The important role of waterbirds, especially waterfowl, as a reservoir for avian influenza viruses of all subtypes is well known from intensive investigations from many regions of the world [14–17]. Avian influenza monitoring of wild birds in natural habitats and in areas at risk of transmission between domestic poultry and wild birds will increase the knowledge of epidemiology, ecology and genetic relationships of AIV infections. This knowledge will facilitate risk assessments concerning poultry and wild bird populations and provides information on currently circulating AIV which might also have the potential to become important for human health. However, little information is available about the circulation of influenza viruses in waterbirds in West and Central Asia and in the Middle East. This is the first study of AIV-investigations in wild birds in Iran and this geographic region.
In a survey during 1999-2000 in Northern Europe, 2.6% of wild ducks and 1.4% of wild geese were positive in rRT-PCR . In more recent monitoring studies of wild birds during 2003-2005 in Italy, 5.1% of Anseriformes were positive in rRT-PCR . Also, in an AIV screening in 2005 in Norway, 13.2% of Anseriformes were positive in rRT-PCR . The prevalence of LPAIV in wild birds in Alaska and Canada seems to be more variable [21–23]. The results from these studies have shown that the prevalence of AIV in wild birds, and especially ducks, depends on various factors, including geographic altitude of sampling area, bird species, seasonal parameters and different sample processing approach. In our investigation, the number of positive birds varied based on species, sampling month and sites. In total 3.4% of all sampled birds were positive, but different families and species had different number of positives. Only two out of 11 investigated families (Anatidae and Rallidae) were AIV positive, and among 17 sampled species of Anatidae, 8 species revealed positive results. Prevalence rates for Mallard, Common Teal and Common Coot, with high sample sizes, were 8.3%, 2.5% and 1.7% respectively (Table 1). No positive samples were found in shorebirds (Recurvirostridae, Charadriidae & Scolpacidae).
Previous studies revealed high virus prevalence during the autumn season in the Northern Hemisphere , whereas the lowest prevalence rates have been measured in early spring. In contrast, 88% of positive samples in our study were found during February and early March. Interestingly, 78% of samples which were collected in this period came from Anatidae.
The geographical distribution of positive samples reveals further significant differences. In Mazandaran (one of the northern provinces), 21 out of 637 samples tested positive (3.3%), while in a small wetland in the southeast of Tehran province, 9 out of 135 sampled birds were positive (6.7%, Table 2). Sample numbers per species largely reflect the proportion of the wintering populations in different geographical regions of Iran. However, with respect to statistical inferences, a bias regarding different species, seasons and geographical regions cannot be excluded.
The results of this study regarding the dominant AIV infected wild bird species are consistent with several other investigations from Europe and the Americas. Dabbling ducks were found infected with LPAIV at higher prevalence rates than other taxonomic groups [23–25]. Similarly, the highest number of positive AIV samples has been reported from Mallard and Common Teal, relative to any other species [20, 23]. Mallards are the most frequently sampled species in most wild birds surveillance studies in the northern hemisphere, though in our study Common Teal and Common Coot occupied a higher ranking (Table 1).
No evidence was found for HPAIV H5N1 circulating in wild birds before (2003-2005) and after introduction of this virus into wild bird and poultry populations in countries around the Caspian Sea in 2006. Nevertheless, the small sample sizes of the species investigated here in comparison to their total populations make it very hard to exclude a low or very low prevalence of HPAIV in wild bird populations. The largest sample sizes in our study belonged to Common Teal, Common Coot and Mallard with 358, 233 and 180 birds, respectively.
The results of recent wild bird surveillance in Sweden indicate that the proportion of positive cloacal samples exceeds positive oropharyngeal samples . Our results show a similar proportion of positive oropharyngeal and cloacal samples. Among 21 positive samples from 2007, nine were only positive in oropharyngeal swabs, 11 were only positive in cloacal swabs, and one was positive in both (Table 3). Only in February 2007 oropharyngeal and cloacal samples were collected, when nearly half of the positive samples were retrieved from oropharyngeal swab samples. In previous years only cloacal swabs had been taken. This may indicate that the number of LPAIV positive birds may be significantly underestimated when relying exclusively on cloacal samples. In addition, it cannot be excluded that different AIV subtypes may exhibit different tissue tropism. For example, out of five H11 subtype viruses detected during 2007, four originated from cloacal samples.
The real-time RT-PCR results of inter-laboratory analyses of 2007 samples were highly concordant, when two identical sets of the samples were tested by laboratories in Germany and France/Italy. This finding shows the robustness of results when using the standard protocols in different laboratories. In addition, it shows sampling of cloaca or oropharyngeal area of the same bird twice, does not cause false negative results of the second sample.
The potentially higher sensitivity of rRT-PCR compared to virus isolation has been mentioned before in other investigations . Although some investigators could demonstrate that avian viruses originating from wild bird samples require several passages in embryonated eggs for adaptation, our investigation during 2003-2005 showed acceptable results even in the first egg passage (Table 3). During the 2007 investigation, only two out of 21 positive samples were also positive in virus isolation. The efficacy of virus isolation was dramatically reduced compared to 2003-2005. This might be due to many factors including: improper sample transfer in 2007 with extended international transport and interruption of the cold chain, quality and preservation of VTM and viral load of the samples.
Validation of a serological assay for detection of AIV-specific antibodies in wild birds is still a matter of debate [15, 18, 27–32]. Only limited information is available about the sensitivity of the HI assay in these species. In a recent study , only 16.9% of wild duck sera which tested positive in a double antibody sandwich blocking ELISA were positive in HI assay. Previous studies showed that competitive ELISAs on the basis of recombinant AIV-NP antigen appear to be a valid and reliable method for testing different bird species for avian influenza infection [15, 32, 34].
In this study, the overall sero-prevalence based on 711 serum samples from different species was 48.5%, and Anseriformes provided 61.45% positive c-ELISA results. Therefore, the overall sero-prevalence could be misleading due to the species composition of samples. Two duck species with high numbers of samples were Mallards with 75% positive of 108, and Common Teal with 57.7% positive of 272 tested birds. These results, together with the virological data, again emphasize the importance of these species for the epidemiology of AIV in the nature, but the high percentage of AIV positive Mallard and Common Teal may just reflect an over-representation of these species in the sample composition due to their large wintering population in Iran. Thus, although the sample size for other members of this order was small, the detected prevalence rates are impressive (Table 3). Interestingly, only one serum sample from 66 Common Coots sampled during 2003-2005 was weakly seropositive, whereas sixteen from 49 Common Coot samples collected in 2007 were seropositive (32%). In addition, in 2003-2005 we could not find any AIV positive samples from 117 Common Coots taken from six different provinces , whereas in 2007 three out of 77 samples from Common Coots were positive in rRT-PCR. The relevance of these findings is not clear. Although seropositive samples were found among only 12% of Non-Anseriformes in 5 different families, some species, such as Greater Flamingo (3/3 positive) and Black-headed Gull (4/10 positive), demonstrated high prevalence rates.