Pharmacokinetics of the analgesic and anti-inflammatory drug meloxicam after administration of multiple doses to nursehound sharks (Scyliorhinus stellaris).

OBJECTIVE: To determine the pharmacokinetics of meloxicam in the nursehound shark (Scyliorhinus stellaris) during multiple dose administration. STUDY DESIGN: Prospective experimental trial. ANIMALS: A total of eight clinically healthy adult nursehounds (four males, four females). METHODS: Meloxicam was administered intramuscularly at a dose of 1.5 mg kg-1 once daily for 7 days. Blood samples were collected from the caudal vein for pharmacokinetic analysis at 2.5 hours and 24 hours after drug administration. After a 4 week washout period, meloxicam was administered orally at the same dose at 12 hour intervals for three repeated doses. Blood samples were collected at 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours after the first administration. Sharks were visually monitored during each study and 4 weeks afterwards for side effects or signs of toxicity. Time required to achieve steady state was assessed by visual inspection and statistical comparison of peak and trough concentrations using a Friedman test; comparison between sexes was performed using a Mann-Whitney U test and p-value was set at 0.05. RESULTS: No animal died or showed clinical signs of toxicity during the study. Meloxicam administered orally did not produce detectable concentrations in plasma. After intramuscular administration, steady state was achieved after five doses, and mean trough and peak plasma concentrations at steady state were 1.76 ± 0.21 µg mL-1 and 3.02 ± 0.23 µg mL-1, respectively. Mean peak concentration accumulation ratio was 2.50 ± 0.22. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that intramuscular posology produces plasma concentrations considered therapeutic for other species. However, meloxicam was not detected in plasma after oral administration. These results suggest that meloxicam administered intramuscularly may be a useful non-steroid anti-inflammatory drug in nursehound sharks. Further pharmacodynamic studies are needed to fully evaluate its clinical use in this species.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus).

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+ , using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+ -sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

First description of a lesion in the upper digestive mucosa associated with a novel gammaherpesvirus in a striped dolphin (Stenella coeruleoalba) stranded in the Western Mediterranean Sea.

BACKGROUND: A wide variety of lesions have been associated with herpesvirus in cetaceans. However, descriptions of herpesvirus infections in the digestive system of cetaceans are scarce. CASE REPORT: A young female striped dolphin stranded in the Valencian Community (Spain) on the 6th August 2021. The animal showed external macroscopic lesions suggestive of an aggressive interaction with bottlenose dolphins (rake marks in the epidermis). Internally, the main findings included congestion of the central nervous system and multiple, well-defined, whitish, irregularly shaped, proliferative lesions on the oropharyngeal and laryngopharyngeal mucosa. Histopathology revealed lymphoplasmacytic and histiocytic meningoencephalitis, consistent with neuro brucellosis. The oropharyngeal and laryngopharyngeal plaques were comprised histologically of focally extensive epithelial hyperplasia. As part of the health surveillance program tissue samples were tested for cetacean morbillivirus using a real-time reverse transcription-PCR, for Brucella spp. using a real-time PCR, and for herpesvirus using a conventional nested PCR. All samples were negative for cetacean morbillivirus; molecular positivity for Brucella spp. was obtained in pharyngeal tonsils and cerebrospinal fluid; herpesvirus was detected in a proliferative lesion in the upper digestive mucosa. Phylogenetic analysis showed that the herpesvirus sequence was included in the Gammaherpesvirinae subfamily. This novel sequence showed the greatest identity with other Herpesvirus sequences detected in skin, pharyngeal and genital lesions in five different species. CONCLUSIONS: To the best of the authors' knowledge, this is the first report of a proliferative lesion in the upper digestive mucosa associated with gammaherpesvirus posititvity in a striped dolphin (Stenella coeruleoalba).

Tension pneumothorax in small odontocetes.

