The authors have declared that no competing interests exist.
Conceived and designed the experiments: JMM NLT NGZ JT. Performed the experiments: JMM NLT. Analyzed the data: JMM NLT. Contributed reagents/materials/analysis tools: JMM NLT. Wrote the paper: JMM.
Major implications on a country's economy, food source, and public health. With recent concern over the highly pathogenic avian influenza outbreaks around the world, government agencies are carefully monitoring and inspecting live bird markets, commercial flocks, and migratory bird populations. However, there remains limited surveillance of non-commercial poultry. Therefore, a cross-sectional study was conducted in backyard poultry flocks using a convenience sampling method across three regions of Maryland from July 2011 to August 2011. The objective of this study was to develop a better understanding of the ecology and epidemiology of avian influenza by investigating the prevalence and seroprevalence in this potentially vulnerable population and by evaluating biosecurity risk factors associated with positive findings. Serum, tracheal, and cloacal swabs were randomly collected from 262 birds among 39 registered premises. Analysis indicated bird and flock seroprevalence as 4.2% (11/262) and 23.1% (9/39), respectively. Based on RT-qPCR analysis, none of the samples were found to be positive for AI RNA and evidence of AI hemagglutinin subtypes H5, H7, or H9 were not detected. Although no statistically significant biosecurity associations were identified (p≤0.05), AI seroprevalence was positively associated with exposure to waterfowl, pest control, and location. AI seropositive flocks exposed to waterfowl were 3.14 times as likely to be AI seropositive than those not exposed (p = 0.15). AI seropositive flocks that did not use pest control were 2.5 times as likely to be AI seropositive compared to those that did and AI seropositive flocks located in the Northern region of Maryland were 2.8 times as likely to be AI seropositive than those that were located elsewhere.
Avian Influenza (AI) is a type A Influenza virus and zoonotic pathogen of significant economic and public health concern. Of particular interest is the highly pathogenic avian influenza (HPAI) H5N1 subtype. Emerging in 1997, it has been responsible for the deaths of millions of birds globally and continues to persist at endemic levels in some countries
This location is of interest when it comes to AI surveillance for several reasons. Delmarva and the Chesapeake Bay coincide with the final significant merging zone of the Atlantic Migratory Flyway serving waterfowl, the natural reservoirs for influenza A viruses, from the far reaches of the Arctic Ocean, Northwest Territories of Canada, and Greenland
Disease surveillance and prevention are critical as the U.S. is the world's leading producer of poultry meat and the second largest poultry meat exporter and egg producer, valuing the industry at over $35.6 billion a year in 2010
At present, only a few studies have evaluated the prevalence of AI in backyard flocks. Government agencies are carefully monitoring and inspecting live bird markets, commercial flocks, and migratory bird populations. However, there remains little surveillance of private poultry flocks which are not confined to the same strict biosecurity practices as their commercial counterparts. Therefore, a cross-sectional study was conducted in non-commercial backyard poultry flocks using a convenience sampling method across three regions of Maryland from July 2011 to August 2011. The objective of this study was to investigate the prevalence and seroprevalence of avian influenza in this potentially vulnerable population and to evaluate biosecurity risk factors associated with positive findings.
This 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-11-27). Written informed consent was obtained from all participants prior to survey and sample collection.
