Chapter 2: Swine Influenza Active Surveillance in the United States Influenza A virus (IAV) in swine continues to be an important swine respiratory agent along with being a source of concern to public health authorities. While veterinary diagnostic laboratories are a valuable source of information with regards to the identification and genetic characterization of newly emerged virus through passive surveillance, there is still a need for additional surveillance programs that can aid in detecting new viruses in a timely manner. An active surveillance program was performed in 32 pig farms throughout the Midwestern United States between June 2009 and December 2011. Thirty nasal swabs were collected from growing pigs on a monthly basis and tested for IAV by RRT-PCR. During sample collection, data on sample collection date, pig age, pig group respiratory signs, clinical status and vaccination history were recorded. A total of 16,170 nasal swabs from 540 groups of growing pigs were collected from which 746 (4.6 %) nasal swabs and 117 (21.7 %) groups tested positive for IAV, respectively. Throughout the study, IAV was consistently detected in at least one farm except in two months. From the positive groups of pigs, H1N1, H1N2, H3N2, 2009 pandemic H1N1were detected in 18%, 16%, 7.6% and 14.5% of the groups, respectively. In seven groups, H1N2 or H3N2 reassortants containing genes from 2009 pandemic H1N1 were found. There were eight groups in which an H1N2 and the 2009 pandemic H1N1 were identified simultaneously. Groups of pigs were more likely to test positive for IAV during the spring and summer seasons compared to the fall. Age and group respiratory clinical signs were not predictors of group IAV status. This active IAV surveillance program provided quality data and increased the understanding of the current situation of circulating viruses in the U.S. pig population. Further studies in swine should be conducted to increase our knowledge regarding the characteristics of IAV. Chapter 3: Swine influenza virus risk factors in growing pigs Influenza A virus (IAV) is an important cause of respiratory disease in swine. Understanding the epidemiology of the disease is in its early stages and is needed to develop effective control and prevention strategies. A study was conducted to assess the relationship between the presence of IAV in growing pig farms and farm level risk factors. Twenty-six pig farms participated in the study from which 30 nasal swabs from growing pigs were collected on a monthly basis for 12 or 24 consecutive months between 2009 and 2011. Nasal swabs were tested for IAV by RRT-PCR. Weather stations were located at every participating farm for monitoring temperature, relative humidity, light intensity, wind speed and wind gusts. Farm level data was obtained through a questionnaire to assess the relationship between the presence of IAV and farm level characteristics. At the individual level, 4.6% of the nasal swabs from growing pigs tested positive for IAV. Of the monthly groups of pigs from which nasal swabs were collected, 20.8% had at least one positive nasal swab. Positive nasal swabs originated from 23 of the 26 participating farms. Farm type, pig flow and gilt source were associated with the presence of IAV. Environmental temperature and wind speed were associated with the presence of IAV. Overall, this study provides insights into the ecology of IAV which can aid in the development of control and prevention strategies. Chapter 4: Prevalence and risk factors for H1N1 and H3N2 influenza A virus infections in Minnesota turkey premisesInfluenza virus infections can cause respiratory and systemic disease of variable severity and also result in economic losses for the turkey industry. Several subtypes of influenza can infect turkeys causing diverse clinical signs. Influenza subtypes of swine origin have been diagnosed in turkey premises. However, it is not known how common these infections are nor the likely routes of transmission. We conducted a cross-sectional study to estimate the seroprevalence of influenza viruses in turkeys and examine factors associated with infection on Minnesota turkey premises. Results for influenza diagnostic tests and turkey and pig premises location data were obtained from the Minnesota Poultry Testing Laboratory (MPTL) and the Minnesota Board of Animal Health (MBAH) respectively from January 2007 to September 2008. Diagnostic data from 356 premises were obtained, of which 17 premises tested positive for antibodies to influenza A virus by agar gel immunodiffusion (AGID) assay and were confirmed as either H1N1 or H3N2 influenza viruses by hemagglutination and neuraminidase inhibition assays. Influenza infection status was associated with proximity to pig premises and flock size. The latter had a sparing effect on influenza status. This study suggests that H1N1 and H3N2 influenza virus infections of turkey premises in Minnesota are an uncommon event. The route of influenza virus transmission could not be determined, however, the findings suggest that airborne transmission should be considered in future studies. Chapter 5: Characterization of the temporal dynamics of airborne influenza A virus detection in acutely infected pigs Influenza