Migration is a common feature of birds living in seasonal environments, and movements range from very short altitudinal movements to trans-hemispherical flights of tens of thousands of kilometres [1], [2]. Bird migration potentially facilitates the spread of pathogens at multiple geographic scales, and could consequently influence disease dynamics [3]–[5]. This is particularly evident for populations of the same species separated during one part of the year but co-occurring in another (cf. “migratory connectivity” in [6]), which not only facilitates population mixing and the maintenance of genetic diversity [6]–[8], but is also likely of great importance for disease transmission [5]. Temporal and spatial variation in the dynamics of disease in relation to migration is a rather young area of research. At present, there are a few model systems in this field, of which influenza A virus dynamics in waterfowl is one. Influenza A viruses are negative-sense RNA viruses capable of infecting a broad range of hosts, most commonly avian, but also mammalian hosts, including man [9]. Dabbling ducks (Anas spp.), and in particular the mallard A. platyrhynchos, have been implicated as the most important reservoir and vector for low-pathogenic avian influenza (LPAI) viruses [9]. Most of the described virus subtypes have been recorded in wild mallards and virus prevalence appears to vary predictably by season and geography [9]–[12]. The virus infects epithelial cells of the gastrointestinal tract and the bursa, but does not appear to induce pathogenic lesions in those tissues [13]. However, studies indicate that influenza A virus infection in mallards could translate into an ecological cost, manifested in reduced body mass [14]. More considerable effects have been observed in wintering Bewick's swans Cygnus columbianus in the Netherlands, where infections with LPAI were associated with reduced foraging and significant delay in the timing of spring migration [15].

Understanding disease dynamics of LPAI viruses is important also for understanding the emergence and spread of virus strains capable of infecting domestic birds. Specific changes in the hemagglutinin (HA) molecule in H5 and H7 subtypes cause increased virulence in poultry, which can lead to outbreaks of highly pathogenic avian influenza (HPAI) [9], [16], [17]. Once started, such outbreaks can spread regionally in poultry [17], or, as in the case of the ongoing HPAI H5N1 epizootic, revert back into wild birds causing deaths in both domestic and wild bird populations [9], [18]. The capacity for wild waterfowl to disperse HPAI viruses through migration is currently a matter of debate. Experimental data clearly show that dabbling ducks are much less affected by HPAI infection than poultry [19], [20] and one study suggest that ducks previously infected with LPAI can subsequently carry HPAI infections asymptomatically [21]. Data from waterfowl migration (counts, band recoveries, transmitters, loggers etc.) and geographic information have been used to model the capacity of waterfowl to transmit HPAI virus through migration [3], [22]–[25]. Such data could also be used to identify areas of higher risk for HPAI and point out relevant sites for influenza A surveillance. However, crucial knowledge is lacking, for example how virus transmission is related to natal or breeding origin of the reservoir hosts. Patterns of LPAI dynamics indeed vary in time and space; prevalence in waterfowl shows distinctive autumn peaks at major stopover sites in Europe [11], and there is also a large-scale latitudinal gradient of generally higher prevalence rates at northern than at southern sites [10]. Local-scale dynamics of LPAI subtypes may also be involved, demonstrated by variations within- as well as between-year at specific sites [11], [26], [27]. One hypothesis to explain these patterns is that hosts from geographically different breeding areas carry different sets of LPAI subtypes and arrive at staging sites at different times in autumn. To date, a general limitation in bridging the ecology and epidemiology of disease in natural systems has been determining the origin of autumn-staging birds, of which most are immuno-naïve hatch-years on their first migration and therefore cannot be readily banded or sampled in their natal areas.

Banding of popular game species, such as dabbling ducks, results in relatively high recovery rates since a large share ends up in hunters' bags [28]. However, since most banding and hunting of migratory species takes place far from the breeding grounds, recoveries give scant information about natal origin, cf. [29]. These and other biases can be overcome by using intrinsic markers such as stable isotope measurements of body tissues such as feathers [30]. Knowledge about origin based on isotopes could then be used to relate, for instance, the prevalence of certain diseases. In this way, LPAI infection in Bewick's swans as well as avian malaria blood parasites in great reed warblers Acrocephalus arundinaceus were found to differ among wintering areas or habitat [31], [32]. One of the more commonly used isotope elements is hydrogen, based on the fact that the relative abundance of deuterium to protium (²H/¹H, measured as δ²H) in annual or growing season precipitation is related, at continental scales, largely to latitude and altitude [33]. The correlation between deuterium in feathers (δ²H_f) grown at specific sites and in long-term amount-weighted precipitation at the same sites (δ²H_p) is often strong [34], [35], allowing inferences about where the feather was grown. Feathers are metabolically inert following growth and so the δ²H_f value conveys information about origin. The recent development of Bayesian assignment approaches to isotope data [36] allows a probabilistic approach to assigning birds to isoscapes including the use of informed priors such as migration directions provided by band recoveries [37].

