Influenza A Prevalence and Subtype Diversity in Migrating Teal Sampled Along the United States Gulf Coast

SUMMARY.

Wild birds in the order Anseriformes are important reservoirs for influenza A viruses (IAVs); however, IAV prevalence and subtype diversity may vary by season, even at the same location. To better understand the ecology of IAV during waterfowl migration through the Gulf Coast of the United States (Louisiana and Texas), surveillance of blue-winged (Spatula discors) and American green-winged (Anas carolinensis) teal was conducted. The surveillance was done annually during the spring (live capture; 2012–17) and fall (hunter harvested; 2007–17) at times inferred to coincide with northward and southward movements, respectively, for these waterfowl species. During spring migration, 266 low pathogenicity (LP) IAV positive samples were recovered from 7547 paired cloacal–oropharyngeal (COP) samples (prevalence, 3.5%; annual range, 1.3%–8.4%). During fall migration, 650 LP IAV-positive samples were recovered from 9493 COP samples (prevalence, 6.8%; annual range, 0.4%–23.5%). Overall, 34 and 20 different IAV subtypes were recovered during fall and spring sampling, respectively. Consistent with previous results for fall migrating ducks, H3 and H4 hemagglutinin (HA) subtypes were most common; however, H4 subtype viruses predominated every year. This is in contrast to the predominance of LP H7 and H10 HA subtype viruses during spring. The N6 and N8 neuraminidase subtypes, which were usually associated with H4, were most common during fall; the N6 subtype was not recovered in the spring. These consistent seasonal trends in IAV subtype detection in teal are currently not understood and highlight the need for further research regarding potential drivers of spatiotemporal patterns of infection, such as population immunity.

Blue-winged teal (BWTE; Spatula discors) are small dabbling ducks common in North and South America (24). The winter distribution of this species extends further south than other ducks that breed in North America, and from late summer to spring, migratory BWTE can be found along the Gulf Coast of Mexico, Venezuela, and Columbia. BWTE also winter in the Caribbean Islands, as well as in Florida and along the United States Gulf Coast in Texas and Louisiana (24). One of the last species of ducks to arrive on northern breeding areas, BWTE initiate spring migration in January, reaching breeding areas in late April and May. Nesting and breeding occurs from May to early July. After the breeding season, adult male BWTE migrate ahead of females in late August through early September, and in mid to late September, flocks of females and immatures begin their fall migration together (24). Although migratory waterfowl in North America use four primary flyways, namely Pacific, Central, Mississippi, and Atlantic (16), BWTE are primarily associated with the Central and Mississippi flyways that include the Texas and the Louisiana Gulf Coasts of the United States.

American green-winged teal (AGWT, Anas carolinensis) are the smallest dabbling duck species in North America. AGWT use all four flyways and are abundant along the Mississippi and Central flyways where the coastal marshes and rice fields of Louisiana and Texas provide ideal habitats (25). AGWT winter in the southern United States, along the western coast of the Yucatan Peninsula and in Cuba. These ducks begin their spring migration in February, traveling north in mated pairs (10), and reach breeding grounds in late April to mid-May. Males migrate first in the fall, around late August–September, followed by females and juveniles in late September–October (23).

Wild birds in the order Anseriformes (ducks, geese, and swans), including BWTE and AGWT, are important reservoirs of avian influenza A virus (IAV) (9,27). Sixteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes of IAV have been detected in wild birds, and seasonal dynamics of IAV prevalence and diversity are likely influenced by a number of factors, including bird density and population-level immunity. In North America, IAV prevalence rates in ducks tend to peak in late summer when waterfowl aggregate prior to and during fall migration (9); the prevalence of infection at that time can be greater than 30% (9,31). Typically, prior to and during fall migration, viruses of the H3 and H4 HA subtypes predominate in duck populations in North America (21,22,31). In contrast, the prevalence of IAV infection in ducks on wintering grounds in the southern United States is generally lower (≤6%) (2,8,28). Teal represent good surveillance targets for IAV for several reasons. These long distant migrants may be exposed to a variety of viruses along their extensive and international migration paths, and given their susceptibility to IAV infection, they may have the ability to move these viruses a great distance. These species are often locally abundant during migration and are targeted by hunters, thus facilitating capture and sampling events. Lastly, previous overall prevalence estimates of IAV in BWTE and AGWT collected from across North America have been reported to be 11.5% and 4.0%, respectively (18).

