Evaluation of a Direct Immunofluorescent Assay and/or Conjunctival Cytology for Detection of Canine Distemper Virus Antigen

Introduction

Canine distemper is caused by the Canine Distemper Virus (CDV), which belongs to the Morbillivirus genus of the Paramyxoviridae family. Canine distemper is a contagious and often lethal viral infection of many carnivore animals, including Canidae, Mustelidae, Felidae, and Procyonidae (15). Canine distemper incidents have also been reported in vaccinated animals (2,19). Disease outbreaks have occurred in many countries, such as the United States (18), Finland (6), Germany (8), and Poland (11). Moreover, CDV is an infectious agent that affects various wildlife populations, especially foxes (1) and ferrets (20) as well as nonhuman primates, which is indicative of a possible adaptation of CDV to humans in the postmeasles eradication era (17).

During early infection, CDV invades macrophages and lymphoid cells of the respiratory tract before spreading to various organ systems, such as the gastrointestinal tract, lymphoid organs, urinary bladder, and central nervous system. A presumptive diagnosis of CDV is usually based on compatible clinical presentation in unvaccinated puppies, but laboratory confirmation is essential for dogs with unknown vaccination history (10). Antibody detection is often not useful because of antibody kinetics and extended vaccinations (14). Thus, antigen detection by cytology, direct immunofluorescent assay (FA), and polymerase chain reaction (PCR) are the most commonly used and clinically relevant methods for the timely diagnosis of the disease using noninvasively acquired specimens.

The purpose of this study is to evaluate the diagnostic performance of FA and cytology to detect CDV antigen in conjunctival specimens compared with an established PCR assay used as the gold standard.

Materials and Methods

A total of 57 samples were collected from young dogs (aged 2–12 months old) with a history of neurological signs and findings upon physical examination that are compatible with canine distemper, such as localized involuntary muscle spasms (myoclonus), focal and generalized seizures, and extraneural signs (mainly respiratory and gastrointestinal). Efforts were made to exclude the main clinical disorders appearing in the list of differentials that might have caused the mentioned signs, including infectious causes of gastrointestinal and respiratory disease (viral, protozoal, and parasitic), dietary indiscretion, and metabolic and toxic encephalopathies. All dogs were either unvaccinated or had an unknown vaccination history.

Exfoliative epithelial cells were collected using a nylon-bristled cytobrush for cytology and sterile cotton-tipped swabs for FA and PCR. After placing the cotton tip of the swab in the lower conjunctival sac, the swabs were rubbed robustly back and forth. The specimen collected with the cytobrush was gently spread, as thinly as possible, onto a clean microscope slide, air dried, and stained with Giemsa before using light microscopy to detect the presence of inclusion bodies in the conjunctival epithelial cells.

For FA to be performed, the swab was used to place a dot of standard diameter within an etched ring on a slide, which was then fixed in 95% ethanol for 10 min, air dried, and placed in a freezer if analysis was not conducted immediately. After removing the slides from the freezer, they were again placed in 95% ethanol for 30 sec and rinsed in deionized water for 5 min. Anti-CDV polyclonal antiserum conjugated to fluorescein isothiocyanate (Canine Distemper FITC conjugated; VMRD, Pullman, Washington) was used to stain the samples, which were then placed in a suitable chamber created by placing a moist paper towel in a standard Petri dish and incubating at 37°C for 1 h. After incubation, the samples were washed several times with phosphate buffered saline (PBS). Mounting fluid was added and the samples were read on a fluorescent microscope.

For PCR determination, the swabs were immersed in 0.5 M sterile PBS in sterile plastic tubes and centrifuged at 1,200 g for 2 min. The tubes were stored at −80°C before analysis. After manual stirring of the swabs, the saline containing the eluted exfoliated cells was transferred into sterile vials for RNA extraction. The viral RNA was isolated from the supernatants after centrifugation using a commercial kit (QIAamp viral RNA Mini Kit; Qiagen, Hilden, Germany) according to the manufacturer's instructions. A commercial kit (QIAGEN OneStep RT-PCR Kit; Qiagen, Hilden, Germany) was used in a continuous reverse transcriptase (RT)-PCR method to perform reverse transcription and amplification of viral RNA with slight modifications. Each 25 μL reaction mixture contained 5 μL 5 × buffer (final MgCl₂ concentration 2.5 mM), 0.4 mM of each deoxynucleotide triphosphate, 1 μL of enzyme mixture, 0.8 μM of each primer, and 3 μL of template RNA. To detect the presence of the viral genome in the conjunctival specimens, primers targeting the CDV large polymerase gene that were reported to attach to the genome of different circulating strains of the virus, including the wild type, were used (5). This primer pair detects a 409 base segment of the conservative region of the large polymerase gene (L) of the virus and has the sequence: 5′-ATCCGCTCATCGATCAAGAC-3′ (nt 12400–12420) and 3′-AAGCCTCTTGCCAAGATTC-5′ (nt 12788–12808) as designed by Demeter et al. (5).

Reverse transcription of the viral RNA was performed at 50°C for 30 min. After an initial denaturation at 95°C for 15 min, the reaction mixtures were subjected to 40 cycles of heat denaturation at 94°C for 45 sec, primer annealing at 52°C for 45 sec, and DNA extension at 72°C for 1 min before a final extension at 72°C for 10 min. The reactions were carried out in a PCR thermocycler. After RT-PCR, electrophoresis was performed using 8 μL of the PCR products in a 1.5% Tris acetate–EDTA–agarose gel. The gel was stained with ethidium bromide and the bands were observed under a UV illuminator. Product sizes were determined using a 100 bp molecular weight ladder.

