Cetacean Intracytoplasmic Eosinophilic Globules: A Cytomorphological, Histological, Histochemical, Immunohistochemical, and Proteomic Characterization.

The nature, etiopathogenesis, and clinicopathologic relevance of the prevalent intracytoplasmic eosinophilic globules (IEGs) within hepatocytes of cetaceans are unknown. This study aims to evaluate the presence and characterize the IEGs in the hepatocytes of cetaceans using histochemical and immunohistochemical electron microscopy, Western blot, lectin histochemistry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry techniques. A total of 95/115 (83%) animals (16 species) exhibited histologically evident intracytoplasmic round to oval, single to multiple, hyaline eosinophilic globules within the hepatocytes. These globules were largely PAS-positive, diastase resistant, and were immunopositive for fibrinogen (FB, 97%), albumin (Alb, 85%), and α1-antitrypsine (A1AT, 53%). The IEG positivity for FB and A1AT were correlated with live-stranding, hepatic congestion and a good nutritional status. The cetaceans lacking IEGs were consistently dead stranded and had poor body conditions. The IEGs in 36 bycaught cetaceans were, all except one, FB-positive and A1AT-negative. The IEGs exhibited morphologic and compositional variations at the ultrastructural level, suggesting various stages of development and/or etiopathogenesis(es). The glycocalyx analysis suggested an FB- and A1AT-glycosylation pattern variability between cetaceans and other animals. The proteomic analyses confirmed an association between the IEGs and acute phase proteins, suggesting a relationship between acute stress (i.e., bycatch), disease, and cellular protective mechanisms, allowing pathologists to correlate this morphological change using the acute hepatocytic cell response under certain stress conditions.

Molecular Characterization of Herpesviral Encephalitis in Cetaceans: Correlation with Histopathological and Immunohistochemical Findings.

Herpesviruses are causative agents of meningitis and encephalitis in cetaceans, which are among the main leading known natural causes of death in these species. Brain samples from 103 stranded cetaceans were retrospectively screened for the presence of herpesvirus DNA in the brain. Molecular detection of Cetacean Morbillivirus was performed in HV positive brain cases. Histopathologic evaluation of brain samples included the presence or absence of the following findings (n = 7): meningitis, perivascular cuffings, microgliosis, intranuclear inclusion bodies, malacia, neuronal necrosis and neurophagic nodules, and haemorrhages. Histological evidence of the involvement of other etiological agents led to complementary analysis. We detected the presence of alpha and gamma-HVs in 12 out of 103 (11.6%) brain samples from stranded cetaceans of five different species: one bottlenose dolphin, six striped dolphins, three Atlantic spotted dolphins, one Cuvier's beaked whale, and one common dolphin. Pathogenic factors such as viral strain, age, sex, and the presence of co-infections were analysed and correlated with the brain histopathological findings in each case. Herpesvirus was more prevalent in males, juveniles, and calves and a 41.6% incidence of co-infections in the brain was detected in our study: three with Dolphin Morbillivirus, one with Staphilococcus aureus septicaemia and one with Brucella spp.

Microscopic Findings in the Cardiac Muscle of Stranded Extreme Deep-Diving Cuvier's Beaked Whales (Ziphius cavirostris).

Considerable information has been gained over the last few decades on several disease processes afflicting free-ranging cetaceans from a pathologist's point of view. Nonetheless, there is still a dearth of studies on the hearts of these species. For this reason, we aimed to improve our understanding of cardiac histological lesions occurring in free-ranging stranded cetaceans and, more specifically, in deep-diving Cuvier's beaked whales. The primary cardiac lesions that have been described include vascular changes, such as congestion, edema, hemorrhage, leukocytosis, and intravascular coagulation; acute degenerative changes, which consist of contraction band necrosis, wavy fibers, cytoplasmic hypereosinophilia, and perinuclear vacuolization; infiltration of inflammatory cells; and finally, the presence and/or deposition of different substances, such as interstitial myoglobin globules, lipofuscin pigment, polysaccharide complexes, and intra- and/or extravascular gas emboli and vessel dilation. This study advances our current knowledge about the histopathological findings in the cardiac muscle of cetaceans, and more specifically, of Cuvier's beaked whales.

