Avian Influenza (H5N1) Susceptibility and Receptors in Dogs

Inoculation of influenza (H5N1) into beagles resulted in virus excretion and rapid seroconversion with no disease. Binding studies that used labeled influenza (H5N1) showed virus attachment to higher and lower respiratory tract tissues. Thus, dogs that are subclinically infected with influenza (H5N1) may contribute to virus spread.

Inoculation of infl uenza (H5N1) into beagles resulted in virus excretion and rapid seroconversion with no disease. Binding studies that used labeled infl uenza (H5N1) showed virus attachment to higher and lower respiratory tract tissues. Thus, dogs that are subclinically infected with infl uenza (H5N1) may contribute to virus spread.
A vian infl uenza (H5N1) virus has been shown to be infectious not only for birds but also for humans and mammals such as mice, ferrets, and cats. Carnivorous mammals that are susceptible to subtype H5N1 may contribute to spread of the virus; shedding of infl uenza (H5N1) by pet carnivores may pose a risk to humans. Cats experimentally inoculated with infl uenza (H5N1) have been shown to be susceptible to infection and to shed virus (1). However, dogs' susceptibility to this virus is unknown. Unpublished studies indicate that a substantial number of dogs tested in Thailand were positive for antibodies against H5N1 subtype (2). Recently, isolation of infl uenza (H5N1) virus from a dog in Thailand has been reported (3). We describe the susceptibility of specifi c pathogen-free (SPF) beagles to avian infl uenza (H5N1) and the presence of receptors for infl uenza (H5N1) in the respiratory tract of these dogs.

The Study
To study the infectivity of avian infl uenza (H5N1) in dogs, we inoculated 3 SPF beagles (HsdCpb:DOBE; Harlan Nederland, Horst, the Netherlands), 16 weeks of age, with 10 6 median tissue culture infectious doses of infl u-enza (H5N1) (A/chicken/GxLA/1204/2004). Half of the dose (0.5 mL) was administered intranasally and the other 0.5mL intratracheally. Body temperature and health status of the dogs were monitored twice a day during the fi rst 5 days after challenge and once a day thereafter. No major changes in body temperature and no clinical signs were noted.
Excretion of virus was monitored daily in swabs from rectum, oropharynx, and nose. The presence of infl uenza (H5N1) virus in these swabs was studied by inoculation into embryonated eggs and by real-time reverse transcription-PCR that targeted the matrix gene. For the PCR, we used the forward primer AI-M-F45 (5′-CTTCTAACC-GAGGTCGAAACGTA-3′, reverse primer AI-M-R251 (5′-CACTGGGCACGGTGAGC-3′) and Taqman probe AI-M-Tqmn1 (5′-6FAM-CTCAAAGCCGAGATCGCG-CAGA-XT-PH) (TIBMolBiol, Berlin, Germany). A calibration curve consisting of serial dilutions of a standard batch of infl uenza (H5N1) virus with a known median 50% egg infectious dose (EID 50 ) titer was included in each PCR. One of the dogs shed virus for several days after challenge. In this dog, virus was demonstrated by PCR in nasal swabs taken on days 1 through 4 after challenge and by virus isolation in embryonated chicken eggs on days 2 and 3 after challenge (Table 1). Quantifi cation by real-time PCR indicated that the amount of virus present in the nasal swabs corresponded to 2.0-3.2 log 10 EID 50 .
Serum samples collected at days 7 and 14 after challenge were tested for antibodies against H5N1 subtype in an infl uenza A nucleoprotein-blocking ELISA as well as in a hemagglutination-inhibition assay. An antibody response against infl uenza (H5N1) was detectable in 1 dog at day 7 after challenge. In all dogs an antibody response against infl uenza (H5N1) was demonstrated in both assays at day 14 after challenge (  the lower part of the respiratory tract where the SA2,3Gal receptors are present (5). To study the attachment pattern of infl uenza (H5N1) in the respiratory tract of dogs, we performed binding experiments with labeled infl uenza (H5N1) virus on formaldehyde-fi xed and paraffi n-embedded tissue sections. We collected tissues directly from a euthanized control dog of the same breed as that used for the infection experiment and fi xed them in 4% buffered formaldehyde solution for several weeks. Virus labeling and histochemical examination were performed according to van Riel et al. (5). Briefl y, infl uenza (H5N1) virus was grown in 10day-old embryonated eggs and inactivated with formalin. After being purifi ed by sucrose gradient centrifugation, the virus was labeled with fl uorescein isothiocyanate (FITC). Staining was performed by incubating tissue sections with FITC-labeled virus and detecting the FITC-label with a peroxidase-labeled rabbit anti-FITC. To create a negative control, staining was performed without prior incubation with labeled virus or after prior incubation with FITC alone. No staining was observed in these negative controls. Chicken tissues were used as positive controls.
Strong virus binding to the epithelia of the chicken respiratory tract tissues was noted. Furthermore, as a specifi city control, preincubation with Maackia amurensis lectin, which specifi cally binds to SA2,3Gal-terminated oligosaccharides, was performed. Moderate particulate binding of infl uenza (H5N1) to canine nasal mucosa, tracheal epithelium, and alveoli was observed. Strong multifocal binding was observed in bronchial epithelium (Figure). This staining pattern was also found after binding of FITC-labeled M. amurensis lectin to canine respiratory tract tissues. Virus binding could be blocked with unlabeled M. amurensis lectin.

Conclusions
Our results demonstrate that dogs are susceptible to infection with avian infl uenza (H5N1) virus and can shed virus from the nose without showing apparent signs of disease. Moreover, receptors for avian (H5N1) virus are present not only in the lower part of the respiratory tract of dogs but also in their trachea and nose, which are potential portals of entry for the virus.
Infl uenza virus infection of dogs was fi rst reported in 2004 (6). Infl uenza (H3N8) of equine origin caused outbreaks in greyhounds in Florida and has since been found in dogs in >20 US states (7). The course of experimental infection of SPF dogs with subtype H5N1 resembles that of the experimental infection of dogs with the subtype H3N8 (6): all dogs seroconverted, and some excreted virus without obvious disease. In contrast to the experimental outcomes, natural infections with infl uenza (H3N8) resulted in serious illness, death, and widespread infection for dogs. This fi nding warrants special attention to the potential course of avian infl uenza (H5N1) infection in dogs. Therefore, dogs' contact with birds and poultry should be avoided in areas with infl uenza (H5N1) outbreaks to prevent possible spread of virus and human exposure to infl uenza (H5N1) virus that might have been adapted to mammals.
The Dutch Ministry of Agriculture fi nancially supported this study.