Avian Influenza Virus Prevalence in Migratory Waterfowl in the United States, 2007–2009 Author(s): Scott R. Groepper, Thomas J. DeLiberto, Mark P. Vrtiska, Kerri Pedersen, Seth R. Swafford, and Scott E. Hygnstrom Source: Avian Diseases, 58(4):531-540. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10849-042214-Reg.1 URL: http://www.bioone.org/doi/full/10.1637/10849-042214-Reg.1

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AVIAN DISEASES 58:531–540, 2014

Avian Influenza Virus Prevalence in Migratory Waterfowl in the United States, 2007–2009 Scott R. Groepper,AD Thomas J. DeLiberto,B Mark P. Vrtiska,C Kerri Pedersen,B Seth R. Swafford,B and Scott E. HygnstromA A

B

415 Hardin Hall, University of Nebraska–Lincoln, 3310 Holdrege Street, Lincoln, NE 68583 United States Department of Agriculture, Animal, Plant Health Inspection Service, Wildlife Services, 4101 LaPorte Avenue, Fort Collins, CO 80521 C Nebraska Game and Parks Commission, 2200 N 33rd Street, Lincoln, NE 68503 Received 24 April 2014; Accepted 16 July 2014; Published ahead of print 18 July 2014

SUMMARY. We analyzed 155,535 samples collected for surveillance of avian influenza viruses (AIVs), in the United States from 2007 to 2009, from migratory waterfowl (ducks, geese, and swans). The goal was to elucidate patterns of prevalence by flyway and functional groups to determine targets for future surveillance. Apparent prevalence of AIV was highest in the Pacific Flyway in 2007–2008 (14.2% and 14.1%, respectively), in the Mississippi Flyway in 2009 (16.8%), and lowest each year in the Atlantic Flyway (range, 7.3%–8.9%). Dabbling ducks had higher apparent prevalence of AIV (12.8%–18.8%) than diving ducks (3.9%– 6.0%) or geese and swans (3.6%–3.9%). We observed highest apparent prevalence in hatch-year waterfowl (15.6%–18.9%). We further analyzed 117,738 of the 155,535 samples to test the hypothesis mallard (Anas platyrhynchos) had highest prevalence of AIV. We compared apparent prevalence and odds ratios for seven species of ducks and one species of goose commonly collected across the United States. Mallards had highest apparent prevalence (15%–26%) in half of comparisons made, whereas American greenwinged teal (Anas creeca, 12%–13%), blue-winged teal (Anas discors, 13%–23%), northern pintail (Anas acuta, 16%–22%), or northern shoveler (Anas clypeata, 15%) had higher apparent prevalence in the remaining comparisons. The results of our research can be used to tailor future surveillance that targets flyways, functional groups, and species with the highest probability of detecting AIV. RESUMEN. Prevalencia del virus de la influenza aviar en aves acua´ticas migratorias en los Estados Unidos, 2007–2009. Se analizaron 155,535 muestras recolectadas para la vigilancia de los virus de influenza aviar (AIV), en los Estados Unidos desde el an˜o 2007 al an˜o 2009, procedentes de aves acua´ticas migratorias (patos, gansos y cisnes). El objetivo fue determinar los patrones de prevalencia por vı´as migratorias y por grupos funcionales para determinar los objetivos de vigilancia en el futuro. La prevalencia aparente de influenza aviar fue ma´s alta en la ruta migratoria del Pacı´fico durante los an˜os 2007 y 2008 (14.2% y 14.1%, respectivamente), en la ruta migratoria del Mississippi en el 2009 (16.8%), y la ma´s baja de cada an˜o fue la ruta migratoria del Atla´ntico (rango 7.3%–8.9%). Los patos chapoteadores mostraron la mayor prevalencia aparente del virus de influenza aviar (12.8%–18.8%) en comparacio´n con los patos buceadores (3.9%–6.0%) o gansos y cisnes (3.6%–3.9%). Se observo´ mayor prevalencia aparente en aves acua´ticas de un an˜o de edad (15.6%–18.9%). Posteriormente, se analizaron 117,738 de las 155,535 muestras para probar la hipo´tesis de que los a´nades reales (Anas platyrhynchos) mostraban la mayor prevalencia de virus de influenza aviar. Se compararon la prevalencia aparente y las razones de momios para siete especies de patos y una especie de ganso comu´nmente recolectados en los Estados Unidos. Los a´nades reales tenı´an una mayor prevalencia aparente (15%–26%) en la mitad de las comparaciones realizadas, mientras que la cerceta comu´n (Anas creeca, 12%–13%), el pato media luna (Anas discors, 13%– 23%), a´nade rabudo (Anas acuta, 16%–22%), o pato cuchara (Anas clypeata, 15%) tuvieron mayor prevalencia aparente en las comparaciones restantes. Los resultados de esta investigacio´n se pueden utilizar para adaptar la vigilancia futura que se enfoque en las vı´as migratorias, grupos funcionales, y las especies con mayor probabilidad de detectar al virus de la influenza aviar. Key words: avian influenza virus, virus prevalence, migratory waterfowl, flyways Abbreviations: AHY 5 after hatch-year; AIC 5 Akaike Information Criterion; AIV 5 avian influenza virus; AUC 5 area under the receiver operating curve; CI 5 confidence interval(s); FG 5 functional group; FW 5 flyway; HY 5 hatch-year; LPAIV 5 low pathogenic avian influenza virus; MO 5 month; USDA 5 United States Department of Agriculture