The aims of the present study were 1) to delineate natal origins of autumn-staging mallards in southern Sweden and 2) to evaluate whether natal origin is related to the variation of influenza A subtypes shed by mallards. We found that birds from different origins had temporal structure in their prevalence of influenza A subtypes. This may have consequences for the likelihood of pathogen transmission throughout the flyway.

The temporal structure in LPAI subtype diversity could have several explanations. Since we found a corresponding temporal effect in the natal origin of the mallards, we propose that migrants from less distant natal areas in the early autumn were infected by virus subtypes different from those found in mallards from more distant origins migrating through southern Sweden in late autumn. Data on influenza A virus infections are present from the staging site at Ottenby only, and not from the breeding grounds, which would be necessary to confirm our proposition. Although other factors (e.g. arrival sequence for other influenza A virus hosts, environmental factors etc., as discussed below) may have induced the temporal pattern in subtype diversity, we consider the ‘natal origin hypothesis’ to be a strong candidate. We therefore believe that our results, confirming the previous suggestion that late-arriving and late-staging autumn mallards in the Northwest European flyway come from more distant breeding areas [11], potentially has several important implications for relationships between avian migration and the epidemiology of common pathogens such as the influenza A virus.

The prevalence of influenza A virus in mallards sampled at Ottenby in 2004 and 2005 was similar to those previously reported within the same flyway (i.e. 10–30% [10], [11]). The exception was late autumn in 2005 with particularly high rates (36.5%). This is not extraordinary though, since it is known that prevalence can be highly variable between years and seasons [11]. Long-term surveillance over nearly ten years has shown that the prevalence of this virus in the mallard populations sampled at Ottenby shows a consistent annual pattern, with little or no virus circulation during spring and summer, with peak viral circulation during staging/migration (September to November), and declining prevalence in December [10], [11]. Precise timing of peaks in autumn is, however, variable between years [11]. This is likely due to between-year weather-related differences in the migration time-table; LPAI dynamics is indeed a large-scale phenomenon with migration influencing disease transmission [3], [4].

We found 32 different influenza A virus subtype combinations. Of these, H4N6 viruses seems to occur frequently and are globally widespread in ducks [10]–[12], [39], whereas HIN1 and H3N8 have been reported as common subtypes in Europe [39] and North America [12], respectively. Low similarity (Sørensen's index) between viral subtype diversity in mallards arriving at Ottenby in early versus late autumn clearly illustrated within-season shifts in virus subtypes. These within-season differences were in fact higher than those between years, corroborating the pattern in Wallensten et al. [11] who included mallard data from Ottenby in 2002–2004 (not autumn 2004; i.e. a different sample compared to the present study). Twenty of the 39 detected subtypes described by Munster et al. [10] were also found in our study (Sørensen similarity index 0.56 when the two studies are compared). Almost all subtypes that were unique for each study were found only once (i.e. each representing only ca 1% of all subtyped viruses). We acknowledge the potential shortcoming of using also adult and unaged birds, in addition to juveniles, in the analyses of subtype data, in contrast to the analyses on natal origins which included juveniles only. We feel however, that this would be unlikely to induce any bias in our comparisons. In fact, for the juveniles only, there would have been only one subtype in 2004 (H1N1) occurring in both early and late autumn periods, in contrast to two subtypes in 2004 and two in 2005 in common for the periods with data on adult and unaged birds. Interpretations would thus have been the same, but with lower statistical power due to less data.

