin fecal smear with modified Ziehl-Neelsen acid-fast stain can help demonstrate Cryptosporidium species oocysts (Figure 1). The oocysts are pink to bright-red spheres about 4 to 6 m in diameter.

A fluorescein-labeled monoclonal antibody-based assay (IFA) that can simultaneously detect Cryptosporidium and Giardia species (Merifluor Cryptosporidium/Giardia—Meridian Bioscience) is available. This test was originally developed for use in human samples, so some infections in cats may not been detected. When a single feline fecal sample was tested, the sensitivity of the IFA was lower than that of the modified Ziehl-Neelsen staining technique.42 However, the sensitivity of the IFA equals that of modified Ziehl-Neelsen acid-fast staining when two to four feline fecal samples collected on consecutive days are tested.42 False negative results can occur with both tests. The same IFA was reported to be more sensitive and specific than zinc sulfate flotation and was comparable to an antigen detection technique when used on refrigerated feline fecal samples.43 Experiments in our laboratory suggest this assay may be the best for secondary screening of cat feces for Cryptosporidium species infections if the results of modified Ziehl-Neelsen acid-fast staining are negative (Lappin MR, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colo: Unpublished data, 2006). However, the assay requires a fluorescence microscope, so most practices must send samples to a clinical pathology laboratory.

A number of point-of-care C. parvum antigen tests are available for use with human feces. The results of these tests with feline feces have been variable.42,43 We recently evaluated two lateral flow devices marketed for the detection of C. parvum in human feces and found them to be inadequate for detecting Cryptosporidium species in feline feces (Bachman D, Lappin MR, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colo: Unpublished data, 2006). This may relate to antigenic differences between C. felis and C. parvum; currently available point-of-care kits use monoclonal antibodies directed against C. parvum.

We optimized a PCR assay used to amplify Cryptosporidium species DNA in feline feces and showed it to be more sensitive than IFA.8 However, positive test results only document infection, not clinical illness due to infection. In general, reserve the use of Cryptosporidium species PCR assays for cats with chronic, unexplained diarrhea that have negative results on other tests. DNA sequencing can be used to differentiate Cryptosporidium and Giardia species.1,5,44

Giardia species

Evaluate the feces of all cats with diarrhea for Giardia species and T. foetus trophozoites. Mix a small amount of mucus or feces collected by loop or from the surface of freshly passed diarrhea with a drop of 0.9% saline solution on a microscope slide, cover it with a coverslip, and examine it immediately. A refrigerated sample or one examined several hours after collection probably contains no living organisms. At 100, the only visible motility may be the flagella.45 Giardia species trophozoites have a falling leaf motility pattern in contrast to the rapid, jerky, forward motion of T. foetus. Structural characteristics such as the concave ventral disk can be observed at 400. Applying Lugol's iodine solution, methylene blue, or acid methyl green to the wet mount helps you see the internal structures of the trophozoites.

As stated above, all cats with diarrhea should have fecal flotation performed. The chances of detecting protozoal cysts are thought to be greatest after using fecal concentration techniques that involve centrifugation, such as sugar centrifugation and zinc sulfate centrifugation.46 Technical information on performing these tests is on the Companion Animal Parasite Council (CAPC) Web site ( http://www.capcvet.org/). Because sugar solution is hypertonic and pulls the cytoplasm of the cysts to one side, making it appear as a half or quarter moon, some parasitologists prefer zinc sulfate solution. After concentration in any solution, the microscope slide should be read within 15 to 20 minutes after being prepared because eventually the cysts will collapse. The feces can be refrigerated, but not frozen, if there is a delay before testing.

The 8-x-12-m and 7-x-10-m cysts (Figure 2) can be can be confused with yeast, but Giardia species cysts should be easily recognized because of their distinct structure. At 100, the cysts are about the size of a neutrophil. At 400 and greater, internal structures such as nuclei can be detected (Figure 2). Because of the intermittent pattern of shedding, examine at least three samples obtained over a period of about one week before ruling out giardiasis. This frequency of examination gave a sensitivity of about 90% in one study in dogs that involved the zinc sulfate centrifugation technique.47

We have used an IFA for simultaneous detection of Cryptosporidium and Giardia species (Merifluor Cryptosporidium/Giardia) in several of our studies.8,37,43 In our opinion, this assay reliably aids in the detection of Giardia species cysts in cat feces. We think the assay results are superior to those of antigen tests because the test is unlikely to give false positive results; not only are positive samples fluorescing, but the cysts can also be measured to make sure they are morphologically consistent with Giardia species. However, the assay requires a fluorescence microscope.

Enzyme-linked immunosorbent assays (ELISAs) titrated for use with human feces have been used to detect Giardia species antigen in dog and cat feces in several studies with variable results.45,48,49 Recently, a point-of-care Giardia species antigen test for use with dog or cat feces (Snap Giardia—IDEXX Laboratories) was introduced. Results of the assay compared favorably with IFA in studies completed by the company.45 In a study recently completed in our laboratory, results of this ELISA and IFA were in agreement for 94.4% of the samples (Bachman D, Lappin MR, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colo: Unpublished data, 2006). If the results of either a wet mount examination or fecal flotation are positive, a fecal antigen test is not needed except as a confirmation test in questionable samples. It is our opinion that if Giardia species antigen testing is considered, the Snap Giardia assay is probably the most logical to use. However, the assay should be a supplemental test and should not replace fecal flotation and wet mount examination.

PCR assays can also be used to amplify Giardia species DNA in feces and are available in some research laboratories. However, we think that a direct smear, the immunologic tests (IFA, antigen tests), and PCR assays should only be used in cats with diarrhea (see "Zoonotic concerns and prevention in people" below).

Tritrichomonas foetus

Perform a direct smear on diarrhea samples from all cats with large bowel diarrhea to detect T. foetus trophozoites by using the technique described for Giardia species. This organism is similar in size to Giardia species but can be differentiated by an undulating membrane, rapid forward motion, the lack of a concave surface, and a single nucleus. The sensitivity of a direct smear using samples from naturally infected cats was only 14% in one study, so results can be falsely negative.50

If you still suspect T. foetus infection after the initial workup, a culture can be performed at a diagnostic laboratory or in-house by using a commercially available culture system (InPouch TF—BioMed Diagnostics). The medium used does not support the growth of Giardia species or P. hominis, so positive test results are likely to correlate to infection by T. foetus.50 A PCR assay is also commercially available.51 Information concerning culture and PCR assay detection of T. foetus infection is available at http://www.cvm.ncsu.edu/mbs/gookin_jody.htm.


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