Histomonosis and Lymphoproliferative Disease Virus in Male Wild Turkeys ( Meleagris gallopavo ) in Alabama, USA Kevin N. Ostrander, 1,5 Matthew S. Day, 1,2 R € udiger Hauck, 3,4 Kellye S. Joiner, 4 and William D. Gulsby 1 1 College of Forestry, Wildlife and Environment, Auburn University, 602 Duncan Drive, Auburn, Alabama 36849, USA 2 West Virginia Department of Environmental Protection, 22288 Northeastern Pike, Romney, West Virginia 26757, USA 3 Department of Poultry Science, College of Agriculture, Auburn University, 260 Lem Morrison Drive, Auburn, Alabama 36849, USA 4 Department of Pathobiology, College of Veterinary Medicine, Auburn University, 1130 Wire Road, Auburn, Alabama 36849, USA 5 Corresponding author (email: kevin.ostrander@auburn.edu) ABSTRACT : Wild Turkeys ( Meleagris gallopavo ) are an important game species in the USA and have experienced population declines in many areas of their range for > 10 yr. Among other hypotheses, increased disease prevalence or novel disease emergence could be contributing factors in Wild Turkey population declines. To address some knowledge gaps and further understand the impacts of two important diseases on Wild Turkey populations, we sought to document the prevalence of histomonosis and lymphoproliferative disease virus (LPDV) in Alabama, USA, and to evaluate the spatial epidemiology of LPDV. We collected hunter-harvested Wild Turkey carcasses and paired observational surveys across Alabama during the 2022 and 2023 spring hunting seasons. During necropsies we collected the ceca, which was frozen and stored at − 20°C, and the spleen, which was stored in 70 % ethanol at 20°C or frozen and stored at − 20°C. We screened cecal walls for Histomonas meleagridis DNA and spleens for LPDV proviral DNA by using quantitative PCR and PCR, respectively. We detected H. meleagridis , the disease-causing protozoan for histomonosis, in 0.7 % (3/ 435) of our samples. We detected LPDV proviral DNA in 88.7 % (416/469) of our sample of frozen spleens. Our results suggest that evaluation of the impact of histomonosis on Wild Turkey populations is dif fi cult through active surveillance alone. We detected proviral LPDV DNA in Wild Turkeys from nearly every county sampled in Alabama (53 of 56 counties); however, a generalized linear mixed model did not reveal a statistically signi fi cant relationship between LPDV and land cover type. Our fi ndings demonstrate that LPDV is widely distributed with high rates of prevalence in Alabama. Because the effects of these two diseases and others on Wild Turkey population vital rates have not been well established, further work is warranted. Key words: Active surveillance, avian pathology, Meleagris gallopavo , spatial epidemiology. INTRODUCTION Wild Turkeys ( Meleagris gallopavo ) are a culturally and economically important game species throughout their range (Dickson 2001; Isabelle et al. 2018; US Fish and Wildlife Ser- vice, US Census Bureau 2018; Chapagain et al. 2020). Multiple long-term datasets indi- cate that Wild Turkey populations in the southeastern USA have declined for > 10 yr, and concerns from primary stakeholders include declining recruitment rates and declining hunter harvest (Byrne et al. 2016; Isabelle et al. 2018). Although several working hypotheses have been proposed to explain all or part of these declines, the relative contribution of dis- eases is not well understood. One hypothesis is that increased disease prevalence, or the emer- gence of novel diseases, may be associated with population declines. Two diseases that have received signi fi cant attention in the recent literature are histomo- nosis and lymphoproliferative disease virus (LPDV). Histomonosis, also known as black- head disease, is caused by the parabasalid pro- tozoan Histomonas meleagridis (Davidson and Wentworth 1992) and typically causes necro- sis with fulminant in fl ammation in the ceca and liver. Advanced histomonosis can be asso- ciated with necrosis in multiple organ systems (Davidson 2008; Clark and Kimminau 2017). When infected with H. meleagridis , Wild Tur- keys experience acute clinical signs, likely fol- lowed by mortality (Davidson 2008). Spillover 1 DOI: 10.7589/JWD-D-24-00150 Journal of Wildlife Diseases , 00(00), 2025, pp. 000 – 000 Ó Wildlife Disease Association 2025 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 