---
id: "edwards-2019-texas-survey"
title: "Survey of Reticuloendotheliosis Virus in Wild Turkeys (Meleagris gallopavo) in Texas, USA"
authors:
  - "Brittany Stewart"
  - "Camille Trautman"
  - "Faith Cox"
  - "Heidi Spann"
  - "Jason Hardin"
  - "Robert Dittmar"
  - "Dustin Edwards"
venue: "Journal of Wildlife Diseases"
year: 2019
date: "2019-07-09"
doi: "10.7589/2018-08-187"
url: "/research/publications/10-7589-2018-08-187/"
pdf: "/research/publications/10-7589-2018-08-187/dustin-edwards-10-7589-2018-08-187.pdf"
openAccess: false
citedBy: 15
citedBySource: "OpenAlex, read 2026-09-12"
---
# Survey of Reticuloendotheliosis Virus in Wild Turkeys (Meleagris gallopavo) in Texas, USA

Survey of 393 wild turkeys in Texas for reticuloendotheliosis virus after an outbreak at Fossil Rim; 5 percent positive in affected counties.

## Abstract

Reticuloendotheliosis virus (REV) is an immunosuppressive and sometimes oncogenic avian retrovirus that establishes lifelong infection in a wide range of avian species. REV-infected wild birds roaming near at-risk captive flocks, such as is the case for the highly endangered Attwater's Prairie Chicken (APC; Tympanuchus cupido attwateri ), could act as a reservoir for viral transmission. In wild birds, prevalence rates of REV are low and appearance of associated disease is uncommon. During 2016-17, nearly half of all captive adult APC mortality at Fossil Rim Wildlife Center captive breeding facility in Glen Rose, Texas, US was attributed to REV infection. The unusually high REV prevalence rate prompted us to survey for this virus in wild galliforms throughout the region. From 2016-17, 393 blood samples collected from two subspecies of Wild Turkeys ( Meleagris gallopavo ) were tested for REV proviral DNA through amplification of the viral 3' long terminal repeat and segments of the viral pol gene. In REV-affected counties, 5% (5/98) of native Rio Grande Wild Turkeys ( Meleagris gallopavo intermedia ) were identified as REV-positive. In addition, we detected REV in one of 62 Eastern Wild Turkeys ( Meleagris gallopavo silvestris ) that had been imported during conservation efforts. To better determine protective measures, continued surveillance, including collection and genetic analysis of REV-infected samples, is necessary to identify sources of REV outbreaks in captive APC flocks.

