Health Assessment of Adult Male Eastern Wild Turkeys (Meleagris gallopavo silvestris) from Western Kentucky, USA.

Wild turkeys (Meleagris gallopavo) are an important game species throughout the geographic range. Populations throughout multiple regions of the US have been declining, including in Kentucky, US, raising concerns among managers and resource users. To better understand the overall population health, we performed postmortem examinations and targeted pathogen, mineral, and toxicant testing on 36 adult male, apparently healthy, wild turkeys that were hunter harvested in western Kentucky during April 2018. We found that birds were in fair to good nutritional condition with no significant gross or microscopic lesions. Ticks (Amblyomma spp.) and lice (three species) were present on 94 and 31% of birds, respectively. We commonly detected intestinal nematodes and cestodes and found coccidian oocysts in 39% and capillarid eggs in 6% of birds. The prevalences of lymphoproliferative disease virus and reticuloendotheliosis virus were 39 and 11%, respectively. Spleen samples tested with PCR were positive for Borrelia burgdorferi, Haemoproteus sp., and Leucocytozoon sp. in 11, 83, and 3%, respectively. Based on a subjective histologic assessment of testis tissues, most birds had widespread and abundant sperm present. Mineral analysis and broad toxicant screening on liver samples from 32 turkeys were unremarkable. Further work is needed to assess potential population risk factors and to determine individual- and population-level impacts of pathogens on adults and poults.

Experimental infection of domestic turkeys with lymphoproliferative disease virus of North American origin.

Lymphoproliferative disease virus (LPDV) was first documented in wild turkeys in North America in 2009. LPDV infection is often subclinical but can manifest as lymphoid proliferation or round cell neoplasia. Despite high prevalence across many sampled areas corresponding to declining populations of wild turkeys, knowledge regarding LPDV pathogenesis, risk factors for disease development, and associated impacts on population dynamics are unknown. To understand transmission, viral shedding, and tissue tropism, we inoculated 21 domestic turkeys via the oral cavity, crop, nasal cavity, subcutis, or coelomic cavity. For 12 weeks, oropharyngeal swabs, cloacal swabs, and whole blood were collected weekly. At 1 week postinoculation, 3 turkeys (3/21; 14%) had detectable LPDV proviral DNA in blood by polymerase chain reaction, and 10 developed DNAemia (50%; 10/20) by 12 weeks. LPDV proviral DNA was intermittently detected in oropharyngeal and cloacal swabs. Splenomegaly was the most consistent gross finding in DNAemic birds (8/11; 73%). Lymphoid hyperplasia in the spleen was the most significant microscopic finding (9/11; 82%). Three turkeys (3/11; 27%) developed round cell neoplasia characterized by sheets of pleomorphic, round to polygonal cells in the adrenal gland, bone marrow, skin, small intestine, and/or spleen. LPDV was detected in the spleen and bone marrow from all turkeys with DNAemia and all neoplasms. Our study establishes that infection and disease with North American LPDV from wild turkeys can be experimentally reproduced in domestic turkeys, laying the groundwork for future investigations into LPDV pathogenesis, development of diagnostic techniques, and understanding the impacts of LPDV on wild turkey populations.

Surveillance for Selected Pathogens and Parasites of Northern Bobwhite (Colinus virginianus) from Western Oklahoma, USA, 2018-20.