Wild waterfowl are reservoirs and an important long-term evolutionary source for low pathogenicity influenza A viruses (LPAIVs; 35,48). Most LPAIVs cause mild respiratory diseases in poultry (1) and in wild waterfowl (18,35) that may be exacerbated by other infections, extreme environmental conditions, or migration. Some LPAIV subtypes harbored in wild waterfowl may become highly pathogenic if introduced to poultry (34). Highly pathogenic avian influenza infection has resulted in human death and billions of dollars of agricultural losses in Asia and Europe (39,40). Influenza A viruses have been isolated from 13 orders of birds, but most have been observed in Anseriformes and Charadriiformes (9,46). Species D

Corresponding author: 300 S. Edgerton Street, Mitchell, SD 57301. E-mail: [email protected]

in these orders may have greater risk of being exposed to AIV due to their affinity to feed in shallow water which is more likely contaminated with high concentrations of infected fecal matter, especially during fall migrations when large numbers of waterfowl congregate in wetlands (9). Species from the family Anatidae pose the highest risk for transmission to other waterfowl because infected individuals can excrete virus and remain healthy while moving long distances (5,8). Concern has been raised about the role wild birds play in harboring, perpetuating, and transmitting AIV to new geographic locations internationally and intercontinentally (24,26), although others have suggested that geographic barriers impede movement of avian influenza virus (AIV) (28). The migratory nature of many waterfowl species and their ability to asymptomatically harbor AIV present a

531

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Fig. 1.

Flyway boundaries established for sampling for AIV in migratory waterfowl in the United States, 2007–2009.

potential vehicle for global dissemination of influenza viruses, as well as a constant source of viruses and genetic material for new strains (6,8). Studies have reported isolation of LPAIVs that show evidence of intercontinental exchange (38,57) which may lead to reassortment or introduction of novel viruses. Prevalence of AIV in waterfowl (0.25%–8%) varies among season, migration patterns, geographic region, habitat preference, and species (34,45) and can be much higher (10%–72%) in premigration juvenile birds (36,37). High prevalence of AIV is typically observed in naı¨ve juveniles that may be attributed to the large congregations of waterfowl before fall migrations (34,53). After nesting, waterfowl commonly congregate on permanent wetlands with dense emergent vegetation where juveniles mature, adults molt, and species mix before migration; these congregations can lead to increased risk of spreading AIV (9,52). Dabbling ducks, particularly those in the genus Anas, are natural reservoirs of influenza A viruses (35). Mallards (Anas platyrhynchos) have been the focus of surveillance efforts because of high apparent prevalence of AIV, and previous studies have implicated mallard as the principle reservoir (4,19). The objectives of this study were to use our surveillance data to determine apparent prevalence of LPAIV in the United States by flyway (FW), functional group (FG), age, and month (MO). In addition, we tested the hypothesis that mallard are the principle reservoir of AIVs by determining differences in apparent prevalence of LPAIV and odds for detecting LPAIV in selected species of waterfowl. Our data set was well suited for comparisons because it was collected over the continental United States and Alaska, included multiple years of surveillance, and had a large number of samples from the eight species of Anatidae that we chose for analysis. Previous studies have reported estimates of prevalence on the species included in our study, but they did not collect all species during the same sampling period or at the spatial scale of our study. The results could be used to modify surveillance efforts depending on emerging spatial and temporal patterns, as well as target functional groups and species important in AIV dissemination.

MATERIALS AND METHODS Field and laboratory methods. Personnel with the United States Department of Agriculture (USDA) and state and tribal wildlife agencies collected 155,535 samples from migratory waterfowl (i.e., dabbling ducks, diving ducks, geese, and swans) in the United States during 2007–2009, using standardized protocols and procedures (49). Functional groups were based on phylogenetic classification of Anseriformes (31). Sampling was conducted by biological year (April 1–March 52). The focus of sampling efforts was on ducks, geese, and swans because of their previously documented role as hosts of AIV (17,43), and they may not exhibit clinical signs of infection (23,54). Most samples were collected from hunter-harvested waterfowl between September and December; consequently, sample sizes were not evenly distributed throughout the year. Cloacal and oropharyngeal samples were collected from each bird using sterile Dacron-tipped swabs (PuritanH; Puritan Medical Products, Guilford, ME), combined in a vial containing 3 ml of brain-heart infusion broth (Becton Dickinson and Co., Sparks, MD) and kept cool until delivery to one of 40 approved laboratories (49). Samples were screened for type A influenza with a matrix real-time reverse transcriptase-PCR (44). Traditional North American Flyway (30) boundaries were modified to include entire states and simplify collection efforts within states (Fig. 1). All necessary collection permits were obtained for the described study through the United States Fish and Wildlife Service, and regulations of the permits were followed. Statistical methods. We used the frequency procedure in SAS 9.3 (PROC FREQ) to calculate apparent prevalence and exact binomial 95% confidence intervals (CI) for all apparent prevalences. We used the logistic procedure (PROC LOGISTIC) in SAS 9.3 to run 38 binomial main effect logistic regression models, including the variables FG, FW, MO, age, and pairwise interactions of the variables and to calculate odds ratios to test association of apparent prevalence. We generated regression models for each biological year to reduce uncertainty in the models due to annual variation in environmental drivers. We used Akaike Information Criterion (AIC) and AIC weights to quantify the strength of competing models. We also calculated the area under the receiver operating curve (AUC; 23) to judge fit of the models. The Atlantic Flyway, dabbling ducks, hatch-year (HY) age class, and MO of May were used as controls for comparing odds ratios. The biological year

533

Prevalence of avian influenza in waterfowl

Table 1. Apparent prevalence of AIV in migratory waterfowl and odds ratios comparing prevalence by FW in the United States, 2007–2009. Year

FW

Positive/total

Prevalence (%)

