Journal of Wildlife Diseases, 46(3), 2010, pp.

981987 # Wildlife Disease Association 2010

Canada Geese and the Epidemiology of Avian Influenza Viruses

Mark T. Harris,1 Justin D. Brown,1 Virginia H. Goekjian,1 M. Page Luttrell,1 Rebecca L. Poulson,1 Benjamin R. Wilcox,1 David E. Swayne,2 and David E. Stallknecht1,3 1 Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA; 2 United States Department of Agriculture, Agricultural Research Service, Southeast Poultry Research Laboratory, Athens, Georgia 30605, USA; 3 Corresponding author (email: dstall@uga.edu)

ABSTRACT:

Canada geese (Branta canadensis) are numerous, highly visible, and widely distributed in both migratory and resident populations in North America; as a member of the order Anseriformes, they are often suggested as a potential reservoir and source for avian influenza (AI) viruses. To further examine the role of Canada Geese in the ecology of AI, we re-evaluated existing literature related to AI virus in this species and tested breeding populations of Canada Geese from three states (Georgia, West Virginia, and Minnesota, USA) by virus isolation and serology. The ability of AI virus to persist in goose feces under experimental conditions also was evaluated as an additional measure of the potential for this species to serve as an AI virus reservoir. Virus was not isolated from 1,668 cloacal swabs and type-specific antibody prevalence was low (4/335, 1.2%). Finally, under experimental conditions, AI virus persistence in goose feces and in water contaminated with goose feces was limited as compared to published estimates from duck feces and water. Our results are consistent with historic reports of a low prevalence of AI virus infection in this species, and we suggest that Canada Geese play a minor, if any, role as a reservoir for low pathogenic AI viruses that naturally circulate in wild bird populations. Key words: Avian influenza virus, Canada Geese, environmental persistence, feces, reservoir.

Avian influenza (AI) virus has been isolated from more than 100 avian species representing 13 orders, and based on historic isolation rates, species in the orders Anseriformes (ducks, geese, and swans) and Charadriiformes (shorebirds, gulls, and terns) are considered as the natural reservoirs for these viruses (Stallknecht and Shane, 1988; Olsen et al., 2006). With regard to understanding AI virus epidemiology, however, it is important to recognize that the species within these orders are diverse, and because of this, each species potential contribution to
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the AI reservoir or ability to serve as a source of infection cannot be generalized. Globally, differences in infection rates within anseriform groups and species exist; differences have been reported between ducks (9.5% of 34,503) and geese (1% of 4,806) and among duck species where global infection rates have ranged from 0.8% to 12.9% (Olsen et al., 2006). The Canada Goose (Branta canadensis) historically was a migratory species that ranged from northern Canada and Alaska to the southern United States and northern Mexico (Bellrose, 1976). Today, Canada Geese are present in northern Europe (Bonner et al., 2004) as a result of introductions from North American populations and exist as resident (nonmigratory) populations throughout much of the United States (Hestbeck, 1995). These changes in range and behavior have resulted in expanding populations, especially in urban, suburban, and agricultural areas. Because this species is a member of the order Anseriformes (a recognized reservoir of AI viruses) and has a highly visible interface with humans and domestic animals, they readily are perceived as a potential risk for AI virus dissemination. The objectives of this study were to evaluate this potential risk through both historic and prospective studies of AI virus in this species. We also attempted to provide a better understanding of potential transmission through goose feces and fecal-contaminated water. An important transmission route for AI viruses among wild waterfowl populations is through an indirect fecaloral route involving contaminated water (Hinshaw et al., 1979). However, there is no information on the persistence of AI viruses in Canada Goose

