Zero Prevalence of Influenza A Virus in Two Raptor Species by Standard Screening

Abstract

Disease can have severe impact on animal populations, especially in rare species. Baseline data for atypical host species are missing for a range of infectious diseases, although such hosts are potentially more affected than the normal vectors and reservoir species. If highly pathogenic avian influenza strikes rare birds of prey, this may have crucial impact on the predator species itself, but also on the food web in which it interacts. Here we present the first large-scale screening of raptors that regularly consume birds belonging to the natural reservoir of influenza A viruses. Influenza A virus prevalence was studied in two rare raptors, the white-tailed sea eagle (Haliaeetus albicilla) and the peregrine falcon (Falco peregrinus). Nestlings were screened for active (181 white-tailed sea eagles and 168 peregrine falcons) and past (123 white-tailed sea eagles and 6 peregrine falcons) infection in 2006–2007, and an additional 20 succumbed adult white-tailed sea eagles were sampled in 2003–2006. Neither high- nor low-pathogenic influenza infections were found in our sample, but this does not rule out that the former may have major impact on rare raptors and their food webs.

Introduction

D isease often influences populations, and may be critical for those already living on the limit of sustainability. Emerging diseases may have especially drastic consequences for wildlife, due to lack of immunity against novel infections (Cleveland et al. 2002). This was exemplified in 2005, when an outbreak of highly pathogenic avian influenza (HPAI) occurred in Qinghai Lake, China, where more than 6000 waterfowl died (Chen et al. 2006). This epizootic was caused by an HPAI-type H5N1, which since 1996 had been circulating among poultry in Southeast Asia (Olsen et al. 2006, Webster and Govorkova 2006). The Qinghai incident raised large concerns. First, it is one of only few examples where an HPAI virus has caused an outbreak outside domestic poultry (Gaidet et al. 2008). Second, it dramatically affected the range-restricted bar-headed goose (Anser indicus), with an estimated 10% reduction of the world population (Chen et al. 2006).

Not only waterfowl have been affected by this virus. During the subsequent spread of HPAI H5N1 in 2005 and onward in Asia, Europe, and Africa, there have been reports of raptors deceased in HPAI H5N1 infection (species listed at www.nwhc.usgs.gov). Although not commonplace, the occurrence of raptors succumbing to this disease is alarming, since these birds are top-consumers in their ecosystems and their populations are often small. Moreover, many raptor species worldwide have decreased in numbers over the last 100 years (e.g., Biljeveld 1974), and emerging diseases may exacerbate such negative trends. Consequences may be far-reaching not only for raptor species directly, but also for trophic webs to which they belong; direct predation on prey species will decrease, but loss of keystone predators can also alter competitive interactions between prey species released from predation (Paine 1969).

The white-tailed sea eagle (Haliaeetus albicilla) and the peregrine falcon (Falco peregrinus) are rare avian top-predators. Both almost went extinct in the Baltic Sea region in the 20th century because of human persecution and the use of organochlorines and mercury. A combination of changed use of chemicals, habitat preservation, management programs, and careful monitoring in several countries successfully helped both species to recover regionally (Helander 2003, Lindberg 2008). However, population sizes are still relatively small (Tjernberg and Svensson 2007), making them sensitive to increased mortality. Among European raptors these two species are probably the ones most likely to encounter influenza A viruses, as they frequently feed on birds that belong to the main reservoir of low-pathogenic avian influenza (LPAI) viruses and that are also susceptible to HPAI H5N1 virus infections (i.e., waterfowl, gulls, and waders) (Helander 1983, Lindberg 1983, Webster et al. 1992). The risk of encountering infected prey is increased by raptor behavior; they are selective hunters, often preying on crippled, sick, or otherwise handicapped individuals. However, to date there are no data about the prevalence of influenza A viruses in these species, nor in raptors in general.

Given (1) the general vulnerability of top-predators, (2) their importance as keystone species in food webs, (3) the regional status of the peregrine falcon and the white-tailed sea eagle, (4) the lack of LPAI prevalence data in raptors, and (5) the current circulation of a potentially devastating HPAI virus, we set out to collect baseline data on the occurrence of influenza A viruses in these potential hosts.

