A time-series analysis of blood-based biomarkers within a 25-year longitudinal dolphin cohort.

Causal interactions and correlations between clinically-relevant biomarkers are important to understand, both for informing potential medical interventions as well as predicting the likely health trajectory of any individual as they age. These interactions and correlations can be hard to establish in humans, due to the difficulties of routine sampling and controlling for individual differences (e.g., diet, socio-economic status, medication). Because bottlenose dolphins are long-lived mammals that exhibit several age-related phenomena similar to humans, we analyzed data from a well controlled 25-year longitudinal cohort of 144 dolphins. The data from this study has been reported on earlier, and consists of 44 clinically relevant biomarkers. This time-series data exhibits three starkly different influences: (A) directed interactions between biomarkers, (B) sources of biological variation that can either correlate or decorrelate different biomarkers, and (C) random observation-noise which combines measurement error and very rapid fluctuations in the dolphin's biomarkers. Importantly, the sources of biological variation (type-B) are large in magnitude, often comparable to the observation errors (type-C) and larger than the effect of the directed interactions (type-A). Attempting to recover the type-A interactions without accounting for the type-B and type-C variation can result in an abundance of false-positives and false-negatives. Using a generalized regression which fits the longitudinal data with a linear model accounting for all three influences, we demonstrate that the dolphins exhibit many significant directed interactions (type-A), as well as strong correlated variation (type-B), between several pairs of biomarkers. Moreover, many of these interactions are associated with advanced age, suggesting that these interactions can be monitored and/or targeted to predict and potentially affect aging.

Evaluation of the safety and efficacy of fecal microbiota transplantations in bottlenose dolphins (Tursiops truncatus) using metagenomic sequencing.

AIMS: Gastrointestinal disease is a leading cause of morbidity in bottlenose dolphins (Tursiops truncatus) under managed care. Fecal microbiota transplantation (FMT) holds promise as a therapeutic tool to restore gut microbiota without antibiotic use. This prospective clinical study aimed to develop a screening protocol for FMT donors to ensure safety, determine an effective FMT administration protocol for managed dolphins, and evaluate the efficacy of FMTs in four recipient dolphins. METHODS AND RESULTS: Comprehensive health monitoring was performed on donor and recipient dolphins. Fecal samples were collected before, during, and after FMT therapy. Screening of donor and recipient fecal samples was accomplished by in-house and reference lab diagnostic tests. Shotgun metagenomics was used for sequencing. Following FMT treatment, all four recipient communities experienced engraftment of novel microbial species from donor communities. Engraftment coincided with resolution of clinical signs and a sustained increase in alpha diversity. CONCLUSION: The donor screening protocol proved to be safe in this study and no adverse effects were observed in four recipient dolphins. Treatment coincided with improvement in clinical signs.

A 25-y longitudinal dolphin cohort supports that long-lived individuals in same environment exhibit variation in aging rates.

While it is believed that humans age at different rates, a lack of robust longitudinal human studies using consensus biomarkers meant to capture aging rates has hindered an understanding of the degree to which individuals vary in their rates of aging. Because bottlenose dolphins are long-lived mammals that develop comorbidities of aging similar to humans, we analyzed data from a well-controlled, 25-y longitudinal cohort of 144 US Navy dolphins housed in the same oceanic environment. Our analysis focused on 44 clinically relevant hematologic and clinical chemistry measures recorded during routine blood draws throughout the dolphins' lifetimes. Using stepwise regression and general linear models that accommodate correlations between measures obtained on individual dolphins, we demonstrate that, in a manner similar to humans, dolphins exhibit independent and linear age-related declines in four of these measures: hemoglobin, alkaline phosphatase, platelets, and lymphocytes. Using linear regressions and analyses of covariance with post hoc Tukey-Kramer tests to compare slopes (i.e., linear age-related rates) of our four aging rate biomarkers among 34 individual dolphins aging from 10 y to up to 40 y old, we could identify slow and accelerated agers and differentiate subgroups that were more or less likely to develop anemia and lymphopenia. This study successfully documents aging rate differences over the lifetime of long-lived individuals in a controlled environment. Our study suggests that nonenvironmental factors influencing aging rate biomarkers, including declining hemoglobin and anemia, may be targeted to delay the effects of aging in a compelling model of human biology.

