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.

Managing the effects of multiple stressors on wildlife populations in their ecosystems: developing a cumulative risk approach.

Assessing cumulative effects of human activities on ecosystems is required by many jurisdictions, but current science cannot meet regulatory demands. Regulations define them as effect(s) of one human action combined with other actions. Here we argue for an approach that evaluates the cumulative risk of multiple stressors for protected wildlife populations within their ecosystems. Monitoring effects of each stressor is necessary but not sufficient to estimate how multiple stressors interact to affect wildlife populations. Examining the mechanistic pathways, from cellular to ecological, by which stressors affect individuals can help prioritize stressors and interpret how they interact. Our approach uses health indicators to accumulate the effects of stressors on individuals and to estimate changes in vital rates, driving population status. We advocate using methods well-established in human health and integrating them into ecosystem-based management to protect the health of commercially and culturally important wildlife populations and to protect against risk of extinction for threatened species. Our approach will improve abilities to conserve and manage ecosystems but will also demand significant increases in research and monitoring effort. We advocate for increased investment proportional to the economic scale of human activities in the Anthropocene and their pervasive effects on ecology and biodiversity.

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.

Linking morbillivirus exposure to individual habitat use of common bottlenose dolphins (Tursiops truncatus) between geographically different sites.

Dolphin morbillivirus (DMV) is a virulent pathogen that causes high mortality outbreaks in delphinids globally and is spread via contact among individuals. Broadly ranging nearshore and open-ocean delphinids are likely reservoir populations that transmit DMV to estuarine populations. We assessed the seroprevalence of DMV antibodies and determined the habitat use of common bottlenose dolphins, Tursiops truncatus truncatus, from two estuarine sites, Barataria Bay and Mississippi Sound, in the northern Gulf of Mexico. We predicted that risk to DMV exposure in estuarine dolphins is driven by spatial overlap in habitat use with reservoir populations. Serum was collected from live-captured dolphins and tested for DMV antibodies. Habitat use of sampled individuals was determined by analysing satellite-tracked movements and stable isotope values. DMV seroprevalences were high among dolphins at Barataria Bay (37%) and Mississippi Sound (44%), but varied differently within sites. Ranging patterns of Barataria Bay dolphins were categorized into two groups: Interior and Island-associated. DMV seroprevalences were absent in Interior dolphins (0%) but high in Island-associated dolphins (45%). Ranging patterns of Mississippi Sound dolphins were categorized into three groups: Interior, Island-east and Island-west. DMV seroprevalences were detected across Mississippi Sound (Interior: 60%; Island-east: 20%; and Island-west: 43%). At both sites, dolphins in habitats with greater marine influence had enriched δ13 C values, and Barataria Bay dolphins with positive DMV titres had carbon isotope values indicative of marine habitats. Positive titres for DMV antibodies were more common in the lower versus upper parts of Barataria Bay but evenly distributed across Mississippi Sound. A dolphin's risk of exposure to DMV is influenced by how individual ranging patterns interact with environmental geography. Barataria Bay's partially enclosed geography likely limits the nearshore or open-ocean delphinids that carry DMV from interacting with dolphins that use interior, estuarine habitats, decreasing their exposure to DMV. Mississippi Sound's relatively open geography allows for greater spatial overlap and mixing among estuarine, nearshore and/or open-ocean cetaceans. The spread of DMV, and likely other diseases, is affected by the combination of individual movements, habitat use and the environment.

A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

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.

Blubber steroid hormone profiles as indicators of physiological state in free-ranging common bottlenose dolphins (Tursiops truncatus).

