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Published in final edited form as: J Wildl Dis. 2018 May 9;54(4):708–715. doi: 10.7589/2017-10-252

NEUTRALIZING ANTIBODIES TO TYPE A INFLUENZA VIRUSES IN SHOREBIRDS AT DELAWARE BAY, NEW JERSEY, USA

Charlie S Bahnson 1,3, Rebecca L Poulson 1, Scott Krauss 2, Robert G Webster 2, David E Stallknecht 1
PMCID: PMC9254111  NIHMSID: NIHMS1817208  PMID: 29741997

Abstract

Influenza A virus (IAV) infections in shorebirds at Delaware Bay, New Jersey, US, have historically included avian hemagglutinin (HA) subtypes H1–13 and H16. In a given year, infections are characterized by a limited number of HA and neuraminidase subtypes and a dominant HA subtype that often represents >50% of all isolates. Predominant HA subtypes shift between consecutive years. In addition, infection prevalence is consistently higher in Ruddy Turnstones (RUTU; Arenaria interpres morinella) compared to Red Knots (REKN; Calidris canutus rufa), despite comparable rates of exposure. To investigate a potential immunological basis for this phenomenon, a virus microneutralization assay was used to detect subtype-specific, neutralizing antibodies to H1–H12 in sera collected from RUTUs from 2012–16 and REKNs in 2012, 2013, and 2016. Neutralizing antibodies to one or more subtypes were detected in 36% (222/611) of RUTUs. Prevalence of antibodies to subtypes H6 and H11 remained high throughout the study, and these virus subtypes were isolated every year, suggesting a continual source of exposure. Antibody prevalence was intermediate for most IAV subtypes that were isolated in 2–3 of 5 yr (H1, H3, H5, H9, H10, and H12) but was low for H7 viruses, despite the isolation of this virus subtype in 3 of 5 yr. This suggests a reduced antigenicity of H7 IAVs compared to other subtypes. Antibody prevalence was low for H4 virus that was isolated once, and H2 and H8 viruses that were never isolated. Neutralizing antibodies were detected in 66% (169/257) of REKNs and subtype-specific antibody prevalences were higher in REKNs than RUTUs with few exceptions. The results suggest that population immunity influences which species is infected at Delaware Bay, indicate that IAV dynamics are subtype-dependent, and demonstrate the utility of the microneutralization assay as a supportive tool for field research.

Keywords: Antibody, Delaware Bay, influenza A virus, microneutralization, Red knot, Ruddy Turnstone

INTRODUCTION

The natural reservoirs of influenza A virus (IAV) are birds of the orders Anseriformes (ducks and geese) and Charadriiformes (gulls and shorebirds; Webster et al. 1992; Krauss et al. 2004; Olsen et al. 2006). Although IAV is routinely isolated from ducks, geese, and gull populations throughout the world, this virus is consistently isolated from shorebirds only at Delaware Bay (DB) in the northeastern US during spring migration when shorebirds stop to refuel on eggs of spawning horseshoe crabs (Hanson et al. 2008; Krauss et al. 2010).

Ruddy Turnstones (RUTUs; Arenaria interpres morinella), Red Knots (REKNs; Calidris canutus rufa), Semipalmated Sandpipers (Calidris pusilla), and Sanderlings (Calidris alba) are the predominant species of shorebirds using beach habitats at DB (Clark et al. 1993). Of these, RUTUs appear to be most important in this local IAV system. Infection rates in RUTUs average 12–14%, the highest of any species at DB (Krauss et al. 2004; Maxted et al. 2012). In contrast, IAV infection rates in any of the other shorebird species feeding alongside RUTUs at DB is less than 2% (Hanson et al. 2008; Maxted et al. 2012). Although IAV hemagglutinin (HA) subtypes H1–H13 and H16 have been variously isolated from RUTUs at DB, annual IAV infections are typically dominated by a single HA subtype that reoccurs in periodic, erratic cycles (Krauss et al. 2004; Stallknecht et al. 2012). Subtype diversity in a given year does not differ between RUTUs and REKNs and, in most years, all of the IAV subtype diversity present at DB can be detected in the RUTU population (Stallknecht et al. 2012).

