Influenza Vaccination of Swine Reduces Public Health Risk at the Swine-Human Interface.

Influenza A viruses (IAV) in swine (IAV-S) pose serious risk to public health through spillover at the human-animal interface. Continued zoonotic transmission increases the likelihood novel IAV-S capable of causing the next influenza pandemic will emerge from this animal reservoir. Because current mitigation strategies are insufficient to prevent IAV zoonosis, we investigated the ability of swine vaccination to decrease IAV-S zoonotic transmission risk. We assessed postchallenge viral shedding in market-age swine vaccinated with either live-attenuated influenza virus (LAIV), killed influenza virus (KV), or sham vaccine (NV). We also assessed postchallenge transmission by exposing naive ferrets to pigs with contact types reflective of those experienced by humans in a field setting. LAIV and KV swine groups exhibited a nearly 100-fold reduction in peak nasal titer (LAIV mean, 4.55 log 50% tissue culture infectious dose [TCID50]/ml; KV mean, 4.53 log TCID50/ml) compared to NV swine (mean, 6.40 log TCID50/ml). Air sampling during the postchallenge period revealed decreased cumulative IAV in LAIV and KV study room air (LAIV, area under the concentration-time curve [AUC] of 57.55; KV, AUC = 24.29) compared to the NV study room (AUC = 86.92). Pairwise survival analysis revealed a significant delay in onset of infection among ferrets exposed to LAIV pigs versus NV pigs (rate ratio, 0.66; P = 0.028). Ferrets exposed to vaccinated pigs had lower cumulative virus titers in nasal wash samples (LAIV versus NV, P < 0.0001; KV versus NV, P= 0.3490) and experienced reduced clinical signs during infection. Our findings support the implementation of preexhibition influenza vaccination of swine to reduce the public health risk posed by IAV-S at agricultural exhibitions. IMPORTANCE Swine exhibited at agricultural fairs in North America have been the source of repeated zoonotic influenza A virus transmission, which creates a pathway for influenza pandemic emergence. We investigated the effect of using either live-attenuated influenza virus or killed influenza virus vaccines as prefair influenza vaccination of swine on zoonotic influenza transmission risk. Ferrets were exposed to the pigs in order to simulate human exposure in a field setting. We observed reductions in influenza A virus shedding in both groups of vaccinated pigs as well as the corresponding ferret exposure groups, indicating vaccination improved outcomes on both sides of the interface. There was also significant delay in onset of infection among ferrets that were exposed to live-attenuated virus-vaccinated pigs, which might be beneficial during longer fairs. Our findings indicate that policies mandating influenza vaccination of swine before fairs, while not currently common, would reduce the public health risk posed by influenza zoonosis.

Genomic Evidence for Sequestration of Influenza A Virus Lineages in Sea Duck Host Species.

Wild birds are considered the natural reservoir of influenza A viruses (IAVs) making them critical for IAV surveillance efforts. While sea ducks have played a role in novel IAV emergence events that threatened food security and public health, very few surveillance samples have been collected from sea duck hosts. From 2014-2018, we conducted surveillance focused in the Mississippi flyway, USA at locations where sea duck harvest has been relatively successful compared to our other sampling locations. Our surveillance yielded 1662 samples from sea ducks, from which we recovered 77 IAV isolates. Our analyses identified persistence of sea duck specific IAV lineages across multiple years. We also recovered sea duck origin IAVs containing an H4 gene highly divergent from the majority of North American H4-HA with clade node age of over 65 years. Identification of IAVs with long branch lengths is indicative of substantial genomic change consistent with persistence without detection by surveillance efforts. Sea ducks play a role in the movement and long-term persistence of IAVs and are likely harboring more undetected IAV diversity. Sea ducks should be a point of emphasis for future North American wild bird IAV surveillance efforts.

Adoption of recommended hand hygiene practices to limit zoonotic disease transmission at agricultural fairs.