## Full text

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DOI: 10.7589/2018-08-187 Journal of Wildlife Diseases, 55(3), 2019, pp. 000–000
Ó Wildlife Disease Association 2019
Survey of Reticuloendotheliosis Virus in Wild Turkeys (Meleagris
gallopavo) in Texas, USA
Brittany Stewart,1 Camille Trautman,1 Faith Cox,1 Heidi Spann,1 Jason Hardin,2 Robert Dittmar,3 and
Dustin Edwards1,4 1Tarleton State University, Department of Biological Sciences, Box T-0100, Stephenville, Texas
76402, USA; 2Texas Parks and Wildlife Department, 2706 W. Commerce, Buffalo, Texas 75831, USA; 3Texas Parks and
Wildlife Department, 309 Sidney Baker South, Kerrville, Texas 78028, USA; 4Corresponding author (email: dcedwards@
tarleton.edu)
ABSTRACT: Reticuloendotheliosis virus (REV) is
an immunosuppressive and sometimes oncogenic
avian retrovirus that establishes lifelong infection
in a wide range of avian species. REV-infected
wild birds roaming near at-risk captive flocks,
such as is the case for the highly endangered
Attwater’s prairie chicken (Tympanuchus cupido
attwateri; APC), could act as a reservoir for viral
transmission. In wild birds, prevalence rates of
REV are low and appearance of associated disease
is uncommon. During 2016–17, nearly half of all
captive adult APC mortality at Fossil Rim Wildlife
Center captive breeding facility in Glen Rose,
Texas, USA was attributed to REV infection. The
unusually high REV prevalence rate prompted us
to survey for this virus in wild galliforms
throughout the region. From 2016–17, 393 blood
samples collected from two subspecies of wild
turkeys (Meleagris gallopavo) were tested for
REV proviral DNA through amplification of the
viral 30 long terminal repeat and segments of the
viral pol gene. In REV-affected counties, 5% (5/
98) of native Rio Grande wild turkeys (Meleagris
gallopavo intermedia) were identified as REV-
positive. In addition, we detected REV in one of
62 eastern wild turkeys (Meleagris gallopavo
silvestris) that had been imported during conser-
vation efforts. To better determine protective
measures, continued surveillance, including col-
lection and genetic analysis of REV-infected
samples, is necessary to identify sources of REV
outbreaks in captive APC flocks.
Key words: Attwater’s prairie chickens, avian
retrovirus, dried blood spots, reticuloendotheliosis
virus, wild turkeys.
Reticuloendotheliosis virus (REV) is an
immunosuppressive and sometimes oncogenic
avian retrovirus that establishes lifelong infec-
tion in Galliformes, Anseriformes, and Passer-
iformes (Ferro et al. 2017). The virus infects
B-lymphocytes, and infected birds can exhibit
clinical abnormalities such as anemia, neopla-
sia, nonneoplastic runting, lymphoma, feath-
e r i n g a b n o r m a l i t y , a n d h e i g h t e n e d
susceptibility to coinfections. However, infec-
tion is rarely associated with clinical disease in
wild birds (Nair et al. 2013). The prevalence
of REV in the wild has been shown to be
generally low, but infected wild birds, such as
wild turkeys (Meleagris gallopavo), roaming
near captive locations could act as reservoirs
for viral transmission (Peterson et al. 2002;
Ingram et al. 2015; Ferro et al. 2017).
Attwater’s prairie chickens (Tympanuchus
cupido attwateri; APC) at the Fossil Rim
Wildlife Center (FRWC) captive breeding
facility in Glen Rose, Texas, USA are tested by
quantitative PCR for REV semiannually
before setting breeding pairs in January and
after breeding season in June. During 2016–
17, nearly half of all adult APC mortality was
attributed to REV infections at this facility,
and most infected individuals were euthanized
before symptoms appeared. The unusually
high REV incidence within the endangered
APC population prompted us to survey the
region for evidence of this virus in related wild
turkeys.
Both Rio Grande wild turkeys (Meleagris
gallopavo intermedia) and eastern wild tur-
keys (Meleagris gallopavo silvestris) have been
found to be infected by REV (Ley et al. 1989;
Hayes et al. 1992; Peterson et al. 2002).
Eastern wild turkeys infected with REV have
been observed in close contact with domestic
poultry, the probable source of infection to
wild birds because domestic flocks have
occasionally been administered REV-contam-
inated fowlpox and Marek’s disease vaccines
(Hayes et al. 1992; Nair et al. 2013). Wild
turkeys were nearly extirpated from Texas by
the early 1900s as a result of overhunting,
habitat degradation, and disease (Rocke and
Yuill 1987; Peterson et al. 2002). Restoration
1

