Identification of Avian Paramyxovirus Serotype-1 in Wild Birds in the USA

Newcastle disease, caused by virulent strains of avian paramyxovirus serotype-1 (APMV-1), is a disease of economic concern for commercial and backyard poultry producers due to high mortality (up to 100%), impact of outbreaks, and trade restrictions associated with infection (Aldous and Alexander 2001). Although infection in wild birds can cause disease (Leighton and Heckert 2007), mortality due to virulent APMV-1 is typically undetected in wild birds compared to poultry (Kim et al. 2007). However, virulent APMV-1 infection in certain species such as cormorants (Family Phalacrocoracidae; Kuiken 1999) and pigeons (Family Columbidae; Kim et al. 2008a) has resulted in large-scale mortality events and occasional spillover into other species, such as pelicans (Family Pelecanidae; USGS 1999), gulls (Family Laridae; Diel et al. 2012), and poultry (Irvine et al. 2011). In addition to virulent forms, a large diversity and wide distribution of low-pathogenicity viruses across different geographic regions exist in waterfowl (Anseriformes) and shorebirds (Charadriiformes; Kim et al. 2007). These low-virulence viruses occasionally spill over into poultry, causing minor respiratory problems (Hamid et al. 1990).

Large-scale efforts to actively identify the species involved in transmission of APMV-1 in the US have been limited to specific species or orders (Kim et al. 2007; Coffee et al. 2010; Pedersen et al. 2014). We evaluated viral shedding, exposure (antibody presence), and host distribution of APMV-1 in a diversity of apparently healthy wild birds across the US.

Wild birds were opportunistically sampled by the US Department of Agriculture's Animal and Plant Health Inspection Service–Wildlife Services during removal of wild birds from airports and other locations that posed a threat to human health and safety. In addition, wild birds were trapped with mist nets, rocket nets, and swim-in traps specifically for sampling and were subsequently released. Samples were collected 1 January 2013 to 30 September 2013 in 44 states (Supplementary Material Table S1).

Cloacal and oropharyngeal swabs were collected from wild birds using sterile Dacron-tipped swabs (Puritan Medical Products Company LLC, Guilford, Maine, USA). Both swabs were placed in a single cryogenic vial containing 3 mL of brain-heart infusion broth (Becton Dickinson and Company, Sparks, Maryland, USA). The vials were immediately placed in a cooler with ice packs, then stored refrigerated, and shipped with ice packs within 3 d of collection.

When possible, blood was collected from swabbed birds via the jugular, brachial, or metatarsal vein, and via postmortem intracardiac puncture from euthanized birds. After centrifugation, ≥1 mL of serum was transferred to a 2-mL cryogenic vial. Sera samples were stored refrigerated and shipped within 3 d of collection.

Swabs were tested at the Michigan State University Genomics Laboratory (East Lansing, Michigan, USA) using multiplex real-time reverse transcription PCR (rRT-PCR), to identify infection with class I and class II APMV-1 (Kim et al. 2008b). If negative, testing was considered complete. Positive samples were forwarded to the US Department of Agriculture's Agricultural Research Services Southeast Poultry Research Laboratory (Athens, Georgia, USA) for confirmatory testing (Pedersen et al. 2014). Briefly, swab medium (0.1 mL) from each sample was inoculated into five 9-to-11-d-old specific-pathogen-free embryonating chicken eggs using standard methods (Senne 2008). Virulence of isolates was assessed by requiring an intracerebral pathogenicity index of ≥0.7 in 1-d-old chickens or multiple basic amino acid residues at the C-terminus of the fusion protein cleavage sites (residues 113–116) and a phenylalanine residue at position 117 (OIE 2012).

Extracted RNA was converted to cDNA and amplified using one-tube reverse-transcriptase PCR (SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase, Life Technologies, Carlsbad, California, USA). The PCR reactions were subjected to electrophoresis in 1% agarose gels, and DNA bands were excised and purified using the QuickClean II Gel Extraction Kit (GenScript, Piscataway, New Jersey, USA). The APMV-1 isolates were sequenced (Miller et al. 2015) and deposited in GenBank (KP780870–KP780878).

