Influenza A virus (IAV) is an RNA, single-stranded, negative-sense virus belonging to the Orthomyxoviridae family. The virus can infect humans and certain domestic and wild animal species, including avian, porcine, equine, canine, feline, and marine mammals. In swine, the virus is considered to play a primary role in polymicrobial respiratory-disease events.1 Swine have been recognized as an important host species for IAV, since they may be potential sources for both zoonotic infections and novel viruses through reassortment.2-4

Among the different IAV subtypes, three (H1N1, H1N2, H3N2) have been circulating in the swine population during recent decades.5-7 Infections in swine are characterized by high morbidity and low mortality. Infected pigs may exhibit sneezing, coughing, lethargy, fever, anorexia, and rhinorrhea.8 Infected pigs start shedding infectious viral particles through their nasal secretions 1 day after infection, and individual pigs can continue to shed for 7 days. Introduction of new viruses into pig farms may accompany infected replacement animals.5,9 Pigs infected with influenza A virus can generate infectious aerosols that may play a role in regional dissemination.10 However, there are gaps in our knowledge of airborne and regional transmission routes and also many unanswered questions regarding the overall epidemiology and possible modes of transmission of influenza in swine.

Risk-factor studies based on serologic data have generated valuable information regarding the epidemiology of influenza in swine. The presence of anti-influenza antibodies has been associated with high farm density, farm type, herd size, female replacement rates, pen density, uncontrolled access of people, and indoor housing.11-16 However, to the authors’ knowledge, there are no risk factor studies associating the presence of the virus itself with farm-level characteristics. Therefore, the objective of this study was to collect virologic and epidemiologic data to investigate whether certain farm-level risk factors are associated with the presence of IAV in growing pigs.

Materials and methods

Procedures performed in this study were approved by the University of Minnesota Institutional Care and Use Committee.

Study population

This study was part of a larger IAV active surveillance study conducted in the midwestern United States that started in June 2009 and ended in December 2011.17 A total of 32 conveniently selected farms were enrolled, for which the primary objective was to actively monitor IAV in growing pigs over time. Six farms withdrew from the surveillance study, leaving 26 farms located in Illinois (n = 4), Indiana (n = 8), Iowa (n = 10), and Minnesota (n = 4) remaining enrolled.

Sample collection and testing

Sample collection began once producers agreed to participate and lasted for 12 or 24 months. On each farm, nasal swabs (Starswab II; Starplex Scientific Inc, Etobicoke, Ontario, Canada) were collected monthly from a convenience sample of 30 growing pigs (95% confident of detecting at least one positive swab when virus prevalence is at least 10%) at approximately 10 weeks old. On pig farms where there were multiple age groups, the investigator selected the age group of pigs that was closest to 10 weeks old. Nasal-swab samples collected during the monthly visit belonged to pigs from the same age-group cohort. Swabs were individually labeled with the farm identification number, state, month, and sample number. Samples were placed in a Styrofoam container with ice packs and shipped overnight to the virology department of St Jude Children’s Research Hospital (Memphis, Tennessee) for IAV testing by real-time reverse transcriptase polymerase chain reaction (RRT-PCR).17-19

Data collection

A questionnaire (Figure 1) containing close-ended questions was designed to capture data on farm characteristics. The survey was modeled after biosecurity questionnaires accepted by the swine industry and available through the American Association of Swine Veterinarians Production Animal Disease Risk Assessment Program (AASV PADRAP at www.padrap.org).

Specifically, the survey assessed farm type, regional farm density, topography surrounding the site, entrance biosecurity measures for employees and visitors, pig flow, origin of pigs, source of gilts, vaccination history, water-treatment protocols, and number of people working at the farm. The person collecting the monthly nasal swabs administered the survey to the owner or farm manager near the time of completion of the study.