95% CI

Odds ratio

2007

Atlantic Mississippi Central Pacific Atlantic Mississippi Central Pacific Atlantic Mississippi Central Pacific Atlantic Mississippi Central Pacific

1204/16,397 1498/14,908 1493/11,937 1646/11,618 1570/17,666 1878/16,308 1690/13,451 1680/11,942 945/11,426 1898/11,461 1410/9951 1115/8470 3719/45,489 5274/42,677 4593/35,339 4441/32,030

7.3 10.1 12.5 14.2 8.9 11.5 12.6 14.1 8.3 16.6 14.2 13.2 8.2 12.4 13.0 13.9

7.0–7.8 9.6–10.5 11.9–13.1 13.5–14.8 8.5–9.3 11.0–12.0 12.0–13.1 13.5–14.7 7.8–8.8 15.9–17.3 13.5–14.9 12.5–13.9 7.9–8.4 12.1–12.7 12.7–13.4 13.5–14.3

Control 1.41 1.80 2.08 Control 1.33 1.47 1.68 Control 2.20 1.83 1.68 Control 1.51 1.59 1.70

2008

2009

All

began in April, but no positive samples were collected in April 2007, so we used May as the control for comparisons in all years. The variable age was divided into three groups: HY, after hatch-year (AHY), and unknown. The other variables in the models were default selections. We were also interested in exploring the strength of the relationship of AIV prevalence among species, so we performed chi-square tests to determine differences (a 5 0.05) in apparent prevalence of LPAIV among eight species selected for comparison, for each FW, and biological years (2007–2009), using PROC FREQ. We stratified the sample by including American green-winged teal (Anas creeca), American wigeon (Anas americana), blue-winged teal (Anas discors), Canada goose (Branta canadensis), gadwall (Anas strepera), mallard, northern pintail (Anas acuta), and northern shoveler (Anas clypeata) in the study because these species were collected in all four FWs throughout the study. Samples of blue-winged teal and cinnamon teal (Anas cyanoptera) were combined in the Central and Pacific FWs because of difficulty distinguishing between them (56). We used the criterion that a random sample of $100 individuals is required to detect AIV in a study population with prevalence of 3% (49). The blue-winged teal/ cinnamon teal did not meet the sample size criterion in the Pacific FW in biological years 2007 and 2009, but we included it in comparisons for data continuity. We calculated exact binomial 95% CI for all estimates of apparent prevalence and odds ratios by biological year using PROC FREQ. Our collection strategy included hunter-collected and live birds spread across both time and space to lessen the influence of environmental or behavioral factors. Herein, we report estimates of apparent prevalence as prevalence.

Table 2.

2.03–2.39 1.68–2.00 1.53–1.85 1.45–1.57 1.53–1.66 1.63–1.77

Apparent prevalence of AIV in migratory waterfowl and odds ratios comparing FGs in the United States, 2007–2009. FG

Positive/total

Prevalence (%)

95% CI

Odds Ratio

Dabbling duck Diving duck Goose/swan Dabbling duck Diving duck Goose/swan Dabbling duck Diving duck Goose/swan Dabbling duck Diving duck Goose/swan

5353/41,756 183/4725 305/8379 8068/42,953 299/5296 433/11,118 4919/31,338 220/3646 229/6324 18,340/116,047 702/13,667 967/25,821

12.8 3.9 3.6 18.8 5.6 3.9 15.7 6.0 3.6 15.8 5.1 3.8

12.5–13.1 3.3–4.5 3.3–4.1 18.4–19.2 5.0–6.3 3.5–4.3 15.3–16.1 5.3–6.9 3.2–4.1 15.6–16.0 4.8–5.5 3.5–4.0

Control 0.27 0.25 Control 0.36 0.25 Control 0.34 0.23 Control 0.33 0.24

All

1.24–1.43 1.37–1.59 1.56–1.81

Sampling distribution and prevalence. Samples were collected in all four FWs throughout 2007–2009, including 54,860 in 2007; 59,367 in 2008; and 41,308 in 2009. The highest proportion of samples collected each year was in the Atlantic FW (30%–33%), followed by the Mississippi (26%–27%), Central (21%–23%), and Pacific (20%) FWs. Average prevalence of LPAIV in waterfowl was highest in the Pacific FW (13.2%–14.2%), followed by the Central (12.5%–14.2%), Mississippi (10.1%–16.6%), and the Atlantic (7.3%–8.9%) FWs during 2007–2009 (Table 1). Prevalence of AIV was highest in dabbling ducks (12.8%–18.8%), followed by diving ducks (3.9%–6.0%) and geese and swans (3.6%–3.9; Table 2), annually. Prevalence of AIV was highest in HY (15.6%– 18.9%), followed by AHY 8.5%–10.7%) and unknown (8.2%– 10.9%; Table 3) age classes, annually. Logistic regression and odds ratios. The regression model that best fit the data for 2007 and 2008 was FW and age interaction + FG and MO interaction (FW | Age + FG | MO; Table 4) and the best fit model for 2009 was FW and MO interaction + FG + age (FW | MO + FG + Age). These models carried .99% of the weight among the top three models in all years, so no averaging or further consideration was given to competing models. All variables and interactions of variables were significant (P , 0.01) in the top models. The variable FG was the most important predictor of prevalence of AIV according to the type 3 analyses of effects, yearly. The calculated AUCs were 0.69 for 2007–2008 and 0.67 in 2009.