Materials and Methods

Study sites and sampling

Samples were collected as part of national Swedish monitoring programs in 2006 and 2007 (6 May–30 June for white-tailed sea eagle, and 1 June–17 July for peregrine falcon). In both programs we visited nests to band and sample nestlings. Four- to 8-week-old eagle nestlings (n = 181) were sampled from 162 nests over most of the species' Swedish range (55°30′ N–68°20′ N)—that is, the Baltic Sea coastline (n₂₀₀₆ =66; n₂₀₀₇ = 58), lakes in southern and central Sweden (n₂₀₀₆ =14; n₂₀₀₇ = 12), and lakes in the northern boreal forest zone of Lapland (n₂₀₀₆ = 27; n₂₀₀₇ = 4) (cf. Table 1). Likewise, we sampled 2- to 3-week-old peregrine falcon nestlings (n = 168) from 70 nests situated in southwestern Sweden (56°00′ N–59°00′ N; n₂₀₀₆ = 55; n₂₀₀₇ = 62), central Sweden (60°50′ N–61°30′ N; n₂₀₀₆ = 11), and northern Sweden (66°00′ N–68°00′ N; n₂₀₀₆ = 28; n₂₀₀₇ = 12) (cf. Table 1).

Table 1.

Numbers of Peregrine Falcon and White-Tailed Sea Eagle Nestlings Sampled for Influenza A Infection in 2006 and 2007

Species	Sex	2006	2007
Peregrine falcon	Female	36	34 (2)
	Male	24	35 (4)
	Not sexed	34	5
White-tailed sea eagle	Female	44 (26)	27 (27)
	Male	44 (22)	29 (24)
	Not sexed	19 (10)	18 (14)

Numbers of birds for which serology was done are given in parentheses. All were uninfected.

Using a sterile cotton swab, a cloacal sample was taken from all nestlings (n = 349; cf. Table 1), and a similar swab was used to sample the oropharyngeal cavity of 42 of the peregrine nestlings. Swabs were immediately put in virus transport medium consisting of Hank's balanced salt solution supplemented with antibiotics and antifungal (see Wallensten et al. 2007). In addition, we sampled blood from the brachial vein of 123 eagle and 6 peregrine nestlings to check for antibodies from previous influenza A virus infection (Table 1). Samples were stored at +4°C in the field (which does not affect virus detection by real-time reverse transcriptase–polymerase chain reaction [RRT-PCR]; see Munster et al. 2009) and transferred within 4 days for storage at −70°C (swabs) or −20°C (plasma).

We also collected cloacal samples from 20 adult white-tailed sea eagle corpses submitted to the Swedish Museum of Natural History, Stockholm, for necropsy. These birds were fresh of near fresh when collected in 2003–2006 (January–December), and had been stored at −20°C upon arrival at the museum.

Virus detection by RRT-PCR and serology

All cloacal and oropharyngeal samples were analyzed for influenza A virus using a one-step RRT-PCR (Spackman et al. 2002), with minor adjustments, detecting a conserved part of the influenza A matrix gene. In brief, we extracted RNA from 100 μL of original samples using the MagAttract^® Virus Mini M48 kit (Qiagen, Solna, Sweden) with the BioRobot^® M48 (Qiagen) set to obtain 75 μL of elution volume. A LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany) performed the thermo-cycling with the following settings: reverse transcription for 30 min at 50°C, activation of HotStart Taq polymerase for 15 min at 95°C, and 45 cycles of 5 s at 95°C and 20 s at 60°C. We used a threshold cut-off value of <40 cycles.

Plasma samples were tested for antibodies targeted for influenza A virus nucleoprotein using a commercial ELISA kit (Avian Influenza A Blocking ELISA, Pourquier, Montpellier, France) known to be sensitive in wild birds (Kalthoff et al. 2008).

Results and Discussion

Despite sampling in several years over large parts of the species' breeding distributions in Sweden, we did not detect any infected nestlings (Table 1) or adults. Likewise, the serological analyses did not yield any antibody-positive birds (Table 1). Both results indicate that (1) the sampled nestlings had not been infected between hatching and sampling, and (2) the adult females in the breeding pairs had not been infected in the near past. Indeed, antibodies from maternal origin remain detectable in the serum of young birds at least for 2 weeks after hatching (Grindstaff et al. 2003, Stout et al. 2005).

The absence of HPAI H5N1 cases was not entirely surprising. The outbreak of this virus in Europe in 2006 was quite restricted geographically, small in terms of documented cases, and mainly noted during the winter period (Webster and Govorkova 2006) before our sampling. Moreover, raptors fatally infected by this virus in 2006 likely died before the breeding season. LPAI, on the other hand, is present year around in waterfowl in the region, although with a pronounced seasonality in prevalence with shedding peaking in autumn and early winter, and being lower in spring. For example, Wallensten et al. (2007) reported means for influenza A prevalence in mallards Anas platyrhynchos in SE Sweden to be 15% (maximum value: 25.7%) in autumn, and 4.0% (maximum value: 9.5%) in spring (see also Krauss et al. 2007 and Munster et al. 2007). In other words, the fact that we did not detect LPAI in the sample was more surprising.

