Detection and description of influenza a virus transmission in commercial weaned pigs in Illinois, USA
Influenza A virus in swine (IAV-S) is a zoonotic and key pathogen in the swine respiratory disease complex. IAV-S is of particular importance because of the implications on human health, as seen during the 2009 swine flu pandemic. Surveillance of IAV-S is useful in monitoring for mutations that confer increased pathogenicity and virulence. Commercial swine are thought to be the diversity reservoir for H1N1 and H3N2 viruses, due to the abundance of strains in circulation. We hypothesize that weaned piglets are the most likely to become infected with influenza A virus; subsequently, this is the age most suitable to target for surveillance. This age of piglets has the challenges of environmental stressors, such as weaning, transportation, comingling, diet change, and environmental temperature change. At the same time, the immune system of mammals at this age is particularly vulnerable since maternally derived immunity is waning while simultaneously interfering with the piglet’s ability to mount an adaptive response. Taken together, weaned piglets represent ideal hosts for infectious pathogen amplification and transmission. Very few point-of-care tests are available for livestock, with only one commercially licensed product for influenza detection in swine. We developed a pandemic Matrix gene detection method via Reverse Transcription Loop-Mediated Isothermal Amplification (LAMP) to help address this problem. The first study was performed to develop the primers and test the assay's sensitivity, limit of detection, and specificity using intercalating dye and fluorescence detection in an RT-qPCR machine. Optimized primers were then tagged with biotin and streptavidin to allow for a point-of-care detection method using a lateral flow dipstick readout. The primer modification integrated an additional specificity step to the assay, subsequently improving the limit of detection from 88 copies per reaction to 4.5 copies per reaction, facilitated by a longer assay incubation. This assay brings nucleic acid detection several steps further to point-of-use testing for swine producers and veterinarians. Subsequent to the diagnostic test development, a surveillance study was performed on three commercial swine farms in Illinois that were naturally infected with IAV-S in order to assess efficacy. Sixty piglets from each farm were sampled weekly from weaning until seven weeks post-weaning to determine IAV-S shedding status. In chapter four, we discuss Farms 1 and 2. Shedding and reinfection incidences were analyzed via RT-PCR of nasal swabs, and whole genome sequencing was performed on all swabs with a CT ≤ 25 to monitor any viral mutations. 97.8% of the piglets shed at least once during the sampling period. No differences were seen between the two farms for initial shedding, with week one post-weaning the most likely period to detect IAV-S. However, Farm 1 was more likely to become reinfected later in the post-weaning period. Sequencing revealed that only one virus was circulating on each farm. Mutations were seen arising independently at different antigenic sites on HA and NA, as well as other mutations previously described to play a role in virulence. Our findings were supportive of previous research in post-weaning swine, such as reinfection occurring in the nursery period and the efficaciousness of screening for IAV-S in post weaning pigs. In chapter five, the third farm’s shedding data is analyzed, as well as the serum antibody data from all farms at three time-points. We found that at weaning, piglets did not have protective levels of antibodies against the homologous strain in circulation. We also found that the H3 maternal vaccine based antibodies were at very low levels, even at two weeks of age. We did see a modest increase in homologous antibody titers at nine weeks of age. Our conclusions from this study were that piglets have a failure of passive transfer of antibodies from their dams, which contributes to their odds of becoming infected and shedding IAV-S at weaning (OR=36) and during the post-weaning period. This was a hypothesis generating study to determine why the failure of passive transfer occurred in the 143 animals included in the study. Our final study was an active surveillance project for SARS-CoV-2 from environmental samples in meatpacking plants in the midwestern USA. The study was conducted due to absenteeism in the meatpacking workforce at the beginning of the SARS-CoV-2 pandemic, which was caused by employee’s fears of becoming infected with SARS-CoV-2 at their workplaces. Due to the high rates of absenteeism in the meatpacking plants, market ready cattle and hogs were unable to be processed, causing massive backlogs of animals along the production chains. The surveillance project was initiated to support the meatpacking plants, with a trickle-down effect to the swine producers, The trial was performed in two phases. Phase one samples were taken by our sampling team, and phase two samples were submitted by the meatpacking plant's health and safety teams. Sites in the plant were sampled to determine areas of potential SARS-CoV-2 fomite transmission and air samples in common areas. No samples were positive during phase one of the trial (Summer 2020), which corresponded to low cases in the surrounding communities. However, after the Thanksgiving holiday, all plants had positive samples in phase 2. This corresponded to a lag in the peak cases reported by the county. We found that environmental and air sampling in the meatpacking plants was a lagging indicator of community cases of SARS-CoV-2. The RT-LAMP Matrix gene detection assay, with further modifications for use with a lateral flow device, has demonstrated the feasibility of point-of-care devices in diagnosing diseases in livestock species. The assay was developed with nasal swabs or nasal wipes in mind, so that veterinarians or producers could take the samples for processing. In addition, the surveillance study showed that IAV-S is most likely to be detected in commercial settings in the first week post weaning. Together, these two pieces of information could allow the diagnosis of IAV-S on farm and could be especially useful in GDU’s where new animals will be isolated before entering as replacements in the herd, or in weaner pens prior to shipment. The final study of environmental surveillance indicated where coronaviruses are most likely to be detected on fomites throughout a meatpacking plant and can help inform future occupational health measures in the event of a zoonotic pathogen, or for the active reduction of human pathogens in the food-processing chain.
Whole Genome Sequencing, Influenza A Virus, Swine, Lamp Assay, Viral Transmission, Serology
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign