The Veterinary Journal 184 (2010) 373–375

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The Veterinary Journal

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Short Communication

Detection of canine parvovirus type 2c by a commercially available

in-house rapid test
Nicola Decaro a,*, Costantina Desario a, Melissa J. Beall b, Alessandra Cavalli a, Marco Campolo a,
Anthony A. DiMarco b, Francesca Amorisco a, Maria Loredana Colaianni a, Canio Buonavoglia a
a
Department of Veterinary Public Health, Faculty of Veterinary Medicine, Strada per Casamassima km 3, 70010 Valenzano (Bari), Italy
b
IDEXX Laboratories, Inc., Westbrook, ME, USA

a r t i c l e i n f o a b s t r a c t

Article history: Diagnosis of canine parvovirus (CPV) infection is usually carried out by means of rapid immunochromato-
Accepted 2 April 2009 graphic assays, but the ability of these tests to detect all CPV variants, including the recently identified
CPV-2c, is still debated. To determine if the assays detect the different CPV variants, 201 CPV PCR-positive
faecal samples or rectal swabs were tested using a commercially available in-house test. Specimens (CPV-
Keywords: 2a, n = 51; CPV-2b, n = 50; CPV-2c, n = 100), containing CPV DNA loads >105 DNA copies/mg faeces, as
Canine parvovirus determined by real-time PCR, were selected from previous studies. The percentage of positive in-house
Diagnosis
tests was 80.4%, 78.0% and 77.0% for CPV types 2a, 2b and 2c, respectively, confirming the ability of
In-house assay
New variant canine parvovirus 2c
the test to detect the new variant CPV-2c. However, considering the sensitivity limits of the in-house tests
that have been observed previously, negative results from the in-house test kit should be confirmed by
PCR-based methods.
Ó 2009 Elsevier Ltd. All rights reserved.

Canine parvovirus (CPV) is one of the main pathogens causing
haemorrhagic gastroenteritis and mortality in young dogs and, de-
spite extensive vaccination, is still widespread worldwide. The ori-
ginal virus type, CPV-2, was first reported in the 1970s, but soon
after its emergence it was replaced in the field by two antigenic
variants, CPV-2a and CPV-2b (Truyen, 2006). A third variant,
CPV-2c, emerged in Italy in 2000 (Buonavoglia et al., 2001) and
rapidly spread to the canine population worldwide (Decaro et al.,
2006, 2007; Kapil et al., 2007). CPV-2c was initially associated with
mild clinical signs and long-term shedding, but subsequently it
was reported to cause severe enteritis and mortality (Kapil et al.,
2007), even in repeatedly vaccinated adult dogs (Decaro et al.,
2008, in press). The three antigenic variants differ from each other
only at residue 426 of the main viral capsid protein VP2, with types
2a, 2b and 2c displaying amino acids Asn, Asp and Glu, respectively
(Truyen, 2006).
Rapid in-house tests are useful for the confirmation of CPV
infection in acute cases presenting to the veterinarian in practice.
However, recent concerns have been expressed about the ability
of such in-house tests to detect the new variant 2c to the same ex-
tent as CPV-2a/2b (Kapil et al., 2007). In this study, we evaluated
the detection rates of the different CPV variants by using a com-
mercially available in-house test.
* Corresponding author. Tel.: +39 0804679832; fax: +39 0804679843.
E-mail address: n.decaro@veterinaria.uniba.it (N. Decaro).
A total of 201 specimens were examined and included 58 faecal
samples and 143 rectal swabs. The samples were recruited from
previous studies (Decaro et al., 2005, 2006, 2007, 2009), including
only those specimens that contained CPV DNA loads >105 DNA
copies/mg faeces, considering that samples with lower titres
mostly were undetected by the in-house test (Desario et al.,
2005). The real-time PCR assay used for assessment of viral DNA
loads has been shown to have equivalent sensitivity for all CPV
variants (Decaro et al., 2005).
The sample distribution according to the virus type was CPV-2a
(n = 51), CPV-2b (n = 50) and CPV-2c (n = 100), as determined by
minor groove binder probe assays able to discriminate variants
(Decaro et al., 2006). Samples were from Italy (n = 125), the United
Kingdom (UK) (n = 56), Spain (n = 5) and Greece (n = 15).
The in-house test kit used in this study was the commercial
SNAP Canine Parvovirus Antigen Test (IDEXX Laboratories) and
all testing was performed blinded to CPV strain and viral load. Per-
formance of the in-house diagnostic test was assessed by calculat-
ing the percentage of positive results for each group of samples
based on CPV type. Confidence intervals about the means were cal-
culated using exact binomial limits (a = 5%; Excel, Microsoft) and
statistical significance was determined with the v2 Square test
for multiple proportions (a = 0.05).
The SNAP Canine Parvovirus Antigen Test was able to detect 41/
51 (80.4%) type 2a, 39/50 (78.0%) type 2b and 77/100 (77.0%) type
2c CPVs (Table 1). The percentages of PCR positive samples de-
tected by the SNAP test according to geographic origin were 95/

