MONITORING WEST NILE VIRUS IN WISCONSIN’S RUFFED GROUSE POPULATION: YEAR ONE
(2018) PRELIMINARY RESULTS
31 January 2020
Lead Project Investigators: Mark Witecha (WDNR), Lindsey Long (WDNR), Nancy Businga
(WDNR), Chris Pollentier (WDNR), Alaina Gerrits (WDNR)
WI Project Collaborators: Tami Ryan (WDNR), Kent Van Horn (WNDR), Scott Hull (WDNR), Betsy
Elsmo (Wisconsin Veterinary Diagnostic Lab)
Regional Collaborators: Kelly Straka (MIDNR), Al Stewart (MIDNR), Thomas Cooley (MIDNR),
Michelle Carstensen (MNDNR), Charlotte Roy (MNDNR), Ted Dick (MNDNR), Brent Rudolph
(Ruffed Grouse Society), Jon Steigerwaldt (Ruffed Grouse Society), Mark Ruder (Southeast
Cooperative Wildlife Disease Study, University of Georgia)
This report examines the first year, preliminary, results (2018) of a three-year, collaborative
study monitoring West Nile virus in Wisconsin’s ruffed grouse.
MONITORING WEST NILE VIRUS IN WISCONSIN’S RUFFED GROUSE POPULATION: YEAR ONE
(2018) PRELIMINARY RESULTS
Abstract
The first year of the Wisconsin Department of Natural Resources three-year monitoring effort
of West Nile virus (WNV) exposure in ruffed grouse (Bonasa umbellus) indicates that of 235
hunter-harvested samples tested, 68 (29%) had antibodies to WNV either confirmed (44 or
19%) or likely (24 or 10%), and viral genetic material was found in two (0.9%) heart samples.
Results from 2018 reveal that although ruffed grouse are exposed to WNV, they can develop
antibodies to the virus and survive exposure. This study is designed to evaluate WNV exposure
in hunter-harvested ruffed grouse over a three-year period (2018–2020). Ruffed grouse
exposure can vary annually based on a number of conditions including but not limited to: virus
prevalence, availability of adept mosquito populations, and weather conditions. This
monitoring effort will be unable to draw conclusions regarding morbidity and mortality of
ruffed grouse due to WNV infections but will allow for a better understanding of the
distribution, prevalence, and scope of WNV exposure in Wisconsin’s ruffed grouse population
from which additional actions can be pursued. Research involving survival analysis of freeranging, ruffed grouse populations would be key to further evaluation of ruffed grouse
response to WNV exposure.
Introduction
In 2017, data from roadside surveys suggested spring ruffed grouse (Bonasa umbellus)
drumming activity increased statewide, and brood observations had also indicated an increase
in production compared to 2016 results. Despite encouraging surveys that suggested a
possibility for increased harvest opportunities for fall 2017, many grouse hunters reported
fewer flushes than anticipated while afield. Similar reports had been conveyed from hunters
and management agency staff in Michigan and Minnesota. Unexpected declines in drumming
survey results in the spring of 2018 escalated concerns regarding recent trends in ruffed grouse
numbers. Furthermore, concurrent research from Pennsylvania has suggested that when taken
together with other supporting evidence, including habitat loss and forest maturation, the
presence of West Nile virus (WNV) has coincided with declines in ruffed grouse abundance.
Given these abrupt decreases in population trends and heightened concerns over perceived
impacts of WNV on ruffed grouse, the Wisconsin Department of Natural Resources (WDNR)
entered a collaborative disease monitoring project with the Michigan and Minnesota
Departments of Natural Resources. This joint effort will provide a more comprehensive view of
the prevalence of exposure of ruffed grouse to WNV across the Upper Great Lakes region than
an individual state could execute alone and demonstrates the interest of regional biologists in
responding to concerns of hunters and the public at large.
The primary objectives of the 3-year study are to:
1. Assess the feasibility of utilizing hunter-harvested ruffed grouse to obtain biological
samples from harvested birds for disease screening and collecting relevant metadata.
2. Determine prevalence of exposure to WNV in ruffed grouse populations and if there is
significant change by year in Wisconsin.