Dietary effects on urinary physicochemistry in Navy bottlenose dolphins (Tursiops truncatus) for the prevention of ammonium urate kidney stones.

Bottlenose dolphins are susceptible to developing ammonium urate (NH4U) kidney stones. The current study was designed to test the hypothesis that diet influences the urinary physicochemistry risk factors associated with nephrolithiasis in dolphins. A comprehensive nutrient analysis was performed revealing that the baseline diet (BD) commonly fed to dolphins under professional care had a greater purine content and a more negative dietary cation-anion difference (DCAD) when compared with a model diet consumed by free-ranging dolphins. A modified diet (MD) was formulated to include free-ranging diet fish species and achieve a more positive DCAD. The BD had a more negative DCAD (-52 mEq/Mcal metabolizable energy) when compared with the MD (+51 mEq/Mcal ME), which more closely approximated the DCAD of the free-ranging model diet (+152 mEq/Mcal ME). Six dolphins (with stones) were fed the BD followed by the MD for a minimum of 4 wk. At the end of each feeding trial, a 6-h continuous urine collection was performed to compare urine parameters of dolphins fed the BD versus MD. Dolphins consuming the MD demonstrated a significant decrease in urinary ammonium, net acid excretion, saturation index of ammonium urate, and phosphorous, and a significant increase in urinary citrate and net gastrointestinal (GI) alkali absorption, as compared with urine parameters assessed when fed the BD. Increasing the proportion of free-ranging diet fish species and optimizing the DCAD positively influenced some of the risk factors believed to be associated with NH4U kidney stone development in bottlenose dolphins under professional care.

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.

Regulation of Osteoclast Differentiation at Multiple Stages by Protein Kinase D Family Kinases.

Balanced osteoclast and osteoblast activity is necessary for skeletal health, whereas unbalanced osteoclast activity causes bone loss in many skeletal conditions. A better understanding of pathways that regulate osteoclast differentiation and activity is necessary for the development of new therapies to better manage bone resorption. The roles of Protein Kinase D (PKD) family of serine/threonine kinases in osteoclasts have not been well characterized. In this study we use immunofluorescence analysis to reveal that PKD2 and PKD3, the isoforms expressed in osteoclasts, are found in the nucleus and cytoplasm, the mitotic spindle and midbody, and in association with the actin belt. We show that PKD inhibitors CRT0066101 and CID755673 inhibit several distinct aspects of osteoclast formation. Treating bone marrow macrophages with lower doses of the PKD inhibitors had little effect on M-CSF + RANKL-dependent induction into committed osteoclast precursors, but inhibited their motility and subsequent differentiation into multinucleated mature osteoclasts, whereas higher doses of the PKD inhibitors induced apoptosis of the preosteoclasts. Treating post-fusion multinucleated osteoclasts with the inhibitors disrupted the osteoclast actin belts and impaired their resorptive activity. In conclusion, these data implicate PKD kinases as positive regulators of osteoclasts, which are essential for multiple distinct processes throughout their formation and function.

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.

Comparison of potential dietary and urinary risk factors for ammonium urate nephrolithiasis in two bottlenose dolphin ( Tursiops truncatus) populations.

Dietary and urinary risk factors have been implicated in conditions favoring ammonium urate nephrolithiasis in managed dolphins compared with free-ranging dolphins. In this study, urine samples were collected from 16 dolphins (8 cases, 8 controls) from the U.S. Navy Marine Mammal Program for the purposes of assessing changes in urinary biomarkers after a large meal. Urinary biomarkers and nephrolithiasis presence were assessed opportunistically in 15 long-term resident free-ranging dolphins living in Sarasota Bay, Florida. Additionally, the total purine contents of fish commonly consumed by each dolphin population were measured to evaluate potential dietary risk factors. Populations were compared for total dietary purine composition, recently fed status, nephrolithiasis presence, and differences in urinary biochemical, acid-base, and physicochemical parameters via Wilcoxon rank sum analysis and least square means. Managed dolphins had higher urinary pH and ammonium ([Formula: see text]) in both pre- and postprandial conditions and higher urinary uric acid and saturation indices of NH4U in the postprandial condition compared with free-ranging dolphins ( P < 0.05). The purine content was greater ( P < 0.0001) in the diet consumed by managed dolphins [7 mmol/Mcal metabolizable energy (ME)] than in the free-ranging dolphin diet (4 mmol/Mcal ME). Free-ranging dolphins did not show evidence of nephrolithiasis. Observed differences in urinary biomarkers and dietary purine content in these two dolphin populations suggest a pathophysiologic basis for the role of fish types on the risk of NH4U stone formation. Future research should investigate fish type and feeding frequency, inhibitors and promoters, and alkalinizing therapy for reducing NH4U nephrolithiasis in dolphins.

