In 2023, an unprecedented outbreak of highly pathogenic avian influenza (HPAI) H5N1 resulted in the death of thousands of pinnipeds along the Argentinean coast, raising concerns about its ecological and epidemiological impact. Here, we present clinical, pathological, and molecular findings associated with HPAI H5N1 infection in pinnipeds from Chubut, Argentina. Necropsies were conducted on three South American Sea Lions (SASLs) ( Otaria flavescens ) and one Southern Elephant Seal (SES) ( Mirounga leonina ), followed by histopathological, immunohistochemical and RT-sqPCR analyses. Neurological clinical signs were observed in two SASLs, with one also exhibiting respiratory distress. Neuropathological findings included lymphoneutrophilic meningoencephalomyelitis and choroiditis, neuronal necrosis, gliosis, hemorrhages, and perivascular cuffing. Viral antigen was localized in neurons, glial cells, choroid plexus epithelial cells, ependymal cells, and the neuropil. Systemic manifestations included HPAI-related necrotizing myocarditis in the elephant seal and placental necrosis in a sea lion, with fetal tissues testing positive for HPAIV. Pulmonary lesions were minimal, limited to bronchial glands in one individual. RT-sqPCR confirmed HPAI H5 in all tested animals. Our findings highlight the neurotropism of HPAI H5N1 in pinnipeds, and expand the known systemic effects of the virus, revealing new tissue tropism and vertical transmission.

The pituitary gland regulates essential physiological processes in mammals. Despite its importance, research on its anatomy and ultrastructure in dolphins remains scarce. Using non-invasive imaging technology (MRI) and a novel skull-opening and dissection protocol, this study characterizes the dolphin pituitary through immunohistochemistry (IHC) and transmission electron microscopy (TEM). A total of 47 pituitaries were collected from stranded common bottlenose dolphins (Tursiops truncatus). common dolphins (Delphinus delphis), and Atlantic spotted dolphins (Stenella frontalis). as well as from captive common bottlenose dolphins. MRI allowed visualization of the gland’s anatomy and its spatial relationship with the hypothalamus and surrounding structures. A modified skull-opening and pituitary extraction protocol ensured the preservation of the adenohypophysis and neurohypophysis for detailed analysis. Histological, immunohistochemical, and ultrastructural studies confirmed the gland’s structural organization, identifying eight distinct adenohypophyseal cell types: corticotrophs (ACTH), somatotrophs (GH), gonadotrophs (FSH and LH), lactotrophs (LTH), melanotrophs (MSH), thyrotrophs (TSH), follicular cells, and capsular cells. This study presents the first immunolabelling of thyrotrophs in cetacean adenohypophysis and the first detailed ultrastructural characterization of adenohypophyseal cells in cetaceans, providing baseline data for future research. By integrating multidisciplinary techniques, it advances the understanding of dolphin neuroendocrinology and highlights broader implications for cetacean health, welfare, and conservation.

The pituitary gland is central to endocrine regulation in vertebrates, coordinating key physiological processes such as growth, reproduction, and stress responses. In cetaceans, and particularly in large odontocetes like orcas (Orcinus orca), understanding pituitary structure is essential for advancing neuroendocrine research and informing welfare and health assessments. Despite their ecological, cognitive, and conservation significance, detailed morphological studies of the orca pituitary gland remain scarce. In this study, we conducted a comprehensive structural and ultrastructural analysis of the orca pituitary gland using postmortem samples from four captive individuals. We combined computed tomography, histology, immunohistochemistry, and transmission electron microscopy to examine the gland’s anatomical organization and cellular composition. Our results reveal features consistent with other cetaceans as well as species-specific characteristics, including the distribution and morphology of endocrine cells within the adenohypophysis and neurohypophysis. These findings provide the first integrated anatomical and ultrastructural reference for the orca pituitary gland, offering valuable insights into cetacean neuroendocrinology and supporting improved species-specific welfare evaluation, health monitoring, and management practices for orcas under human care.