Pneumothorax, the accumulation of air in the pleural cavity, occurs when air enters the pleural space by the pleuro-cutaneous, pleuro-pulmonary, or pleuro-oesophageal-mediastinal route. Tension pneumothorax is an infrequent and severe form of pneumothorax where a positive pressure in the pleural space is built up during at least part of the respiratory cycle, with compression of both lungs and mediastinal vessels, and, if unilateral, with midline deviation towards the unaffected hemithorax. We describe 9 cases of tension pneumothorax in 3 species of small cetaceans (striped dolphin Stenella coeruleoalba, common dolphin Delphinus delphis, and common bottlenose dolphin Tursiops truncatus) from the western Mediterranean coast of Spain, and one case from a dolphinarium. Computed tomography (CT) imaging performed in 2 carcasses before necropsy showed lung compression, midline deviation, and pressure on the diaphragm, which was caudally displaced. Tension pneumothorax was recognized at necropsy by the presence of pressurized air in one of the hemithoraces. Seven of the pneumothorax cases were spontaneous (2 primary and 5 secondary to previous lung pathology). In the other 2 dolphins, the pneumothorax was traumatic, due to oesophageal-pleural perforation or rib fractures. We hypothesize that pneumothorax in dolphins is predominantly tensional because of their specific anatomical and physiological adaptations to marine life and the obligate exposure to extreme pressure changes as diving mammals.

Hepatitis E Virus Infections in Free-Ranging and Captive Cetaceans, Spain, 2011-2022.

Epidemiologic surveillance of hepatitis E virus in over 300 free-ranging and captive cetaceans in waters off Spain revealed extensive exposure to this pathogen. We suggest the persistent and widespread presence of hepatitis E in the marine environment off the coast of Spain may be driven by terrestrial sources of contamination.

Absence of atrial smooth muscle in the heart of the loggerhead sea turtle (Caretta caretta): a re-evaluation of its role in diving physiology.

Contraction of atrial smooth muscle in the hearts of semi-aquatic emydid turtles regulates ventricular filling, and it has been proposed that it could regulate stroke volume during characteristic rapid transitions in cardiac output associated with diving. For this hypothesis to be supported, atrial smooth muscle should be widely distributed in diving Testudines. To further understand the putative function and evolutionary significance of endocardial smooth muscle in Testudines, we studied the hearts of loggerhead sea turtles, Caretta caretta (n=7), using immunohistochemistry and histology. Surprisingly, we found no evidence of prominent atrial smooth muscle in C. caretta. However, smooth muscle was readily identified in the sinus venosus. Our results suggest that atrial smooth muscle does not contribute to the diving capabilities of C. caretta, indicating that the possible roles of smooth muscle in emydid turtle hearts require a re-evaluation. In sea turtles, the sinus venosus may instead contribute to regulate cardiac filling.

Increasing the data on elasmobranch plasma protein electrophoresis: electrophoretogram reference values determination in the undulate skate (Raja Undulata) and the nursehound shark (Scyliorhinus stellaris) maintained under human care.

BACKGROUND: This study determined plasma protein electrophoresis (PPE) reference intervals in two elasmobranch species: the undulate skate (Raja undulata) and the nursehound shark (Scyliorhinus stellaris), using a reference population of 48 undulate skates (27 males, 21 females) and 62 nursehounds (32 males, 30 females), considered to be clinically healthy. Plasma samples were analyzed using capillary zone electrophoresis (CZE). RESULTS: The undulate skate electrophoretogram resembled those previously reported in other batoids and could be divided into seven consistent fractions. No statistically significant differences were detected between sexes and developmental stages. The nursehound electrophoretogram was similar to that previously described in other shark species and could be divided into eight consistent fractions. Fraction 5% was significantly higher in juvenile nursehounds when compared to adults, while fraction 6 concentration and percentage were significantly higher in adults. Fraction 4% was higher in males than in females. Albumin band was not detected, and pre-albumin was negligible in both studied species. Alpha-globulins were predominant in the undulate skate, while beta-globulins were predominant in nursehounds. Statistically significant differences were found in all electrophoretogram fraction percentages and concentrations between the two species. CONCLUSION: To the authors knowledge, this is the first study reporting PPE values in undulate skates and nursehounds, and the first study using CZE in elasmobranch plasma. These findings can serve as a primary reference for health monitoring in both species and will add to the limited data available on PPE in elasmobranchs.

PHARMACOKINETICS OF MELOXICAM AFTER A SINGLE 1.5 MG/KG INTRAMUSCULAR ADMINISTRATION TO NURSEHOUND SHARKS (SCYLIORHINUS STELLARIS) AND ITS EFFECTS ON HEMATOLOGY AND PLASMA BIOCHEMISTRY.

