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.

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.

The role of drought as a determinant of hemorrhagic disease in the eastern United States.

Bluetongue virus and epizootic hemorrhagic disease (HD) virus are globally distributed, vector-borne viruses that infect and cause disease in domestic and wild ruminant species. The forces driving increases in resulting HD may be linked to weather conditions and increasing severity has been noted in northerly latitudes. We evaluated the role of drought severity in both space and time on changes in HD reports across the eastern United States for a recent 15 year period. The objectives of this study were to: (a) develop a spatiotemporal model to evaluate if drought severity explains changing patterns of HD presence; and (b) determine whether this potential risk factor varies in importance over the present range of HD in the eastern United States. Historic data (2000-2014) from an annual HD presence-absence survey conducted by the Southeastern Cooperative Wildlife Disease Study and from the United States Drought Monitor were used for this analysis. For every county in 23 states and for each of 15 years, data were based on reported drought status for August, wetland cover, the physiographic region, and the status of HD in the previous year. We used a generalized linear mixed model to explain HD presence and evaluated spatiotemporal predictors across the region. We found that drought severity was a significant predictor of HD presence and the significance of this relationship was dependent on latitude. In more northerly latitudes, where immunological naivety is most likely, we demonstrated the increasing strength of drought severity as a determinant of reported HD and established the importance of variation in drought severity as a risk factor over the present range of HD in the eastern United States. Our research provides spatially explicit evidence for the link between climate forces and emerging disease patterns across latitude for a globally distributed disease.

Evaluation of 2012 US EHDV-2 outbreak isolates for genetic determinants of cattle infection.

Following a summer of severe drought and abnormally high temperatures, a major outbreak of EHDV occurred during 2012 in the USA. Although EHDV-1, -2 and -6 were isolated, EHDV-2 was the predominant virus serotype detected during the outbreak. In addition to large losses of white-tailed deer, the Midwest and northern Plains saw a significant amount of clinical disease in cattle. Phylogenetic analyses and sequence comparisons of newly sequenced whole genomes of 2012 EHDV-2 cattle isolates demonstrated that eight of ten EHDV-2 genomic segments show no genetic changes that separate the cattle outbreak sequences from other EHDV-2 isolates. Two segments, VP2 and VP6, did show several unique genetic changes specific to the 2012 cattle outbreak isolates, although the impact of the genetic changes on viral fitness is unknown. The placement of isolates from 2007 and 2011 as sister group to the outbreak isolates, and the similarity between cattle and deer isolates, point to environmental variables as having a greater influence on the severity of the 2012 EHDV outbreak than viral genetic changes.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus.

UNLABELLED: Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. IMPORTANCE: The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

Genetic characterization of epizootic hemorrhagic disease virus strains isolated from cattle in Israel.

Epizootic hemorrhagic disease virus (EHDV), a member of the genus Orbivirus not reported previously in Israel, was isolated from Israeli cattle during a 'bluetongue-like' disease outbreak in 2006. To ascertain the origin of this new virus, three isolates from the outbreak were fully sequenced and compared with available sequences. Whilst the L2 gene segment clustered with the Australian EHDV serotype 7 (EHDV-7) reference strain, most of the other segments were clustered with EHDV isolates of African/Middle East origin, specifically Bahrain, Nigeria and South Africa. The M6 gene had genetic relatedness to the Australian/Asian strains, but with the limited data available the significance of this relationship is unclear. Only one EHDV-7 L2 sequence was available, and as this gene encodes the serotype-specific epitope, the relationship of these EHDV-7 L2 genes to an Australian EHDV-7 reflects the serotype association, not necessarily the origin. The genetic data indicated that the strains affecting Israel in 2006 may have been related to similar outbreaks that occurred in North Africa in the same year. This finding also supports the hypothesis that EHDV entered Israel during 2006 and was not present there before this outbreak.

Effect of Temperature on Replication of Epizootic Hemorrhagic Disease Viruses in Culicoides sonorensis (Diptera: Ceratopogonidae).

