Beluga whale and bottlenose dolphin ACE2 proteins allow cell entry mediated by spike protein from three variants of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses infect numerous non-human species. Spillover of SARS-CoV-2 into novel animal reservoirs may present a danger to host individuals of these species, particularly worrisome in populations already endangered or threatened by extinction. In addition, emergence in new reservoirs could pose spillback threats to humans, especially in the form of virus variants that further mutate when infecting other animal hosts. Previous work suggests beluga whales (Delphinapterus leucas) and bottlenose dolphins (Tursiops truncatus) may be at risk owing to their formation of social groups, contact with humans, exposure to contaminated wastewater, and structure of their angiotensin-converting enzyme 2 (ACE2) proteins, which SARS-CoV-2 uses as a cellular receptor. We examined marine-mammal susceptibility to virus infection by challenging 293T cells expressing beluga or dolphin ACE2 with pseudovirions bearing the SARS-CoV-2 spike protein. Beluga and dolphin ACE2 were sufficient to allow cell entry by an early pandemic isolate (Wuhan-Hu-1) and two evolved variants (Delta B.1.617.2 and Omicron BA.1 strains). We conclude that SARS-CoV-2 poses a potential threat to marine mammal reservoirs that should be considered in surveillance efforts.

Forward shift of feeding buzz components of dolphins and belugas during associative learning reveals a likely connection to reward expectation, pleasure and brain dopamine activation.

For many years, we heard sounds associated with reward from dolphins and belugas. We named these pulsed sounds victory squeals (VS), as they remind us of a child's squeal of delight. Here we put these sounds in context with natural and learned behavior. Like bats, echolocating cetaceans produce feeding buzzes as they approach and catch prey. Unlike bats, cetaceans continue their feeding buzzes after prey capture and the after portion is what we call the VS. Prior to training (or conditioning), the VS comes after the fish reward; with repeated trials it moves to before the reward. During training, we use a whistle or other sound to signal a correct response by the animal. This sound signal, named a secondary reinforcer (SR), leads to the primary reinforcer, fish. Trainers usually name their whistle or other SR a bridge, as it bridges the time gap between the correct response and reward delivery. During learning, the SR becomes associated with reward and the VS comes after the SR rather than after the fish. By following the SR, the VS confirms that the animal expects a reward. Results of early brain stimulation work suggest to us that SR stimulates brain dopamine release, which leads to the VS. Although there are no direct studies of dopamine release in cetaceans, we found that the timing of our VS is consistent with a response after dopamine release. We compared trained vocal responses to auditory stimuli with VS responses to SR sounds. Auditory stimuli that did not signal reward resulted in faster responses by a mean of 151 ms for dolphins and 250 ms for belugas. In laboratory animals, there is a 100 to 200 ms delay for dopamine release. VS delay in our animals is similar and consistent with vocalization after dopamine release. Our novel observation suggests that the dopamine reward system is active in cetacean brains.

Effects of health status on pressure-induced changes in phocid immune function and implications for dive ability.

The ability of marine mammals to cope with environmental challenges is a key determining factor in strandings and successful release of rehabilitated animals. Dive behavior is related to foraging and thus survival. While dive adaptations have been well studied, it is unknown how the immune system responds to diving and whether health status impacts immune function during diving. This study investigated the functional response of ex situ immune cells from stranded phocids to in vitro increased pressure, over the course of rehabilitation. Blood samples were drawn from stranded harbor seals (Phoca vitulina), gray seals (Halichoerus grypus) and harp seals (Phoca groenlandica) at the time of admit to the Mystic Aquarium, Mystic, CT and again after rehabilitation (pre-release). Phagocytosis, lymphocyte proliferation and immune cell activation were measured in vitro, with and without exposure to 2000 psi (simulated dive depth of 1360 m). Plasma epinephrine and norepinephrine, and serum cortisol were measured in vivo. All hormone values decreased between admit and release conditions. Under admit or release conditions, pressure exposures resulted in significant changes in granulocyte and monocyte phagocytosis, granulocyte expression of CD11b and lymphocyte expression of the IL2 receptor (IL2R). Overall, pressure exposures resulted in decreased phagocytosis for admit conditions, but increased phagocytosis in release samples. Expression of leukocyte activation markers, CD11b and IL2R, increased and the response did not differ between admit and release samples. Specific hematological and serum chemistry values also changed significantly between admit and release and were significantly correlated with pressure-induced changes in immune function. Results suggest (1) dive duration affects the response of immune cells, (2) different white blood cell types respond differently to pressure and (3) response varies with animal health. This is the first study describing the relationship between diving, immune function and health status in phocids.

Noradrenergic and peptidergic innervation of lymphoid organs in the beluga, Delphinapterus leucas: an anatomical link between the nervous and immune systems.