A single-dose meloxicam pharmacokinetic (PK) study was performed with eight clinically healthy nursehound sharks (Scyliorhinus stellaris) maintained under human care. Meloxicam was administered IM at a dosage of 1.5 mg/kg to six animals; two animals were administered elasmobranch physiological saline solution (EPSS) IM as a negative control group. Blood samples were obtained prior to and at 12 predetermined times during the first 36 h after administration. Effects on hematology and plasma biochemistry were compared prior to and 24 h after administration. No animal died or showed clinical signs during the study. A significant increase in creatinine kinase and aspartate aminotransferase was found in both EPSS and meloxicam groups and could be considered a direct consequence of sampling and handling required for the PK study. Observed mean time to maximum plasma concentration ± SEM was 2.58 ± 0.47 h and observed mean maximum plasma concentration ± SEM was 806 ± 66 ng/ml; mean terminal half-life ± SEM was 15.97 ± 1.20 h; mean residency time ± SEM was 23.40 ± 2.25 h. Area under the plasma concentration-versus-time curve extrapolated to infinity ± SEM was 15.52 ± 1.70 h·µg/ml. This study suggests that meloxicam 1.5 mg/kg IM in nursehound sharks is likely to result in clinically relevant plasma levels for periods of 24 h without producing significant alterations in blood analytics, although further PK studies with meloxicam IV in sharks are needed. Future PK and pharmacodynamic studies with different drugs and doses are needed in elasmobranchs to establish safe and effective treatment protocols.

HEMATOLOGY AND PLASMA BIOCHEMISTRY REFERENCE VALUES OF JUVENILE UNDULATE RAYS (RAJA UNDULATA) UNDER HUMAN CARE.

Despite the paucity of published literature on elasmobranch hematology and biochemistry, great interspecific diversity has been observed. Blood samples from 43 undulate rays (Raja undulata) (23 males, 20 females) hatched and raised at Oceanogràfic Aquarium, were analyzed for hematology and plasma biochemistry. Animals were divided into two age groups: 1 yr old (28 skates) and 2 yr old (15 skates). All individuals were clinically healthy on physical examination. Weight, total length, standard length, and disc width were recorded. No statistically significant differences were observed between male and female juvenile skates for the evaluated morphometric, hematologic, and plasma biochemical values. Once reference intervals (RI) were determined, blood samples from seven healthy adult skates housed at the same aquarium were collected for comparison. Statistically significant differences were observed in cholesterol, triglycerides, alkaline phosphatase, blood urea nitrogen, chloride, and sodium between juvenile and adult skates. This is the first article describing hematological and plasma biochemical RI for this species, increasing the clinical knowledge on elasmobranch blood analytics. These data will serve as a valuable diagnostic and research tool for professionals working with undulate rays and closer relatives in aquariums and in the field. Further studies using larger elasmobranch sample sizes are needed to determine reliable species-specific baseline health values and to evaluate the effect of intrinsic and extrinsic parameters on blood analytics more accurately.

Pathological findings and husbandry management in captive Chrysaora spp. medusae affected by umbrellar ulcerative lesions.

During a 12 month period, a group of 14 medusa-stage jellies of the genus Chrysaora, including Pacific sea nettle (Chrysaora fuscescens, n = 11) and Japanese sea nettle (Chrysaora pacifica, n = 3), that were maintained in a public aquarium developed progressive ulcerative umbrellar lesions. In 6 cases (42.9%), ulceration was deep, transmural, and perforated through the mesoglea and subumbrella. In 6 cases (42.9%), ciliated protozoa histomorphologically consistent with scuticociliates were observed in the mesoglea and gastrovascular cavity. In 2 cases (14.3%), commensal dinoflagellates (zooxanthellae) were in the mesoglea and in the cytoplasm of the scuticociliates. During this period, water quality parameters including temperature [°C], pH, oxidation-reduction potential (ORP) [mV], salinity [psu], dissolved oxygen [%], ammonia (NH3), and nitrite(NO2) levels were monitored daily or weekly. The main water quality abnormalities were increased NO2 and pH levels above recommended reference ranges for C. fuscescens and elevated temperature above recommended reference ranges for C. pacifica tank. After correction of water quality parameters, apparent improvement of jellies was observed. In this case, environmental factors were considered the most likely predisposing factors for the development of ulcerative lesions, and ciliated protozoa were considered secondary rather than primary pathogens.

Pharmacokinetics of single dose oral Terbinafine in common shelducks (Tadorna tadorna).