Meteorological data collection

A weather station (HOBO; Onset Computer Corporation, Bourne, Massachusetts), set up to log data every hour, was placed 20 to 30 m away from the pig barns on each of the participating farms. Meteorological data recorded included temperature (°C), relative humidity (%), sunlight intensity (watts per m2), wind direction (degrees), wind speed (km per hour), and wind-gust speed (km per hour). Data were downloaded into a portable computer from the weather station monthly on the day nasal swabs were collected.

Data analysis

For the purpose of this study, a group of pigs was defined as the sample set of 30 growing pigs selected for monthly monitoring. A group was considered positive if at least one nasal swab tested RRT-PCR-positive for IAV.

Repeated measures logistic regression with an autoregressive correlation matrix was used to assess the relationship between farm IAV status and farm-level risk factors. Farm was included in the model as a random effect. One logistic model examined the relationship between IAV status of the group and farm-level characteristics, and a second assessed the weather data.

Univariate analysis was performed to screen variables, and those with a P value < .25 were considered for further analysis. A multivariable model was built by forcing all variables that met the screening criteria, and a stepwise backward elimination procedure was employed for model simplification by removing variables with P ≥ .05. Farm was included in the model as a random effect to account for clustering of the groups of pigs per farm. Month was included in the model as the repeated measure under an autoregressive correlation structure matrix. P < .05 was considered statistically significant in all analyses. SAS 9.2 (SAS Institute Inc, Cary, North Carolina) was used for all statistical procedures.

Results

A total of 522 groups with a mean and median age of 13.5 and 13.0 weeks, respectively, were screened for IAV. Age ranged from 3.5 to 31.0 weeks of age. Eight farms were finishing farms, eight were wean-to-finish farms, four were farrow-to-finish farms, four were nursery-to-finisher farms, one was a nursery, and one was a gilt developer unit.

The number of visits per farm was not constant due to absence of pigs, time constraints, or farms withdrawing from the study. Of the 26 farms enrolled in the study, one was visited 25 times, 14 were visited 24 times, one was visited 23 times, one was visited 21 times, two were visited 17 times, six were visited 12 times, and one was visited 11 times between June 2009 and December 2011. Thus, 32.1%, 24.7%, 18.3%, 15.5%, 4.6%, and 4.6% of the samples originated from groups of pigs in finisher, wean-to-finish, farrow-to-finish, nursery-finisher, nursery, and gilt developer unit farms, respectively.

At the individual level, 730 of 15,630 nasal swabs (4.7%) tested positive for IAV, whereas at the group level, 110 of 522 groups of pigs (21.1%) had at least one RRT-PCR-positive nasal swab. All but three farms had at least one IAV-positive group. The three farms with no IAV-positive groups were monitored for 12 months or less.

Farm-level factors

For the univariate analysis, the odds of testing positive for IAV were 3.05 and 16.69 times higher for farrow-to-finish and nursery farms, respectively, than for finishing farms (Table 1). Pig farms that managed their pigs all-in, all-out (AIAO) by barn or by site, compared to farms that managed pigs in a continuous flow, had lower odds of testing positive for IAV (0.31 for AIAO by barn, 0.35 for AIAO by site). Growing pigs born in sow farms in which gilts originated from an off-site facility had lower odds (0.17 if gilts were born at a breeding site, moved to another site and later returned; 0.25 if gilts originated from a multiplier) than did pigs born in sow farms in which replacement gilts were born at the breeding site and never moved from that site. The presence at the site of other animals, such as dogs and cats, was not identified as a significant risk factor for detection of IAV in pigs. Of the four variables included in the multivariable model (ie, farm type, pig flow, gilt source, and presence of other animals), only farm type remained significant.

Meteorological factors

All measures met the multivariable model inclusion criteria (Table 2). Temperature and wind speed remained in the final multivariable model after backwards stepwise elimination. Each degree increase in temperature increased the likelihood of a group of pigs testing positive for IAV by 1.04 (95% CI, 1.01-1.07). Similarly, the likelihood of testing positive for IAV increased 1.24 times with every km per hour increase in wind speed (95% CI, 1.08-1.43).

