S Ethics StatementThis study was approved in accordance with the University

S Ethics StatementThis study was approved in accordance with the University of Maryland’s Institutional Review Board (IRB #11-0335), Federal Policy for the Protection of Human Subjects (45 CFR 46), and Institutional Animal Care and Use Committee (IACUC # R-1127). Written informed consent was obtained from all participants prior to survey and sample collection.Hemagglutination (HA) and Hemagglutination Inhibition (HI) AssaysHA titers were determined using 50 ul of 0.5 chicken red blood cells in PBS to 50 ul of a two-fold serial dilution of virus and PBS. Microtiter plates were incubated for 30 minutes at room temperature. HA titers were subsequently calculated 12926553 as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titrations were calculated by performing a serial two-fold dilution of 25 ul of Receptor Destroying Enzyme (RDE) treated sample and control serum with 25 ul of PBS. Twenty five ul of virus dilution containing 4 HA units/25 ul was then added to each well. Wells were incubated at room temperature for 30 minutes and 50 ul of 0.5 chicken red blood cell suspension was added. After 30 minutes HI titers were calculated as the reciprocal of the serum dilution that inhibited hemagglutination. A titer of 1:128 was used to define the reactivity of samples. This was the titer of the last well in a serial dilution of the positive control column that completely inhibited hemagglutination [14].Study ��-Sitosterol ��-D-glucoside site Design and PopulationThis study used a cross-sectional survey design and convenience sampling method to determine biosecurity risk factors and disease prevalence among Maryland non-commercial poultry flocks. Surveillance included active observational, active serologic, and active antigen methods. Counties were chosen based on the proportion of registered backyard flock owners and location of commercial industries and auction markets. In May 2011, the Maryland Department of Agriculture (MDA) confidentially mailed 1,000 informational letters and return postcards to poultry owners enrolled in the Maryland Poultry Registration Program. Participants were eligible for the study if they lived in Maryland, owned domesticated fowl, and maintained a flock size fewer than 1,000 birds.Study SitesStudy sites were designated by counties within three regions of Maryland: Northern (Frederick Carroll), Southern (St. Mary’s Charles), and Eastern Shore (Caroline, Dorchester, Talbot, Wicomico, Worcester) (Table 1).Antigen AssaysRNA Purification. Swabs were removed from the BHI transport media and samples vortexed for 5 seconds followed by centrifugation for 5 minutes at 5,0006 g. Supernatant was processed following the purchase Lecirelin organic method protocol [15]. RNA samples were stored at 280uC while awaiting RT-qPCR analysis.Biosecurity QuestionnaireUpon state and academic review, a four page questionnaire and information sheet was mailed to backyard flock owners. Participants were asked to self-report information on the number and species of poultry reared, presence of other animals, animal husbandry, opportunities for interaction between wild birds and poultry, flock biosecurity measures, and health status of poultry. Questionnaire is available upon request.Reverse Transcription Quantitative PCR (RT-qPCR)RT-qPCR was conducted on the Bio-Rad (Hercules, CA) CFX96 Real-Time thermal cycler and analyzed with CFX Manager Software using the one-step QuantiTect SYBRH green RT-PCR kit (Qiagen, Valencia, CA). For gallinaceous poultry (chickens,.S Ethics StatementThis study was approved in accordance with the University of Maryland’s Institutional Review Board (IRB #11-0335), Federal Policy for the Protection of Human Subjects (45 CFR 46), and Institutional Animal Care and Use Committee (IACUC # R-1127). Written informed consent was obtained from all participants prior to survey and sample collection.Hemagglutination (HA) and Hemagglutination Inhibition (HI) AssaysHA titers were determined using 50 ul of 0.5 chicken red blood cells in PBS to 50 ul of a two-fold serial dilution of virus and PBS. Microtiter plates were incubated for 30 minutes at room temperature. HA titers were subsequently calculated 12926553 as the reciprocal value of the highest dilution that caused complete hemagglutination. HI titrations were calculated by performing a serial two-fold dilution of 25 ul of Receptor Destroying Enzyme (RDE) treated sample and control serum with 25 ul of PBS. Twenty five ul of virus dilution containing 4 HA units/25 ul was then added to each well. Wells were incubated at room temperature for 30 minutes and 50 ul of 0.5 chicken red blood cell suspension was added. After 30 minutes HI titers were calculated as the reciprocal of the serum dilution that inhibited hemagglutination. A titer of 1:128 was used to define the reactivity of samples. This was the titer of the last well in a serial dilution of the positive control column that completely inhibited hemagglutination [14].Study Design and PopulationThis study used a cross-sectional survey design and convenience sampling method to determine biosecurity risk factors and disease prevalence among Maryland non-commercial poultry flocks. Surveillance included active observational, active serologic, and active antigen methods. Counties were chosen based on the proportion of registered backyard flock owners and location of commercial industries and auction markets. In May 2011, the Maryland Department of Agriculture (MDA) confidentially mailed 1,000 informational letters and return postcards to poultry owners enrolled in the Maryland Poultry Registration Program. Participants were eligible for the study if they lived in Maryland, owned domesticated fowl, and maintained a flock size fewer than 1,000 birds.Study SitesStudy sites were designated by counties within three regions of Maryland: Northern (Frederick Carroll), Southern (St. Mary’s Charles), and Eastern Shore (Caroline, Dorchester, Talbot, Wicomico, Worcester) (Table 1).Antigen AssaysRNA Purification. Swabs were removed from the BHI transport media and samples vortexed for 5 seconds followed by centrifugation for 5 minutes at 5,0006 g. Supernatant was processed following the organic method protocol [15]. RNA samples were stored at 280uC while awaiting RT-qPCR analysis.Biosecurity QuestionnaireUpon state and academic review, a four page questionnaire and information sheet was mailed to backyard flock owners. Participants were asked to self-report information on the number and species of poultry reared, presence of other animals, animal husbandry, opportunities for interaction between wild birds and poultry, flock biosecurity measures, and health status of poultry. Questionnaire is available upon request.Reverse Transcription Quantitative PCR (RT-qPCR)RT-qPCR was conducted on the Bio-Rad (Hercules, CA) CFX96 Real-Time thermal cycler and analyzed with CFX Manager Software using the one-step QuantiTect SYBRH green RT-PCR kit (Qiagen, Valencia, CA). For gallinaceous poultry (chickens,.