Merging Metagenomics and Spatial Epidemiology To Understand the Distribution of Antimicrobial Resistance Genes from Enterobacteriaceae in Wild Owls.

Antimicrobial resistance (AMR) is a well-documented phenomenon in bacteria from many natural ecosystems, including wild animals. However, the specific determinants and spatial distribution of resistant bacteria and antimicrobial resistance genes (ARGs) in the environment remain incompletely understood. In particular, information regarding the importance of anthropogenic sources of AMR relative to that of other biological and ecological influences is lacking. We conducted a cross-sectional study of AMR in great horned owls (Bubo virginianus) and barred owls (Strix varia) admitted to a rehabilitation center in the midwestern United States. A combination of selective culture enrichment and shotgun metagenomic sequencing was used to identify ARGs from Enterobacteriaceae Overall, the prevalence of AMR was comparable to that in past studies of resistant Enterobacteriaceae in raptors, with acquired ARGs being identified in 23% of samples. Multimodel regression analyses identified seasonality and owl age to be important predictors of the likelihood of the presence of ARGs, with birds sampled during warmer months being more likely to harbor ARGs than those sampled during cooler months and with birds in their hatch year being more likely to harbor ß-lactam ARGs than adults. Beyond host-specific determinants, ARG-positive owls were also more likely to be recovered from areas of high agricultural land cover. Spatial clustering analyses identified a significant high-risk cluster of tetracycline resistance gene-positive owls in the southern sampling range, but this could not be explained by any predictor variables. Taken together, these results highlight the complex distribution of AMR in natural environments and suggest that both biological and anthropogenic factors play important roles in determining the emergence and persistence of AMR in wildlife.IMPORTANCE Antimicrobial resistance (AMR) is a multifaceted problem that poses a worldwide threat to human and animal health. Recent reports suggest that wildlife may play an important role in the emergence, dissemination, and persistence of AMR. As such, there have been calls for better integration of wildlife into current research on AMR, including the use of wild animals as biosentinels of AMR contamination in the environment. A One Health approach can be used to gain a better understanding of all AMR sources and pathways, particularly those at the human-animal-environment interface. Our study focuses on this interface in order to assess the effect of human-impacted landscapes on AMR in a wild animal. This work highlights the value of wildlife rehabilitation centers for environmental AMR surveillance and demonstrates how metagenomic sequencing within a spatial epidemiology framework can be used to address questions surrounding AMR complexity in natural ecosystems.

Sublethal and Lethal Methods to Detect Recent Imidacloprid Exposure in Birds with Application to Field Studies.

We used domestic chickens (Gallus gallus domesticus) as a model for granivorous birds to identify methods to detect recent imidacloprid exposure in wild birds. We conducted dosing experiments of 1, 5, 10, and 20% of a reported median lethal dose for domestic chickens using repeated daily exposures over 7 d, at dosages equating to 1.04, 5.2, 10.4, and 20.8 mg/kg/d. We examined the parent compound and metabolites in serial collections of feces and blood during exposures and for 15 d after exposures. We also collected liver, kidney, brain, muscle, and spleen at the experiment end. Mean concentrations of parent compound at 15 d postexposure were highest in the feces and brain, followed by the liver, muscle, spleen, and kidney; but mean concentrations of metabolites 5-OH-imidacloprid and imidacloprid-olefin were highest in feces; then liver, spleen, muscle, and kidney; and then brain. Imidacloprid was rapidly cleared from blood, with only one individual in any dose group having detectable concentrations after 48 h. In contrast, fecal pellets had the highest frequency of imidacloprid detection after 15 d. Concentrations of metabolites were higher than those of the parent compound at all sampling times examined but provided no information about time since exposure. Feces may provide a reliable nonlethal method for detection of recent imidacloprid exposure in wild birds. Additional work is needed to disentangle exposure dose concentration and time since exposure in field-collected samples. Environ Toxicol Chem 2020;39:1355-1366. © 2020 SETAC.

Evaluation of neurobehavioral abnormalities and immunotoxicity in response to oral imidacloprid exposure in domestic chickens (Gallus gallus domesticus).

