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2006 SOUTHEASTERN NATURALIST 5(3):561–565
Evaluating Red-cockaded Woodpeckers for Exposure to
West Nile Virus and Blood Parasites
Robert J. Dusek1,*, David Richardson2, Kristina F. Egstad1,
and Dennis M. Heisey1
Abstract - A marked decline in the Picoides borealis (Red-cockaded Woodpecker
[RCW]) population at Noxubee National Wildlife Refuge, MS, was observed in
2002. Demographic changes—including absence of hatch-year birds, decreases in
size of known groups, and loss of known groups—were identified during annual
fall surveys and are uncharacteristic of RCW populations. In 2003, a serosurvey
of 28 adult RCWs was conducted to investigate the presence of West Nile virus
(WNV) exposure in the population, possibly providing insight into whether WNV
may have been responsible for this decline. Blood smears were also examined
from these birds for blood parasites. We found no evidence of West Nile virus
exposure or blood parasites in any of the RCWs sampled. Further monitoring of
the RCW population and WNV activity in other species at Noxubee NWR is
recommended to further evaluate the potential role of WNV and blood parasites
in their decline.
Picoides borealis Vieillot (Red-cockaded Woodpecker [RCW]) is an
endangered species endemic to the southeastern United States (US Fish
and Wildlife Service 2003). They nest and roost in tree cavities, do not
migrate, and hold a defendable territory around their cavity trees (see US
Fish and Wildlife Service  for a complete discussion of RCW ecology).
At Noxubee National Wildlife Refuge (NWR), Brooksville, MS, the
RCW population had grown from 16 groups (breeding pair and helper
birds) in 1989 to 37 groups in 2002 (Richardson and Stockie 1995, US
Fish and Wildlife Service 2003). This population is demographically isolated
and receives no detectable immigration of birds from outside the
population. In 2002, the population had good reproduction, as evidenced
by monitoring of nests and banding of 35 nestlings by refuge personnel
following recommendations of the RCW recovery plan (US Fish and
Wildlife Service, Noxubee NWR, unpubl. data). Based on this monitoring
effort, refuge personnel estimated that 50 nestlings should have fledged
that year. However, during scheduled fall and early winter surveys in
2002, only 2 hatch-year (HY) birds were found, 3 of the 37 RCW groups
could not be located, and 8 of the remaining 34 groups had decreased in
1US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road,
Madison WI, 53711. 2US Fish and Wildlife Service, Noxubee National Wildlife
Refuge, 2970 Bluff Lake Road, Brooksville, MS, 39739. *Corresponding author -
562 Southeastern Naturalist Vol. 5, No. 3
size. The lack of HY birds and the loss of all birds from established
groups are atypical for the population dynamics for this species (R.
Costa, US Fish and Wildlife Service, Clemson, SC, pers. comm.).
West Nile virus (WNV) (Flaviviridae, Flavivirus) was first discovered in
the United States in the fall of 1999 (Centers for Disease Control and
Prevention 2000, Lanciotti et al. 1999). Since that time, WNV has spread
rapidly in the Western Hemisphere (Peterson et al. 2004). West Nile virus
was first reported in Mississippi in September 2001; by fall 2002, nearly
every county in Mississippi reported WNV-positive birds (Mississippi Department
of Health, Jackson, MS, unpubl. data). There were also numerous
reports of WNV-positive Corvus sp. (crows) and Cyanocitta cristata
Linnaeus (Blue Jays) near the Noxubee NWR (Mississippi Department of
Health, unpubl. data).
West Nile virus is a mosquito-borne virus that was first identified in the
West Nile region of Uganda in 1937 (Smithburn et al. 1940). The natural
transmission cycle of WNV is principally a mosquito-bird cycle (see Komar
2003). West Nile virus has been reported in five woodpecker species, including
two members of the same genus as RCW: P. pubescens Linnaeus
(Downy Woodpecker) and P. villosus Linnaeus (Hairy Woodpecker) (E.
Saito, National Wildlife Health Center, Madison, WI, pers. comm.).
Due to the dramatic apparent loss of RCWs at Noxubee NWR in 2002,
the possibility of a disease such as WNV being responsible for the observed
decline was considered. As a part of the investigation into the
disappearance of RCWs at Noxubee NWR, a survey of apparently healthy
RCWs was conducted at Noxubee NWR during the spring and summer of
2003 to determine the prevalence of blood parasites and specific WNVneutralizing
Red-cockaded Woodpeckers were captured between 11 April 2003 and
24 July 2003 at Noxubee NWR in accordance with guidelines established by
the Red-cockaded Woodpecker Recovery Team (US Fish and Wildlife Service
2003). Captures were made at dusk and dawn, as RCWs went to their
roost in the evening or left their roost in the morning, using a net attached to
a telescoping pole that was placed over the entrance to the roost cavity.
