A novel nested PCR for the diagnosis of calicivirus infections in the cat

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Abstract

A novel nested PCR (nPCR) assay is reported on the diagnosis of the feline calicivirus (FCV) infection. The test was performed on 47 ocular and 40 pharyngeal swabs collected from 47 cats with respiratory syndrome; among the 87 samples examined, 18 ocular and 23 pharyngeal swabs were positive in nPCR.

The nPCR sensitivity was compared to other diagnostic techniques such as virus isolation on cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). The nPCR was more sensitive than the virus isolation and RT-PCR; therefore, it can be used for calicivirosis diagnosis in cats.

Introduction

Feline calicivirus (FCV) is considered the most common upper respiratory tract disease (URTD) associated pathogen in cats, especially those living in colonies (Gaskell and Dawson, 1994).

The main reasons are: (i) the presence of carriers, since the recovered animals can be virus-persistent eliminators for many years (Gaskell and Dawson, 1998); (ii) the FCV resistance in the environment (Doultree et al., 1999).

Even though FCV infection can have a subclinical course, it often shows URTD symptoms such as sneeze and conjunctivitis. Oral cave ulcerations, chronic gingivitis and temporary limp are often observed (Dawson et al., 1994). The disease usually shows a benignant course and possible complications may be due to the contemporary presence of immunosuppressor agents like feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) or secondary bacterial infections.

The diagnosis of FCV infection is very difficult only by the anamnesis and the clinical signs because there are no pathognomonic clinical signs useful to differentiate calicivirosis from other respiratory infections. Therefore, it is necessary to refer to laboratory methods such as isolation on cell cultures, immunofluorescence (IF) test and nucleic acid amplifications by polymerase chain reaction (PCR) for the pathogen detection.

Viral isolation from nasal, conjunctival and oro-pharingeal swabs is easily performed but some false negatives may sometimes occur due to a very small number of virions in the sample or to the presence of antibodies in extracellular fluids able to stop virus replication in vitro. IF test is considered less sensitive than virus isolation mainly during chronic or recurrent infections, whereas it is reliable to identify viral strains isolated in cell cultures (Gaskell et al., 1985). The conventional reverse transcriptase-polymerase chain reaction (RT-PCR) sensitivity to detect little quantity of viruses in the mucosal swabs can show decreases due to: (i) the presence of ribonucleases in mucosal secretions able to degrade viral RNA; and (ii) the typical genomic variability of caliciviruses (Sykes et al., 2001, Ratcliff et al., 2002). The nested PCR (nPCR) can increase sensitivity (Radford et al., 1997).

In the present note, we report the development and use of a novel nested PCR assay on mucosal swabs from cats affected by URTD. The nPCR was compared to other techniques for the diagnosis of FCV infection such as virus isolation and conventional RT-PCR.

Section snippets

Virus

The F9 vaccinal strain of FCV cultivated in Crandell Feline Kidney (CrFK) cell line in minimum essential medium modified by Dulbecco (D-MEM) with 10% of foetal calf serum was used in the study. When the cytopathic effect was observed, cells were kept at −80 °C until their use for the nucleic acid extraction.

Mucosal swabs

From 47 cats affected by URTD-related respiratory symptoms no. 87 mucosal samples (pharyngeal and conjunctival swabs) were collected from October 2002 through March 2003. It was not possible

Results

Eighteen swabs produced cpe referable to FCV. In particular, FCV was isolated from both conjunctival and pharyngeal swabs in four animals; the virus was isolated only from the pharyngeal ones in nine cats and FCV was isolated from the conjunctival swab in one cat, from which the pharyngeal swab was not collected. The isolates were identified as FCV strains by IFA test.

FCV was detected by one-step RT-PCR in 5 conjunctival and 12 pharyngeal swabs and by n-PCR in 18 conjunctival and 23 pharyngeal

Discussion

The main problems for FCV detection in feline mucosal swabs are: (i) the very small number of virus particles; (ii) the presence of ribonucleases in mucosal excreta; and (iii) FCV genetic variability. We performed a high sensitive nPCR able to overcome these limitations. As already reported by other authors (Sykes et al., 1998), no difference in the sensitivity was observed between isolation in cell cultures and RT-PCR, whereas the difference was found using the nPCR. In addition, the time

Acknowledgements

We are grateful to Mr. Ottavio Palucci for the exellent technical assistance. Draft of the manuscript was revised by Drs. Stella Iurlaro, Francesca Rosati and Clara Di Giuseppe.

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