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feces, whether deposited on land or in water. Studies consistently have reported a low prevalence of AI virus infection in Canada Geese; in nine studies conducted in North America during the past 42 yr, 3,777 geese were tested by virus isolation and 19 (0.5%) were positive for AI virus (Table 1). Reported AI virus antibody prevalence in Canada Geese also is low; previous studies report an overall antibody prevalence of 4.7% (70 positive/1,494 tested; Table 2). Discrepancies exist between serologic results derived from hemagglutination inhibition or agar gel immunodiffusion (AGID) test formats used in these previous studies, but individual antibody prevalence estimates from these studies, regardless of assay used, remain low (Table 2). These studies demonstrate that Canada Geese can be infected naturally with low-pathogenic avian influenza (LPAI) viruses, but the reported prevalence of infection is well below that expected to occur in North American ducks (Olsen et al., 2006). There have been relatively few experimental infections of Canada Geese with AI viruses (Homme and Easterday, 1970; Winkler et al., 1972; Pasick et al., 2007), but results are consistent with the low prevalence of AI virus isolations and AI virus antibodies reported for free-living Canada Geese. These studies have been restricted to a few AI viruses but have demonstrated no or very low rates of viral shedding. In the most recent study, virus detection by polymerase chain reaction (PCR) from Canada Geese challenged with A/Mallard/British Columbia/373/ 2005 (H5N2) was restricted to 3 days postinfection (DPI) in juvenile birds; AI virus was not detected from challenged adult birds (Pasick et al., 2007). These results are in contrast to experimental infection studies of mallards (Anas platyrhynchos) with LPAI virus; in this species, viral shedding consistently can be demonstrated from 1 hr postinoculation to more than 10 DPI (Homme and Easterday,

TABLE 1. Summary of historic prevalence data reported in nine studies on avian influenza viruses in Canada Geese. Total is reported as positive isolations/number sampled (percentage).

Total

Pooled cloacal and tracheal swabs Tracheal swabs unknown Cloacal swabs Pooled cloacal and tracheal swabs Pooled cloacal and tracheal swabs Maryland, USA Minnesota, USA Quebec and Ontario, Canada New York, USA Pennsylvania and Maryland, USA Pennsylvania, USA Rosenberger et al., 1974 Bahl et al., 1977 Boudreault et al., 1980 Diebel et al., 1985 Nettles et al., 1985 Hinshaw et al., 1986 November 1974 December 1975 July and August 1975 July 19781980 November 1983May 1984 JuneNovember 1984

Samples tested

Study site(s)

Graves, 1992 Slemons et al., 1991 Ito et al., 1995

Study

April 19771979 Maryland, USA Spring, Summer, and Fall 19861988 Ohio, USA July 19911994 Alaska, USA

Dates of study

Cloacal swabs Cloacal swabs Fecal material swabs Virus Isolation Results

1/52 0/65 4/7 0/261 0/1,504 Juvenile Adult 0/348 0/315 4/663 19/3,777

(1.9%) (0%) (57.1%) (0%)

9/311 (2.9%) 1/251 (0.4%) (0%) (0%) (0.6%) (0.5%)

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TABLE 2. Summary of serologic studies where Canada Geese were tested for antibodies to AI viruses. Total is reported as positive isolations/number sampled (percentage).
Study Dates of study Study site(s) Testa Total

Winkler et al., Summer and Winter 1972 19661969 Bahl et al., 1977 December 1975 Graves, 1992 April 19771979 Overall Total
a b

Michigan, Missouri, Wisconsin, Illinois, and New York, USA Minnesota, USA Maryland and North Carolina, USA

HI 66/1,401 (4.7%) AGP (8/1,359 [0.6%])b HI and AGP 0/65 (0%) HI and EI 4/28 (14%) 70/1,494 (4.7%)

HA 5 hemagglutination inhibition; AGP 5 agar gel precipitin; EI 5 elution inhibition. The 1,359 samples were part of the 1,401 tested by HI.

