An expert-based system to predict population survival rate from health data.

Timely detection and understanding of causes for population decline are essential for effective wildlife management and conservation. Assessing trends in population size has been the standard approach, but we propose that monitoring population health could prove more effective. We collated data from 7 bottlenose dolphin (Tursiops truncatus) populations in the southeastern United States to develop a method for estimating survival probability based on a suite of health measures identified by experts as indices for inflammatory, metabolic, pulmonary, and neuroendocrine systems. We used logistic regression to implement the veterinary expert system for outcome prediction (VESOP) within a Bayesian analysis framework. We fitted parameters with records from 5 of the sites that had a robust network of responders to marine mammal strandings and frequent photographic identification surveys that documented definitive survival outcomes. We also conducted capture-mark-recapture (CMR) analyses of photographic identification data to obtain separate estimates of population survival rates for comparison with VESOP survival estimates. The VESOP analyses showed that multiple measures of health, particularly markers of inflammation, were predictive of 1- and 2-year individual survival. The highest mortality risk 1 year following health assessment related to low alkaline phosphatase (odds ratio [OR] = 10.2 [95% CI: 3.41-26.8]), whereas 2-year mortality was most influenced by elevated globulin (OR = 9.60 [95% CI: 3.88-22.4]); both are markers of inflammation. The VESOP model predicted population-level survival rates that correlated with estimated survival rates from CMR analyses for the same populations (1-year Pearson's r = 0.99, p = 1.52 × 10-5 ; 2-year r = 0.94, p = 0.001). Although our proposed approach will not detect acute mortality threats that are largely independent of animal health, such as harmful algal blooms, it can be used to detect chronic health conditions that increase mortality risk. Random sampling of the population is important and advancement in remote sampling methods could facilitate more random selection of subjects, obtainment of larger sample sizes, and extension of the approach to other wildlife species.

Un sistema basado en conocimiento experto para predecir la tasa de supervivencia a partir de datos de salud Resumen La detección y el entendimiento oportunos de la declinación poblacional son esenciales para que el manejo y la conservación de fauna tengan efectividad. La evaluación de las tendencias en el tamaño poblacional ha sido la estrategia estándar, pero proponemos que el monitoreo de la salud poblacional podría ser más efectivo. Recopilamos datos de siete poblaciones de delfines (Tursiops truncatus) en el sureste de Estados Unidos para desarrollar un método de estimación de la probabilidad de supervivencia con base en un conjunto de medidas sanitarias identificadas por expertos como índices para los sistemas inflamatorio, metabólico, pulmonar y neuroendocrino. Usamos la regresión logística para implementar el sistema de expertos veterinarios para la predicción de resultados (SEVPR) en un análisis bayesiano. Ajustamos los parámetros con los registros de cinco sitios que contaban con una buena red de respondientes a los varamientos de mamíferos marinos y censos de identificación fotográfica (foto-ID) que documentaron los resultados de supervivencia definitivos. También realizamos análisis de marcaje-recaptura (MR) en los datos de identificación fotográfica para obtener estimados separados de las tasas de supervivencia poblacional para compararlos con los estimados del SEVPR. Los análisis del SEVPR mostraron que varias medidas sanitarias, particularmente los marcadores de inflamación son buenos predictores de la supervivencia individual para uno y dos años. El riesgo de mortalidad más alto un año después de la valoración sanitaria se relacionó con una fosfatasa alcalina baja (cociente de probabilidades de 10.2 [95% CI 3.41-26.8]), mientras que la mortalidad a los dos años estuvo más influenciada por una globulina elevada (9.60 [95% CI 3.88-22.4]); ambas son marcadores de la inflamación. El modelo del SEVPR predijo las tasas de supervivencia a nivel poblacional en correlación con las tasas estimadas de supervivencia de los análisis de MR para las mismas poblaciones (Pearson de un año r = 0.99, p = 1.52e-05; dos años r = 0.94, p = 0.001). Aunque nuestra propuesta no detecta las amenazas agudas de mortalidad que en su mayoría son independientes de la salud animal, como la proliferación de algas nocivas, puede usarse para detectar las condiciones crónicas de salud que incrementan el riesgo de mortalidad. Es importante el muestreo aleatorio de la población y los avances en los métodos de muestreo remoto podrían facilitar una selección más aleatoria de los sujetos, la obtención de muestras de mayor tamaño y la expansión de la estrategia a otras especies de fauna.

A Review of Circumpolar Arctic Marine Mammal Health-A Call to Action in a Time of Rapid Environmental Change.