Long-term surveillance programs targeting birds through space and time are critical to better understanding the ecology and natural history of IAV in waterfowl hosts. For example, over 30 years of waterfowl surveillance in Alberta, Canada, has greatly informed our knowledge about the maintenance and diversity of IAV on breeding grounds (1). In this investigation, we targeted teal species at a geographic location in the United States that commonly supports large numbers of birds at two stages of migration: during spring with presumed movement of birds northward toward breeding grounds and during fall with presumed movement of large numbers of ducks southward toward wintering locations. Thus, the objective of this long-term surveillance effort was to investigate seasonal trends in IAV subtype diversity and prevalence in BWTE and AGWT during migratory periods by comparing results from spring and fall collections.

MATERIALS AND METHODS

Study sites and sample collection.

Surveillance for IAV from BWTE and AGWT was carried out at various locations along the Texas and Louisiana Gulf Coast (Fig. 1); sites included flooded rice fields, freshwater marshes, brackish marshes, and commercial crawfish farms. Hunter-harvested samples were collected in September of 2007 and 2010–17. In 2008, fall sampling was delayed until November due to the September landfall of Hurricane Ike on the Gulf Coast; in 2009, sample collection took place in September, November, and December. Fall sample collections took place at central locations where ducks were brought in from the field by hunters; samples were collected within several hours of hunter harvest. In the spring, live-captured duck samples were obtained from 2012–16 in mid-March through early April via prebaiting of sites and capture by rocket net. Rocket netting was conducted by trained personnel under U.S. Geological Survey Federal Bird Banding Permit numbers 23792 and 09702. In the spring, the sample collection took place within 90 min of capture, and at both times of year, paired cloacal–oropharyngeal (COP) swab samples were collected from target species and placed into 2 ml of sterile brain heart infusion viral transport medium supplemented with antibiotics as previously described (7,25). Field-collected samples were kept on ice or ice packs or stored at 4 C until received in the lab where they were kept at −80 C until processing.

Fig. 1.

Location of collection sites at county (Texas) or parish (Louisiana) level for this study. Shaded and numbered counties or parishes designate locations where collections occurred, based on time of year (black, fall only; black and white hatch, spring only; grey, at least one spring and at least one fall). Locations in Texas are as follows: 1) Brazoria County (Co.), 2) Chambers Co., and 3) Jefferson Co. Collection sites in Louisiana, by parish, include the following: 4) Cameron, 5) Calcasieu, 6) Evangeline, 7) La Salle, 8) Avoyelles, 9) Concordia, 10) St. Landry, 11) St. Martin, 12) Iberia, 13) St. Mary, 14) Lafourche, and 15) Plaquemines.

Virus isolation and subtyping.

Prevalence estimates were based on virus isolation, which was performed as previously described (27,29). Briefly, COP swabs were thawed, vortexed for 15 sec, and centrifuged at 1,500 × g for 15 min. Three to four 9-day-old specific-pathogenic-free embryonated chicken eggs were then inoculated (0.25 ml/egg) with the supernatant and incubated at 37 C for 5 days, at which point amnio-allantoic fluid was harvested and tested by hemagglutination assay. Viral RNA was extracted (QIAamp Viral RNA Mini Kit, Qiagen Inc., Valencia, CA) from all hemagglutination assay-positive samples as per the manufacturer’s instructions and tested by IAV matrix real-time reverse transcriptase–PCR (RRT-PCR) as previously described (5,26). Resultant HA and NA subtypes of IAV isolates were identified by serological (19,20) and/or molecular methods (15,22).