events into Wild Turkey populations have often been linked to domestic chickens ( Gal- lus domesticus ; Davidson 2008; Cupo 2018). When domestic chickens are infected with H. meleagridis , they typically survive infection and do not show clinical signs (McDougald 2005). This asymptomatic infection enables H. meleagridis to complete its lifecycle and infect an intermediate host, the cecal worm Heterakis gallinarum , before being shed into the environment (Davidson 2008). To survive outside a primary host, H. meleagridis must infect its cecal worm intermediate host. Wild Turkeys are infected by H. meleagridis when they consume a cecal worm – infected paratenic host, typically an earthworm (McDougald 2005; Davidson 2008; Gerhold et al. 2011). Accord- ingly, some researchers have suspected that the use of commercial and noncommercial chicken litter as a fertilizer contributes to H. meleagridis infection in Wild Turkey populations (Waters et al. 1994). Previous research has found that his- tomonosis does occur in Wild Turkey popula- tions (passive surveillance studies with ≥ 50 samples: 5.4 – 10.2 % prevalence; Table 1). How- ever, active surveillance data documenting H. meleagridis infection remain unavailable; therefore, persistent knowledge gaps exist about histomonosis, including regarding its spatial epi- demiology (MacDonald et al. 2022). The oncogenic avian retrovirus LPDV (Payne and Venugopal 2000) is sometimes fatal to Wild Turkeys. This retrovirus can pro- duce fatal lymphoid tumors in internal organs such as the spleen and liver (Biggs et al. 1978; Allison et al. 2014). Previous studies with ≥ 50 samples by using active surveillance strategies have found LPDV prevalences across the Wild Turkey ’ s range from 29 % to 82 % , although documented prevalences tend to have been higher in the northeastern USA and Canada and lower in the southeastern and western USA (Table 2). These data have led some to hypothe- size that prevalence increases latitudinally (Thomas et al. 2015; Kreh and Palamar 2022) or may be in fl uenced by landscape composition. For example, in New York, USA, Alger et al. (2017) found a positive relationship between LPDV prevalence and the agriculture-to-forest land cover ratio and Shea et al. (2022) found that the odds of LPDV infection increased with percent forest cover in winter home ranges in Maine, USA. However, relationships between LPDV and environmental factors such as land- scape composition remain unexplored in the lit- erature for the southeastern USA. It is also poorly understood how frequently clinical signs occur from LPDV infection (MacDonald et al. 2022), or how it affects population vital rates. To address infection path- ways, researchers recently inoculated domestic turkey ( Meleagris gallopavo domestica ) poults with North American LPDV strains from Wild Turkeys and reported a 100 % detection rate of lymphoid aggregates at euthanasia (Goodwin et al. 2024). However, LPDV detection rates fl uctuated over the 12-wk period and most of the poults ( n = 21) did not have clinical signs before euthanasia (Goodwin et al. 2024). These data show that LPDV can become widespread within populations, but individual and popula- tion-level effects remain uncertain. Other evi- dence suggests that LPDV infection may reduce clutch size in females (Shea 2021), a par- ticularly concerning fi nding given that decreas- ing recruitment has been widely cited as the primary factor driving Wild Turkey population declines (Byrne et al. 2016; Londe et al. 2023). Collectively, these data demonstrate that LPDV can rapidly spread among individuals (Goodwin et al. 2024) and reach high prevalence in popu- lations (Table 2); however, knowledge gaps about the spatial epidemiology and larger impacts of LPDV remain. Despite the possibility that histomonosis or LPDV may be in fl uencing Wild Turkey demography, active surveillance for either dis- ease has never been conducted in Alabama, USA, where spring Wild Turkey harvest per hunter declined by > 40 % from 2004 to 2019 (Tapley et al. 2005; Chamberlain et al. 2022). Furthermore, studies documenting the spatial epidemiology of common diseases impacting Wild Turkey populations in Alabama are absent in the literature. These data would enable area-speci fi c and land cover – based risk assessments for