Second North American Sea Duck Conference - Patuxent Wildlife ...

SECOND NORTH AMERICAN SEA DUCK CONFERENCE
DETECTION AND CHARACTERIZATION OF A NOVEL ADENOVIRUS
FROM STELLER’S EIDERS BY PCR
Mary Bozza and Tuula Hollmen
Alaska SeaLife Center; mary_bozza@alaskasealife.org
The Alaskan population of Steller’s eiders (Polysticta stelleri) has undergone drastic declines
in nesting ranges and was listed as threatened under the U.S. Endangered Species Act in 1997.
As part of an investigation to evaluate the role of diseases in eider declines, we are developing
molecular techniques for detection and characterization of viruses in Steller’s eiders. Detection
and characterization of pathogens from field samples is a first step in a multi-faceted effort toward
understanding disease ecology in declining sea duck populations. This effort is often hampered by a
lack of reagents specific for the species or for the disease agent. A species-specific, virus-specific PCR
(polymerase chain reaction) assay was developed as an additional tool to complement cell culture and
serological assays to detect adenoviruses in Steller’s eiders and other declining sea duck populations
in Alaska. An adenovirus was isolated from a cloacal swab of a Steller’s eider sampled in 2003 at the
Alaska Peninsula. Preliminary identification in cell culture was followed by molecular analyses to
further characterize the isolate. PCR using viral DNA and primers from conserved regions of the fowl
adenovirus genome produced an approximately 900 base pair fragment, and DNA sequence analysis
of the amplified fragment suggested a novel adenovirus. Four oligonucleotide primers were designed
from the DNA sequence of the newly isolated adenovirus, resulting in PCR bands of predicted sizes.
Using these primers a virus-specific PCR assay was developed to detect and characterize the Steller’s
eider adenovirus and related adenoviruses in both field samples and cell cultures. DNA extraction
followed by PCR from dilutions of titrated virus stocks demonstrated a dramatic increase (over 10fold)
in sensitivity as compared to the cell culture assay. This increase may be due to detection of
non-infectious virus in our stocks by PCR. To assess the sensitivity of the PCR assay on samples from
wild birds, PCR was performed on the original cloacal swab that the virus was isolated from, resulting
in a detectable band of predicted size. This demonstrates potential for more sensitive detection in
field samples by PCR, since the virus was undetectable in the original inoculation of the cell culture
assay. Additional cloacal swabs will be analyzed by PCR to determine if we can detect the presence of
virus in cloacal swabs that were negative in cell culture assays. The PCR assay will be used to screen
for viruses in cloacal swabs from Steller’s eiders and other sea ducks that share breeding, molting or
wintering grounds. PCR assays will be valuable in determining specificity to related adenoviruses from
other species, and will facilitate virus tracking and determination of prevalence. PCR will also allow a
fast response and characterization in case of a suspected viral outbreak.
NOV. 7-11, 2005 ANNAPOLIS, MARYLAND, USA
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