PHARMACOKINETICS OF CEFTIOFUR CRYSTALLINE-FREE ACID ADMINISTERED VIA INTRAMUSCULAR INJECTION IN WHOOPING CRANES (GRUS AMERICANA).

The objective of this study was to evaluate the pharmacokinetic properties of a single dose of ceftiofur crystalline-free acid (CCFA) in whooping cranes (Grus americana). Ceftiofur crystalline-free acid is a long-acting, injectable, third-generation cephalosporin antibiotic drug. A preliminary study evaluated CCFA administered intramuscularly in the pectoral or thigh muscle at 20 or 30 mg/kg IM to a single adult whooping crane for each dose. On the basis of these data, a dose of 30 mg/kg IM of CCFA was administered to five additional whooping cranes, and blood was collected at various time points from 0 to 288 h. Pharmacokinetic parameters for ceftiofur equivalents were determined and reached concentrations above minimum inhibitory concentrations of various bacteria in other avian species (>1 µg/ml) for at least 96 h in all birds, and for 144 h in two birds. From these findings, ceftiofur crystalline-free acid appears to be a long-acting antibiotic option for whooping cranes and may be dosed every 96 h; however, additional multidose studies are needed.

Blood Mercury in Three Populations of Endangered Whooping Crane (Grus americana).

We investigated concentrations of blood total mercury (THg) in three extant populations of endangered Whooping Crane (Grus americana). Blood THg was greater in cranes reintroduced during 2001-2008 that range in the eastern US (median = 0.31 ug/g ww) than both wild cranes in central North America (median = 0.11 ug/g ww) and from captivity (median = 0.01 ug/g ww). The median THg blood concentrations in the two free-ranging populations of Whooping Cranes were low compared to reproductive toxic thresholds suggested for other large bird species (> 4.3 ug/g ww), but a singular elevated concentration was observed in one crane (1.04 ug/g ww; 6% of samples from the reintroduced population). These results likely reflect variable dietary Hg exposure among these populations, but should prompt additional biomonitoring to discern risk of greater Hg exposure nearer to the time of breeding and potential effects on productivity.

NEOPLASIA IN CAPTIVE CRANES.

A retrospective study of neoplasia was conducted from necropsy and histologic reports of 446 cranes representing all 15 extant species. Cases were obtained from the International Crane Foundation (ICF), Northwest ZooPath (NWZP), and six other zoologic institutions in the United States during 1993 to 2019. Only reports from ICF (n = 61) and NWZP (n = 374) were used for estimates of disease prevalence. Overall prevalence of neoplasia was 7.35% (32 of 435), with a metastasis rate of 31.8%. Seventeen types of neoplasms were identified. Geriatric cranes were the most common age class affected (60%). The digestive system was most frequently involved (n = 12; 27.3%), followed by urinary (n = 8; 18.2%) and integumentary systems (n = 6; 13.6%). Carcinoma was the most common tumor type across all species (n = 15; 34.0%) followed by benign epithelial tumors (adenomas; n = 11; 25.0%). Multiple neoplasms were observed in only one crane. Of the cases with metastasis, hematopoietic, reproductive, and respiratory tumors had 100% metastasis (2 of 2, 2 of 2, and 3 of 3, respectively), whereas tumors of the integumentary, nervous, and musculoskeletal systems had no evidence of metastasis (0 of 7, 0 of 3, and 0 of 1, respectively). Overall, Gruidae species were found to have a high prevalence of neoplasia but a low rate of metastasis compared with other avian species with the highest species-specific rates in Eurasian (Grus grus), demoiselle (Anthropoides virgo), and wattled cranes (Bugeranus carunculatus) (40.0%, 21.4%, and 19.0%, respectively). This is the first large-scale study of neoplasia in cranes.

Emergency tracheotomy and subsequent tracheal resection and anastomosis in a blue crane (Anthropoides paradiseus).