The impacts of climate change on the health of marine mammals are increasingly being recognised. Given the rapid rate of environmental change in the Arctic, the potential ramifications on the health of marine mammals in this region are a particular concern. There are eleven endemic Arctic marine mammal species (AMMs) comprising three cetaceans, seven pinnipeds, and the polar bear (Ursus maritimus). All of these species are dependent on sea ice for survival, particularly those requiring ice for breeding. As air and water temperatures increase, additional species previously non-resident in Arctic waters are extending their ranges northward, leading to greater species overlaps and a concomitant increased risk of disease transmission. In this study, we review the literature documenting disease presence in Arctic marine mammals to understand the current causes of morbidity and mortality in these species and forecast future disease issues. Our review highlights potential pathogen occurrence in a changing Arctic environment, discussing surveillance methods for 35 specific pathogens, identifying risk factors associated with these diseases, as well as making recommendations for future monitoring for emerging pathogens. Several of the pathogens discussed have the potential to cause unusual mortality events in AMMs. Brucella, morbillivirus, influenza A virus, and Toxoplasma gondii are all of concern, particularly with the relative naivety of the immune systems of endemic Arctic species. There is a clear need for increased surveillance to understand baseline disease levels and address the gravity of the predicted impacts of climate change on marine mammal species.

Modeling population effects of the Deepwater Horizon oil spill on a long-lived species.

The 2010 Deepwater Horizon (DWH) oil spill exposed common bottlenose dolphins (Tursiops truncatus) in Barataria Bay, Louisiana to heavy oiling that caused increased mortality and chronic disease and impaired reproduction in surviving dolphins. We conducted photographic surveys and veterinary assessments in the decade following the spill. We assigned a prognostic score (good, fair, guarded, poor, or grave) for each dolphin to provide a single integrated indicator of overall health, and we examined temporal trends in prognostic scores. We used expert elicitation to quantify the implications of trends for the proportion of the dolphins that would recover within their lifetime. We integrated expert elicitation, along with other new information, in a population dynamics model to predict the effects of observed health trends on demography. We compared the resulting population trajectory with that predicted under baseline (no spill) conditions. Disease conditions persisted and have recently worsened in dolphins that were presumably exposed to DWH oil: 78% of those assessed in 2018 had a guarded, poor, or grave prognosis. Dolphins born after the spill were in better health. We estimated that the population declined by 45% (95% CI 14-74) relative to baseline and will take 35 years (95% CI 18-67) to recover to 95% of baseline numbers. The sum of annual differences between baseline and injured population sizes (i.e., the lost cetacean years) was 30,993 (95% CI 6607-94,148). The population is currently at a minimum point in its recovery trajectory and is vulnerable to emerging threats, including planned ecosystem restoration efforts that are likely to be detrimental to the dolphins' survival. Our modeling framework demonstrates an approach for integrating different sources and types of data, highlights the utility of expert elicitation for indeterminable input parameters, and emphasizes the importance of considering and monitoring long-term health of long-lived species subject to environmental disasters. Article impact statement: Oil spills can have long-term consequences for the health of long-lived species; thus, effective restoration and monitoring are needed.

El derrame de petróleo Deepwater Horizon (DWH) en 2010 expuso gravemente a este hidrocarburo a los delfines (Tursiops truncatus) de la Bahía Barataria, Luisiana, causando un incremento en la mortalidad y en las enfermedades crónicas, y deteriorando la reproducción de los delfines sobrevivientes. Realizamos censos fotográficos y evaluaciones veterinarias durante la década posterior al derrame. Asignamos un puntaje pronóstico (bueno, favorable, moderado, malo, o grave) a cada delfín para proporcionar un indicador integrado único de la salud en general. También examinamos las tendencias temporales de estos puntajes. Usamos información de expertos para cuantificar las implicaciones de las tendencias para la proporción de delfines que se recuperaría dentro de su periodo de vida. Integramos esta información, junto con información nueva, a un modelo de dinámica poblacional para predecir los efectos sobre la demografía de las tendencias observadas en la salud. Comparamos la trayectoria poblacional resultante con aquella pronosticada bajo condiciones de línea base (sin derrame). Las condiciones de enfermedad persistieron y recientemente han empeorado en los delfines que supuestamente estuvieron expuestos al petróleo de DWH: 78% de aquellos evaluados en 2018 tuvieron un pronóstico moderado, malo o grave. Los delfines que nacieron después del derrame contaron con mejor salud. Estimamos que la población declinó en un 45% (95% CI 14-74) relativo a la línea base y tardará 35 años (95% CI 18-67) en recuperar el 95% de los números de línea base. La suma de las diferencias anuales entre el tamaño poblacional de línea base y el dañado (es decir, los años cetáceos perdidos) fue de 30,993 (95% CI 6,607-94,148). La población actualmente está en un punto mínimo de su trayectoria de recuperación y es vulnerable a las amenazas emergentes, incluyendo los esfuerzos de restauración ambiental planeada que probablemente sean nocivos para la supervivencia de los delfines. Nuestro marco de modelado demuestra una estrategia para la integración de diferentes fuentes y tipos de datos, resalta la utilidad de la información de expertos para los parámetros de aportación indeterminable, y enfatiza la importancia de la consideración y el monitoreo de la salud a largo plazo de las especies longevas sujetas a los desastres ambientales. Modelado de los Efectos Poblacionales del Derrame de Petróleo Deepwater Horizon sobre Especies Longevas.

