Assessment of 2 Treatment Methods for Ulna Fractures With an Intact Radius in Raptors: Conservative Management and Surgical Fixation With a Type I External Skeletal Fixator Intramedullary Pin Tie-in.

Several methods have been reported for treating avian antebrachial fractures, yet the quantitative information regarding their success is limited. This retrospective study applied data from raptors admitted to a wildlife rehabilitation center to provide metrics associated with the treatment of ulna fractures with an intact radius by either conservative management or a single surgical method: a type I external skeletal fixator tied in to an intramedullary pin (ESF IMP tie-in). Postfledging wild raptors with an ulna fracture admitted to The Raptor Center at the University of Minnesota College of Veterinary Medicine (St Paul, MN, USA) between January 1, 2007, and December 31, 2017, that survived for at least 7 days after admission were included in the study. Information regarding signalment, fracture characteristics, treatment method, outcome, and complications were recorded. The study included 110 raptors representing 15 species. The majority of birds (69%, 76/110) were treated with conservative management. Conservative management was used more often in cases of chronic (P = .02) and closed (P = .02) fractures, whereas fractures with poor alignment at the time of admission were frequently treated through surgical means (P=.001). Alignment was more likely to improve with an ESF IMP tie-in (P=1.01 ×10-9). The majority of cases (65%, 72/110) had a successful outcome. Birds with closed fractures were more likely to have a successful outcome (P=.03). Birds in which fracture alignment became exacerbated with either treatment method were more likely to have a poor outcome (P=.002). The results of this retrospective study found that conservative management and surgical fixation by the ESF IMP tie-in technique are both viable treatment options for ulna fractures with an intact radius in raptors. The choice of treatment method is dependent on the clinician's assessment of the fracture characteristics and individual patient.

Distraction Osteogenesis in Two Wild Raptors.

Distraction osteogenesis was used in 2 wild raptor patients for the repair of tibiotarsal fractures. The first case was a hatching year female peregrine falcon (Falco peregrinus) admitted with an open oblique right distal tibiotarsal fracture. The fracture was surgically managed with the external skeletal fixator intramedullary pin tie-in technique (ESF-IM pin tie-in). Appropriate healing of the fracture site occurred with the ESF-IM pin tie-in. However, there was significant pelvic limb shortening of the affected leg, and the bird consequently developed pododermatitis on the contralateral foot. The second case was an adult female great horned owl (Bubo virginianus) that was admitted with a closed, partially healed, overriding right tibiotarsal fracture. Because of the advanced stage of this fracture, which presented with a fibrous callus, and the already apparent pelvic limb shortening, the ESF-IM pin tie-in was not used. Both patients were fitted with a circular external skeletal fixator (CESF), and distraction osteogenesis was performed until the length of the pelvic limb was deemed anatomically adequate. It is critical that rehabilitated raptors be released without any physical conditions that may reduce their ability to survive and reproduce in the wild. Pelvic limb shortening can potentially predispose a raptor patient to pododermatitis, even with fatal consequences, in both captive and wild environments. The orthopedic technique used here proved useful to repair the limb shortening in both raptor cases, and each bird fully recovered and was released.

Patterns and trends in lead (Pb) concentrations in bald eagle (Haliaeetus leucocephalus) nestlings from the western Great Lakes region.

Most studies examining bald eagle (Haliaeetus leucocephalus) exposure to lead (Pb) have focused on adults that ingested spent Pb ammunition during the fall hunting season, often at clinical or lethal levels. We sampled live bald eagle nestlings along waterbodies to quantify Pb concentrations in 3 national park units and 2 nearby study areas in the western Great Lakes region. We collected 367 bald eagle nestling feather samples over 8 years during spring 2006-2015 and 188 whole blood samples over 4 years during spring 2010-2015. We used Tobit regression models to quantify relationships between Pb concentrations in nestling feathers and blood using study area, year, and nestling attributes as covariates. Pb in nestling feather samples decreased from 2006 to 2015, but there was no trend for Pb in blood samples. Pb concentrations in nestling feather and blood samples were significantly higher in study areas located closer to and within urban areas. Pb in feather and blood samples from the same nestling was positively correlated. Pb in feathers increased with nestling age, but this relationship was not observed for blood. Our results reflect how Pb accumulates in tissues as nestlings grow, with Pb in feathers and blood indexing exposure during feather development and before sampling, respectively. Some nestlings had Pb concentrations in blood that suggested a greater risk to sublethal effects from Pb exposure. Our data provides baselines for Pb concentrations in feathers and blood of nestling bald eagles from a variety of waterbody types spanning remote, lightly populated, and human-dominated landscapes.