This shifting infection pattern might have an immunologic basis. Natural and experimental studies in Mallards (Anas platyrhynchos) have suggested that homo- and heterosubtypic immunity induced through infection with IAV might drive population infection dynamics (Fereidouni et al. 2010; Latorre-Margalef et al. 2013; Segovia et al. 2017). The immune response to single or multiple IAV infections is not as well understood in shorebirds. Ruddy turnstones and REKNs are relatively long-lived and individuals remain at wintering grounds for their first year (Nettleship 2000; Baker et al. 2013). As a result, the population of shorebirds at DB consists of older individuals that are likely to have been previously exposed to one or more IAV subtypes.

Previous serologic investigations at DB reported that the prevalences of IAV antibodies in RUTUs and REKNs are comparable. Antibodies to the IAV nucleoprotein (NP) were detected in 55–65% and 54–86% of RUTUs and REKNs at DB, respectively (Brown et al. 2010; Maxted et al. 2012; Stallknecht et al. 2012). Exposure and seroconversion do not appear to occur at the same time for both species, however. By using date and body weight as a proxy for time spent at DB, Maxted et al. (2012) determined that IAV antibody prevalence in RUTUs increased from less than 40% at arrival to over 95% at departure, implying that over half the population seroconverts while at DB. In contrast, the antibody prevalence in REKNs was 82% at arrival and slowly declined throughout the remainder of the season, a period that lasts from mid-May to early June. This provides valuable insight into species-specific IAV exposures within the DB system, but because NP is conserved between IAV subtypes, these antibody patterns are of limited use when considering virus-host interactions at a subtype-specific level. In this study, we sought to broaden our understanding of IAV infection dynamics at DB by employing a microneutralization (MN) assay to detect HA-specific neutralizing antibodies.

We hypothesized that subtype-specific antibody patterns, as determined by MN, could be used to explain annually shifting IAV infection patterns. Our objectives were to: 1) compare IAV antibody patterns in RUTUs to REKNs; 2) determine if annual subtype-specific IAV antibody patterns in RUTUs reflect the observed IAV subtype diversity at DB; and 3) demonstrate the utility of the MN assay as a supportive tool for field research.

MATERIALS AND METHODS

Sample collection

Fieldwork was conducted during 16–29 May 2012, 15–29 May 2013, 14–27 May 2014, 12–28 May 2015, and 16–22 May 2016, at DB, New Jersey under federal scientific collection permit number MB779238 and New Jersey scientific collection permit numbers 2012029, 2013037, 2014057, 2015003, and 2016006. Ruddy turnstones and REKNs were captured with cannon nets as part of a long-term population study. Blood samples were collected by jugular venipuncture at a total volume less than or equal to 1% of the bird’s body mass. Blood samples were not collected from REKNs in 2014 and 2015. Samples were kept on ice until they could be centrifuged the same day of collection. The serum fraction was aliquoted and stored at −20 C until testing by blocking enzyme-linked immunosorbent assay (bELISA), and then stored at −20 C until testing by MN. Research was approved by the University of Georgia Animal Care and Use Committee AUP numbers A2010 06-101, A2013 05-021, and A2016 05-020. The number of serum samples tested was: 128 RUTUs and 102 REKNs in 2012; 116 RUTUs and 104 REKNs in 2013; 115 RUTUs in 2014; 112 RUTUs in 2015; and 140 RUTUs and 49 REKNs in 2016.

bELISA

All sera were tested by bELISA (IDEXX Laboratories, Westbrook, Maine, USA) for antibodies to the NP (Brown et al. 2010). Sera were considered positive if the serum-sample-to-negative-control absorbance value was less than 0.50. The negative control was provided by the manufacturer and consisted of dilute chicken (Gallus gallus domesticus) serum that was not reactive to IAV.