Blubber has been proposed as a possible alternative to blood in the assessment of endocrine physiology in marine mammals because it can be collected via remote biopsy, which removes some of the confounding variables and logistical constraints associated with blood collection. To date, few studies have directly assessed the relationships between circulating versus blubber steroid hormone profiles in marine mammals, and these studies have been limited to a small subset of steroid hormones, which collectively limit the current utility of blubber steroid hormone measurements. In this study, we used liquid-chromatography tandem-mass spectrometry (LC-MS/MS) to screen for 16 steroid hormones in matched blood and blubber samples from free-ranging common bottlenose dolphins (Tursiops truncatus). Seven steroid hormones were detected and quantified, including two progestogens, two androgens, and three corticosteroids. Using principal components analysis (PCA), we explored relationships between hormones in both matrices and three physiological states: sexual maturity, pregnancy, and acute stress response. Plasma and blubber testosterone and its precursors, 17-hydroxyprogesterone and androstenedione, loaded to the first principal component (PC1), and PC1 scores were higher in mature males. Plasma and blubber progesterone loaded to PC2, and pregnant/probable pregnant females had significantly higher PC2 scores. Pregnant females also had higher PC1 scores than other females, suggesting differences in androgen profiles between these groups. There was disagreement between plasma and blubber corticosteroid profiles, as indicated by their loading to different PCs; plasma corticosteroids loaded to PC3 and blubber corticosteroids to PC4. PC3 scores were significantly predicted by elapsed time to blood collection (i.e., time between initiating the capture process and blood collection), while elapsed time to blubber collection significantly predicted PC4 scores, indicating that corticosteroid profiles shift in both tissues during acute stress. Corticosteroid profiles were not related to demographic group, site-month, body mass index, water temperature, or time spent outside of the water on the processing boat. Overall, these results demonstrate that blubber steroid hormone profiles reflect changes in endocrine function that occur over broad temporal scales.

Remote blubber sampling paired with liquid chromatography tandem mass spectrometry for steroidal endocrinology in free-ranging bottlenose dolphins (Tursiops truncatus).

Liquid chromatography tandem mass spectrometry allows for the measurement of steroid hormone suites in the blubber of marine mammals. By combining this technology with minimally invasive techniques such as remote biopsy, endocrine profiles can be assessed, allowing for studies of hormonal profile variation over time. In this study, we explored associations among different steroidogenic pathways and seasonal differences in blubber hormone profiles of free-ranging common bottlenose dolphins along the coast of South Carolina, USA. Male dolphins experience a peak in testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone in the spring, likely related to an upregulation of the androgen steroidogenic pathway during mating season. We also observed increased cortisol concentrations during summer compared to winter. Among females, there was an increase in androstenedione with elevated progesterone concentrations indicative of pregnancy, highlighting another potential endocrine marker for pregnancy in free-ranging dolphins. This work emphasizes the importance of selecting the appropriate season for studies on endocrine status to effectively uncover physiological variation or disruption in free-ranging cetaceans.

Characterization of circulating steroid hormone profiles in the bottlenose dolphin (Tursiops truncatus) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Systemic steroid hormone measurements are often used in the assessment of reproductive, developmental, and stress physiology in vertebrates. In protected wildlife, such as the common bottlenose dolphin (Tursiops truncatus), these measures can provide critical information about health and fitness to aid in effective conservation and management. Circulating steroid hormone concentrations are typically measured by immunoassays, which have imperfect specificity and are limited to the measurement of a single hormone per assay. Here we demonstrate that reverse phase solid phase extraction (SPE) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) allows for the simultaneous, precise (<15% relative standard deviation), and accurate (between 70% and 120% recovery of spiked quantities) measurement of at least seven steroid hormones in dolphin plasma. These seven steroid hormones include three hormones that have been measured previously in bottlenose dolphin blood (progesterone, testosterone, and cortisol) and three hormones which have never been quantified in dolphin blood (17-hydroxyprogesterone, androstenedione, cortisone, and corticosterone). While 17ß-estradiol was not detected endogenously, we were able to accurately and precisely measure spiked quantities estradiol. Measures from plasma were more precise (i.e., lower RSD) than serum, and thus we recommend plasma as the preferred matrix for this analytical method. In order to facilitate comparison of current and future plasma-based studies to previous serum-based studies, we characterized the relationships between hormone measurements in matched plasma and serum, and found that measurements across matrices are significantly and positively correlated. Lastly, to demonstrate potential applications of this method, we examined how steroid hormone profiles vary by pregnancy, sexual maturity, and stress status - pregnancy was associated with elevated progesterone, adult males had higher testosterone, and capture stress was associated with elevated corticosteroids. Overall, we conclude that this method will enable investigators to more thoroughly and efficiently evaluate steroid hormone homeostasis in bottlenose dolphins compared to immunoassay methods. These methods can potentially be applied to the assessment of sexual maturity/seasonality, pregnancy status, and stress in free-ranging bottlenose dolphins as well as those maintained under human care, and potentially other marine mammals.

