Times as likely to be AI seropositive than those from Southern or Eastern Maryland (C.I. = 0.7?1.7; p = 0.12). Five out of 11 flocks (46 ) that were AI seropositive had also experienced diarrhea in the past six months compared to 16 (4/25) of AI seropositive flocks that did not exhibit diarrhea. Seropositive flocks that experienced diarrhea within the past six months were 2.8 times more likely to be AI seropositive than those that did not experience diarrhea (C.I. = 0.9?.6; p = 0.08). Results from statistical analysis may be found in Tables 5, 6, and 7.DiscussionThis study suggests that backyard flocks are no exception to avian influenza exposure and that Maryland flocks may have been exposed to AI from wild birds or pests. Pests are defined as both mammals and invertebrates. AI vaccination was ruled out based on survey data, as all owners denied vaccinating flocks once on the premises. AI vaccination practices are also rare in the U.S. and require USDA licensure and approval from both state and federal governments prior to field deployment [18]. To date, only a handful of studies based in industrialized countries have evaluated the KN-93 (phosphate) site seroprevalence of avian influenza in unvaccinated backyard flocks. While one study in New Zealand found a flock seroprevalence of 20.8 (5/24), comparable to 23.1 (9/39) in this study, a Minnesota team only detected one flock out of 150 (0.66 ) forFigure 1. Sample site locations with AI seropositive backyard flocks. Poultry were grouped by size based on number of commercial houses within a 15 km radius. doi:10.1371/journal.pone.0056851.gBiosecurity in Maryland Backyard PoultryTable 4. Results of antigen and serological screenings of 262 birds from 39 backyard flocks.Titer Distribution,1,000 7 1,000?,999 2 2,000?,999 2 4,000Seropositive birds/total birds11/262 (4.2 )Seropositive flocks/total flocks9/39 (23.1 )Positive flocks/total flocksdoi:10.1371/journal.pone.0056851.tAI buy KB-R7943 antibodies [19,20]. In Switzerland, researchers reported a higher seroprevalence of AI at 37.5 (15/40) in fancy breeding flocks [21]. However, many variables contribute to sample prevalence rates such as testing method, time of year, climate differences, migratory trends, species and age of waterfowl, and backyard flock exposure and management practices. Earlier studies focusing on the Delaware Bay and Maryland’s Eastern shore have shown the prevalence of AI reservoir species ranging from May to November. The Delaware Bay has been identified as a “hotspot” for AIV prevalence, from May to June, in shore birds, particularly the ruddy turnstone, however, the surveying time period excludes this population. Migratory waterfowl also travel up the Atlantic Flyway and arrive late July through October with peak AIV prevalence detected in August [22,23]. A study on the Eastern Shore of Maryland sampled cloacal swabs from resident ducks for 3 weeks between May 28 and Sept 2, 1998. Results suggested that influenza A viruses were introduced or increased in prevalence in resident waterfowl between July 15 and Aug 27 as AIV positives were detected from August 27 to September 2 at a prevalence of 13.9 [8]. While no AI RNA was detected in backyard poultry flocks, serological analysis indicated that almost a quarter of flocks had been previously exposed. Detection of antibodies against AI also allowed for screening of poultry that were infected prior to the sampling period. Detectable levels of antibodies against AI appear one to two weeks after infec.Times as likely to be AI seropositive than those from Southern or Eastern Maryland (C.I. = 0.7?1.7; p = 0.12). Five out of 11 flocks (46 ) that were AI seropositive had also experienced diarrhea in the past six months compared to 16 (4/25) of AI seropositive flocks that did not exhibit diarrhea. Seropositive flocks that experienced diarrhea within the past six months were 2.8 times more likely to be AI seropositive than those that did not experience diarrhea (C.I. = 0.9?.6; p = 0.08). Results from statistical analysis may be found in Tables 5, 6, and 7.DiscussionThis study suggests that backyard flocks are no exception to avian influenza exposure and that Maryland flocks may have been exposed to AI from wild birds or pests. Pests are defined as both mammals and invertebrates. AI vaccination was ruled out based on survey data, as all owners denied vaccinating flocks once on the premises. AI vaccination practices are also rare in the U.S. and require USDA licensure and approval from both state and federal governments prior to field deployment [18]. To date, only a handful of studies based in industrialized countries have evaluated the seroprevalence of avian influenza in unvaccinated backyard flocks. While one study in New Zealand found a flock seroprevalence of 20.8 (5/24), comparable to 23.1 (9/39) in this study, a Minnesota team only detected one flock out of 150 (0.66 ) forFigure 1. Sample site locations with AI seropositive backyard flocks. Poultry were grouped by size based on number of commercial houses within a 15 km radius. doi:10.1371/journal.pone.0056851.gBiosecurity in Maryland Backyard PoultryTable 4. Results of antigen and serological screenings of 262 birds from 39 backyard flocks.Titer Distribution,1,000 7 1,000?,999 2 2,000?,999 2 4,000Seropositive birds/total birds11/262 (4.2 )Seropositive flocks/total flocks9/39 (23.1 )Positive flocks/total flocksdoi:10.1371/journal.pone.0056851.tAI antibodies [19,20]. In Switzerland, researchers reported a higher seroprevalence of AI at 37.5 (15/40) in fancy breeding flocks [21]. However, many variables contribute to sample prevalence rates such as testing method, time of year, climate differences, migratory trends, species and age of waterfowl, and backyard flock exposure and management practices. Earlier studies focusing on the Delaware Bay and Maryland’s Eastern shore have shown the prevalence of AI reservoir species ranging from May to November. The Delaware Bay has been identified as a “hotspot” for AIV prevalence, from May to June, in shore birds, particularly the ruddy turnstone, however, the surveying time period excludes this population. Migratory waterfowl also travel up the Atlantic Flyway and arrive late July through October with peak AIV prevalence detected in August [22,23]. A study on the Eastern Shore of Maryland sampled cloacal swabs from resident ducks for 3 weeks between May 28 and Sept 2, 1998. Results suggested that influenza A viruses were introduced or increased in prevalence in resident waterfowl between July 15 and Aug 27 as AIV positives were detected from August 27 to September 2 at a prevalence of 13.9 [8]. While no AI RNA was detected in backyard poultry flocks, serological analysis indicated that almost a quarter of flocks had been previously exposed. Detection of antibodies against AI also allowed for screening of poultry that were infected prior to the sampling period. Detectable levels of antibodies against AI appear one to two weeks after infec.