The Northern Bobwhite (Colinus virginianus) has been undergoing a range-wide population decline. Potential causes for declines across its historic range have been investigated for decades and include habitat loss and fragmentation and a variety of parasitic and infectious diseases. Although there have been studies on bobwhite ecology in Oklahoma, USA, relatively little is known about parasites and pathogens in the region. We evaluated the health of free-ranging bobwhites from nine sites in western Oklahoma. From 2018 to 2020, 206 bobwhites were evaluated for gross and microscopic lesions and tested for selected pathogens. In general, bobwhites were in good nutritional condition with ample muscle mass and fat stores. No significant gross lesions were observed in any bobwhite and no significant histologic lesions were detected in a subset. There was no evidence of infection with or exposure to reticuloendotheliosis virus, West Nile virus, respiratory Mycoplasmataceae species, Pasteurella multocida, intestinal Eimeria spp., or oral Trichomonas spp. Several pathogens of potential concern were detected, including avian adenovirus (8.6%), Toxoplasma gondii (2.3%), and haemosporidians (a Haemoproteus sp. (1.5%), Leucocytozoon schoutedeni (1.5%), and Plasmodium homopolare haplotype 2 [lineage LAIRI01; 3.6%]). Physaloptera sp. (12%) and Sarcocystis sp. (1%) were detected in the breast muscle. Low intraspecific genetic diversity was noted for Physaloptera sp., and sequences were most similar to Physaloptera sequences from bobwhites and grasshoppers (Orthoptera) in Texas. Low intensities of chewing lice, chiggers, and ticks were observed. A subset of bobwhites had evidence of exposure to selected toxicants and heavy metals; a small number had low levels of iron, manganese, zinc, molybdenum, and copper, which were not considered diagnostically relevant. In general, bobwhites from western Oklahoma appeared to be in good health with a low diversity of pathogens detected, but future work is needed to understand potentially changing disease risks for this population.

Lymphoproliferative Disease Virus and Reticuloendotheliosis Virus Detection and Disease in Wild Turkeys (Meleagris gallopavo).

Lymphoproliferative disease virus (LPDV) and reticuloendotheliosis virus (REV) are oncogenic retroviruses that can cause disease in wild and domestic fowl. Lymphoproliferative disease virus infections are common and widespread in Wild Turkeys (Meleagris gallopavo) in the US and east-central Canada, while REV has been detected worldwide in numerous avian host species. We tested tissues (spleen, liver, and/or bone marrow, plus neoplastic tissue, if present) from 172 Wild Turkeys that underwent necropsy from December 2018 through October 2021 for both viruses using PCR. We evaluated demographic, geographic, temporal, and seasonal data by chi-square test of independence and logistic regression for turkeys infected with LPDV and/or REV. At least one of these retroviruses was detected in 80.8% (139/172) of Wild Turkeys from 15 US states, with significantly more turkeys being positive for LPDV (72.1%, 124/172) versus REV (43.6%, 75/172; P<0.001). Both viruses (coinfections) were detected in 34.9% (60/172) of turkeys. Among LPDV-infected turkeys (including coinfections), bone marrow had the highest detection rate (38/58, 65.5%), significantly higher than spleen (30/58, 51.7%) and liver (20/58, 34.5%; P<0.001). In REV-infected turkeys, bone marrow had the highest detection rate (24/58, 41.4%). All three tissues (spleen, liver, bone marrow) concurrently tested positive in most (15/25, 60%) REV-infected turkeys. These results suggest LPDV tissue tropism for bone marrow, whereas REV may have broader tissue tropism. Histopathology consistent with lymphoid proliferation and/or neoplasia characteristic of lymphoproliferative disease was evident in 29/172 (16.9%) turkeys assessed, including two REV-only-infected turkeys. Season was significantly associated with LPDV prevalence (highest in winter); year and season were both significantly associated with REV prevalence (highest in 2020 and winter). These data contribute to optimizing diagnostic strategies that may aid in pathogen monitoring and improve detections to increase our understanding of the potential impacts of these viruses on Wild Turkey populations.

Susceptibility of wild turkeys (Meleagris gallopavo) to experimental West Nile virus infection.