This 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 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) (
Date of Sample Collection | Flock ID | Region |
Sampled Species | Total Birds Sampled | ||||
Chicken | Turkey | Duck | Guinea Fowl | Pheasant | ||||
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1 | (N) Frederick | 6 | 6 | ||||
2 | (N) Frederick | 5 | 5 | |||||
3 | (N) Frederick | 7 | 7 | |||||
4 | (N) Frederick | 6 | 6 | |||||
5 | (N) Frederick | 12 | 12 | |||||
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6 | (N) Frederick | 21 | 21 | ||||
7 | (N) Frederick | 3 | 3 | |||||
8 | (N) Frederick | 8 | 8 | |||||
9 | (N) Frederick | 2 | 2 | 2 | 6 | |||
10 | (N) Frederick | 6 | 6 | |||||
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11 | (S) St. Mary's | 2 | 2 | 2 | 6 | ||
12 | (S) St. Mary's | 3 | 3 | |||||
13 | (S) St. Mary's | 6 | 6 | |||||
14 | (S) St. Mary's | 4 | 2 | 6 | ||||
15 | (S) St. Mary's | 4 | 2 | 6 | ||||
16 | (S) St. Mary's | 6 | 6 | |||||
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17 | (E) Wicomico | 3 | 3 | ||||
18 | (E) Wicomico | 10 | 10 | |||||
19 | (E) Wicomico | 6 | 6 | |||||
20 | (E) Wicomico | 3 | 3 | |||||
21 | (E) Wicomico | 6 | 6 | |||||
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22 | (N) Frederick | 6 | 6 | ||||
23 | (N) Frederick | 4 | 1 | 5 | ||||
24 | (N) Frederick | 8 | 8 | |||||
25 | (N) Frederick | 6 | 6 | |||||
26 | (N) Frederick | 6 | 6 | |||||
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27 | (S) Charles | 8 | 8 | ||||
28 | (S) Charles | 4 | 2 | 6 | ||||
29 | (S) Charles | 4 | 2 | 2 | 8 | |||
30 | (S) Charles | 2 | 4 | 6 | ||||
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31 | (E) Dorchester | 4 | 4 | ||||
32 | (E) Talbot | 4 | 4 | 8 | ||||
33 | (E) Caroline | 6 | 6 | |||||
34 | (E) Talbot | 4 | 4 | |||||
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35 | (N) Frederick | 10 | 6 | 2 | 18 | ||
36 | (N) Carroll | 6 | 6 | |||||
37 | (N) Carroll | 6 | 6 | |||||
38 | (N) Carroll | 4 | 4 | |||||
39 | (N) Frederick | 6 | 6 | |||||
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Upon 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.
Blood (1–3 ml) was collected from the brachial vein of each bird and placed in a serum separator vacutainer. Tracheal and cloacal swabs were also collected, and stored in vials containing 2.5 ml of protein based brain-heart infusion (BHI) transport media. All tubes were labeled with date, species, sample type, and location. Once samples were collected, they were stored at 4°C (24–48 hours) until processed.
Serum was separated from the clot by centrifugation at 1,300× g for 10 minutes in a swinging bucket centrifuge and stored at −20°C. Evaluation for antibodies to influenza A viruses in sera was carried out using Synbiotics USDA-licensed screening kit,
Influenza virus strains A/Mallard/PA/10218/84 (H5N2), A/Mallard/Alberta/24/01 (H7N3), and A/Quail/Arkansas/20209-1/93 (H9N2) were generously provided by Dr. Daniel Perez from the University of Maryland (College Park, MD). Viruses were propagated in nine day-old embryonated chicken eggs for 48 hours as previously described
HA 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 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
Swabs were removed from the BHI transport media and samples vortexed for 5 seconds followed by centrifugation for 5 minutes at 5,000× g. Supernatant was processed following the organic method protocol
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 SYBR® green RT-PCR kit (Qiagen, Valencia, CA). For gallinaceous poultry (chickens, turkey, quail, pheasant) tracheal RNA swab samples were used for AIV RT-qPCR analysis as these viruses primarily replicate in the respiratory tract. For waterfowl, cloacal RNA swab samples were used as AI virus primarily replicates in the intestinal tract of these birds
After descriptive data analysis (mean, median, and range), univariate and multivariate statistical analyses were carried out. The association of the independent variables elucidated from the questionnaire, such as biosecurity practices and the dependent variables (bird or flock disease positive) were analyzed using Fisher's exact test, (right sided) for the categorical variables due to small counts (
Biosecurity risk factor | Description |
Housing (HOUSING) | Free range vs. coop |
