Newcastle disease is a highly contagious, infectious disease in birds with recurrent outbreaks in poultry all over the world. The infection is caused by the Newcastle disease virus (NDV), more formally termed avian paramyxovirus type I (APMV-1), which is a member of the Avulavirus genus in the Paramyxoviridae family. This virus family is composed of ten different serotypes (APMV-1 to APMV-10), most of which are exclusive to birds [1]. Members of Paramyxoviridae have a single stranded, non-segmented, negative-sense RNA genome containing six open reading frames (ORFs) that encode a nucleocapsid protein (NP), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutinin-neuraminidase (HN), and the RNA-dependent RNA polymerase (L). HN and F proteins are involved in host cell attachment and membrane fusion, respectively, during infection [2].

The F protein is expressed as a precursor (F0) that needs to be activated by proteolytic processing into active F1 and F2 [3]. The primary structure of the F0 cleavage site is an important factor associated with pathogenicity, which in turn is closely related to viral tissue tropism [4]. NDV are further divided into three different pathotypes, with classification criteria partly based on amino acid sequence of the F0 cleavage site. Velogenic and mesogenic pathotypes have several basic amino acid residues at their F0 cleavage site, which enable furin-like proteases expressed in multiple tissue types of the host to provide the maturation cleavage that is essential for viral progeny to become infectious [5, 6]. Lentogenic viruses, on the other hand, encode F0 precursors with a cleavage site that lacks basic residues, and consequently, these viruses are restricted to replication in tissues of the infected birds that express trypsin-like enzymes, including the respiratory and gastro-intestinal tracts [7]. If introduced into chicken flocks, velogenic and mesogenic strains can cause severe outbreaks with high mortality rates, sometimes as high as 100%. These highly virulent strains are considered such a risk for the agricultural economy that NDV is included in the OIE-list of diseases that are considered ‘specific hazards’ by the World Organization for Animal Health.

There is substantial genetic variation within the APMV-1 serotype, and viral strains are subdivided into two major clades termed class I and class II [8, 9]. One feature that distinguishes these clades is genome size, where class I viruses have a slightly larger genome (15 198 nucleotides) compared to class II strains (15 186 or 15 192 nucleotides). Previously, class I and II viruses have, based on sequence variation of the F gene, been subdivided into nine and eleven genotypes, respectively [8]. However, according to a recent proposal of a unified nomenclature for APMV-1 based on the F gene sequence, the class I clade consist of one single genotype while class II is composed of fifteen genetic groups [10]. Other features that differ between the classes of APMV-1 are pathogenicity and host range. Class I viruses are almost exclusively of the lentogenic type and detected in wild waterfowl, whereas a majority of velogenic strains, often isolated during outbreaks in poultry, belong to class II [11]. The reservoir hosts and transmission routes of viruses that become highly virulent in chicken are not well characterized, although reports from Australia seem to indicate that lentogenic strains circulating in wild birds may transform into velogenic variants when introduced into poultry [12, 13]. In addition, the transition from a lentogenic to a velogenic pathotype in chickens has been demonstrated experimentally [14].

Wild waterfowl is also the main reservoir for influenza A virus (IAV). Naturally, there is a significant chance of birds being co- or superinfected with both AMPV-1 and IAV. The dynamics between these two viruses has been investigated experimentally in embryonated chicken eggs [15], but much less is known about corresponding dynamics in the wild bird population. Up until now, the prevalence of APMV-1 in wild birds has often been discovered as a consequence of failed attempts to determine the subtype of isolated IAV strains [16], rather than actively screening birds for the virus. We have conducted long-term surveillance for IAV in waterfowl, particularly Mallards (Anas platyrhynchos), in Sweden from 2002 to present [17, 18]. Similar to other study systems, putative APMV-1 viruses have been isolated occasionally when propagating IAV-positive samples in embryonated chicken eggs, but the prevalence and patterns of occurrence of APMV-1 has not been investigated in detail. Prompted by the isolation of APMV-1 from IAV samples, a real-time reverse transcription PCR-based surveillance for APMV-1 in wild migratory waterfowl was set up to gain information regarding virus prevalence in these birds. The goals were to answer (A) how prevalent APMV-1 are in migratory Mallards on this migration route, (B) to investigate the pathotypes and phylogenetic relatedness of isolated APMV-1 viruses, and (C) assess the frequency of Mallards co-infected with APMV-1 and IAV.

