---
id: "edwards-2022-lymphoproliferative"
title: "Molecular Surveillance for Lymphoproliferative Disease Virus and Reticuloendotheliosis Virus in Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) in Texas, USA"
authors:
  - "Faith Cox"
  - "Jason Hardin"
  - "Robert Dittmar"
  - "Dustin Edwards"
venue: "Journal of Wildlife Diseases"
year: 2022
date: "2022-11-30"
doi: "10.7589/JWD-D-22-00023"
url: "/research/publications/10-7589-jwd-d-22-00023/"
pdf: "/research/publications/10-7589-jwd-d-22-00023/dustin-edwards-10-7589-jwd-d-22-00023.pdf"
openAccess: false
citedBy: 6
citedBySource: "OpenAlex, read 2026-09-12"
---
# Molecular Surveillance for Lymphoproliferative Disease Virus and Reticuloendotheliosis Virus in Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) in Texas, USA

First detection of lymphoproliferative disease virus in Texas wild turkeys; 373 birds tested, about 4 percent positive in affected counties.

## Abstract

Reticuloendotheliosis virus (REV) and lymphoproliferative disease virus (LPDV) are avian retroviruses that can cause neoplastic disease and present with similar pathologies. Lymphoproliferative disease virus has been reported in the Eastern US and states bordering Texas, USA, but has not been previously detected within the state. In a prior study, we detected REV in native Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) and an Eastern Wild Turkey (Meleagris gallopavo silvestris) originating from West Virginia. Given LPDV detection in states bordering Texas and our finding of an REV-positive Eastern Wild Turkey imported from a LPDV endemic region, we sought to determine LPDV prevalence in Texas and continue surveillance for REV. During 2018-20, dried blood spots from 373 individual Rio Grande Wild Turkeys from 20 different counties were tested for the presence of proviral REV or LPDV DNA. In affected counties, approximately 4% of individuals were infected with REV (7/197) or LPDV (10/273) and one bird was coinfected with both viruses. Phylogenetic analysis indicated a close relationship of the LPDV isolates to variants from other Southern and Central states. This study provides molecular evidence of LPDV in Texas, and continued surveillance is necessary to determine the potential effects of the virus on reproductive success, coinfections, and overall health of Wild Turkey populations.

## Full text

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DOI: 10.7589/JWD-D-22-00023 Journal of Wildlife Diseases, 58(4), 2022, pp. 909–913
Ó Wildlife Disease Association 2022
Molecular Surveillance for Lymphoproliferative Disease Virus and
Reticuloendotheliosis Virus in Rio Grande Wild Turkeys (Meleagris
gallopavo intermedia) in Texas, USA
Faith Cox,1 Jason Hardin,2 Robert Dittmar,3,4 and Dustin Edwards1,5 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 S, Kerrville, Texas
78028, USA; 4Deceased; 5Corresponding author (email: dcedwards@tarleton.edu)
ABSTRACT: Reticuloendotheliosis virus (REV) and
lymphoproliferative disease virus (LPDV) are
avian retroviruses that can cause neoplastic
disease and present with similar pathologies.
Lymphoproliferative disease virus has been re-
ported in the Eastern US and states bordering
Texas, USA, but has not been previously detected
within the state. In a prior study, we detected
REV in native Rio Grande Wild Turkeys (Melea-
gris gallopavo intermedia) and an Eastern Wild
Turkey (Meleagris gallopavo silvestris) originating
from West Virginia. Given LPDV detection in
states bordering Texas and our finding of an REV-
positive Eastern Wild Turkey imported from a
LPDV endemic region, we sought to determine
LPDV prevalence in Texas and continue surveil-
lance for REV. During 2018–20, dried blood spots
from 373 individual Rio Grande Wild Turkeys
from 20 different counties were tested for the
presence of proviral REV or LPDV DNA. In
affected counties, approximately 4% of individuals
were infected with REV (7/197) or LPDV (10/
273) and one bird was coinfected with both
viruses. Phylogenetic analysis indicated a close
relationship of the LPDV isolates to variants from
other Southern and Central states. This study
provides molecular evidence of LPDV in Texas,
and continued surveillance is necessary to deter-
mine the potential effects of the virus on
reproductive success, coinfections, and overall
health of Wild Turkey populations.
