During June–August 2013, 553 paired nasal swabs and snout wipes were taken congruently from pigs at the end of 29 agricultural fairs across Ohio and Indiana. Each pig was restrained with a snare and a polyester-tipped swab was inserted into both nares of the pig after which the nasal swab was placed in an individual vial containing 1·8 ml of VTM. A snout wipe was collected from the same pig by wiping a 2 in. × 2 in (5·08 cm × 5·08 cm) sterile cotton gauze pad across the pig's snout with a gloved hand (Figure1 ). Gauze was placed in a sterile HDPE storage pot containing 5 ml VTM. Gloves were changed between pigs, and the snare was disinfected with Wexcide (Wexford Labs, Kirkwood, MO, USA). The order of nasal swab and snout wipe was alternated between pigs to correct for bias toward the sampling method used first on each pig. Nasal swab and snout wipe vials were frozen in the field on dry ice and stored at ≤−70°C until testing was conducted. Animals used in this study were included in protocol number 2009A0134-R1, which was approved by Institutional Animal Care and Use Committee of The Ohio State University.
The nasal swab samples were thawed in a 37°C dry bead bath, treated with amphotericin B (20 μg/ml), gentamicin sulfate (1000 μg/ml), and kanamycin sulfate (325 μg/ml) and vigorously agitated before centrifugation at 1200g for 30 minutes at 4°C. Snout wipe samples were also thawed at 37°C after which they were treated with amphotericin B (22·5 μg/ml), gentamicin sulfate (1000 μg/ml), and kanamycin sulfate (325 μg/ml). A slightly higher concentration of amphotericin B was used for the snout wipes due to increased risk of fungal contamination from environmental debris. Due to the flat bottom of the HDPE vials and the volume of VTM, the snout wipes were not centrifuged like the nasal swabs.
Viral transport media supernatants from the nasal swab and snout wipes underwent testing by real-time reverse transcription PCR (rRT-PCR) using the VetMAX™-Gold SIV Detection Kit (Life Technologies) and were inoculated in quadruplicate onto monolayers of serum-free adapted and maintained Madin-Darby canine kidney cells, on 24-well plates.15 The rRT-PCR and virus isolation results from nasal swabs and snout wipes were cross-tabulated and compared using the kappa static.17
The nasal swab samples were thawed in a 37°C dry bead bath, treated with amphotericin B (20 μg/ml), gentamicin sulfate (1000 μg/ml), and kanamycin sulfate (325 μg/ml) and vigorously agitated before centrifugation at 1200
Viral transport media supernatants from the nasal swab and snout wipes underwent testing by real-time reverse transcription PCR (rRT-PCR) using the VetMAX™-Gold SIV Detection Kit (Life Technologies) and were inoculated in quadruplicate onto monolayers of serum-free adapted and maintained Madin-Darby canine kidney cells, on 24-well plates.15 The rRT-PCR and virus isolation results from nasal swabs and snout wipes were cross-tabulated and compared using the kappa static.17
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