Microneutralization

Serum samples were tested for antibodies against H1–H12 by virus MN (Wong et al. 2016), with the exception that serum and antigen were allowed to incubate at room temperature for 1.5 h rather than 2 h. Viruses used as antigens included A/mallard/NJ/AI12-4823/2012 (H1N1), A/mallard/MN/AI08-2755/2008 (H2N3), A/mallard/MN/AI10-2593/2010 (H3N8), A/mallard/MN/AI10-3208/2010 (H4N6), A/mallard/MN/AI11-3933/2011 (H5N1), A/mallard/MN/Sg-00796/2008 (H6N1), A/mallard/MN/AI08-3770/2009 (H7N9), A/mallard/MN/SG-01048/2008 (H8N4), A/RUTU/DE/AI11-809/2011 (H9N2), A/mallard/MN/SG-00999/2008 (H10N7), A/mallard/MN/SG-00930/2008 (H11N9), and A/mallard/MN/AI07-3285/2007 (H12N5). Subtypes H13 and H16 were not included in testing because adequate viral titers could not be achieved in Madin-Darby canine kidney cells (American Type Culture Collection, Manassas, Virginia, USA) through conventional methods, and neither has been a dominant IAV subtype in RUTUs in previous years (Stallknecht et al. 2012).

Virus isolation

Virus isolation data were provided by St. Jude Children’s Research Hospital (SJCRH) and the University of Georgia (UGA). Samples were collected at DB in May 2012–16 and were tested for IAVs by virus isolation (Stallknecht et al. 2012). The HA of each isolated IAV was determined by hemagglutination inhibition (Hanson et al. 2008). Samples tested by SJCRH consisted of shorebird fecal swabs collected on the beaches of DB and totaled 610 in 2012, 600 in 2013, 600 in 2014, 600 in 2015, and 672 in 2016. Samples tested by UGA consisted of oropharyngeal and cloacal swabs collected from RUTUs at the time of capture, as well as RUTU fecal swabs collected from the beaches of DB. The number of samples collected and tested each year by UGA was 1,087 in 2012, 1,002 in 2013, 823 in 2014, 978 in 2015, and 697 in 2016. The prevalence of IAVs was calculated from the UGA dataset. The proportion of H1–H12 viral isolates was calculated from the combined datasets of SJCRH and UGA.

Statistical analysis

The mean prevalences of antibodies and IAVs were calculated for 2012–16 and 2012, 2013, and 2016 by dividing the total number of positive samples from that time period by the number of samples tested. Ninety-five percent confidence intervals for prevalences were calculated using the Wilson method. A Fisher’s exact test was used to compare the prevalences of neutralizing antibodies to one or more HA subtypes, neutralizing antibodies to two or more HA subtypes, and antibodies to NP between RUTUs and REKNs. Mean, subtype-specific prevalences were organized into three class intervals designated “highest,” “intermediate,” and “lowest” based on the range of prevalences recorded for each species. Comparisons were made between species for individual years (2012, 2013, 2016) when sera were available for both species as well as the mean prevalence for these 3 yr. A Fisher’s exact test was also used to compare the annual change in prevalence of neutralizing antibodies for each HA subtype in RUTUs. The difference between antibody prevalence was considered significant if P<0.05. The proportion of viral isolates in a given year was calculated by dividing the number of IAVs of each HA isolated by the total number of H1–H12 IAVs isolated that year. Calculations were performed in STATA 13.1 (StataCorp LP, College Station, Texas, USA).

RESULTS

The prevalence of IAVs in RUTU samples at DB from 2012–16 was 14% (642/4,587; 95% confidence interval [CI]: 13–15%) and ranged from 9% (74/823; 95% CI: 7–11%) in 2014 to 20% (139/697; 95% CI: 17–23%) in 2016 (Table 1). The mean prevalence of antibodies to the NP, as determined by bELISA, in 2012, 2013, and 2016, for RUTUs and REKNs was 62% (238/383; 95% CI: 57–67%) and 69% (172/251; 95% CI: 63–74%), respectively, and was not statistically different between species (P=0.100). Neutralizing antibodies to one or more subtype of IAV were detected by MN in 36% (222/611; 95% CI: 33–40%) of 611 RUTUs sampled from 2012–16 and in 66% (169/257; 95% CI: 60–72%) of 255 REKNs sampled in 2012, 2013, and 2016. For the same years, neutralizing antibodies to two or more subtypes were detected by MN in 18% (108/611; 95% CI: 15–21%) of RUTUs and 49% (126/257; 95% CI: 43–55%) of REKNs. The mean MN prevalence across 2012, 2013, and 2016 was significantly different by species for both ≥1 and ≥2 subtypes (P<0.001).

Table 1.

