Experimental infection of dogs and ferrets

To examine the pathogenicity and transmissibility of AS-01/09, AS-05/12, AS-11/13, LPM91/06, and each of the reassortant viruses between AS-01/09 and LPM91/06 viruses, influenza antibody-free beagles (two beagles per virus per cage/in duplicate) aged nine to ten weeks were intranasally inoculated with 10^5.5 EID₅₀ of the virus in a volume of 1 mL (500 µL per nostril). The next day, two naïve dogs were introduced into the adjacent cage, spatially separated from the infected dogs by two stainless steel grids 5 cm apart. Nasal secretion swabs were collected from infected dogs at 1, 3, 5, and 7 days post infection (dpi) and from contact dogs daily until 9 days post contact (dpc). Rectal temperatures were measured with a digital predictive thermometer with the use of lubricant. The rectal digital thermometer (RDT) was inserted into the rectum for a length of about 2 cm. Time of a single rectal measurement for each dog was 10–20 s. In between dogs, RDT was disinfected with 70% ethanol. Five days after infection, two inoculated dogs from each group were euthanized for virus titration in the trachea and lungs. Sera were collected at 14 and 21 dpi, and seroconversion was analyzed using a hemagglutination inhibition (HI) assay. To rule out other secondary Mycoplasma species infection during the study, we tested all the collected lung tissues for species-specific PCR reactions for M. arginini, M. canis, M. cynos, M. edwardii, M. felis, M. gateae, M. maculosum, M. molare, M. opalescens, M. spumans, Mycoplasma sp. HRC 689, and M. collis as previously described²⁹. Further, Canine parvovirus, Canine distemper virus, and seasonal H1N1 and H3N2 influenza A viruses were tested for any opportunistic infection by (RT)-PCR with each specific primer set. To assess the transmissibility of the three viruses in ferrets, groups of ferrets (n = 4) (Mustela putorius furo) (Marshall Bio Resources, NY, USA) seronegative for influenza virus antibody were intranasally inoculated with 10^7.0 EID₅₀ of each H3N2 virus in a volume of 1 mL (500 µL per nostril). The next day, two naïve ferrets were introduced into the adjacent cage and spatially separated from infected ferrets by two stainless steel grids, 5 cm apart. Five days after infection, two of the infected ferrets in each group were euthanized for viral titration in the brain, trachea, lung, kidney, spleen, and intestines (rectum). Sera were collected 14 and 21 dpi, and seroconversion was analyzed using a HI assay.