INFLUENCE OF SNOWFALL ON BLOOD LEAD LEVELS OF FREE-FLYING BALD EAGLES (HALIAEETUS LEUCOCEPHALUS) IN THE UPPER MISSISSIPPI RIVER VALLEY.

Lead poisoning of scavenging raptors occurs primarily via consumption of game animal carcasses containing lead, which peaks during fall firearm hunting seasons. We hypothesized that snowfall would mitigate exposure by concealing carcasses. We categorized blood lead level (BLL) for a subsample of Bald Eagles (Haliaeetus leucocephalus) from the Upper Mississippi River Valley and described BLL with respect to age, sex, and snowfall. We captured Bald Eagles overwintering in the Upper Mississippi River Valley (n=55) between December 1999 and January 2002. Individual BLL ranged from nondetectable to 335 µg/dL, with 73% of the samples testing positive for acute exposure to lead. Eagle BLL did not significantly differ between age or sex, but levels were higher immediately following the hunting season, and they were lower when the previous month's snowfall was greater than 11 cm. This study suggests a window of time between the white-tailed deer (Odocoileus virginianus) hunting season and the onset of snow when the population experienced peak exposure to lead. Combining these findings with existing research, we offer a narrative of the annual lead exposure cycle of Upper Mississippi River Valley Bald Eagles. These temporal associations are necessary considerations for accurate collection and interpretation of BLL.

Correlation of brain Magnetic Resonance Imaging of spontaneously lead poisoned bald eagles (Haliaeetus leucocephalus) with histological lesions: A pilot study.

Six bald eagles with severe, acute lead poisoning based on blood lead values were analyzed by Magnetic Resonance Imaging (MRI) of the brain and histopathology. The aims of the study were to use MRI to locate brain lesions and correlate the changes in MRI signal with the histological character of the lesions at necropsy. All of the bald eagles presented with neurologic and non-neurologic signs suggestive of severe lead poisoning and had blood lead levels in excess of 1.0 ppm. Areas of change in image intensity in the brainstem, midbrain and cerebellum were detected in the MRI scans. Histopathology confirmed the presence of all suspected lesions. The character of the lesions suggested vascular damage as the primary insult. MRI was useful for detecting lesions and defining their three-dimensional distribution and extent. Future studies are needed to evaluate the utility of MRI for detection of lesions in less severely lead poisoned eagles and determining prognosis for treatment.

External skeletal fixator intramedullary pin tie-in for the repair of tibiotarsal fractures in raptors: 37 cases (1995-2011).

OBJECTIVE: To evaluate the outcome of the application of an external skeletal fixator intramedullary pin tie-in (TIF) to tibiotarsal fractures in raptors. DESIGN: Retrospective case series. ANIMALS: Thirty-four raptors with 37 tibiotarsal fractures. PROCEDURES: Medical records and radiographs for raptors with tibiotarsal fractures that were treated at The Raptor Center at the University of Minnesota between 1995 and 2011 were reviewed. Descriptive statistics were generated and univariate logistic regression analyses were used to assess whether age, sex, body weight, location and nature of the fracture, and type of surgical reduction were significantly associated with whether the fracture healed following surgical reduction and TIF application. RESULTS: 31 of 37 (84%) tibiotarsal fractures successfully healed following surgical reduction and TIF application. The mean healing time was 38 days (range, 15 to 70 days). None of the variables assessed were significantly associated with whether the tibiotarsal fracture healed. Twenty of the 34 (59%) raptors were eventually rehabilitated and released. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that most tibiotarsal fractures were successfully managed by surgical reduction and stabilization with a TIF. However, other comorbidities (eg, systemic infections and visual deficits) negatively affected the rehabilitation of raptors and sometimes resulted in euthanasia despite the fact that the tibiotarsal fracture had healed, and those comorbidities, along with the variables evaluated (eg, age, sex, and nature of the fracture), should be used as triage criteria and prognostic indicators.