Domestic chickens (Gallus gallus domesticus) were exposed to imidacloprid by gavage once daily for 7 consecutive days at 0, 0.03, 0.34, 3.42, 10.25, and 15.5 mg/kg/day (n = 20 per group; 5 6-week-old males, 5 6-week-old females, 5 9-week-old males, and 5 9-week-old females). The severity and duration of neurobehavioral abnormalities were recorded. Components of the innate and adaptive immune system were assessed with 7 standard functional assays. Temporary neurobehavioral abnormalities were observed in a dose-dependent manner, including muscle tremors, ataxia, and depressed mentation. Based upon mean clinical severity scores, the no observed adverse effect level (NOAEL) was 3.42 mg/kg/day, and the lowest observed adverse effect level (LOAEL) was 10.25 mg/kg/day. The effective dose value for the presence of any neurobehavioral abnormalities in 50% of the test group (ED50) was 4.62 ± 0.98 mg/kg/day. The ED50 for an adjusted score that included both severity and duration of neurobehavioral abnormalities was 11.24 ± 9.33 mg/kg/day. These ED50 values are equivalent to a 1 kg bird ingesting 29 or 70 imidacloprid treated soybean seeds respectively. Immunotoxicity was not documented, possible causes include the assays were insensitive, relevant immune functions were not examined, or imidacloprid is not immunotoxic at this dosing schedule in this species. Neurobehavioral abnormalities were a more sensitive indicator of the sublethal effects of imidacloprid than immunotoxicity.

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.

Auditory performance in bald eagles and red-tailed hawks: a comparative study of hearing in diurnal raptors.

Collision with wind turbines is a conservation concern for eagles with population abundance implications. The development of acoustic alerting technologies to deter eagles from entering hazardous air spaces is a potentially significant mitigation strategy to diminish associated morbidity and mortality risks. As a prelude to the engineering of deterrence technologies, auditory function was assessed in bald eagles (Haliaeetus leucocephalus), as well as in red-tailed hawks (Buteo jamaicensis). Auditory brainstem responses (ABRs) to a comprehensive battery of clicks and tone bursts varying in level and frequency were acquired to evaluate response thresholds, as well as suprathreshold response characteristics of wave I of the ABR, which represents the compound potential of the VIII cranial nerve. Sensitivity curves exhibited an asymmetric convex shape similar to those of other avian species, response latencies decreased exponentially with increasing stimulus level and response amplitudes grew with level in an orderly manner. Both species were responsive to a frequency band at least four octaves wide, with a most sensitive frequency of 2 kHz, and a high-frequency limit of approximately 5.7 kHz in bald eagles and 8 kHz in red-tailed hawks. Findings reported here provide a framework within which acoustic alerting signals might be developed.

Multi-scale availability of neonicotinoid-treated seed for wildlife in an agricultural landscape during spring planting.

Neonicotinoid pesticides are applied to seeds and are known to cause lethal and sub-lethal effects in birds and mammals. Neonicotinoid-treated seeds could be available to wildlife through spillage or exposed seeds near or at the soil surface due to incomplete or shallow drilling. We quantified seed spills that may occur during loading or refilling the hopper at a landscape-scale using road-based surveys. We also quantified undrilled seeds in 1-m2 frames on the soil in the center and corner of fields to obtain estimates at the field scale. We broadcast seeds on the soil surface of a tilled field and left them for 0, 1, 2, 4, 8, 16, and 30 days to quantify the decrease of neonicotinoids under field conditions. Lastly, we documented wildlife at neonicotinoid-treated seed spills with trail cameras. We estimated the number of spills during planting to be 3496 (95% CI: 1855-5138) and 2609 (95% CI: 862-4357) for corn, 11,009 (95% CI: 6950-15,067) and 21,105 (95% CI: 6162-36,048) for soybean, and 830 (95% CI: 160-1500) and 791 (95% CI: 0-1781) for wheat in 2016 and 2017, respectively. Exposed seeds were present at the soil surface in 35% of 71 fields. The probability that seeds were present on the soil surface was higher for soybeans (18.8 and 49.4% in the center and corners, respectively) than for corn (1.6 and 2.7%, respectively), and seed densities were also higher (1.04 vs 0.07 seeds/m2, respectively). Neonicotinoids decreased rapidly on seeds on the soil surface but persisted as long as 30 days. Over a dozen species of birds and mammals consumed seeds at simulated spills, with an average time for birds to find spills of 1.3 ±â€¯1.5 days and an average time to consumption of 4.1 ±â€¯3.4 days. Seeds are abundant on the soil surface for wildlife to consume during the spring planting season and should be considered in pesticide risk assessments.