RESULTS

During 11 yr of fall surveillance, 8792 BWTE samples and 696 AGWT samples were collected. In total, 650 low pathogenicity (LP) IAV were isolated from teal samples (6.8% overall prevalence), and in 6 yr of spring surveillance, 6844 BWTE samples and 702 AGWT samples were collected, yielding a total of 266 LP IAV (3.5% overall prevalence). The prevalence of IAV and subtype diversity in BWTE or AGWT within a given season and year were similar (unpubl. data). Some of these spring (2012, 2013), and fall (2007–11) data have been previously published (22). In combined teal species in the fall, IAV prevalence ranged from 0.4% in November 2008 to 23.5% in September 2011; in the spring, the IAV prevalence in live-captured teal was lower overall and ranged from 1.3% in 2016 to 8.4% in 2014 (Fig. 2). Influenza viral diversity was higher in the fall with 34 HA and NA subtype combinations detected compared with that of the spring, from which 20 combined IAV subtypes were identified (Fig. 3). Furthermore, of the 46 different HA and NA combinations recovered, 26 (56.5%) and 12 (26.1%) HA and NA combinations were unique to fall or spring collections, respectively (Table 1; Fig. 3). All representative North American HA subtypes normally associated with waterfowl (H1–H12, H14; Table 1) and all nine NA subtypes (Table 2) were recovered over the course of this study. Several IAVs were isolated from nonteal species collected as bycatch in both the fall and the spring (Supplemental Table S1).

Fig. 2.

Overall prevalence of IAVs in BWTE (Spatula discors) and AGWT (Anas carolinensis) sampled in Texas and Louisiana in the spring (2012–17; grey filled diamonds) and fall (2007–17; black filled circles).

Fig. 3.

Relative distribution of IAV HA and NA subtypes in BWTE (Spatula discors) and AGWT (Anas carolinensis) sampled in Texas and Louisiana in the spring (2012–17; grey bars) and fall (2007–17; black bars). Bar height reflects the percent contribution of a given subtype combination to all IAV recovered in each season, spring or fall. Bar width below subtype combinations reflects relative number of NA subtypes detected per HA subtype.

Table 1.

Total number of IAVs of each HA subtype isolated from samples from teal species collected in the spring and fall along the Gulf Coasts of Texas and Louisiana

	NA subtypes
Seasons and parameters	N1	N2	N3	N4	N5	N6
FallA 2007–17  No. of IAV isolates (% of all isolates)	14 (2.2)	30 (4.6)	12 (1.8)	0	5 (0.8)	300 (46.2) B
No. years detected (range as % of contribution to all subtypes within years detected)	7 (1.5–7.1)	8 (1.3–19.8)	6 (1.3–5.2)	0	3 (0.6–3.1)	11 (19.2–100.0)
SpringC, 2012–17  No. of IAV isolates (% of all isolates)	20 (7.5)	19 (7.1)	73 (27.4)	2 (0.8)	1 (0.4)	0
No. years detected (range as % of contribution to all subtypes within years detected)	4 (2.8–80.0)	3 (2.8–16.7)	6 (8.3–69.8)	2 (1.6–2.8)	1 (2.8)	0

^AFall collections occurred in September, except for 2008 when collection occurred in November and 2009 when collections also occurred in November and December.

^BBold entries reflect annual dominant or codominant HA subtypes.

^CSpring collections occurred in March every year except for 2015 when collections spanned the time period March and April.

Table 2.

Total number of IAVs of each NA subtype isolated from samples from teal species collected in the spring and fall along the Gulf Coasts of Texas and Louisiana

NA subtypes
N7	N8	N9	Mixed/unknown
9 (1.4)	240 (36.9)	14 (2.2)	26 (4.0)
3 (1.5–77.8)	10 (14.3–61.5)	7 (1.2–7.8)	6 (1.6–7.7)
132 (49.6)	3 (1.1)	14 (5.3)	2 (0.8)
5 (18.8–77.8)	1 (2.9)	5 (1.0–12.5)	1 (12.5)

