This study was conducted to characterize NDVs isolated from waterfowl in the Upper Midwest region of the US. The initial aim of this study was to isolate and characterize AIV from waterfowl. During the study period, 7458 cloacal samples were collected and of these, 11.9% samples were AIV positive by rRT-PCR. Inoculation of these AIV positive samples in embryonated chicken eggs yielded hemagglutinating viruses and of these, 43 were identified as NDVs by RT-PCR using primer specific for F gene. We were expecting the isolation of AIV rather than NDV on inoculation in embryonated eggs as the samples were initially positive for AIV by rRT-PCR. The possibility of the presence of other hemagglutinating virus(es) in HA positive-AIV negative (by RT-PCR for matrix gene)-NDV negative (by RT-PCR for F gene) allantoic fluid cannot be ruled out and testing of such allantoic fluid is underway in our laboratory. The isolation of NDV from samples that were rRT-PCR positive for AIV indicates that the cloacal sample may have mixed infection with NDV and AIV with concentration of NDV being higher than that of AIV. Hence, the NDV probably overgrew AIV upon inoculation in embryonated chicken eggs. It is to be noted that we tested only AI rRT-PCR positive samples by inoculation in embryonated eggs; testing of more samples might have led to isolation of more NDVs. The isolation of NDV from AIV positive samples indicates the presence of both viruses (AIV and NDV) in waterfowl. The AIV positive allantoic fluid by RT-PCR was not tested for NDV; this testing might provide a better picture of mixed infection of both NDV and AIV. Mixed infection of AIV and NDV in waterfowl has been reported earlier [17, 18].
A large amount of sequence data on NDVs isolated throughout the world has been published over the years and is now available for sequence comparison and phylogenetic analysis which can be used to predict the pathotypes and to determine the origin of NDV outbreaks. It has been well established that cleavage of NDV fusion protein is a major determinant for viral virulence. In this study, the F gene sequence of NDVs was used for pathotyping as well as their characterization into different classes and genotypes. None of the isolates was found to be velogenic on the basis of sequence motif of F gene cleavage site. It has been reported that virulent virus has at least one pair of basic amino acids at residues 115 and 116 plus a phenylalanine at residue 117 and a basic amino acid (R) at 113 at the cleavage site whereas lentogenic strains lack dibasic amino acids . All NDV isolates of this study had lentogenic motif at the cleavage site. These results are in agreement with previous studies reporting the detection of lentogenic NDVs in wild birds and domestic ducks [4, 9, 15, 20, 21]. None of the isolates had the sequence motif of 111GERQE/DRL117 of class I isolates, although the latter have been reported in wild birds and domestic ducks [4, 21]. For example,  reported seven of the nine genotypes of class I NDVs in waterfowl and shore birds in the US while  reported the presence of class I genotype 2 NDVs in domestic ducks in Korea.
Of the 43 isolates, 42 had the sequence motif of 111GG/EKQGRL117 at the cleavage site and were phylogenetically similar to either genotype I or genotype II within class II. This sequence motif has been reported earlier in genotypes I and II of class II NDVs . However, a different sequence motif (111GRRQRRF117) was reported in the lentogenic strains from Australia . One of the isolates had the sequence motif of 111GERQGRL117 and this isolate also clustered with class II genotype I strains. This isolate differed from other 42 isolates in the sense that the amino acid lysine was replaced by arginine at position 113.
Overall genotype II viruses were more predominant than genotype I viruses in this study. This finding has the support of  who also observed more genotype IIa viruses than genotype I viruses within class II. The NDV isolates in this study were derived only from rRT-PCR AIV positive samples, the possibility of presence of genotypes of both classes (that were not detected in this study) in rRT-PCR AIV negative samples cannot be ruled out. Within class II, the NDV sequences clustered into two different groups. None of the isolates was phylogenetically close to vaccine strains used for comparison. This indicates that in spite of the regular use of live vaccines in poultry throughout the world, their transmission to wild birds may not be a common phenomenon. In an earlier study,  also did not detect any vaccine strains in wild birds in the US. Since wild birds have been reported to be a reservoir of NDV [16, 23], the mixing of different species at stop-overs during migration and the sharing of common wintering and breeding areas may provide opportunity for virus spread within and between countries and may help perpetuate different genotypes and classes of NDVs in these birds. The phylogenetic proximity of our isolates with those from the US, China, Korea, and Ireland points to this likelihood.
The presence of class II viruses in wild birds is of concern because this class of viruses has been responsible for several panzootics of Newcastle disease in poultry [24, 25]. There are reports suggesting that velogenic NDVs might arise from lentogenic NDVs in nature [23, 26]. Further, studies have also suggested that point mutation, and not gene recombination, may be responsible for generating virulent and avirulent strains. For example, the NDV outbreak in Australian poultry during 1998-2000 was caused by a virulent NDV that originated due to mutation in a class II genotype I virus . These authors were of the opinion that lentogenic viruses have the potential to become virulent with the passage of time. Even passaging of NDVs from one host to another has been reported to increase their virulence [16, 27]. In addition, the selective forces imposed by a new host environment may also play a role in acquisition of virulence . These findings suggest that the lentogenic strains from wild birds may acquire virulence by waterfowl-to-domestic poultry transmission in nature. In such a scenario we may encounter an NDV outbreak in domestic poultry.
Similar to low pathogenic AIV, the lentogenic NDVs in wild bird populations invariably do not cause obvious disease. Even virulent strains of NDVs that are lethal to chickens, have been isolated from apparently healthy domestic ducks [14, 29, 30]. Though virulent strains of NDVs were not detected in this study, their presence in the population cannot be ruled out in view of the potential created by the comingling nature and migration patterns of wild birds within and across continents. Thus, continuous surveillance for NDV in wild birds is essential for better understanding of its epidemiology. In conclusion, the present study reveals the circulation of class II (genotypes I and II) lentogenic strains of NDVs in wild birds in the Upper Midwest region of the US. Further studies are needed to determine the true prevalence and implications of various genotypes of NDV within wild bird population.