Rapid transmission and spread of infectious pathogens are enhanced by the agricultural fair environment, where large numbers of livestock and people from numerous backgrounds congregate for several days. The transmission of influenza A virus and zoonotic enteric pathogens to fairgoers is a considerable risk and has occurred several times over the past decade. In an effort to mitigate zoonotic disease transmission in these settings, public health guidelines and recommendations including hand sanitation stations have been implemented. While hand hygiene recommendations to prevent the spread of zoonotic disease are well communicated, it is hypothesized that the adoption of these recommendations by agricultural fairs and fairgoers is low. To test this hypothesis, hand hygiene data collected from 658 agricultural fairs between 2012 and 2019 was analyzed to determine frequency and function of hand sanitation stations at the fairs, as well as utilization of educational signage. In addition, an observational study was performed to calculate the proportion of fairgoers who use hand sanitation stations at the fair. Lastly, samples were taken from working hand sanitation stations present at the exits of livestock barns and tested for the presence of influenza A virus and antimicrobial resistant coliform bacteria. Hand sanitation stations were present at most fairs (77.4 %) as recommended, but only 142 out of 2021 (7.0 %) visitors were observed using the stations. Health risk signage was displayed at more than half of fairs while the proper wash procedure was displayed at less than half. No influenza A virus was detected on any of the hand sanitation stations, however antimicrobial resistant coliform bacteria were recovered from 75.5 % of the sampled hand sanitation stations. Fairs should employ educational material along with functional hand sanitation stations in order to promote hand hygiene at fairs. Stations should be maintained and cleaned often to ensure effectiveness, as hand hygiene continues to be recommended to fairgoers when exiting animal barns to reduce zoonotic disease transmission.

Year­Round Influenza A Virus Surveillance in Mallards (Anas platyrhynchos) Reveals Genetic Persistence During the Under­Sampled Spring Season.

Active influenza A virus (IAV) surveillance in wild waterfowl in the United States has revolved around convenience-based sampling methods, resulting in gaps in surveillance during the spring season. We conducted active IAV surveillance in mallards continuously from July 2017 to July 2019 in the coastal marshes of Lake Erie near Port Clinton, Ohio. We aimed to understand ecological and evolutionary dynamics of IAV across multiple seasons, including the under­sampled spring season. We collected 2096 cloacal swabs and estimated a 6.1% (95% confidence interval (CI): 0.050-0.071) prevalence during the study period. Prevalence was lowest during spring (1.0%, 95% CI: 0.004-0.015). Time­stamped phylogenetic analyses revealed local persistence of genetic lineages of multiple gene segments. The PA segment consists of a lineage detected in multiple seasons with a time to most recent common ancestor of 2.48 years (95% highest posterior density: 2.16-2.74). Analysis of the H3 and H6 segments showed close relation between IAVs detected in spring and the following autumn migration. Though the mechanisms behind viral persistence in a single location are not well understood, we provide evidence that viruses can persist across several seasons. Current surveillance methods should be evaluated to ensure they are capturing the breadth of genetic diversity of IAV in waterfowl and prepare for IAV outbreaks in both animals and humans.

Using Environmental Sampling Techniques to Conduct Influenza A Virus Surveillance in Poultry and Waterfowl at Ohio Agricultural Exhibitions.

The outbreak of highly pathogenic H5Nx influenza A viruses (IAVs) in the United States during 2014-2015 caused devastating economic losses; therefore, several measures were established to control and eliminate highly pathogenic H5Nx from U.S. poultry flocks. One such measure was a temporary ban on poultry exhibitions during 2015, and this decision dramatically affected youth raising poultry as part of agricultural education programs. During the summer of 2016, surveillance of the environment was conducted at 20 Ohio agricultural fairs to estimate the prevalence of IAV in exhibition poultry to determine the baseline during nonoutbreak exhibition seasons. Of the 400 total samples collected, two were positive by real-time reverse transcription-PCR; however, virus isolation attempts with both embryonating chicken eggs and cell culture were unsuccessful. The detection of nucleic acid highlights the risk exhibition poultry could play in the transmission and spread of IAVs between humans, swine, wild birds, and domestic poultry during low or highly pathogenic IAV outbreaks. Additional surveillance at agricultural fairs and biosecurity education for youth exhibitors in this setting are warranted to reduce risk.