Replication of arboviruses, including orbiviruses, within the vector has been shown to be temperature dependent. Cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Previous research with epizootic hemorrhagic disease virus (EHDV) serotype 1 demonstrated that higher temperatures were associated with shorter extrinsic incubation periods in Culicoides sonorensis Wirth & Jones, a confirmed vector of EHDV in North America. To further our understanding of the effect of temperature on replication of EHDV within the vector, C. sonorensis were experimentally infected with one of three EHDV strains representing three serotypes (1, 2, and 7). Midges were fed defibrinated white-tailed deer (Odocoileus virginianus) blood spiked with EHDV (≥10(6.5) TCID(50)/ml) through a parafilm membrane using an artificial feeding device and were then held at 20, 25, or 30°C. In addition to this in vitro method, a white-tailed deer experimentally infected with EHDV-7 was used to provide an infectious bloodmeal to determine if the results were comparable with those from the in vitro feeding method. Whole midges were processed for virus isolation and titration at regular intervals following feeding; midges with ≥10(2.7) TCID(50) were considered potentially competent to transmit virus. The virus recovery rates were high throughout the study and all three viruses replicated within C. sonorensis to high titer (≥ 10(2.7) TCID(50)/midge). Across all virus strains, the time to detection of potentially competent midges decreased with increasing temperature: 12-16 d postfeeding (dpf) at 20°C, 4-6 dpf at 25°C, and 2-4 dpf at 30°C. Significant differences in replication of the three viruses in C. sonorensis were observed, with EHDV-2 replicating to a high titer in a smaller proportion of midges and with lower peak titers. The findings are consistent with previous studies of related orbiviruses, showing that increasing temperature can shorten the apparent extrinsic incubation period for multiple EHDV strains (endemic and exotic) in C. sonorensis.

Evidence of Active Orbivirus Transmission in 2016 in Kansas and Nebraska.

Retrospective serological and case diagnostic data of endemic bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) provide evidence of viral transmission among livestock and wildlife from 2016 in Kansas and Nebraska. Serological testing of mature cattle in nine distinct regional zones of Kansas revealed 76% to 100% had detectable antibodies to BTV and/or EHDV. Specimens tested in the Kansas Veterinary Diagnostic Laboratory (55 submissions) were 51% test positive for antibodies to BTV and/or EHDV. Specimens tested in the Nebraska Veterinary Diagnostic Center (283 submissions) were 25% test positive for antibodies to BTV and/or EHDV. Low disease incidence in white-tailed deer and other susceptible wild ungulates was observed during 2016. However, there were no confirmed reports of disease in livestock in either state. The reasons for emergence of significant clinical disease in livestock and wildlife populations remain undefined.

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.

Species composition of Culicoides (Diptera: Ceratopogonidae) in the Ridge and Valley region of Tennessee, USA.

Biting midges in the genus Culicoides Latreille (Diptera: Ceratopogonidae) are known to transmit many pathogens of veterinary and medical concern. Although much work has been done globally and in certain regions of North America, Culicoides spp. research in rural Appalachia is limited. To begin characterizing the distribution and community structure of Culicoides spp. in Appalachia, we surveyed 2 distinct sites in the Ridge and Valley ecoregion of northeastern Tennessee, USA, from April 2021-September 2021. Culicoides spp. were sampled using 2 methods: Centers for Disease Control ultraviolet LED light traps and potential larval habitat substrate collection (coupled with water chemistry values). Site 1 was dominated by natural features, and Site 2 was a beef cattle operation. During 96 trap nights, a total of 1,568 Culicoides were collected, representing 24 species. Site 1 yielded the highest diversity, with 24 species, while Site 2 yielded 12 species. Overall, the most abundant species in light traps were C. stellifer Coquillett (44%), C. bergi Cochrane (18%), C. haematopotus Malloch (12%), and C. debilipalpis Lutz (11%). From substrate sampling, 8 species were identified. Culicoides haematopotus was the most abundant and was collected during each sampling period. Water chemistry values taken at the time of substrate collection were not significantly related to which Culicoides spp. emerged from a given substrate. Our results indicate a diverse community of Culicoides spp. in our study area, however, further work is needed to identify Culicoides species composition across a variety of landscapes in Appalachia and inform research on vector presence and associated vector disease dynamics.