3. Evaluate if samples can be collected in sufficient numbers to assess prevalence across
different regions of the state.
4. Examine submitted samples for evidence of clinical disease associated with WNV
infection.
Methods
For the first year of the monitoring project in 2018, the goal for Wisconsin was to distribute 500
self-sampling kits to ruffed grouse hunters, with priority being to collect samples from the
primary grouse range in northern and central regions of Wisconsin. After successfully
harvesting a grouse, hunters were asked to collect blood onto Nobuto blood filter strips, to
remove and collect the heart, and to complete a datasheet with other pertinent information
including location of harvest, body condition of the grouse, and age and sex of the bird.
Hunters were then asked to send all samples and the datasheet to the WDNR Science
Operations Center in Madison.
Once received at the WDNR Science Operation Center, samples were processed and readied for
shipment to the Southeastern Cooperative Wildlife Disease Study (SCWDS) to be tested for
West Nile virus. Samples from each of the three participating states were sent to SCWDS to
ensure consistent testing protocols. Once samples were received by SCWDS they underwent
two different forms of testing. Blood samples were evaluated for the presence of antibodies to
West Nile virus using a plaque reduction neutralization test. Heart samples were tested by reverse
transcription polymerase chain reaction (rtPCR) for viral genetic material. Results were also
analyzed by age and sex of the bird, location of harvest, and hunter-reported body condition.
Results
Wisconsin submitted and received results for 235 ruffed grouse samples from 20 counties
across the northern region of the state (Table 1). Sixty-eight (29%) samples had antibodies to
WNV either confirmed (44 or 19%) or likely (24 or 10%; Fig. 1). The 24 “likely WNV” results
were samples where virus could only be identified to the genus Flavivirus, which includes WNV
among several other viruses. The presence of antibodies indicates that the individual was
exposed to WNV and developed an immune response to the virus. Viral genetic material was
found in 2 (0.9%) of the 235 heart samples (Fig. 2). Fragments of viral genetic material may
indicate many things – such as that birds had fragments of the virus enter their system or been
exposed to the virus recently, or that they had an ongoing exposure. Michigan submitted 213
samples and WNV antibodies were detected in 28 (13%) blood samples with exposure to the
virus either confirmed (9 or 4%) or likely (19 or 9%); viral genetic material was found in 4 heart
samples. Minnesota submitted 273 samples and WNV antibodies were detected in 34 (12%) of
the samples with exposure either confirmed (10 or 3%) or likely (24 or 9%). Viral genetic
material was not found in any of the Minnesota heart samples.
Samples from Wisconsin were analyzed by sex (female, male, unknown) and age (adult,
juvenile, unknown) to evaluate potential differences in WNV exposure. No differences were
found in the presence of antibodies in blood samples between sexes (?4
2
= 5.24, P = 0.26), nor
was there a difference between sexes regarding the presence of viral genetic material from the
rtPCR test (?4
2
= 5.22, P = 0.27; Table 2). Likewise, no differences were found between age
classes for presence of antibodies (?4
2
= 0.26, P = 0.99) or presence of viral genetic material (?4
2
= 0.81, P = 0.94; Table 2). Hunter-reported body condition (fat, normal, thin) was also
evaluated, but no differences were found amongst condition levels for the presence of
antibodies (?4
2
= 3.35, P = 0.50) or presence of viral genetic material (?4
2
= 0.33, P = 0.99; Table
3).
Eastern Equine Encephalitis virus (EEEV)
Three ruffed grouse heart samples from Wisconsin tested positive for Eastern equine
encephalitis virus (EEEV), a similar virus to WNV.
EEEV can be found in states throughout the eastern portion of the U.S. It was first documented
in Wisconsin’s ruffed grouse population in the 1950s, when 50% of grouse sampled tested
positive for antibodies to EEEV. Like WNV, testing has been limited and opportunistic in nature
to this point. Given that the virus was first identified as present in the 1950s and likely still
present through normal population cycles and historic highs in the 1970s–1990s, it is believed
that EEEV may affect individual birds but likely does not influence grouse populations.
Opportunistic sampling for EEEV in ruffed grouse is ongoing in Wisconsin.