A viruses infect many species including avians, mammals and humans. Aerosol transmission is one route that enables the virus to infect populations. This study explored the relationship between number of infected pigs and the probability of detecting influenza virus RNA in bioaerosols through the course of an acute infection. Bioaerosols were collected using a cyclonic collector in two groups of seven week-old pigs that were experimentally infected upon exposure with a contact infected pig (seeder pig). After contact exposure, individual pig nasal swab samples were collected daily and air samples were collected three times per day for eight days. All samples were tested for influenza by RRT-PCR targeting the influenza virus matrix gene. All pigs' nasal swabs became influenza virus RRT-PCR positive upon exposure to the infected seeder pig. Airborne influenza was detected in 58% (25/43) of the air samples collected. Temporal dynamics of influenza virus detection in air samples were in close agreement with the nasal shedding pattern in the infected pigs. First detection of positive bioaerosols occurred 2 days post contact (DPC). Positive bioaerosols were consistently detected between 3 and 6 DPC, a time when most pigs were also shedding virus in nasal secretions. Overall, the odds of detecting a positive air sample increased 2.2 times with every additional nasal swab positive pig in the group. In summary, there was a strong relationship between the number of pigs shedding influenza virus in nasal secretions and the detection of bioaerosols during the course of an acute infection in non-immune population. Chapter 6: Detection of airborne influenza A virus in experimentally infected pigs with maternally derived antibodies.This study assessed whether recently weaned piglets with maternally derived antibodies were able to generate infectious influenza aerosols. Three groups of piglets were assembled based on the vaccination status of the dam. Sows were either non vaccinated (CTRL) or vaccinated with the same (VAC-HOM) strain or a different (VAC-HET) strain than the one used for challenge. Piglets acquired the maternally derived antibodies by directly suckling colostrum from their respective dams. At weaning, pigs were challenged with influenza virus by direct contact with an infected pig (seeder pig) and clinical signs were evaluated. Air samples, collected using a liquid cyclonic air collector, and individual nasal swabs were collected daily for 10 days from each group and tested by matrix real-time reverse transcriptase polymerase chain reaction (RRT-PCR) assay. Virus isolation and titration were attempted for air samples on Madin-Darby canine kidney (MDCK) cells. All individual pigs from both VAC-HET and CTRL groups tested positive during the study but only one pig in the VAC-HOM group was positive by nasal swab RRT-PCR. Influenza virus could not be detected or isolated from air samples from the VAC-HOM group. Influenza A virus was isolated from 3.2% and 6.4% air samples from both the VAC-HET and CTRL groups, respectively. Positive RRT-PCR air samples were only detected in VAC-HET and CTRL groups on day 7 post-exposure. Overall, this study provides evidence that recently weaned pigs with maternally derived immunity without obvious clinical signs of influenza infection can generate influenza infectious aerosols which is relevant to the transmission and the ecology of influenza virus in pigs. Chapter 7: Detection of airborne swine influenza A virus in air samples collected inside, outside and downwind from swine barns
Airborne transmission of influenza A virus (IAV) in swine is speculated to be an important route of virus dissemination, but data are scarce. This study attempted to detect airborne IAV by virus isolation and RRT-PCR in air samples under field conditions. This was accomplished by collecting air samples from four acutely infected pig farms and locating air samplers inside the barns, at the external exhaust fans and downwind from the farms and at distances up to 2.1 km. Weather data was also collected to explore the relationship between detection of IAV and temperature, relative humidity and sunlight intensity. IAV was detected in air samples collected in all the farms included in the study. On average, 96% and 85% of the air samples collected inside and at the exhaust fans from positive farms tested positive through RRT-PCR, respectively. Isolation of IAV was possible from air samples collected inside the barn at two of the farms and in one farm from the exhausted air. Influenza virus RNA was detected in air samples collected between 1.5 and 2.1 Km away from the farms. The odds of detecting IAV decreased with distance from the farm and greater levels of sunlight intensity. The results from this study prove evidence of the risk of aerosol transmission in pigs under field conditions and perhaps to other species as well.
University of Minnesota Ph.D. dissertation. August 2012. Major: Veterinary Medicine. Advisors:Dr. Robert Morrison and Dr. Marie Gramer. 1 computer file (PDF); xii, 146 pages.
Corzo, Cesar Agustin.
Epidemiology of influenza A viruses of swine: surveilance, airborne detection and dissemination.
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