Three dogs (beagles, 11
months old) and three Thoroughbred horses (18–19
months old) were studied. All the animals were healthy and showed no serological evidence of prior H3N8 virus infection or vaccination in haemagglutination inhibition (HI) tests (HI titres <10) for antibodies to CO06 and EIV (A/equine/Ibaraki/1/2007, H3N8) (See below). CO06 was isolated from a diseased dog showing an acute respiratory sign in the United States. Each dog was randomly paired with a horse giving a total of three pairs. The dogs were inoculated with CO06 [108.3
50% egg infective doses (EID50
)] by inhalation using an ultrasonic nebulizer (Soniclizer305, ATOM, Tokyo, Japan) connected to a facemask on Day 0 under sedation by intramuscular administration of medetomidine hydrochloride (10
μg/kg bodyweight; Domitor, Zenoaq, Fukushima, Japan).
Each pair remained in the same stall (3
m wide, 6.05
m deep and 4.1
m high) continuously from Day 0 to Day 14 as previously reported [11
]. On Day 14, the dogs were euthanized. From Days 15 to 21, each horse was kept alone in the same stall. The rectal temperatures of all the animals were measured each morning during the experiment. A dog and a horse with rectal temperatures exceeding 39.5 and 38.8°C, respectively, were defined as having significant pyrexia in this study. We performed daily physical observations of the dogs and horses from Days −1 to 14 and from Days −1 to 21, respectively. The observation records for each animal were assigned scores as previously described by Jirjis et al. [12
] and Toulemonde et al. [13
] with slight modifications (Table
). We calculated the total clinical scores for each animal each day.
Clinical score assignment of respiratory disease
Nasal samples were collected from the dogs using 3.0
mm absorbent cotton swabs (1P1501, JCB Industry, Tokyo, Japan) and from the horses using 1.0
cm absorbent cotton swabs (JMS menbou, Japan Medical Supply, Hiroshima, Japan) on a daily basis (from Days -1 to 14 and Days -1 to 21, respectively) and their extracts were titrated in 10-day-old embryonated hen’s eggs as previously described [11
]. Briefly, the swabs collected from the horses and the dogs were immersed in 2.5 and 1.0
ml of transport medium [phosphate buffered saline (PBS, pH 7.2) supplemented with 0.6% tryptose phosphate broth, 500 unit/ml penicillin, 500
μg/ml streptomycin and 1.25
μg/ml amphotericin B), respectively. The swab samples in the transport medium were vortexed and briefly centrifuged to precipitate debris. Then, 200
μl of the supernatants that had been diluted at 1:10 (v/v) in transport medium were injected into the allantoic cavities of embryonated hen’s eggs (four eggs per sample). The allantoic fluid was harvested after 3
days of incubation at 34.0°C and examined for the presence of influenza A virus in a hemagglutination test using 0.5% hen’s red blood cells. The virus titres (log10
μl) were determined for nasal swab samples that were haemagglutination-positive [14
The HI titres to CO06 of the sera collected from each dog or horse on Days −1, 9 and 14 or Days −1, 9, 14 and 21, respectively, were also measured as previously described [15
]. Briefly, the antisera were treated with trypsin, heat and potassium metaperiodate to remove non-specific inhibitors. Then the required final dilution of the treated antiserum (1:10) was prepared and adsorbed with packed chicken erythrocytes. Two-fold dilutions of the antiserum with PBS were prepared; 25
μl of the diluted serum was used in each well of a microplate. 25
μl of virus containing 4 haemagglutination units was added to each well, and the microplate was incubated at room temperature for 30
min. Then 50
μl of 0.5% chicken erythrocytes was added to each well. The results were read after incubation at room temperature for 60
min. The HI antibody titres were determined by the reciprocal of the highest serum dilution that exhibited no haemagglutination.
The dogs and horses were examined by gross pathology on Days 14 and 21, respectively, after the euthanasia. If lesions were observed by gross pathology, they were subjected to further bacterial examinations. The samples were aseptically trimmed and then homogenized with nine times their volume of sterile distilled water. Subsequently, 100
μl of each sample was inoculated onto a blood agar plate and a MacConkey agar plate, and then incubated aerobically for 24
h at 37°C or anaerobically for 48
h also at 37°C. Bacterial identification was performed by employing Gram staining, morphological features, the catalase test, the oxidase test and commercial identification test kits (API, SYSMEX).
All experimental procedures were conducted in a biosafety level-3 facility and approved by the Animal Care Committee of the Equine Research Institute of the Japan Racing Association.