RAPID POINT-OF-CARE TESTING FOR DETECTION OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK VULTURES AND RING-BILLED GULLS FROM PENNSYLVANIA.

Toxoplasma gondii is a zoonotic protozoan parasite that infects most warm-blooded animals, including birds. Scavenging birds are epidemiologically important hosts because they can serve as indicators of environmental T. gondii levels. A rapid point-of-care (POC) test that detects antibodies to T. gondii in humans is commercially available. In this research, we assessed the ability of the human POC test to detect anti-T. gondii antibodies in 106 black vultures (Coragyps atratus) and 23 ring-billed gulls (Larus delawarensis) from Pennsylvania, USA. Serum samples were tested with the POC test and compared to the modified agglutination test (MAT) in a blinded study. Overall, anti-T. gondii antibodies were detected in 2.8% (3/106) of black vultures and 60.9% (14/23) of ring-billed gulls by the POC test. One false-positive POC test occurred in a black vulture that was negative by MAT. False-negative results were obtained in 2 black vultures and 4 ring-billed gulls that had MAT titers of 1:25 or 1:50. The sensitivity and specificity of the POC for both black vultures and ring-billed gulls combined were 95.7% and 95.5%, respectively. This is the first study using human POC tests to detect antibodies to T. gondii in birds. Further study of the rapid test as a screening tool for serological surveillance of T. gondii in birds is warranted.

A De Novo Whole Genome Assembly and Annotation of Parelaphostrongylus tenuis.

Parelaphostrongylus tenuis causes ungulate morbidity and mortality in eastern and central North America, but no reference genome sequence exists to facilitate research. Here, we present a P. tenuis genome assembly and annotation, generated with PacBio and Illumina technologies. The assembly is 491 Mbp, with 7285 scaffolds and 185 kb N50.

Toxoplasma gondii Survey in Waterfowl and Gulls from Eight USA States.

Sera from 391 waterbirds from eight USA states were tested for Toxoplasma gondii antibodies using the modified agglutination test. Fifteen different waterbird species (26.6%; n=104) were seropositive. Of the adults, 25.4% (n=52) showed a significantly higher T. gondii seroprevalence compared with juveniles (13.4%; n=17); however, sex was not a significant factor.

Isolation of Toxoplasma gondii in cell culture: an alternative to bioassay.

Toxoplasma gondii is an apicomplexan protozoan parasite that can infect mammals and birds. The infection can cause acute toxoplasmosis and death in susceptible hosts. Bioassay using cats and mice has been the standard for the isolation of T. gondii from infected hosts for the past several decades. However, bioassay is labor-intensive, expensive, and involves using laboratory animals. To search alternative approaches and o work towards replacement of animal experiments, we summarized the key literature and conducted four experiments to isolate T. gondii in vitro by cell culture. A few heart tissue samples from animals with the highest antibody titers in a given collection were used for T. gondii isolation. These experiments included samples from five out of 51 wild ducks, four of 46 wild turkeys, six of 24 white-tailed deer, as well as from six kangaroos that had died with acute toxoplasmosis in a zoo. These experiments resulted in three isolates from five chronically infected wild ducks (60%), four isolates from four chronically infected wild turkeys (100%), one isolate from six chronically infected white-tailed deer (17%), and four isolates from six kangaroos with acute toxoplasmosis (67%). In addition, five isolates from the five chronically infected wild ducks were obtained by bioassay in mice, showing a 100% success rate, which is higher than the 60% rate by direct cell culture. These T. gondii isolates were successfully propagated in human foreskin fibroblast (HFF) or Vero cells, and genotyped by multilocus PCR-RFLP markers. The results showed that it is practical to isolate T. gondii directly in cell culture. Although the cell culture approach may not be as sensitive as the bioassay, it does provide an alternative that is simple, cost-effective, ethically more acceptable, and less time-sensitive to isolate T. gondii. In this paper we propose a procedure that may be applied and further optimized for isolation of T. gondii.