Discussion

Fully understanding the ecology, evolution, and transmission of influenza A viruses requires both virological detection techniques and epidemiological investigation methods such as we have described in the present study. Although previous studies have associated farm-level characteristics with increased risk of seropositivity for IAV, to the authors’ knowledge, this is the first study in which the presence of the virus in pig farms has been associated with farm-level and meteorological risk factors in swine.

The infection dynamics of the IAV are farm-type dependent. Farm type has been found as a significant risk factor according to our study reported here and in a previously published serological risk factor study.14 Both studies concluded that finisher pigs were more likely to be IAV-positive when sows were on site than were finisher pigs raised on farms separated from the sow herd. It has been reported that pigs become infected at an earlier age in farrow-to-finish farms than in finisher-only herds.20 In farrow-to-finish farms, which contain all the different age groups of pigs on the same site, the virus is allowed to perpetuate due to the constant presence of susceptible individuals with waning maternal antibodies (ie, suckling piglets approaching weaning age). The existence of susceptible individuals of varying ages is absent in finishing farms that contain pigs only in the later stages of growth. Nursery farms, like farrow-to-finish farms, also have a constant influx of recently weaned pigs which provides the necessary conditions for IAV to maintain transmission between older pigs and the incoming pigs. Additionally, it has been reported that recently weaned pigs themselves can be a source of virus by introducing new viruses into recipient barns.21,22 Furthermore, personnel and equipment shared between these different age groups may play a role in IAV transmission, since it is known that the virus can survive outside the swine host.23-27 Since introduction of infected animals is one of the most important risk factors for new pathogen introduction into a farm,28 it is not surprising that pig movement within a farm is an important factor to consider when assessing risk of IAV in pigs. There are two main types of animal flows: AIAO and continuous flow. In an AIAO management system, the entire building is emptied out at one time, and then one age group of pigs enters the building, moving through the production system together. Conversely, continuous flow means that pigs are constantly entering and exiting the building, and pigs of different age groups are housed in the same building. It has been reported that AIAO pig flow has a significant positive impact on growth rate, since pigs are not being challenged with infectious agents from older pigs.28-30 Our data show that pigs were less likely to test positive for IAV if they were raised under an AIAO system as analyzed by barn (OR = 0.31; 95% CI, 0.14-0.66) or by site (OR = 0.35; 95% CI, 0.15-0.88) than if they were raised under continuous-flow management. Pigs of different age groups are not exposed to one another when utilizing AIAO flow, which precludes horizontal transmission of infectious agents from older to younger pigs, a known mechanism for IAV maintenance in pig populations.5

In addition to pig flow, gilt replacement source was also associated with IAV positivity. Groups of pigs born in farms where the gilt source was an off-site facility had a lower likelihood of testing IAV- positive than did groups of pigs born in sow farms where gilts were born and raised on-site. This finding contrasts with what has been previously published, in that farms introducing replacement animals were reported to be at higher risk of being IAV-seropositive.5,14-16 Furthermore, introduction of replacement animals should also maintain the circulation of IAV in the population due to the influx of susceptible animals and new virues.5 Yet this appeared not to be the case in our study: detection of IAV in growing pigs was less frequent on farms that introduced gilts raised in off-site facilities. In today’s swine farms, veterinarians are aware of the importance of controlling gilt introduction to reduce disease transmission. It will be important in future studies to determine if incoming gilts from high-health multiplier systems with no detectable IAV, or even from AIAO gilt developer units with infrequent detection of IAV, affect the number of viral introductions into the recipient farms.