Year

2009

1.30–1.53 1.66–1.96 1.92–2.26

RESULTS

2007 2008

95% CI

95% CI

0.24–0.32 0.22–0.28 0.32–0.41 0.22–0.27 0.30–0.40 0.47–0.70 0.30–0.35 0.22–0.25

534

S. R. Groepper et al.

Table 3. Apparent prevalence of AIV in migratory waterfowl and odds ratios comparing age groups in the United States, 2007–2009. Year

Age

Positive/total

Prevalence (%)

95% CI

Odds Ratio

2007

HY AHY Unknown HY AHY Unknown HY AHY Unknown HY AHY Unknown

2629/16,840 2305/27,040 907/10,980 2889/15,587 2966/32,089 963/11,691 2178/11,533 2188/20,545 1002/9230 7696/43,960 7459/79,674 2872/31,901

15.6 8.5 8.8 18.5 9.2 8.2 18.9 10.7 10.9 17.5 9.4 9.0

15.1–16.2 8.2–8.9 8.3–9.4 17.9–19.2 8.9–9.6 7.8–8.8 18.2–19.6 10.2–11.9 10.2–11.5 17.2–17.9 9.2–9.6 8.7–9.3

Control 0.50 0.49 Control 0.45 0.40 Control 0.51 0.52 Control 0.53 0.51

2008 2009 All

Odds of detecting AIV in waterfowl were highest in the Pacific FW in 2007 and 2008 (2.08 and 1.68, respectively) and in the Mississippi FW in 2009 (2.20; Table 1). Throughout the study, odds of detecting AIV were lowest in the Atlantic FW. Odds of detecting AIV in dabbling ducks were higher than diving ducks (0.27–0.36) or geese and swans (0.23–0.25) in all FWs during the study period (Table 2). Odds of detecting AIV in HY waterfowl were higher than AHY (0.45–0.51) or unknown age (0.40–0.52) in all FWs during the study (Table 3). Odds of detecting AIV in waterfowl were highest from August through December, annually (Table 5). Species analysis. Atlantic FW. In 2007, we used 9425 samples and found differences in prevalence of AIV among species (x2 5 301.6, P , 0.001). Highest prevalence was in mallard (15.1, 95% CI 5 13.8–16.3; Table 1) and lowest prevalence and odds of detection were in gadwall (2.9, 95% CI 5 1.5–5.2 and 0.17, 95% CI 5 0.09–0.32, respectively). In 2008, we used 10,234 samples and observed differences among species (x2 5 769.2, P , 0.001). The highest prevalence was in mallard (21.2, 95% CI 5 19.9–22.7; Table 6), whereas lowest prevalence and odds were in Canada goose (1.1, 95% CI 5 0.8–1.5 and 0.04, 95% CI 5 0.03–0.06, respectively). In 2009, we used 6501 samples and discovered differences among species (x2 5 307.3, P , 0.001). Highest prevalence was in mallard (16.6, 95% CI 5 14.8–18.4; Table 6), whereas lowest prevalence and odds were detected in Canada goose (1.0, 95% CI 5 0.6–1.5 and 0.05, 95% CI 5 0.03–0.08, respectively) and gadwall (1.4, 95% CI 5 0.3–3.5 and 0.07, 95% CI 5 0.02–0.20, respectively). Mississippi FW. In 2007, we used 11,960 samples and found differences in prevalence of AIV among species (x2 5 266.4, P , 0.001). The highest prevalence and odds of detection were in northern pintail (18.6, 95% CI 5 16.5–21.4 and 1.66, 95% CI 5 1.38–1.99, respectively; Table 6), whereas lowest prevalence and

95% CI

0.47–0.54 0.45–0.53 0.42–0.47 0.37–0.43 0.48–0.55 0.48–0.57 0.52–0.55 0.49–0.54

odds of detecting AIV were in Canada goose (1.4, 95% CI 5 0.8– 2.3 and 0.10, 95% CI 5 0.06–0.17, respectively). In 2008, we used 13,015 samples and observed differences among species (x2 5 420.0, P , 0.001). Prevalence and odds of detection were highest in northern pintail (21.6, 95% CI 5 18.8–24.6 and 1.38, 95% CI 5 1.14–1.67, respectively; Table 6), whereas lowest prevalence and odds of detection were in Canada goose (1.0, 95% CI 5 0.6–1.6 and 0.05, 95% CI 5 0.03–0.08, respectively). In 2009, we used 8513 samples and discovered differences among species (x2 5 305.1, P , 0.001). Prevalence was similar in mallard and northern pintail (27.2, 95% CI 5 25.5–28.9 and 27.3, 95% CI 5 24.2–30.6, respectively; Table 6). The species with lowest prevalence and odds were detected in Canada goose (3.5, 95% CI 5 1.5–3.8 and 0.07, 95% CI 5 0.04–0.11, respectively). Central FW. In 2007, we used 10,548 samples and found differences of prevalence of AIV among species (x2 5 312.3, P , 0.001). The highest prevalence and odds of detecting AIV was in blue-winged teal (20.6, 95% CI 5 18.7–22.5 and 2.16, 95% CI 5 1.83–2.54, respectively; Table 6). The lowest prevalence and odds were detected in Canada goose (3.1, 95% CI 5 2.0–4.5 and 0.27, 95% CI 5 0.17–0.40, respectively) and American wigeon (4.3, 95% CI 5 2.9–6.0 and 0.37, 95% CI 5 0.25–0.55, respectively). In 2008, we used 11,581 samples and observed differences among species (x2 5 266.0, P , 0.001). Highest prevalence and odds were detected in blue-winged teal (21.7, 95% CI 5 19.7–23.8 and 1.64, 95% CI 5 1.41–1.91, respectively; Table 6). Lowest prevalence and odds were detected in gadwall (5.0, 95% CI 5 3.6–6.8 and 0.31, 95% CI 5 0.22–0.44, respectively) and Canada goose (5.2, 95% CI 5 4.1–6.4 and 0.33, 95% CI 5 0.25–0.42, respectively). In 2009, we used 8942 samples and discovered differences in prevalence of AIV among species (x2 5 220.9, P , 0.001). Prevalence was similar in blue-winged teal and northern shoveler (22.2, 95% CI 5 20.0– 24.6 and 20.2, 95% CI 5 15.4–25.6, respectively; Table 6), but

