Influenza A Virus Surveillance in Underrepresented Avian Species in Ohio, USA, in 2015

Abstract

We surveyed passerines and other terrestrial avian species for influenza A virus, resulting in molecular detection of virus from 1.5% of the 615 birds. However, no viral isolates were recovered. Little is known about the role that these undersurveilled avian species play in the ecology of influenza A virus.

Influenza A viruses (IAVs) have been isolated from wild birds in the orders Anseriformes (ducks, geese, and swans) and Charadriiformes (gulls, terns, and shorebirds), which are considered to be the main reservoirs of the virus (Webster et al. 1992; Stallknecht and Shane 1998). However, investigation of the 1968 outbreak of highly pathogenic H7N7 IAV in Australia showed the susceptibility of starlings and sparrows to infection, indicating these species had potential to spread IAV (Webster et al. 1992).

Passeriformes, known as perching birds, is the largest order of birds, with more than half of the nearly 10,000 known species (Poetranto et al. 2011). With the exception of Antarctica, passerines have habitats across the world and are potentially important in IAV ecology because many of them freely intermingle with wild and domestic populations of waterfowl and poultry. Their habitats also often put them into close association with humans, suggesting that passerine species might be important from a zoonotic disease perspective.

Experimentally, passerine species have been infected with IAV (Perkins and Swayne 2003; Boon et al. 2007; Brown et al. 2009; Nemeth et al. 2013), and several publications reported natural infections of passerine species with highly pathogenic H5N1 IAV (Ellis et al. 2004; Kou et al. 2005; Poetranto et al. 2011). Additionally, peridomestic avian species using habitats closely associated with poultry IAV outbreaks (i.e., commercial poultry farms, live bird markets, etc.) have been reported to shed IAV (Ellis et al. 2004; Ellis et al. 2009; Siengsanan et al. 2009; Slusher et al. 2014). However, previous surveillance efforts rarely isolated IAV from passerines (Deibel et al. 1985; Webster et al. 1992), making the role of passerines and other peridomestic birds in the spread of IAV unclear.

To further explore the role of undersurveilled species in the ecology of IAV, passerines and other terrestrial birds were sampled in Ohio, US from March 2015 to October 2015. All bird handling and sample collection procedures were approved by The Ohio State University’s Institutional Animal Care and Use Committee (Protocol 2007A0148-R2) and, where applicable, under US Fish and Wildlife Service permit MB66162B-0 and US Geological Survey federal bird banding permit 23781.

Cloacal swabs were collected as previously described (Slemons et al. 2003) from birds at zoos and wildlife rehabilitation centers, birds trapped on agricultural properties, and birds trapped in marshes for banding by wildlife biologists. Swab samples were collected from birds in five counties across Ohio. Each sampled bird was visually examined to determine species. Viral RNA extraction was performed using Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek, Norcross, Georgia, USA), following the manufacturer’s protocol with the addition of 50 mg/mL bovine serum albumin and 17% sodium sulfate solution to the TNA lysis buffer (Spackman and Suarez 2008). The presence or absence of IAV RNA was determined using real-time reverse transcriptase (rRT) PCR (VetMAX^™—Gold SIV Detection Kit, Life Technologies, Austin, Texas, USA) per the manufacturer’s instructions. This kit targets a conserved region of the IAV genome and is highly sensitive for the detection of diverse influenza A viruses. Virus isolation was attempted in parallel with molecular testing for each sample using 10-d-old specific-pathogen-free embryonated eggs as previously described (Slemons et al. 2003), with rRT-PCR–positive samples receiving a second passage.

A total of 615 cloacal swabs were collected from 94 different species (Table 1 and the Supplementary Table). A majority of the samples (453) were collected from passerine birds, with 37% of all samples coming from four species: Gray Catbird (Dumetella carolinensis; n=104), House Sparrow (Passer domesticus; n=48), American Robin (Turdus migratorius; n=47), and Song Sparrow (Melospiza melodia; n=31). The remaining 162 (26.3%) samples were taken from birds from other orders (i.e., Falconiformes, Suliformes, Piciformes, etc.). Of the 615 samples, 9 (1.5%) tested positive for IAV nucleic acid using rRT-PCR (Table 1); however, no virus was isolated. The rRT-PCR–positive samples were collected from Gray Catbird (n=3), Common Yellowthroat (Geothlypis trichas; n=1), White-throated Sparrow (Zonotrichia albicollis; n=1), Black-capped Chickadee (Poecile atricapillus; n=2), House Wren (Troglodytes aedon; n=1), and Swainson’s Thrush (Catharus ustulatus; n=1).

Table 1.

Distribution of 153 cloacal swab samples from six bird species from which influenza A virus was detected with real-time reverse transcriptase PCR. These samples are part of a collection of 615 samples from passerine and other terrestrial bird species in Ohio, USA that were collected in 2015. Although influenza A virus was detected molecularly in nine samples (1.5%), no influenza A virus isolates were recovered.

Species	Common name	n	n PCR positive	% PCR positive
Catharus ustulatus	Swainson’s Thrush	5	1	20
Dumetella carolinensis	Gray Catbird	104	3	3
Geothlypis trichas	Common Yellowthroat	25	1	4
Poecile atricapillus	Black-capped Chickadee	7	2	29
Troglodytes aedon	House Wren	5	1	20
Zonotrichia albicollis	White-throated Sparrow	7	1	14

Interestingly, all of the rRT-PCR–positive samples were collected from the same location across a period of 30 d in autumn 2015. This location is a wildlife sanctuary and environmental educational center that includes ponds, marshes, and wetlands. The rRT-PCR–positive samples might indicate true infections from which we were unable to recover virus, or these samples may represent nonviable virus passing through the passerine birds as a result of drinking water contaminated by migrating waterfowl.

The findings of the current study are comparable to the results of other investigators where IAV was uncommonly isolated from passerine and other terrestrial birds (Deibel et al. 1985; Webster et al. 1992; Slusher et al. 2014). Sampling efforts to date, including those undertaken as part of this study, have failed to identify a clear role of passerine and other underrepresented bird species in the epidemiology and ecology of IAV.

Additional epidemiologic investigation is needed to determine whether these bird populations are mixing with reservoir species more frequently than at other sites, which could provide further evidence that passerine species are truly a spillover host. Although we failed to isolate viable IAV, passerine species could still be integral in the transmission of IAV from wild waterfowl to domesticated species; thus, additional surveillance efforts are warranted.