both pathogens. Therefore, 2 JOURNAL OF WILDLIFE DISEASES, VOL. 00, NO. 00, MONTH 2025 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 our fi rst objective was to determine the preva- lence and distribution of histomonosis and LPDV in male Wild Turkeys in Alabama. Our second objective was to test for morphometric differences between infected and uninfected hunter-harvested male Wild Turkeys. We pre- dicted that our active surveillance strategy would enable detection of H. meleagridis in hunter-harvested male Wild Turkeys at simi- lar or lower rates than have been previously reported in the literature, due to the high mortality rate of histomonosis (Davidson 2008) and the low reported prevalences from passive surveillance strategies (Table 1). We predicted that LPDV prevalence in Alabama would be lower than has been reported from similar studies at the Wild Turkey ’ s northern range extent (Alger et al. 2017; Shea et al. 2022), based on previous suggestions that LPDV prevalence tends to be positively corre- lated with latitude (Thomas et al. 2015; Kreh and Palamar 2022). MATERIALS AND METHODS Study area We collected hunter-harvested male Wild Tur- key carcasses from across Alabama during the 2022 and 2023 spring hunting seasons. The spring hunting season during our study period opened 1 wk earlier on private land than on public land and varied according to location within the state (Alabama Department of Conservation and Natu- ral Resources 2021, 2022). Speci fi cally, the season on private land began on 25 March in zones 1 and 3 but on 1 April in zone 2. Zone 2 included the following counties: Colbert, Franklin, Lauderdale, Lawrence, Limestone, Madison, Morgan, and Winston (Fig. 1). Zone 2 also included the area in T ABLE 1. Prevalence rates of Histomonas meleagridis and Heterakis gallinarum in Wild Turkeys ( Meleagris gallopavo ) from North American active and passive observational studies. Study Surveillance years Study area a Organism Prevalence ( % ) n b Maxfield et al. 1963 c 1960 – 61 Alabama, Arkansas, Flor- ida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, South Carolina, Tennessee, Virginia H. gallinarum 62.3 243/390 Hurst 1980 d 1978 – 79 Mississippi H. meleagridis 100 3/3 Davidson et al. 1985 d 1972 – 84 Alabama, Arkansas, Florida, Georgia, South Carolina, Tennessee, Virginia, West Virginia H. meleagridis 10.2 11/108 Ley et al. 1989 d 1988 North Carolina H. meleagridis 100 1/1 Thogmartin et al. 1999 d 1992 – 97 Arkansas H. meleagridis 10.5 2/19 Oates et al. 2005 c 1997 – 98 Nebraska H. gallinarum 9.4 10/106 Martinez-Guerrero et al. 2010 c 2007 Durango H. gallinarum 76.2 16/21 MacDonald et al. 2016 d 1992 – 2014 Ontario H. meleagridis 5.4 3/56 Greenawalt et al. 2020 c,d 2015 – 18 Pennsylvania H. gallinarum 60.4 55/91 MacDonald et al. 2022 d 2008 – 18 Pennsylvania H. meleagridis 5.8 7/121 MacDonald et al. 2022 c 2013 – 18 Pennsylvania H. gallinarum 61.9 52/84 Adcock et al. 2024 c 2018 – 21 Multiple e H. meleagridis 34.6 9/26 a States in the USA except for Durango (a state in Mexico) and Ontario (a province in Canada). b n = number detected/number sampled. c Samples were collected with an active surveillance strategy, as defined by MacDonald et al. (2022). d Samples were collected with a passive surveillance strategy, as defined by MacDonald et al. (2022). e Multiple = multiple USA states. OSTRANDER ET AL.—HISTOMONOSIS AND LPDV IN ALABAMA ’ S MALE WILD TURKEYS 3 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 T ABLE 2. Lymphoproliferative disease virus (LPDV) prevalence in Wild Turkeys ( Meleagris gallopavo ) from previous research, with region-wide prevalence calculated from studies with ≥ 50 samples and USA region clas- sifications based on Adcock et al. (2024). Region Study a Study area Prevalence ( % ) n b Canada MacDonald et al. 2019a c Manitoba 30.8 20/65 MacDonald et al. 2019a c Québec 100.0 4/4 MacDonald et al. 2019b c,d Ontario 65.0 119/183 Total 56.0 139/248 Northeast USA Thomas et al. 2015 d Maine 82.0 50/61 Thomas et al. 2015 d Massachusetts 55.6 5/9 Thomas et al. 2015 d New Hampshire 83.3 25/30 Thomas et al. 2015 d New Jersey 45.8 22/48 Thomas et al. 2015 d New York 48.4 132/273 Thomas et al. 2015 d Rhode Island 33.3 3/9 Thomas et al. 2015 d Vermont 71.4 20/28 Thomas et al. 2015 d West Virgina 55.3 26/47 Alger et al. 2017 d