Using RRT-PCR analysis and virus isolation to determine the prevalence of avian influenza virus infections in ducks at Minto Flats State Game Refuge, Alaska, during August 2005

Abstract

Summary-This study describes surveillance for avian influenza viruses (AIV) in the Minto Flats State Game Refuge, high-density waterfowl breeding grounds in Alaska. Five hundred paired cloacal samples from dabbling ducks (Northern Pintail, Mallard, Green Wing Teal, and Widgeon) were placed into ethanol and viral transport medium (VTM). Additional ethanol-preserved samples were taken. Of the ethanol-preserved samples, 25.6% were AIV RNApositive by real-time RT-PCR. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined for 38 of the first-passage isolates, and four first-passage isolates could not be definitively subtyped. Five influenza Avirus HA-NA combinations were identified: H3N6, H3N8, H4N6, H8N4, and H12N5. Differences in the prevalence of AIV infections by sex and by age classes of Northern Pintail and Mallard ducks were detected, but the significance of these differences is undefined. In the 500 paired samples, molecular screening detected positive birds at a higher rate than viral isolation (χ 2 = 8.35, p = 0.0035, df = 1); however, 20 AIV isolates were recovered from PCR-negative ducks. Further research is warranted to compare the two screening protocols' potential for estimating true prevalence in wild birds. Our success during 2005 indicates Minto Flats will be a valuable study site for a longitudinal research project designed to gain further insight into the natural history, evolution, and ecology of AIV in wild birds.

Prevalence of avian influenza viruses,Borrelia garinii,Mycobacterium avium, andMycobacterium aviumsubsp.paratuberculosisin waterfowl and terrestrial birds in Slovakia, 2006

“…However, we can exclude the contamination of our samples for several reasons: the samples were collected in three different localities, the samples were collected at different times, the samples were not tested in one laboratory at one time, the negative controls were always negative, and the determined subtypes of HA and NA were not the same. It has been already published that the molecular screening detected positive birds at a much higher rate than viral isolation (Runstadler et al, 2007). The prevalence of AIV in passerines (based on virus isolation) has previously been reported as being particularly low (Morishita et al, 1999;Fouchier et al, 2003;Schnebel et al, 2005;Lebarbenchon et al, 2007), although use of nested reverse transcriptase-PCR may have increased the sensitivity of virus detection (Gronesova et al, 2007;Mizakova et al, 2008).…”

Cross-Seasonal Patterns of Avian Influenza Virus in Breeding and Wintering Migratory Birds: A Flyway Perspective

“…The refuge is an expanse of remote shallow ponds and waterways drained by the Chatanika and Tolovana Rivers in the boreal forest of Interior Alaska. Ducks were either live-trapped (August 2007 and or hunterharvested (September 2007 and. Live-trapping was conducted using swim-in traps made of 30 · 60 cm welded wire fencing baited with corn and barley (Hunt and Dahlka 1953).…”

“…In brief, viral RNA was extracted from VTM (M4RT; Remel) using the MagMAX-96 Viral Isolation Kit (Ambion Inc.). RNA was screened for AIV with a two-step rRT-PCR targeting the matrix gene (Runstadler et al 2007). PCR assays 244 HILL ET AL.…”

“…This proposed AIV cycle, where birds act as agents for the spread of the virus along a migratory flyway has become widely accepted (Stallknecht and Brown 2008). However, evidence for this cycle of dispersal is fragmented because surveillance studies of waterfowl have been conducted independently at either wintering (Stallknecht et al 1990c, Sharp et al 1993, Hanson et al 2005, Ferro et al 2008 or breeding grounds , Ito et al 1995, Runstadler et al 2007, Ip et al 2008) but never at both. Studies of geese (genus: Anser) conducted throughout the annual cycle have provided insights into the antibody dynamics of AIV (Hoye et al 2010).…”

Virulence of H5n1 Influenza Virus in Cattle Egrets (Bubulcus Ibis)

“…RNA extraction and realtime RT-PCR were performed using a QIA-GEN RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) for RNA extraction as recommended by the manufacturer. Real-time RT-PCR assays were performed on a Bio-Rad iQ5 Sequence Detection System machine (Bio-Rad Laboratories, Inc., Hercules, California, USA) using the OneStep RT-PCR Kit (QIAGEN), and the AIV matrix assay of Runstadler et al (2007).…”