Noninvasive Respiratory Metabolite Analysis Associated with Clinical Disease in Cetaceans: A Deepwater Horizon Oil Spill Study.

Health assessments of wild cetaceans can be challenging due to the difficulty of gaining access to conventional diagnostic matrices of blood, serum and others. While the noninvasive detection of metabolites in exhaled breath could potentially help to address this problem, there exists a knowledge gap regarding associations between known disease states and breath metabolite profiles in cetaceans. This technology was applied to the largest marine oil spill in U.S. history (The 2010 Deepwater Horizon oil spill in the Gulf of Mexico). An accurate analysis was performed to test for associations between the exhaled breath metabolome and sonographic lung abnormalities as well as hematological, serum biochemical, and endocrine hormone parameters. Importantly, metabolites consistent with chronic inflammation, such as products of lung epithelial cellular breakdown and arachidonic acid cascade metabolites were associated with sonographic evidence of lung consolidation. Exhaled breath condensate (EBC) metabolite profiles also correlated with serum hormone concentrations (cortisol and aldosterone), hepatobiliary enzyme levels, white blood cell counts, and iron homeostasis. The correlations among breath metabolites and conventional health measures suggest potential application of breath sampling for remotely assessing health of wild cetaceans. This methodology may hold promise for large cetaceans in the wild for which routine collection of blood and respiratory anomalies are not currently feasible.

Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4 g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations. Graphical abstract The steroid hormone profile is quantifiable from a single sample of bottlenose dolphin blubber using liquid chromatography tandem mass spectrometry. This profile can be applied to remotely collected dart biopsies and be used to determine reproductive or stress status of a wild-living dolphin.

RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins.

BACKGROUND: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1 year to establish baseline information on the content and variation of the dolphin blood transcriptome. RESULTS: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1 year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22 % of variance). Limited seasonal change was observed, with < 2.5 % of genes differentially expressed between winter and summer months (FDR < 0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters. CONCLUSIONS: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Detection of Brucella spp. in bottlenose dolphins Tursiops truncatus by a real-time PCR using blowhole swabs.

Blowhole swabs are a simple and non-invasive method for collecting samples from cetaceans and can be used for screening large numbers of animals in the field. This study reports a real-time PCR assay for the detection of Brucella spp. using blowhole swab samples from bottlenose dolphins Tursiops truncatus stranded in the coastal region of Virginia, South Carolina and northern Florida, USA, between 2013 and 2015. We used real-time PCR results on lung samples from the same dolphins in order to estimate the relative sensitivity and specificity of real-time PCR of blowhole swabs. Brucella DNA was detected in lung tissue of 22% (18/81) and in blowhole swabs of 21% (17/81) of the sampled dolphins. The relative sensitivity and specificity of real-time PCR on blowhole swabs as compared to the real-time PCR on lung samples was 94% (17/18) and 100% (63/63), respectively. These results indicate that real-time PCR on blowhole swabs may be used as a non-invasive test for rapid detection of Brucella spp. in the respiratory tract of dolphins. To our knowledge, this is the first report on the use of blowhole swabs for detection of bacterial pathogens by real-time PCR in bottlenose dolphins.

Reproductive outcome and survival of common bottlenose dolphins sampled in Barataria Bay, Louisiana, USA, following the Deepwater Horizon oil spill.

Common bottlenose dolphins (Tursiops truncatus) inhabit bays, sounds and estuaries across the Gulf of Mexico. Following the Deepwater Horizon oil spill, studies were initiated to assess potential effects on these ecologically important apex predators. A previous study reported disease conditions, including lung disease and impaired stress response, for 32 dolphins that were temporarily captured and given health assessments in Barataria Bay, Louisiana, USA. Ten of the sampled dolphins were determined to be pregnant, with expected due dates the following spring or summer. Here, we report findings after 47 months of follow-up monitoring of those sampled dolphins. Only 20% (95% CI: 2.50-55.6%) of the pregnant dolphins produced viable calves, as compared with a previously reported pregnancy success rate of 83% in a reference population. Fifty-seven per cent of pregnant females that did not successfully produce a calf had been previously diagnosed with moderate-severe lung disease. In addition, the estimated annual survival rate of the sampled cohort was low (86.8%, 95% CI: 80.0-92.7%) as compared with survival rates of 95.1% and 96.2% from two other previously studied bottlenose dolphin populations. Our findings confirm low reproductive success and high mortality in dolphins from a heavily oiled estuary when compared with other populations. Follow-up studies are needed to better understand the potential recovery of dolphins in Barataria Bay and, by extension, other Gulf coastal regions impacted by the spill.