Since the introduction of West Nile virus (WNV) to North America in 1999, WNV is estimated to have contributed to population-level declines in numerous avian species. However, the potential impacts of this virus on many free-ranging upland game bird species, including the wild turkey (Meleagris gallopavo), which is undergoing regional population declines, remain unknown. Herein, two age groups (∼5 to 6 weeks and ∼15 to 16 weeks post-hatch) of juvenile wild turkeys were subcutaneously inoculated with WNV, sampled daily from 1 to 7 days post-inoculation (dpi), and euthanized on 14 dpi. No clinical signs and minimal gross lesions were attributable to WNV infection. Peak viraemia titres were similar between age groups (<101.7 to 104.6 plaque-forming units [PFU]/ml), but the duration of viraemia was longer in the old group (3-4 days) than in the young group (0-3 days). Intermittent oral and/or cloacal viral shedding from 2 to 7 dpi was detected in both age groups. No infectious virus was detected in the heart, brain, kidney, skeletal muscle, spleen, and feathers from WNV-inoculated turkeys euthanized on 14 dpi. All WNV-inoculated birds seroconverted by 14 dpi, as well as two co-housed sham-inoculated birds. The most consistent microscopic lesions among all WNV-inoculated birds were mild lymphoplasmacytic myocarditis and encephalitis. Minimal immunohistochemical labelling was detected in tissues in addition to scant macrophages within the blood, spleen, and bone marrow. These data suggest WNV is unlikely to pose a significant risk to wild turkey populations, although the possibility remains that WNV may indirectly decrease fitness or predispose wild turkeys to other health stressors.RESEARCH HIGHLIGHTS Clinical disease was not observed in wild turkeys experimentally infected with WNV.Pathology attributed to WNV was mild and included brain and heart inflammation.Viraemias suggest WNV-infected wild turkeys do not play a role in WNV transmission.No age-associated differences in WNV clinical disease or pathology were observed.

An Investigation of the Cause of Wild Turkey Mortality in Mississippi.

Lymphoproliferative disease virus (LPDV) is an exogenous alpharetrovirus that sporadically causes fatal lymphoid neoplasia in affected turkeys. Previous studies of wild turkeys (Meleagridis gallopavo) in the United States have demonstrated geographically widespread LPDV infection and frequent coinfection with avian poxvirus (APV) and reticuloendotheliosis virus (REV). This study was conducted to better understand health risks to Mississippi wild turkeys, including the relative importance of LPDV, APV, and REV in contributing to mortality. Thirteen wild turkeys, which died naturally or were euthanized due to illness, were submitted to Mississippi State University's Poultry Research and Diagnostic Laboratory for postmortem examinations. Birds originated from nine counties across the state over the past 5 yr. Carcasses were submitted as fresh (nonfrozen) or frozen. At autopsy, 9 of 13 turkeys had severe, proliferative cutaneous lesions on the head and neck, with diphtheritic or proliferative oral and esophageal lesions. Samples were collected for molecular diagnostic testing (LPDV and REV PCR), histopathology, and bacterial culture and isolation. External and internal parasites were preserved in formalin for identification. APV (cutaneous and/or diphtheritic forms) was diagnosed in 9 of 13 birds by identification of pathognomonic histologic lesions (including intracytoplasmic inclusion bodies). Interestingly, all birds with APV were also REV PCR positive. Furthermore, eight turkeys were positive for LPDV, and LPDV was commonly associated with coinfections with APV and REV.

El virus de la enfermedad linfoproliferativa (LPDV) es un Alfaretrovirus exógeno que esporádicamente provoca una neoplasia linfoide mortal en los pavos afectados. Estudios previos de pavos salvajes (Meleagridis gallopavo) en los Estados Unidos han demostrado que la infección por la enfermedad linfoproliferativa está geográficamente extendida y es una coinfección frecuente con el virus de la viruela aviar (APV) y el virus de la reticuloendoteliosis (REV). Este estudio se realizó para comprender mejor los riesgos para la salud de los pavos salvajes de Mississippi, incluida la importancia relativa de enfermedad linfoproliferativa, el virus de la viruela aviar y el virus de la reticuloendoteliosis en la contribución a la mortalidad. Trece pavos salvajes, que murieron naturalmente o fueron sacrificados por enfermedad, fueron enviados al Laboratorio de Investigación y Diagnóstico Avícola de la Universidad Estatal de Mississippi para exámenes post-mortem. Las aves provenían de condados de todo el estado durante los últimos cinco años. Las canales se enviaron tanto frescas (no congeladas) como congeladas. A la necropsia, 9 de 13 pavos mostraron lesiones cutáneas proliferativas graves en la cabeza y el cuello, con lesiones orales y esofágicas diftéricas o proliferativas. Se recolectaron muestras para pruebas de diagnóstico molecular (LPDV y REV PCR), histopatología y cultivo y aislamiento bacterianos. Los parásitos externos e internos se conservaron en formalina para su identificación. Se diagnosticó viruela aviar (formas cutáneas y/o diftéricas) se diagnosticó en 9 de 13 aves mediante la identificación de lesiones histológicas patognomónicas (incluidos los cuerpos de inclusión intracitoplasmáticos). Curiosamente, todas las aves con viruela aviar también fueron positivas a la presencia del virus de la reticuloendoteliosis por PCR. Además, ocho pavos fueron positivos para el virus de la enfermedad linfoproliferativa, y se asoció comúnmente con coinfecciones con viruela aviar y con el virus de la reticuloendoteliosis.

