913526
research-article2020
VDIXXX10.1177/1040638720913526Serology of influenza A virus infection in dogs in PolandKwasnik et al.
Brief Communication
Serologic investigation of influenza A virus
infection in dogs in Poland
Malgorzata Kwasnik,1
Wojciech Rozek
Journal of Veterinary Diagnostic Investigation
2020, Vol. 32(3) 420­–422
© 2020 The Author(s)
Article reuse guidelines:
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https://doi.org/10.1177/1040638720913526
DOI: 10.1177/1040638720913526
jvdi.sagepub.com
Marcin Smreczak, Jerzy Rola, Kinga Urbaniak,
Abstract. The 2 predominant circulating subtypes of influenza A virus in the dog population, equine-origin H3N8 and
avian-origin H3N2, constitute a potential zoonotic risk. We determined the prevalence of influenza A antibodies in 496 dogs
in Poland and found 2.21% of sera positive by commercial ELISA. Hemagglutination inhibition (HI) assays indicated 7.25%
of sera positive using equine H3N8, swine H3N2, and pandemic H1N1 antigens, with the most frequently detected immune
response being to H3N2. Considering interspecies transfer, reassortment ability, and close contact between dogs and humans,
infections of dogs with influenza A virus should be monitored.
Key words: canine diseases; influenza A virus; serologic tests; zoonotic infections.
Influenza A viruses (IAVs) are highly contagious pathogens
that can cause isolated infections or large outbreaks in a wide
variety of animals. Dogs have not typically been considered
a natural host of this orthomyxovirus. Canine influenza A
virus (CIV) subtype H3N8 was first identified in 2004 in the
United States, but retrospective studies have suggested that
H3N8 CIV emerged in dogs in ~ 1999.3 Further studies have
shown that H3N8 CIV was present in many places across the
United States.2 Molecular analysis of the viral genome
revealed 96% homology with equine influenza A virus (EIV)
subtype H3N8, suggesting direct transmission from horses to
dogs.3 Some years later, the H3N2 subtype of CIV of avian
origin was identified in South Korea,14 but subsequent data
showed the H3N2 CIV to be widely distributed in East
Asia.22 H3N2 CIV emerged in the United States and Canada
through the importation of infected dogs.18 Given the spread
of CIV in the United States and East Asia, serologic surveillance is being maintained.16,21 In Europe, an outbreak of
influenza caused by H3N8 EIV was confirmed in English
foxhounds in 2002.4 No other outbreaks in Europe have been
recorded to date, but seroprevalence data for H3N8, H3N2,
and pandemic H1N1 (H1N1pdm) viruses have been reported
in Germany and Italy.5–7,12 Recognizing the deficiency of
information in Europe, our aim was to determine the seroprevalence of IAV infections in dogs in Poland.
The canine sera used in our study were originally submitted to the National Veterinary Research Institute in Pulawy,
Poland (2016–2017) for mandatory verification of the effectiveness of rabies vaccination of dogs under the aegis of the
Pet Travel Scheme and, therefore, these dogs were not
selected for testing because of their demonstration of clinical
signs of respiratory diseases. The animals came from all
parts of the country, thus representing the general population
of pet dogs in Poland. We tested 496 canine sera by both a
hemagglutination inhibition (HI) test and enzyme-linked
immunosorbent assay (ELISA). HI tests were performed for
3 Polish isolates of IAV: equine A/equine/Pulawy/1/2008
(H3N8), swine A/swine/Poland/04711/2013 (H3N2), and
pandemic A/swine/Poland/15620/2012 (H1N1pdm).11 The
homology of hemagglutinin (HA) sequences between H3N8
or H3N2 strains used in HI tests and CIV strains (isolated in
the United States or China) is presented in supplementary
materials (Supplementary Table 1). Before the HI test, all
sera were treated with receptor-destroying enzyme (RDE;
Denka Seiken, Tokyo, Japan) to remove nonspecific reactions. Briefly, 3 volumes of RDE were added to each serum
for 16 h at 37°C. A HI titer ≥ 16 was treated as a positive
result. The use of cutoff 32 for positive results is recommended and ensures high sensitivity and specificity of the HI
test.1 We adopted a cutoff of ≥ 16 to increase detection of
weak-positive sera, at the expense of specificity. The procedure was carried out according to the instructions in the OIE
Manual of Diagnostic Tests and Vaccines for Terrestrial Animals using chicken erythrocytes.19 An ELISA was used to
screen samples for the presence of influenza antibodies (ID
Screen influenza A antibody competition assay; IDVet,
Montpellier, France) according to the manufacturer’s instructions. This assay detects antibodies to the conserved nucleoprotein (NP) antigen of the IAV, regardless of HA or
neuraminidase subtype.
Departments of Virology (Kwasnik, Smreczak, Rola, Rozek) and
Swine Diseases (Urbaniak), National Veterinary Research Institute,
Pulawy, Poland.