CASE DESCRIPTION: A 7-year-old female blue crane (Anthropoides paradiseus) was initially evaluated after it had suddenly developed signs of respiratory distress following aspiration of a rock. Emergency tracheotomy had been performed, and the rock had been removed from the proximal cervical portion of the trachea. Fifty-one days later, the clinical signs had returned and the crane was reevaluated. CLINICAL FINDINGS: On reevaluation, no obvious external abnormalities were appreciated at the previous surgical site and no discharge was observed from the glottis. Computed tomography and tracheoscopy revealed marked tracheal stenosis and architectural collapse of the trachea at the previous surgery site. TREATMENT AND OUTCOME: Tracheal resection and anastomosis was performed to remove the stenotic tracheal segment. Histologic examination of the resected tracheal segment revealed pyogranulomas with intralesional coccobacilli, fungal hyphae consistent with Aspergillus spp, possible parasitic ova, and features suggestive of mild to moderate heterophilic and lymphoplasmacytic tracheitis. The crane was treated with piroxicam, ceftiofur crystalline free acid, terbinafine, and itraconazole. At a follow-up examination 12 weeks later, no abnormalities were appreciated, and the surgical site had completely healed. CLINICAL RELEVANCE: To the authors' knowledge, this is the first reported case of successful tracheal resection and anastomosis in a bird of the order Gruiformes. The surgical approach used for the blue crane may be useful for removal of tracheal foreign bodies in this and other long-necked avian species.

Comparison of fecal glucocorticoid metabolite concentrations in hand- versus parent-reared whooping cranes (Grus americana).

Endangered whooping cranes (Grus americana) have been produced in captivity for reintroduction programs since the 1980s, using techniques such as artificial insemination, multiple clutching, and captive-rearing to speed recovery efforts. Chicks are often hand-reared (HR) by caretakers in crane costumes, socialized into groups and released together, unlike parent-reared (PR) cranes that are raised individually by a male/female crane pair and released singly. HR cranes historically exhibit greater morbidity rates during development than PR cranes, involving musculoskeletal and respiratory system disease, among others. We hypothesized that HR crane chicks exhibit a higher baseline fecal glucocorticoid metabolite (FGM) concentrations during the development compared with PR chicks. Fecal samples were collected between 15 and 70 days of age from HR (n = 15) and PR (n = 8) chicks to test for differences in FGM concentrations using a radioimmunoassay technique following ethanol extraction for steroids. Linear mixed model analysis suggests increasing age of the chick was associated with an increase in FGM (p < .001). Analysis also supported the interaction between rearing strategy and sex of the crane chick (p < .01). Female PR chicks had greater FGM concentrations than all other groups (PR male, p < .01; HR female, p < .001; and HR male, p < .001). This result suggests that there may be an effect of rearing strategy on stress physiology of whooping crane chicks, especially among females. Further research is needed to investigate whether the FGM concentrations are reflective of true differences in stress physiology of young cranes and whether this may impact health and conservation success.

A bird's-eye view of phylosymbiosis: weak signatures of phylosymbiosis among all 15 species of cranes.

In numerous animal clades, the evolutionary history of host species drives patterns of gut microbial community structure, resulting in more divergent microbiota with increasing phylogenetic distance between hosts. This phenomenon, termed phylosymbiosis, has been observed in diverse evolutionary lineages, but has been difficult to detect in birds. Previous tests of phylosymbiosis among birds have been conducted using wild individuals, and thus interspecific differences in diet and environment may have masked a phylogenetic signal. Therefore, we tested for phylosymbiosis among all 15 species of cranes (family Gruidae) housed in the same captive environment and maintained on identical diets. 16S rRNA sequencing revealed that crane species harbour distinct gut microbiota. Overall, we detected marginally significant patterns of phylosymbiosis, the strength of which was increased when including the estimates of absolute microbial abundance (rather than relative abundance) derived from microbial densities determined by flow cytometry. Using this approach, we detected the statistically significant signatures of phylosymbiosis only after removing male cranes from our analysis, suggesting that using mixed-sex animal cohorts may prevent the detection of phylosymbiosis. Though weak compared with mammals (and especially insects), these results provide evidence of phylosymbiosis in birds. We discuss the potential differences between birds and mammals, such as transmission routes and host filtering, that may underlie the differences in the strength of phylosymbiosis.

