Serosurvey of pandemic H1N1 influenza A virus in dogs in Andalusia (southern Spain)

In April 2009, a new influenza A virus (IAV) subtype (A(H1N1)pdm09) spread worldwide and triggered the first human influenza pandemic of the 21st century. Since then, exposure to the pandemic H1N1 IAV has been confirmed in different animal species. Serological evidence and clinical infection with A(H1N1)pdm09 have been reported in canines, but the information available about the role of dogs in the epidemiology of this IAV subtype is still very limited in Europe. A cross‐sectional study was carried out to determine the seroprevalence of A(H1N1)pdm09 in dogs in southern Spain, a region with endemic seasonal circulation in human. Sera from 750 companion dogs were collected during the period 2013–2016. Antibodies against pandemic H1N1 IAV were analysed using the haemagglutination inhibition test. Positive samples were also tested by single radial haemolysis assay. Seropositivity was only confirmed by both methods in one (0.13%; 95% CI: 0.00–0.38) adult animal sampled in 2013. To the best of the authors’ knowledge, this is the first report of A(H1N1)pdm09 exposure in dogs in Spain. The low seroprevalence obtained indicates a limited exposure history to A(H1N1)pdm09 IAV in dogs in this country and suggests a low risk of transmission of this zoonotic IAV subtype between humans and dogs.


| INTRODUC TI ON
Influenza A viruses (IAV; family Orthomyxoviridae) are among the most important emerging pathogens worldwide, affecting a wide range of animal species, including human beings. Although wild birds are considered the main reservoir of these viruses, mammalian species can also be implicated in the transmission of IAV (Chen et al., 2018).
In April 2009, a new swine-origin H1N1 IAV subtype labelled A(H1N1)pdm09 was first reported in Mexico and United States.
The virus spread rapidly worldwide causing the first influenza pandemic of the 21st century. During the first year, more than 575,000 human deaths were associated with this emergent subtype around the world, and in the United States alone between 43.3 and 89.3 million cases and 12,469 deaths were linked to this virus (CDC, 2019).
Nowadays, the A(H1N1)pdm09-like viruses have an endemic seasonal circulation in Europe and represent almost the 40% of the identified IAV in humans (ECDC, 2019).
Influenza A(H1N1)pdm09-like viruses have been confirmed or suggested to be transmitted from humans to different wild and domestic species (Britton et al., 2019;Keenliside, 2013).
Furthermore, exposure to this IAV has been detected in different pet animals in several countries (Martínez-Orellana et al., 2015;Tangwangvivat et al., 2019;Zhao et al., 2014). The close contact between companion dogs and humans could be an important interface for the transmission of IAVs with zoonotic potential.
Canids have been shown to be susceptible to swine-, equine-, avian-and human-origin IAV (Dubovi, 2010). Moreover, evidence of genetic reassortment between canine H3N2 and human-H1N1 IAV including the pandemic H1N1 subtype has been documented (Chen et al., 2018;Song et al., 2012), which raises a public health concern that dogs may become intermediate hosts for novel emergent IAV.
A(H1N1)pdm09 infections have been reported in dogs in natural and experimental conditions (Lin et al., 2012). Although dogto-dog transmission of this subtype has been shown to be limited (Lin et al., 2012;Song et al., 2015), infections of dogs with human A(H1N1)pdm09 acquired from their owners were previously documented (Keenliside, 2013;Lin et al., 2012). Serological evidence of A(H1N1)pdm09 has also been reported in dogs worldwide (Chanvatik et al., 2016;Jang et al., 2017;Su et al., 2019;Sun et al., 2014). However, the information available about the role of canines in the epidemiology of this IAV subtype is still very limited in Europe (Damiani, Kalthoff, Beer, Müller, & Osterrieder, 2012;Dundon, De Benedictis, Viale, & Capua, 2010). Hence, we aimed to assess exposure to A(H1N1)pdm09 influenza virus in domestic dogs in Spain, where this subtype was predominately circulating in humans at the time of sampling (ISCIII, 2020).