New York 55.0 1,396/2,538 Niedringhaus et al. 2019 c Kentucky 100.0 2/2 Niedringhaus et al. 2019 c Michigan 0.0 0/1 Niedringhaus et al. 2019 c New Jersey 100.0 1/1 Niedringhaus et al. 2019 c Pennsylvania 100.0 1/1 Niedringhaus et al. 2019 c West Virginia 50.0 4/8 Shea et al. 2022 d Maine 58.5 409/699 MacDonald et al. 2022 c Pennsylvania 3.3 4/121 MacDonald et al. 2022 d Pennsylvania 75.3 61/81 Haynes et al. 2024 d Kentucky 38.9 14/36 Adcock et al. 2024 c Multiple e 77.9 60/77 Total 54.9 2,112/3,850 Southeast USA Allison et al. 2014 d South Carolina 44.6 33/74 Thomas et al. 2015 d Florida 45.0 77/171 Thomas et al. 2015 d Georgia 41.7 20/48 Thomas et al. 2015 d Louisiana 59.4 57/96 Thomas et al. 2015 d North Carolina 39.1 34/87 Thomas et al. 2015 d Virginia 28.8 17/59 Niedringhaus et al. 2019 c Alabama 100.0 2/2 Niedringhaus et al. 2019 c Florida 100.0 1/1 Niedringhaus et al. 2019 c Georgia 37.5 3/8 Niedringhaus et al. 2019 c Louisiana 33.3 1/3 Niedringhaus et al. 2019 c Mississippi 50.0 1/2 Niedringhaus et al. 2019 c North Carolina 90.9 10/11 Niedringhaus et al. 2019 c South Carolina 0.0 0/1 Niedringhaus et al. 2019 c Tennessee 25.0 1/4 Kreh and Palamar 2022 d North Carolina 46.1 382/829 Thiemann et al. 2022 c Mississippi 51.1 24/47 Adcock et al. 2024 c Multiple e 69.1 56/81 Total 47.0 656/1,397 West USA Thomas et al. 2015 d Kansas 34.8 8/23 Thomas et al. 2015 d Missouri 29.7 22/74 Thomas et al. 2015 d Oklahoma 25.9 7/27 Niedringhaus et al. 2019 c Arkansas 100.0 2/2 4 JOURNAL OF WILDLIFE DISEASES, VOL. 00, NO. 00, MONTH 2025 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 Cullman County west of Interstate 65 and north of Lewis Smith Lake and Cullman County Road 437. Zone 3 included Clarke, Clay, Covington, Monroe, Randolph, and Talladega counties. Zone 1 covered the remainder of the state. Every zone had a youth season on the weekend preceding the regular season opening, and all regular seasons concluded on 8 May (Alabama Department of Conservation and Natural Resources 2021, 2022). Located in the eastern USA, Alabama is approximately 340 km E to W and 530 km N to S from the Gulf of Mexico to the Appalachian Mountains. Its landscape is comprised of the Lower and Upper Coastal Plain, Piedmont, Ridge and Val- ley, Southwestern Appalachian Mountains, and Inte- rior Low Plateau physiographic regions (Shankman and Hart 2007). Common land cover types include natural and planted pine ( Pinus spp.) forests, urban areas, row crop agriculture, and mixed and hardwood forests (Chen 2007; Dewitz 2023). During this study, Alabama experienced a moist subtropical midlatitude climate, with approximately 140.8 cm of annual pre- cipitation and average monthly temperatures ranging from − 1.1°C to 33.1°C (Li et al. 2009; National Cen- ters for Environmental Information 2022). Carcass collection, hunter observations, and necropsy We established a network of Wild Turkey hunt- ers throughout Alabama to aid in carcass collection during the 2022 and 2023 spring hunting seasons. Team leaders across the state collected and stored carcasses at − 20°C until the end of each hunting season, at which time we collected them. We asked each participating hunter to complete a survey (Auburn University Institutional Review Board pro- tocol no. 22-033 ex. 2201) for each donated carcass that included questions related to harvest date, har- vest county, behaviors observed, number of conspe- ci fi cs accompanying the harvested male, body mass, and equipment used by the hunter. We performed necropsies on each donated car- cass after con fi rming the age based on the pres- ence or absence of barring on the 9th and 10th primary feathers (Lewis and Breitenbach 1966). We collected the liver, spleen, and ceca. Portions of the liver and spleen were frozen at − 20°C. A second portion of the liver and spleen were also stored at 20°C in 10 % neutral buffered formalin and 70 % ethanol (ETOH), respectively. Ceca F IGURE 1. Map of Wild Turkey ( Meleagris gallo- pavo ) hunting zones in Alabama, USA, during the 2021 – 22 and 2022 – 23 hunting seasons (Alabama Department of Conservation and Natural Resources 2021, 2022). T ABLE 2. Continued. Region Study a Study area Prevalence ( % ) n b Niedringhaus et al. 2019 c Missouri 88.9 8/9 Smith 2022 d Iowa 42.9 391/912 Adcock et al. 2024 c Multiple e 57.1 8/14 Total 41.9 413/986 a Some studies may have a high prevalence rate due to low sample size and a passive surveillance strategy, as defined by MacDonald et al. (2022). b n = number detected/number sampled. c Samples were collected with a passive surveillance strategy, as defined by MacDonald et al. (2022). d Samples were collected with an active surveillance strategy, as defined by MacDonald et al. (2022). e Multiple = multiple USA states. OSTRANDER ET AL.