Nota de Investigación- Aplicación de técnicas de muestreo ambiental para realizar la vigilancia del virus de la influenza A en aves domésticas de corral y en aves acuáticas en exposiciones agrícolas de Ohio. El brote con virus de la influenza A H5Nx altamente patógenos (IAVs) en los Estados Unidos durante los años 2014 y 2015 causó pérdidas económicas devastadoras; por lo tanto, se establecieron varias medidas para controlar y eliminar estos virus H5Nx altamente patógenos de las granjas avícolas estadounidenses. Una de esas medidas fue la prohibición temporal de las exhibiciones de aves de durante el año 2015, esta decisión afectó drásticamente a la juventud criadora de aves de corral que forma parte de los programas de educación agrícola. Durante el verano del año 2016, se realizó vigilancia del medio ambiente en 20 ferias agrícolas de Ohio para estimar la prevalencia del virus de la influenza en aves de corral de exhibición para determinar la línea base durante las temporadas de exposición sin presencia de brotes. Del total de 400 muestras recolectadas, dos fueron positivas por transcripción inversa y PCR en tiempo real; sin embargo, los intentos de aislamiento del virus mediante huevos embrionados de pollo y cultivos celulares no tuvieron éxito. La detección de ácido nucleicos resalta el riesgo que pueden tener las aves de corral de exhibición en la transmisión y propagación del virus de la influenza aviar entre humanos, cerdos, aves silvestres y aves domésticas durante brotes de influenza de baja o alta patogenicidad. La vigilancia adicional en ferias agrícolas y la educación sobre bioseguridad para los expositores jóvenes en este entorno son necesarias para reducir el riesgo.

Assessing exhibition swine as potential disseminators of infectious disease through the detection of five respiratory pathogens at agricultural exhibitions.

Widespread geographic movement and extensive comingling of exhibition swine facilitates the spread and transmission of infectious pathogens. Nasal samples were collected from 2862 pigs at 102 exhibitions and tested for five pathogens. At least one pathogen was molecularly detected in pigs at 63 (61.8%) exhibitions. Influenza A virus was most prevalent and was detected in 498 (17.4%) samples. Influenza D virus was detected in two (0.07%) samples. More than one pathogen was detected in 165 (5.8%) samples. Influenza A virus remains a top threat to animal and human health, but other pathogens may be disseminated through the exhibition swine population.

Identifying Gaps in Wild Waterfowl Influenza A Surveillance in Ohio, United States.

The Mississippi Flyway is of utmost importance in monitoring influenza A viral diversity in the natural reservoir, as it is used by approximately 40% of North American migratory waterfowl. In 2008, influenza A virus (IAV) surveillance was initiated in eight states within the flyway during annual southern migration, to gain better insight into the natural history of influenza A viruses in the natural reservoir. More than 45,000 samples have been collected and tested, resulting in hundreds of diverse influenza A viral isolates, but seasonal sampling may not be the best strategy to gain insight into the natural history of IAV. To investigate the progress of this sampling strategy toward understanding the ecology of IAV in wild waterfowl, data from mallard ducks (Anas platyrhynchos) sampled nearly year-round in Ohio were examined. Overall, 3,645 samples were collected from mallards in Ohio from 2008 to 2016, with IAV being recovered from 13.6% of all samples collected. However, when data from each month are examined individually, it becomes apparent that the aggregated summary may be providing a misleading view of IAV in Ohio mallards. For instance, in August the frequency of viral recovery is 29.8%, with isolates representing at least 47 hemagglutinin/ neuraminidase (HA/NA) combinations. In November, during the height of southern migration, IAV isolation drops to 6.2%, with only 25 HA/NA combinations being represented. Our biased sampling towards convenience and high IAV recovery has created gaps in the data set, which prohibit a full understanding of the IAV ecology in this waterfowl population.