Hepatozoon spp. infection in wild canids in the eastern United States.

BACKGROUND: Hepatozoon spp. are apicomplexan parasites known to cause musculoskeletal disease in a variety of animals. Two species are known to infect wild and domestic canids in the US: Hepatozoon canis and H. americanum. METHODS: In this study, blood, heart, and/or spleen samples were collected from 278 wild canids (180 coyotes, 93 red foxes, and 5 gray foxes) in the eastern US and tested via PCR for Hepatozoon. Histology slides of heart and skeletal muscle were assessed for Hepatozoon cysts and associated inflammation when fresh tissue was available (n = 96). RESULTS: Hepatozoon spp. were found in 24.2% (59/278) of individuals, with Hepatozoon canis in 14.0% (34/278) and H. americanum in 10.7% (26/278). One coyote was positive for both H. canis and H. americanum. Foxes were more likely to be positive for H. canis than coyotes (23% and 7% respectively, P = 0.0008), while only coyotes were positive for H. americanum. Of the eight sampled states, H. canis was present in six (Louisiana, North Carolina, Pennsylvania, South Carolina, Tennessee, and Virginia) while H. americanum was found in two southern states (South Carolina and Louisiana). Infection status was positively correlated with myositis and myocarditis, and heart or muscle cysts were found in 83% (5/6) of H. americanum-positive coyotes. CONCLUSION: This survey showed a moderate prevalence of H. canis and H. americanum in states where the parasite was previously unrecorded including South Carolina and Pennsylvania.

Persistent Trichomonas vaginalis infections and the pseudocyst form.

Recent studies have proposed that Trichomonas vaginalis, the causative agent of trichomoniasis [the most common nonviral sexually transmitted infection (STI) in humans] can establish persistent infections in the vagina. T. vaginalis infections are often asymptomatic but can have adverse consequences such as increased risk of HIV-1 infection and cervical cancer. Despite this, it remains an understudied infection. A potential agent of persistent infections is the 'pseudocyst', a spherical form of T. vaginalis identified by several laboratories and linked to persistence in related species such as the avian parasite Trichomonas gallinae and cattle parasite Tritrichomonas foetus. Additional robust and reproducible research on pseudocysts and persistent T. vaginalis infections is required, which may ultimately shed light on how to better diagnose and treat trichomoniasis.

North American wintering mallards infected with highly pathogenic avian influenza show few signs of altered local or migratory movements.

Avian influenza viruses pose a threat to wildlife and livestock health. The emergence of highly pathogenic avian influenza (HPAI) in wild birds and poultry in North America in late 2021 was the first such outbreak since 2015 and the largest outbreak in North America to date. Despite its prominence and economic impacts, we know relatively little about how HPAI spreads in wild bird populations. In January 2022, we captured 43 mallards (Anas platyrhynchos) in Tennessee, USA, 11 of which were actively infected with HPAI. These were the first confirmed detections of HPAI H5N1 clade 2.3.4.4b in the Mississippi Flyway. We compared movement patterns of infected and uninfected birds and found no clear differences; infected birds moved just as much during winter, migrated slightly earlier, and migrated similar distances as uninfected birds. Infected mallards also contacted and shared space with uninfected birds while on their wintering grounds, suggesting ongoing transmission of the virus. We found no differences in body condition or survival rates between infected and uninfected birds. Together, these results show that HPAI H5N1 clade 2.3.4.4b infection was unrelated to body condition or movement behavior in mallards infected at this location during winter; if these results are confirmed in other seasons and as HPAI H5N1 continues to evolve, they suggest that these birds could contribute to the maintenance and dispersal of HPAI in North America. Further research on more species across larger geographic areas and multiple seasons would help clarify potential impacts of HPAI on waterfowl and how this emerging disease spreads at continental scales, across species, and potentially between wildlife and domestic animals.