—HISTOMONOSIS AND LPDV IN ALABAMA ’ S MALE WILD TURKEYS 5 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 were stored whole at − 20°C. We measured the whole mass of the liver and spleen, as well as any subsamples, to the nearest 0.1 g. We calculated the relative spleen and liver mass post hoc by using the hunter-reported body mass. Further- more, both histomonosis and LPDV are often associated with gross hepatic lesions that can range from subtle pale pink discoloration to overt well-delineated pale-gray-to-tan foci interspersed throughout the liver (Abdul-Aziz and Barnes 2018). Similar discolorations and abnormalities may be present on spleens and heads due to LPDV infection (Allison et al. 2014; MacDonald et al. 2022). Accordingly, we also noted whether there were any discoloration, lesions, or abnor- mally colored, irregular foci and/or nodules on the head, spleen, and liver. Finally, between each necropsy, we sterilized surfaces with a 10 % bleach solution and replaced scalpel blades. Laboratory diagnoses We followed the quantitative PCR (qPCR) pro- cess described by Hauck and Hafez (2012) with 0.02 – 0.03 g of cecal wall tissue to detect H. meleagridis DNA. We fi rst extracted DNA fol- lowing a similar process to that used to extract LPDV DNA. With the extracted DNA, ampli fi ca- tions were performed with the master mix Per- feCTa qPCR FastMix II, ROX (Quantabio, Beverly, Massachusetts, USA) in a qTOWER 3 G thermal cycler (Analytik Jena, Jena, Germany). In each reac- tion, working solutions for probe HmqS (1 μ L) and primer (2 μ L) were combined with cecal wall DNA template (1 μ L), master mix (10 μ L), and nuclease- free water (6 μ L). As described in Hauck and Hafez (2012), we used the probe HmqS 5 ′ -[6FAM] CTGCACGCGCGCTACAATGTTAAA[BHQ1]-3 ′ and the primer mix comprising HmqF2 5 ′ - CCGTGATGTCCTTTAGATGC-3 ′ and HmqR 5 ′ - GATCTTTTCAAATTAGCTTTAAATTATTC-3 ′ to amplify a segment of the histomonal ribosomal RNA (87 base pairs [bp] in length) at a concentra- tion of 10 pmol/ μ L. The thermal pro fi le and method that we used to collect the fl uorescence data to detect DNA are described in Hauck and Hafez (2012). We diagnosed LPDV with PCR by using 0.01 – 0.02 g of spleen tissue stored in 70 % ETOH at 20°C or frozen at − 20°C to detect proviral DNA. Tissues were incubated at 56°C for 1 h in 180 μ L of ATL buffer and 20 μ L of Proteinase K. Next, DNA was extracted using QIAamp DNA Mini QIAcube kits (Qiagen, Hilden, Germany) follow- ing the manufacturer ’ s guidelines. We ampli fi ed the extracted DNA with the qTOWER 3 G (Analy- tik Jena), a real-time PCR thermal cycler, by using 1 μ L of template DNA, 2 μ L of primer, 10 μ L of AccuStart II PCR SuperMix (Quantabio), and 7 μ L of nuclease-free water. Speci fi cally, a 413-bp segment from the retroviral gag gene (Allison et al. 2014) of the virus was ampli fi ed with the primers described by Alger et al. (2015): LPDTf 5 ′ -ATGAGGACTTGTTAGATTGGTTAC-3 ′ and LPDTr 5 ′ -TGATGGCGTCAGGGCTATTTG-3 ′ The ampli fi cations began with an initial denatur- ation of 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 54°C for 30 s, and 68°C for 1 min, and a fi nal step of 72°C for 10 min. Ampli fi cations also included positive and negative controls. Finally, we used 1 % agarose gels, with GelRed nucleic acid gel stain (Biotium, Hayward, California, USA), to detect LPDV proviral DNA in amplicons. Prevalence rates and land cover classification We fi rst compared LPDV prevalence rate between the two spleen storage mechanisms with a Pearson ’ s chi-square test. After detecting a sig- ni fi cant difference in LPDV detection between the two storage methods ( χ 2 = 133.8, df = 1, P < 0.001), we removed the ethanol-stored spleens from further analysis due to sample degradation. We summarized histomonosis and LPDV prevalence by age class, har- vest year, and county in R 4.5.1 (R Core Team 2025). We used