Myoglobin Concentration and Oxygen Stores in Different Functional Muscle Groups from Three Small Cetacean Species.

Compared with terrestrial mammals, marine mammals possess increased muscle myoglobin concentrations (Mb concentration, g Mb · 100g-1 muscle), enhancing their onboard oxygen (O2) stores and their aerobic dive limit. Although myoglobin is not homogeneously distributed, cetacean muscle O2 stores have been often determined by measuring Mb concentration from a single muscle sample (longissimus dorsi) and multiplying that value by the animal's locomotor muscle or total muscle mass. This study serves to determine the accuracy of previous cetacean muscle O2 stores calculations. For that, body muscles from three delphinid species: Delphinus delphis, Stenella coeruleoalba, and Stenella frontalis, were dissected and weighed. Mb concentration was calculated from six muscles/muscle groups (epaxial, hypaxial and rectus abdominis; mastohumeralis; sternohyoideus; and dorsal scalenus), each representative of different functional groups (locomotion powering swimming, pectoral fin movement, feeding and respiration, respectively). Results demonstrated that the Mb concentration was heterogeneously distributed, being significantly higher in locomotor muscles. Locomotor muscles were the major contributors to total muscle O2 stores (mean 92.8%) due to their high Mb concentration and large muscle masses. Compared to this method, previous studies assuming homogenous Mb concentration distribution likely underestimated total muscle O2 stores by 10% when only considering locomotor muscles and overestimated them by 13% when total muscle mass was considered.

Histopathological Differential Diagnosis of Meningoencephalitis in Cetaceans: Morbillivirus, Herpesvirus, Toxoplasma gondii, Brucella sp., and Nasitrema sp.

Infectious and inflammatory processes are among the most common causes of central nervous system involvement in stranded cetaceans. Meningitis and encephalitis are among the leading known natural causes of death in stranded cetaceans and may be caused by a wide range of pathogens. This study describes histopathological findings in post-mortem brain tissue specimens from stranded cetaceans associated with five relevant infectious agents: viruses [Cetacean Morbillivirus (CeMV) and Herpesvirus (HV); n = 29], bacteria (Brucella sp.; n = 7), protozoa (Toxoplasma gondii; n = 6), and helminths (Nasitrema sp.; n = 1). Aetiological diagnosis was established by molecular methods. Histopathologic evaluations of brain samples were performed in all the cases, and additional histochemical and/or immunohistochemical stains were carried out accordingly. Compared with those produced by other types of pathogens in our study, the characteristic features of viral meningoencephalitis (CeMV and HV) included the most severe and frequent presence of malacia, intranuclear, and/or intracytoplasmic inclusion bodies, neuronal necrosis and associated neuronophagia, syncytia and hemorrhages, predominantly in the cerebrum. The characteristic features of Brucella sp. meningoencephalitis included the most severe and frequent presence of meningitis, perivascular cuffing, cerebellitis, myelitis, polyradiculoneuritis, choroiditis, ventriculitis, vasculitis, and fibrinoid necrosis of vessels. The characteristic features of T. gondii meningoencephalitis included lymphocytic and granulomatous encephalitis, tissue cysts, microgliosis, and oedema. In the case of Nasitrema sp. infection, lesions are all that we describe since just one animal was available. The results of this study are expected to contribute, to a large extent, to a better understanding of brain-pathogen-associated lesions in cetaceans.

Retrospective Study of Fishery Interactions in Stranded Cetaceans, Canary Islands.