Evaluation of a genus-specific ELISA and a commercial Aspergillus Western blot IgG® immunoblot kit for the diagnosis of aspergillosis in common bottlenose dolphins (Tursiops truncatus).

Aspergillosis is a fungal infection with high mortality and morbidity rates. As in humans, its definitive diagnosis is difficult in animals, and thus new laboratory tools are required to overcome the diagnostic limitations due to low specificity and lack of standardization. In this study of common bottlenose dolphins (Tursiops truncatus), we evaluated the diagnostic performance of a new commercial immunoblot kit that had been initially developed for the serologic diagnosis of chronic aspergillosis in humans. Using this in a quantitative approach, we first established its positive cutoff within an observation cohort of 32 serum samples from dolphins with "proven" or "probable" diagnosis of aspergillosis and 55 negative controls. A novel enzyme-linked immunosorbent assay (ELISA) test was also developed for detecting anti-Aspergillus antibodies, and results were compared between the two assays. Overall, the diagnostic performance of immunoblot and ELISA were strongly correlated (P < .0001). The former showed lower sensitivity (65.6% versus 90.6%), but higher specificity (92.7% vs. 69.1%), with no cross-reaction with other fungal infections caused by miscellaneous non-Aspergillus genera. When assessing their use in a validation cohort, the immunoblot kit and the ELISA enabled positive diagnosis before mycological cultures in 42.9% and 33.3% subjects addressed for suspicion of aspergillosis, respectively. There was also significant impact of antifungal treatment on the results of the two tests (P < .05). In all, these new serological methods show promise in aiding in the diagnosis of aspergillosis in dolphins, and illustrate the opportunity to adapt commercial reagents directed for human diagnostics to detect similar changes in other animals.

Clinical management of Candida albicans keratomycosis in a bottlenose dolphin (Tursiops truncatus).

OBJECTIVE: Corneal ulceration secondary to trauma commonly affects marine mammals, often with opportunistic secondary bacterial or fungal infections. This report characterizes the combined use of auriculopalpebral and ophthalmic nerve blocks, adipose-derived stem cells, and subconjunctival injections for successful treatment of corneal trauma and infection in dolphins. ANIMAL STUDIED: An 11-year-old, female bottlenose dolphin (Tursiops truncatus) presented with bilateral diffuse corneal opacities, which progressed to keratomycosis caused by Candida albicans. PROCEDURE: Aggressive medical management was employed, including the use of subconjunctival injections of adipose-derived stem cells, plasma, topical and oral antifungals and antibiotics, and anti-inflammatory and pain medications. Anesthetic block of the auriculopalpebral and ophthalmic nerves was employed to evaluate the corneas. CONCLUSION: Subconjunctival injections were employed over 52 days, followed by topical drops for 5 months. At last evaluation, there was no evidence of blepharospasm bilaterally. Only a faint superficial gray corneal opacity remained OS. A temporal paraxial corneal opacity was present OD, with receding inactive vascularization and a small amount of melanosis temporally.

The Function of Twisted Gastrulation in Regulating Osteoclast Differentiation is Dependent on BMP Binding.