Fungal disease is a major cause of morbidity and mortality in avian species; thus, antifungals are the treatment of choice. Despite widely used in clinical practice, terbinafine pharmacokinetic studies are scarce in literature and only cover some avian families, with marked differences between them. This study evaluates the pharmacokinetic behaviour of terbinafine after a single oral administration of 60 mg/kg in 7 healthy adult common shelducks (Tadorna tadorna) by measuring plasma concentrations through high-performance liquid chromatography (HPLC) at times 0, 30 min, 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 hr postadministration. Noncompartmental analyses of the data showed a Cmax (geometric mean) of 5.43 µg/ml, tmax (median) 1.0 hr and AUC0-∞ 29.70 mg h/L. Elimination half-life was 6.33 hr and MRT 6.61 hr. Plasma concentrations remained above previously described MIC for terbinafine in some fungal species for at least 6 to 8 hr. A single oral administration of 60 mg/kg terbinafine did not produce adverse effects and could be a good treatment choice for fungal diseases in anatids.

OPTIMIZING A PLATELET-RICH PLASMA CONCENTRATION PROTOCOL FOR SOUTH AMERICAN SEA LIONS (OTARIA FLAVESCENS).

Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.

RESPIRATORY CHANGES IN STRANDED BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS).

Lung function (breath duration, respiratory flow [V̇], and tidal volume [VT]), and end-expiratory O2 were measured in 19 adult bottlenose dolphins (Tursiops spp.) while at rest in water or beached for up to 10 min. The results show that inspiratory VT, expiratory VT, or inspiratory V̇ did not differ on land or in water. The average expiratory V̇ for all dolphins on land decreased by 16%, and the expiratory and total breath durations increased by 5% and 4%, respectively, compared with in water. There were temporal changes observed during beaching, where expired and inspired VT and inspired V̇ decreased by 13%, 16%, and 9%, respectively, after 10 min on land. These data suggest that dolphins compensate for the effect of gravity by adjusting respiration to maintain alveolar ventilation and gas exchange, but during extended durations, the increased work of breathing may impede ventilation and gas exchange. Continuous monitoring of lung function and gas exchange may help prevent long-term damage during out-of-water medical procedures, optimize animal transport conditions, and improve survival during stranding events.

Alpha- and gammaherpesviruses in stranded striped dolphins (Stenella coeruleoalba) from Spain: first molecular detection of gammaherpesvirus infection in central nervous system of odontocetes.

BACKGROUND: Herpesvirus infections in cetaceans have always been attributed to the Alphaherpesvirinae and Gammaherpesvirinae subfamilies. To date, gammaherpesviruses have not been reported in the central nervous system of odontocetes. CASE PRESENTATION: A mass stranding of 14 striped dolphins (Stenella coeruleoalba) occurred in Cantabria (Spain) on 18th May 2019. Tissue samples were collected and tested for herpesvirus using nested polymerase chain reaction (PCR), and for cetacean morbillivirus using reverse transcription-PCR. Cetacean morbillivirus was not detected in any of the animals, while gammaherpesvirus was detected in nine male and one female dolphins. Three of these males were coinfected by alphaherpesviruses. Alphaherpesvirus sequences were detected in the cerebrum, spinal cord and tracheobronchial lymph node, while gammaherpesvirus sequences were detected in the cerebrum, cerebellum, spinal cord, pharyngeal tonsils, mesenteric lymph node, tracheobronchial lymph node, lung, skin and penile mucosa. Macroscopic and histopathological post-mortem examinations did not unveil the potential cause of the mass stranding event or any evidence of severe infectious disease in the dolphins. The only observed lesions that may be associated with herpesvirus were three cases of balanitis and one penile papilloma. CONCLUSIONS: To the authors' knowledge, this is the first report of gammaherpesvirus infection in the central nervous system of odontocete cetaceans. This raises new questions for future studies about how gammaherpesviruses reach the central nervous system and how infection manifests clinically.

Gas embolism and massive blunt force trauma to sea turtles entrained in hopper dredges in North and South Carolina, USA.