1
Corresponding author: Malgorzata Kwasnik, National Veterinary
Research Institute, Al. Partyzantow 57, 24-100 Pulawy, Poland.
malgorzata.kwasnik@piwet.pulawy.pl
Serology of influenza A virus infection in dogs in Poland
421
Figure 1. Hemagglutination inhibition results of 496 canine sera, with 3 influenza A virus subtypes: equine H3N8, swine H3N2, and
swine H1N1pdm.
Figure 2. Hemagglutination inhibition titers of canine sera
for equine H3N8, swine H3N2, and swine H1N1pdm subtypes of
influenza A virus. Median value and 95% confidence intervals are
indicated by long and short horizontal lines, respectively.
We identified 36 of 496 (7.25%) sera as positive for IAV
using the HI test. The percentages of sera with antibodies by
subtype were 1.41% for equine H3N8, 4.23% for swine
H3N2, and 1.61% for swine H1N1pdm (Fig. 1). The median
HI titers were 64, 32, and 16 for the H3N8, H3N2, and
H1N1pdm subtypes, respectively. The range of HI titers for
all positive samples was 16–256 (Fig. 2). Of the 36 HI positive sera, 10 had reactions with more than one antigen. In
these cases, sera were considered positive for the subtype
with which they had the highest reactivity (Supplementary
Fig. 1). In the ELISA, 11 of 496 sera tested were positive
(2.21%), of which 6 were also positive in HI (Table 1). No
subtype was identified for 5 of the sera positive in the ELISA.
Both tests used are not specific to CIV. The CIV designation
should be restricted only to those IAV strains that can propagate in dogs and have a unique genetic signature.
The seroprevalence of influenza in dogs in Europe has
been studied in Italy and Germany. In Italy, the positive
results obtained in a NP ELISA were 0.5–3.5%. HI positive
reactions were obtained for H3N8, H3N2, and H1N1pdm
subtypes of IAV (using canine H3N8, equine H3N8, swine
H3N2, and H1N1pdm antigens).6,7,12 Seroprevalence was
lower In Germany, 7 of 736 sera (0.95%) were positive in an
NP ELISA, and antibodies against H1N1pdm were detected.5
We found a reaction with the swine H1N1pdm strain in a
small number of dogs (1.6%) in our current study; infections
with H1N1pdm have been confirmed in Poland, both in
swine and humans.11,13 Generally, in Europe, the percentage
of dogs seropositive in NP ELISA is low, whereas the results
of the HI test indicate a higher percentage of seropositive
animals.
The seroprevalence of CIV has been studied worldwide,
notably in countries in which outbreaks of canine influenza
have been reported, such as the United States or China. In a
2019 report of testing for canine H3N2 in pet dogs in the
United States, 3.53% of sera were positive in ELISA and
2.21% in HI.8 Although only a low seroprevalence of canine
influenza is usually recorded in the United States, seroprevalence can reach high levels in influenza-endemic areas.8,9
Studies carried out among dogs in Hong Kong have shown
that seroprevalence rates of canine H3N2 or H3N8 (0.9%)
were lower than those of human IAVs H1N1pdm or H3N2
(7.5%), indicating that humans may serve as the major
source of exposure to IAV for dogs in a densely populated
city.17
Among the ELISA-negative samples, we detected positive sera in the HI test. A similar tendency has been observed
in previous studies.9,12,17 This divergence might arise from
the kinetics of anti-NP and anti-HA antibodies; anti-NP titers
detected by ELISA decrease over time, as observed for avian
influenza.10,15 Additionally, ELISA for swine influenza
detects mainly IgG antibodies, whereas the HI test detects
IgM as well as IgG antibodies; hence the ELISA may not
422
Kwasnik et al.
Table 1. Subtypes of influenza A virus among ELISA-positive (n = 11) and -negative (n = 485) samples.
ELISA-positive
ELISA-negative
H3N8
H3N2
H1N1pdm
H3N8
H3N2
H1N1pdm
1
1
4
6
20
4
HI positive
HI = hemagglutination inhibition.
identify positive animals at the early stage of infection.20 NPbased ELISA is therefore indicated as a suitable tool for surveillance purposes, but it can give false-negative results. On
the other hand, not all sera positive in ELISA have been
assigned a specific serotype of IAV. It is possible that the
strains used in HI are incompatible with strains circulating in
the dog population. Given the limitations of the tests used, it
seems advisable to use both tests in parallel to check the
seroprevalence of IAV in dogs.
Although rather low seroprevalence is noted generally, it
seems advisable to monitor the seroprevalence of IAV infection in both pet and farmed dogs, especially in the context of
H1N1pdm distribution. Given the population of dogs estimated at 700 million worldwide, the close contact between
humans and dogs, and the susceptibility of dogs to infection
by IAVs of both mammalian and avian origin, serosurveillance in dogs may be useful.
Acknowledgments
We thank Urszula Bocian for excellent technical assistance.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The study was supported by KNOW, Ministry of Science and
Higher Education, Poland [05-1/KNOW2/2015 (K/02/1.0)].
ORCID iD
Malgorzata Kwasnik
https://orcid.org/0000-0002-8689-6906
Supplementary material
Supplementary material for this article is available online.
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