Seasonal differences in subtype diversity were also observed. In the fall, viruses of the H3 and H4 subtype were the most common, representing 26.9% and 63.2% of all viruses recovered in this study, respectively (Table 1). Interestingly, H4 viruses were the predominant virus in every fall season of this study except in November 2008 when early migrating teal were not sampled and only one H6N6 virus was recovered (Supplemental Tables S2, S3). This is in contrast to the spring, when H7 and H10 viruses made up 58.6% and 27.1% of total viruses recovered, respectively; H7 viruses were predominant from 2012–15, but in 2016 and 2017, H10 was the most common subtype identified (Supplemental Table S2). Viruses of H2, H6, H9, and H12 HA subtypes were identified only in fall collections, whereas H8 viruses were only recovered in the spring (Table 1; Fig. 2). In the fall, N6 and N8 subtypes were most common, accounting for 48.0% (n = 300) and 38.6% (n = 241) of the viruses detected during this season, respectively; combined, they represented between 75.0%–96.6% of all NA subtypes recovered in any given autumn (Table 2). In the spring, viruses of the N7 (n = 132; 49.6%) and N3 (n = 73; 27.4%) NA subtypes were most common overall (Table 2); however, viruses of the N1 NA subtype were the most common in the spring of 2012. Although the N4 subtype was only isolated from samples collected in the spring, the N6 subtype was only detected in fall collections; it was the dominant NA subtype in all years except 2009 and 2010 when the N8 subtype predominated (Supplemental Table S3).

DISCUSSION

Data from this study support the premise that IAV prevalence and subtype diversity in wild birds of the order Anseriformes may vary by season, even in the same location. A higher IAV prevalence was estimated for both teal species sampled at similar locations in the fall versus spring, which is likely a function of the congregation of immunologically naïve hatch year ducks prior to and during southward fall migration. Thus, IAV prevalence in teal sampled along the U.S. Gulf Coast followed a seasonal pattern in prevalence consistent with previous reports (12,17). The highest fall IAV prevalence in this study (September 2011, 23.9%) may partially be a function of environmental factors. More than 70% of the state of Texas was under exceptional drought conditions, including all areas in this study, from June 21, 2011 to October 18, 2011 (29); in all other years of surveillance, such extreme drought was not recorded. In 2011, it is possible that the lack of available open water led to the congregation of large numbers of ducks in smaller areas, which amplified the overall prevalence.

The prevalence of IAV in late fall and early winter months in 2008 and 2009 (November 2008, 0.4%; November 2009, 1.5%; and December 2009, 2.6%) were the lowest estimates for late-season samples and comparable to rates previously reported for waterfowl sampled during these months and in this area (3,4,27). Low prevalence has also been previously described for BWTE wintering in Guatemala during this same period (6). Lower incidence of IAV in teal during this time of year could be related to the development of population immunity, as birds infected with IAV in autumn and winter may have homo- and heterosubtypic immune responses that carry over into spring.

Decreasing trends in prevalence have been previously described from BWTE wintering in Guatemala during this same period (6). Although the predominance of H3 and H4 IAV has been well described, especially in waterfowl sampled during the late summer and early fall (7,12,21,22), the consistent predominance of H4 viruses in fall-sampled ducks during every year of this study is interesting. This is not the normal pattern described for waterfowl on staging areas where the predominance of H3 or H4 viruses varies annually (12,30). It has been suggested that this cyclic pattern is related to population immunity, but to date it has not been possible to measure such immunity. As teal migrate south in late summer–early fall, they are likely in contact and infected with both H3 and H4 IAV subtypes and would likely have some existing homo- or heterosubtypic immunity to both. As noted experimentally (13), heterosubtypic immunity can confer protection to mallards sequentially challenged with heterologous Has, and it is possible that patterns toward the dominance of H4 viruses as seen at these southern sites is influenced by a population’s immunity. However, an annual pattern described with H13 and H16 viruses in gulls may deserve attention (29). These viruses are primarily associated with fledging gulls on breeding grounds, and it has been reported that peak H13 infections precede H16 infection annually. This same short-term pattern may exist with H3 and H4 viruses in waterfowl, and this possibility should be explored. It was not surprising that the N6 and N8 NA subtypes were most common during the fall, as these subtypes are frequently associated with H4 and H3 subtypes (30).