Decompression sickness (DCS) has been described mainly in loggerhead turtles Caretta caretta bycaught in trawls and gillnets. Here we present cases of gas emboli (GE) in 8 green turtles Chelonia mydas and 2 Kemp's ridleys Lepidochelys kempii entrained in hopper dredges that were working at 8.8-15.2 m depths during shipping channel maintenance or beach renourishment activities. Turtle weights ranged from 2.2 to 6.7 kg. All were found alive with blunt force injuries from passage through the dredge and were taken to rehabilitation facilities. Four green turtles died or were euthanized within 24 h. Six turtles survived. Radiographic or ultrasonographic evidence of GE was detected in 4 turtles, including 3 mortalities. Computed tomography (CT) revealed perirenal and cervical GE in 4 turtles, including 1 mortality. No GE were detected in 2 of the survivors. Upon necropsy, GE were found in mesenteric vessels, the right atrium, and kidneys. Histopathology confirmed that tissues were in a good state of preservation without evidence of bacterial overgrowth or putrefactive gas formation. Death likely resulted primarily from massive tissue trauma from the dredge, but moderate GE could have led to DCS and complicated recovery. The surviving turtles weighed less than those that did not survive. Besides hypothesized stress/exercise-induced circulatory changes of blood through the lungs and pressure reduction of forced surfacing from depth, drastic pressure change within the dredge pipes before and after the pump could contribute to GE. Hopper dredge entrainment is an additional cause of GE and potential DCS in sea turtles.

Pulmonary ventilation-perfusion mismatch: a novel hypothesis for how diving vertebrates may avoid the bends.

Hydrostatic lung compression in diving marine mammals, with collapsing alveoli blocking gas exchange at depth, has been the main theoretical basis for limiting N2 uptake and avoiding gas emboli (GE) as they ascend. However, studies of beached and bycaught cetaceans and sea turtles imply that air-breathing marine vertebrates may, under unusual circumstances, develop GE that result in decompression sickness (DCS) symptoms. Theoretical modelling of tissue and blood gas dynamics of breath-hold divers suggests that changes in perfusion and blood flow distribution may also play a significant role. The results from the modelling work suggest that our current understanding of diving physiology in many species is poor, as the models predict blood and tissue N2 levels that would result in severe DCS symptoms (chokes, paralysis and death) in a large fraction of natural dive profiles. In this review, we combine published results from marine mammals and turtles to propose alternative mechanisms for how marine vertebrates control gas exchange in the lung, through management of the pulmonary distribution of alveolar ventilation ([Formula: see text]) and cardiac output/lung perfusion ([Formula: see text]), varying the level of [Formula: see text] in different regions of the lung. Man-made disturbances, causing stress, could alter the [Formula: see text] mismatch level in the lung, resulting in an abnormally elevated uptake of N2, increasing the risk for GE. Our hypothesis provides avenues for new areas of research, offers an explanation for how sonar exposure may alter physiology causing GE and provides a new mechanism for how air-breathing marine vertebrates usually avoid the diving-related problems observed in human divers.

Deciphering function of the pulmonary arterial sphincters in loggerhead sea turtles (Caretta caretta).

To provide new insight into the pathophysiological mechanisms underlying gas emboli (GE) in bycaught loggerhead sea turtles (Caretta caretta), we investigated the vasoactive characteristics of the pulmonary and systemic arteries, and the lung parenchyma (LP). Tissues were opportunistically excised from recently dead animals for in vitro studies of vasoactive responses to four different neurotransmitters: acetylcholine (ACh; parasympathetic), serotonin (5HT), adrenaline (Adr; sympathetic) and histamine. The significant amount of smooth muscle in the LP contracted in response to ACh, Adr and histamine. The intrapulmonary and systemic arteries contracted under both parasympathetic and sympathetic stimulation and when exposed to 5HT. However, proximal extrapulmonary arterial (PEPA) sections contracted in response to ACh and 5HT, whereas Adr caused relaxation. In sea turtles, the relaxation in the pulmonary artery was particularly pronounced at the level of the pulmonary artery sphincter (PASp), where the vessel wall was highly muscular. For comparison, we also studied tissue response in freshwater sliders turtles (Trachemys scripta elegans). Both PEPA and LP from freshwater sliders contracted in response to 5HT, ACh and also Adr. We propose that in sea turtles, the dive response (parasympathetic tone) constricts the PEPA, LP and PASp, causing a pulmonary shunt and limiting gas uptake at depth, which reduces the risk of GE during long and deep dives. Elevated sympathetic tone caused by forced submersion during entanglement with fishing gear increases the pulmonary blood flow causing an increase in N2 uptake, potentially leading to the formation of blood and tissue GE at the surface. These findings provide potential physiological and anatomical explanations on how these animals have evolved a cardiac shunt pattern that regulates gas exchange during deep and prolonged diving.