1970; Slemons and Easterday, 1978; Webster et al., 1978). To provide current information on the prevalence of AI virus infection in Canada Geese in the United States, including the nonmigratory portion of this population, cloacal swabs (n51,668) were collected for virus isolation from Canada Geese in Georgia, Minnesota, and West Virginia, USA (Table 3) as previously described (Hanson et al., 2005) and stored at 280 C until virus isolation was performed. Samples were collected in Georgia during molting season in the summer from 2005 to 2007. In West Virginia and Minnesota, samples were collected in summer 2007. Virus isolation was performed in 9- to 11day-old embryonating specific pathogen free (SPF) chicken eggs using standard methods (Swayne et al., 2008). Additionally, 335 serum samples (283 from Georgia, 52 from West Virginia) were collected from Canada Geese; samples were stored

at 220 C until tested, using the AGID test as described (Swayne et al., 2008). Reagents for the AGID were obtained from National Veterinary Services Laboratory (NVSL, Ames, Iowa, USA). To provide preliminary data on the duration of AI virus in goose feces and contaminated water, two LPAI viruses were used, A/Mallard/MN/199036/99 (H3N2; MN99-68) and A/Northern Pintail/TX/421716/01 (H8N4; TX01-67). Viruses were propagated and stored as described (Brown et al., 2009). The persistence of these AI viruses in Canada Goose feces was evaluated in two separate laboratory trials: 1) the persistence of AI virus in a fecal-water slurry under varying temperatures, and 2) persistence of AI virus in fecal material under varying degrees of desiccation (to simulate fecal deposition on terrestrial habitats). Fresh Canada Goose feces were collected in Clarke County, Georgia, USA. Fecal

TABLE 3. Sites in Georgia (GA), West Virginia (WV), and Minnesota (MN), USA, where Canada Geese were captured for virus isolation by cloacal swabs. Sites are divided into counties, followed by number of samples taken from each county in parentheses.
Year Location Total

2005 2006

2007 2007 2007

GA: Fulton (35) GA: Butts (3), Carroll (126), Clayton (16), Coweta (52), Dekalb (89), Douglass (21), Elbert (11), Greene (8), Gwinnett (12), Henry (20), Macon (10), Morgan (3), Oconee (6), Rockdale (4), White (4) GA: Barrow (14), Clarke (17), Cobb (29), Dekalb (88), Gwinnett (20), Hart (47), McDuffie (36), Monroe (72), Rockdale (20) MN: Beltrami (174), Cass (52), Clearwater (40), Hubbard (48), Marshall (1), Polk (36) WV: Grant (71), Hampshire (55), Harrison (110), Pleasants (151), Preston (38), Raleigh (52), Summers (77)

35 385

343 351 554

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material was allowed to dry in sterile Petri dishes for 48 hr at room temperature, ground into fine pieces, and sterilized in an autoclave for 25 min at 121 C. For the persistence in fecal slurry trials, four 5% fecal slurries (20 ml each) were created by combining sterile feces with distilled water buffered with 2 mM HEPES in sterile 100 ml glass beakers. Slurries were inoculated with AI virus-infected allantoic fluid at a 1:100 dilution, covered, and stored at 4 C and 17 C in temperature-controlled chambers. Water samples (1 ml) for titration, were collected at 0, 4, and 7 DPI from the slurries at 17 C and 0, 7, 14, 26, and 33 DPI from the slurries at 4 C. The pH of the slurry at 17 C was measured at 0, 3, 7, and 10 DPI using commercially available pH strips (Whatman International Ltd., Maidstone, England; Electron Microscopy Sciences, Hatfield, Pennsylvania, USA). The experiment was repeated in two separate trials, with both viruses; one with feces as described above, and one in distilled water without feces that was buffered with 2 mM HEPES and adjusted to pH of 6.6 and 7.2. Infectivity of AI virus in fecal slurry supernatants were quantified using microtiter endpoint titration as described in MadinDarby Canine Kidney (MDCK) cells (Brown et al., 2009). The minimal detectable value for this assay is log10 2.47 median tissue culture infective doses (TCID50)/ml sample. Results from infectivity assays were log10-transformed and subjected to linear regression analysis using Microsoft Excel (Microsoft Office Excel 2004, Microsoft, Redmond, Washington, USA). The slope of the linear regression models were then used to determine the estimated time, in days, for the viral concentration to be reduced by 90% (1 log10), which was reported as the RT value (time in days required to reduce virus infectivity by 90% [1 log10]). For the second laboratory trial, fecal material was dried, ground, and sterilized as described above. Five samples were created by adding 2 g of fecal material to 5 ml polystyrene tubes (BD Falcon, Two