The adrenal glands are central regulators of endocrine function and stress physiology, yet detailed species-specific anatomical baselines remain limited in cetaceans. This study provides a comprehensive gross, histological, morphometric, and ultrastructural characterization of the adrenal glands in 55 short-beaked common dolphins (Delphinus delphis) examined postmortem in the Canary Islands. Adrenal glands were evaluated macroscopically and microscopically, and histological corticomedullary ratios were calculated from mid-transverse sections. Associations with body length, sexual maturity, and cause-of-death category were assessed statistically. Transmission electron microscopy was performed to characterize cortical and medullary cellular ultrastructure. Adrenal weight showed a positive correlation with body length. Histological corticomedullary ratio showed no lateral asymmetry but differed significantly between sexually immature and mature individuals, indicating ontogenetic remodeling of adrenal architecture. In contrast, corticomedullary ratio did not differ significantly between adult dolphins that died from acute events and those with more progressive pathological conditions. Ultrastructural analysis identified characteristic steroidogenic cortical cells and two chromaffin cell populations in the medulla. These findings establish the first integrated anatomical baseline for the adrenal gland in Delphinus delphis, providing essential reference data for comparative anatomy, veterinary pathology, and interpretation of endocrine-related findings in cetaceans.

In 2023, high pathogenicity avian influenza (HPAI) H5N1 outbreaks caused mass mortality events affecting pinnipeds throughout South America. Here, we present the clinical, pathological, immunohistochemical, and molecular findings associated with HPAI H5N1 infection in 3 South American sea lions ( Otaria flavescens ) and 1 southern elephant seal ( Mirounga leonina ) stranded during the outbreak in Chubut, Argentina. Neurological signs were observed in 2 sea lions, with 1 also exhibiting respiratory distress. Necropsies were conducted on all 4 animals. Neuropathology revealed mild-to-severe lymphohistiocytic (2/4) to mixed neutrophilic (2/4) meningoencephalitis with neuronal necrosis, neuronophagia, glial cell proliferation, multifocal hemorrhage, and perivascular cuffing in all animals. Novel findings for HPAI H5N1-infected pinnipeds included choroid plexitis (2/4) and myelitis (2/4). Immunohistochemistry for viral nucleoprotein was positive in 3 of 4 animals and involved neurons (3/3), glial cells (3/3), and ependymal cells (2/3). Viral-related systemic findings included multifocal necrotizing myocarditis in the elephant seal and multifocal necrotizing placentitis in a sea lion, with HPAI virus detected within fetal tissues. Pulmonary lesions were minimal and were limited to multifocal necrosis of bronchial glands in 1 sea lion. HPAI H5 clade 2.3.4.4b virus was confirmed by polymerase chain reaction and sequencing in all animals. These findings indicate a predominance of central nervous system involvement in HPAI H5N1-infected pinnipeds and expand the recognized spectrum of virus-associated lesions, identifying novel tissue tropisms and transmission routes.

The adrenal glands are central regulators of endocrine function and stress physiology, yet detailed species-specific anatomical baselines remain limited in cetaceans. This study provides a comprehensive gross, histological, morphometric, and ultrastructural characterization of the adrenal glands in 55 short-beaked common dolphins (Delphinus delphis) examined postmortem in the Canary Islands. Adrenal glands were evaluated macroscopically and microscopically, and histological corticomedullary ratios were calculated from mid-transverse sections. Associations with body length, sexual maturity, and cause-of-death category were assessed statistically. Transmission electron microscopy was used to characterize cortical and medullary cellular ultrastructure. Adrenal weight showed a positive correlation with body length. The histological corticomedullary ratio showed no lateral asymmetry but differed significantly between sexually immature and mature individuals, indicating ontogenetic remodeling of adrenal architecture. In contrast, the corticomedullary ratio did not differ significantly between adult dolphins that died from acute events and those that died following more progressive pathological conditions. Ultrastructural analysis identified characteristic steroidogenic cortical cells and two distinct chromaffin cell populations in the medulla. These findings establish the first integrated anatomical baseline for the adrenal gland in Delphinus delphis, providing essential reference data for comparative anatomy, veterinary pathology, and the interpretation of endocrine-related findings in cetaceans.