Cardiac assessments of bottlenose dolphins (Tursiops truncatus) in the Northern Gulf of Mexico following exposure to Deepwater Horizon oil.

The Deepwater Horizon (DWH) oil spill profoundly impacted the health of bottlenose dolphins (Tursiops truncatus) in Barataria Bay, LA (BB). To comprehensively assess the cardiac health of dolphins living within the DWH oil spill footprint, techniques for in-water cardiac evaluation were refined with dolphins cared for by the U.S. Navy Marine Mammal Program in 2018 and applied to free-ranging bottlenose dolphins in BB (n = 34) and Sarasota Bay, Florida (SB) (n = 19), a non-oiled reference population. Cardiac auscultation detected systolic murmurs in the majority of dolphins from both sites (88% BB, 89% SB) and echocardiography showed most of the murmurs were innocent flow murmurs attributed to elevated blood flow velocity [1]. Telemetric six-lead electrocardiography detected arrhythmias in BB dolphins (43%) and SB dolphins (31%), all of which were considered low to moderate risk for adverse cardiac events. Echocardiography showed BB dolphins had thinner left ventricular walls, with significant differences in intraventricular septum thickness at the end of diastole (p = 0.002), and left ventricular posterior wall thickness at the end of diastole (p = 0.033). BB dolphins also had smaller left atrial size (p = 0.004), higher prevalence of tricuspid valve prolapse (p = 0.003), higher prevalence of tricuspid valve thickening (p = 0.033), and higher prevalence of aortic valve thickening (p = 0.008). Two dolphins in BB were diagnosed with pulmonary arterial hypertension based on Doppler echocardiography-derived estimates and supporting echocardiographic findings. Histopathology of dolphins who stranded within the DWH oil spill footprint showed a significantly higher prevalence of myocardial fibrosis (p = 0.003), regardless of age, compared to dolphins outside the oil spill footprint. In conclusion, there were substantial cardiac abnormalities identified in BB dolphins which may be related to DWH oil exposure, however, future work is needed to rule out other hypotheses and further elucidate the connection between oil exposure, pulmonary disease, and the observed cardiac abnormalities.

Biochemical and hematological biomarkers of reproductive failure in bottlenose dolphins Tursiops truncatus.

The physiological demands of pregnancy inevitably result in alterations in both biochemical and hematological parameters as fetal development occurs. The shifts observed in successful pregnancy in bottlenose dolphins Tursiops truncatus to support both fetal physiological needs and maternal basal requirements have been established according to each trimester. Detecting aberrations in blood-based biomarkers could help facilitate diagnosis of gestational abnormalities, improve our understanding of factors influencing reproductive outcomes and aid in prediction of reproductive failure. This study retrospectively analyzed 263 blood samples from 15 bottlenose dolphins in 21 failed pregnancies over 28 yr (1989-2017). Most samples remained within normal pregnancy reference ranges; however, significant shifts were observed between trimesters. Hematological alterations, compared to successful pregnancy reference ranges from previously published data, were consistent across failed pregnancies and included an increased prevalence of elevated 2nd and 3rd trimester neutrophils, elevated 2nd trimester monocytes and decreased 3rd trimester eosinophils. In addition, low hematocrit and low red blood cells were more prevalent in the 2nd trimester. Biochemical shifts included an increased prevalence of elevated creatine phosphokinase in the 3rd trimester outside of the normal reference ranges. Across failed pregnancies, calcium and iron were decreased in the 3rd trimester. Significantly decreased progesterone in the 3rd trimester was a negative prognostic indicator of pregnancy outcome with decreasing 3rd trimester progesterone associated with failed pregnancy. This study demonstrates the use of blood-based biomarkers as possible predictors of pregnancy outcome in bottlenose dolphins.

SAFETY OF MULTIPLE-DOSE INTRAMUSCULAR KETOPROFEN TREATMENT IN LOGGERHEAD TURTLES (CARETTA CARETTA).

Sea turtles are frequently presented for rehabilitation with injuries for which analgesic treatment is warranted. Ketoprofen is a nonsteroidal anti-inflammatory drug (NSAID) widely used in clinical veterinary medicine for musculoskeletal pain relief. Pharmacokinetics of 2 mg/kg IM have been studied in loggerhead sea turtles (Caretta caretta) as a single and a repeated dose q24hr for 3 days. Safety of longer term administration has not been performed, however, and NSAID use carries a risk of potential complications, including gastrointestinal ulceration, kidney damage, and bleeding. The objective of the current study was to determine the effects of a 5-day course of ketoprofen on thromboelastography (TEG) and hematological (including thrombocytes) and plasma biochemical analytes in loggerheads. A secondary objective was to determine 24-hr trough concentrations of ketoprofen after 5 days of treatment. Eight loggerheads were treated with ketoprofen 2 mg/kg IM q24hr for 5 days, and TEG, hematology, and plasma biochemistry panels were performed before and at the conclusion of treatment. Eight controls were treated with an equivalent volume of saline intramuscularly. Virtually no changes were detected before and after treatment or between treatment and control groups in any of the 24 endpoints evaluated, and marginal differences were not considered clinically relevant. Plasma ketoprofen concentrations after 5 days of treatment indicated no accumulation over that duration. Ketoprofen at 2 mg/kg IM q24hr for up to 5 days in loggerheads appears safe with respect to blood clotting and blood data, although other potential effects were not evaluated.

Standardization of Dolphin Cardiac Auscultation and Characterization of Heart Murmurs in Managed and Free-Ranging Bottlenose Dolphins (Tursiops truncatus).

Cardiac auscultation is an important, albeit underutilized tool in aquatic animal medicine due to the many challenges associated with in-water examinations. The aims of this prospective study were to (1) establish an efficient and repeatable in-water cardiac auscultation technique in bottlenose dolphins (Tursiops truncatus), (2) describe the presence and characterization of heart murmurs detected in free-ranging and managed dolphins, and (3) characterize heart murmur etiology through echocardiography in free-ranging dolphins. For technique development, 65 dolphins cared for by the Navy Marine Mammal Program (Navy) were auscultated. The techniques were then applied to two free-ranging dolphin populations during capture-release health assessments: Sarasota Bay, Florida (SB), a reference population, and Barataria Bay, LA (BB), a well-studied population of dolphins impacted by the Deepwater Horizon oil spill. Systolic heart murmurs were detected at a frequent and similar prevalence in all dolphin populations examined (Navy 92%, SB 89%, and BB 88%), and characterized as fixed or dynamic. In all three populations, sternal cranial and left cranial were the most common locations for murmur point of maximal intensity (PMI). An in-water transthoracic echocardiogram technique was refined on a subset of Navy dolphins, and full echocardiographic exams were performed on 17 SB dolphins and 29 BB dolphins, of which, 40 had murmurs. Spectral Doppler was used to measure flow velocities across the outflow tracts, and almost all dolphins with audible murmurs had peak outflow velocities ≥1.6 m/s (95%, 38/40); three dolphins also had medium mitral regurgitation which could be the source of their murmurs. The presence of audible murmurs in most of the free-ranging dolphins (88%) was attributed to high velocity blood flow as seen on echocardiography, similar to a phenomenon described in other athletic species. These innocent murmurs were generally characterized as Grade I-III systolic murmurs with PMI in the left or sternal cranial region. This study is the first to describe an efficient technique for in-water dolphin cardiac auscultation, and to present evidence that heart murmurs are common in bottlenose dolphins.

Ultrasonographic findings associated with normal pregnancy and fetal well-being in the bottlenose dolphin (Tursiops truncatus).

Reproductive success is vital in sustaining free-ranging and managed bottlenose dolphin (Tursiops truncatus) populations. Ultrasonography is an invaluable, non-invasive tool in assessing the fetomaternal unit in humans and animals, including dolphins and horses. The purpose of this prospective longitudinal cohort study was to develop a protocol for fetomaternal ultrasonographic monitoring in dolphins and to report normal measurements and descriptive findings correlated with a positive outcome. From 2010 to 2017, serial ultrasonographic evaluations of 12 healthy dolphins were performed over the course of 16 pregnancies. A total of 203 ultrasound examinations were included in the study. Several metrics were accurate in predicting fetal age. Fetal biparietal diameter (BPD), thoracic width in dorsal and transverse planes, thoracic height in a sagittal plane, aortic diameter, and blubber thickness all demonstrated high correlation with gestational age (r > 0.94, P < .00001). Regional uteroplacental thickness significantly increased with each trimester (range 0.22-0.40 cm; P < .00011 cranial uterus, P < .00057 mid, and P < .000011 caudal). Lung:liver mean pixel intensity was 2.57 ± 0.46 (95% confidence interval 2.47-2.67). Ultrasonographic characteristics of normal pregnancy in dolphins are described and an equation for prediction of parturition date in Tursiops is reported: days to parturition = 348.16 - (26.03 × BPD(cm)) (R2  = 0.99). Future applications of these normal data will help identify in utero abnormalities indicative of fetal morbidity, and improve understanding of reproductive failure in wild and managed populations.

XANTHINE NEPHROLITHIASIS IN JUVENILE CAPTIVE GIANT OTTERS (PTERONURA BRASILIENSIS).

Nephrolithiasis has been reported in several aquatic mammals including bottlenose dolphins (Tursiops truncatus), small clawed otters (Amblonyx cinereus), European river otters (Lutra lutra), North American river otters (Lontra canadensis), northern elephant seals (Mirounga angustirostris), Florida manatees (Trichechus manatus latirostris), and California sea lions (Zalophus californianus). Compositions of calculi in previous cases were predominantly calcium oxalate or ammonium acid urate. Xanthine urolithiasis is rare in veterinary medicine. Primary cases (without exposure to xanthine dehydrogenase inhibitors) occur as a consequence of hereditary xanthinuria, although the causal mutation has only been discovered in a subset of cases. Five captive juvenile giant otters (Pteronura brasiliensis) from two facilities were diagnosed with nephrolithiasis: three siblings from one set of parents and two siblings from another pair. Serum analyte assays revealed renal compromise in affected individuals. Computed tomography (CT) confirmed the presence of nephrolithiasis in one individual. Postmortem evaluation identified extensive bilateral nephrolithiasis on gross necropsy in four of five cases. Calculus analyses identified 100% xanthine composition. Histologic examination revealed marked nephrolithiasis with associated tubular necrosis and gastric mineralization. Nutrient composition of the diet including mineral and purine content was assessed. No association between diet and nephroliths was found in this study. This is the first report of xanthine nephrolithiasis in aquatic mammals. The potential role of diet and genetics in xanthine nephrolithiasis in the small inbred population of giant otters under human care needs further investigation to assess the implications of this disease process for the long-term captive management of this species.

Pregnancy profiles in the common bottlenose dolphin (Tursiops truncatus): Clinical biochemical and hematological variations during healthy gestation and a successful outcome.

The physiological demands of pregnancy inevitably result in changes of both biochemical and hematological parameters as the fetus develops. Alterations in blood parameters have been observed to shift according to both trimester and species, to support fetal physiological needs and maternal basal requirements. Establishing normal reference ranges for each stage in gestation is important to facilitate diagnosis of underlying health concerns and prevent over-diagnosing abnormalities. Despite bottlenose dolphins (Tursiops truncatus) being one of the most highly studied cetaceans, the blood profile changes occurring as a result of pregnancy have not been previously described. A retrospective analysis was performed from blood samples obtained from 42 successful pregnancies from 20 bottlenose dolphins in a managed population over 30 years. Samples were compared to non-pregnant states and among trimesters of pregnancy. Blood profile fluctuations occurred throughout gestation, however significant alterations predominantly occurred between the 2nd and 3rd trimester. Hematological changes from the 2nd to the 3rd trimester included a decrease in lymphocytes, decrease in platelet count, and hemoconcentration with increased hematocrit and hemoglobin. Biochemical changes in the 3rd trimester included significant reductions in ALKP (alkaline phosphatase), ALT (alanine aminotransferase) and AST (aspartate aminotransferase) with significant increases observed in albumin, globulins, total protein, cholesterol, triglycerides and CO2. It's important to note that despite significant shifts occurring between the 2nd and 3rd trimester, there was no significant change in platelets, hematocrit, hemoglobin, lymphocytes or CO2 between non-pregnant and 3rd trimester blood samples. The normal reference ranges for each trimester established herein, will enable future identification of abnormalities occurring during pregnancy and help improve our understanding of factors potentially influencing a failed or successful pregnancy outcome.

Radiographic assessment of pectoral flipper bone maturation in bottlenose dolphins (Tursiops truncatus), as a novel technique to accurately estimate chronological age.

Accurate age estimation in wildlife conservation is an important diagnostic tool in the interpretation of biological data, necropsy examination, reproductive status and population demographics. The most frequently utilized methods to age bottlenose dolphins (Tursiops truncatus) include tooth extraction; counting dental growth layer groups and dental radiography. These methods are inaccurate in dolphins > 13 years old, due to overlapping of the growth layer groups in dolphins and worn teeth. Establishing a non-invasive method of accurately aging bottlenose dolphins across the entire age range is important to long term conservation efforts to understand health status, lifespan, reproduction and survivability. A database of 126 radiographs from 94 dolphins of known chronological age was utilized to establish the stages of skeletal ossification over time. A numerical score from -1 to 8 was assigned to 16 anatomic locations on the pectoral radiograph, to create a formula to estimate age. The most informative areas to evaluate morphologically were the metaphyseal regions of the radius and ulna, and the proximal and distal epiphysis of metacarpals II and III. Third order polynomial regression calculated separate age predictor formulas for male and female dolphins, with females reaching sexual maturity earlier than males. Completion of epiphyseal closure of the long bones correlated with average sexual maturity. Managed care dolphin ages could be properly estimated with decreasing precision from within 3 months in animals < 5 years old, to within 5 years in animals > 30 years old. This diagnostic tool could also be applied to diagnose atypical ossification patterns consistent with nutritional, developmental or growth abnormalities, and identifying subclinical health issues. In conclusion, knowledge of the lifespan and the onset of sexual maturity for each species will allow this model to be applied to other cetaceans, facilitating age estimation via pectoral radiography in future research.

BASELINE PLASMA THROMBOELASTOGRAPHY IN KEMP'S RIDLEY (LEPIDOCHELYS KEMPII), GREEN (CHELONIA MYDAS) AND LOGGERHEAD (CARETTA CARETTA) SEA TURTLES AND ITS USE TO DIAGNOSE COAGULOPATHIES IN COLD-STUNNED KEMP'S RIDLEY AND GREEN SEA TURTLES.

Cold-stunning in sea turtles is a frequent natural cause of mortality and is defined as a hypothermic state due to exposure to water temperatures <12°C. Derangements of biochemistry and hematology data by cold stunning have been well documented, although the effects on coagulation have not yet been investigated. The objectives of this study were to characterize the hemostatic state of non-cold-stunned sea turtles and to compare cold-stunned sea turtles at admission and after successful rehabilitation via a sea turtle-specific thromboelastography (TEG) protocol. TEG enables evaluation of the entire coagulation process, and the methodology has recently been established in sea turtles. Initially, 30 wild and apparently healthy sea turtles were sampled as controls: loggerhead sea turtles (Caretta caretta), n =17; Kemp's ridley sea turtles (Lepidochelys kempii), n = 8; and green turtles (Chelonia mydas), n = 5. In addition, paired TEG samples were performed on 32 Ch. mydas and 14 L. kempii at admission and prerelease after successful rehabilitation from cold stunning. Statistically significant differences in reaction time, kinetics, angle, and maximum amplitude parameters in L. kempii and Ch. mydas species demonstrated that the time taken for blood clot formation was prolonged and the strength of the clot formed was reduced by cold stunning. These findings indicate that cold stunning may cause disorders in hemostasis that can contribute to the severity of the condition. Early diagnosis of coagulopathies in the clinical assessment of a cold-stunned sea turtle may influence the treatment approach and clinical outcome of the case.

Health Assessments of Common Bottlenose Dolphins (Tursiops truncatus): Past, Present, and Potential Conservation Applications.

The common bottlenose dolphin (Tursiops truncatus) is a global marine mammal species for which some populations, due to their coastal accessibility, have been monitored diligently by scientists for decades. Health assessment examinations have developed a comprehensive knowledge base of dolphin biology, population structure, and environmental or anthropogenic stressors affecting their dynamics. Bottlenose dolphin health assessments initially started as stock assessments prior to acquisition. Over the last four decades, health assessments have evolved into essential conservation management tools of free-ranging dolphin populations. Baseline data enable comparison of stressors between geographic locations and associated changes in individual and population health status. In addition, long-term monitoring provides opportunities for insights into population shifts over time, with retrospective application of novel diagnostic tests on archived samples. Expanding scientific knowledge enables effective long-term conservation management strategies by facilitating informed decision making and improving social understanding of the anthropogenic effects. The ability to use bottlenose dolphins as a model for studying marine mammal health has been pivotal in our understanding of anthropogenic effects on multiple marine mammal species. Future studies aim to build on current knowledge to influence management decisions and species conservation. This paper reviews the historical approaches to dolphin health assessments, present day achievements, and development of future conservation goals.

Establishing a protocol for thromboelastography in sea turtles.

Thromboelastography (TEG) provides a global evaluation of haemostasis. This diagnostic test is widely used in mammals but has not previously been performed in reptiles, mainly due to the limited availability of taxon-specific reagents. The objective of this pilot study was to establish a protocol to perform TEG in sea turtles. Pooled citrated plasma, stored at -80°C, from four green turtles (Chelonia mydas) was assayed on a TEG 5000. Several initiators were evaluated: kaolin (n=2), RapidTEG (n=2), fresh (n=2) and frozen (n=6) thromboplastin extracted from pooled brain tissue from several chelonian species, human recombinant tissue factor at 1:100 (n=1), Reptilase (n=2), and rabbit thromboplastin (n=1). Both fresh and frozen chelonian thromboplastin were superior in producing quantifiable TEG reaction time compared with all other reagents. These findings are consistent with the lack of an intrinsic pathway in turtles and confirmed a lack of coagulation in the turtle samples in response to mammalian thromboplastin. A TEG protocol was subsequently established for harvested species-specific frozen thromboplastin. The frozen thromboplastin reagent remained stable after one year of storage at -80°C. The developed protocol will be useful as a basis for future studies that aim to understand the pathophysiology of haemostatic disorders in various stranding conditions of sea turtles.

Thromboelastography in wild Florida manatees (Trichechus manatus latirostris).

BACKGROUND: Thromboelastography (TEG) provides a comprehensive evaluation of blood clot formation. This test can be used to identify abnormalities in coagulation by assessing multiple aspects of the clotting cascade, including the speed of clot initiation and formation, clot strength, and ultimately fibrinolysis. Thromboembolic disease has been hypothesized to play a role in the pathophysiology of cold stress syndrome (CSS), an important cause of mortality in the threatened Florida manatee (Trichechus manatus latirostris). OBJECTIVES: The objective of this study was to establish thromboelastography RIs using the TEG 5000 with citrated whole blood samples and kaolin activation in wild, healthy manatees. METHODS: In December 2014 and January 2015, 29 wild manatees (17 male and 12 female) were blood sampled as part of the annual wild manatee health assessments organized by United States Geological Survey (USGS). TEG was performed using citrated kaolin-activated samples. RESULTS: The samples were obtained from manatees caught in Crystal River, Citrus County, and used to identify the mean ± SD of normal TEG parameters: R = reaction time 2.1 minutes (0.8), K = clot formation time 0.8 min (0), α angle = 83.1° (2), MA = maximum amplitude 75 mm (7.6), and LY30 = clot lysis 0.41% (0.68). No significant differences were found between manatee size, sex, or time between sampling and running the test. CONCLUSIONS: Manatee TEG parameters demonstrate a relatively hypercoagulable condition when compared to other mammals. This information will facilitate detection of changes in hemostasis during injury and disease and provide a valuable reference range.

Identifying coagulopathies in the pathophysiology of cold stress syndrome in the Florida manatee Trichechus manatus latirostris.

Cold stress syndrome (CSS) in the Florida manatee Trichechus manatus latirostris has been defined as morbidity and mortality resulting from prolonged exposure to water temperatures <20°C. The pathophysiology is described as multifactorial, involving nutritional, immunological and metabolic disturbances; however, the exact mechanisms are unknown. We hypothesized that thromboembolic complications contribute to the pathophysiology of CSS in addition to the previously described factors. During the winter of 2014-2015, 10 Florida manatees with clinical signs of CSS were presented to Lowry Park Zoo, Tampa, FL, USA. Thromboelastography (TEG) and coagulation panels were performed at admission. In addition, coagulation panel data from 23 retrospective CSS cases were included in the analyses. There were numerous differences between mean values of TEG and coagulation parameters for healthy manatees and those for CSS cases. Among TEG parameters, reaction time (R), clot formation time (K) and percentage of clot lysed after 30 min (LY30) values were significantly different (p < 0.05) between the 2 groups. CSS cases also had significantly higher mean D-dimer concentration and coagulation factor XI activity, prolonged mean activated partial thromboplastin time (aPTT) and significantly decreased mean antithrombin activity. These combined abnormalities include clinicopathologic criteria of disseminated intravascular coagulation, indicating an increased risk of thromboembolic disease associated with manatee CSS.

PROPOSAL AND APPLICATION OF A NOVEL DISSEMINATED INTRAVASCULAR COAGULATION SCORING SYSTEM IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS).

Disseminated intravascular coagulopathy (DIC) is an acquired disorder of hemostasis resulting in activation of the coagulation and fibrinolytic pathways. It is reported secondarily to multiple disease processes and can be associated with increased mortality. Previous research at Tampa's Lowry Park Zoo (LPZ) demonstrated that Florida manatees ( Trichechus manatus latirostris) with cold stress syndrome (CSS) demonstrated thromboembolic disease. The object of this retrospective study was to establish the presence and clinical relevance of DIC in Florida manatees admitted to LPZ for rehabilitation from 07 March 2010 to 15 August 2015. A coagulation panel, including prothrombin time, partial thromboplastin time, platelet count, fibrinogen level, and D-dimer level was used to diagnose DIC. There were 100 cases identified in the study period: 35 trauma, 43 CSS, 17 secondary to harmful algae blooms (HAB), and five miscellaneous. Manatees with CSS had the highest incidence of DIC with 24 of 43 cases (56%) affected, followed by trauma with 18 of 35 cases (52%) affected. None of the manatees with HAB were found to have DIC. Manatees that developed DIC during rehabilitation or when DIC progressed did not survive. Due to the clinical implications of DIC, identifying its presence and recognizing its severity could improve clinical outcomes by enabling more intensive treatment protocols.

COAGULATION ASSESSMENT: UNDERUTILIZED DIAGNOSTIC TOOLS IN ZOO AND AQUATIC ANIMAL MEDICINE.

Veterinarians specializing in nondomestic species are faced with unique challenges regarding research and diagnostic capabilities given the wild and frequently dangerous nature of their patients. Standard diagnostic techniques used in small or large animal practice are not always possible due to anatomical constraints, size, tractability, or the inherent risk of anesthesia in highly valued, rare species. Diagnostic modalities that utilize simple, relatively noninvasive techniques show promise in evaluating nondomestic species and elucidating the pathophysiology behind poorly characterized disease processes in both wild and captive populations. Coagulation profiles, which may include prothrombin time (PT), partial thromboplastin time (PTT), D-dimer concentration, platelet count, and thromboelastography (TEG) are frequently used in domestic species but often overlooked in exotic medicine due to lack of normal reference values and/or availability. Whenever possible, coagulation profiles should be utilized in the evaluation of various disease processes including neoplasia, sepsis, trauma, inflammation, toxin exposure, and envenomation. There are several reports of coagulopathies in both wild and captive species; however, few studies on coagulation profiles have been published on nondomestic species. Clinicians should consider coagulation testing as part of the diagnostic work-up in nondomestic species. A review of available coagulation diagnostic tests is provided here in addition to summarizing the pertinent coagulation disorders currently established in the literature.

Normal Hemostatic Profiles and Coagulation Factors in Healthy Free-Living Florida Manatees ( Trichechus manatus latirostris).

Hemostatic disorders presumptively play an important role in the pathophysiology of several important disease conditions in the Florida manatee ( Trichechus manatus latirostris). Prior to pursuing such clinical implications, it is essential to establish normal hemostatic profiles in clinically healthy animals. During annual health assessments of free-living manatees organized by the US Geological Survey, blood samples were collected from 12 healthy animals from the Atlantic coast and 28 from the Gulf of Mexico coast of Florida, with body lengths of 210-324 cm. The following analyses were performed on citrated plasma: prothrombin (PT), partial thromboplastin time (PTT), and concentrations of fibrinogen, D-dimers, and coagulation factors VII, VIII, IX, X, XI, and XII. Compared to other mammalian species, manatees had short PT (9.2±1.5 s) and PTT (10.7±0.5 s), fibrinogen was 369±78.7 mg/dL, antithrombin III was 132±11%, and D-dimer was 142±122 ng/mL. Baseline concentrations for the listed coagulation factors were established. When comparing coagulation factors between locations, Atlantic coast manatees had significantly higher factors VIII, IX, and X than did Gulf Coast manatees. This finding may reflect differences in water salinity, diet, or genetics. There were no differences in coagulation factors when among sexes and sizes. These baselines for hemostatic profiles and coagulation factors in healthy free-living manatees lay the foundation for diagnosis and future research of hemostatic disorders and contribute to understanding their role in the pathophysiology of manatees affected by various diseases.