Key words: Avian retrovirus, lymphoprolifer-
ative disease virus, reticuloendotheliosis virus,
Wild Turkeys.
Reticuloendotheliosis virus (REV) and lym-
phoproliferative disease virus (LPDV) are
avian retroviruses associated with neoplastic
disease (Fadly 1997). Reticuloendotheliosis
virus is an oncogenic gammaretrovirus that
infects B-lymphocytes of Galliformes, Anser-
iformes, and Passeriformes and can cause
immunosuppression (Nair et al. 2013; Ferro et
al. 2017). Clinical abnormalities associated
with REV infection include anemia, non-
neoplastic runting, lymphoma, and feathering
abnormalities (Nair et al. 2013). Lymphopro-
liferative disease virus, an alpharetrovirus,
infects Wild Turkeys (Meleagris gallopavo)
and is associated with lymphoid tumors
(Biggs et al. 1978). Reticuloendotheliosis
virus and LPDV are rarely associated with
clinical disease. However, retrovirus infec-
tions are associated with decreased repro-
ductive success, hatchability, and sexual
maturation in domestic poultry (Payne
1998; Wei et al. 2012). First identified in
the UK in 1972, LPDV has been credited
with neoplastic disease outbreaks in Europe
and Israel (Biggs et al. 1978; Ianconescu et
al. 1983). Lymphoproliferative disease virus
proviral DNA was first detected in the US in
2009 in a Wild Turkey in Arkansas and has
been detected in Wild Turkeys from 24 US
states, including Oklahoma (prevalence 26%;
7/27), Louisiana (57%; 57/96), and West
Virginia (55%; 26/47; Allison et al. 2014;
Thomas et al. 2015; Alger et al. 2017). In a
2016–17 survey, we identified five REV-
positive Rio Grande Wild Turkeys (Meleagris
gallopavo intermedia) and one REV-positive
Eastern Wild Turkey (Meleagris gallopavo
silvestris) imported from West Virginia
(Stewart et al. 2019). Given LPDV detection
in states bordering Texas and this finding of
an REV-positive imported Eastern Wild
Turkey, our objectives were to determine
REV and LPDV distribution and prevalence
in Texas, USA, continue surveillance for
REV, and add to our understanding of LPDV
host range within a subspecies of Wild
Turkeys.
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Outwardly healthy Rio Grande Wild Tur-
keys (adult¼272, juvenile¼63; female¼202,
male¼133; unknown age or sex¼38) were
captured from 20 different counties of Texas,
with walk-in funnel traps, drop nets, or rocket
nets by Texas Parks and Wildlife Department
(TPWD) biologists. Capture was authorized
by an Employee Scientific Collection Autho-
rization Permit as part of the TPWD Eastern
Wild Turkey restoration program. Dried
blood spots were collected on sampling cards
constructed from Whatman grade 3MM
paper (GE Healthcare, Chicago Illinois,
USA) prelabeled with a unique identification
number. Sterile lancets were used to puncture
the wing vein and blood was spotted on the
cards and allowed to dry for approximately 1
h. From each dried blood spot, 5-mm squares
were cut, mixed with 75-lL HotSHOT
alkaline lysis reagent, and incubated for 30
min at 95 C while shaking, then cooled on ice
for 5 min before the addition of HotSHOT
neutralization buffer (Truett et al. 2000).
Extracted genomic DNA was stored at 20
C until initial screening by quantitative PCR
(qPCR) assay for REV env and LPDV env
genes to test for viral infection and the pan-
avian GAPDH gene, which served as a DNA
extraction control. Reaction conditions were
optimized (see Supplementary Methods 1).