Health of common bottlenose dolphins ( Tursiops truncatus ) in Barataria Bay, Louisiana, following the deepwater horizon oil spill.

The oil spill resulting from the explosion of the Deepwater Horizon drilling platform initiated immediate concern for marine wildlife, including common bottlenose dolphins in sensitive coastal habitats. To evaluate potential sublethal effects on dolphins, health assessments were conducted in Barataria Bay, Louisiana, an area that received heavy and prolonged oiling, and in a reference site, Sarasota Bay, Florida, where oil was not observed. Dolphins were temporarily captured, received a veterinary examination, and were then released. Dolphins sampled in Barataria Bay showed evidence of hypoadrenocorticism, consistent with adrenal toxicity as previously reported for laboratory mammals exposed to oil. Barataria Bay dolphins were 5 times more likely to have moderate-severe lung disease, generally characterized by significant alveolar interstitial syndrome, lung masses, and pulmonary consolidation. Of 29 dolphins evaluated from Barataria Bay, 48% were given a guarded or worse prognosis, and 17% were considered poor or grave, indicating that they were not expected to survive. Disease conditions in Barataria Bay dolphins were significantly greater in prevalence and severity than those in Sarasota Bay dolphins, as well as those previously reported in other wild dolphin populations. Many disease conditions observed in Barataria Bay dolphins are uncommon but consistent with petroleum hydrocarbon exposure and toxicity.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

Survey of antibiotic-resistant bacteria isolated from bottlenose dolphins Tursiops truncatus in the southeastern USA.

Contamination of coastal waters can carry pathogens and contaminants that cause diseases in humans and wildlife, and these pathogens can be transported by water to areas where they are not indigenous. Marine mammals may be indicators of potential health effects from such pathogens and toxins. Here we isolated bacterial species of relevance to humans from wild bottlenose dolphins Tursiops truncatus and assayed isolated bacteria for antibiotic resistance. Samples were collected during capture-release dolphin health assessments at multiple coastal and estuarine sites along the US mid-Atlantic coast and the Gulf of Mexico. These samples were transported on ice and evaluated using commercial systems and aerobic culture techniques routinely employed in clinical laboratories. The most common bacteria identified were species belonging to the genus Vibrio, although Escherichia coli, Shewanella putrefaciens, and Pseudomonas fluorescens/putida were also common. Some of the bacterial species identified have been associated with human illness, including a strain of methicillin-resistant Staphylococcus aureus (MRSA) identified in 1 sample. Widespread antibiotic resistance was observed among all sites, although the percentage of resistant isolates varied across sites and across time. These data provide a baseline for future comparisons of the bacteria that colonize bottlenose dolphins in the southeastern USA.

Spatial Variation in Mercury Accumulation in Bottlenose Dolphins (Tursiops spp.) in Southeastern U.S.A.

Bottlenose dolphins (Tursiops spp.) inhabit bays, sounds, and estuaries (BSEs) throughout the southeast region of the U.S.A. and are sentinel species for human and ecosystem-level health. Dolphins are vulnerable to the bioaccumulation of contaminants through the coastal food chain because they are high-level predators. Currently, there is limited information on the spatial dynamics of mercury accumulation in these dolphins. Total mercury (THg) was measured in dolphin skin from multiple populations across the U.S. Southeast Atlantic and Gulf of Mexico coasts, and the influence of geographic origin, sex, and age class was investigated. Mercury varied significantly among sampling sites and was greatest in dolphins in St. Joseph Bay, Florida Everglades, and Choctawhatchee Bay (14,193 ng/g ± 2196 ng/g, 10,916 ng/g ± 1532 ng/g, and 7333 ng/g ± 1405 ng/g wet mass (wm), respectively) and lowest in dolphins in Charleston and Skidaway River Estuary (509 ng/g ± 32.1 ng/g and 530 ng/g ± 58.4 ng/g wm, respectively). Spatial mercury patterns were consistent regardless of sex or age class. Bottlenose dolphin mercury exposure can effectively represent regional trends and reflect large-scale atmospheric mercury input and local biogeochemical processes. As a sentinel species, the bottlenose dolphin data presented here can direct future studies to evaluate mercury exposure to human residents in St. Joseph Bay, Choctawhatchee Bay, and Florida Coastal Everglades, as well as additional sites with similar geographical, oceanographic, or anthropogenic parameters. These data may also inform state and federal authorities that establish fish consumption advisories to determine if residents in these locales are at heightened risk for mercury toxicity.

