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Experiments using ferrets was conducted with approval from the CSL Limited/Pfizer Animal Ethics Committee (project license number 868) in strict accordance with the Australian Government, National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes (8th edition). Animal studies were conducted at CSL Limited using services provided under a Support Service Agreement between CSL Limited and WHO Collaborating Centre for Reference and Research on Influenza.
Adult male and female ferrets weighing 700–1300 g were used. Serum samples from ferrets were tested by hemagglutination inhibition (HI) assay against the reference strains A/California/7/2009 A(H1N1)pdm09, A/Victoria/361/2011 (H3N2), B/Wisconsin/1/2010 (B/Yamagata-lineage) and B/Brisbane/60/2008 (B/Victoria-lineage) to ensure seronegativity against currently circulating influenza subtypes and lineages.
Virus infection and oseltamivir treatment of ferrets: Ferrets were anaesthetized [50:50 mix of Ketamine (100 mg/mL): Ilium Xylazil (Xyalazine; 20 mg/mL)] and infected artificially by intranasal inoculation with either 1×103 or 1×105 TCID50 (median tissue culture infectious dose) of MDCK (Madin-Darby canine kidney (MDCK; ATCC CCL-34)-propagated A/Perth/265/2009 A(H1N1)pdm09 (2.0 × 106 TCID50/mL). A 5 mg/kg dose of oseltamivir phosphate in ferrets has been considered to be equivalent (based on weight) to the standard human adult dose of 75 mg, which is normally delivered twice daily for treatment [23]. Oseltamivir phosphate (kindly provided by Hoffmann-La Roche Ltd., Basel, Switzerland) was prepared at a concentration of 10 mg/mL in a sterile 0.5% (v/v) sugar/phosphate-buffered solution (PBS) solution and 5 mg/kg was delivered orally to conscious ferrets twice daily with the volume adjusted based on weight (e.g. 1200 g ferret received 600 μL). Ferrets were administered oseltamivir twice daily starting with the first dose 2 hours prior to infection. All oseltamivir treatment was carried out for a total of 5 days. Ferrets were housed individually in high efficiency particulate air filtered cages with free access to food, water and enrichment equipment such as toys throughout the experimental period. A single room was used exclusively to house the ferret cages (n = 12) for this study to maintain minimal disturbance from the main animal holding facility. Infected ferrets were observed for a 10-day period with the infection day of all experiments occurring on a Monday (day 0 post-infection) and the last day of the experiment on a Friday (day 11 post-infection).
Ferret monitoring, measurement and sample collection: Body temperature, weight and nasal washes of all ferrets were collected daily post-infection. Temperatures were measured daily using implanted temperature transponders fitted to identification chips (LifeChip Bio-Thermo, Digivet, Australia). Nasal washes were collected daily from sedated ferrets (intramuscular injection of Xylazine at 5 mg/kg) by instilling 1 mL of sterile PBS into one nostril and allowing the liquid to flow out of the other nostril into a collection tube. The number of cells in the nasal washes was counted immediately after collection. Aliquots of nasal washes were stored at -80°C prior to determining the virus titres and protein concentrations. Blood samples were collected 10 days post-infection and sera stored at -80°C prior to testing for influenza-specific antibodies. Ferrets were sacrificed 10 days post-infection by intramuscular injection of anaesthesia [50:50 mix of Ketamine (100 mg/mL): Ilium Xylazil (Xyalazine; 20 mg/mL)] followed by an overdose of pentobarbitone sodium (Lethabarb; 0.5 mL/kg).
Inflammatory, virological and serological analysis: Inflammatory cell count in the nasal wash was determined by Trypan blue exclusion using a Countess automated cell counter (Life technologies, Australia). Protein concentration in the nasal wash was determined using Coomassie Plus (Bradford) assay as according to the manufacturer’s instructions (Thermoscientific, Australia). Titres of infectious virus in the nasal washes were quantified by viral infectivity assay and a TCID50 determined [24]. Briefly, samples were serially diluted 10-fold in PBS and each dilution added in triplicate to flat-bottom 96-well plates containing a confluent monolayer of MDCK cells. Infected cells were incubated for 4 days at 35°C, 5% CO2 followed by detection of virus by addition of 25 μL of 1% Turkey red blood cells (RBCs) to 25 μL of infected cell supernatant (wells containing fully hemagglutinated RBCs were scored as positive for influenza virus). Virus titres were calculated as described by Reed and Muench [25]. The influenza-specific antibodies in sera were assessed by HI assay [26]. Briefly, receptor destroying enzyme (RDE; Denka Seiken, Japan)-treated sera were serially 2-fold diluted from a starting dilution of 1:20 in V-bottom 96-well plates. Virus (4 hemagglutination units) was added to all wells and 1% Turkey red-blood cells were added after one hour incubation at room temperature. Positive wells were defined as those where there was complete inhibition of hemagglutination.