Previous studies show that the HPAI H5N1 virus is able to infect a wide range of avian species and that symptoms differ among infected hosts. For example, infection experiments indicate high mortality in geese and swans (Brown et al. 2008, Kalthoff et al. 2008) and that diving ducks of the genus Aythya seem to be more adversely affected than dabbling ducks (genus Anas) (Keawcharoen et al. 2008). Death caused by HPAI infection seems to be frequent also in atypical hosts like raptors. For example, a peregrine falcon that died in the United Arab Emirates in 2000 was positive for HPAI H7N3 (Manvell et al. 2000), and during the HPAI H5N1 epizootic, succumbed wild peregrines infected with the virus were found in Hong Kong, Germany, and Denmark (Webster et al. 2005, Bragstad et al. 2007, Lierz et al. 2007). Worth noting is also the experimental study by Lierz et al. (2007), who infected gyr-saker hybrid falcons (F. rusticolus × F. cherrug; i.e., closely related to the peregrine) with HPAI H5N1. Falcons initially given an H5N2 vaccine all survived the inoculation, in contrast to the total death observed in the nonvaccinated birds. Eagle species have also been infected by HPAI H5N1 (e.g., Hodgon's hawk eagle [Spizaetus nipalensis]; van Borm et al. 2005), but to date there are no reported cases of infected white-tailed sea eagles.

Although we got a zero-prevalence result for both raptor species, we nevertheless acknowledge that (1) the sampled birds did not excrete any virus, but influenza A infection may theoretically still be possible since viral excretion by infected birds may be intermittent (Keawcharoen et al. 2008), (2) as maternal antibodies usually remain detectable for only 3 weeks after hatching (Grindstaff et al. 2003), eagle nestlings sampled by us were possibly too old to still carry such antibodies, (3) the sampling for an active influenza A infection included only very few fledged birds, which possibly may face a higher risk of encountering the virus than nestlings, and we therefore cannot draw any final conclusion about the populations as a whole, and (4) host species should preferably be sampled in thousands to get estimates reflecting the true prevalence. For raptors, and especially for rare ones, the latter is not possible. Still, with the present sample size of 349, prevalence is at the most 1.1% assuming infinite populations and following binomial law. In conclusion, it seems unlikely, but we cannot totally rule out the possibility that influenza A viruses may have affected the peregrine falcon and the white-tailed sea eagle populations studied by us.

To our knowledge there is no previous extensive sampling of LPAI infection in raptors, making our study unique. Concerning LPAI, the present study concurs with previous influenza A surveillance studies in that other hosts than aquatic birds only rarely are found infected (Olsen et al. 2006, Munster et al. 2007). If our zero-prevalence result is due to raptors not encountering the virus in nature or to the inability of the virus to replicate in epithelial cells in these birds, remains to be elucidated, as are the possible sublethal effects of LPAI infection in wildfowl. The latter issue has received increased interest in recent years (e.g., van Gils et al. 2007, Latorre-Margalef et al. 2009), but there is still no consensus. This is partly due to complex patterns; differences surely exist among species, but also among conspecifics (cf. Mutinelli et al. 2003, Kalthoff et al. 2008). In HPAI virus infections, consequences to individuals are often more clearly severe, and although relatively few waterfowl died from HPAI H5N1 in Europe in the winter of 2006 (Olsen et al. 2006), some raptors were indeed among the victims. Important to note is that consequences for top-predators will certainly be worse if future HPAI outbreaks affect larger proportions of prey populations. This could have far-reaching consequences for biodiversity per se, at the same time triggering cascading effects at other trophic levels (Paine 1969).

Footnotes

Acknowledgments

This study was funded by grants from the Swedish Research Council (2007-2774), the Swedish Research Council FORMAS (2006-297), the Swedish Environmental Protection Agency (V-162-05 and 212-0817), the Swedish Society for Nature Conservation/SNF, “Fondation pour la Recherche Médicale,” Göran Gustafsson Foundation, and Kalmar University. We would like to thank all persons involved in the field work and in analyzing the samples in the laboratory.

Disclosure Statement

No competing financial interests exist.

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