The prevalence of hemagglutinin subtype 1–12 influenza A viruses isolated from Ruddy Turnstones (RUTU; Arenaria interpres morinella) and RUTU feces, and percentage of RUTU and Red Knot (REKN; Calidris canutus rufa) sera that were antibody-positive by blocking enzyme-linked immunosorbent assay (bELISA) or microneutralization (MN) positive for neutralizing antibodies to one or more influenza A virus hemagglutinin subtypes at Delaware Bay, New Jersey, USA, from 2012–16.a

bELISA
 MN
Virus isolation
 RUTU
 REKN
 RUTU
 REKN
Year Percent positive (n) P valueb Percent positive (n) P valueb
2012 10 (1,087) 69 (128) 65 (102) 0.517 36 (128) 61 (102) <0.001
2013 15 (1,002) 60 (115) 73 (104) 0.041 31 (116) 66 (106) <0.001
2014    9 (823) 57 (115) — — 28 (115) — —
2015  17 (978) 68 (111) — — 36 (112) — —
2016  20 (697) 58 (140) 67 (45) 0.294 49 (140)   78 (49) <0.001
Subtotalc  14 (2,786) 62 (383) 69 (251) 0.100 39 (384) 66 (257) <0.001
Totald  14 (4,587) 62 (609) — — 36 (611) — —
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a

— = not applicable.

b

P value for difference in percent positive between each species for given assay and year.

c

Mean percent positive from 2012, 2013, and 2016.

d

Mean percent positive for 2012–16.

Over the 5 yr surveyed, HA-specific antibodies to H1–12 were detected in RUTUs with H6, H9, and H11 detected in the highest prevalence (12, 11, 10%, respectively), followed by H1, H3, H5, H10, and H12 at an intermediate prevalence (6–8%), and H2, H4, H7, and H8 at the lowest prevalence (<2%). Prevalence varied by year and subtype with the highest single-year prevalence of seropositive RUTUs recorded in 2016 for H1 (19%, Fig. 1), whereas no antibodies were detected for H4 or H8 in multiple years.

Figure 1.

Figure 1.

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The prevalence of antibodies to influenza A viruses (IAVs) in Ruddy Turnstones (RUTU; Arenaria interpres morinella, gray bars) and Red Knots (Calidris canutus rufa, white bars) as determined by microneutralization assay and distribution of hemagglutinin (HA) subtype IAVs isolated from RUTUs and shorebird fecal samples (asterisk) from 2012–16 in Delaware Bay, New Jersey, USA. The number adjacent to asterisks is the percentage of IAVs of a given HA subtype, calculated by divided the number of isolated viruses of a given HA subtype by the total number of IAVs isolated that year. Any number between 0% and 1% was rounded up to 1%. Subtypes marked with a dagger (†) were significantly different between species (P≤0.05) for that year. Subtypes marked with a double asterisk (**) were significantly higher in RUTUs compared to the previous year. Error bars represent 95% confidence limits.

The degree to which annual RUTU serologic results corresponded to IAV isolation data varied by IAV subtype (Fig. 1). Although they were never dominant subtypes, H6 and H11 IAVs were isolated every year surveyed. Antibodies to these subtypes, along with H9, were detected at the highest mean rates. Subtypes H1, H10, and H12 were each dominant or codominant IAVs in 2/5 yr of sampling. The annual prevalence of antibodies to these subtypes varied, but the mean prevalence over the study period was intermediate compared to other subtypes. Subtypes H3, H5, and H9 IAVs were each isolated in 2 of 5 yr but these were never dominant subtypes. However the mean antibody prevalence to these subtypes was also intermediate (H3 and H5) or high (H9) compared to other subtypes. The prevalence of antibodies to H7 fit isolation data the least well. Despite the repeated isolation of H7, which was also the dominant subtype in 2015, antibody levels remained low (<2%) throughout the study. One H4 IAV was isolated in 2014 and subtypes H2 and H8 were never isolated. Prevalence of antibodies to these subtypes was also low (mean <2%). A significant increase in neutralizing antibodies was detected for H1 from 2015 to 2016 (P=0.003) and corresponded with the detection of H1 IAVs in 2015. The number of neutralizing antibodies to H3 increased significantly from 2013 to 2014 (P=0.006) and viruses of this subtype were isolated in 2014. The prevalence of neutralizing antibodies to H10 increased significantly from 2015 to 2016 (P=0.005) and this was a dominant IAV subtype in 2016. A significant decrease in neutralizing antibodies was detected for H9 from 2012 to 2013 (P=0.018). Subtype H9 IAVs were detected in 2012, but not again until 2016. No other single-year change in antibody prevalence was found to be significant at a level of 5%.