Previous surveillance studies indicate that waterfowl are an important reservoir of lentogenic class I and class II APMV-1 strains world-wide. All Scandinavian countries aside from Denmark employ a NDV non-vaccination policy, and rely instead on high biosecurity with poultry separated from wild birds, in addition to veterinary inspections and rapid diagnostics techniques for virus detection. Ongoing outbreaks are stamped out by euthanizing infected flocks, followed by disinfection of animal facilities. Despite the ban on vaccination, there have been few outbreaks in Scandinavia compared to other parts of the world, including Asia and Africa, where NDV are endemic in poultry. Two relatively recent NDV outbreaks in Sweden, one in 1995 and another one in 1997, were caused by velogenic class II strains of genotype VIc and genotype XIII, respectively ([31, 32], see also in Figure 1). These particular genotypes have been circulating world-wide for decades, causing numerous outbreaks. Sweden is located in the north-western Baltic Sea region, and has a long coast-line along the Western European waterfowl flyway. It has previously been shown that Mallards from the eastern Baltic region migrate along the Swedish coast to their wintering grounds in Western Europe [33].

During the sampling period from October to November 2010, just over 2300 samples were collected and screened for presence of APMV-1. In total, less than one percent of the samples were APMV-1 positive, hence, a prevalence considerably lower than the total prevalence of 11.8% observed in waterfowl in Finland in 2010 [34]. In the same study it was shown that 4.8% of Mallards were infected. Possibly, observed differences in prevalence may in part be due to the fact that different rRT-PCR methods were used in our study [21] and the study by Lindh et al. [35]. It is also interesting to note, based on the results reported in the Finnish study, that the prevalence in Eurasian teal Anas crecca was higher than in Mallards [34]. It is not known whether this is due to differences in host susceptibility to the virus, or if it has more to do with differences in behaviour, including migration and breeding.

Comparisons of APMV-1 prevalence in Mallards of different age classes indicated that there is no significant difference in the probability of being infected. This observation does not agree with results obtained in a recent Australian study examining the relation between age and infection frequency [36]. In the Australian study, based on multivariate analysis of NDV infections in Plumed whistling ducks (Dendrocygna eytoni), for which the overall virus prevalence was 4.2%, the odds of being infected were approximately three times higher in juveniles compared to adult birds. It is possible that the limited dataset in our study, especially the number of APMV-1 infected adult birds, affects the outcome of the statistical analysis.

One advantage with the sampling system used at Ottenby, with daily sampling and subsequent release of sampled birds, is that it makes it possible to recapture individual birds and sample them several times during their stopover at the site. Among sampled birds in this study, four individuals that were positive on the first sampling occasion were later resampled within a two to four day period. Thus, in more extensive studies in the future, resampling systems may contribute to give a better understanding of the progression of infections in wild birds. Sequence analyses of the F gene sequence of isolates obtained from one of the ducks in this study seem to indicate that the same viral strain was shed during a four day period. To our knowledge this is the first observation of an APMV-1 infection in wild birds followed for several days.

Virus isolates were generated from ten out of twenty APMV-1 positive samples. As indicated by the deduced amino acid sequence of the F0 cleavage site, none of the APMV-1 isolates seem to be of the velogenic type. Corresponding results from screenings of wild birds have been reported previously, both in North America, Japan and Europe [16, 37, 38]. However, for an absolute confirmation of virus pathotype, tests in chicken embryos or chickens such as MDT (mean death time) and IVPI (intravenous pathogenicity index), respectively, needs to be performed [39].