Isolation of Heartland Virus from Lone Star Ticks, Georgia, USA, 2019.

Report of a human death and exposure of white-tailed deer to Heartland virus (HRTV) in Georgia, USA, prompted the sampling of questing ticks during 2018-2019 in 26 sites near where seropositive deer were captured and the residence of the human case-patient. We processed 9,294 Amblyomma americanum ticks in pools by virus isolation in Vero E6 cells and reverse transcription PCR. Positive pools underwent whole-genome sequencing. Three pools were positive for HRTV (minimum infection rate 0.46/1,000 ticks) and none for Bourbon virus. Cell cultures confirmed HRTV presence in 2 pools. Genome sequencing, achieved for the 3 HRTV isolates, showed high similarity among samples but marked differences with previously sequenced HRTV isolates. The isolation and genomic characterization of HRTV from A. americanum ticks in Georgia confirm virus presence in the state. Clinicians and public health professionals should be aware of this emerging tickborne pathogen.

Experimental West Nile Virus Infection in Northern Bobwhite Quail (Colinus virginianus).

West Nile virus (WNV) has been implicated in regional declines of numerous North American bird species, although its potential impact upon many species, including some game birds, remains unknown. Specifically, information about susceptibility to infection and infection outcome are crucial to assessing health risks. Northern bobwhite quail (Colinus virginianus) are a popular and common game bird across much of the United States, as well as in captive breeding programs and as backyard birds. Two age groups of bobwhites were subcutaneously inoculated with WNV and euthanatized on 15 days postinoculation (DPI). Three of 10 inoculated 5-wk-old and 4/10 inoculated 15-wk-old birds developed detectable viremia titers during 1-5 DPI, with low peak titers (101.7-103.0 plaque-forming units [PFU]/ml). Three of 10 inoculated 5-wk-old and 1/10 inoculated 15-wk-old birds shed low viral titers (peak 100.7-101.8 PFU/swab) either orally or cloacally or both for limited periods from 2 to 6 DPI. All inoculated birds (n = 20) remained apparently healthy and seroconverted by 15 DPI. No infectious virus was detected in select tissues: heart, kidney, brain, skeletal muscle, spleen (15-wk-old group only), and feathers from any of the bobwhites. No sham-inoculated, contact control birds (n = 8) became viremic or had virus isolated from tissues or swabs. The most consistent microscopic lesion was minimal to mild, lymphoplasmacytic myocarditis (6/10 in 5-wk-olds; 5/10 in 15-wk-olds). Immunohistochemical labeling was most often in macrophages in spleen and bone marrow, likely reflective of clearance of infection. There were no statistically significant differences in the peak viremia and shedding titers between age groups and no differences in the development of WNV-associated lesions between the two age groups. These results suggest that WNV is unlikely to pose a health risk to bobwhites and that bobwhites likely are an incompetent reservoir host species in WNV transmission.