Novel methods of immunogenic antigen selection for serological diagnosis of Parelaphostrongylus tenuis infection.

This paper outlines methods used to identify novel antigens for use in the development of serological assays. Specifically, we applied these methods to a neurogenic parasitic nematode of cervids called Parelaphostrongylus tenuis. This parasite is of particular concern in both wild and domestic ungulates as it causes significant neurological signs and definitive diagnosis is only possible post-mortem, necessitating the development of serologic assays for antemortem diagnosis. Proteins extracted from P. tenuis organisms were affinity isolated using antibodies enriched from seropositive moose (Alces alces). The proteins were analyzed using mass spectrometry and liquid chromatography to obtain amino acid sequences that were then cross-referenced to open reading frames predicted from an assembled transcriptome. An antigen of interest was assessed for immunogenic epitopes and subsequently synthesized into 10-mer synthetic overlapping peptides representing these regions. These synthetic peptides were then assessed for reactivity against positive and negative moose sera and demonstrated potential use as a serological assay in diagnostic laboratories. Known negative moose sera revealed significantly lower optical density when compared to the positive samples (p < 0.05). This method serves as a pipeline for the construction of diagnostic assays of pathogens in both human and veterinary medicine.

MORTALITY, SURVIVAL, AND SEROLOGIC RESULTS FOR ELK (CERVUS CANADENSIS) IN THE CUMBERLAND MOUNTAINS OF TENNESSEE, USA.

Comprehensive disease surveillance has not been conducted in elk (Cervus canadensis) in Tennessee, US, since their reintroduction to the state 20 yr ago. We identified causes of death, estimated annual survival, and identified pathogens of concern in elk at the North Cumberland Wildlife Management Area (NCWMA), Tennessee, US. In 2019 and 2020, we captured 29 elk (21 females, eight males) using chemical immobilization and fitted individuals with GPS collars with mortality sensors. Elk that died between February 2019 and February 2022 were necropsied to identify causes of death; these included disease associated with meningeal worm (Parelaphostrongylus tenuis; n=3), poaching (n=1), vehicular collision (n=1), legal hunter harvest (n=1), and unknown due to carcass degradation (n=3). Using data from GPS collars and known-fate survival models, we estimated an average yearly survival rate of 80.2%, indicating that survival had not significantly increased from soon after elk reintroduction (79.9%). We collected blood, tissue, feces, and ectoparasites opportunistically from anesthetized elk for health surveillance. We identified lone star ticks (Amblyomma americanum; n=53, 85.5%; 95% confidence interval [CI], 73.72-92.75), American dog ticks (Dermacentor variabilis; n=8, 12.9%; 95% CI, 6.13-24.40), and black-legged ticks (Ixodes scapularis; n=1, 1.6%; 95% CI, 0.08-9.83). We detected evidence of exposure to Anaplasma marginale (100%; 95% CI, 84.50-100.00), Leptospira interrogans (70.4%; 95% CI, 49.66-85.50), Toxoplasma gondii (55.6%; 95% CI, 35.64-73.96), epizootic hemorrhagic disease virus (51.9%; 95% CI, 32.35-70.84), and Theileria cervi (25.9%; 95% CI, 11.78-46.59). Johne's disease (Mycobacterium avium subsp. paratuberculosis) is potentially established within the population, but has not been previously documented in eastern elk populations. Disease associated with P. tenuis was a primary cause of death, and more research is needed to understand its ecology and epidemiology. Research to determine population implications of other detected pathogens at the NCWMA is warranted.

USE OF A POINT OF CARE TEST TO DETERMINE THE PREVALENCE OF ANTIBODIES TO TOXOPLASMA GONDII IN BLACK BEARS FROM NORTH CAROLINA AND PENNSYLVANIA.