Proper regulation of osteoclast (OCL) function is critical for normal bone homeostasis. Bone morphogenetic protein (BMP) signaling and its regulation have been shown to have direct effects on OCL differentiation and activity. One of the major modulators of BMP signaling in the extracellular space is the secreted protein twisted gastrulation (TWSG1), which can inhibit BMP signaling and OCL differentiation. In this study we examine specific N-terminal regions of TWSG1 protein that have been previously proposed as BMP binding sites to determine whether TWSG1 binding to BMPs is required for its inhibitory effects on OCLs. We demonstrate that overexpression of wild type TWSG1 suppresses osteoclastogenesis, while overexpression of mutant TWSG1 proteins (W66A and N80Q/N146Q mutants), which cannot bind BMPs, leads to increased BMP signaling, enhanced osteoclastogenesis, increased resorptive activity, and expression of OCL-specific genes. Our results show that BMP binding is required for TWSG1-mediated inhibition of OCL formation and function, and validate the critical functional regions within the TWSG1 protein for these interactions.

Smad1/5 and Smad4 expression are important for osteoclast differentiation.

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP's effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity.

Protein kinase D promotes in vitro osteoclast differentiation and fusion.

Although PKD is broadly expressed and involved in numerous cellular processes, its function in osteoclasts has not been previously reported. In this study, we found that PKD2 is the main PKD isoform expressed in osteoclastic cells. PKD phosphorylation, indicative of the activated state, increased after 2-3 days of treatment of bone marrow macrophages with M-CSF and RANKL, corresponding to the onset of preosteoclast fusion. RNAi against PKD2 and treatment with the PKD inhibitor CID755673 showed that PKD activity is dispensable for induction of bone marrow macrophages into tartrate-resistant acid phosphatase-positive preosteoclasts in culture but is required for the transition from mononucleated preosteoclasts to multinucleated osteoclasts. Loss of PKD activity reduced expression of DC-STAMP in RANKL-stimulated cultures. Overexpression of DC-STAMP was sufficient to rescue treatment with CID755673 and restore fusion into multinucleated osteoclasts. From these data, we conclude that PKD activity promotes differentiation of osteoclast progenitors through increased expression of DC-STAMP.

Bone morphogenetic proteins signal via SMAD and mitogen-activated protein (MAP) kinase pathways at distinct times during osteoclastogenesis.

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII(fl/fl);lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.

Pathophysiological and physicochemical basis of ammonium urate stone formation in dolphins.

PURPOSE: Nephrolithiasis is increasingly reported in bottle-nosed dolphins. All cases to date have been ammonium urate nephrolithiasis. MATERIALS AND METHODS: A case-control study was performed in dolphins with and without evidence of nephrolithiasis to identify biomarkers and risk factors associated with stone formation in a managed population. Dolphins were sampled in fasting and postprandial states to study the effect of dietary factors on serum and urinary biochemistry. Urine was continuously collected for 6 hours via catheter and divided into 3, 2-hour collections with a bolus fish meal given after completing the first collection. Blood was sampled at the beginning of the fasting period and the end of the postprandial period. RESULTS: There were no significant differences in serum and urine chemistry or acid-base profiles between dolphins with vs without stones at baseline or postprandially. This suggests that cases and controls represent a continuum of stone risk. On analysis combining cases and controls in a single cohort we noted significant postprandial increases in urinary uric acid, sulfate and net acid excretion accompanied by increased urinary ammonium excretion and a commensurate increase in urine pH. The supersaturation index of ammonium urate increased more than twofold postprandially. CONCLUSIONS: These findings suggest that dolphins are susceptible to ammonium urate nephrolithiasis at least in part because a high dietary load of acid and purines results in a transient but marked increase in the urinary supersaturation of the sparingly soluble ammonium urate salt.

Pharmacokinetics of single dose oral meloxicam in bottlenose dolphins (Tursiops truncatus).

The objective of this study was to investigate the pharmacokinetics of meloxicam in bottlenose dolphins (Tursiops truncatus). Ten adult bottlenose dolphins were used for the study. Each animal received a single oral dose of meloxicam at 0.1 mg/kg. Two to seven serial blood samples were collected per animal, at one of fourteen time points between T = 0 and T = 240 hr. Complete blood count and serum chemistry analysis were performed prior to drug administration, as well as at the final time point for each individual. Plasma drug concentrations were determined by high-pressure liquid chromatography. No adverse hematological, biochemical or clinical changes were noted during the study period. After oral administration, a peak plasma concentration of 1.03 microg/mL was achieved at approximately 11 hr. This suggests that a single oral dose of 0.1 mg/kg provides a peak plasma level similar to what is considered therapeutic in other species. However, the elimination of meloxicam in cetaceans was slower than in other species, with an elimination half-life of almost 70 hr, and detectable drug concentrations up to 7 days. A single oral dose of 0.1 mg/kg appears safe for use in this species, but caution in repeated dosing must be used, due to the prolonged elimination, until multi-dose pharmacokinetic studies are determined.