Discussion
Findings thus far suggest that while ruffed grouse are exposed to WNV, individuals are capable
of surviving exposure and developing an immune response. This monitoring study is providing
important baseline data on prevalence of exposure to WNV in ruffed grouse, something that
until now has not been evaluated in Wisconsin. Not only are there many factors that can affect
WNV prevalence, but there are also many factors that can determine an individual’s response
to a viral exposure and the competency of their immune system. Further evaluation of
morbidity and mortality associated with WNV on ruffed grouse should incorporate a full
survival analysis that considers not only presence and absence of the virus but also key factors
that may affect immune responses such as nutrition, available cover, weather-related impacts,
etc.
There are potentially several reasons the higher exposure rates that occurred in Wisconsin
compared to our neighboring states (i.e., localized and variable levels of mosquito activity), and
we plan to examine these possibilities further after the 3-year sampling period has concluded.
Some have speculated that juvenile ruffed grouse or birds in poor condition may be more
susceptible to WNV exposure. After one year of sampling, differences in WNV exposure among
hunter-harvested ruffed grouse have not been detected by age-class or between males and
females. Body condition would also not be expected to be a reliable indicator of WNV exposure
– some ruffed grouse in seemingly excellent condition also tested positive for WNV exposure. It
is pertinent to note that only one year of sampling has been completed, and analyses will be
more robust after the second and third years of sampling have been finalized and trends can be
assessed across the entire dataset.
Furthermore, it is important to remember the limitations of this current monitoring effort.
Because samples are being taken from hunter-harvested ruffed grouse, and not from birds that
are captured and radio-marked, like in an intensive demographic study, it is not possible to
discern any population-level impact WNV may be having on ruffed grouse at this time. The
current monitoring effort will be able to shed light on the prevalence and overall distribution of
WNV exposure across Wisconsin and the Upper Great Lakes region.
Sampling efforts continued in 2019 and all 500 kits were allocated to hunters. The 2019 season
has recently concluded, and samples are currently being processed for testing. To gather more
accurate data on the age and sex of ruffed grouse, WDNR requested hunters submit rump and
primary wing feathers for identification in 2019. An additional 500 kits are scheduled to be
allotted for the 2020 hunting season, the final year of the monitoring effort.
Research has indicated that the best method for maintaining healthy ruffed grouse populations
is to manage appropriate habitat. With higher quantities and quality of habitat, the more
suited grouse will be to overcome environmental stressors; not only disease but also weather,
predation and others.
Acknowledgements
We are grateful to the Ruffed Grouse Society and Wisconsin Conservation Congress for their
collaboration and assistance in distributing sampling kits. We are grateful to the grouse hunting
community for taking time to provide the department with samples from their harvested birds.
Without their support, this study would not be possible.
Figure 1. Distribution of hunter submitted ruffed grouse (n = 235) from the fall 2018 hunting
season across northern Wisconsin, USA, tested for West Nile virus exposure. Serology tests
examined the blood samples and results indicate whether ruffed grouse had confirmed West
Nile virus exposure (n = 44, yellow dots) or likely exposure which were confirmed only to the
genus level of Flavivirus (n = 24, black triangles). Flavivirus is a genus of viruses that includes
West Nile virus among several other viruses. Negative test results (n = 167, gray dots) indicate
those individual birds likely had not been exposed and antibodies to West Nile virus were not
present.
Figure 2. Distribution of hunter submitted ruffed grouse (n = 235) from the fall 2018 hunting
season across northern Wisconsin, USA, tested for West Nile virus (WNV) exposure. Heart
samples were tested for presence of WNV by reverse transcription polymerase chain reaction
(rtPCR), and results indicate whether detectable fragments of viral genetic material were
present in the heart samples. Positive samples (n = 2, yellow dots) indicate those individual
ruffed grouse had recent exposure to WNV but does not directly indicate these birds were sick
at time of harvest. Three other birds showed positive test results for eastern equine
encephalitis virus (EEEV, black triangles), which is also spread by the bite of a mosquito and is
known to infect a wide range of animals including birds. Negative test results (n = 230, gray
dots) indicate virus was not present in those individual birds.