Estimating cetacean interactions with fishery activities is challenging. Bycatch and chronic entanglements are responsible for thousands of cetacean deaths per year globally. This study represents the first systematic approach to the postmortem investigation of fishery interactions in stranded cetaceans in the Canary Islands. We retrospectively studied 586 cases necropsied between January 2000 and December 2018. Of the cases with a known cause of death, 7.4% (32/453) were due to fishery interactions, and the Atlantic spotted dolphin (Stenella frontalis) was the most affected species [46.9% (15/32)]. Three types of fishery interactions were recognized by gross findings: bycatch [65.6% (21/32)], chronic entanglements [18.8% (6/32)], and fishermen aggression [15.6% (5/32)]. Among the bycaught cases, we differentiated the dolphins that died because of ingestion of longline hooks [23.8% (5/21)] from those that died because of fishing net entrapments [76.2% (16/21)], including dolphins that presumably died at depth due to peracute underwater entrapment (PUE) [37.5% (6/16)], dolphins that were hauled out alive and suffered additional trauma during handling [43.8% (7/16)], and those that were released alive but became stranded and died because of fishery interactions [18.7% (3/16)]. Gross and histologic findings of animals in each group were presented and compared. The histological approach confirmed gross lesions and excluded other possible causes of death. Cetaceans in good-fair body condition and shallow diving species were significantly more affected by fishery interactions, in agreement with the literature. Low rates of fishery interactions have been described, compared with other regions. However, within the last few years, sightings of entangled live whales, especially the minke whale (Balaenoptera acutorostrata) and Bryde's whale (B. edeni), have increased. This study contributes to further improvement of the evaluation of different types of fishery interactions and may facilitate the enforcement of future conservation policies to preserve cetacean populations in the Canary Islands.

Comparison of Methods for the Histological Evaluation of Odontocete Spiral Ganglion Cells.

Cetaceans greatly depend on their hearing system to perform many vital activities. The spiral ganglion is an essential component of the auditory pathway and can even be associated with injuries caused by anthropogenic noise. However, its anatomical location, characterized by surrounding bony structures, makes the anatomical and anatomopathological study of the spiral ganglion a difficult task. In order to obtain high-quality tissue samples, a perfect balance between decalcification and the preservation of neural components must be achieved. In this study, different methodologies for spiral ganglion sample preparation and preservation were evaluated. Hydrochloric acid had the shortest decalcification time but damaged the tissue extensively. Both formic acid and EDTA decalcification solutions had a longer decalcification time but exhibited better preservation of the neurons. However, improved cell morphology and staining were observed on ears pretreated with EDTA solution. Therefore, we suggest that decalcifying methodologies based on EDTA solutions should be used to obtain the highest quality samples for studying cell morphology and antigenicity in cetacean spiral ganglion neurons.

Increased Plasma Cardiac Troponin I in Live-Stranded Cetaceans: Correlation with Pathological Findings of Acute Cardiac Injury.

Capture myopathy (CM), is a syndrome that occurs as the result of the stress during and after capture, handling, restraint, and transport of wild animals. Although CM has been described for many species of cetaceans, characterization of the acute cardiac injury - an important component of this syndrome - are still scarce. In this study, we firstly estimated a normal range for cardiac troponin I (cTnI) on cetaceans. Here, through biochemical analysis (especially of cTnI) and histopathological, histochemical, and immunohistochemical correlations with decreased troponin immunolabelling, we studied the cardiac injury in live-stranded cetaceans. Nine cetaceans which stranded alive on the Canary Islands (January 2016 - June 2019) were included in this study. Sampled individuals presented elevated values of plasma cTnI, which were correlated to histopathological lesions comprised of vascular changes and acute degenerative lesions. Immunohistochemically, injured cardiomyocytes showed a decreased intrafibrillar troponin immunoreaction. This is the first attempt to establish a normal baseline range for cTnI in cetaceans, and the first study comparing plasma biomarkers values with histopathological and immunohistochemical findings. This approach allowed us to demonstrate the degree of cardiac damage as a result of injury, consistent with ischemia-reperfusion lesions. The knowledge gained here could improve decision-making procedures during stressful situations, mainly in live-strandings, handling, and rehabilitation, thereby reducing the mortality of cetaceans.