Disease risk in a dynamic environment: the spread of tick-borne pathogens in Minnesota, USA.

As humans and climate change alter the landscape, novel disease risk scenarios emerge. Understanding the complexities of pathogen emergence and subsequent spread as shaped by landscape heterogeneity is crucial to understanding disease emergence, pinpointing high-risk areas, and mitigating emerging disease threats in a dynamic environment. Tick-borne diseases present an important public health concern and incidence of many of these diseases are increasing in the United States. The complex epidemiology of tick-borne diseases includes strong ties with environmental factors that influence host availability, vector abundance, and pathogen transmission. Here, we used 16 years of case data from the Minnesota Department of Health to report spatial and temporal trends in Lyme disease (LD), human anaplasmosis, and babesiosis. We then used a spatial regression framework to evaluate the impact of landscape and climate factors on the spread of LD. Finally, we use the fitted model, and landscape and climate datasets projected under varying climate change scenarios, to predict future changes in tick-borne pathogen risk. Both forested habitat and temperature were important drivers of LD spread in Minnesota. Dramatic changes in future temperature regimes and forest communities predict rising risk of tick-borne disease.

Clinical, pathological, and immunohistochemical findings in bald eagles (Haliaeetus leucocephalus) and golden eagles (Aquila chrysaetos) naturally infected with West Nile virus.

Fifteen bald eagles (Haliaeetus leucocephalus) and 3 golden eagles (Aquila chrysaetos) were diagnosed with West Nile disease based on 1) presence of lesions in brain, eyes, and heart, 2) viral antigen detection in brain, eyes, heart, kidney, and/or liver by immunohistochemical staining, 3) detection of viral RNA in tissue samples and/or cerebrospinal fluid (CSF) by polymerase chain reaction, and/or 4) detection of West Nile virus (WNV)-specific antibodies in CSF by serum neutralization assay. West Nile virus-associated gross lesions included cerebral pan-necrosis with hydrocephalus ex vacuo (7/15 bald eagles), fibrin exudation into the fundus in 1 golden eagle, retinal scarring in 1 bald eagle, and myocardial pallor and rounded heart apex in 4 bald eagles. Histologic lesions included lymphoplasmacytic encephalitis, most prominently in the cerebrum (17 eagles), lymphoplasmacytic pectenitis and choroiditis (15 and 8 eagles, respectively), and myocarditis (12 eagles). West Nile virus antigen was detected in the majority of the eagles in neurons of the brain (cerebrum and cerebellum), and less commonly present in neurons of the retina, tubular epithelial cells of the kidney, and cardiomyocytes. West Nile disease was diagnosed in 2 bald eagles based on the presence of cerebral pan-necrosis and WNV-specific antibodies in the CSF despite lacking viral antigen and RNA. In conclusion, WNV infection causes a fatal disease in bald and golden eagles. A variety of gross and histologic lesions are highly suggestive of WN disease in most eagles. A combination of detection of viral antigen and/or RNA or virus-specific antibodies proved useful in confirming the diagnosis.

Serologic evidence of exposure of raptors to influenza A virus.

Serum or plasma samples from raptors that prey or scavenge upon aquatic birds were tested by a commercially available blocking enzyme-linked immunosorbent assay for the evidence of antibodies to influenza A virus. Samples were taken from birds (n = 616) admitted to two rehabilitation centers in the United States. In addition, samples from 472 migrating peregrine falcons (Falco peregrinus) trapped on autumnal and vernal migrations for banding purposes were also tested. Only bald eagles were notably seropositive (22/406). One each of peregrine falcon, great horned owl (Bubo virginianus), and Cooper's hawk (Accipiter cooperi) from a total of 472, 81, and 100, respectively, were also positive. None of the turkey vultures (n = 21) or black vultures (n = 8) was positive. No clinical signs referable to avian influenza were seen in any bird at the time of capture. These data indicate that, among raptors, bald eagles do have exposure to influenza A viruses.