Electrocardiograms of bottlenose dolphins (Tursiops truncatus) out of water: habituated collection versus wild postcapture animals.

Electrocardiography (ECG) was performed on captured free-ranging bottlenose dolphins (Tursiops truncatus) during a health assessment exercise and compared with that of a Navy collection of dolphins habituated to handling out of water in order to assess possible cardiovascular impacts of capture and handling. Six-lead recordings (I, II, III, aVr, aVl, and aVf) in the frontal plane and direct thorax leads were collected from both groups, with a modified base-apex lead additionally employed with the Navy collection dolphins. Measured and calculated parameters included amplitudes of P, R, S, and T waves and total QRS complex; T:S and T:QRS ratios; heart rate; durations of P wave; QRS complex, PR, QT, and RR intervals; maximum minus minimum RR interval; ST segment elevation-depression; and mean electrical axis (MEA). Physiologically minor but statistically significant differences were detected in S wave amplitude, PR interval, QRS duration, and MEA. The PR interval, QRS duration, and S wave amplitude were slightly greater and the MEA oriented slightly rightward in wild postcapture dolphins compared to Navy collection dolphins. There were no differences in heart rate or maximum minus minimum RR interval, which serves as a proxy for the expected sinus arrhythmia of dolphins. The base-apex lead resulted in greater QRS amplitude than lead II, as expected for the category B ventricular activation of dolphins. The left-side direct thorax lead was more consistent than that of the right side. Clinically, ECG was a useful adjunct to auscultation and thoracic palpation for monitoring heart rate and rhythm and generated a record for archiving. Safe capture and handling protocols in place, under which dolphins are immediately returned to the water at progressive signs of distress, may make cardiovascular decompensation less likely to be detected by ECG. It appears that the dolphin cardiovascular system compensates suitably well to capture, as measured by ECG under the conditions of this study.

Using Blood Gas Analysis and Capnography to Determine Oxygenation Status in Bottlenose Dolphins (Tursiops truncatus) Following the Deepwater Horizon Oil Spill.

Following the Deepwater Horizon (DWH) oil spill in 2010, poor pulmonary health and reproductive failure in bottlenose dolphins (Tursiops truncatus) in the northern Gulf of Mexico were well-documented. One postulated etiology for the increased fetal distress syndrome and pneumonia found in affected perinatal dolphins was maternal hypoxia caused by lung disease. The objective of this study was to evaluate the utility of blood gas analysis and capnography in determining oxygenation status in bottlenose dolphins with and without pulmonary disease. Blood and breath samples were collected from 59 free-ranging dolphins in Barataria Bay, Louisiana (BB), during a capture-release health assessment program, and from 30 managed dolphins from the U.S. Navy Marine Mammal Program in San Diego, CA. The former was the oil-exposed cohort and the latter served as a control cohort with known health histories. Capnography and select blood gas parameters were compared based on the following factors: cohort, sex, age/length class, reproductive status, and severity of pulmonary disease. Animals with moderate-severe lung disease had higher bicarbonate concentrations (p = 0.005), pH (p < 0.001), TCO2 (p = 0.012), and more positive base excess (p = 0.001) than animals with normal-mild disease. Capnography (ETCO2) was found to have a weak positive correlation with blood PCO2 (p = 0.020), with a mean difference of 5.02 mmHg (p < 0.001). Based on these findings, indirect oxygenation measures, including TCO2, bicarbonate, and pH, show promise in establishing the oxygenation status in dolphins with and without pulmonary disease.