In the 3 yr that REKNs were surveyed, HA-specific antibodies to H1–H12 were detected with H6, H9, H11, and H1 detected in the highest prevalence (36, 35, 32, and 28%, respectively). Antibodies to H5 H12, H2, and H10 were detected at an intermediate rate (23, 22, 15, and 15%, respectively). The prevalence of antibodies to the H7 was among the lowest (11%), as were the prevalences of H3, H4, and H8 (all less than 2%). The highest antibody prevalence recorded for a single year was H9 in 2016 (43%, Fig. 1).

Because serological data from 2014 and 2015 are unavailable for REKNs, pairing antibody prevalences in REKNs to virus isolation data is challenging. The subtype specific antibody patterns in REKNs mirrored that described for RUTUs with two exceptions. Although an intermediate prevalence of neutralizing antibodies to H3 was detected in RUTUs, only 1% of REKNs had antibodies to H3 despite this subtype being isolated in 2016. In contrast, the prevalence of antibodies to H2 was intermediate in REKNs but low in RUTUs. No H2 IAVs were ever isolated.

For the 3 yr when data were available for both species, the mean antibody prevalence was significantly different by species for every subtype with the exception of H4 (P=0.219). For individual years, antibody prevalence was significantly higher in REKNs for most subtypes and most years (Fig. 1). Exceptions include H3, which was significantly higher in RUTUs in 2016 but not different in 2012 or 2013 (P=0.104, P=0.626, respectively); H4, which was not significantly different in 2012 (P=0.262) and wasn’t detected in either species in 2013 and 2016; H5, which was not significantly different in 2016 (P=0.090); H8, which was not detected in either species in 2013 and not significantly different in 2016 (P=0.090); and H10, which was not significantly different in 2012 or 2016 (P=0.085, P=0.343, respectively).

DISCUSSION

Of the several shorebird species that feed alongside one another at DB, RUTUs are the only species where IAV is consistently isolated at high rates (Kawaoka et al. 1988; Hanson et al. 2008). Previous authors have suggested this might be attributed to subtle behavioral differences such as feeding strategies or roost-site selection that lead to higher rates of exposure (Hanson et al. 2008; Brown et al. 2010; Maxted et al. 2012). The comparatively high rate of neutralizing antibodies in REKNs versus RUTUs supports our hypothesis that population immunity might be an additional factor in determining which species is infected at DB.

The extent of immunity that develops as a result of infections at DB versus infections prior to arrival is unclear. Both species are long-distance migrants that could potentially be infected at diverse sites and at different times in North and South America. Unfortunately, the limited surveillance efforts in shorebirds and the few IAV detections reported from these species outside of DB provide an inadequate basis to determine where or how population immunity develops or persists in them (Kawaoka et al. 1988; Krauss et al. 2004; Hanson et al. 2008). Regardless, our data suggest that, as compared to RUTUs, REKNs at DB have antibody profiles that are more robust in neutralizing antibody subtype prevalence and diversity.

We expected to detect a prodigious population of subtype-specific, neutralizing IAV antibodies within the sampled RUTUs that would partially account for the shifting IAV infection patterns. Although it is unknown what antibody prevalence would be necessary to drive the shift in annual dominant virus subtype, our data do not seem to sufficiently account for this phenomenon. Nevertheless, they do reveal heretofore uncharacterized patterns that might be related to the combined effects of IAV exposure rates and antibody response. With regard to exposure as determined by the annual IAV isolation results, several patterns were evident: rarely detected viral isolates with low antibody prevalence (H2, H4, H8), occasionally detected viral subtypes with intermediate prevalence (H1, H3, H5, H9, H10, H12), and routinely detected viral isolates with a high antibody prevalence (H6, H11).

