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.

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.

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.

Echinococcus Species Infections among Wild Canids in Pennsylvania, USA.

Echinococcus species are zoonotic tapeworms that can impact the health of wildlife, domestic animals, livestock, and humans. Two species of interest in North America are Echinococcus multilocularis and Echinococcus canadensis (Echinococcus granulosus sensu lato). The primary wildlife definitive hosts for E. multilocularis and E. canadensis are similar, including red foxes (Vulpes vulpes), gray foxes (Urocyon cinereoargenteus), coyotes (Canis latrans), and wolves (Canis lupus). These two Echinococcus spp. use different intermediate hosts, including small mammals for E. multilocularis and artiodactylids for E. canadensis. Although historically absent from much of the eastern US, recent reports in new US states (e.g., Virginia, Vermont, Maine, Missouri) highlight the need for Echinococcus spp. surveillance in this region. During 2019-2020, 308 gastrointestinal tracts were collected from wild canids in Pennsylvania and microscopically screened for adult Echinococcus species. Two coyotes (2/155) were co-infected with both E. multilocularis and E. canadensis as determined by molecular confirmation. No red foxes (n=137) or gray foxes (n=16) were positive. These data indicate both Echinococcus species are present in Pennsylvanian coyotes, highlighting the need to better understand the ecological and epidemiological consequences for human and animal health.

High Prevalence of Antibodies against Canine Parvovirus and Canine Distemper Virus among Coyotes and Foxes from Pennsylvania: Implications for the Intersection of Companion Animals and Wildlife.

Canine distemper virus (CDV) and Canine parvovirus (CPV) can cause deadly infections in wildlife and companion animals. In this report, we screened serum from free-ranging eastern coyotes (Canis latrans; N = 268), red foxes (Vulpes vulpes; N = 63), and gray foxes (Urocyon cinereoargenteus; N = 16) from Pennsylvania, USA, for antibodies (Abs) to CDV and CPV. This comprehensive screening was achieved using a commercially available enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay. Abs to CDV and CPV were detected in 25.4% and 45.5% of coyotes, 36.5% and 52.4% of red foxes, and 12.5% and 68.8% of gray foxes, respectively. Abs to both viruses were detected in 9.7% of coyotes, 19.1% of red foxes, and 12.5% of gray foxes. This study demonstrates significant wildlife exposure in a northeastern state to CDV and CPV. As wildlife species continue to urbanize, the probability of spillover between domestic animals and wildlife will increase. Ongoing surveillance of wildlife for CDV and CPV exposure is warranted. IMPORTANCECanine distemper virus (CDV) and Canine parvovirus (CPV) are significant health threats to domestic dogs (Canis familiaris) and wildlife. CDV and CPV have been identified in diverse vertebrates, including endangered wildlife species. Susceptibility to these viral pathogens varies significantly among geographic regions and between host species. High morbidity and mortality have been reported with infection by either virus in susceptible species, including dogs. As humans and companion animals encroach on wildlife habitat, and as wildlife becomes increasingly urbanized, the potential for transmission between species increases. This study assessed CPV and CDV Ab prevalence in wild canids (eastern coyotes, red foxes, and gray foxes) harvested in Pennsylvania between 2015 and 2020. High Ab prevalence was demonstrated for both viruses in each species. Ongoing monitoring of CPV and CDV in wildlife and increased efforts to vaccinate dogs and prevent spillover events are essential.

Toxoplasma gondii prevalence in carnivorous wild birds in the eastern United States.

Toxoplasma gondii is an important zoonotic protozoan parasite that can infect all warm-blooded animals including mammals and birds. Raptors can be intermediate hosts for T. gondii and the infection may be dependent on their feeding habits. In this study, we investigated the seroprevalence of T. gondii in ten raptor species from Florida, Pennsylvania, and Tennessee followed by a parasite bioassay on select seropositive samples. From a total of 155 raptors, we detected T. gondii antibodies using a modified agglutination test (cutoff 1:25) in 32 (20.6%) birds. The T. gondii seroprevalence was 44.8% in Falconiformes (13/29), 75% in Strigiformes (15/20), and 3.8% in Ciconiiformes (4/106). All Ciconiiformes samples (hearts and sera) were collected from Pennsylvania during nuisance wildlife removal projects and all birds were apparently healthy. Falconiform and Strigiform samples were collected from an exotics clinic in Tennessee and a rehabilitation center in Florida. All sampled birds were dead or euthanatized due to failure of rehabilitation or treatment. There was no statistically significant difference in T. gondii seroprevalence between Tennessee and Florida in the tested raptors. There was also no statistically significant difference in T. gondii exposure between males and females or adults and subadults. Mice bioassay attempts using fresh brain and/or heart tissue were performed on four seropositive birds. We isolated viable T. gondii tachyzoites from one red-shouldered hawk (Buteo lineatus) and genotyped the isolate using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ten genetic markers. The isolated strain was designated as TgHawkFL1, which is ToxoDB PCR-RFLP genotype #28. Further research is needed to investigate the prevalence of T. gondii in raptors in the United States to obtain a better understanding of the life cycle, wildlife population impacts, and transmission dynamics of the parasite.