the Wilson calculation method to calculate 95 % con fi dence intervals (CIs) for prevalence esti- mates in the Hmisc package in R (Harrell 2024). In ArcGIS 3.2.2 (ESRI, Redlands, California, USA), we mapped the distribution of histomonosis-positive counties and the county-level LPDV prevalence. We also examined data from the hunter surveys (e.g., har- vest county) and necropsy examinations (e.g., organ lesion presence) for relationships with H. meleagridis DNA and LPDV proviral DNA detection by using two sample t -tests, Wilcoxon rank-sum tests, Pear- son ’ s chi-square tests, and Fisher ’ s exact tests ( α = 0.05; R Core Team 2025). We used land cover data (Dewitz 2023) in Arc- GIS 3.2.2 to calculate the percent area of land cover by type in each county (Alabama Geographic Infor- mation Of fi ce 2018). The cover types we included in our analysis were as follows: open (open developed + herbaceous + hay-pasture), deciduous forest, 6 JOURNAL OF WILDLIFE DISEASES, VOL. 00, NO. 00, MONTH 2025 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 evergreen forest, mixed forest, and shrub-scrub. Based on the literature, these land cover catego- ries are broadly used by Wild Turkeys for forag- ing, reproduction, and roosting (Speake et al. 1975; Kennamer et al. 1980; Everett et al. 1985; Dickson 2001), leading to a higher selection than other available cover types. Accordingly, we con- structed a generalized linear mixed model (GLMM) that included these cover types as pre- dictors, county-level LPDV prevalence as the response variable, and ‘ County ’ as a random effect ( α = 0.05; Bates et al. 2015). We hypothe- sized that if land cover type(s) consolidated male Wild Turkey space use, our model would detect a signi fi cant relationship between land cover type(s) and county-level LPDV prevalence. We did not evaluate spatial relationships to histomo- nosis due to the low number of positive samples. RESULTS We collected 955 male Wild Turkey car- casses from 59 of 67 Alabama counties during spring 2022 and 2023. Because of tissue deg- radation and sample quality, we could not use every carcass and organ for disease analysis. We detected H. meleagridis DNA in 3 of 435 (0.7 % ; Wilson 95 % CI, 0.5 – 1.0; Table 3) of our samples. We detected LPDV proviral DNA in 416 or 469 (88.7 % ; CI, 87.7 – 89.6; Table 3) of our samples frozen at − 20°C. We detected H. meleagridis DNA in 3 of 56 and LPDV in 53 of 56 sampled counties (Figs. 2, 3). County-level LPDV prevalence ranged from 0 % to 100 % (Fig. 3; see Supplementary Material Table S1). From our sample, we detected histomonosis-LPDV coinfection in 2 of 411 males. We did not detect a difference in H. meleagridis prevalence between the sub- adult and adult age classes by using Fisher ’ s exact test (Table 3; P = 1.00). Because of a lack of subadults in the sample, we did not exam- ine the relationship between H. meleagridis or LPDV status and hunter-reported body mass for this age class. We did not fi nd an associa- tion between adult hunter-reported body mass and H. meleagris status (Wilcoxon rank- sum test: W = 165, P = 0.27). We did not detect a difference in relative spleen mass (two-sample t -test: t = − 1.14, df = 205, P = 0.25) or relative liver mass (Wilcoxon rank-sum test: W = 259.5, P = 0.06) between age classes. Therefore, we pooled across age classes to test for differences in these variables by H. meleagridis status. Using Wilcoxon rank-sum tests, we did not detect differences in relative spleen mass (W = 47, P = 0.35) or relative liver mass (W = 35, P = 0.26) due to H. meleagridis infection. We did not detect any foci or discolored lesions on the heads (0/3), spleens (0/3), or livers (0/3) of any H. meleagridis – positive males, limiting our abil- ity to test for relationships between these vari- ables and H. meleagridis detection. We did not fi nd a difference in LPDV pro- viral DNA detection between age classes (Table 3; P = 0.61). We also did not fi nd a sta- tistically signi fi cant relationship between detection of LPDV proviral DNA and hunter- reported adult body mass (two-sample t -test: T ABLE 3. Histomonosis and lymphoproliferative dis- ease virus (LPDV) a prevalence rates by age class and year in hunter-harvested male Wild Turkey ( Melea- gris gallopavo ) from 2022 and 2023 spring hunting seasons (March – May) in