Identificación de brechas en la vigilancia de la influenza aviar en aves acuáticas silvestres en Ohio, Estados Unidos. La ruta migratoria de Mississippi es de suma importancia para monitorear la diversidad del virus de la influenza A en los reservorios naturales, ya que es utilizada por aproximadamente el 40% de las aves acuáticas migratorias de América del Norte. En el año 2008, se inició la vigilancia del virus de la influenza A (IAV) en ocho estados dentro de dicha ruta migratoria durante la migración anual al sur, para obtener una mejor comprensión de la historia natural de los virus de la influenza A en el reservorio natural. Se han recolectado y analizado más de 45,000 muestras, lo que dio como resultado cientos de diversos aislados virales de influenza A, pero el muestreo estacional puede no ser la mejor estrategia para conocer la historia natural del virus de la influenza aviar. Para investigar el progreso de esta estrategia de muestreo para comprender la ecología del virus de la influenza aviar en aves acuáticas silvestres, se examinaron datos de patos de collar (Anas platyrhynchos) muestreados durante casi todo el año en Ohio. En total, se recolectaron 3,645 muestras de patos silvestres en Ohio desde el año 2008 hasta el 2016, y se recuperó al virus de la influenza aviar en el 13.6% de todas las muestras recolectadas. Sin embargo, cuando los datos de cada mes se examinaron individualmente, se hace evidente que el resumen agregado puede proporcionar un panorama engañoso del virus de la influenza aviar en los patos silvestres de Ohio. Por ejemplo, en agosto, la frecuencia de recuperación viral fue del 29.8%, con aislamientos que representan al menos 47 combinaciones de hemaglutininas (HA) y neuraminidasas (NA). En noviembre, durante el apogeo de la migración hacia el sur, el aislamiento del virus de la influenza se redujo a 6.2%, con solo 25 combinaciones de HA y NA representadas. El muestreo sesgado hacia la conveniencia y la alta recuperación del virus de influenza ha creado brechas en el conjunto de datos, que prohíbe una comprensión completa de. Abbreviations: HA = hemagglutinin; IAV = influenza A virus; NA = neuraminidase.

Evaluation of a Field-Deployable Insulated Isothermal Polymerase Chain Reaction Nucleic Acid Analyzer for Influenza A Virus Detection at Swine Exhibitions.

Rapid detection of influenza A virus (IAV) at swine exhibitions, where zoonotic transmission has occurred, can allow exhibition officials to quickly implement mitigation strategies and reduce public health risk. While laboratory diagnostic methods using PCR exist, pen-side detection of IAV can reduce lag time between sample collection and results. Portable insulated isothermal PCR (RT-iiPCR) has been used for point-of-care pathogen detection in veterinary medicine. This study compared laboratory methods of real-time reverse transcription PCR (rRT-PCR) to RT-iiPCR to determine the potential effectiveness of RT-iiPCR for detection of IAV in swine in the field. Two methods of extraction (magnetic bead and spin-column) and the two PCR platforms were used in a crossover study design to detect IAV in nasal wipes of 150 individual swine from one exhibition. Magnetic bead extraction is considered the laboratory gold standard while spin-column purification is considered the field-deployable method. IAV RNA was detected in 17 samples using Mag/rRT-PCR (reference assay) and 16 samples using Mag/RT-iiPCR (Sensitivity-S 76.5%), whereas only 14 samples using Spin/rRT-PCR (S 88.2%) and 12 samples using Spin/RT-iiPCR (field method) (S 58.8%) were positive, demonstrating a reduction in detection of viral RNA using column purification. There is moderate agreement (Cohen's kappa = 0.6575) between Mag/rRT-PCR and Spin/RT-iiPCR. There is good agreement between both PCR assays when using the same method of extraction (Mag: Cohen's kappa = 0.8203, Spin: Cohen's kappa = 0.7642). RT-iiPCR requires testing of 10 more samples than the rRT-PCR to detect disease at the 95% confidence level in a population of 300 animals with a disease prevalence of 20%. In conclusion, although there is some reduction in sensitivity, RT-iiPCR used in conjunction with spin-column purification is an acceptable method of IAV in swine detection at exhibitions where it may help reduce lag time and allow for rapid control of an IAV outbreak.