The shedding time of the influenza A virus is relatively short in naturally infected mallards, normally lasting 3–8 days in autumn birds at Ottenby [14]. However, from experimental infections it seems that primary infection can last longer, with virus shedding up to two or three weeks [40], [41]. Moreover, mallards are frequently reinfected by different subtypes during their stopover [14]. Virus prevalence and diversity in the sampled staging populations will therefore be a function of virus-related factors, such as the number and prevalence of virus subtypes present, their infectivity and virulence, and host-related factors such as animal numbers, animal condition, pre-existing immunity and stopover duration. Several of these factors could also be influenced by seasonal factors such as temperature, productivity or day length. The large turnover of virus subtypes within seasons suggests that most subtypes are not capable of remaining in circulation in the staging populations of mallards at Ottenby for extended periods of time. Changes in subtypes could then theoretically be explained by cohorts of ducks bringing influenza A virus specific to their area of origin to the sampled staging site. However, although numbers of trapped mallards showed significant variation within seasons (Figure 4), suggesting distinct influx and emigration of birds, there are also large numbers of other susceptible host species in the area, such as swans, geese, other dabbling ducks, diving ducks, gulls and waders. Some species within these groups are captured at Ottenby during autumn, but in lower numbers: for example Eurasian wigeon (Anas penelope, 343 samples, 32 positive) common teal (Anas crecca, 124 samples, 24 positive) and northern pintails (Anas acuta, 58 samples, 3 positives). The limited available data from these species do not suggest marked deviations in the timing of influenza A virus prevalence peaks compared to mallards, but timing of migration may differ; e.g. the peak of common teals is usually one month before that of mallard [42]. The role of non-mallard waterbirds in influenza A virus transmission is still poorly understood, except for gulls, which tend to carry gull-specific virus subtypes H13 and H16 [43]. However, two recent studies on migratory geese in Europe (white-fronted goose Anser albifrons and pink-footed goose Anser brachyrhynchus) indicate that most transmission is primarily from ducks to geese, rather than the geese being significant reservoirs themselves [17], [44], [45]. At our study site, the mallard is the most common dabbling duck species during nearly the entire autumn period, and also the most sampled species. Increased trapping efforts for other species are warranted to determine the potential influence of non-mallard waterbirds to influenza A virus dynamics in this system.

Abiotic factors, such as pH, salinity and temperature, certainly affect virus tenacity in the environment [46]. If these factors vary temporally, or, if there are subtype-specific differences in virus persistence [47], [48], they could affect the rate of subtype turnover in hosts that are utilizing a common environment.. Our study was conducted in a seasonal environment with progressively shorter days and lower water temperatures in the autumn. However, a drop in prevalence rates is normally not seen until ice formation. In laboratory studies, virus tenacity in water range from several weeks to years depending on virus and experimental setup [49]. Field data from North America suggest that virus can persist in lakes over the course of a winter and infect birds in spring [48]. Between-year persistence of virus is not clearly evident in our study system, as naïve lure ducks, introduced to the duck trap at Ottenby in the spring to attract wild ducks, do not tend to be infected until migratory birds have appeared in larger numbers (J. Waldenström unpublished data). At subtype level, the available data suggest that most subtypes occur in rather short time frames, indicative of smaller, passing outbreaks rather than a constant circulation that would be expected if infection from an abiotic water reservoir occurred frequently.

The subtype diversity was higher in late autumn than in early autumn in both years. We speculate that mallards arriving in late autumn not only bring subtypes specific to their natal area, but also pick up “new” ones from conspecifics along the migration route, leading to higher subtype diversity on the population level. An alternative explanation for different patterns of subtype occurrence in early versus late autumn would be that immunity against certain subtypes will change the subtype composition over the season. In experimental studies, infected mallards show homosubtypic immunity (short-term [21], [40], as well as long-term [50]), and even heterosubtypic effects, where partial or complete immunity derived from the infection of a certain subtype may be active also against other subtypes [21], [40].

Waterfowl, and especially mallards, are among the most frequently recovered species in European band recovery efforts [29]. Our study shows that when combined with migration directions based on band recoveries, δ²H_f measurements of mallards emphasize the importance of natal areas in Finland and northwestern Russia. This is consistent with previous studies [11], [29] and suggests further that almost all hatch-year mallards staging at Ottenby in autumn are foreign birds, cf. [11]. The likely linkage between the epidemiology of influenza A virus, mallard migration and natal area as suggested by our data, lead to possibilities in predicting influenza virus dynamics within a flyway. In more general terms, migration has indeed been shown to be important for the dynamics of several common pathogens in the wild (e.g. Lyme disease, West Nile virus, and avian malaria parasites) [5], [31], and identifying spatiotemporal transmission routes of pathogens is therefore of great and wide interest. Naturally, a prerequisite of this approach is the ability to reliably link an individual, or sets of co-occurring individuals, to distinct populations with known natal, stopover and wintering areas. In this respect, the mallard seem to be a good model species given its large population size, a distribution covering large geographical areas with varying baseline isotopic signatures, and its status as a common game species. The δ²H measurements presented here co-varied with temporal trends in timing of migration and the virus subtypes infecting them. Clearly, a structured sampling of feathers from mallards at breeding sites across the distribution is warranted, providing a refined feather isoscape to which samples can be probabilistically assigned. Recent outbreaks of HPAI H5N1 in Asia, Europe and Africa, provide a concrete example of how such data could prove useful. Such outbreaks are potentially devastating to the poultry industry, and pose risks to human health. Thus, improved knowledge of migratory connectivity between populations and timing of migration in relation to climatic factors would allow improved prediction of disease transmission and potential for HPAI outbreaks, facilitating risk assessment and management in Europe and elsewhere.