Alabama, USA. Disease Year Prevalence ( % ) (95 % CI) b n c Histomonosis Adult 2022 0 (0.0 – 10.2) 0/4 2023 0.8 (0.5 – 1.1) 3/395 Unknown 0 (0.0 – 8.3) 0/5 Subadult 2023 0 (0.0 – 6.1) 0/7 Unknown 2023 0 (0.0 – 8.3) 0/5 Unknown 0 (0.0 – 2.3) 0/19 Total 0.7 (0.5 – 1.0) 3/435 LPDV Adult 2022 100 (91.7 – 100) 5/5 2023 88.5 (87.4 – 89.5) 377/426 Unknown 100 (93.0 – 100) 6/6 Subadult 2023 100 (94.6 – 100) 8/8 Unknown 2023 100 (91.7 – 100) 5/5 Unknown 78.9 (72.0 – 84.5) 15/19 Total 88.7 (87.7 – 89.6) 416/469 a LPDV prevalence rates calculated from spleen samples stored at − 20°C. b The 95 % confidence interval (CI) calculated with the Wilson method (Harrell 2024). c n = number detected/number sampled. OSTRANDER ET AL.—HISTOMONOSIS AND LPDV IN ALABAMA ’ S MALE WILD TURKEYS 7 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 t = 1.35, df = 220, P = 0.18). We did not detect a difference in relative spleen (two-sample t - test: t = − 1.26, df = 225, P = 0.21) or liver mass (Wilcoxon rank-sum test: W = 360, P = 0.18) by age class. Thus, we pooled liver and spleen mass data across age classes and found that spleen mass was greater for LPDV-positive males (Wil- coxon rank-sum test: W = 1366; P = 0.01; 95 % CI, − 6.74 3 10 − 5 to − 4.51 3 10 − 4 ). We did not detect a signi fi cant difference in relative liver mass based on LPDV proviral DNA detection (two-sample t -test: t = − 0.29, df = 224, P = 0.77). We did not detect any foci or discolored lesions on the heads of LPDV-positive males (0/366). Although we did note LPDV-positive spleens (15/394) and livers (30/398) with foci or discolor- ation, neither was signi fi cantly related to LPDV proviral DNA detection (Fisher ’ s exact test: spleen, P = 1.00; liver, P = 0.27). Our GLMM did not reveal a signi fi cant relationship between any land cover type and LPDV prevalence. DISCUSSION We observed an H. meleagridis infection rate (0.7 % ) lower than those that have been reported in previous studies (Table 1), sug- gesting histomonosis may occur at a low rate in Alabama. However, due to the lethality of H. meleagridis ( ≤ 90 % mortality, 12 – 21 d to death; Hauck and Hafez 2013), the preva- lence in our study was probably an underesti- mate. The acute nature of histomonosis would result in mortality of most infected individuals before hunter harvest, possibly leading to biased interpretations when using active surveillance data alone. Although previous research has found that histomonosis is a source of mortality in Wild Turkeys (Hurst 1980; Davidson et al. 1985), our study was the fi rst intensive, active sampling effort that attempted to measure histo- monosis prevalence across a large scale. Thus, although we found that H. meleagridis infection F IGURE 2. Histomonas meleagridis DNA (histomo- nosis) was detected by quantitative PCR in 3 of 435 (0.7 % ; Wilson 95 % confidence interval, 0.5 – 1.0) hunter- harvested male Wild Turkeys ( Meleagris gallopavo ) in 3 of 56 sampled counties (Crenshaw [1/1], Randolph [1/5], and Sumter [1/8]) during the spring hunting 2022 – 23 seasons (March – May) in Alabama, USA. F IGURE 3. Prevalence of lymphoproliferative dis- ease virus (LPDV) in 53 of 56 sampled counties (0 [0/ 1] – 100 [26/26] % ) during the spring hunting 2022 – 23 seasons (March – May) in Alabama, USA. LPDV was detected by PCR in 416 of 469 (88.7 % ; Wilson 95 % confidence interval, 87.7 – 89.6) hunter-harvested male Wild Turkeys ( Meleagris gallopavo ). 8 JOURNAL OF WILDLIFE DISEASES, VOL. 00, NO. 00, MONTH 2025 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 occurred at low rates among hunter-harvested male Wild Turkeys (Table 3), the annual H. meleagridis infection rate affecting Wild Tur- key populations is probably higher than our reported infection rate of 0.7 % Contrary to our prediction, we detected a higher LPDV infection rate from Wild Turkey spleens in Alabama than in most other areas of this species ’ range (Tables 2, 3). This was surprising because several recent studies have suggested that LPDV tends to be more preva- lent in northern populations. Speci fi cally, the three closest prevalences to that of our study (89 % ) have been reported from Pennsylvania, USA (75 % ; MacDonald et al. 2022), Vermont, USA (71 % ; Thomas et al. 2015), and Ontario, Canada (65 % ; MacDonald et al. 2019b), whereas the mean prevalence