Avian influenza viruses used in this study originated from ruddy turnstones (Arenaria interpres) and were originally isolated from cloacal swabs in 9-day-old embryonated chicken eggs (ECE) incubated at 37°C for 72 hours. Viruses were minimally passaged (3 or fewer passages) in ECEs. A PFU titer was established for each virus via plaquing isolates on Madin-Darby canine kidney cells with a 1.2% Avicel overlay (FMC biopolymer, Philadelphia, PA), incubating for 72 hours at 37°C, 5% CO₂, then removing the overlay, fixing cells and visualizing plaques with crystal violet. Select isolates were screened in a previous study in BALB/c mice.⁴ Two isolates that exhibited efficient pulmonary replication and induced pulmonary lesions in BALB/c mice were selected for in vivo studies in cats. Isolates included A/Ruddy Turnstone/DE/650625/02 (H6N4) (abbreviated RT/625) and A/Ruddy Turnstone/DE/650645/02 (H1N9) (abbreviated RT/645).

The study included 6 5-month-old domestic cats (Liberty Research, Inc, Waverly, NY); all were seronegative to circulating H1N1 and H3N2 influenza viruses as determined by hemagglutination inhibition assay. Sera samples collected from each cat prior to infection were treated with receptor-destroying enzyme (RDE) and tested for serum antibodies with a hemagglutination inhibition assay using 0.5% chicken red blood cells as previously described.²¹ Viruses were diluted to contain 4 agglutinating units in sterile PBS. Additionally, pre and post inoculation sera were tested for serum antibodies by microneutralization assay using experimental viruses RT/625 and RT/645 as previously described, starting at a 1:20 dilution.¹ All cats had a pre inoculation sera microneutralization titer of less than 1:20 against these experimental LPAIVs. Cats were housed in open caging (Allentown, Allentown, NJ) in ABSL-3 facilities. A subcutaneous temperature transponder (BMDS) was implanted in each cat for identification and temperature measurement. Cats were observed twice daily by animal care staff and once daily by the veterinarian conducting the study (EA Driskell). Disease was not expected in inoculated cats; therefore, no clinical scoring system was applied to the study. Studies were conducted under guidelines approved by the Institutional Animal Care and Use Committee of the University of Georgia and the Centers for Disease Control and Prevention.

For each of the viruses used, 3 cats were anesthetized with acepromazine (Boehringer Ingelheim Vetmedica, St. Joseph, MO), butorphanol (Fort Dodge Animal Health, Fort Dodge, IA), and dexmedetomidine (Pfizer Animal Health, Monmouth Junction, NJ) and intratracheally inoculated with 10⁶ PFU of virus in allantoic fluid diluted in 500 μL of sterile PBS. Temperature, weights, complete blood counts, and nasal, pharyngeal, and rectal swabs were sampled on days 0, 1, 3, 5, and 7 post inoculation (pi) under sedation with acepromazine and butorphanol. On day 7 pi, all cats were sedated and euthanized. A necropsy was performed with gross examination of thoracic and abdominal viscera, brain, and nasal turbinates. Lung and trachea were sampled and frozen at −80°C for virus isolation. Sections of cranial lung lobes, caudal lung lobes, trachea, tracheobronchial lymph nodes, heart, thymus, spleen, liver, kidney, stomach, small intestine, colon, pancreas, mesenteric lymph nodes, adrenal gland, nasal turbinates, and brain were fixed in 10% neutral buffered formalin for histopathology and immunohistochemistry. One section from each of the following lung lobes were examined on each cat: cranial left lobe, cranial right lobe, caudal left lobe, caudal right lobe, and accessory lobe. Virus isolation was performed for all swabs and fresh lung and trachea. Swabs (in 2 μL PBS) and tissues were frozen at −80°C for later virus isolation. Tissues were then homogenized in 1 μL PBS, clarified by centrifugation, and samples from clarified tissue homogenate and PBS from swabs were inoculated into 9-day-old ECEs (4 ECEs per sample) and incubated 72 hours for virus isolation. Complete blood counts were performed using Vet Scan analyzer (Abaxis, Union City, CA).