Postmortem Evaluation of Reintroduced Migratory Whooping Cranes (Grus americana) in Eastern North America.

We reviewed necropsy records of 124 Whooping Cranes (Grus americana) recovered following reintroduction of 268 individuals from 2001 to 2016 in the eastern US. Causes of death were determined in 62% (77/124) of cases facilitated by active monitoring that limited decomposition and scavenging artifact. The greatest proportions of mortality were caused by predation (0.468; 95% confidence interval 0.356-0.580; 36/77), collision with power lines or vehicles (0.260; 0.162-0.358; 20/77), and gunshot (0.169; 0.085-0.253; 13/77). Six deaths were attributed to infection (0.078; 0.018-0.138; 6/77), including bacterial and fungal etiologies. Lead analysis of 50 liver samples yielded two results with elevated concentrations (3.65 and 10.97 ppm wet weight), and 10 bone samples from partial carcasses lacking suitable liver tissue resulted in one elevated result (48.82 ppm dry weight). These data indicate that underlying subclinical or clinical lead toxicosis may be a factor in up to 5% of deaths attributed to predation or impact trauma. Brain cholinesterase activity testing indicated no exposure to organophosphate or carbamate pesticides (mean±SD=17.32±2.90 µmol/min/g, 31/71). The causes of death and potential underlying factors summarized in this study constitute the first definitive mortality survey of migratory Whooping Cranes based on a high carcass recovery rate. Causes of death by infectious etiologies remained comparatively rare in this study, and occurred as single cases with no evidence of sustained transmission among reintroduced Whooping Cranes.

Conservation biology needs a microbial renaissance: a call for the consideration of host-associated microbiota in wildlife management practices.

The central aim of conservation biology is to understand and mitigate the effects of human activities on biodiversity. To successfully achieve this objective, researchers must take an interdisciplinary approach that fully considers the effects, both direct and indirect, of anthropogenic disturbances on wildlife physiology and health. A recent surge in research has revealed that host-associated microbiota-the archaeal, bacterial, fungal and viral communities residing on and inside organisms-profoundly influence animal health, and that these microbial communities can be drastically altered by anthropogenic activities. Therefore, conservation practitioners should consider the disruption of host-associated microbial diversity as a serious threat to wildlife populations. Despite the tremendous potential for microbiome research to improve conservation outcomes, few efforts have been made to truly integrate these fields. In this review, we call for the microbial renaissance of conservation biology, where biodiversity of host-associated microbiota is recognized as an essential component of wildlife management practices. Using evidence from the existing literature, we will examine the known effects of anthropogenic activities on the diversity of host-associated microbial communities and integrate approaches for maintaining microbial diversity to successfully achieve conservation objectives.

Serum Biochemical Analytes and Trace Elements in Juvenile Whooping Cranes (Grus americana).

Biochemical and trace element analyses of blood from wild Whooping Cranes (Grus americana) were performed to assess the health of the only self-sustaining, migratory population in North America. Juvenile cranes (n=31) approximately 49-70 d-old were sampled at Wood Buffalo National Park, Northwest Territories, Canada, in midsummer from 2010 to 2012. Archived serum (n=24) and whole blood (n=31) samples from captive juvenile cranes were selected as age-matched controls. Reference values were calculated for serum biochemical analytes and trace elements in whole blood from the captive juvenile Whooping Cranes reared under controlled conditions and with known health histories. Several statistical differences among blood biochemical and trace element values of the wild and captive juveniles were identified and were likely attributable to dietary differences between the populations.

MANIFESTATIONS OF HYPERPARATHYROIDISM IN JUVENILE WHOOPING CRANES (GRUS AMERICANA).

Juvenile whooping cranes (Grus americana) raised for wild release were found to have an increased incidence of rib fractures at fledging in 2017 compared with the previous 16 years. Serum analysis showed 30-day-old juveniles in 2017 (n = 12) had significantly lower 25-hydroxyvitamin D3 and significantly higher parathyroid hormone concentrations than juveniles in 2010 (n = 6) with no history of rib fractures. Increased serum parathyroid hormone concentrations in the 2017 juveniles persisted to fledging age. Review of dietary and environmental management revealed that juveniles in 2017 were provided a commercial diet with a lower, and perhaps suboptimal, calcium:phosphorus ratio and experienced reduced time outdoors in the first month after hatch, presumably resulting in less ultraviolet B radiation exposure. Mild hyperparathyroidism in precocial whooping cranes may result when dietary constraints and/or outdoor access is compromised and manifest as rib fractures in the absence of traumatic injury.

PHARMACOKINETICS OF PIROXICAM IN CRANES (FAMILY GRUIDAE).

To investigate the pharmacokinetics of the nonsteroidal anti-inflammatory drug (NSAID) piroxicam in cranes, three brolgas (Antigone rubicunda) were administered piroxicam as a single oral dose at 0.5 mg/kg and 1.0 mg/kg during separate trials. Serial blood samples were collected for quantification of piroxicam in plasma. Piroxicam was readily absorbed at both dosages, and no adverse effects were observed. Plasma concentrations peaked at 3.67 hr with a concentration of 4.00 µg/ml for the lower dosage, and at 0.83 hr at 8.77 µg/ml for the higher dosage. Piroxicam may exhibit linear kinetics and dose proportionality in brolgas, but will require further study. Mean peak plasma concentrations in brolgas were comparable to concentrations demonstrated to be analgesic in humans. To the authors' knowledge, this study represents the first pharmacokinetic investigation of piroxicam in an avian species.

Haemosporida prevalence and diversity are similar in endangered wild whooping cranes (Grus americana) and sympatric sandhill cranes (Grus canadensis).

The population growth of endangered whooping cranes (Grus americana) is not consistent with species recovery goals, and the impact of parasite infection on whooping crane populations is largely unknown. Disease ecology and epidemiology research of endangered species is often hindered by limited ability to conduct invasive sampling on the target taxa. Accordingly, we hypothesized that sandhill cranes (Grus canadensis) would be a useful surrogate species to investigate the health impacts of Haemosporida infection in whooping cranes. Our goal was to compare the prevalence and diversity of Haemosporida infection between whooping cranes and sandhill cranes. We detected an overall infection prevalence of 83·6% (n = 61) in whooping cranes and 59·6% (n = 47) and 63·6 (n = 22) in two sympatric sandhill crane populations captured in Texas. Prevalence was significantly lower in allopatric sandhill cranes captured in New Mexico (12·1%, n = 33). Haemoproteus antigonis was the most abundant haemoparasite in cranes, present in 57·4% of whooping cranes and 39·2% of sandhill cranes; Plasmodium and Leucocytozoon were present at significantly lower levels. The high prevalence of Haemosporida in whooping cranes and sympatric sandhill cranes, with shared parasite lineages between the two species, supports sandhill cranes as a surrogate species for understanding health threats to endangered whooping cranes.

A novel Haemosporida clade at the rank of genus in North American cranes (Aves: Gruiformes).

The unicellular blood parasites in the order Haemosporida are highly diverse, infect many vertebrates, are responsible for a large disease burden among humans and animals, and have reemerged as an important model system to understand the evolutionary and ecological dynamics of host-parasite interactions. The phylogenetics and systematics of Haemosporida are limited by poor sampling of different vertebrate host taxa. We surveyed the Haemosporida of wild whooping cranes (Grus americana) and sandhill cranes (Grus canadensis) (Aves: Gruiformes) using a combination of morphological and molecular approaches. We identified Haemoproteus antigonis in blood smears based on published morphological descriptions. Phylogenetic analysis based on partial cytochrome b (cytb) and cytochrome oxidase (coI) sequences placed H. antigonis parasites in a novel clade, distinct from all avian Haemosporida genera for which cytb and/or coI sequences are available. Molecular clock and divergence estimates suggest this crane clade may represent a new genus. This is the first molecular description of H. antigonis and the first report of H. antigonis in wild whooping cranes, an endangered bird in North America. Further sampling of Haemosporida, especially from hosts of the Gruiformes and other poorly sampled orders, will help to resolve the relationship of the H. antigonis clade to other avian Haemosporida genera. Our study highlights the potential of sampling neglected host species to discover novel lineages of diverse parasite groups.