reported from previous studies throughout the southeastern USA was 47 % (Table 2). Although previous research has suggested LPDV prevalence may be correlated with latitude, Adcock et al. (2024) also found a prevalence (69 % ) for Wild Turkeys in the southeastern USA that was higher than previously reported prevalences. Interestingly, Adcock et al. (2024) reported that LPDV prevalence was lower during spring than in winter. However, Shea et al. (2022) reported the opposite — that the risk of LPDV infection was greater in spring than winter in Maine. Many studies (e.g., Alger et al. 2017; Shea et al. 2022; Thiemann et al. 2022) have also reported higher LPDV preva- lence among females; however, because our sample was comprised of males, we did not assess this relationship in our study area. When examined together, our results and those of pre- vious studies suggest that there are still many knowledge gaps regarding LPDV, its viral spread, and its impacts on Wild Turkey popula- tion dynamics. We were surprisingly unable to detect a relationship between county-level LPDV prevalence and land cover. By contrast, land- scape composition has been documented to affect LPDV prevalence in both New York (Alger et al. 2017) and Maine (Shea et al. 2022). Speci fi cally, Shea et al. (2022) reported that LPDV infection was positively correlated with the amount of forest cover in Wild Tur- key winter home ranges. Alger et al. (2017) reported higher LPDV prevalence in areas with a higher ratio of agriculture:forest land cover in New York. We suggest that variation in sampling period timing could explain these differences. Increased spleen mass in LPDV-positive males from our study suggests that male Wild Turkeys were actively responding to infection (Smith and Hunt 2004). This raises questions about the physiological costs of mounting an immune response to infection, and the result- ing impacts on individual fi tness and popula- tion vital rates. To address similar questions, researchers recently examined the relation- ship between Wild Turkey spring harvest den- sity, annual recruitment, and LPDV in North Carolina, USA (Kreh and Palamar 2022). Although they did not detect a signi fi cant rela- tionship between LPDV prevalence and vital rates, another recent study in Maine showed that clutch size was reduced, on average, by 1.43 eggs for LPDV-positive females (Shea 2021). Although our study was not designed to measure the effects of disease on Wild Tur- key vital rates, these data collectively suggest that additional research into the effects of LPDV, and other diseases, on population vital rates is warranted. Considering recent Wild Turkey declines in Alabama and the southeastern USA overall, concomitant with decreasing recruitment (Byrne et al. 2016) and decreasing hunter har- vest (Chamberlain et al. 2022), the impact of increasing disease prevalence or new disease emergence on Wild Turkey populations needs to be considered. The population-level impacts of our fi ndings and those of these other recent studies are uncertain, and data from manipulative experiments on the impacts of either histomonosis or LPDV on Wild Turkey vital rates are unavailable in the literature. Accordingly, we recommend addi- tional research on the effects of histomonosis, LPDV, and other common diseases on Wild Turkey demography. OSTRANDER ET AL.—HISTOMONOSIS AND LPDV IN ALABAMA ’ S MALE WILD TURKEYS 9 Downloaded from http://meridian.allenpress.com/jwd/article-pdf/doi/10.7589/JWD-D-24-00150/3543813/10.7589_jwd-d-24-00150.pdf by University of Florida user on 17 November 2025 ACKNOWLEDGMENTS Funding for this project was provided by the Alabama Wildlife Federation, Turkeys for Tomor- row, and the Alabama Chapter of the National Wild Turkey Federation. We thank the team lead- ers for their support and the hunters who donated their Wild Turkey carcasses to this project. We thank the faculty members, technicians, and grad- uate students who assisted with necropsies, logis- tics, and laboratory work: J. Alford, C. Bedoian, H. Day, T. Dormitorio, G. Goto, J. Kading, K. Smelter, R. Stogner, T. Swartout, and W. Tabish, as well as S. Shea for providing an LPDV-positive sample. We are grateful to two anonymous reviewers whose suggestions improved this manu- script. 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