Subclinical Influenza Virus A Infections in Pigs Exhibited at Agricultural Fairs, Ohio, USA, 2009–2011

Close contact between pigs and humans could result in zoonotic transmission.

A wareness of bidirectional zoonotic transmission of infl uenza virus A between pigs and humans was heightened by the emergence of the infl uenza A(H1N1)pdm09 virus, which resulted in an infl uenza pandemic among humans starting in 2009. Interspecies transmission of infl uenza virus A is believed to be a principal mechanism contributing to the emergence of novel infl uenza virus A strains that pose a threat to human and swine health (1,2). Pig respiratory tracts have receptors for swine-, human-, and avian-origin infl uenza virus A, which facilitates genomic reassortment among viruses from multiple host species. As a result, swine have been identifi ed as mixing vessels for infl uenza virus A and a source of emergence for novel viruses (3).
For >60 years after its identifi cation as a swine pathogen, infl uenza virus A circulating among North American swine was predominantly the H1N1 subtype (4). In 1998, triple-reassortant infl uenza virus A (H3N2), containing genes originating from swine-, human-, and avian-origin infl uenza virus A, was identifi ed among swine in the United States (5). This lineage quickly became established among North American swine (6), and the 6 gene segments coding for internal proteins, including the matrix (M) gene, subsequently served as a common backbone for many new reassortant viruses appearing among pigs (7). Various subtype H1N1, H1N2, and H3N2 infl uenza virus A lineages continue to cocirculate and evolve among North American swine (6)(7)(8)(9).
Swine are a source of novel and existing infl uenza virus A strains that infect humans (10)(11)(12)(13). These strains pose a pandemic threat if they become capable of being transmitted effi ciently from person to person and if limited protective immunity exists in the human population. Bidirectional zoonotic transmission of infl uenza virus A strains usually involves close contact between humans and swine. The United States has 3 major swine-human interfaces: commercial swine production, abattoirs, and agricultural fairs. Agricultural fairs are unique because they facilitate prolonged commingling of pigs from numerous sources raised under varied management programs with millions of persons who have widely disparate histories of exposure to various infl uenza viruses. This situation creates an environment conducive to zoonotic transmission of infl uenza virus A.
More persons come in contact with live swine at agricultural fairs than in any other setting in the United States, and several human cases of infl uenza A have been linked to swine exposure occurring at fairs. In 1988, a woman died of infection with a variant infl uenza virus A (H1N1) that she acquired while attending a Wisconsin fair where numerous pigs showed signs of infl uenza-like illness (ILI); a follow-up investigation identifi ed more human infections (14). In Ohio, human infections with variant infl uenza virus A after exposure to pigs with ILI were detected at the 1988 Ohio State Fair, 2 weeks before the Wisconsin case was reported (R.D. Slemons, unpub. data), and more recently at the 2007 Huron County Fair (15).
Because of dynamic human and swine populations at fairs and the number of human infl uenza A cases associated with swine exposure that occurs at fairs (13)(14)(15), we hypothesized that infl uenza virus A infections in swine occur undetected at agricultural fairs. This study was initiated after the emergence of infl uenza A(H1N1)pdm09 to actively monitor the antigenic and genomic properties of infl uenza virus A among pigs at agricultural fairs in Ohio, with a goal of protecting the health of swine and the public.

Study Sites and Samples
During each year of this 3-year study, 2009-2011, agricultural fairs in Ohio were strategically recruited to participate on the basis of the size of the county's commercial swine industry, the number of 4-H/FFA swine exhibitors, the number of pigs previously exhibited, or the geographic proximity to study sites used for infl uenza virus A surveillance in wild birds. Selection criteria were chosen to provide a diverse representation of Ohio's exhibition swine and the infl uenza virus A strains they might harbor. Before visiting the fair, the study team provided the leaders of each participating agricultural fair with an educational fact sheet on swine infl uenza.
The agricultural fair season in Ohio begins in June and continues into October; the fairs participating in this study occurred throughout the fair season ( Figure). Fairs were visited at the end of the swine exhibition period, at which time pigs were visually examined for signs of ILI, and nasal swab specimens were collected from 20 selected pigs representing all areas of the exhibit, without consideration for individual pig health status (healthy or ill). Each nasal swab was placed in an individual vial containing brain-heart infusion broth supplemented with penicillin and streptomycin (16). The samples were frozen at -70°C until testing was initiated. The Institutional Animal Care and Use Committee of The Ohio State University approved protocol no. 2009A0134 for the use of animals in this study.