Even in today’s swine farms, where pigs are raised entirely indoors, weather conditions still influence the environment of the pig inside the barn (for example, low outside temperatures trigger the need to provide a heat source to increase barn temperature). Data on the relationship between environmental conditions and the presence of IAV in pigs is scarce. However, there is data regarding environment and disease transmission in other species or involving different microbiological agents. Specifically, meteorological conditions have been associated with regional dissemination of equine IAV in Australia31 and with airborne detection of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae in pigs.32,33 Our study detected an association, albeit with a measurably small effect with OR near 1.0, between two meteorological variables and the presence of IAV in groups of growing pigs. As outside temperature and wind speed increased, the likelihood of a group of pigs being infected with IAV increased. This may seem counter-intuitive, as some experimental work with guinea pigs has shown that at high temperatures (30°C) influenza aerosol transmission ceased.34 However, follow-up studies on the effects of temperature and influenza transmission showed that while aerosol transmission decreased at 30°C, direct-contact transmission was still maintained in the experimentally infected guinea pigs.34,35 A reasonable interpretation for the relationship between temperature increase and presence of IAV in swine farms may be that as environmental temperature increases, the temperature of the barn increases as well, creating the need for increased air movement to reduce ambient temperature. A higher rate of air exchange in the building can be accomplished either by increasing exhaust fan speed or by lowering the curtains, increasing the entry of external air particles that may include airborne pathogens. However, it has been reported36 that higher rates of air exchange in pig units have a protective effect on pneumonia lesions at slaughter, suggesting that as the concentration of housing-unit air particles decreases, respiratory lesions decrease. On the other hand, increasing wind speed may reduce external temperatures, but it is not clear what impact this has on pig-barn environmental conditions. Also unclear is the effect of wind direction on the odds of being IAV-positive. In particular, it would be interesting, and perhaps enlightening, to correlate not only wind direction but direction of the nearest farm in relationship to wind direction (eg, downwind or upwind). In this study, we recorded only the distance to the nearest farm and not the location of the nearest farm. Even though nearest farm location was not recorded, neither wind direction nor nearest neighbor nor number of pigs farms nearby were significant. Thus, directionality or predominant wind direction may be a moot point. Finally, one could speculate that at higher wind speeds, virus particles would become disrupted, and a so-called “viral cloud” could not stay intact. These associations and speculations require further investigation to deepen our understanding of the impact of meteorological conditions on disease within the barn. Until more studies are performed examining larger numbers of farms, complete risk factor analyses that include not only the farm characteristics presented here but also meteorological data, efforts at controlling IAV may best be focused on the more impactful variables of pig flow, gilt source, and farm type rather than environmental conditions and weather. The small number of farms in this study was likely a limitation.

Farm-level risk factors identified in this study provide insights into understanding the epidemiology of influenza in swine. Virological detection coupled with risk-factor identification through epidemiological investigations such as this are encouraged as part of a global surveillance effort not only to more fully understand IAV in pigs, but also to assess the impact swine IAV may have on human health.37 In addition, determining virus genetic characteristics is also encouraged in an attempt to further elucidate possible virus-level characteristics important for IAV control in swine populations. This study emphasizes the importance of population dynamics, in that certain farm-type facilities (nursery and farrow-to-finish) and pig flow (continuous flows) play a role in the epidemiology of the disease due to the constant entry of animals into a population, providing the necessary susceptible hosts for virus maintenance and increased likelihood of IAV detection. Therefore, efforts should be made to decrease IAV transmission and endemic IAV infections by managing closed populations and pig movements AIAO, which will benefit not only pigs but also possibly humans.

Implications

•  Growing pigs on farms where sows are present or where replacement gilts are raised on site are at higher risk of testing positive for IAV than pigs in finishing farms or farms that introduce replacement gilts from outside sources.

•  Management practices such as AIAO may reduce the likelihood that pigs test positive for IAV.

•  More research is needed to fully understand the relationships between weather and IAV in pig populations.

Acknowledgements

This research was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under Contract No. HHSN266200700005C and by the American Lebanese Syrian Associated Charities.

Conflict of interest

None reported.

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