Comparative effectiveness of isolation techniques for contemporary Influenza A virus strains circulating in exhibition swine

Abstract

Abstract. The current study sought to compare the effectiveness of 2 virus isolation methods for the recovery of contemporary Influenza A virus (FLUAV) strains circulating in swine at agricultural exhibitions. Following the emergence of the influenza A (H1N1)pdm09 virus, increased surveillance of FLUAV strains among swine was recommended for early detection of emerging strains that threaten animal and human health. The increase in genetic drift and genomic reassortment among FLUAV strains infecting swine since 1998 necessitates that detection protocols be periodically validated for contemporary FLUAV strains. During 2009, nasal swabs were collected from 221 clinically healthy pigs at 12 agricultural exhibitions in Ohio. Nasal swabs were tested in parallel for the presence of FLUAV strains using 3 methodologies: 2 passages through Madin-Darby canine kidney (MDCK) cells adapted to serum-free medium (SFM), 2 passages through embryonated chicken eggs (ECEs), and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Of the 221 samples, 40 (18.1%) were positive for FLUAV recovery in MDCK cell culture and 13 (5.9%) were positive in ECEs (P = 0.015). All samples positive in ECEs were also positive in MDCK cell culture. MDCK cell culture virus isolation results were in perfect agreement with results of the realtime RT-PCR. Hemagglutinin and neuraminidase combinations of the recovered isolates were H1N2 and H3N2, which were consistent with FLUAV strains circulating in U.S. pigs. Effectiveness and cost savings justify the use of SFM-adapted MDCK cell culture over ECEs for the recovery of contemporary FLUAV strains from exhibition swine.

Swine-to-Human Transmission of Influenza A(H3N2) Virus at Agricultural Fairs, Ohio, USA, 2012

“…Detection and characterization of influenza A virus from nasal swab samples were performed as previously described ( 21 , 22 ). Briefly, samples were screened by real-time reverse transcription PCR (rRT-PCR) for influenza A virus (VetMAX-Gold SIV Detection Kit; Applied Biosystems, Austin, TX, USA).…”

“…Briefly, samples were screened by real-time reverse transcription PCR (rRT-PCR) for influenza A virus (VetMAX-Gold SIV Detection Kit; Applied Biosystems, Austin, TX, USA). If > 1 sample from a fair was positive for influenza A virus, then viral transport medium for all nasal swab samples from that fair was inoculated individually into serum-free medium-adapted MDCK cells for virus isolation ( 21 ). Cells were observed daily for 72 hours, at which time cell culture supernatant was tested for hemagglutination activity.…”

The Inability to Screen Exhibition Swine for Influenza A Virus Using Body Temperature

“…The scientific community currently has little knowledge about IAV activity among exhibition swine at the beginning of agricultural fairs because the majority of the IAV surveillance at agricultural fairs has occurred at the end of the exhibition period, when it is common to find >75% of the pigs infected when IAV is detected at the fair. (Bowman et al , 2013b, Bowman et al , 2014). The working assumption has been that a small number of pigs enter the fair infected with IAV and the virus subsequently spreads within the comingled population of pigs during the course of the fair or exhibition.…”

Utility of snout wipe samples for influenza A virus surveillance in exhibition swine populations

“…Serial dilutions of VTM supernatant were inoculated in triplicate onto 96‐well plates of serum‐free adapted and maintained Madin‐Darby canine kidney cells . TCID 50 titers were calculated with the Reed & Muench method .…”