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.

Patterns of Hemorrhagic Disease in White-Tailed Deer (Odocoileus virginianus) in the Great Plains of the USA, 1982-2020.

Hemorrhagic disease (HD) of deer is caused by epizootic hemorrhagic disease virus (EHDV) or bluetongue virus (BTV) and is considered one of the most important viral diseases of white-tailed deer (Odocoileus virginianus). Despite evidence of changing patterns of HD in the northeastern and upper midwestern US, the historical and current patterns of HD in the Great Plains remain poorly described. We used results from an annual survey documenting HD mortality to characterize historic and current patterns of HD in the northern and central Great Plains (North Dakota, South Dakota, Nebraska, Kansas, and Oklahoma), US, between 1982 and 2020. Further, we assessed temporal change using linear regression to determine change in annual reporting intensity (percentage of counties in a state with reported HD) and change in reporting frequency (the number of years a county or state reported HD) during each decade between 1982 and 2020. Across the 38-yr study period, HD reports expanded northeast across latitude and longitude. Intensity of HD reports significantly increased during this period for three (North Dakota, South Dakota, Kansas) of five states examined. Frequency of reports also increased for all five states. Such changes in northern latitudes might lead to increased deer mortality in regions where HD epizootics have been historically less frequent. Understanding how patterns of HD are changing on the landscape is important when considering future deer management in the face of other mortality factors.

Demographic Risk Factors Vary in the Invasion Front of Chronic Wasting Disease in West Virginia, USA.

After detecting chronic wasting disease (CWD) in white-tailed deer (Odocoileus virginianus) in Hampshire County, West Virginia, USA, in 2005, we investigated the change of CWD apparent prevalence and potential factors influencing infection risk during the invasion front. Over eight sampling years (2006-2012 and 2017) during a 12-yr period within a 101-km2-area monitoring zone, we sampled and tested a total of 853 deer for CWD by ELISA and immunohistochemistry. Bayesian logistic regression of risk factors included collection year, age class, sex, and adjusted body weight (weight after accounting for sex, age, kidney fat index, and number of fetuses). In the whole-herd model (n=634), collection year, age, and adjusted body weight were associated with increased odds of CWD, whereas an age-weight interaction had a negative relationship. We found that males drove the positive associations with age and adjusted body weight, whereas females were responsible for the negative interaction effect. These findings suggest potential behavioral and physiological mechanisms related to sex that may influence CWD exposure. Older males exhibited higher CWD prevalence, aligning with previous studies. Notably, the novel finding of adjusted body weight as a risk factor in males warrants further investigation, and this study highlights the need for future research on social behavior and its role in CWD transmission within white-tailed deer populations.

Causes of morbidity and mortality in wild cottontail rabbits in the eastern United States, 2013-2022.

Interest in causes of mortality of free-ranging, native North American lagomorphs has grown with the emergence of rabbit hemorrhagic disease virus 2 (RHDV2). Over the years 2013-2022, the Southeastern Cooperative Wildlife Disease Study received 119 Sylvilagus spp. case submissions from the central and eastern United States, comprising 147 rabbits. Most (86%) of these submissions occurred after detecting RHDV2 in the United States in 2020. Laboratory data from these rabbits were retrospectively evaluated for major causes, contributors to mortality, and pathogen detections. Gross and histologic examination was performed for 112 rabbits. Common primary causes of death included trauma (n = 49), bacterial disease (n = 31), emaciation (n = 6), and parasitism (n = 6). Among the 32 rabbits with bacterial disease, 12 were diagnosed with tularemia and 7 with pasteurellosis. Rabbits with pasteurellosis had disseminated abscessation, septicemia, and/or polyserositis. Less commonly, cutaneous fibroma (n = 2), notoedric mange (n = 2), encephalitozoonosis (n = 2), neoplasia (round-cell sarcoma; n = 1), and congenital abnormalities (n = 1) were diagnosed. RHDV2 was not detected in 123 rabbits tested. Although RHDV2 has not been detected in wild lagomorphs in the eastern United States, detections in domestic rabbits from the region emphasize the need for continued surveillance. Furthermore, continued surveillance for Francisella tularensis informs public health risk. Overall, increased knowledge of Sylvilagus spp. health furthers our understanding of diseases affecting these important prey and game species.