Improved method for the isolation and sub-typing of avian influenza viruses from oropharyngeal samples of ducks.

Waterfowl are the natural reservoirs of avian influenza viruses (AIVs), from which the virus can spread to other species including humans, poultry, and swine. For the surveillance of AIV in their natural reservoir, most laboratories initially screen the samples using real-time reverse-transcriptase-polymerase chain reaction because of its high speed and sensitivity. Thereafter, virus isolation is used to isolate viruses from positive samples. Although many studies point to the need of testing both cloacal and oropharyngeal (OP) samples in AIV surveillance programs, most laboratories focus only on cloacal samples. This study was undertaken to determine the utility of OP samples as target samples in AIV surveillance programs under a strict cold chain of samples from the field to the laboratory. A total of 16 AIV (15.1%) were isolated from the 106 OP samples examined. Upon subtyping, four hemagglutinin subtypes (H1, H3, H4, and H6) and three neuraminidase subtypes (N1, N2, and N8) were detected in nine different combinations. Mixed infection with two different subtypes was found in four samples. No AIVs were isolated from the corresponding cloacal samples. These results highlight the fact that testing of properly frozen OP samples could add value to the understanding of the epidemiology and ecology of AIV in waterfowl populations.

Effect of West Nile virus DNA-plasmid vaccination on response to live virus challenge in red-tailed hawks (Buteo jamaicensis).

OBJECTIVE: To evaluate the safety and efficacy of an experimental adjuvanted DNA-plasmid vaccine against West Nile virus (WNV) in red-tailed hawks (Buteo jamaicensis). ANIMALS: 19 permanently disabled but otherwise healthy red-tailed hawks of mixed ages and both sexes without detectable serum antibodies against WNV. PROCEDURES: Hawks were injected IM with an experimental WNV DNA-plasmid vaccine in an aluminum-phosphate adjuvant (n = 14) or with the adjuvant only (control group; 5). All birds received 2 injections at a 3-week interval. Blood samples for serologic evaluation were collected before the first injection and 4 weeks after the second injection (day 0). At day 0, hawks were injected SC with live WNV. Pre- and postchallenge blood samples were collected at intervals for 14 days for assessment of viremia and antibody determination; oropharyngeal and cloacal swabs were collected for assessment of viral shedding. RESULTS: Vaccination was not associated with morbidity or deaths. Three of the vaccinated birds seroconverted after the second vaccine injection; all other birds seroconverted following the live virus injection. Vaccinated birds had significantly less severe viremia and shorter and less-intense shedding periods, compared with the control birds. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the WNV DNA-plasmid vaccine in red-tailed hawks was safe, and vaccination attenuated but did not eliminate both the viremia and the intensity of postchallenge shedding following live virus exposure. Further research is warranted to conclusively determine the efficacy of this vaccine preparation for protection of red-tailed hawks and other avian species against WNV-induced disease.

Isolation of H5 avian influenza viruses from waterfowl in the upper Midwest region of the United States.

In recent years, the H5N1 subtype of avian influenza virus (AIV) has become an important zoonotic pathogen. The surveillance of AIV in its natural host, the waterfowl, is crucial to monitoring and controlling the disease in poultry and other species. In this study, we report on the isolation of H5 AIV from cloacal swabs of waterfowl captured in Minnesota and South Dakota. We screened a total of 7260 cloacal samples from waterfowl using matrix gene-directed, real-time reverse transcription-(rRT-PCR) and H5-specific rRT-PCR and found 148 samples to be positive for the H5 subtype. On inoculation of 71 of these samples in embryonated chicken eggs, 25 samples yielded H5 AIV. On subtyping with N-specific primers, we detected a mixture of subtypes in 15 isolates. Molecular pathotyping confirmed the isolated H5 subtypes to be low pathogenicity avian influenza. Continuation of AIV surveillance programs should help in understanding the epidemiology and ecology of AIV.