Evaluation of nonwoven fabrics for nasal wipe sampling for influenza A virus in swine.

Influenza A virus (IAV) is a zoonotic pathogen threatening animal and public health; therefore, detection and monitoring of IAV in animal populations are critical components of a surveillance program. Swine are important hosts of IAV, wherein the virus can undergo rapid evolution. Several methods (i.e., nasal swabs, nasal wipes, and oral fluids) have been used to collect samples from swine for IAV surveillance. We utilized nasal wipes made from cotton gauze and multiple, polyester or mixed polyester fabrics to compare performance in the molecular detection and isolation of IAV. In vitro experiments revealed that no polyester or mixed polyester fabric was superior to cotton gauze for molecular IAV detection; however, 3 polyester or mixed polyester fabrics yielded significantly more viable IAV than cotton. In a field trial, both cotton gauze and the polyester or mixed polyester fabric yielded similar proportions of IAV isolates from swine. The results indicate that cotton gauze remains a practical and useful material for swine nasal wipes.

Detection of influenza A virus from agricultural fair environment: Air and surfaces.

Agricultural fairs facilitate an environment conducive to the spread of influenza A virus with large numbers of pigs from various different locales comingling for several days (5-8 days). Fairs are also associated with zoonotic transmission of influenza A virus as humans have unrestricted contact with potentially infected swine throughout the fair's duration. Since 2005, the Centers for Disease Control and Prevention has reported 468 cases of variant influenza A virus, with most cases having had exposure to swine at agricultural fairs. Many mechanisms have been proposed as potential direct and indirect routes of transmission that may be enhancing intra- and inter-species transmission of influenza A virus at fairs. This study examines airborne respiratory droplets and portable animal-care items as potential routes of transmission that may be contributing to enhanced viral spread throughout the swine barn and the resulting variant cases of influenza A. Air samples were taken from inside swine barns at 25 fairs between the years 2013 and 2014. Influenza A virus was detected molecularly in 11 of 59 (18.6%) air samples, representing 4 of the 25 fairs. Viable H1N1 virus, matching virus recovered from swine at the fair, was recovered from the air at one fair in 2013. During the summer of 2016, 75 of 400 (18.8%) surface samples tested positive for molecular presence of influenza A virus and represented 10 of 20 fairs. Seven viral isolates collected from four fairs were recovered from the surfaces. Whole genome sequences of the viruses recovered from the surfaces are >99% identical to the viruses recovered from individual pigs at each respective fair. The detection and recovery of influenza A virus from both the air and surfaces found within the swine barn at agricultural fairs provide evidence for potential viral transmission through these routes, which may contribute to both intra- and inter-species transmission, threatening public health. These findings reinforce the need for new and improved mitigation strategies at agricultural fairs in order to reduce the risk to animal and public health.

Influenza A(H3N2) Virus in Swine at Agricultural Fairs and Transmission to Humans, Michigan and Ohio, USA, 2016.

In 2016, a total of 18 human infections with influenza A(H3N2) virus occurred after exposure to influenza-infected swine at 7 agricultural fairs. Sixteen of these cases were the result of infection by a reassorted virus with increasing prevalence among US swine containing a hemagglutinin gene from 2010-11 human seasonal H3N2 strains.