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.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

SUPERFICIAL CERVICAL LYMPHADENITIS ASSOCIATED WITH STREPTOCOCCUS PHOCAE IN FIVE COMMON BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS): A CASE SERIES.

Between 2009 and 2018, five common bottlenose dolphins (Tursiops truncatus) at the US Navy Marine Mammal Program presented with superficial cervical lymphadenitis. Clinical findings included ultrasonographic evidence of cervical lymph node enlargement, severe leukocytosis, elevated erythrocyte sedimentation rates, and reduced serum iron. Three of the dolphins presented with clinicopathologic changes without presence of clinical signs, and the other two cases additionally presented with partial to complete anorexia, lethargy, and refusal to participate in training sessions. Ultrasound-guided fine needle aspiration or biopsy of the affected lymph nodes yielded Streptococcus phocae by PCR in all cases, and the organism was cultured in one of five cases. Animals were treated with a combination of enteral, parenteral, intralesional antimicrobial, or a combination of those therapies and supportive care. Time to resolution of clinical disease ranged between 62 and 188 days. To the authors' knowledge, this is the first report of Streptococcus phocae cervical lymphadenitis in cetaceans. Streptococcus phocae lymphadenitis should be a differential for cervical lymphadenopathy in this species, especially when associated with pronounced systemic inflammation and a history of potential exposure.

URINARY BLADDER TRANSITIONAL CELL CARCINOMA AND CARCINOMATOSIS IN A LINNAEUS'S TWO-TOED SLOTH, CHOLOEPUS DIDACTYLUS.

A 19-yr-old female Linnaeus's two-toed sloth (Choloepus didactylus) with a history of urinary incontinence, ascites, and behavioral changes was euthanized after diagnostic imaging revealed a large bladder mass. On gross necropsy, the sloth had a severely thickened bladder mucosa, partial urinary obstruction, and nonseptic exudate in the peritoneal cavity. Histopathology showed a malignant and highly invasive transitional cell (urothelial) carcinoma with transmural and intra-abdominal invasion and diffuse carcinomatosis. Immunohistochemistry for expression of pancytokeratin (AE1/AE3), cytokeratin 7 (CK7), cytokeratin 20 (CK20), and uroplakin III was performed to confirm the diagnosis of transitional cell carcinoma. Neoplastic cells had a strong cytoplasmic immunoreactivity with the antipancytokeratin antibody clone AE1/AE3, which was consistent with a neoplasm of epithelial origin. Neoplastic cells were negative for expression of CK20. This is the first detailed report describing the antemortem diagnosis of urinary bladder transitional cell carcinoma with carcinomatosis in a two-toed sloth.

Normal pre- and post-prandial bile acids and protein C values vary by age in harbor seal pups (Phoca vitulina richardsi) undergoing rehabilitation.

OBJECTIVE: To establish normal values for pre- and post-prandial bile acids and protein C in Pacific harbor seal (Phoca vitulina richardsi) pups. ANIMALS: 45 harbor seals undergoing rehabilitation at the Vancouver Aquarium Marine Mammal Rescue Centre, 0 to 16 weeks, and deemed healthy aside from malnutrition or maternal separation. PROCEDURES: Venous blood was collected from the intervertebral extradural sinus in fasted seals and again 2 hours after a fish meal. RESULTS: The reference interval (90% CL, confidence limit) for pre-prandial (fasting) bile acids was 17.2 µmol/L to 25.4 µmol/L, post-prandial bile acids were 36.9 µmol/L to 46.4 µmol/L, and protein C was 72.3% to 85.4%, across ages. For comparison between developmental ages, pups were grouped into 3 age classes: < 14 days, 5 to 8 weeks, and 10 to 16 weeks. Age affected pre- and post-prandial bile acids; pups < 14 days had significantly higher pre-prandial bile acids (36.0 µmol/L ± 16.5 µmol/L; P < .0001) than other age groups and pups 5 to 8 weeks had significantly higher post-prandial bile acids (50.4 µmol/L ± 21.9 µmol/L; P < .001). Protein C was also affected by age, with seals < 14 days having significantly lower values (mean, 51.8% ± 16.7%; P < .0001). CLINICAL RELEVANCE: This study established normal reference intervals for bile acids in harbor seal pups and offered a preliminary investigation into protein C in pinnipeds. The bile acid values from 0- to 16-week-old seal pups were well above established normal ranges for domestic species, highlighting the utility of age- and species-specific reference ranges. The values presented here and the differences across age classes will aid clinicians in accurately diagnosing hepatobiliary disease in harbor seal pups.

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.

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.