efforts have involved translocation of turkeys
from areas of higher concentration to other
portions of the USA (Van Why et al. 2001).
The ‘‘Super-Stocking’’ plan of translocating
240 individuals to restore eastern wild turkey
populations in Texas is an example of this
effort (Alldredge et al. 2014). Translocated
birds are tested for Mycoplasma gallisepticum,
Mycoplasma synoviae, Mycoplasma meleagri-
dis, Salmonella pullorum, Salmonella typhi-
murium, Newcastle disease virus, and avian
influenza virus prior to transport, but testing
for REV is not standard (Charlton 2000). A
study conducted in 2002 identified two REV-
infected Rio Grande wild turkeys within Kerr
County in central Texas; however, there have
been no published reports of testing for REV
in eastern wild turkeys imported into the state
(Peterson et al. 2002). Our purpose was to
analyze the current occurrence and location of
REV in both Rio Grande and imported
eastern wild turkeys in Texas.
Samples were collected from 15 January
2016 to 10 March 2016 and again from 25
January 2017 to 1 April 2017. Wild turkeys
were captured using a combination of walk-in
funnel traps, drop nets, and rocket nets by
state wildlife agency staff authorized by an
Employee Scientific Collection Authorization
Permit as part of the Texas Parks and Wildlife
Department eastern wild turkey restoration
program and statewide banding efforts to
assess harvest rates in Texas. Texas Parks
and Wildlife Department biologists in 17
Texas counties collected blood on sampling
cards constructed from Whatman grade 3MM
paper (GE Healthcare, Chicago, Illinois,
USA) prelabeled with a unique identification
number. The wing-vein was punctured using a
sterile lancet and blood was spotted onto a
blood collection card and allowed to dry for 1
hr. Wild turkeys involved in this study were
released alive and unharmed. Additional dried
blood spot samples were prepared from APCs
originating at FRWC and Abilene Zoological
Gardens (Abilene, Texas, USA) previously
tested by Texas A&M Veterinary Medical
Diagnostic Laboratory (College Station, Tex-
as, USA) and used as positive (APC B14125)
and negative (APC 2909) controls for the
presence of REV proviral DNA. Five-milli-
meter squares were cut from each dried blood
spot and washed with 1 mL of phosphate-
buffered saline with 0.1% Tween 20 (PBS-T;
Bio-Rad, Hercules, California, USA) in a 1.7
mL tube while rocking. The PBS-T solution
was aspirated and 75 lL of HotSHOT alkaline
lysis reagent was added for genomic DNA
extraction (Truett et al. 2000). The samples
were incubated at 95 C for 30 min and then
cooled on ice for 5 min before addition of 75
lL of HotSHOT neutralization solution.
Samples were centrifuged for 10 s and the
supernatant from each was transferred to a
new microcentrifuge tube. Samples were
assembled for PCR in 25 ll reactions, using
5.5 ll nuclease-free water, 12.5 ll OneTaq
Hot Start 2X Master Mix (New England
Biolabs, Ipswich, Massachusetts, USA), 1 ll
each of forward and reverse primers (10 lM
starting concentration), and 5 ll of the eluted
DNA sample (see Supplementary Materials).
We used REV-specific forward and reverse
primers that targeted the virus 3 0 long
terminal repeat (LTR) to amplify proviral
DNA by PCR (Aly et al. 1993). Pan-avian
GAPDH DNA was amplified as a genomic
DNA extraction control (Olias et al. 2014).
For samples with detectable REV 30 LTR
PCR products, we amplified REV pol gene
segments 2500–3075 and 4777–5575 by a
touchdown PCR cycle (Barbosa et al. 2007).
All PCR products were resolved using gel
electrophoresis in a 2% agarose gel with Tris
Borate EDTA buffer (see Supplementary
Materials). The PCR products of REV pol
gene segments from identified REV-positive
samples were sequenced, using the same
forward and reverse primers, at Texas A&M
University Corpus Christi Genomics Core
Laboratory (Corpus Christi, Texas, USA).
Similarity to published REV proviral sequenc-
es from APC (GenBank DQ387450) and
turkey (GenBank KJ756349) samples was
determined by BLASTn and sequences were
aligned for analysis of nucleotide variation
using MEGA X software (Altschul et al. 1990;
Kumar et al. 2018).
A total of 331 Rio Grande wild turkeys were
tested: 155 samples were from 15 Texas
2 JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 3, JULY 2019