The phylogenetic analysis of APMV-1 isolates showed that F gene sequences clustered with those of class II viruses. This is consistent with previous reports of a predominance of class II viruses in wild birds [8, 16, 34, 36]. According to a recently suggested nomenclature of virus genotypes, class II viruses are divided into fifteen distinct clades [10]. Using this classification, the F0 sequences obtained in this study belong to genotype Ib, and cluster, within this clade, most closely with viral strains from wild waterfowl sampled in Luxembourg from 2006 to 2008 [40], and to viral sequences from birds sampled at live bird markets in China [41]. It is not presently known whether this vast geographic distribution of genetically related sequences is primarily a product of bird migration, or if lentogenic APMV-1 of particular genotypes are constantly circulating at different locations world-wide. However, the integration of several different migration routes of, for example Eurasian teal [42] carrying class II viruses of genotype I [34], including the north-west European flyway and the Mediterranean flyway, might, at least partly, contribute to the wide-spread distribution of certain virus genotypes.

It is worth pointing out that the detection of predominantly genotype Ib viruses is different from observations in recent North American studies detecting predominantly genotype II viruses in wild birds [8, 16]. Possibly, differences dominating genotypes circulating in Northern Europe and North America is related to the geographical distance and the obstacle imposed by the Atlantic Sea, hence a phenomenon partly related to the well-established distinction between North American and Eurasian IAV sequences [43]. Among viral strains previously reported from Sweden, the sequences of this study is most closely related to a strain from a Black-Headed Gull (Larus ridibundus), found in the Ia clade, sampled at the Ottenby Bird Observatory in 1994 [44].

Superinfections and co-infections of the host with two or more pathogens may complicate studies of a certain pathogen’s effect on its host, but can also offer a valuable opportunity to explore particular properties unique to one of the pathogens during co-infection, and to evaluate the contribution of such factors to observed prevalence and virulence. Co-infections involving APMV-1 and IAV have been observed previously in North America and Europe [16, 34]. In this study, more than half of the APMV-1 infected Mallard were simultaneously infected with IAV and statistical analyses suggest, although barely significant in this limited dataset, that the frequency of co-infection might be higher than expected. This is in contrast to previous observations with virus cross-species inhibition between NDV and IAV during co- and superinfection in experiments using embryonated chicken eggs [15, 45]. Although these studies fail to provide a conclusive picture, the order and time of infection as well as the virulence of included virus strains seem to be significant for the outcome of experimental co-infections. A number of mechanisms have been put forward as possible explanations to observed virus induced viral cross-species inhibition, including attachment interference, differences in replication speed and intracellular interference in the form of virus-induced interferon production [46]. However, in wild birds infected with low pathogenic viruses, the cross-species relationship between viruses in a particular host might be different.

Another noticeable observation is the significant difference in APMV-1 and IAV prevalence in sampled ducks. APMV-1 and IAV have both negative-sense single-stranded RNA genomes with hemagglutinin and neuraminidase virion surface structures that attach to sialic acid moieties of host cell surface molecules. Also, for both viruses, waterfowl is a central reservoir for maintaining a constitutive circulation of virus. However, an important distinction between APMV-1 and IAV is their genome organization: The APMV-1 genome is consists of one continuous, linear RNA molecule, whereas IAV has a segmented genome consisting of eight linear RNA molecules. Possibly, the advantages with a segmented genome, including the capacity for genome reassortment, is a contributing factor to the higher IAV prevalence compared to that of APMV-1 in Mallards.

In conclusion, approximately one percent of the Mallard population sampled during autumn migration at Öland, Sweden, was infected with class II APMV-1s. All virus isolates were of the lentogenic type and phylogenetically closely related to genotype I viruses from central and northern Europe, but clustered also with viruses isolated at live bird markets in the south eastern parts of China. Additional studies examining the circulation of APMV-1s in different wild avian species are essential in order to understand viral evolution and epidemiology.