Selective isolation of Avian influenza virus (AIV) from cloacal samples containing AIV and Newcastle disease virus.

Avian influenza viruses (AIVs) are important zoonotic pathogens whose natural reservoir is waterfowl. In addition to AIV, waterfowl are often coinfected with other viruses, such as the paramyxoviruses, of which Newcastle disease virus (NDV) is of particular importance because of the highly virulent nature of certain strains of this virus for domestic poultry. In routine surveillance of waterfowl for AIV, a number of cloacal samples were encountered that were positive for AIV by real-time reverse transcription polymerase chain reaction (RT-PCR), but did not yield AIV by inoculation in embryonated chicken eggs. On further testing, these samples were also positive for NDV by conventional RT-PCR. It was hypothesized that if both NDV and AIV are present in a sample, the former may overgrow AIV yielding false-negative AIV results. Such samples were treated with chicken anti-NDV polyclonal antiserum and then inoculated in embryonated chicken eggs. Several samples were found to be positive for different subtypes of AIV, indicating that, in the presence of mixed infection with NDV and AIV, it is imperative to remove the influence of NDV, so a true picture of AIV prevalence emerges. An additional benefit is that information on the circulation of NDV in these birds sheds light on their epidemiologic and ecologic significance.

Isolation of Avian influenza virus from polymerase chain reaction-negative cloacal samples of waterfowl.

Avian influenza virus (AIV) is one of the most important zoonotic pathogens because of its potential to cause severe disease outbreaks in avian and human hosts. Virus isolation in embryonated chicken eggs (ECEs) remains a gold standard technique for AIV detection. However, some laboratories prefer molecular methods, such as real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), for initial sample screening because of their high throughput sample processing and rapid results. Samples found positive on real-time qRT-PCR are then inoculated in ECEs for virus isolation and characterization. This approach is based on the premise that real-time qRT-PCR will detect all AIV-positive samples. The current study aimed to determine if AIV can be isolated from cloacal samples of waterfowl that were initially found to be negative by real-time qRT-PCR screening. Quantitative RT-PCR-negative cloacal samples (1,369) were inoculated for virus isolation in commercial nonspecific pathogen-free ECEs. After 4 days of incubation, the allantoic fluids were harvested and inoculated in fresh ECEs for a second passage. Allantoic fluids from 147 samples were positive for hemagglutination with chicken erythrocytes. Of the 147 hemagglutination-positive allantoic fluids, 82 were AIV positive when confirmed with real-time qRT-PCR. Ten isolates were subtyped as H7N2 (n  =  7), H7N1, H1N2, and H2N2. In addition, N subtype could be determined for isolates from an additional 25 samples. These results highlight the fact that screening by real-time qRT-PCR may result in some false-negative cloacal samples for AIV.

Isolation and molecular characterization of Newcastle disease viruses from raptors.

The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry.

Isolation of mixed subtypes of influenza A virus from a bald eagle (Haliaeetus leucocephalus).

From April 2007 to March 2008, cloacal swabs were obtained from 246 casualty raptors recovered by various wildlife rehabilitation centers in the United States. The swabs were placed in a virus transport medium and transported to the laboratory on ice packs. At the laboratory, the samples were pooled with each pool consisting of five samples. All pools (n = 50) were screened for the presence of avian influenza virus (AIV) using a real time reverse transcription-polymerase chain reaction (rRT-PCR); one of the pools was found positive. All five samples in this pool were tested individually by rRT-PCR; one sample from a bald eagle was found positive. This sample was inoculated in embryonated chicken eggs for virus isolation and a hemagglutinating virus was isolated. Complete genome sequencing of the isolate revealed a mixed infection with H1N1 and H2N1 subtypes. Further analysis revealed that the PB1-F2 gene sequence of H1N1 virus had the N66S virulence-associated substitution. Further studies on ecology and epidemiology of AIV in raptors are needed to help understand their role in the maintenance and evolution of AIV.