Manual activity scoring of individually housed ferrets was performed in a ‘blinded’ manner at the same time each day by an experienced animal technician. A seven-level arbitrary activity scale was used according to a previously published study of A(H1N1)pdm09 virus [7]: 0: alert and fully playful; 0.5: alert but slightly less playful than usual; 1: alert and playful when encouraged to play; 1.5: alert and slightly playful when encouraged to play; 2: alert and slightly playful with strong encouragement; 2.5: alert but not playful, and 3: neither alert nor playful (required to be euthanized). Ferrets were ‘encouraged’ to play by firstly tapping the cage window gently to gain their attention, followed by wiggling the technician’s fingers as an encouragement to play. All attempts of encouragement were carried out while the ferrets were in the cages. Manual activity scoring of the ferrets was carried out at least 3 hours after filming to minimise unnecessary disturbance to the ferrets which may affect either activity scores. Mean activity scores for ferrets in each experimental group were calculated as described previously [16].
Activity measurement: video filming and software analysis: Video analysis of ferret activity was assessed daily at the same time by placing a single ferret into a 400 litre plastic box (length 97.5cm × width 75.5cm × height 67.6cm; Theplasticman, Australia; referred to as the ‘activity box’) and recording movement for 1 to 5 minutes after 7 seconds of acclimatisation on a video camera (MAGINON digital full HD camcorder; Supra, Germany) positioned above the box. Longer acclimatisation periods (1, 2, 3 and 4 minutes), prior to commencing filming decreased the overall activity rate with each additional minute of acclimatisation and thus 7 seconds was chosen as the optimal acclimatisation time (S1 Fig.). Relative to the size of a typical ferret, the 400 litre box was considered a better option than a 100 litre box as there was a larger area for movement and therefore we would expect a more accurate activity measurement (S2 Fig.). The interior of the 400 litre box was painted grey matt (colour code 3102; PlastiKote, USA) to enable a good contrast between both the white and dark-coloured ferrets used in the experiments, and the background colour. Prior to painting, the box was gloss white in colour which was highly reflective and resulted in reflections of the animal on the side wall of the box which misled the video analysis software and led to inaccurate activity measurements. Between the filming of each ferret, the box was decontaminated with 80% (v/v) ethanol to remove residual smell and/or viral contamination from the previous ferret. This cleaning protocol was effective in preventing viral transmission from one ferret to another [non-infected ferrets remained infection-free (absence of virus shedding and influenza-specific antibodies) even when they were filmed directly after an infected ferret for 10 consecutive days]. The filming sequence of individual ferrets in all groups was randomly assigned and filming was carried out once in the morning prior to other procedures such as oseltamivir dosing or nasal washing.
EthoVision settings for activity, distance and velocity analyses: Activity (pixel change), distance moved (m) and velocity (m/s) were measured using an automated video-tracking and motion analysis program, EthoVision XT 10.0 (Noldus IT, Netherlands). Video files (AVI. format, filmed at 30 frames/second, 640 × 480 resolution) were uploaded into the program and analysed for activity, distance and velocity using program settings as outlined in Table 1. Program settings for activity measurement included ‘gray scaling’ as the detection method, and activity threshold and noise filter adjusted to 6 and 2 respectively to achieve minimal background noise and zero activity detection when the animal was stationary (Table 1; S1 Video). Activity level of ferrets was determined by the software as a function of pixel changes, where ferret movement results in pixel changes as represented by the purple colour pixels in S1 Video. Specific measurement of pixel changes involved analysing video films at a sampling rate of 30 samples/second (as the maximum rate for video filmed at 30 frames/second). The gray scale of each pixel of the film image was compared from one sample to the next, and the proportion of pixel changes was calculated (the number of pixels where the gray scale has changed from one sample to the next divided by the overall number of pixels in the image). An overall mean pixel change was finally determined by taking an average of pixel changes in the total samples within the film period (e.g. 1800 samples per minute of film). To investigate how the sampling rate may affect the mean activity result, we compared the maximum rate (30 samples/second) with two slower rates (15 samples/second and 1 sample/second) in the same set of ferret films. The maximum sampling rate was found to be the most sensitive, resulting in the greatest number of days where ferret activity was determined to be significantly different from baseline levels following influenza infection (S3 Fig.). To accurately locate both white and dark-coloured ferrets for distance and velocity analyses (the program automatically locates the centre point of the animal as represented by the red dot in S2 Video and tracks the distance and velocity), ‘differencing’ was used as the other detection methods (static subtraction, dynamic subtraction and gray scaling) were not able to detect the ferret and distinguish ferrets with different coloured coats. Sensitivity settings for ‘differencing’ were adjusted to 17 with subject size between 159 and 14436 for optimal animal detection (Table 1; S2 Video).
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0118780.t001 Program settings for measuring activity, distance and velocity in EthoVision software. NA: Not applicable Mann-Whitney U non-parametric test was used to compare day-to-day differences in mean activity, distance, velocity and body temperature to mean baseline value. Kruskal-Wallis non-parametric test was used to compare nasal wash cell concentration, protein concentration, percentage weight loss and virus titre among the different groups. A P-value of <0.05 was considered statistically significant.
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