counties in 2016 and 176 samples were from
12 Texas counties in 2017 (Table 1). In
affected counties, REV was detected in 5%
(5/98) of wild turkeys. One REV-positive bird
(TSU 0208) was sampled in 2016 in Mason
County, and four additional REV-positive
individuals were identified in 2017 from
Cottle (TSU 0079), Gillespie (TSU 0580 and
1129), and Mason (TSU 1089) counties. Fifty
eastern wild turkeys were surveyed during
2016, with 45 imported from Iowa, USA and
five from West Virginia, USA. In the 2016
cohort, one REV-positive bird (TSU 0015)
was detected and originated from West
Virginia. The 2017 cohort included 12 eastern
wild turkeys (10 imported from Missouri, USA
and two imported from West Virginia, USA),
none of which tested positive for REV. All
imported birds appeared in good health and
tested negative for avian influenza virus and
Pullorum-Typhoid at the Iowa State Univer-
sity Veterinary Diagnostic Laboratory (Ames,
Iowa, USA) or Texas A&M Veterinary Med-
ical Diagnostic Laboratory prior to transport
by air to Dallas-Fort Worth International
Airport. Detection of REV proviral sequence
in positive samples was further confirmed by
sequencing and analysis of PCR-amplified
R E V p o l g e n e s e g m e n t s ( G e n B a n k
MK033877–MK033890). The REV sequences
from the eastern wild turkey sample had
nucleotide variations, as compared to refer-
ence sequences, at genome nucleotides
2762G.A, 2728G.A, 4924C.T, and
5198G.A.
Our purpose was to further understand the
prevalence of REV in wild turkeys in Texas,
both native and imported, and their potential
role in REV infections in at-risk captive
populations. Although REV is thought to be
a low-level health threat to wild birds, infected
birds can be a source of infection for the
highly endangered Attwater’s prairie chicken.
A recent study, conducted near the Attwater
Prairie Chicken National Wildlife Refuge,
which is bordered by Gulf Prairies and
Marshes and Post Oak Savannah ecoregions
in south Texas, showed REV to be at low
prevalence in 32 species of birds within close
proximity of a free-range APC flock (Ferro et
al. 2017). In this study, none of the wild
turkeys from these regions tested positive for
REV. Our study detected four infected Rio
Grande wild turkeys in the Edwards Plateau
and Llano Uplift ecoregions in central Texas,
counties contiguous to an area previously
determined to contain REV-infected wild
turkeys (Peterson et al. 2002). This is also
the first report of a REV-infected Rio Grande
wild turkey in the Rolling Plains ecoregion in
north Texas, which is near captive APCs at the
TABLE 1. Results of reticuloendotheliosis virus by
Texas county and year of reticuloendotheliosis virus
proviral DNA PCR testing of dried blood spot samples
collected from Rio Grande wild turkeys (Meleagris
gallopavo intermedia; n¼331). Percent positive and
frequency was determined by the number of REV-
positive samples divided by the total number of
samples.
County Year
Reticuloendotheliosis virus
Percent positive Frequency
Bell 2016 0 0/12
Blanco 2016 0 0/3
Brooks 2016 0 0/2
Comal 2016 0 0/2
Cottle 2016 0 0/33
Gillespie 2016 0 0/11
Hodges 2016 0 0/4
Jones 2016 0 0/2
Kendall 2016 0 0/3
Lampasas 2016 0 0/16
Llano 2016 0 0/22
Mason 2016 3 1/30
San Saba 2016 0 0/1
Scurry 2016 0 0/5
Williamson 2016 0 0/3
Unknown 2016 0 0/6
Bell 2017 0 0/8
Blanco 2017 0 0/24
Childress 2017 0 0/4
Cottle 2017 13 1/8
Gillespie 2017 4 2/48
Jones 2017 0 0/15
Kent 2017 0 0/5
Kerr 2017 0 0/20
Llano 2017 0 0/29
Mason 2017 8 1/12
Menard 2017 0 0/2
Scurry 2017 0 0/1
STEWART ET AL.—SHORT COMMUNICATIONS 3