Pre-anal gland abscess in a male Atlantic bottlenose dolphin (Tursiops truncatus).

OBJECTIVE: Anal glands have been identified in a variety of terrestrial and aquatic mammalian species, but there are few accounts describing their presence in cetaceans. To our knowledge, this report describes the first documented case of a pre-anal gland abscess in an Atlantic bottlenose dolphin (Tursiops truncatus). ANIMAL: A 9-year-old male bottlenose dolphin (T truncatus) part of the US Navy Marine Mammal Program in San Diego Bay, California. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: The patient presented for a 3-day history of lethargy, failure to perform voluntary behaviors, and an elevated respiratory rate. Complete blood count and serum biochemistry results showed an inflammatory hemogram. Physical examination revealed a 4-cm circular swelling at the right pre-anal gland pore. The swelling was warm and erythematous, with multifocal pinpoint ulcerations. An abscess of the pre-anal gland was diagnosed using cytology, culture, and ultrasound. TREATMENT AND OUTCOME: Treatment included systemic oral antibiotic and antifungal therapy, along with daily lavage and warm compress of the gland. Treatment was successful, and the abscess resolved. CLINICAL RELEVANCE: This case provides insight into a previously unreported disease process in bottlenose dolphins and encourages veterinarians to evaluate the pre-anal gland during routine physical examinations and complete further work-up if swelling or clinical signs associated with this region are present.

An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Assessing the potential health impacts of the 2003 and 2007 firestorms on bottlenose dolphins (Tursiops trucatus) in San Diego Bay.

CONTEXT: Firestorms negatively affected air quality throughout San Diego County during 2003 and 2007, including the San Diego Bay, which houses the Navy's bottlenose dolphins (Tursiops truncatus). OBJECTIVE: To assess the potential impact of the 2003 and 2007 fires on dolphin health. MATERIALS AND METHODS: Hematology and serum chemistry values were evaluated retrospectively among Navy dolphins the year and month before; during; and the month after the 2003 and 2007 fires. RESULTS: Both 2003 and 2007 fires were associated with lower calcium either during or the month post-fire compared to the control periods. During and the month following the 2003 fire, dolphins had higher serum carbon dioxide compared to the control periods. Dolphins during and the month following the 2007 fire had lower absolute or percent neutrophils and higher chloride. The 2007 fire was also associated with increased percent eosinophils during the fire and higher percent monocytes and bilirubin the month following the fire compared to the control periods. DISCUSSION AND CONCLUSION: Consistent with what has been previously reported in humans and other animals, this study supports that fire smoke inhalation may have mild effects on dolphin physiology, including calcium homeostasis, lung function and immune response.

Diagnosis and successful treatment of a lung abscess associated with Brucella species infection in a bottlenose dolphin (Tursiops truncatus).

This brief communication describes the clinical presentation, antemortem diagnosis, and successful treatment of a pulmonary abscess associated with a Brucella sp. in a 27-yr-old female bottlenose dolphin (Tursiops truncatus). Ultrasound revealed a 3-cm diameter hypoechoic mass deep to the pleural lining in the left lung field. Multiple ultrasound-guided fine-needle aspirates were performed and tested for bacterial and fungal etiology. All cultures were negative, but the infectious agent was identified by MicroSEQ analysis in two samples and confirmed with real-time polymerase chain reaction (PCR) amplification using known Brucella sp. primers. Amikacin was infused into the abscess and was followed by an oral doxycycline and rifampin protocol. Follow-up diagnostic imaging, including radiographs and computed tomography, revealed a resolved lesion with minimal mineralization within the affected lung fields. Brucellosis should be considered for pulmonary disease in dolphins, and personnel who interact with marine animals should use caution to prevent zoonotic brucellosis.