The source of the IAVs that contribute to annual infections at DB remains unknown. Some subtypes might be maintained in the population at low prevalence and occasionally emerge as a dominant subtype before receding to low, maintenance levels of infection. This is possible with the majority of subtypes, particularly those within the first two categories: rarely detected viral isolates with intermediate prevalence and occasionally detected viral subtypes with intermediate prevalence. By contrast, many have suggested migrating and resident ducks and gulls serve as a continual source of IAV infection (Hanson et al. 2008; Guinn et al. 2016; Maxted et al. 2016). This is plausible with subtypes that are represented nearly every year in low isolation prevalence and high antibody prevalence (i.e., H6 and H11). In fact, Guinn et al. (2016) detected the highest prevalence of antibodies to H6 and H11 in gulls at DB, although the directionality of viral transmission cannot be determined.

A final pattern observed was with neutralizing antibodies to H7. Viruses of this subtype were isolated at DB in 3 of the 5 yr and in 54% of viruses isolated in 2015. Despite this, the annual prevalence of neutralizing antibodies to H7 in RUTUs was never above 2%. In recent years, H7 subtype viruses have received attention within human health research because they appear to generate an antibody response that is either reduced or not detectable by conventional hemagglutination inhibition or MN assays, despite confirmed or suspected exposures of and infections in humans (Puzelli et al. 2005; Skowronski et al. 2007; Guo et al. 2014). Humanized mice injected with recombinant hemagglutinin from H7 viruses generated an antibody response that was similar by bELISA, but significantly lower as measured by hemagglutination inhibition and MN compared to mice injected with hemagglutinin from H1 and H3 viruses (Blanchfield et al. 2014). Infections with H7 IAVs in shorebirds at DB might also generate fewer neutralizing antibodies or antibodies that were less detectable by our MN assay as compared to other subtypes.

In a disease system involving long-lived hosts that are exposed to multiple IAVs, the potential exists for a manifestation of “original antigenic sin” (OAS) to be influencing antibody patterns. Under this framework, an imprinting occurs in a host upon first exposure to an IAV and subsequent exposures generate a humoral response more tailored to the original IAV than the challenge IAV (Fazekas de St. Groth and Webster 1966). Subtle evidence of OAS was recently observed in a Mallard challenge study (Latorre-Margalef et al. 2017). Currently, an effect of OAS on wild bird infection dynamics, including those at DB, is plausible, but its magnitude remains unknown.

Finally, our study demonstrates the importance to field research of assessing subtype-specific antibody patterns. As demonstrated here, the infection status of one species within an IAV system can reveal an incomplete story that is likely obscured by heterosubtypic immunity. For example, viral shedding was abrogated, reduced, or shortened in previously infected Mallards challenged with other subtypes of IAV, whereas antibodies to the challenge IAV were often still detected by MN (Segovia et al. 2017). Extrapolating this to shorebirds at DB validates MN and other serologic tools as a valuable component of IAV surveillance. In this study, it revealed unique patterns related to the potential contribution of specific species and nondominant viral subtypes to IAV dynamics that would not be captured with infection data alone.

Interpretation of serologic data comes with limitations and is particularly complex in relation to IAV in wild birds where multiple IAV subtypes annually infect numerous avian species. The extent of cross reactive HA antibodies related to repeated infections with different subtypes, the potential effects of antibodies to neuraminidase subtypes on test specificity, and the duration of the detectable immune response are not well established. In addition, many of these questions cannot be addressed in exhaustive challenge studies. In its current form, serologic testing cannot replace virus detection, but with technical refinement and improved interpretive guidelines, serologic data has the potential to provide an additional and valuable perspective in our efforts to unravel the epidemiology of IAV in wild bird populations.

ACKNOWLEDGMENTS

We thank Larry Niles, Amanda Dey, and the numerous volunteers for capturing the shorebirds in this study and for their ongoing conservation efforts at Delaware Bay. We thank Deborah Carter, Nicholas Davis-Fields, Alinde Fojtik, Laura Hollander, and Clara Kienzle for sample collection and processing. We thank Roy Berghaus for help with statistics. We thank personnel at St. Jude Children’s Research Hospital, Memphis, Tennessee for collection and sample processing. This project was funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract HHSN272201400006C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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