Lead in the Red-Crowned Cranes (Grus japonensis) in Zhalong Wetland, Northeastern China: A Report.

The dietary uptake of Cd and Pb may contribute to the decline of migratory red-crowned cranes (Grus japonensis) on the Asian mainland. To uncover the relevance of this hypothesis, we determined the concentrations of Pb and Cd as well as further macro and trace elements (Ca, Mg, Cu, Zn, Hg and As) in the gastric contents, gastric wall, intestinal wall, liver, kidney, muscle, and feathers of two individuals found dead in Zhalong Wetland in Northeastern China. Indeed, the Pb concentrations in the liver and kidney tissues was with 31.4 and 60.3 mg kg(-1) dry weight (dw), respectively, above concentrations considered as potentially toxic level in common birds (i.e. 30 mg kg(-1)). These Pb concentration may have possibly been associated with lethal toxicosis in this endangered species suggesting Pb as major threat for G. japonensis populations. Thus, the inputs of Pb into Zhalong Wetland should be reduced to maintain and reestablish environmental conditions supporting the population development of these migratory red-crowned cranes in the Zhalong Wetland, a critical crane habitat for the long-term sustainability of this species.

COMPUTED TOMOGRAPHIC ANATOMY AND CHARACTERISTICS OF RESPIRATORY ASPERGILLOSIS IN JUVENILE WHOOPING CRANES.

Respiratory diseases are a leading cause of morbidity and mortality in captivity reared, endangered whooping cranes (Grus americana). Objectives of this retrospective, case series, cross-sectional study were to describe computed tomography (CT) respiratory anatomy in a juvenile whooping crane without respiratory disease, compare CT characteristics with gross pathologic characteristics in a group of juvenile whooping cranes with respiratory aspergillosis, and test associations between the number of CT tracheal bends and bird sex and age. A total of 10 juvenile whooping cranes (one control, nine affected) were included. Seven affected cranes had CT characteristics of unilateral extrapulmonary bronchial occlusion or wall thickening, and seven cranes had luminal occlusion of the intrapulmonary primary or secondary bronchi. Air sac membrane thickening was observed in three cranes in the cranial and caudal thoracic air sacs, and air sac diverticulum opacification was observed in four cranes. Necropsy lesions consisted of severe, subacute to chronic, focally extensive granulomatous pathology of the trachea, primary bronchi, lungs, or air sacs. No false positive CT scan results were documented. Seven instances of false negative CT scan results occurred; six of these consisted of subtle, mild air sacculitis including membrane opacification or thickening, or the presence of small plaques found at necropsy. The number of CT tracheal bends was associated with bird age but not sex. Findings supported the use of CT as a diagnostic test for avian species with respiratory disease and tracheal coiling or elongated tracheae where endoscopic evaluation is impractical.

Comparison of Serum Protein Electrophoresis Values in Wild and Captive Whooping Cranes ( Grus americana ).

Protein electrophoresis of serum samples from endangered, wild whooping cranes ( Grus americana ) was performed to help assess the health of the only self-sustaining, migratory population in North America. Serum samples from wild adult cranes (n = 22) were taken at Aransas National Wildlife Refuge, Texas, USA during winter. Wild juvenile cranes (n = 26) were sampled at Wood Buffalo National Park, Northwest Territories, Canada, in midsummer. All captive crane samples were acquired from the International Crane Foundation, Baraboo, WI, USA. Captive adult cranes (n = 30) were sampled during annual examinations, and archived serum samples from captive juvenile cranes (n = 19) were selected to match the estimated age of wild juveniles. Wild juveniles had significantly lower concentrations of all protein fractions than wild adults, except for prealbumin and γ globulins. All protein fraction concentrations for wild juveniles were significantly lower compared with captive juveniles, except for prealbumin and γ globulins, which were higher. Wild adults had significantly greater γ globulin concentrations than captive adults. Captive juveniles had significantly lower prealbumin and albumin concentrations and albumin : globulin ratios than captive adults. The higher γ globulin concentrations in wild versus captive cranes are likely because of increased antigenic exposure and immune stimulation. Protein fraction concentrations vary significantly with age and natural history in this species. Reference intervals for serum protein electrophoresis results from captive adult whooping cranes are provided in this study.