Species Separate (SPECSEP) | Together vs. separate |
Owner exp wild waterfowl (OWNWFOWL) | Exposed vs. not exposed |
Owner exp wild birds (OWNWDBRD) | Exposed vs. not exposed |
Owner exp neighbor birds (OWNNEBRD) | Exposed vs. not exposed |
Owner exp rodents (OWNRODNT) | Exposed vs. not exposed |
Owner exp wild carnivore (OWNCARN) | Exposed vs. not exposed |
Owner exp livestock (OWNLVSTK) | Exposed vs. not exposed |
Bird exp wild waterfowl (BRDWFOWL) | Exposed vs. not exposed |
Bird exp wild birds (BRDWDBRD) | Exposed vs. not exposed |
Bird exp pets (BRDPETS) | Exposed vs. not exposed |
Bird exp rodents (BRDRODNT) | Exposed vs. not exposed |
Bird exp wild carnivore (BRDCARN) | Exposed vs. not exposed |
Bird exp livestock (BRDLVSTK) | Exposed vs. not exposed |
Allow visitors (ALLVIS) | Allow visitors vs. no visitors |
Isolate new birds (ISONWBRD) | No isolation vs. isolation |
Disease mortality (DIESICK) | Deaths vs. no deaths |
Diarrhea (DIARRHEA) | Sick vs. not sick |
Respiratory disease (RESPDIS) | Sick vs. not sick |
Neurologic disease (NEURODIS) | Sick vs. not sick |
Weight loss (WGTLOSS) | Sick vs. not sick |
Footbath/footwear (FOOTBATH) | No footbath vs. footbath |
Clean and disinfect (CLEAN) | Don't clean vs. do clean |
Pest control (PESTCON) | No pest control vs. pest control |
Region (REGION) | North, South, or East vs. other regions |
Biosecurity risk factor | Description |
Commercial farms (COMMFARM) | Number of farms within 1/4 mile |
Backyard flocks (BACKFLCK) | Number of backyard flocks within 1/4 mile |
Years of ownership (YEAROWN) | Number of years kept poultry |
Flock size (FLCKSZE) | Number of birds in flock |
Visit commercial (VISCOMM) | Number of times visit commercial farm (1 yr) |
Visit backyard flocks (VISBKYD) | Number of times visit backyard flock (1 yr) |
The overall survey response rate was 4.1% (41/1000). Two backyard flock owners of the 41 could not be reached for testing arrangements. From July 15–August 25, 2011, 262 birds from 39 backyard flocks were sampled. The sampled poultry population consisted of various ages and species including 227 chickens (
Poultry were grouped by size based on number of commercial houses within a 15 km radius.
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11/262 (4.2%) | 9/39 (23.1%) | 0 |
7 | 2 | 2 | 0 |
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.] = 1.1–8.9; 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. = 0.6–10.4; p = 0.17). 35% (7/20) of seropositive flocks were from Northern Maryland while 13% (2/16) were from other regions. Seropositive birds from Northern Maryland were 2.8 times as likely to be AI seropositive than those from Southern or Eastern Maryland (C.I. = 0.7–11.7; 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. = 0.9–8.6; p = 0.08). Results from statistical analysis may be found in
Variable | Description | Prevalence Ratio | 95% Confidence Interval | P-value |
Diarrhea | Reported within past 6 mo. | 2.84 | 0.939–8.596 | 0.075 |
Location | North vs. other regions | 2.80 | 0.672–11.670 | 0.122 |
Pest control | Implemented pest control | 2.50 | 0.601–10.394 | 0.165 |
Waterfowl | Exposed to wild waterfowl | 3.14 | 1.116–8.853 | 0.148 |
Variable | Description | Coefficient | P-value |
Time owned | How many years kept poultry | 0.613 | 0.133 |
Visit comm. | How often visit commercial sites | 2.701 | 0.104 |
Diarrhea | Reported within past 6 mo. | −1.314 | 0.380 |
Location | North vs. other regions | 2.500 | 0.204 |
Pest control | Implemented pest control | −0.107 | 0.942 |
Waterfowl | Exposed to wild waterfowl | 18.377 | 0.736 |
Variable | Description | Coefficient | P-value |
Time owned | How many years kept poultry | 0.154 | 0.127 |
Visit comm. | How often visit commercial sites | 0.713 | 0.080 |
Location | North vs. other regions | 2.379 | 0.102 |
This 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
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
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 infection and can last for several months
It is believed that all of the AI seropositive chickens identified in this study were exposed to LPAI viruses as the birds survived the infection and owners did not report any significant mortalities in their flocks as a result of disease. The majority of circulating strains are low pathogenic viruses which may produce subtle or no signs of clinical infection to mild respiratory distress. Other signs may include diarrhea, decrease in egg production, and inactivity. However, these signs are not specific to AI infection and are often present in other poultry diseases