Capture Myopathy and Stress Cardiomyopathy in a Live-Stranded Risso's Dolphin (Grampus griseus) in Rehabilitation.

Capture myopathy (CM) is described in wild animals as a metabolic syndrome resulting from the extreme stress suffered during and after capture, handling, restraint, and transport. Although CM has been characterized in many species of cetaceans, descriptions of cardiac injury-an important component of this syndrome, and, according to previous authors, comparable to the existing human pathology so-called stress cardiomyopathy (SCMP)-are still rare. Therefore, the main aim of this report is to illustrate, for the first time, the biochemical analysis, and gross, histopathological, histochemical and immunohistochemical features of CM, and more specifically of the SCMP involved in this syndrome, caused by the live-stranding and consequent rehabilitation attempt, for a certain period of time, in a juvenile male Risso's dolphin (Grampus griseus). The animal presented elevated values of creatine kinase, cardiac troponin I and blood urea nitrogen, with some variations during the rehabilitation period. Histologically, we detected vascular changes and acute degenerative lesions analogous to the ones observed in humans with SCMP. We consider this study to be an important contribution to the study of cetaceans since it could help in decision-making and treatment procedures during live-strandings and improve conservation efforts by reducing the mortality of these animals.

Stress cardiomyopathy in stranded cetaceans: a histological, histochemical and immunohistochemical study.

BACKGROUND: Free-living cetaceans are exposed to a wide variety of stressful situations, including live stranding and interaction with human beings (capture myopathy), vessel strikes, and fishing activities (bycatch), which affect their wellbeing and potentially lead to stress cardiomyopathy (SCMP). METHODS: Here, the authors aimed to characterise SCMP of stranded cetaceans as an injury resulting from extreme stress responses, based on pathological analyses (histological, histochemical and immunohistochemical). Specifically, the authors examined heart samples from 67 cetaceans found ashore (48 live strandings, seven dead from ship collision and 12 dead from bycatch) on the coast of Spain, more specifically in the Canary Islands from 2000 to 2016 and Andalusia from 2011 to 2014. RESULTS: The microscopic findings were characterised by vascular changes, acute or subacute cardiac degenerative necrotic lesions, interstitial myoglobin globules, and infiltration of inflammatory cells. Immunohistochemically, cardiac troponin I, cardiac troponin C and myoglobin were depleted, along with fibrinogen being expressed in the degenerated/necrotic cardiomyocytes. A perivascular pattern was also identified and described in the damaged cardiomyocytes. CONCLUSIONS: This study advances current knowledge about the pathologies of cetaceans and their implications on conserving this group of animals by reducing mortality and enhancing their treatment and subsequent rehabilitation to the marine environment.

Skeletal and Cardiac Rhabdomyolysis in a Live-Stranded Neonatal Bryde's Whale With Fetal Distress.

The main objective of wildlife forensic investigation is to recognize pathologic changes and cause of death. Even though it may not always be possible to determine the specific illness and/or etiology, the description and subsequent interpretation of the injuries provide an invaluable understanding of pathology in cetacean post-mortem investigations. Although pathological studies have been previously reported in various cetacean species, such descriptions of the infraorder Mysticeti remain rare. A live-stranded neonatal Bryde's whale (Balaenoptera edeni) which subsequently died soon after the stranding, was assessed by physical exam, blood examination, gross necropsy evaluation, histopathology, and immunohistochemistry. It presented with elevated serum levels of creatine kinase, cardiac troponin I, urea, and creatinine. Microscopically, we observed keratin spicules (squamous epithelial cells) and areas of atelectasis in the lungs. Acute degeneration in the myocytes and cardiomyocytes were comparable to the findings previously described in cases of capture myopathy in live-stranded cetaceans. Immunohistochemistry biomarkers such as myoglobin, fibrinogen, and troponin were analyzed. Skeletal and myocardial damage has been documented in several cetacean species. However, this is the first reported case of skeletal and cardiac rhabdomyolysis associated with live-stranding in a newborn Bryde's whale that suffered from fetal distress.