Comparison of cloacal and oropharyngeal samples for the detection of avian influenza virus in wild birds.

This study was conducted to compare oropharyngeal (OP) and cloacal samples of wild birds (n = 137) for the detection and isolation of avian influenza virus (AIV). A total of 39 (28.5%) cloacal and 85 (62.0%) OP samples were positive for AIV by real-time reverse transcription-PCR (RRT-PCR). The AIV nucleic acid was detected in both cloacal and OP samples from 27 (19.7%) birds, in cloacal samples only from 12 (8.8%) birds, and in OP samples only from 58 (42.3%) birds. Thus, a total of 97 (70.8%) birds were AIV positive by RRT-PCR. The cycle threshold values for the cloacal samples ranged from 16.6 to 36.9 (mean 31.5), and those for OP samples ranged from 18 to 38.9 (mean 34.9). Of the cloacal samples, 12 were positive for H5 subtype influenza virus by RRT-PCR, with one being low pathogenic H5N1. In contrast, five of the OP samples were H5 positive, but none was H5N1. None of the cloacal or OP samples was H7 positive. Eight cloacal samples yielded AIV on inoculation in embryonated chicken eggs, while only one isolate was obtained from OP samples. Thus, from testing of 137 birds, only nine (6.6%) AIV isolates were obtained. The isolates from cloacal samples were subtyped as H6N1 (n = 5), H3N8 (n = 2), and H4N8 (n = 1), and the isolate from OP sample was subtyped as H6N1. No virus was isolated from the corresponding cloacal sample of the bird whose OP sample yielded AIV on virus isolation. These results suggest that surveillance programs for detection of AIV by RRT-PCR may include both sample types (cloacal and OP) to obtain a better picture of AIV prevalence, and OP samples may yield additional isolates of AIV when tested in conjunction with cloacal samples.

Phylogenetic analysis of Newcastle disease viruses isolated from waterfowl in the upper midwest region of the United States.

BACKGROUND: This study was conducted to characterize Newcastle disease virus (NDV) isolates obtained from waterfowl from the Upper Midwest region of the United States. A total of 43 NDVs were isolated by inoculation of cloacal samples in embryonated chicken eggs. These isolates were obtained from 24 mallards, seven American green-winged teals, six northern pintails, four blue-winged teals, and two wood ducks. Partial sequences of fusion gene were analyzed to determine the pathotypes and genotypes involved. RESULTS: Deduced amino acid sequence of the cleavage site of fusion (F) protein revealed that all isolates had avirulent motifs. Of the 43 isolates, 23 exhibited sequence motif of (111)GGKQGRL(117) at the cleavage site, 19 exhibited (111)GEKQGRL(117) while one isolate showed (111)GERQGRL(117). Phylogenetic analysis based on comparison with different classes of NDVs revealed that all 43 isolates clustered with class II NDVs and none with class I NDVs. Within class II, five isolates were phylogenetically close to genotype I NDVs while the remaining 38 were close to genotype II. CONCLUSION: We conclude that more than one genotype of NDV circulates in waterfowl in the Upper Midwest region of the US. Continuous surveillance may help better understand the epidemiology of NDVs maintained in wild bird populations and their relationship to NDVs in domestic poultry, if any.

Management of a cutaneous squamous cell carcinoma in an American flamingo (Phoenicopterus ruber).

A 32-year-old female American flamingo (Phoenicopterus ruber) was presented with a squamous cell carcinoma of the middle digit of the right foot. No clinical, hematologic, or radiologic evidence of metastasis was present. Salvage amputation of the digit resulted in complete cure, whereas previous electrosurgery and radiation therapy were unsuccessful. Three years later, another squamous cell carcinoma was diagnosed in the middle digit of the left foot. The digit was also amputated. Seven months after the second amputation, the bird did not have any recurrence or signs of metastasis.