Toxoplasma gondii is an important protozoan parasite of humans and animals throughout the world. Black bears are among the animals with the highest seroprevalence of T. gondii in the United States. A rapid point of care (POC) test is commercially available to detect antibodies to T. gondii in humans. We evaluated the utility of the POC test to detect anti-T. gondii antibodies in 100 wild black bears from North Carolina (n = 50) and Pennsylvania (n = 50). In a blind study, sera were tested by the POC test, and results were compared to the modified agglutination test (MAT). Overall, anti-T. gondii antibodies were detected in 76% (76/100) black bears by both MAT and POC tests. One false positive and one false negative result in the POC test were obtained in bears from Pennsylvania. The sensitivity and specificity of the POC test were both 99% when compared to the MAT. Results from our study indicate the POC test could be a useful screening tool for serological surveillance of T. gondii in black bears.

Parasite Prevalence in Feral Swine (Sus scrofa) from the Great Smoky Mountains National Park, USA.

Feral swine (Sus scrofa) are an introduced species to the Great Smoky Mountains National Park (GSMNP), US, and serve as carriers of several diseases that are considered a threat to other wildlife, domestic animals, and humans. During 2013 and 2015, fecal samples from 67 feral swine from the GSMNP within both Tennessee and North Carolina, US, were opportunistically collected as part of a feral swine removal program and submitted to the University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee, for parasite screening by centrifugal sugar flotation. Ten taxa from the phyla Acanthocephala, Apicomplexa, and Nematoda were identified: Ascaris spp., Strongylid-type spp., Capillaria spp., Trichuris suis, Metastrongylus spp., Macracanthorhynchus spp., Coccidia, Sarcocystis spp., and Cryptosporidium spp. In 98.5% of samples, at least one parasite was found. No differences in parasite prevalence or species diversity were noted based on state of collection (Tennessee or North Carolina), sex, or age. The high prevalence of gastrointestinal parasites in these feral swine, some of which are zoonotic, represents a potential public health risk as well as a concern for free-range swine farmers.

Coccidiosis in Wild Eastern Newts (Notophthalmus viridescens): An Unexpected Finding in Consecutive Mortality Events in Tennessee, USA.

Wildlife diseases are a major threat for species conservation, and there is a growing need to implement more comprehensive disease response programs to better identify these diseases of concern. During March 2017, we observed moribund and dead eastern newts, Notophthalmus viridescens, in a single pond in middle Tennessee. All moribund individuals were emaciated. We euthanized and processed all individuals immediately on-site and later performed histopathology and quantitative PCR for ranavirus, the protist Perkinsea, and chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans. One newt was positive for ranavirus. Histopathology showed no evidence of ranavirosis but did reveal overwhelming coccidiosis. Overlapping partial sequences of coccidian 18S subunit DNA showed a 96.4% match with Eimeria steinhausi, suggesting that lesions were due to a previously undescribed Eimeria sp. In 2019, two more moribund newts were encountered at the same pond. Histopathology revealed the same suspicious parasitic organisms, and one individual was positive for B. dendrobatidis. Further research on how seasonal and other environmental parameters may influence coccidia-associated morbidity and mortality is warranted. These events highlight the importance of histopathologic evaluation of mortality events and provide guidance for investigation of future outbreaks.

Case report: Halicephalobus gingivalis in a Tennessee pony.

A 17-year-old female grade pony presented to University of Tennessee Veterinary Medical Center in May of 2021 for evaluation of multifocal, firm, sessile, circular lesions of various diameters on the ventrum and flank. The lesions had been present for two weeks at presentation. An excisional biopsy found numerous adult and larval rhabditid nematodes most consistent with Halicephalobus gingivalis. PCR targeting a portion of the large ribosomal subunit confirmed this diagnosis. The patient was treated with a high dose course of ivermectin followed by fenbendazole. The patient began showing neurologic signs five months after initial diagnosis. Due to the poor prognosis, euthanasia was elected. PCR of CNS tissues confirmed the presence of H. gingivalis in the brain, and one adult worm and several larvae were found on histologic sections of the cerebellum. H. gingivalis is a rare but lethal disease of horses and people.

