Onsisted of various ages and species including 227 chickens (Gallus GW-0742 custom synthesis domesticus), 16 turkeys (Meleagris gallopavo), 15 ducks (Anas platyrhynochos, Cairina moschata), 2 guinea fowl (Numida meleagris), andTable 2. Categorical variables examined for association with AI seropositive flocks.Biosecurity risk factor Housing (HOUSING) Species Separate (SPECSEP) Owner exp wild waterfowl (OWNWFOWL) Owner exp wild birds (OWNWDBRD) Owner exp neighbor birds (OWNNEBRD) Owner exp rodents (OWNRODNT) 1655472 Owner exp wild carnivore (OWNCARN) Owner exp livestock (OWNLVSTK) Bird exp wild waterfowl (BRDWFOWL) Bird exp wild birds (BRDWDBRD) Bird exp pets (BRDPETS) Bird exp rodents (BRDRODNT) Bird exp wild carnivore (BRDCARN) Bird exp livestock (BRDLVSTK) Allow visitors (ALLVIS) Isolate new birds (ISONWBRD) Disease mortality (DIESICK) Diarrhea (DIARRHEA) Respiratory disease (RESPDIS) Neurologic disease (NEURODIS) Weight loss (WGTLOSS) Footbath/footwear (FOOTBATH) Clean and disinfect (CLEAN) Pest control (PESTCON) Region (REGION) doi:10.1371/journal.pone.0056851.tDescription Free range vs. coop Together vs. separate Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Allow visitors vs. no visitors No isolation vs. isolation Deaths vs. no deaths Sick vs. not sick Sick vs. not sick Sick vs. not sick Sick vs. not sick No footbath vs. footbath Don’t clean vs. do clean No pest control vs. pest control North, South, or East vs. other regionsBiosecurity in Maryland Backyard Poultrypheasants (Phasianus colchicus). Seroprevalence of AI in backyard birds was 4.2 (11/262), while the overall flock seroprevalence was 23.1 (9/39) (Table 4). HI tests did not detect H5, H7, or H9 subtype-specific antibodies among the ELISA-positive sera. Chickens were the only species that were seropositive on nine premises among four counties. One bird from each premises was seropositive except one Frederick and one St. Mary’s flock which had two seropositive birds each (Fig. 1). Based on RT-qPCR analysis, none of the samples were found to be positive for AI RNA. All seropositive Clavulanic acid potassium salt biological activity flocks were reported and subsequently tested by the MDA, all of which were determined to be negative for current infection. In this cross-sectional study we also evaluated transmission pathways and biosecurity risk factors that may be associated with seropositives. Of the 39 flocks sampled, 36 completed the survey and were analyzed for statistically significant associations. No significant associations (p#0.05) were identified; however, some risk factors showed a positive association after relative risk calculations. 67 (2/3) of seropositive flocks were exposed to waterfowl compared to 21 (7/33) that were not exposed. Seropositive flocks exposed to waterfowl were therefore 3.14 times as likely to be AI seropositive than those not exposed to waterfowl (95 confidence interval [C.I.] = 1.1?.9; p = 0.15). 33 (7/21) of seropositive flocks did not use pest control compared to 13 (2/ 15) that did. Seropositive flocks that did not use pest control were 2.5 times as likely to be AI seropositive than those that did (C.I. = 0.6?0.4; p = 0.17). 35 (7/20) of seropositive flocks werefrom Northern Maryland while 13 (2/16) were from other regions. Seropositive birds from Northern Maryland were 2.8.Onsisted of various ages and species including 227 chickens (Gallus domesticus), 16 turkeys (Meleagris gallopavo), 15 ducks (Anas platyrhynochos, Cairina moschata), 2 guinea fowl (Numida meleagris), andTable 2. Categorical variables examined for association with AI seropositive flocks.Biosecurity risk factor Housing (HOUSING) Species Separate (SPECSEP) Owner exp wild waterfowl (OWNWFOWL) Owner exp wild birds (OWNWDBRD) Owner exp neighbor birds (OWNNEBRD) Owner exp rodents (OWNRODNT) 1655472 Owner exp wild carnivore (OWNCARN) Owner exp livestock (OWNLVSTK) Bird exp wild waterfowl (BRDWFOWL) Bird exp wild birds (BRDWDBRD) Bird exp pets (BRDPETS) Bird exp rodents (BRDRODNT) Bird exp wild carnivore (BRDCARN) Bird exp livestock (BRDLVSTK) Allow visitors (ALLVIS) Isolate new birds (ISONWBRD) Disease mortality (DIESICK) Diarrhea (DIARRHEA) Respiratory disease (RESPDIS) Neurologic disease (NEURODIS) Weight loss (WGTLOSS) Footbath/footwear (FOOTBATH) Clean and disinfect (CLEAN) Pest control (PESTCON) Region (REGION) doi:10.1371/journal.pone.0056851.tDescription Free range vs. coop Together vs. separate Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Exposed vs. not exposed Allow visitors vs. no visitors No isolation vs. isolation Deaths vs. no deaths Sick vs. not sick Sick vs. not sick Sick vs. not sick Sick vs. not sick No footbath vs. footbath Don’t clean vs. do clean No pest control vs. pest control North, South, or East vs. other regionsBiosecurity in Maryland Backyard Poultrypheasants (Phasianus colchicus). Seroprevalence of AI in backyard birds was 4.2 (11/262), while the overall flock seroprevalence was 23.1 (9/39) (Table 4). HI tests did not detect H5, H7, or H9 subtype-specific antibodies among the ELISA-positive sera. Chickens were the only species that were seropositive on nine premises among four counties. One bird from each premises was seropositive except one Frederick and one St. Mary’s flock which had two seropositive birds each (Fig. 1). Based on RT-qPCR analysis, none of the samples were found to be positive for AI RNA. All seropositive flocks were reported and subsequently tested by the MDA, all of which were determined to be negative for current infection. In this cross-sectional study we also evaluated transmission pathways and biosecurity risk factors that may be associated with seropositives. Of the 39 flocks sampled, 36 completed the survey and were analyzed for statistically significant associations. No significant associations (p#0.05) were identified; however, some risk factors showed a positive association after relative risk calculations. 67 (2/3) of seropositive flocks were exposed to waterfowl compared to 21 (7/33) that were not exposed. Seropositive flocks exposed to waterfowl were therefore 3.14 times as likely to be AI seropositive than those not exposed to waterfowl (95 confidence interval [C.I.] = 1.1?.9; p = 0.15). 33 (7/21) of seropositive flocks did not use pest control compared to 13 (2/ 15) that did. Seropositive flocks that did not use pest control were 2.5 times as likely to be AI seropositive than those that did (C.I. = 0.6?0.4; p = 0.17). 35 (7/20) of seropositive flocks werefrom Northern Maryland while 13 (2/16) were from other regions. Seropositive birds from Northern Maryland were 2.8.