An inter-rater reliability analysis using the Kappa statistic (Cohen's κ-value) was performed to determine the agreement between readouts for FA and PCR, cytology and PCR, and cytology and FA. Kappa values >0.7 indicated a very good level of agreement. In this study, κ-values were calculated using commercial software (Graph Pad Prism v.6 Graph Pad, Inc., San Diego, CA). The sensitivity and specificity (SE, SP) as well as positive likelihood ratio (PLR) and negative likelihood ratio (NLR), which allows assessment of the diagnostic performance value of the tests (cytology, FA, and both methods in series and in parallel against PCR as the gold standard) using SE and SP, were calculated. These calculations were done using commercial software (Calc v. 12.3.0.0; MedCalc Software, Ostend, Belgium).

All procedures were done according to the ethical standards of the Helsinki Declaration of 1975, as revised in 2000, as well as national law and the guidelines of our Institutional Animal Care and Use Committee. According to the regulations, a formal approval from the Ethical Committee was not required as long as the samples were acquired for diagnostic or monitoring purposes under informed owner consent.

Results

CDV viral RNA was found in 19 of 57 (33.33%) specimens using a continuous RT-PCR analysis. Cytological examination of conjunctival samples revealed sheets of nonkeratinized epithelial cells with large, round, homogeneous nuclei, and abundant cytoplasm. Inclusion bodies were found in conjunctival epithelial cells from 4 of the 57 dogs (7.02%) tested. One of these four dogs was PCR negative. The κ-value between the cytology and PCR is shown in Table 1, whereas SE, SP, PLR, and NLR of cytology against PCR are presented in Table 2.

Among the 57 samples tested, 15 (26.3%) were found to be positive using the FA. The κ-value between the FA and PCR assay and that between cytology and FA are shown in Table 1. SE, SP, PLR, and NLR of FA against PCR as a gold standard are presented in Table 2.

SE, SP, PLR, and NLR of cytology and FA used in combination in series and in parallel against PCR as a gold standard are also presented in Table 2.

Discussion

Despite vaccination efforts, canine distemper continues to present a serious threat to dog populations, and is characterized by high morbidity and mortality rates, particularly in young puppies (16). CDV and the measles virus belong to the genus Morbillivirus and share many structural and functional features. Therefore, canine distemper has also been used as an animal model to study the development and progression of human measles (3). A variety of specimens can be used for the detection of CDV, including whole blood, sera, cerebrospinal fluid, skin, and stomach biopsy specimens, as well as cells from tracheal washings and blood smears (10).

High viral loads of CDV can also be found in conjunctival scrapings (7). In this study, conjunctival scrapings were used because of their clear advantages. Such samples can be easily and conveniently collected by clinicians and stored with refrigeration for long periods. The sampling procedure is quick, noninvasive, and is not painful or expensive. Although swabs and cytobrushes can be used for conjunctival specimen collection, cytobrushes form more even monolayers and result in superior cell quality and yield relative to swab samples (20). Conjunctival scrapings are also simultaneously suitable for RNA isolation of the virus after extraction and for FA when the conjunctival material is placed on a slide. Meanwhile, cytology occasionally results in detection of cytoplasmic or intranuclear inclusion bodies and is a simple, low cost and noninvasive diagnostic test that is frequently used in advance of other examinations. However, our results indicated that cytology is not sensitive and is less specific than FA. Furthermore, the diagnostic performance of the cytology and FA used in combination in series and in parallel was equal to that of cytology and FA, respectively, relative to when each method was used alone. Moreover, conjunctival histopathology is very similar in dogs with noninfectious and CDV-induced keratoconjunctivitis (4).

In this study, the results for FA and cytology were compared with PCR analysis of conjunctival scrapings from 57 dogs with clinical signs compatible with canine distemper. We found good agreement between FA and PCR in that PCR detected viral RNA in 19 samples and FA detected CDV antigen in 15 samples. A similar deviation was previously observed and the difference was attributed to the disappearance of CDV antigens from epithelial cells 3 weeks after infection (12). Therefore, for subacute and chronic infections, FA may yield negative results. PCR may thus be better suited for confirmation of these infections (12). Both methods agreed for negative results.

Recent vaccination may have been a source of disagreement between the two methods, since some dogs sampled in this study had an unknown vaccination history and recent vaccination could have interfered with the PCR results. Therefore, FA could be used instead of PCR to reduce the incidence of false positive results, despite its lower SE (13).

The SP of FA was calculated to be 100%, and supports that direct FA is an extremely specific method for diagnosing CDV in conjunctival scrapings. This statistical measure indicated that the negative samples found using this method are likely true negatives. Moreover, SE was calculated to be 78.95%, which means that FA is adequately sensitive and usually detects the presence of CDV antigen. However, a negative FA result cannot exclude CDV. Positive and negative predictive values were not used during the statistical analysis because they depend on disease prevalence (9), which for canine distemper in Greece is unknown.

However, likelihood ratios are very useful measures of expressing diagnostic accuracy and are easily applied to clinical practice. These measures are independent of disease prevalence and allow the quantitation of the probability of a condition for every patient, because they are calculated using a ratio of SP and SE (9). Given the 100% SP for FA, a PLR could not be measured, but the NLR states that a negative result is truly negative and thus a negative result suggests that disease can be ruled out. The NLR value in this study indicated that a negative result is more likely to show the absence of canine distemper.

The overall diagnostic performance of cytology was poor, whereas FA was shown to be a very specific and adequately sensitive method. A negative FA result does not always preclude the possibility that a dog has canine distemper, whereas a positive result almost certainly indicates the presence of CDV in the conjunctiva.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.