A blocking enzyme-linked immunosorbent assay (ID VET, Montpellier, France) was used to detect antibodies against APMV-1 using multiple positive and negative controls according to the manufacturer's instructions. Percent inhibition values >40% were considered positive, <30% were considered negative, and values 30–40% were considered undetermined and were retested once.

The majority (68%) of the 3,789 wild birds sampled were after-hatch-year (AHY) birds, 14.5% were hatch year (HY), and the remainder were of undetermined (UND) age. Viral shedding was slightly higher in HY birds (1.3%; 95% confidence limits [CL] 0.62–2.6) compared to AHY birds (0.58%; 95% CL: 0.35–0.96) or UND birds (0.3%; 95% CL: 0.08–1.1). Antibody prevalence was highest in AHY (19.2%; 95% CL: 17.69–20.81), followed by UND (17.4%; 95% CL: 14.61–20.7), and HY (9.9%; 95% CL: 7.65–12.78).

Although recorded for each bird, sex was undetermined for 74.4%. Viral shedding was similar for females (1.4%; 95% CL: 0.63–2.98) and males (1.1%; 95% CL: 0.53–2.48) where sex was identified (n=963). Antibody prevalence was also similar in females (21.9%; 95% CL: 18.17–26.05) and males (19.8%; 95% CL: 16.58–23.48).

We tested 3,779 swabs for APMV-1, and 3,578 sera samples for antibody to APMV-1. The majority (3,568) of these were paired samples from the same birds. Antibody prevalence was highest in wild birds of Order Suliformes (44.9%), followed by Pelecaniformes (24.4%), Anseriformes (22.7%), and Columbiformes (11.7%; Table 1). We isolated virus from 24 samples that were positive on the initial rRT-PCR (Table 2).

The seven class II (virulent) viruses we identified were classified as pigeon paramyxovirus serotype-1 (PPMV-1), or genotype VI, from Rock Pigeons (Columba livia; Table 2), and they were phylogenetically similar to other paramyxoviruses isolated from pigeons in the US. PPMV-1 is a genetic variant of APMV-1 adapted to pigeons and doves that contains a virulent cleavage site and can have variable pathogenicity in chickens (Aldous et al. 2004). Although PPMV-1 is considered endemic in pigeons and doves in the US, it is a concern to poultry (Kommers et al. 2001) because spillover can result in severe economic consequences.

Class I viruses are common to wild birds, are typically of low virulence, and have been identified in many species, such as Mute Swans (Cygnus olor; Pedersen et al. 2014), Northern Pintails (Anas acuta; Ruenphet et al. 2011), and other waterfowl and shorebirds (Kim et al. 2007). The two class I viruses we identified were in an apparently healthy Mallard (Anas platyrhynchos) and American Green-winged Teal (Anas crecca; Table 2).

Antibody prevalence of APMV-1 was much higher than virus prevalence (Table 1), which is not surprising because virus is shed for a limited, but variable time, depending on the species infected (Kuiken et al. 1998), while antibodies persist much longer and vary depending on many factors related to the host (Staszewski et al. 2007) and the amount of antigen to which they are exposed (Broggi et al. 2013). There was very little concordance between the 24 rRT-PCR-positive samples and the serology results (Table 2). This is most likely because antibodies had not yet developed, had dropped below detectable levels, or had developed as a result of exposure to other APMVs. In addition, because none of our samples was collected in the fall (October through December), when viral transmission might be facilitated by the concentration of birds preceding and during migration (Vickers and Hanson 1982), antibody prevalence and viral shedding prevalence were likely skewed.

Our results suggest that wild birds are commonly exposed to APMV-1, but active viral shedding in apparently healthy birds is relatively uncommon, and consequently the risk to poultry appears low. Further studies focusing on pigeons are warranted because of their greater risk of contact with poultry (Gilchrist 2005), and because all virulent viruses we identified originated from Rock Pigeons.

We thank the wildlife biologists and technicians who collected samples for this study. We also thank the Michigan State University Genomics laboratory for conducting rRT-PCR for the samples, and Sarah Bevins for assistance with statistical analysis. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

Supplementary material for this article is online at http://dx.doi.org/10.7589/2015-10-278.