EVALUATING THE EFFICACY OF NONINVASIVE FECAL SAMPLING FOR PREGNANCY DETECTION IN ELK (CERVUS CANADENSIS).

Elk (Cervus canadensis) were reintroduced to Tennessee, USA in the early 2000s, with limited reproductive monitoring since initial release. We assessed the efficacy of noninvasive sampling for determining pregnancy using invasive (capture) and noninvasive (fecal collection in the field) techniques at the North Cumberland Wildlife Management Area (NCWMA), Tennessee. We captured 20 female elk 2019-2020, used pregnancy-specific protein B (PSPB) in blood to determine pregnancy and compared results to fecal progesterone metabolite (FPM) concentrations using two commercially available enzyme immunoassay (EIA) kits. Based on PSPB concentrations, 8/11 and 3/4 of captured adult elk (≥2.5 yr of age) were pregnant in 2019 and 2020, respectively; no 1.5-yr-old elk were pregnant (n=5). Using the progesterone EIA kit, FPM concentrations were x̄=192.84±38.63 ng/g (95% CI, 96.48-289.20) for nonpregnant and x̄=536.17±74.98 ng/g (95% CI, 375.97-696.36) for pregnant captured females. For the progesterone metabolite kit, FPM concentrations were x̄=188.16±43.39 ng/g (95% confidence interval [CI], 76.63-299.69) for nonpregnant and x̄=693.52±126.52 ng/g (95% CI, 407.31-979.72) for pregnant captured females. From February to May 2019, we collected 357 fecal samples in 65 areas across 489.62 km2 of the NCWMA. Using extracted DNA and analysis of 15 microsatellites, we identified 62 unique individuals from 128 female fecal samples collected on the landscape. We categorized females from landscape-collected feces as nonpregnant (35.5-40.3%; Metabolite-EIA kits), undetermined (1.6-6.5%; Metabolite-EIA kits), or pregnant (62.9-53.2%; Metabolite-EIA kits) based on a 95% CI of captured female FPM concentrations, giving an overall pregnancy rate of 53.2% using the recommended EIA kit. The pregnancy rate in sexually mature females may be higher, as it was not possible to distinguish age classes of landscape-collected fecal samples; therefore, some may have been from younger age classes not expected to be pregnant. Analysis of FPM may be useful at a population level to detect pregnancy.

Prevalence and Molecular Analysis of Hellbender (Cryptobranchus alleganiensis) Trypanosomes in Tennessee.

Populations of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have been declining for multiple decades because of a variety of stressors associated with anthropogenic habitat disturbance followed by riparian sedimentation. The influence of parasite-associated morbidity and mortality on wild hellbender populations is poorly understood. Research has detected widespread trypanosome infection in hellbenders in Virginia, US, with no other reported detections within the eastern hellbender's extensive range. In our study, trypanosomes mostly closely resembling Trypanosoma cryptobranchi were observed in a blood smear of a hellbender from Tennessee during a population survey. Banked whole blood from this hellbender along with 51 other hellbenders was molecularly tested for the 18s rRNA gene of amphibian trypanosomes using newly designed PCR primers. In total, 3/52 (5.8%) hellbenders were PCR- and sequence-positive for trypanosomes. This is the first report of the partial 18s rRNA sequence from hellbender trypanosomes from North America. Further research into trypanosome epidemiology and hellbender health implications is warranted.

MORBIDITY AND MORTALITY OF FREE-RANGING AMERICAN BLACK BEARS (URSUS AMERICANUS) UNDERGOING REHABILITATION IN EASTERN TENNESSEE, USA, 1996-2021.