Abilene Zoological Gardens. Conservation
efforts would benefit from continued surveil-
lance for REV-positive wild birds, both
resident and migratory, within reachable
proximity to APCs, and further genetic
analysis to determine if the viral strains found
in wild birds match those in captive flocks. We
also report the first case of a REV-positive
eastern wild turkey that was imported into the
Piney Woods ecoregion of Texas for conser-
vation purposes. Sequence alignments of the
well-conserved REV pol gene demonstrated a
nucleotide difference in the sample that
originated from West Virginia compared to
those in Texas and could be used to determine
the origin of new infections within the region.
Although REV is currently infrequently de-
tected in the wild, areas in which potentially
infected eastern wild turkeys might be im-
ported could be at risk for introduction of
REV or increased transmission. Additional
studies are necessary to determine whether
imported birds should be REV-tested prior to
transport. Future regional surveys of wild
turkeys for other emerging retroviruses, in-
cluding lymphoproliferative disease virus,
which has similar pathology that is nearly
indistinguishable from REV, would be valu-
able in APC and eastern wild turkey conser-
vation efforts (Allison et al. 2014).
We thank the Texas Parks and Wildlife
Department biologists who collected the
wild turkey blood samples, Fossil Rim
Wildlife Center, Abilene Zoological Gar-
dens, and US Fish and Wildlife Service for
Attwater’s prairie chicken blood samples,
and P. Sudman, H. Haefele, J. Swenson,
and P. Ferro for their technical guidance.
This work was a collaboration between
Tarleton State University and Texas Parks
and Wildlife Department with support from
Tarleton State University Undergraduate
Research Assistantships and Student Re-
search Grants.
SUPPLEMENTARY MATERIAL
Supplementary material for this article is online
at http://dx.doi.org/10.7589/2018-08-187.
LITERATURE CITED
Alldredge BE, Hardin JB, Whiteside J, Isabelle JL,
Parsons S, Conway WC, Cathey JC. 2014. Eastern
wild turkeys in Texas: Biology and management.
Texas A&M AgriLife Extension WF-011. Texas A&M
University, College Station, Texas, 20 pp.
Allison AB, Keel MK, Philips JE, Cartoceti AN, Munk
BA, Nemeth NM, Welsh TI, Thomas JM, Crum JM,
Lichtenwalner AB, et al. 2014. Avian oncogenesis
induced by lymphoproliferative disease virus: A
neglected or emerging retroviral pathogen? Virology
450–451:2–12.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ.
1990. Basic local alignment search tool. J Mol Biol
215:403–410.
Aly MM, Smith EJ, Fadly AM. 1993. Detection of
reticuloendotheliosis virus infection using the poly-
merase chain reaction. Avian Pathol 22:543–554.
Barbosa T, Zavala G, Cheng S, Villegas P. 2007. Full
genome sequence and some biological properties of
reticuloendotheliosis virus strain APC-566 isolated
from endangered Attwater’s prairie chickens. Virus
Res 124:68–77.
Charlton KG. 2000. Antibodies to selected disease agents
in translocated wild turkeys in California. J Wildl Dis
36:161–164.
Ferro PJ, Morrow ME, Flanagan JP, Ortego B, Chester
RE, Mueller JM, Lupiani B. 2017. Wild birds, a
source of reticuloendotheliosis virus infection for the
endangered Attwater’s prairie-chicken (Tympanu-
chus cupido attwateri)? J Wildl Dis 53:586–590.
Hayes LE, Langheinrich KA, Witter RL. 1992. Reticulo-
endotheliosis in a wild turkey (Meleagris gallopavo)
from coastal Georgia. J Wildl Dis 28:154–158.
Ingram DR, Miller DL, Baldwin CA, Turco J, Lockhart
JM. 2015. Serologic survey of wild turkeys (Meleagris
gallopavo) and evidence of exposure to avian
encephalomyelitis virus in Georgia and Florida,
USA. J Wildl Dis 51:374–379.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018.
MEGA X: Molecular evolutionary genetics analysis
across computing platforms. Mol Biol Evol 35:1547–
1549.
Ley DH, Ficken MD, Cobb DT, Witter RL. 1989.
Histomoniasis and reticuloendotheliosis in a wild
turkey (Meleagris gallopavo) in North Carolina. J
Wildl Dis 25:262–265.
Nair V, Zavala G, Fadly AM. 2013. Reticuloendotheliosis.
In: Diseases of poultry, 13th Ed, Swayne DE, Glisson
JR, McDougald LR, Nolan JV, Suarez DL, and Nair
V, editors. Wiley-Blackwell, Ames, Iowa. pp. 593–
604.
Olias P, Adam I, Meyer A, Scharff C, Gruber AD. 2014.
Reference genes for quantitative gene expression
studies in multiple avian species. PLoS One 9:
e99678.
Peterson MJ, Aguirre R, Ferro PJ, Jones DA, Lawyer TA,
Peterson MN, Silvy NJ. 2002. Infectious disease
4 JOURNAL OF WILDLIFE DISEASES, VOL. 55, NO. 3, JULY 2019

survey of Rio Grande wild turkeys in the Edwards
Plateau of Texas. J Wildl Dis 38:826–833.
Rocke TE, Yuill TM. 1987. Microbial infections in a
declining wild turkey population in Texas. J Wildl
Manag 51:778–782.
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA,
Warman ML. 2000. Preparation of PCR-quality
mouse genomic DNA with hot sodium hydroxide
and tris (HotSHOT). Biotechniques 29:52–54.
Van Why KR, Applegate RD, Cable TT, Gipson PS.
2001. Survey of Kansas wild turkey hunters:
Experiences, opinions, and satisfactions. Kansas
State University, Agriculture Experiment Station
and Cooperative Extension Service, Manhattan,
Kansas. 27 pp.
Submitted for publication 1 August 2018.
Accepted 29 October 2018.
STEWART ET AL.—SHORT COMMUNICATIONS 5