Assembled 20-lL reactions contained 500 nM
forward and reverse primer and 200 nM
probe for each target, 10 lL PrimeTimet
Gene Expression 2X MasterMix (Integrated
DNA Technologies, Coralville, Iowa, USA), 5
lL nuclease-free water, and 3 lL DNA in
each well. Standard PCR (see Supplementary
Methods 2) was used to verify samples that
were REV- or LPDV-positive by qPCR and
was assembled in 25-lL reactions using 5.5
lL nuclease-free water, 12.5 lL OneTaq Hot
Start 2X MasterMix (New England Biolabs,
Ipswitch, Massachusetts, USA), 2 lL of the
eluted DNA, and 1 lL of the forward and
reverse primers (200–400 nM final concen-
tration) targeting either the REV 30 long
terminal repeat, LPDV p31/CA, or pan-avian
GAPDH (Aly et al. 1993; Allison et al. 2014;
Olias et al. 2014). Detectable PCR products
were submitted to Texas A&M–Corpus Chris-
ti Genomics Core Laboratory (Corpus Christi,
Texas, USA) for Sanger sequencing (GenBank
accession no. OL960637–OL960653) using
the same forward and reverse primers. The
similarity in nucleotide sequence identity to
published REV and LPDV proviral sequences
was determined by BLASTn (Altschul et al.
1990).
Evolutionary analyses were conducted in
MEGA X (version 11.0.11) using the Maxi-
mum Likelihood method based on the Gen-
eral Time Reversible model (Nei and Kumar
2000; Kumar et al. 2018). Initial trees for the
heuristic search were obtained automatically
by applying Neighbor-Join and BioNJ algo-
rithms to a matrix of pairwise distances
estimated using the Maximum Composite
Likelihood approach and then selecting the
topology with a superior log-likelihood value.
A discrete gamma distribution was used to
model evolutionary rate differences among
sites (5 categories [þG, parameter¼0.4955]).
The initial analysis involved 195 nucleotide
sequences previously published from the US
and Israel (Allison et al. 2014; Thomas et al.
2015). Redundant sequences were removed
from the dataset and the remaining sequences
were re-analyzed. The final analysis involved
98 nucleotide sequences, with a total of 423
positions in the final dataset.
We identified seven REV-positive and 10
LPDV-positive birds (see Supplementary
Figs. S1 and S2). In affected counties, 7/197
(4%) of individuals were infected with REV
and 10/273 (4%) with LPDV (Table 1). Two
infected Wild Turkeys, one REV-positive and
the other LPDV-positive, were identified in
Cottle County within the Rolling Plains
ecoregion in North Texas. In the Edwards
Plateau ecoregion in Central Texas, we
identified four REV-infected and eight
LPDV-positive Wild Turkeys, as well as one
individual that was co-infected. Within South
Texas, we identified one REV-positive Wild
Turkey in Live Oak County. Of the REV-
positive Wild Turkeys, 5/7 (71%) were adults,
as were 9/10 (90%) of those infected with
LPDV. The sex ratio (female:male) was 5:2 for
REV-infected birds and 4:1 for LPDV-infect-
ed. Phylogenetic analysis revealed that most
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(7/10) LPDV p31/Ca sequences from Texas
grouped with sequences from other Southern
and Central states (Fig. 1), suggesting a
potential origin for LPDV strains from Texas.
Three sequences grouped separately from
previously published US sequences, while all
were distinct from the Israeli strain.
This study, our previous survey in 2016–17,
and a 2002 report indicate that REV is
circulating within Rio Grande Wild Turkey
populations in Texas (Peterson et al. 2002;
Stewart et al. 2019). Our current study reports
the largest detected geographic range of REV
in Wild Turkeys within Texas. In 2002, REV-
positive Wild Turkeys were identified in the
Edwards Plateau ecoregion (Peterson et al.