Table 1. Number of hunter-submitted ruffed grouse samples collected in 2018 from northern
Wisconsin, USA, and tested for West Nile virus (WNV) exposure. Hunters submitted blood
samples (serology) to test for presence of antibodies which indicated whether birds had been
exposed to WNV, and the heart was tested for presence of WNV genetic material by reverse
transcription polymerase chain reaction (rtPCR).
Serology rtPCR
County No. of samples Negative WNV Flavivirusa Negative WNV EEEV
b
Ashland 10 6 4 0 10 0 0
Barron 1 1 0 0 1 0 0
Bayfield 14 12 0 2 14 0 0
Burnett 6 5 1 0 6 0 0
Douglas 10 7 1 2 10 0 0
Florence 4 1 2 1 4 0 0
Forest 14 10 2 2 14 0 0
Iron 9 7 2 0 9 0 0
Langlade 7 2 3 2 7 0 0
Lincoln 14 11 1 2 14 0 0
Marinette 14 11 3 0 14 0 0
Oconto 1 0 0 1 1 0 0
Oneida 30 21 3 6 30 0 0
Polk 1 0 0 1 1 0 0
Price 45 33 9 3 43 1 1
Rusk 6 3 2 1 6 0 0
Sawyer 15 10 5 0 15 0 0
Taylor 8 6 2 0 8 0 0
Vilas 12 9 2 1 11 1 0
Washburn 14 12 2 0 12 0 2
Totals 235 167 44 24 230 2 3
a Flavivirus is a genus of viruses that includes West Nile virus. During lab testing, the antibodies present in some
blood samples were consistent with flavivirus and are likely to be West Nile virus (though not confirmed).
b During lab testing, 3 ruffed grouse heart samples tested positive for eastern equine encephalitis virus (EEEV),
which is also spread by the bite of a mosquito and is known to infect a wide range of animals including birds.
Table 2. Sex and age of hunter-submitted ruffed grouse samples (n = 235) collected in 2018
from northern Wisconsin, USA, and tested for West Nile virus (WNV) exposure. Hunters
submitted blood samples (serology) to test for presence of antibodies which indicated whether
birds had been exposed to WNV, and the heart was tested for presence of WNV genetic
material by reverse transcription polymerase chain reaction (rtPCR).
Serology rtPCR
Parameter Negative WNV Flavivirusa Negative WNV EEEV
b
Sex
Female 54 15 11 80 0 0
Male 73 24 9 102 1 3
Unknown sex 40 5 4 48 1 0
Age
Adult 68 19 10 95 1 1
Juvenile 81 21 12 111 1 2
Unknown age 18 4 2 24 0 0
a Flavivirus is a genus of viruses that includes West Nile virus. During lab testing, the antibodies present in some
blood samples were consistent with flavivirus and are likely to be West Nile virus (though not confirmed).
b During lab testing, 3 ruffed grouse heart samples tested positive for eastern equine encephalitis virus (EEEV),
which is also spread by the bite of a mosquito and is known to infect a wide range of animals including birds.
Table 3. Body condition of hunter-submitted ruffed grouse samples (n = 235) collected in 2018
from northern Wisconsin, USA, and tested for West Nile virus (WNV) exposure. Body condition
was evaluated by hunters after successfully harvesting a ruffed grouse and was determined by
the overall size of the breast muscles and whether the keel was visible. Hunters submitted
blood samples (serology) to test for presence of antibodies which indicated whether birds had
been exposed to WNV, and the heart was tested for presence of WNV genetic material by
reverse transcription polymerase chain reaction (rtPCR).
Serology rtPCR
Body condition Negative WNV Flavivirusa Negative WNV EEEV
b
Fat 8 5 2 15 0 0
Normal 151 38 22 207 2 2
Thin 2 0 0 2 0 0
Not indicated 6 1 0 6 0 1
a Flavivirus is a genus of viruses that includes West Nile virus. During lab testing, the antibodies present in some
blood samples were consistent with flavivirus and are likely to be West Nile virus (though not confirmed).
b During lab testing, 3 ruffed grouse heart samples tested positive for eastern equine encephalitis virus (EEEV),
which is also spread by the bite of a mosquito and is known to infect a wide range of animals including birds.