1090-0233/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2009.04.006
374 N. Decaro et al. / The Veterinary Journal 184 (2010) 373–375
Fig. 1. Distribution of in-house ELISA results by viral load (DNA copies/mg of faeces) and CPV strain. Horizontal line indicates mean of each group.
Table 1
Percentage of faecal samples testing positive for canine parvovirus using an in-clinic
ELISA assay.
Type Positive (%) Confidence interval (95%)
CPV-2a 80.4 67.5–88.9
CPV-2b 78.0 64.7–87.2
CPV-2c 77.0 67.8–84.1
Detection did not differ between CPV strains (P = 0.997).
125 (76.0%), 45/56 (80.4%), 12/15 (80.0%) and 5/5 (100%) for Italian,
UK, Greek and Spanish samples, respectively.
The distribution of the CPV samples by SNAP test result and vir-
al titre showed that samples with a viral load >109 DNA copies/mg
faeces were generally detected by the in-house assay, with the
exception of three UK CPV-2b samples that gave a negative reac-
tion despite very high CPV loads (109–1011 DNA copies/mg faeces)
calculated by real-time PCR (Fig. 1). The three high-titre CPV-2b
samples (all from the UK) were inoculated on A-72 cells (Desario
et al., 2005) and virus isolation was successful with two strains.
The two CPV-2b isolates were amplified through serial passages
on cell cultures and, when cytopathic effects were visible in
approximately 80% of cells, the supernatants from the cell cultures
were submitted to the SNAP test, giving positive results with both
viruses.
To assess the ability of the SNAP test to detect the CPV antigenic
variants, we tested samples representative of the three CPV types,
including 100 samples containing the recently detected CPV-2c.
We selected samples of the different CPV types containing viral
loads >105 DNA copies/mg faeces to minimise the influence of viral
titre on the SNAP test results. The detection rate of CPV-2c was not
different from those of the other two CPV types, although a defin-
itive comparison of sensitivity of the test for detection of different
variants should be obtained by testing samples with lower viral
titres.
While the SNAP test routinely detected samples containing viral
loads >105 DNA copies/mg faeces (>75%), we were surprised to find
three British CPV-2b samples containing very high viral loads that
tested negative by the in-house assay. To rule out a possible test
failure, CPV-2b viruses isolated in cell culture from these SNAP
test-negative faecal samples were both positive when tested using
the rapid assay. This suggested the presence of high titres of inter-
fering antibodies in the samples or, less likely, degradation of the
capsid antigens due to long-term storage.
The TaqMan real-time PCR assays for canine parvovirus have
proven to be highly sensitive diagnostic tests which provide CPV
typing information. For that reason, samples characterised by
real-time PCR were ideal for this comparative study. Previous stud-
ies have shown that the in-house assays, as well as more tradi-
tional diagnostic methods commonly used for CPV detection,
including haemagglutination and virus isolation, are less sensitive
than real-time PCR, but are highly specific and unlikely to detect
modified live vaccines (Desario et al., 2005).
In conclusion, we have shown that the SNAP Parvo test is able to
detect the new variant CPV-2c. However, considering the higher
sensitivity of the molecular methods, the recommended approach
for parvovirus diagnosis is to utilise the in-house assay first, fol-
lowed by submission of faecal samples to the laboratory for the
PCR-based assays in questionable cases.
Conflict of interest statement
The study was equally funded by IDEXX Laboratories and by
grants from University of Bari, Italy: project ex 60% 2006 ‘Caratter-
izzazione delle varianti di campo del parvovirus del cane mediante
real-time PCR con sonde minor groove binding (MGB)’. M.J. Beall
and A.A. DiMarco are employees of IDEXX Laboratories.
Acknowledgements
The authors would like to acknowledge Michael Monn for tech-
nical support, Jason Aguire for statistical assistance and Dr. Phil
Andersen and Nancy Leonard for project support.
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