To the authors' knowledge, this is the first study to report associations between biosecurity management practices and disease prevalence/seroprevalence of AI among backyard flocks located within close proximity to the Delmarva commercial poultry region. However, this study was subject to some limitations. The overall response rate of this study (4.1%) was relatively poor, but believed to stem from the concern over the mandatory reporting of flock positives to the State Veterinarian and potential repercussions, such as “Hold Orders” that restrict the movement of birds onto or off the premises, as well as the stigma attached to having an infectious disease. A larger sample size may have also increased the ability of this study to detect significant associations between biosecurity risk factors and disease prevalence. While association could be hypothesized based on proportional analysis, wide confidence intervals indicate that these estimates have low precision from an inadequate sample size and therefore associated risk results should be interpreted cautiously in this preliminary study. Although methods of convenience sampling are often assumed to be representative of a population, sampling biases (most notably selection bias) do occur, making it difficult to develop statistically valid estimates of disease prevalence, regardless of how many birds are sampled. Another constraint was the lack of detail collected in the wild bird-domestic poultry interface such as type of wild bird/waterfowl species identified on the property as well as the means of exposure (i.e. nose to nose, adjacent habitat, droppings only) which may have provided greater insight to the exposure risk and should be included in future studies. Widening the sample collection time frame from May to October could have improved the chances of obtaining a more representative data set in relation to the transmission of AI from wild birds to poultry. This study was also limited to a population of backyard flock owners that had registered with the MDA. It is believed that AI prevalence estimates reported in this study are lower than the true population as most owners with clinically ill birds would be reluctant to participate. Due to the low response rate and potential biases, this study cannot be generalized to other backyard flock populations.
Surveillance is a dynamic process that requires continuous observation, collection, and analysis of data in order to identify the presence of a disease and contain its spread. While migratory waterfowl have been the main target of disease investigations, domesticated poultry warrant consideration as well. This surveillance study aimed to capture the prevalence and seroprevalence of AI during an outbreak-free period and to illustrate baseline levels of exposure in this growing population. As a result, data from this project has provided a better understanding of AI ecology and transmission relationships within backyard flocks. As demonstrated in this study, education is essential for backyard flock owners especially with non-commercial poultry ownership's recent increase in popularity. Several flock owners did not practice biosecurity methods, many of which are simple, practical, and affordable. Therefore, it is recommended that proactive biosecurity education highlight prevention measures such as protecting poultry from wild birds and waterfowl particularly during the spring and summer months when migration season is at its peak and implementing a pest control plan. Targeted education and surveillance strategies will help protect the health of U.S. poultry flocks, minimize economic effects of the disease, and greatly reduce the health risks to the U.S. public.
We would like to express our gratitude to all those at the Maryland Department of Agriculture who helped make this project possible as well as the Maryland backyard flock owners who participated in the study. Thank you to Dr. Daniel Perez and his lab for providing the avian influenza positive controls and to the Synbiotics lab for generously providing the ELISA kits.