Genetic heterogeneity of dolphin morbilliviruses detected in the Spanish Mediterranean in inter-epizootic period.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the three dolphin morbillivirus (DMV) epizootics of 1990-1992, 2006-2008 and 2011 that affected Mediterranean striped dolphins (Stenella coeruleoalba). Systemic infection caused by DMV in the Mediterranean has been reported only during these outbreaks. RESULTS: We report the infection of five striped dolphins (Stenella coeruleoalba) stranded on the Spanish Mediterranean coast of Valencia after the last DMV outbreak that ended in 2011. Animal 1 stranded in late 2011 and Animal 2 in 2012. Systemic infection affecting all tissues was found based on histopathology and positive immunohistochemical and polymerase chain reaction positive results. Animal 3 stranded in 2014; molecular and immunohistochemical detection was positive only in the central nervous system. Animals 4 and 5 stranded in 2015, and DMV antigen was found in several tissues. Partial sequences of the DMV phosphoprotein (P), nucleoprotein (N), and hemagglutinin (H) genes were identical for Animals 2, 3, 4, and 5, and were remarkably different from those in Animal 1. The P sequence from Animal 1 was identical to that of the DMV strain that caused the epizootic of 2011 in the Spanish Mediterranean. The corresponding sequence from Animals 2-5 was identical to that from a striped dolphin stranded in 2011 on the Canary Islands and to six dolphins stranded in northeastern Atlantic of the Iberian Peninsula. CONCLUSIONS: These results suggest the existence of an endemic infection cycle among striped dolphins in the Mediterranean that may lead to occasional systemic disease presentations outside epizootic periods. This cycle involves multiple pathogenic viral strains, one of which may have originated in the Atlantic Ocean.

Respiratory function and mechanics in pinnipeds and cetaceans.

In this Review, we focus on the functional properties of the respiratory system of pinnipeds and cetaceans, and briefly summarize the underlying anatomy; in doing so, we provide an overview of what is currently known about their respiratory physiology and mechanics. While exposure to high pressure is a common challenge among breath-hold divers, there is a large variation in respiratory anatomy, function and capacity between species - how are these traits adapted to allow the animals to withstand the physiological challenges faced during dives? The ultra-deep diving feats of some marine mammals defy our current understanding of respiratory physiology and lung mechanics. These animals cope daily with lung compression, alveolar collapse, transient hyperoxia and extreme hypoxia. By improving our understanding of respiratory physiology under these conditions, we will be better able to define the physiological constraints imposed on these animals, and how these limitations may affect the survival of marine mammals in a changing environment. Many of the respiratory traits to survive exposure to an extreme environment may inspire novel treatments for a variety of respiratory problems in humans.

Fluorescent microbead-based immunoassay for anti-Erysipelothrix rhusiopathiae antibody detection in cetaceans.

A fluorescent microbead-based immunoassay (FMIA) for detection of anti-Erysipelothrix rhusiopathiae antibodies in pigs was adapted for use in cetaceans. The FMIA was validated and adjusted using serum samples from 10 vaccinated captive bottlenose dolphins Tursiops truncatus collected between 1 and 13 mo after immunization. The technique was then used to analyze specimens from 15 free-ranging cetaceans stranded alive on the Valencian Mediterranean coast between 2006 and 2014: 11 striped dolphins Stenella coeruleoalba, 3 Risso's dolphins Grampus griseus and 1 bottlenose dolphin Tursiops truncatus. One of these wild animals was confirmed to have died from E. rhusiopathiae septicemia, but no anti-E. rhusiopathiae antibodies were detected in its serum, pericardial fluid or milk samples. Another free-ranging individual, which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection, showed high fluorescence intensity similar to that measured in captive dolphins at 6-13 mo after vaccination. These results suggest that this animal underwent an E. rhusiopathiae infection several months before stranding. The findings in the present study suggest that FMIA can be useful for detecting anti-E. rhusiopathiae antibodies in cetaceans, and its application to free-ranging animals is particularly interesting because of the great value of these specimens. Furthermore, the FMIA can be multiplexed to allow the determination of up to 100 analytes per sample in a single well, thereby reducing the cost, time and sample volume needed.