2002). During 2016–17, we identified addi-
tional REV-positive Wild Turkeys in the
Edwards Plateau and Rolling Plains ecore-
gions (Stewart et al. 2019). In this study, we
detected REV-positive Wild Turkeys not only
in these previously tested ecoregions, but also
in the South Texas Brush Country where
turkeys had not previously been tested for
REV (Ferro et al. 2017).
Our finding that LPDV is present in Texas
Wild Turkeys supports previous studies indi-
cating that LPDV is widespread across the
Eastern and Central US and that Central
states have lower prevalence than the North-
eastern, Mid-Atlantic, and Southeastern states
(Thomas et al. 2015; Alger et al. 2017). Wild
Turkey susceptibility to LPDV may vary by
subspecies (McDougall et al. 1978). Preva-
lence of LPDV correlates with Wild Turkey
subspecies, and Rio Grande Wild Turkeys are
the predominant subspecies in Texas, Okla-
homa, and Kansas as compared to the Eastern
Wild Turkey, which is most common in the
Northeastern, Mid-Atlantic, and Southeastern
US. A limitation of our study was the use of
dried blood spots, which may decrease assay
sensitivity as compared to whole blood
samples (Smit et al. 2014). We found that
adult female Wild Turkeys had a higher
proportion of LPDV and REV infections than
did juveniles or males. Other reports suggest-
ed that LPDV is more likely to be found in
adult birds and is fatal to young (4–16 wk)
birds. However, infection in young poultry
may be more prevalent than adult surveys
suggest, as a result of increased mortality and
scavenging of carcasses (Biggs et al. 1978;
McDougall et al. 1978; Thomas et al. 2015;
Niedringhaus et al. 2019). Although we
detected the majority of LPDV infections in
females, evidence of sex as a predictor of
infection varies (Alger et al. 2017; Niedring-
haus et al. 2019). While subclinical infections
with LPDV may not pose an immediate
threat, the potential effects on reproductive
success, coinfections, and the overall health of
Wild Turkey populations warrant further
surveillance.
We thank the Texas Parks and Wildlife
Department biologists that collected the Wild
Turkey blood samples, Nicole Nemeth for LPDV-
TABLE 1. Reticuloendotheliosis virus (REV) and
lymphoproliferative disease virus (LPDV) prevalence
in Rio Grande wild turkeys (Meleagris gallopavo
intermedia; n¼373) in Texas, USA, by county. Percent
positive and frequency was determined by the number
of REV- and LPDV- positive samples divided by the
total number of samples.
County
REV LPDV
Percent
positive Frequency
Percent
positive Frequency
Bell 0 0/11 0 0/11
Comal 0 0/2 50 1/2
Cottle 1 1/97 1 1/97
Gillespie 0 0/43 2.3 1/43
Karnes 0 0/4 0 0/4
Kendall 10 1/10 20 2/10
Kerr 0 0/36 2.8 1/36
La Salle 0 0/5 0 0/5
Lampasas 0 0/19 0 0/19
Live Oak 20 1/5 0 0/5
Llano 0 0/2 0 0/2
Mason 0 0/19 0 0/19
McMullen 0 0/1 0 0/1
Menard 0 0/8 0 0/8
Navarro 0 0/3 0 0/3
Palo Pinto 0 0/6 0 0/6
Schleicher 4.7 4/85 4.7 4/85
Shackelford 0 0/5 0 0/5
Williamson 0 0/3 0 0/3
Wise 0 0/8 0 0/8
Unknown 0 0/1 0 0/1
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positive control DNA, and Camila Romano for
technical support. This work was supported by
Tarleton State University Undergraduate Re-
search Assistantships and Student Research
Grants.
SUPPLEMENTARY MATERIAL
Supplementary material for this article is online
at http://dx.doi.org/10.7589/JWD-D-22-00023.
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