Coccidian Parasites and Conservation Implications for the Endangered Whooping Crane (Grus americana).

While the population of endangered whooping cranes (Grus americana) has grown from 15 individuals in 1941 to an estimated 304 birds today, the population growth is not sufficient to support a down-listing of the species to threatened status. The degree to which disease may be limiting the population growth of whooping cranes is unknown. One disease of potential concern is caused by two crane-associated Eimeria species: Eimeria gruis and E. reichenowi. Unlike most species of Eimeria, which are localized to the intestinal tract, these crane-associated species may multiply systemically and cause a potentially fatal disease. Using a non-invasive sampling approach, we assessed the prevalence and phenology of Eimeria oocysts in whooping crane fecal samples collected across two winter seasons (November 2012-April 2014) at the Aransas National Wildlife Refuge along the Texas Gulf coast. We also compared the ability of microscopy and PCR to detect Eimeria in fecal samples. Across both years, 26.5% (n = 328) of fecal samples were positive for Eimeria based on microscopy. Although the sensitivity of PCR for detecting Eimeria infections seemed to be less than that of microscopy in the first year of the study (8.9% vs. 29.3%, respectively), an improved DNA extraction protocol resulted in increased sensitivity of PCR relative to microscopy in the second year of the study (27.6% and 20.8%, respectively). The proportion of positive samples did not vary significantly between years or among sampling sites. The proportion of Eimeria positive fecal samples varied with date of collection, but there was no consistent pattern of parasite shedding between the two years. We demonstrate that non-invasive fecal collections combined with PCR and DNA sequencing techniques provides a useful tool for monitoring Eimeria infection in cranes. Understanding the epidemiology of coccidiosis is important for management efforts to increase population growth of the endangered whooping crane.

Capture of sandhill cranes using alpha-chloralose: a 10-year follow-up.

Seasonal adjustment of alpha-chloralose captures of sandhill cranes was associated with a modest increase in capture efficacy (+13%), decreased morbidity from exertional myopathy (-1.4%), and overall mortality (-1.7%) rates despite little change in sedation scores. Postcapture fluid administration also decreased confinement times by several hours over most sedation scores.

Reintroduction medicine: whooping cranes in Wisconsin.

This paper presents veterinary management strategies and diagnostic findings in the reintroduction of the endangered whooping crane (Grus americana). Between 2005 and 2010, 63 (27 male, 36 female) hatchling whooping cranes were assigned to a reintroduction project involving autumn release of costume-reared chicks in Wisconsin. Veterinary care included preventive measures and comprehensive pre-release evaluations to improve fitness and reduce translocation of potential disease agents to native habitats. A total of 44 clinically normal birds were released (70% of assigned individuals). Cases of morbidity were classified according to primary body system affected. Musculoskeletal disorders were described in 57 birds (90%); five birds were removed from the project prior to release (8%), all for abnormalities that prevented normal function. Fourteen birds died or were euthanized prior to release (22%); pre-release mortality was attributed to developmental abnormality, predation, trauma or infectious disease. Chronic respiratory aspergillosis, diagnosed in seven birds (11%), was the most common infectious disease of concern. Predation and trauma were primary causes of post-release mortality; no evidence of infectious disease of captive origin was detected in the study population by the end of 2010. The assessment of data accumulated by this project helped to outline successful health management strategies, as well as identify and mitigate ongoing risks to captive whooping cranes that impede reintroduction efforts and achieving management goals for species recovery.