Oak Park, Bedford, Massachusetts, USA), followed by 1 ml of distilled water buffered with 2 mM HEPES, and 200 ml of MN99-68 virus. Samples were stored at three temperatures, 4, 17, and 37 C. One sample was sealed with a snap cap at each temperature. At 17 and 37 C; one sample was left uncapped to simulate natural desiccation. Feces in all of the tubes were swabbed every 24 DPI, starting at 0 DPI, stored, and processed for virus isolation in SPF chicken eggs as described (Hanson et al., 2005). No AI viruses were isolated from cloacal swabs, and only four geese were positive (three weak positives and one positive, 1.2%) for antibodies to AI virus. Virus in water supplemented with goose fecal material had a lower RT value than did virus in water alone that was adjusted to the same pH range (6.6 to 7.2; Table 4). In a second experiment with AI virusinoculated feces, simulating fecal deposition in a terrestrial environment, a starting titer of 107.52 TCID50/ml, infective virus was detected by virus isolation in eggs from ,1 to 4 days at 37 C, 9 days at 17 C, and 14 days at 4 C. These results were consistent with previous findings that a negative correlation exists between duration of AI virus infectivity and increasing temperatures and aridity (Table 5; Brugh and Johnson, 1986; Brown et al., 2009). Under a controlled laboratory setting, the persistence of AI virus in fecal material of Canada Geese is lower than that reported for fecal material of mallards (Webster et al., 1978), and this might relate to variations in pH (AI virus persistence has been shown to decrease with lower pH) (Stallknecht et al., 1990); the Canada Goose feces evaluated in this study were more acidic (pH 6.56.8), than reported for mallard feces (pH 7.68; Webster et al., 1978). However, the fact that both viruses tested persisted for less time in fecal slurries than in distilled water of similar pH, suggests that factors other than pH contribute to the limited persistence of AI virus in fecal material of

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TABLE 4. Persistence of the avian influenza (AI) viruses A/Mallard/MN/199036/99 (H3N2; MN99-68) and A/Northern Pintail/TX/421716/01 (H8N4; TX01-67) in fecal slurries and distilled water at 4 C and 17 C. RT value5time (in days) for the starting viral concentration to be reduced by 90% (1 log10).
Feces/Water slurrya (RT Value) Distilled water pHb RT Value

Treatment and temp (C)

MN99-68 4 17 17 TX01-67 4 17 17
a b

23.42 NDc 4 18.35 ND 5.51

7.2 7.2 6.6 7.2 7.2 6.6

94.64 31.82 13.5 92.42 31.82 26.74

pH dropped to 6.6 with the addition of the goose feces to distilled water at pH 7.2. All distilled water samples were done at pH 7.2. For each virus (at 17 C only) pH in the distilled water system was also done at pH 6.6 to match the pH in the fecal slurry. ND 5 not done.