Table 4. Summary of binomial logistic regression models testing influence of variables on apparent prevalence of AIV in migratory waterfowl in the United States, 2007–2009. Year

Model

KA

AIC

Weight

P

2007

FW | Age + FG | MO FW | Age + FG + MO FW | MO + FG | Age FW | Age + FG | MO FG | MO + FW + Age FG | MO + Age FW | MO + FG + Age FW | MO + FG | Age FW | Age + FG | MO

16 9 8 16 7 4 11 6 10

34,999 35,023 35,048 39,410 39,538 39,542 30,091 30,093 30,269

0.99 ,0.01 ,0.01 0.99 ,0.01 ,0.01 0.99 ,0.01 ,0.01

,0.0001

2008 2009

A

K 5 degrees of freedom.

,0.0001 ,0.0001

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Prevalence of avian influenza in waterfowl

Table 5.

Odds ratios comparing apparent prevalence of AIV in migratory waterfowl by MO in the United States, 2007–2009.

MO

2007 Odds ratio

95% CI

2008 Odds ratio

95% CI

2009 Odds ratio

95% CI

May June July August September October November December January February March April

Control 1.79 10.0 34.7 29.9 22.2 17.9 8.89 0.20 0.08 0.14 1.01

0.42–7.59 2.47–40.7 8.62–139.6 7.44–120.4 5.53–89.4 4.45–72.1 2.21–35.8 0.05–0.80 0.02–0.31 0.03–0.59 1.00–1.02

Control 1.10 12.9 46.0 38.0 31.8 21.4 10.0 0.09 0.07 0.23 0.40

0.33–3.68 4.12–40.7 14.7–143.4 12.2–118.5 10.2–99.0 6.85–66.6 3.22–31.4 0.03–0.27 0.02–0.22 0.06–0.84 0.09–1.80

Control 1.98 6.13 35.0 35.5 32.4 22.0 18.1 0.08 0.05 0.08 0.20

0.47–8.39 1.49–25.3 8.70–141.2 8.82–142.7 8.06–130.3 5.47–88.5 4.50–73.0 0.02–0.31 0.01–0.20 0.02–0.33 0.04–0.98

odds of detecting AIV was highest in blue-winged teal (1.34, 95% CI 5 1.14–1.57). The lowest prevalence and odds were in American wigeon (4.5, 95% CI 5 4.6–8.2 and 0.21, 95% CI 5 0.14–0.33, respectively) and Canada goose (6.2, 95% CI 5 3.0–6.4 and 0.31, 95% CI 5 0.22–0.43, respectively). Pacific FW. In 2007, we used 9552 samples and found differences in prevalence of AIV among species (x2 5 419.6, P , 0.001). The highest prevalence was in mallard (24.8, 95% CI 5 23.4–26.3) and the lowest prevalence and odds were in Canada goose (1.5, 95% CI 5 0.6–3.1 and 0.05, 95% CI 5 0.02–0.10, respectively; Table 6). In 2008, we used 9991 samples and observed differences among species (x2 5 545.1, P , 0.001). Highest prevalence was in mallard (25.5, 95% CI 5 24.1–26.9; Table 6), whereas lowest prevalence and odds of detection were in CAGO (2.2, 95% CI 5 1.0–4.2 and 0.07, 95% CI 5 0.03–0.13, respectively). In 2009, we used 7476 samples and discovered differences among species (x2 5 206.0, P , 0.001). The highest prevalence was in mallard (21.2, 95% CI 5 19.5–23.0; Table 6) and lowest prevalence and odds of detection was in Canada goose (1.4, 95% CI 5 0.6–2.8 and 0.05, 95% CI 5 0.02–0.12, respectively). DISCUSSION

Our study indicates that prevalence of LPAIV in selected FGs of waterfowl, in the United States, generally increases from east to west. One possible explanation for lower prevalence in the Atlantic FW is the number of mallard. Estimates of mallard harvest in the Atlantic FW, from 1999–2011, were ,50% of the other FWs (27). Mallard was the most collected species during the study (33%–36%) due in part to targeted surveillance, but mallard also is the most abundant duck in North America (,12.5 million; 50). Other studies have concluded mallard is the principle host of AIV in North America (2,17,18), and mallard has the ability to spread AIVs along migration routes (22,29), so higher populations of mallard may lead to higher prevalence of AIV at the FW scale. A phylogeographic analysis suggested AIVs were strongly spatially structured (28) and further noted that any novel AIV in the wild bird population would follow a predictable route of dispersal and that crossover between FWs was rare. Our results of prevalence at the FW scale agree with Lam et al. (28) that prevalence of AIV differs by FW, likely due in part to differing habitat or species compositions. We observed dabbling ducks were more likely to be AIV positive than diving ducks or geese and swans. Other studies had similar conclusions (6,42). Although dabbling ducks were most commonly collected (72%–76%) during our study, large numbers of diving ducks and geese and swans were also collected (,10,000–16,400, annually). Abundance and propensity for LPAIV in dabbling ducks