Captured birds were held for the minimum amount of time necessary (not
longer then 30 min) to obtain a blood sample and band them. Birds were then
released at the site of capture.
Blood samples (0.25 ml) were obtained from the brachial vein by piercing
the vein with a 27-gauge needle, then using a capillary tube to collect
the blood coming from the vein. Blood smears were prepared in the field
immediately after sample collection and air dried. The remaining blood
sample was then placed in a cooler with blue ice until returning to the lab.
In the lab, blood samples were centrifuged for 10 minutes (at approximately
100 x g) to separate the serum from the red blood cells. The serum
2006 R.J. Dusek, D. Richardson, K.F. Egstad, and D.M. Heisey 563
was then removed and dispensed into a labeled cryovial and placed into a -
70 °C freezer until shipped on dry ice to the National Wildlife Health
Center for testing. The sera were tested for specific WNV-neutralizing
antibodies using a microneutralization test (Beaty et al. 1995). Viral titer
and determination of specificity for WNV were performed according to the
current CDC guidelines (Centers for Disease Control and Prevention
2003). Briefly, equal volumes (0.05 mls) of WNV (50 infectious units) and
each serum, diluted 1/5 and heat inactivated at 56 °C for 30 minutes, were
added to a 96-well microtiter plate in duplicate. After a 30-minute incubation
at 37 °C, VERO (Chlorocebus aethiops L. [African green monkey]
kidney) cells were added to each test well (0.15 mls of a 1.67x105-cells/ml
suspension). Appropriate serum, cell, and WNV test-dose controls were
included in the test. Cultures were then observed for virus neutralization
over a one-week period. In the lab, blood smears were fixed with methanol,
stained with Giemsa, and examined for the presence of blood parasites for
≥ 10 minutes at 400x using a compound microscope. Upper 95% confidence
bounds were calculated on results based on estimated populations of
90 and 100 RCWs (Lindgren 1976).
Twenty-eight adult woodpeckers were captured and blood sampled. Of
these, one sample did not have sufficient serum for testing, and a blood
smear was not prepared for one bird. All samples tested were negative for
specific WNV-neutralizing antibody (0% prevalence). No blood parasites
were detected on the blood smears (0% prevalence). An upper confidence
bound was calculated, resulting in a maximum prevalence of 7.8% (N = 90)
and 9% (N = 100).
We were unable to detect specific WNV-neutralizing antibodies in any of
the 27 RCWs sampled in this study. Although the sample size of 27 may
seem modest, it constitutes about 1/3 of the remaining RCW population on
Noxubee NWR during this investigation. This sample size is sufficient for us
to have statistical confidence that the seroprevalence at Noxubee, even if not
exactly 0, is indeed low.
It is almost a statistical impossibility to ever ascertain that the prevalence
in a population is 0, but our results show convincingly that prevalence is
low, especially in light of what might be expected to produce the sorts of
declines thought to have occurred. We consider four possibilities for the low
prevalence: 1) RCWs at Noxubee NWR may not have had much exposure to
WNV, although WNV was diagnosed in sick and dead Blue Jays from
Starkville, MS, (approximately 10 miles from Noxubee NWR) and nearly all
counties in Mississippi reported WNV-positive birds in 2002 (Mississippi
Department of Health, unpubl. data.); 2) WNV may be unevenly distributed
564 Southeastern Naturalist Vol. 5, No. 3
across the landscape, and unknown landscape factors at Noxubee NWR may
not have been favorable for the maintenance and subsequent detection of
WNV; 3) Any RCWs infected with WNV may have died prior to our survey;
West Nile virus is reported to cause high mortality in some species of birds,
particularly crows, where > 90% of experimentally infected individuals died
(Komar 2003, McLean et al. 2001); and 4) We may have been unable to
detect antibody presence in RCWs because birds previously infected had lost
detectable specific WNV-neutralizing antibodies prior to sampling. Currently
the only data available describing long-term (ca. 1 yr) duration of
detectable specific WNV-neutralizing antibody in birds is limited to Columbia
livia Gmelin (Rock Doves) (Gibbs et al. 2005). It is not clear which, if
any, of these four possibilities affected our findings.
We also were unable to detect blood parasites in the RCWs sampled.
Others have found unidentified microfilaria and two species of Haemoproteus
in RCWs (Luttrell et al. 1995, Pung et al. 2000). However, Love et al.
(1953) reported no blood parasites in RCW blood smears they examined. In
general, reports of mortality due to blood-parasite infection are mostly
confined to domesticated birds or birds held in exotic environments, or
mortality caused by introduced parasites (Bennett et al. 1993).
Many individuals assisted in this research. Kelly Meeks and Julie Bennett were
instrumental in helping to capture birds and collect samples and process them for
analysis. Margaret Copeland surveyed the population in spring and fall, documenting
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