| Study design and sample collection
A cross-sectional study was carried out to determine the prevalence of antibodies against A(H1N1)pdm09 IAV in domestic dogs from Andalusia (southern Spain: 36°N-38°60′N, 1°75′W-7°25′W), the Spanish region with the highest census of this species (MAPA, 2019). The sample size was estimated assuming a prevalence of 50% (the highest sample size for studies with unknown prevalence), with a 95% confidence level and a desired precision of ± 3.5% (Thrusfield, 2018). A stratified sampling design was adopted based on the proportion of dogs in the eight provinces of Impacts • This is the first report of A(H1N1)pdm09 exposure in dogs in Spain.
• Our results indicate a limited A(H1N1)pdm09 circulation in dogs in Spain.
• The risk of A(H1N1)pdm09 transmission from dogs to humans can be considered low. Andalusia. Within each province, animals were selected randomly. A total of 750 blood samples from dogs admitted into veterinary clinics in 129 municipalities were finally collected between 2013 and 2016 ( Figure 1). Samples were obtained by cephalic or jugular vein puncture using a sterile collection system. Sera were separated by centrifugation at 400 g for 15 min and stored at −20°C until analysis. Epidemiological data, including sex, age (<12, 12-24, 25-36 and >36 months), location, whether pure-or crossbred, activity (pet, hunting or watchdog) and size (height at the withers: small (<40 cm), medium (41-60 cm) or large (>60 cm)), were recorded for each animal (Table 1). Afterwards, 50 μl of 0.5% packed cell volume chicken red blood cells was added into each well and incubated at room temperature for 30 min. HI antibody titre was defined as the reciprocal of the last dilution that showed complete absence of agglutination. Samples with HI titres ≥40 were considered positive.

| Serological analysis
Screened positive and doubtful samples by HI were further examined for antibodies against A(H1N1)pdm09 influenza subtype using single radial haemolysis assay (SRH) as previously described without modification (OIE, 2016). Samples with a radial zone of lysis around the well were considered positive. The diameter of the zone of haemolysis was measured with digital callipers, and the strain-specific antibody level was expressed as the area (mm 2 ). Serum from a hyper-immunized experimental pony (Scott et al., 2012) was used as positive control in both diagnostic tests.

| Statistical analyses
The overall prevalence of antibodies was estimated from the ratio of positives to both HI and SRH to the total number of analysed samples, with 95% CI. spss 22.0 software (IBM Corp.) was used for statistical analyses.

| Ethical considerations
Serum samples were opportunistically collected from animals subjected to health programmes, medical check-ups or surgical interventions during the study period; therefore, no ethical approval was necessary for this study.    To the author's knowledge, this is the first surveillance of A(H1N1)pdm09 in dogs in Spain. The low seroprevalence obtained is in accordance with those previously reported in this species
Dogs have been found to be clinically infected with pandemic H1N1 influenza virus (Lin et al., 2012), and significantly higher seropositivity to A(H1N1)pdm09 was observed in dogs with respiratory illness than in healthy animals (Jang et al., 2017). However, the occurrence of A(H1N1)pdm09 in dogs is likely to be overlooked because most infections in this species are mild or subclinical (Lin et al., 2012;Sun et al., 2014;Yin et al., 2014), which may lead in under-diagnosis of cases. Unfortunately, data on the clinical history of respiratory disease could not be gathered in the tested animals in the present study.
In conclusion, the results obtained indicate limited A(H1N1) pdm09 IAV circulation in domestic dogs in Spain during the period 2013-2016. To the best of our knowledge, this is the first report of A(H1N1)pdm09 exposure in dogs in Spain, which may be of public health concern. Despite this being the main subtype of human IAV causing outbreaks in the study country (ISCIII, 2020) and the high census of companion dogs in the study area, the low seroprevalence obtained suggests that the risk of A(H1N1)pdm09 transmission from dogs to other sympatric species, including humans, can be considered low. Nonetheless, due to the presence

ACK N OWLED G EM ENTS
We are grateful to the clinic vets for provision of serum samples.

CO N FLI C T O F I NTE R E S T
None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.