FPL virus can be detected with a transmission electron microscope (TEM) by using fecal or intestinal tissue samples. This
method can be used to confirm clinical cases or necropsy cases when classic gross and histopathologic lesions are not identified.
Tissue samples are scraped and pulverized to release virus particles from cells. The particles are concentrated by ultracentrifugation,
treated with 4% phosphotungstic acid, and then aerosolized onto a carbon-coated grid. The grid is placed in the TEM and screened
for viral particle morphology (Figure 3). This form of microscopy has the advantage of enabling screening for more than one type of virus at a time. It is a good
method for FPL virus detection because it can be done on fecal material, is a technique offered by most diagnostic laboratories,
and allows the detection of all strains of parvovirus, including CPV2a and CPV2b. A drawback to the test is a relatively low
sensitivity. The concentration of viral particles must be in the 106 to 107 range to be detected,12 and in fecal sample submissions the concentration of virus depends on the level of shedding of the virus at the time the
samples are collected. Maximal virus shedding corresponds with the onset of diarrhea, so sending fresh fecal samples from
this phase in the disease can increase the sensitivity of the test.
3. An electron micrograph of parvovirus particles measuring 20 to 24 nm.
Several conventional polymerase chain reaction (PCR) tests have a sensitivity level of 103 viral particles per gram of raw feces.13 A nested set PCR assay has also been developed that can detect between 101 and 102 viral particles.14 These assays have been standardized for detection in feces and are not affected by PCR inhibitors in feces. In addition,
methods that use formalin-fixed tissue have also been reported3,15,16 for use with postmortem samples. Using PCR tests requires considerable expertise. While they are highly sensitive, they
are also easily contaminated; this is especially true with nested set assays.
Immunofluorescent antibody staining (IFA) is another useful diagnostic tool, which detects FPL viral antigens in cryostat
tissue sections. The preferred tissue for this technique is fresh intestine, but spleen and thymus may also be used. A specific
mouse monoclonal antibody directed against FPL virus proteins is applied to the cryosections and incubated in a humidified
chamber. After unbound monoclonal antibody is washed away, fluorescein isothiocyanate conjugated to anti-mouse secondary antibody
is added to the section, followed by incubation and a wash cycle. Epifluorescence microscopy is then used to visualize FPL-specific,
apple-green fluorescence. Because the virus replicates in the rapidly dividing epithelial cells of the mucosal crypts, these
sites appear as localized areas of specific fluorescence (Figure 4). The IFA procedure is used when a rapid, sensitive, and specific test is required. However, the results depend on tissue
samples, and the tissue quality greatly affects results. Highly autolyzed or degraded tissues rarely yield definitive staining.
4. Epifluorescence microscopy of small intestinal mucosa from a cat heavily infected with FPL virus. Note the concentration
of fluorescent signal in the mucosa and lack of fluorescence in the smooth muscle wall (lower right) (20X).