Virus Isolation from Swine Nasal Swab Specimens
Samples were thawed and treated with amphotericin B, gentamicin sulfate, and kanamycin sulfate (17); they then underwent centrifugation at 1,200 × g for 30 min at 4°C. The brain-heart infusion broth supernatant was added to 24-well plates containing monolayers of Madin-Darby canine kidney (MDCK; catalog no. 84121903, Sigma-Aldrich Co., St. Louis, MO, USA), adapted to and maintained in serum-free medium (A.S. Bowman et al., unpub. data). MDCK monolayers were examied for cytopathic effect (CPE) daily for 3 days after the supernatant was added, at which time the cell culture supernatant was tested for hemagglutination activity by using 0.5% turkey erythrocytes (18)  Infl uenza virus A isolates were further characterized by using real-time reverse transcription PCR (rRT-PCR) assays.

RNA Extraction and rRT-PCR
RNA was extracted from original samples and infl uenza virus A isolates by using the PrepEase RNA Spin Kit (Affymetrix, Inc. Cleveland, OH, USA) according to the manufacturer's instructions. Pan-infl uenza virus A rRT-PCR (19,20) was used to screen all original samples for infl uenza virus A. Hemagglutinin (HA) and neuraminidase (NA) subtypes of the infl uenza isolates were determined by using rRT-PCR assays specifi c for swine-origin infl uenza A virus H1 and H3 HA genes and N1 and N2 NA genes by using either a previously published protocol (21)  The M gene of the infl uenza A virus isolates was further characterized by differentiating between the North American swine triple-reassortant and the infl uenza A(H1N1) pdm09 virus M genes by using an rRT-PCR protocol (22) adapted to laboratory conditions. The reactions were carried out by using the QuantiFast Multiplex RT-PCR +R Kit (QIAGEN, Valencia, CA, USA) in a 20-μL reaction mixture containing 10 μL 2× quantitative RT-PCR master mix, 7.5 pmol of forward primer, 2.5 pmol of each reverse primer, 0.125 μmol/L EA minor groove binder probe, 0.0625 μmol/L of each NA minor groove binder probe, 0.4 μL 50× ROX reference dye, 0.2 μL of reverse transcription product, and 5 μL of extracted RNA. The reactions were performed on an Mx3000P QPCR System (Agilent Technologies, Inc., Santa Clara, CA, USA) under these thermocycling conditions: 50°C for 20 min, then 95°C for 5 min, followed by 50 cycles of 97°C for 2 s and 60°C for 40 s. Cycle threshold values were calculated for each sample automatically by the QPCR System's software by using the background-based method. Samples with cycle threshold <40 were considered positive.