The springtime circulation of H7 and H10 viruses has been previously documented in migrating BWTE from 2012 to 2013 at our study site (22). With the additional data from 2014 to 2017, this trend has been consistently demonstrated for 6 consecutive years. Although not consistent between years, H7 and H10 viruses also are commonly isolated from Charadriiformes (shorebirds) at Delaware Bay during May (11,12). It is unknown why these consistent seasonal trends with H7 and H10 viruses in teal occur, and it is possible that waning heterosubtypic immunity to H3 and H4 obtained during the fall provides an opportunity for these seasonal infections. Regardless of the mechanisms driving these patterns, sampling teal during spring migration provides a reliable source for representative North American H7 viruses. Viruses of the H7 subtype have caused outbreaks in poultry in both Mexico and the United States, and previous analyses have indicated a shared ancestry of H7 IAVs in poultry with North American waterfowl (14). As such, continued surveillance of these springtime migrating teal is important for monitoring the circulation of viruses with outbreak potential, especially as related to the movement and migration of such viruses with their hosts.

Seasonal dynamics in both prevalence and subtype diversity are influenced by indirect and direct factors at the host, pathogen, and environmental levels. Species susceptibility, density, age, immune status, and previous IAV exposure histories probably play a combined role in observed IAV prevalence and subtype diversity trends. Ongoing annual surveillance efforts will continue to inform our understanding of IAV prevalence and subtype diversity in these long-distant migrants, as well as provide a better understanding of the potential effects of population immunity related to these unique seasonal patterns.

Extended Data

Table 1.

Extended.

HA subtypes
LPA H7	H8	H9	H10	H11	H12	H14	Mixed/unknown
5 (0.8)	0	3 (0.5)	10 (1.5)	9 (1.4)	2 (0.3)	1 (0.2)	7(1.1)
2 (0.6–4.1)	0	3 (1.0–3.6)	4 (1.6–14.6)	6 (1.5–5.1)	2 (1.0–1.6)	1 (1–0)	3 (2.1–2.6)
156 (58.6)	1 (0.4)	0	72 (27.1)	7 (2.6)	0	3 (1.1)	0
6 (17.6–83.3)	1 (1.6)	0	6 (5.6–73.5)	4 (1.0–8.8)	0	2 (2.8–12.5)	0

Table 2.

Extended.

	HA subtypes
Seasons and parameters	H1	H2	H3	H4	LPA H5	H6
FallB, 2007–17  No. of IAV isolates (% of all isolates)	12 (1.8)	2 (0.3)	175 (26.9)	411 (63.2) C	4 (0.6)	9 (1.4)
No. of years detected (range as % of contribution to all subtypes within years detected)	6 (0.6–6.3)	2 (1.2–3.6)	10 (7.1–44.6)	10 (47.7–78.6)	2 (2.0–7.9)	6 (1.0–100.0)
SpringD, 2012–17  No. of IAV isolates (% of all isolates)	3 (1.1)	0	22 (8.3)	1 (0.4)	1 (0.4)	0
No. years detected (range as % of contribution to all subtypes within years detected)	1 (2.9)	0	2 (12.5–19.6)	1 (2.8)	1 (1–6)	0

^AAll H5 and H7 subtypes were confirmed to be LP IAV.

^BFall collections occurred in September, except for 2008 when collection occurred in November and 2009 when collections also occurred in November and December.

^CBold entries reflect annual dominant or codominant HA subtypes.

^DSpring collections occurred in March every year except for 2015 when collections spanned the time period March and April.

Abbreviations:

AGWT

American green-winged teal

BWTE

blue-winged teal

COP

cloacal–oropharyngeal

Co.

county

HA

hemagglutinin

IAV

influenza A virus

LP

low pathogenicity

NA

neuraminidase