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.

High Prevalence and Broad Distribution of Trichomonas gypaetinii in Bald Eagles (Haliaceetus leucocephalus) in the USA.

Trichomonas gypaetinii was detected in 117 (88%) of 133 Bald Eagles (Haliaceetus leucocephalus) and in 0 of 7 Golden Eagles (Aquila chrysaetos) in the USA, with no sex or age prevalence difference. All eagles lacked associated lesions. This study indicated that T. gypaetinii is common and widespread in Bald Eagles, but rarely associated with disease.

Role of multiple hosts in the cross-species transmission and emergence of a pandemic parvovirus.

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Understanding rabbit owners' willingness to engage in disease prevention behaviors.

Rabbit hemorrhagic disease virus 2 (RHDV2) is a fatal, highly contagious pathogen that infects wild and domestic lagomorphs (rabbits and hares). RHDV2 is an important cause of disease in pet and companion rabbits, has resulted in economic losses for the commercial rabbit industry, and has caused declines of wild lagomorph populations. It is essential for domestic rabbit owners to engage in appropriate actions (e.g., using effective disinfectants, creating secure barriers between domestic and wild rabbits) to protect the health and welfare of their rabbits and reduce the risk of human-mediated spread of RHDV2. Thus, we investigated rabbit owners' stated willingness to engage in nine commonly recommended biosecurity practices and their support for seven potential government-implemented management actions. We administered an online survey to 1790 rabbit owners in the United States between April and August 2021. Respondents were likely to engage in all biosecurity measures and were supportive of most management actions that could be implemented by government agencies. Respondents' willingness to engage in and support biosecurity measures was positively correlated with their perceptions of the importance of biosecurity, risk perceptions pertaining to the impact of RHDV2 on lagomorphs and rabbit-related industries, knowledge of RHDV2, and trust in government to manage RHDV2. Respondents' motivations for owning rabbits, husbandry behaviors, and demographic characteristics also influenced their willingness to engage in or support biosecurity measures. Engaging domestic rabbit owners in collaborative biosecurity measures is critical for protecting domestic rabbit health and preventing potential spillover between domestic and free-roaming lagomorphs, as there are still many uncertainties about how RHDV2 is spreading across the United States and the world. Implementing outreach strategies that communicate the importance and effectiveness of biosecurity practices in protecting rabbit welfare, rabbit-related activities, and wild lagomorph populations may increase the likelihood of rabbit owners adopting biosecurity measures.

Storage time and temperature of filter paper strips affect anti-West Nile virus antibody detection in 2 galliform species.

The effects of filter paper strip (FPS) storage time and temperature on antibody detection are poorly understood despite widespread use in wildlife research. We collected sera and FPSs from 23 wild turkeys (Meleagris gallopavo) and 20 northern bobwhites (Colinus virginianus) experimentally infected with West Nile virus (WNV) to compare FPS storage methods with WNV plaque reduction neutralization test (PRNT) sensitivity. FPS storage methods included: immediate elution after drying, and storage at -20°C for 3 mo, -20°C for 6 mo, room temperature (RT) for 3 mo, and RT for 6 mo prior to elution. FPS eluates and sera were co-titrated to determine endpoint antibody titers, which were compared between FPS sera eluted immediately and sera, and among FPS eluates that underwent different storage conditions. Antibody titers were ~4-fold less in FPS sera eluted immediately versus sera, and dropped more frequently below PRNT detection threshold in northern bobwhites, which had ~10-fold lower serum antibody titers than wild turkeys. Antibody titers were lower in FPS samples stored at RT and for 6 mo. WNV serologic surveys may result in falsely low seroprevalence estimates if FPSs are stored at RT for ≥ 3 mo before elution.