Infección experimental por el virus del Nilo occidental en codornices de Virginia (Colinus virginianus). El virus del Nilo Occidental (con las siglas en inglés) se ha visto implicado en la disminución regional de numerosas especies de aves en América del Norte, aunque se desconoce su impacto potencial sobre muchas especies, incluidas algunas aves de caza. Específicamente, la información sobre la susceptibilidad a la infección y el resultado de la infección es crucial para evaluar los riesgos para la salud. La codorniz de Virginia (Colinus virginianus) es un ave de caza popular y común en gran parte de los Estados Unidos, así como en programas de cría en cautiverio y como aves de traspatio. Dos grupos de codornices de diferentes edades fueron inoculadas por vía subcutánea con el virus del Nilo Occidental y fueron sacrificadas 15 días después de la inoculación (DPI). Tres de diez aves inoculadas de cinco semanas y cuatro de diez aves inoculadas de 15 semanas desarrollaron títulos de viremia detectables durante uno a cinco días después de la inoculación, con bajos títulos durante la cima de la curva (101.7­103.0 unidades formadoras de placa [PFU]/ml). Tres de diez aves inoculadas de cinco semanas de edad y una de diez aves inoculadas a las 15 semanas de edad mostraron títulos virales bajos (pico 100.7­101.8 PFU/hisopo) ya sea por vía oral, cloacal o ambas durante períodos limitados de dos a seis días después de la inoculación. Todas las aves inoculadas (n = 20) permanecieron aparentemente sanas y mostraron seroconversión a los 15 días después de la inoculación. No se detectó ningún virus infeccioso en tejidos seleccionados: corazón, riñón, cerebro, músculo esquelético, bazo (solo en el grupo de 15 semanas de edad) y plumas de cualquiera de las codornices. Ninguna ave no inoculada o de control por contacto (n = 8) se volvió virémica o mostró aislamiento viral de los tejidos o hisopos. La lesión microscópica más consistente fue miocarditis linfoplasmocítica de mínima a leve (6/10 en aves de cinco semanas; 5/10 en aves de 15 semanas). La tinción inmunohistoquímica se observó con mayor frecuencia en macrófagos del bazo y la médula ósea, lo que probablemente refleja la eliminación de la infección. No hubo diferencias estadísticamente significativas en el pico de viremia y los títulos de eliminación entre los grupos de edad y no hubo diferencias en el desarrollo de lesiones asociadas con el virus del Nilo Occidental entre los dos grupos de edad. Estos resultados sugieren que es poco probable que el virus del Nilo Occidental represente un riesgo para la salud de las codornices de Virginia y que estas aves probablemente sean una especie hospedadora reservorio incompetente en la transmisión del virus del Nilo Occidental.

Experimental Susceptibility of North American Raccoons (Procyon lotor) and Striped Skunks (Mephitis mephitis) to SARS-CoV-2.

Recent spillback events of SARS-CoV-2 from humans to animals has raised concerns about it becoming endemic in wildlife. A sylvatic cycle of SARS-CoV-2 could present multiple opportunities for repeated spillback into human populations and other susceptible wildlife. Based on their taxonomy and natural history, two native North American wildlife species -the striped skunk (Mephitis mephitis) and the raccoon (Procyon lotor) -represent a high likelihood of susceptibility and ecological opportunity of becoming infected with SARS-CoV-2. Eight skunks and raccoons were each intranasally inoculated with one of two doses of the virus (103 PFU and 105 PFU) and housed in pairs. To evaluate direct transmission, a naïve animal was added to each inoculated pair 48 h post-inoculation. Four control animals of each species were handled like the experimental groups. At predetermined intervals, we collected nasal and rectal swabs to quantify virus shed via virus isolation and detect viral RNA via rRT-PCR and blood for serum neutralization. Lastly, animals were euthanized at staggered intervals to describe disease progression through histopathology and immunohistochemistry. No animals developed clinical disease. All intranasally inoculated animals seroconverted, suggesting both species are susceptible to SARS-CoV-2 infection. The highest titers in skunks and raccoons were 1:128 and 1:64, respectively. Low quantities of virus were isolated from 2/8 inoculated skunks for up to day 5 post-inoculation, however no virus was isolated from inoculated raccoons or direct contacts of either species. Neither species had gross lesions, but recovering mild chronic pneumonia consistent with viral insult was recorded histologically in 5/8 inoculated skunks. Unlike another SARS-CoV-2 infection trial in these species, we detected neutralizing antibodies in inoculated raccoons; thus, future wildlife serologic surveillance results must be interpreted with caution. Due to the inability to isolate virus from raccoons, the lack of evidence of direct transmission between both species, and low amount of virus shed by skunks, it seems unlikely for SARS-CoV-2 to become established in raccoon and skunk populations and for virus to spillback into humans. Continued outbreaks in non-domestic species, wild and captive, highlight that additional research on the susceptibility of SARS-CoV-2 in wildlife, especially musteloidea, and of conservation concern, is needed.