Discussion
Our fi ndings highlight the limitations of relying on visual examination for ILI to identify pigs infected with infl uenza virus A at agricultural fairs. Subclinical infl uenza virus A infections predominated among the pigs we tested, with subclinical infections detected among pigs at 10/53 (18.9%) participating fairs during 2009-2011. These fi ndings may explain the frequency of variant infl uenza virus A infections among humans who have only been exposed to apparently healthy swine at fairs.
Agricultural fairs are often the face of agriculture to the general public. The International Association of Fairs and Expositions estimates annual attendance at fairs in North America to be 150 million persons (The Association, pers. comm.). Agricultural fairs have been occurring in the United States since 1811 (23) and are special community events with a strong tradition and history of celebrating agricultural heritage and achievement (24). As the agricultural workforce has decreased to <2% of the US population (25), fairs have added educational programs to showcase advancements in food production systems in an effort to maintain attendance (26) and meet societal needs. These much-needed educational efforts often provide an opportunity for attendees to have direct contact with all facets of agriculture, including pork production. Many of these persons would not otherwise have any exposure to swine and the pathogens they harbor, so their close contact with pigs at fairs may play multiple roles in the transmission of infl uenza A viruses: they may pass human-origin infl uenza virus A to swine, leading to novel reassortant viruses; they may serve as early sentinels by becoming infected fi rst with a novel swine-origin infl uenza A virus; or they may disseminate a novel swine-origin infl uenza virus A in their local communities (27). The long duration of many agricultural fairs (3-10 days) is distinctly different than other swine concentration points or commingling events (i.e., abattoirs, markets, auctions, or shows), where interactions are limited to hours. In addition to their long duration, agricultural fairs also enable the comingling of pigs from multiple locations and various production systems (backyard to intensive commercial) at 1 site. Exhibition swine are commonly a unique population of noncommercial swine, reared by the use of management practices that differ greatly from standard commercial swine production practices (28). These pigs likely have varying levels of immunity to infl uenza virus A and may bring a variety of infl uenza virus A strains with them to the fair, where the viruses can spread to other pigs, possibly reassort, and potentially transmit to humans.
Swine-to-human transmission of infl uenza virus A has been sporadically reported worldwide (11), but the true incidence of this transmission is unknown. The Centers for Disease Control and Prevention reported that 36 humans were infected with variant infl uenza virus A in the United States during December 2005-April 2012 (29). Of these cases, 15 occurred after July 2011, and 6 cases, all involving infection with infl uenza A (H3N2) viruses containing the M gene from the infl uenza A(H1N1)pdm09 virus (H3N2v), were associated with exposure to swine at agricultural fairs. However, none of the implicated fairs reported signs of ILI in the pigs, and infl uenza virus A could not be isolated from the pigs that were suspected to be the sources because of delays and lack of the availability of the pigs. Nonetheless, it is possible that subclinical infl uenza infections in pigs at these swine-human interfaces played a key role in zoonotic infections.
The increased swine-human exposure occurring at agricultural fairs may also facilitate human-origin infl uenza A virus transmission to swine. The earliest reports of introductions of the infl uenza A(H1N1)pdm09 virus into the US swine herd occurred at the state fairs in Minnesota and South Dakota (30,31). Human-to-swine transmission is credited as a primary source of the genetic diversity seen in currently circulating swine infl uenza virus strains (32)(33)(34). Human-to-swine transmission of infl uenza virus A can be economically devastating for the pork industry because of decreased domestic sales, restrictions imposed by export partners, and production losses due to disease. Agricultural fairs may provide a conduit to introduce human-origin infl uenza virus A into the US swine herd.
No human cases of variant infl uenza A associated with any of the agricultural fairs included in this study were reported, even though infl uenza A (H3N2) viruses containing the M gene from the infl uenza A(H1N1)pdm09 virus were recovered from pigs at 6 of the participating fairs in 2011. However, the number of confi rmed H3N2v cases dramatically increased during the summer of 2012, with most cases epidemiologically linked to swine exposure occurring at agricultural fairs (35,36).
The HA, NA, and M gene combinations of the infl uenza virus A isolates recovered from 155/1,073 (14.4%) sampled pigs were consistent with infl uenza virus A concurrently circulating in the US swine population (37,38). The high frequency of virus isolation from the pigs at the 12 fairs at which infl uenza virus A was found is likely due to sample collection occurring at the end of the exhibition period, ≈5-7 days after arrival, which probably coincided with peak viral shedding in the swine population.
A limitation of the study is that extrapolating the fi ndings to other Ohio fairs and fairs in other states may not be possible because of the selection bias and inherent variability among agricultural fairs. Although the fairs where  infl uenza virus A was recovered were diverse regarding the predetermined selection criteria (data not shown), the participating fairs were included in the study because they were ranked relatively high among Ohio fairs within >1 selection category. Expanded surveillance efforts for agricultural fairs are underway to more accurately estimate the true prevalence of infl uenza virus A infections among swine at agricultural fairs in Ohio. Recognized risk factors and accurate prevalence estimates are needed to lay the foundation for studies investigating potential interventions to decrease the probability of swine-to-human and human-to-swine transmission of infl uenza virus A at agricultural fairs. The subclinical infl uenza virus A infections identifi ed in this study would not be detected by the current national swine infl uenza virus surveillance program (39), which is passive and focuses on swine showing signs of ILI and on reacting to reports of variant infl uenza A cases in humans (39). Thus, subclinical infl uenza virus A infections among pigs are likely underreported. This passive surveillance strategy does not adequately describe the breadth of infl uenza virus A circulating in swine because it does not identify less virulent strains of infl uenza virus A (40) and does not collect metadata on host, environmental, and agent factors that affect severity of illness. Therefore, to accurately capture the risk infl uenza virus A in swine populations presents to swine and public health, surveillance efforts should include healthy and clinically ill pigs.
Reducing bidirectional zoonotic transmission of infl uenza virus A between pigs and humans is crucial to agriculture and biomedicial science. Unfortunately, little scientifi c evidence exists on which to base changes in policies and management practices to reduce the risk for interspecies transmission of infl uenza virus A between pigs and humans. This investigation highlights the need for additional studies to quantify the risk for interspecies infl uenza A virus transmission at fairs and to evaluate interventions to mitigate the risk.
Potential strategies to mitigate the risk for intra-and interspecies transmission of infl uenza virus A at fairs on the swine side of the human-swine interface include shortening the swine exhibition period, preventing interfair movement of pigs, and vaccinating exhibition swine for appropriate infl uenza A viruses. Recommendations have previously been made for mitigating risk on the human side of the human-swine interface (www.cdc.gov/fl u/swinefl u). Expanded risk assessments at agricultural fairs will provide animal and public health offi cials with scientifi c data that will enable them to make appropriate decisions to protect animal and public health while still furthering appreciation and understanding of agriculture and ensuring our future food security.