make them a high-quality candidate for surveillance. Factors contributing to the role of primary host by dabbling ducks include population size and feeding strategy (34). Data from the United States in 1999–2011 indicated a 4 and 10 times larger harvest of dabbling ducks than geese or diving ducks, respectively (27), suggesting much smaller populations of geese and diving ducks. Also, diving ducks, geese, and swans are less likely to contract AIV, in part because they use deeper water areas of wetlands and lakes that may not concentrate virus in the water, whereas geese and swans tend to feed on waste grains in fields (3). HY birds collected in our study were 1.9–2.5 times more likely to be positive for AIV than AHY or unknown age birds. This relationship was expected and has been noted in studies in North America and worldwide (33,55). Our odds ratio estimate is somewhat lower than other estimates between age classes, but we had a large sample (21%) of unknown age class birds that may have biased the estimate, although the estimates for unknown birds were similar to estimates of AHY birds. Parmley et al. (36) reported 55% prevalence of AIV in live, HY dabbling and diving ducks collected from the Pacific FW of Canada compared to our estimates of 16%– 19%. Higher prevalence than we report could be expected when studies are designed to sample in areas where large congregations of immunologically naı¨ve, HY birds are present during late summer or early fall. Based on our results, samples are more likely to be AIV positive from August to December and are easily obtained from hunters, thus simplifying surveillance efforts and reducing costs. Previous analysis of our data (11) showed inconclusive effect of latitude on prevalence of AIV. The timing of the southward waterfowl migration is somewhat predictable (3), so we believe that MO is a suitable substitute for latitude. Some CIs overlapped in our odds ratio calculations, but trends of AIV prevalence pointed to sampling during this period. Due to uneven sampling and the large number of samples from hunter-harvested birds our results for February–June may not represent true prevalence. Other studies have reported decreasing prevalence of AIV in winter (December–January) in the Mississippi, Central, and Pacific FWs (7,16,47); prevalence of AIV is thought to decline on wintering grounds as immunity builds in flocks (12,26). Mallard had highest prevalence in the coastal FWs yearly, but American green-winged teal, blue-winged teal, northern pintail, and northern shoveler had similar or higher prevalence in the inland FWs. In a previous study conducted in Alaska, mallard also had higher prevalence of AIV than northern pintail or American greenwinged teal (41). Populations of blue-wined teal (,9 million) are second only to mallard; American green-winged teal, northern pintail, and northern shoveler (,3–5 million) are also relatively

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Table 6. Summary of number AIV samples, apparent prevalence, and odds ratio differences by FW, year, and species of Anatidae in the United States, 2007–2009. FW

Year

SpeciesA

Positive/total

Prevalence (%)

95% CI

Odds ratio

95% CI

Atlantic

2007

AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO

190/1993 32/430 20/402 105/2994 11/373 496/3296 37/289 14/348 257/1928 13/344 36/396 37/3355 6/334 717/3377 25/238 19/262 145/1428 15/242 44/531 18/1856 4/293 289/1746 18/165 20/240 592/5349 60/1016 100/1329 160/7505 21/1000 1502/8419 80/692 53/850 217/1614 30/367 172/1353 15/1071 39/1173 549/4472 194/1030 129/880 308/2237 70/461 159/1395 18/1788 53/1073 763/4588 175/810 117/663 305/1402 38/230 249/1471 20/807 83/676 728/2676 207/758 106/493 830/5253 138/1058 580/4219 53/3666 175/2922 2040/11,736 576/2598 352/2036

9.5 7.4 5.0 3.5 2.9 15.1 12.8 4.0 13.3 3.8 9.1 1.1 1.8 21.2 10.5 7.3 10.2 6.2 8.3 1.0 1.4 16.6 10.9 8.3 11.1 5.9 7.5 2.1 2.1 17.8 11.6 6.2 13.4 8.2 12.7 1.4 3.3 12.3 18.8 14.7 13.8 15.2 11.4 1.0 4.9 16.6 21.6 17.7 21.8 16.5 16.9 3.5 12.3 27.2 27.3 21.5 15.8 13.0 13.7 1.5 6.0 17.4 22.2 17.3

8.3–10.9 5.2–10.3 3.1–7.6 2.9–4.2 1.5–5.2 13.8–16.3 9.2–17.2 2.2–6.7 11.8–14.9 2.0–6.4 6.5–12.4 0.8–1.5 0.7–3.9 19.9–22.7 6.9–15.1 4.4–11.1 8.6–11.8 3.5–10.0 6.1–11.0 0.6–1.5 0.3–3.5 14.8–18.4 6.6–16.7 5.2–12.6 10.2–11.9 4.6–7.5 6.2–9.1 1.8–2.5 1.4–3.2 17.0–18.7 9.4–14.2 4.8–8.1 11.8–15.2 5.6–11.5 11.0–14.6 0.8–2.3 2.4–4.5 11.3–13.3 16.5–21.4 12.4–17.2 12.4–15.3 12.0–18.8 9.8–13.2 0.6–1.6 3.7–6.4 15.6–17.7 18.8–24.6 14.8–20.8 19.6–24.0 12.0–22.0 15.0–19.0 1.5–3.8 9.9–15.0 25.5–28.9 24.2–30.6 18.0–25.4 14.8–16.8 11.2–15.2 12.7–14.8 1.1–1.9 5.2–6.9 16.7–18.1 20.6–23.8 15.7–19.0

0.60 0.61 0.30 0.21 0.17 Control 0.83 0.24 0.56 0.14 0.36 0.04 0.07 Control 0.43 0.28 0.57 0.33 0.46 0.05 0.07 Control 0.66 0.46 0.62 0.33 0.42 0.12 0.12 Control 0.65 0.35 1.11 0.64 1.04 0.10 0.25 Control 1.66 1.23 0.80 0.90 0.65 0.05 0.26 Control 1.38 1.07 0.74 0.53 0.55 0.07 0.38 Control 1.01 0.73 0.91 0.75 0.79 0.08 0.34 Control 1.28 0.99