Serologic Evidence for the Exposure of Eastern Coyotes (Canis latrans) in Pennsylvania to the Tick-Borne Pathogens Borreliella burgdorferi and Anaplasma phagocytophilum.

Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes (Canis latrans) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum, respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively.IMPORTANCE The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.

Serosurvey for Influenza Virus Subtypes H3N8 and H3N2 Antibodies in Free-Ranging Canids in Pennsylvania, USA.

Canine influenza virus (CIV) subtypes H3N8 and H3N2 are endemic among domestic dog ( Canis lupus familiaris ) populations in the northeastern US. Infection of free-ranging carnivores with influenza virus has been sporadically reported. Generalist mesocarnivores that exploit anthropogenic, peri-urban habitats share a wide interface with domestic dogs that allows for the transmission of infectious disease. To investigate the potential exposure of free-ranging canids to CIV in Pennsylvania, US, serum samples were obtained from freshly killed coyotes ( Canis latrans, n=67), grey foxes ( Urocyon cinereoargenteus, n=8), and red foxes ( Vulpes vulpes, n=5) from 24 counties. Animals were harvested during the January-February 2017 hunting season. We failed to detect antibodies to CIV subtypes H3N2 and H3N8 by using hemagglutination inhibition assays validated for domestic dogs. Results suggest CIV was not endemic in free-ranging canid populations in Pennsylvania or that prevalence was too low to be detected by our limited sample size.

Demographic history influences spatial patterns of genetic diversityin recently expanded coyote (Canis latrans) populations.

Human-mediated range expansions have increased in recent decades and represent unique opportunities to evaluate genetic outcomes of establishing peripheral populations across broad expansion fronts. Over the past century, coyotes (Canis latrans) have undergone a pervasive range expansion and now inhabit every state in the continental United States. Coyote expansion into eastern North America was facilitated by anthropogenic landscape changes and followed two broad expansion fronts. The northern expansion extended through the Great Lakes region and southern Canada, where hybridization with remnant wolf populations was common. The southern and more recent expansion front occurred approximately 40 years later and across territory where gray wolves have been historically absent and remnant red wolves were extirpated in the 1970s. We conducted a genetic survey at 10 microsatellite loci of 482 coyotes originating from 11 eastern U.S. states to address how divergent demographic histories influence geographic patterns of genetic diversity. We found that population structure corresponded to a north-south divide, which is consistent with the two known expansion routes. Additionally, we observed extremely high genetic diversity, which is atypical of recently expanded populations and is likely the result of multiple complex demographic processes, in addition to hybridization with other Canis species. Finally, we considered the transition of allele frequencies across geographic space and suggest the mid-Atlantic states of North Carolina and Virginia as an emerging contact zone between these two distinct coyote expansion fronts.

Serologic Evidence of Various Arboviruses Detected in White-Tailed Deer (Odocoileus virginianus) in the United States.

White-tailed deer (Odocoileus virginianus) are an abundant mammal with a wide geographic distribution in the United States, which make them good sentinels for monitoring arboviral activity across the country. Exposure to various arboviruses has been detected in white-tailed deer, typically in conjunction with another diagnostic finding. To better assess the exposure of white-tailed deer to seven arboviruses, we tested 1,508 sera collected from 2010 to 2016 for antibodies to eastern equine encephalitis (2.5%), Powassan (4.2%), St. Louis encephalitis, (3.7%), West Nile (6.0%), Maguari (19.4%), La Crosse (30.3%), and bluetongue (7.8%) viruses. At least one arbovirus was detected in 51.3%, and exposure to more than one arbovirus was identified in 17.6% of the white-tailed deer sampled.

Subtype-specific influenza A virus antibodies in Canada geese (Branta canadensis).