All tissues collected were routinely processed for light microscopy. Briefly, sections of formalin-fixed, paraffin-embedded tissues were deparaffinized and stained with HE or processed for immunohistochemistry. Immunohistochemistry was performed on lung, trachea, and tracheobronchial lymph node for all cats. Immunohistochemistry was performed on an autostainer (Dako Cytomation, Carpinteria, CA) with DAB chromagen (Dako Cytomation) or Vulcan fast red chromagen (Biocare Medical, Concord, CA). Tissues were blocked with Dako Biotin Blocking System (Dako Cytomation). To detect viral antigen, a goat polyclonal antibody to the nucleoprotein of influenza A virus (Biodesign International, Sako, Maine; diluted 1:10,000) and rabbit biotinylated anti-goat IgG as a secondary antibody (Vector Laboratories, Burlingame, CA) were used. For double labeling, a mouse monoclonal antibody for cytokeratins AE1/AE3 (Biogenex, San Ramon, CA; diluted 1:200) with Biogenex Multilink kit with HRP label (Biogenex) and a mouse monoclonal antibody to the nucleoprotein of influenza A (Biodesign International; diluted 1:1000) with Biogenex Multilink kit with alkaline phosphatase label (Biogenex) were used. Hematoxylin counterstain was used for all immunohistochemistry. Sections of formalin-fixed, paraffin-embedded heart from a chicken infected with H5N1 influenza virus incubated with the goat polyclonal or mouse monoclonal primary antibodies for influenza virus antigen served as a positive control. The same tissue (chicken heart) was used as a negative control for the single labeling for influenza A by replacing the primary antibody with isotype-matched control antibody. For the double labeling immunohistochemistry, lung sections from a domestic cat that died due to trauma were treated with the same protocol as double-labeled experimental tissues served as negative controls.

Cats inoculated with either RT/625 or RT/645 had no clinical evidence of disease; all cats remained alert and playful with minimal weight change. For RT/625 inoculated cats, no weight loss occurred post-inoculation compared to pre-inoculation weights, and for RT/645 there was an average of 3% weight loss on day 1 pi, but an average weight gain for days 3 through 7 pi. There was no significant group, day, or group by day interaction effect on counts of total leukocytes, lymphocytes, monocytes, or granulocytes (Table 1).

Despite absence of clinical disease, all cats seroconverted to either RT/625 or RT/645. For RT/645, virus was not isolated from either antemortem swabs or postmortem tissues, but all cats seroconverted by day 7 pi determined by microneutralization assay (Table 2). For RT/625, all cats seroconverted and virus was isolated from the lung of 1 of 3 cats on day 7 pi. Additionally, 2 out of 3 cats inoculated with RT/625 had evidence of pharyngeal shedding based on positive virus isolation from pharyngeal swabs taken on days 3 and 5 pi for one cat and on days 3, 5, and 7 pi for another cat (Table 2). Virus was not isolated from the tracheal tissue or nasal or rectal swabs for RT/645- or RT/625-inoculated cats.

No gross lesions were observed. Microscopic lesions were primarily restricted to the lung in both RT/625- and RT/645-inoculated cats and were usually more extensive and severe in the cranial lung lobes, possibly a result of the inoculation method. Most cats had only a small percentage of lung involvement microscopically, although up to 50% pulmonary involvement of examined sections was present in a few of the cats (Table 2). In the lung on day 7 pi, bronchioles were filled by primarily macrophages and few neutrophils, often with adjacent alveoli filled with macrophages (Figs. 1, 3, 4). There was bronchiolar epithelial regeneration as affected bronchioles were lined by large, plump epithelial cells (Fig. 1). Frequently, vessels were surrounded by thick cuffs of lymphocytes (Fig. 2). Clear evidence of alveolar involvement was present, with prominent, extensive type II pneumocyte hyperplasia of alveolar epithelium (Figs. 3, 4). No lesions were observed in the intrapulmonary bronchial or bronchiolar glands.

There was sinus histiocytosis present in the tracheobronchial lymph nodes of all inoculated cats. All RT/625- and RT/645-inoculated cats had strong intracytoplasmic immunoreactivity in pulmonary and tracheobronchial lymph node macrophages for the nucleoprotein of influenza A virus (NP) using immunohistochemistry, suggestive of phagocytosis of influenza antigen (Fig. 5). There were also rare alveolar cells with strong intranuclear and some intracytoplasmic immunoreactivity for the NP of influenza for both RT/625- and RT/645-inoculated cats (Table 2). These cells were confirmed as pneumocytes with double staining for the NP of influenza virus A and cytokeratin AE1/AE3 (Fig. 6). The morphology of these pneumocytes were flat, suggesting they were type I pneumocytes. No immunoreactivity for the NP of influenza was observed in type II pneumocytes.