Canada Geese. The environmental persistence trials conducted in this study were artificial and only were done to provide an example of how environmental conditions, relative to species behavior, could potentially affect transmission efficiency. Results were consistent with published results that demonstrate reduced infectivity of AI viruses related to increased temperature and lower pH; these relationships are reported for AI viruses originating from both ducks and shorebirds (Brown et al., 2009). The persistence estimates presented here should not be applied directly to the field, because other variables, such as fecal flora (bacterial) and fecal structure (not disrupted through

autoclaving and mechanical manipulation) could decrease or increase the actual duration of infectivity. To gain field validity, trials need to be done with infective feces from geese. This could be a logistical challenge based on biocontainment requirements for both birds and feces, but the preliminary results presented here justify additional work. The results of this study confirm a limited role for Canada Geese as a potential reservoir for AI virus. AI viruses are not prevalent in Canada Goose populations, as shown by the 0% isolation rate in this study and the very low isolation rate (0.5%) in nine previous studies (Table 1). The low prevalence of AI virus among

TABLE 5. Summary of results for avian influenza virus persistence in Canada Goose feces in a terrestrial habitat. Covered treatment signifies a test tube with a cover and uncovered signifies a test tube left open. Results are reported as positive (+) or negative (2) for virus isolation in eggs. RT5time (in days) for the starting viral concentration to be reduced by 90% (1 log10).
Days postinoculation Treatment 0 2 4 7 9 11 14 16 Estimated RT

Covered 4 C Covered 17 C Uncovered 17 C Covered 37 C Uncovered 37 C

a

+ + + + +

+ + + 2 2

+ + + + 2

+ + + 2 2

+ + + 2 2

+ 2 2 2 2

+ 2 2 2 2

2 2 NDa NDa NDa

1.8 1.2 1.2 0.5 ,0.2

ND 5 not done.

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Canada Geese we tested might be attributed to seasonal variation as reported in ducks. The highest rate of infection in ducks occurs during premigration staging in the late summer and fall and, at this time, prevalence in juvenile birds can reach 60% (Olsen et al., 2006). All of the geese in this study were sampled during the molting season from late June to early July, prior to the seasonal peak prevalence reported for ducks. However, among studies conducted, reported prevalence was low regardless of season. Another explanation for the low prevalence could relate to the migration habits of the Canada Geese. Most of the geese tested in this study, especially in Georgia and West Virginia, are nonmigratory, which could limit contact with other infected waterfowl. However, results from previous studies (Table 1) and isolation rates from other goose species (Olsen et al., 2006) have reported the same low isolation rate from migratory geese. Canada Geese are not refractory to AI infection, as evidenced by reported virus isolations and the detection of antibodies to AI viruses. However, the low prevalence of infection (both historic and current) and the limited shedding documented in experimental infections (Homme and Easterday, 1970; Winkler et al., 1972; Pasick et al., 2007) suggests that they contribute minimally to LPAI virus transmission and maintenance. The low prevalence consistently observed in this species might represent the combined effects of reduced host susceptibility and behavior that indirectly affects transmission. In this regard, the limited persistence of AI virus in goose feces mighty be significant. Canada Geese feed extensively in terrestrial habitats (including many human-modified habitats such as recreational sports fields). Based on our results that demonstrated limited viral shedding and a rapid loss of infectivity in goose feces under experimental conditions, AI virus transmission might be greatly reduced on such habitats. This reduced

environmental persistence could lessen potential risks associated with indirect contact between geese and domestic animals and humans using these same habitats. Potential public health risks associated with goose feces or fecalcontaminated water could be further limited due to rapid environmental degradation during seasons with warmer temperature when recreational activity and human contact can increase. Although we do not believe that Canada Geese significantly contribute to the transmission or maintenance of these viruses, reported virus isolations and the detection of antibodies to AI viruses does indicate that this species can be infected. Canada Geese have been shown to be highly susceptible to Eurasian lineage H5N1 highly pathogenic avian influenza (HPAI) viruses in both experimental (Pasick et al., 2007) and natural infections (Teifke et al., 2007) and potentially could be utilized as a sensitive detection system for H5N1 HPAI viruses in North America. Funding for this work was provided by CDC Cooperative Agreement 5U19Cl000401-02 and Specific Cooperative Agreement 58-6612-2-0220 between the Southeast Poultry Research Laboratory and the Southeastern Cooperative Wildlife Disease Study.
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