Historically, surveillance for influenza A viruses (IAVs) in wild birds has relied on viral detection assays. This was largely due to poor performance of serological assays in wild birds; however, recently developed commercial serological assays have improved the ability to detect IAV antibodies in wild birds. Serological surveillance for IAV antibodies in Canada geese (Branta canadensis) has shown that, despite a low prevalence of virus isolations, Canada geese are frequently exposed to IAVs and that exposure increases with latitude, which follows virus isolation prevalence patterns observed in dabbling ducks. The objectives of this study were to further evaluate IAV antibodies in Canada geese using a subtype-specific serological assay to determine if Canada geese are exposed to subtypes that commonly circulate in dabbling ducks. We collected serum samples from Canada geese in Minnesota, New Jersey, Pennsylvania, and Wisconsin and tested for antibodies to IAVs using a blocking ELISA. Positive samples were further tested by hemagglutination inhibition for 10 hemagglutinin IAV subtypes (H1-H10). Overall, we detected antibodies to NP in 24% (714/2919) of geese. Antibodies to H3, H4, H5, and H6 subtypes predominated, with H5 being detected most frequently. A decrease in H5 HI antibody prevalence and titers was observed from 2009 to 2012. We also detected similar exposure pattern in Canada geese from New Jersey, Minnesota, Washington and Wisconsin. Based on the published literature, H3, H4, and H6 viruses are the most commonly reported IAVs from dabbling ducks. These results indicate that Canada geese also are frequently exposed to viruses of the same HA subtypes; however, the high prevalence of antibodies to H5 viruses was not expected as H5 IAVs are generally not well represented in reported isolates from ducks.

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania.

Toxoplasma gondii infects virtually all warm-blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture-derived tachyzoites of these isolates was characterized using multilocus PCR-RFLP markers. Three genotypes were revealed, including ToxoDB PCR-RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat.

Serologic survey of antibodies to Trypanosoma cruzi in coyotes and red foxes from Pennsylvania and Tennessee.

Trypanosoma cruzi is a zoonotic parasite of humans and other mammalian hosts with distribution throughout the Americas. Domestic and wild canine species are reservoirs for human T. cruzi infections. The present study examined the prevalence of antibodies to T. cruzi in wild canids from the United States. Sera from 13 red foxes (Vulpes vulpes) and 263 coyotes (Canis latrans), originating in Pennsylvania and Tennessee, were assayed for antibodies to T. cruzi with immunochromatographic tests. Antibodies to T. cruzi were found in 2 of 276 (0.72%) of all wild canids tested. Both T. cruzi-positive wild canids were coyotes and represented 2 of 21 (9.52%) wild canids assayed from Tennessee. Antibodies to T. cruzi were not detected in red fox. Anti-T. cruzi antibodies were not found in any wild canids from Pennsylvania. These results suggest that coyotes are exposed to T. cruzi in Tennessee but not in Pennsylvania.

Survey of antibodies to Leishmania spp. in wild canids from Pennsylvania and Tennessee.

Visceral leishmaniasis (VL) is a zoonosis with worldwide distribution. Infections with the Leishmania donovani complex, including Leishmania infantum, cause the VL. Domestic dogs are the most important reservoir host for human VL, and wild canids are also susceptible. In the United States, infections with L. infantum are common in the foxhound dog breed. Little information is available regarding L. infantum in wild canids in the Unites States. Sera from 11 foxes and 256 coyotes originating in Pennsylvania and Tennessee (USA) were tested for antibodies to visceralizing Leishmania spp. with rapid immunochromatographic dipstick assays, which utilize recombinant antigen K39. Anti-Leishmania spp. antibodies were found in 5 of 267 (1.9%) of wild canids from Pennsylvania, including four coyotes and one red fox. These results suggest that wild canids are exposed to Leishmania spp. at a low level in the United States.

Antibodies to avian influenza viruses in Canada geese (Branta canadensis): a potential surveillance tool?

Traditionally, the epidemiology of avian influenza viruses (AIVs) in wild birds has been defined by detection of virus or viral RNA through virus isolation or reverse-transcription polymerase chain reaction. Our goals were to estimate AIV antibody prevalence in Canada geese (Branta canadensis) and measure effects of age and location on these estimates. We collected 3,205 samples from nine states during June and July 2008 and 2009: Georgia, Massachusetts, Minnesota, Mississippi, New Jersey, North Carolina, Pennsylvania, Washington, and West Virginia. Serum samples were tested for AIV antibodies with the use of a commercial blocking enzyme-linked immunosorbent assay. Overall, 483 (15%) Canada geese had detectable antibodies to AIV. Significantly higher prevalences were detected in geese collected from northeastern and upper midwestern states compared with southeastern states. This trend is consistent with results from virus isolation studies reporting AIV prevalence in North American dabbling ducks. Within Pennsylvania, significantly higher antibody prevalences were detected in goose flocks sampled in urban locations compared to flocks sampled in rural areas. Antibody prevalence was significantly higher in after-hatch-year geese compared to hatch-year geese. No significant differences in prevalence were detected from 10 locations sampled during both years. Results indicate that Canada geese are frequently exposed to AIVs and, with resident populations, may potentially be useful as sentinels to confirm regional AIV transmission within wild bird populations.