0.50–0.71 0.41–0.90 0.18–0.48 0.16–0.26 0.09–0.32

Atlantic

Atlantic

Atlantic

Mississippi

Mississippi

Mississippi

Mississippi

2008

2009

All

2007

2008

2009

All

0.57–1.20 0.13–0.42 0.48–0.65 0.08–0.26 0.25–0.52 0.03–0.06 0.03–0.15 0.27–0.66 0.17–0.46 0.46–0.71 0.19–0.58 0.32–0.64 0.03–0.08 0.02–0.20 0.40–1.09 0.28–0.75 0.57–0.68 0.26–0.42 0.35–0.51 0.10–0.14 0.08–0.18 0.52–0.80 0.27–0.46 0.93–1.32 0.43–0.95 0.86–1.26 0.06–0.17 0.17–0.35 1.38–1.99 0.99–1.52 0.69–0.93 0.68–1.18 0.54–0.78 0.03–0.08 0.19–0.35 1.14–1.67 0.86–1.34 0.64–0.87 0.36–0.77 0.46–0.64 0.04–0.11 0.29–0.48 0.84–1.21 0.58–0.93 0.84–0.98 0.64–0.88 0.73–0.86 0.06–0.11 0.30–0.40 1.17–1.38 0.90–1.10

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Prevalence of avian influenza in waterfowl

Table 6. FW

Central

Central

Central

Central

Pacific

Pacific

Pacific

Pacific

A

Continued. Year

SpeciesA

Positive/total

Prevalence (%)

95% CI

Odds ratio

95% CI

2007

AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO AGWT AMWI BWTE CAGO GADW MALL NOPI NSHO

146/1210 31/727 354/1722 26/843 45/846 370/3454 192/1039 53/507 200/1508 56/727 352/1622 79/1522 41/814 571/3952 172/1067 56/369 115/901 27/605 282/1268 45/723 43/650 639/3633 133/904 52/258 461/3619 114/2059 988/4612 150/3088 129/2310 1780/11,239 497/3010 161/1134 199/1382 64/1096 10/74 7/462 42/542 886/3567 156/1420 174/1009 181/1551 58/1209 11/109 9/401 30/594 954/3745 148/1442 154/940 151/1362 96/797 6/84 7/505 21/346 468/2208 178/1519 104/655 531/4295 218/3102 27/267 23/1368 93/1482 2308/9520 482/4381 432/2604

12.1 4.3 20.6 3.1 5.3 10.7 18.5 10.5 13.3 7.7 21.7 5.2 5.0 14.5 16.1 15.2 12.8 4.5 22.2 6.2 6.6 17.6 14.7 20.2 12.7 5.5 21.4 4.9 5.6 15.8 16.5 14.2 14.4 5.8 13.5 1.5 7.8 24.8 11.0 17.2 11.7 4.8 10.1 2.2 5.1 25.5 10.3 16.4 11.1 12.1 7.1 1.4 6.1 21.2 11.7 15.9 12.4 7.0 10.1 1.7 6.3 24.2 11.0 16.6

10.3–14.0 2.9–6.0 18.7–22.5 2.0–4.5 3.9–7.1 9.7–11.8 16.2–21.0 7.9–13.5 11.6–15.1 6.2–10.3 19.7–23.8 4.1–6.4 3.6–6.8 13.4–15.6 14.0–18.5 11.7–19.3 10.7–15.1 3.0–6.4 20.0–24.6 4.6–8.2 4.8–8.8 16.4–18.9 12.5–17.2 15.4–25.6 11.7–13.9 4.6–6.6 20.3–22.6 4.2–5.7 4.7–6.6 15.2–16.5 15.2–17.9 12.3–16.4 12.6–16.4 4.5–7.4 6.7–23.5 0.6–3.1 5.6–10.3 23.4–26.3 9.4–12.7 15.0–19.7 10.1–13.4 3.7–6.2 5.2–17.3 1.0–4.2 3.4–7.1 24.1–26.9 8.7–12.0 14.1–18.9 9.5–12.9 9.9–14.5 2.7–14.9 0.6–2.8 3.8–9.1 19.5–23.0 10.1–13.4 13.2–18.9 11.4–13.4 6.2–8.0 7.0–14.3 1.1–2.5 5.2–7.6 23.4–25.1 10.1–12.0 15.2–18.1

1.14 0.37 2.16 0.27 0.47 Control 1.89 0.97 0.91 0.49 1.64 0.33 0.31 Control 1.14 1.06 0.69 0.22 1.34 0.31 0.33 Control 0.81 1.18 0.80 0.35 1.35 0.31 0.35 Control 1.04 0.90 0.51 0.19 0.47 0.05 0.25 Control 0.37 0.63 0.39 0.15 0.33 0.07 0.16 Control 0.34 0.57 0.46 0.51 0.29 0.05 0.24 Control 0.49 0.70 0.51 0.29 0.42 0.07 0.26 Control 0.45 0.68