Free-ranging American black bears (Ursus americanus) often share habitat with humans and domestic animals, predisposing them to anthropogenic conflicts. Rehabilitation under professional care is a management option for orphaned, injured, and/or ill bears. Across several southeastern states, rescued bears are assessed and treated at the University of Tennessee and rehabilitated at Appalachian Bear Rescue (ABR). Records from 1996-2021 showed 337 bears (170 males, 166 females, 1 unknown) from nine states were admitted to ABR. Three bears were admitted twice, resulting in 340 admissions (42 neonates <3 mo old, 206 cubs 3-12 mo, 87 yearlings 1-2 yr, and 5 adults >2 yr). Bears presented as orphans (58%), malnourished (24%), injured or ill (12%), or confiscated/other (6%). Individuals were returned to the wild (85%); died or were euthanized (12%); or were placed into professional care (3%). Of released bears, 195 had complete medical records available for evaluation; 31% were healthy upon intake while the remaining were treated successfully for malnutrition and internal parasites (49%), orthopedic (9%) and soft tissue injuries (5%), or other diseases (5%). Causes of death determined during necropsies performed (n=30) were classified as trauma (50%), developmental (13%), undetermined (13%), malnutrition (13%), infectious or inflammatory (7%), and toxicosis (3%). Despite the lack of maternal care and high prevalence of malnutrition and trauma, most bears recovered to release with appropriate husbandry and medical care. This study provides a foundation for research to further improve care of rehabilitating black bears.

Possible Cross-Reactivity of Feline and White-Tailed Deer Antibodies against the SARS-CoV-2 Receptor Binding Domain.

In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2's RBD is antigenically distinct from common human and animal coronaviruses, allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2's RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2's RBD. Surprisingly prepandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were ∼50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing, as cross-reactive antibodies toward SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species. IMPORTANCE We report cross-reactive antibodies from prepandemic cats and postpandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2's RBD region. However, we lack conclusive evidence of prepandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

Toxoplasma gondii contamination at an animal agriculture facility: Environmental, agricultural animal, and wildlife contamination indicator evaluation.

Toxoplasma gondii is a parasite of significant public health importance. We attempted to detect T. gondii contamination and assess advantages and disadvantages of contamination indicators through surveilling soil, wildlife, cats (Felis catus), and cows (Bos taurus) on a farm in Tennessee, U.S. in 2016 and 2017. Twenty-two soil samples were collected from the farm and subjected to oocyst flotation, DNA extraction, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting 18S ribosomal RNA (18S rRNA) gene to detect and identify T. gondii. Three samples (13.6%) were positive for the parasite; however, T. gondii DNA was not consistently detected from repeated tests. Blood samples were collected from small mammals, cats, and mesopredators live-trapped on the farm, and serum from 30 of the farm's cows were obtained. Serological testing by the modified agglutination test (MAT; cutoff 1:50) found 2.5% (1/40) of small mammals, 52.9% (9/17) of raccoons (Procyon lotor), and 50% (1/2) of domestic cats were seropositive for T. gondii antibodies. No antibodies were found in 16 opossums (Didelphis virginiana), two skunks (Mephitis mephitis), and 30 cows. Small mammal tissue samples were subjected to PCR-RFLP detection. Four out of 29 (13.7%) tissue samples were positive for T. gondii; however, T. gondii DNA was not consistently detected during repeated PCR-RFLP testing. Our results indicate the ability to detect T. gondii varies greatly by contamination indicator. We found detection of soil oocysts to be challenging, and results suggest limited utility of the method performed. The ability to detect T. gondii in animals was highly variable among species. Our research emphasizes the importance of a holistic approach when surveilling for T. gondii to compensate for shortcomings of each contamination indicator. Future research should be conducted to further investigate the most effective T. gondii surveillance methods and species with increased sample sizes at other agricultural facilities.

Correction: Why Lyme disease is common in the northern US, but rare in the south: The roles of host choice, host-seeking behavior, and tick density.

[This corrects the article DOI: 10.1371/journal.pbio.3001066.].