Raptor Acupuncture for Treating Chronic Degenerative Joint Disease.

A permanently captive 21-year-old male bald eagle was diagnosed with chronic degenerative joint disease in the right stifle with severe lameness (Grade 5) based on radiography. Clinical signs included decreased movement, vocalization, non weight-bearing on the affected limb, inappetence, depression, and pododermatitis on the left foot (bumblefoot, Grade 3). The eagle was treated with anti-inflammatory or analgesic drugs including carprofen and celecoxib. As there was no observed clinical improvement with any of the treatments, acupuncture treatment was provided. The eagle was treated with dry needle acupuncture once per week for 2 months and biweekly for another 2 months. The Traditional Eastern Medicine diagnosis of this eagle was Bony Bi syndrome. The selected acupuncture points were ST 36, LI 4, BL 40, BL 60, GB 34, and Ba Feng (Table 3). The lameness score improved from Grade 5 to Grade 1 after 4 months of acupuncture treatment. The observed pododermatitis improved from Grade 3 to Grade 0. Symptoms including inappetence and vocalizations were significantly reduced over the 4 month period. There was no significant improvement in the radiographic signs. In conclusion, acupuncture may be a potential medical option for permanently captive raptors having musculoskeletal conditions, such as degenerative joint disease.

Management of select bacterial and parasitic conditions of raptors.

Raptors are susceptible to a broad array of established and emerging bacterial and parasitic diseases, including babesiosis, chlamydiosis, clostridiosis, coccidiosis, cryptosporidiosis, malaria, mycobacteriosis, pasteurellosis, salmonellosis, trichomoniasis, and pododermatitis. Many of these conditions are opportunistic and can be easily managed or averted with proper preventive measures related to captive management, husbandry and diet, and veterinary care. Once infected, treatment must be prompt, appropriate, and judicious. This article examines the significance, diagnosis, management, and prevention of select bacterial and parasitic pathogens of raptors.

Ophthalmologic and oculopathologic findings in red-tailed hawks and Cooper's hawks with naturally acquired West Nile virus infection.

OBJECTIVE: To assess ophthalmologic features and ocular lesions in red-tailed hawks and Cooper's hawks naturally infected with West Nile virus (WNV). DESIGN: Original study. ANIMALS: 13 hawks. PROCEDURES: All hawks underwent complete ophthalmic examinations including slit lamp biomicroscopy and binocular indirect ophthalmoscopy. Eleven hawks were euthanized because of a grave prognosis; complete necropsies were performed. Eyes, brain, heart, and kidneys were processed for histologic and immunohistochemical examinations. Pooled tissue homogenates and aqueous humor samples were assessed for WNV nucleic acid via PCR assay, and anti-WNV antibody titers in aqueous humor and plasma were determined. RESULTS: All birds had similar funduscopic abnormalities including exudative chorioretinal lesions and chorioretinal scarring in a geographic or linear pattern. Eleven birds were euthanized, and 2 birds were released. Plasma from both released hawks and plasma and aqueous humor of all euthanized hawks that were evaluated contained anti-WNV antibodies. Except for 1 hawk, all euthanized hawks had WNV-associated disease (determined via detection of WNV antigen or nucleic acid in at least 1 organ). Histopathologic ocular abnormalities, most commonly pectenitis, were detected in all euthanized birds; several birds had segmental choroiditis, often with corresponding segmental retinal atrophy. West Nile virus antigen was detected in the retinas of 9 of the euthanized birds. In 2 hawks, WNV antigen was detected in the retina only. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that funduscopically detectable chorioretinal lesions appear to be associated with WNV disease in hawks. Detection of ocular lesions may aid in antemortem or postmortem diagnosis of this condition.