0.93–1.41 0.25–0.55 1.83–2.54 0.17–0.40 0.34–0.65

2008

2009

All

2007

2008

2009

All

1.56–2.30 0.71–1.33 0.76–1.08 0.37–0.66 1.41–1.91 0.25–0.42 0.22–0.44 0.94–1.38 0.78–1.44 0.55–0.85 0.14–0.33 1.14–1.57 0.22–0.43 0.24–0.46 0.66–1.00 0.85–1.64 0.73–0.88 0.29–0.42 1.26–1.45 0.26–0.36 0.30–0.42 0.95–1.14 0.77–1.04 0.43–0.60 0.14–0.25 0.23–0.96 0.02–0.10 0.18–0.36 0.31–0.45 0.52–0.76 0.32–0.46 0.11–0.20 0.17–0.63 0.03–0.13 0.11–0.23 0.28–0.40 0.47–0.69 0.38–0.57 0.40–0.65 0.11–0.69 0.02–0.12 0.15–0.39 0.41–0.60 0.55–0.89 0.46–0.56 0.25–0.33 0.29–0.60 0.05–0.10 0.22–0.32 0.41–0.50 0.62–0.75

AGWT 5 American green-winged teal, AMWI 5 American wigeon, BWTE 5 blue-winged/cinnamon teal; CAGO 5 Canada goose; GADW 5 gadwall; MALL 5 mallard; NOPI 5 northern pintail; NSHO 5 northern shoveler.

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abundant (50). Robust populations along with high prevalence make those species attractive targets for future surveillance efforts. The blue-winged teal is an early migrator and often moves south from breeding grounds in southern Canada and the Dakotas in early September (3) during the time when prevalence of LPAIV is highest in waterfowl and may be a vehicle for dissemination of AIV. Odds of detecting AIV in Texas, 2006–2009, were highest in blue-winged teal (2.18; 6). Although that study (3) did not make comparisons directly with mallard, blue-winged teal were compared to American green-winged teal, gadwall, and northern shoveler, with results similar to our study. Reported prevalence of northern pintail in previous studies is generally within ranges we report (35,36,41). They are one of the most abundant duck species at high latitudes and are sympatric with a diverse array of waterbirds, thus transfer of AIVs is likely (24). The northern shoveler is an important host for LPAIV throughout the year (16). They are efficient filter feeders (12), a behavior that may increase the risk of AIV contact during foraging. Wintering northern shoveler in the Atlantic FW had higher prevalence (8%) than all other species of birds sampled (10). Mallard had higher prevalence than northern shoveler during our study in the Atlantic FW, but generally northern shoveler was collected in low numbers, so increased sampling may give further insight to this species’ role in AIV epidemiology. Hunter-harvested northern shoveler collected in California and Mexico had higher prevalence than all other species of dabbling ducks, diving ducks, geese, or other waterbirds (16,32). Due to their high prevalence and abundance, they are attractive targets for surveillance. The American wigeon also had high prevalence in the Mississippi FW, but low prevalence in other FWs. American wigeon may have a higher degree of habitat overlap with other species in the Mississippi FW, thus increasing probability of contact with infectious environments. The harvest of American wigeon and age ratio estimates were at or below average for the Mississippi FW in 2007–2009 (27), so we did not expect a bias in the prevalence data based on an influx of HY birds, which may be more likely AIV positive (14,20,33). Canada goose and gadwall had lower prevalence than other species in all FWs and years. Geese are equally susceptible to AIV as dabbling ducks, but feeding habits may lead to less efficient transmission (51). Frequency of AIV infection in waterfowl may be more related to behavior than population levels (33). Canada geese on eight national refuges in all FWs except the Pacific were tested for AIV, and prevalence was 4.7% (58). In addition, Harris et al. (15) compiled AIV surveillance data from Canada goose in previous studies and reported prevalence of 0.5% in 3777 samples collected over 42 yr. Both studies report prevalence within ranges we report and agree with our findings that Canada goose has lower prevalence than other targeted species. Gadwall prefer to feed on aquatic plant material and are often found in deeper water than other dabbling ducks (3). Similar to the Canada goose, they may not be exposed to contaminated environments as often as other species of dabbling ducks. American wigeon often had low prevalence of AIV compared with other species, with the Mississippi FW being the exception, a finding that may be due in part to their feeding habits; they are aquatic grazers, foraging on grasses and sedges in wet meadows (3). Previous studies also indicated relatively low prevalence of LPAIV in American wigeon and gadwall (4,16,42). The understanding of AIV in migratory waterfowl in the United States has increased considerably over the past 20 yr. Although other studies have been conducted on AIV in waterfowl in North America, most have been of limited spatial and temporal scales. FW-specific collection strategies can be developed to target species most likely to be AIV positive. We suggest future AIV surveillance focus on

mallard, but also include American green-winged teal, blue-winged teal, northern pintail, and northern shoveler, all members of the dabbling duck FG, because of high prevalence of LPAIV and their availability for surveillance across North America. The targeting of species with highest prevalence may aid in detecting novel strains, should they appear in the United States. We further suggest future research explore interactions of the environment and AIV, such as breeding populations of waterfowl, number of HY birds in fall migrations, habitat availability, and interactions of FGs. We may further increase our understanding of annual cycles of AIV by increasing sampling effort before fall migration to obtain estimates of prevalence in previously underrepresented time periods. Our data provide a comprehensive analysis of LPAIV in the United States over a 3-yr period. These findings lend insight into the annual cycle of AIV in wild waterfowl at a scale that has previously not been reported and is unlikely to be replicated.

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ACKNOWLEDGMENTS We thank USDA, state, and tribal employees and volunteers who collected samples for this research. This effort would not have been possible without cooperation of waterfowl hunters who allowed us to sample birds. We thank L. Powell and three anonymous reviewers for comments on early drafts of the manuscript and E. Blankship and T. Buckley for statistical advice. Funding for this research was provided by the USDA, the Nebraska Game and Parks Commission, and the Federal Aid in Wildlife Restoration W-15-R. Additional funding was provided by the Shikar-Safari Club International Foundation and the Nebraska Chapter of the Safari Club International.