Morbillivirus infection in Risso's dolphin Grampus griseus: a phylogenetic and pathological study of cases from the Canary Islands.

The earliest evidence of cetacean morbillivirus (CeMV) infection dates from 1982, when the dolphin morbillivirus strain (DMV) was identified in bottlenose dolphins Tursiops truncatus stranded in the mid-Atlantic region. Since then, CeMV has been detected globally in at least 26 species of mysticetes and odontocetes, causing widespread mortality and a wide range of pathological effects. In the Canary Islands, DMV and pilot whale morbillivirus have been detected in cetacean species, including short-finned pilot whales Globicephala macrorhynchus and bottlenose dolphins. Risso's dolphins Grampus griseus have been reported year-round in waters of the Canary Islands and are considered a resident species. No information is currently available on CeMV prevalence in this species in this ocean region. We searched for evidence of CeMV infection in 12 Risso's dolphins stranded in the Canary Islands from 2003 to 2015 by means of histopathology, PCR and immunohistochemistry. PCR revealed 2 CeMV-positive animals (16.6%). Phylogenetic analysis showed that the strains from the 2 positive specimens were phylogenetically quite distant, proving that more than 1 strain infects the Risso's dolphin population in this region. We also determined that the strain detected in one of the specimens mainly circulated in the northeastern Atlantic Ocean from 2007 to 2013.

Prostatic Lesions in Odontocete Cetaceans.

The prostate is the only accessory male genital gland described in cetaceans. Although few studies describe the gross and histologic anatomy of the prostate in cetaceans, there is no information on pathological findings involving this organ. The prostate glands of 45 cetaceans, including 8 different odontocete species ( n = 44) and 1 mysticete, were evaluated. The main pathologic diagnoses were verminous prostatitis, septic prostatitis, viral prostatitis, benign prostatic hyperplasia, and prostatitis of unknown etiology. Verminous prostatitis ( n = 12) was caused by nematodes of the genus Crassicauda, and different presentations were observed. Septic prostatitis, identified in 2 cases, both involved nematode infestation and Clostridium spp coinfection. One case of viral prostatitis was identified and was associated with morbillivirus infection. In prostatitis of unknown cause ( n = 7), varying degrees of prostatic lesions, mostly chronic inflammation, were identified. Impacts at individual levels (eg, localized disease, loss of reproductive capacity) and population levels (eg, decreased reproductive success) are plausible. Our results indicate a high occurrence of prostatic lesions in free-ranging odontocetes. For this reason, the prostate should be routinely inspected and sampled during necropsy of odontocete cetaceans.

Pulmonary carcinoma with metastasis in a long-finned pilot whale (Globicephala melas).

BACKGROUND: Lung cancer is the most commonly diagnosed neoplasm in humans, however this does not apply to other animal species. Living in an aquatic environment the respiratory system of cetaceans had to undergo unique adaptations in order to them to survive and cope with totally different respiratory pathogens and potentially carcinogens from those affecting humans. CASE PRESENTATION: This article discusses not only macroscopical, histopathological and immunohistochemical features of a pulmonary carcinoma with disseminated metastases in a long-finned pilot whale (Globicephala melas), as well as the immunohistochemical analysis performed on various tissues of cetaceans belonging to the genus Globicephala. On the necropsy examination of the carcass, multiple pulmonary nodules and generalised thoracic lymphadenomegaly were noted. Histologically, a malignant epithelial neoplasia was identified in the lung, thoracic lymph nodes, and adrenal gland. Immunohistochemical analysis revealed a pulmonary carcinoma. Vasculogenic mimicry and epithelial-to-mesenchymal transition phenotype, as suggested by cytomorphological and immunohistochemical characteristics, were observed. CONCLUSIONS: A diagnosis of metastatic pulmonary carcinoma was determined, which to the author's knowledge, appears to be not previously recorded in long-finned pilot whale species. This is also the first report of vasculogenic mimicry and epithelial-to-mesenchymal transition event in a spontaneous cancer from a cetacean species.