The presence of peptidergic and noradrenergic sympathetic nerve fibers in specific compartments of both primary and secondary lymphoid organs of the rodent is well established. These nerve fibers directly contact lymphocytes and macrophages, as well as vascular and trabecular smooth muscle. We investigated the noradrenergic and neuropeptide-Y innervation of lymphoid organs in the cetacean, Delphinapterus leucas (beluga whale). The spleen, thymus, tonsil, gut-associated lymphoid tissue, and assorted lymph nodes were collected from five belugas, obtained during sanctioned hunts, and processed for catecholamine fluorescence histochemistry and for tyrosine hydroxylase and neuropeptide-Y immunocytochemistry. Innervation studies revealed fluorescent nerve fibers, tyrosine hydroxylase, and neuropeptide-Y positive nerve fibers in parenchymal lymphoid compartments, where they were closely associated with cells of the immune system, and in vascular and trabecular compartments. In lymphoid zones, tyrosine hydroxylase and neuropeptide-Y positive nerve fibers were observed in the periarteriolar lymphatic sheath and marginal zone of the spleen; in the outermost portion of the cortex, the corticomedullary zone, and medulla of the lymph nodes; in the parafollicular zones, and diffuse lymphocyte layer below the epithelium of the tonsil; in the outermost portion of some thymic lobules; and in the lamina propria of the gut. These findings are similar to those described for other mammals and substantiate an anatomical link between the nervous and immune systems in the beluga, whereby central nervous system activity may influence autonomic outflow to lymphoid organs and effect immunologic reactivity.

A microscopic investigation of the lymphoid organs of the beluga, Delphinapterus leucas.

Lymphoid organs from belugas, Delphinapterus leucas, ranging in age from less than one to 16 years, were harvested during a sanctioned hunt to investigate morphology. The spleen is divisible into red and white pulp and a stroma consisting of a reticular network, a collagenous capsule, and trabeculae containing smooth muscle bundles. White pulp areas appear to be devoid of follicles and consist mainly of periarteriolar lymphatic sheaths (PALS), that are larger in younger than in older belugas. Definitive marginal zones between red and white pulp are difficult to discern in older belugas. Lymph nodes are similar to those of other mammals; they possess a follicular cortex surrounding a vascular medulla composed of lymphatic cords and sinuses. Smooth muscle is abundant in the medullary region, usually in close proximity to sinuses. The expansive nodular mass at the root of the mesentery, often referred to as the "pseudopancreas," is similar to lymph nodes in microscopic architecture. Pharyngeal tonsils and gut-associated lymphoid tissue (GALT) are found along the digestive tract and display an "active" morphology. Tonsils are comprised of lobules of follicles separated by vascular connective tissue. Epithelial-lined crypts communicate with the pharyngeal lumen. GALT consists of diffuse and follicular lymphocytes within the intestinal mucosa and submucosa. The thymus is well developed in the younger belugas, with lobules divisible into densely packed cortical zones of thymocytes and more loosely arranged medullary lymphocytes. Hassall's corpuscles are occasionally visible within the medulla. Cetaceans diverged evolutionarily from other mammals over 55 million years ago. This study investigates changes in lymphoid organ morphology in a species that now inhabits a unique ecological niche. This study also lays the groundwork for functional investigation of the beluga immune system, particularly as it relates to differences between healthy and stranded animals.

Simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (Tursiops truncatus) utilizing flow cytometry.

Phagocytic and respiratory burst activity was simultaneously measured by flow cytometry in polymorphonuclear leukocytes (PMN) and monocytes in whole blood from bottlenose dolphins (Tursiops truncatus). Blood was collected from 16 adult dolphins, 12 males (6-34 years of age) and 4 females (11-30 years) and subsequently incubated with a bacteria-to-leukocyte ratio of 25:1 and 10 µl of 500 µM 2',7'-dichlorofluorescein diacetate for 70 min at 37°C. PMN (44.5 ± 3.2%) and monocytes (33.5 ± 3.0%) were positive for propidium iodide-labeled Staphylococcus aureus, indicating phagocytosis. Respiratory burst activity after 70 min as measured by the mean fluorescence intensity (MFI) was 68.0 ± 14.4 in PMN and 47.0 ± 10.3 in monocytes. There were no significant differences in MFI or percentage of phagocytizing PMN (p > 0.094) or monocytes (p > 0.275) after storage at 4°C for 24h when compared to activity measured in fresh blood. Nor was there an effect of storage on respiratory burst activity (MFI or percentage) in PMN (p > 0.420) or monocytes (p > 0.301). This assay may be particularly useful to assess the ability of dolphins to effectively combat bacterial pathogen challenges with minimal amounts of blood.

MHC class II molecules and immunoglobulins on peripheral blood lymphocytes of the bottlenosed dolphin, Tursiops truncatus.

The immune system of marine mammals is of comparative interest because of its adaptation to the aquatic environment. Little information, however, is available on its cellular and molecular components. Here, we used a cross-reactive antibody to MHC class II molecules and an immunoglobulin-specific antiserum for identifying these molecular species on lymphocytes of the bottlenosed dolphin, Tursiops truncatus. Limited structural analyses indicated that class II molecules and immunoglobulins of dolphin closely resemble those of other vertebrates. In the peripheral blood of most land mammals both class II and immunoglobulins are usually found on B but not T lymphocytes. Expression of immunoglobulins on dolphin peripheral blood lymphocytes suggests a ratio of B cells to T cells comparable to that of land mammals. However, unlike the majority of land mammals, virtually 100% of the peripheral T cells display pronounced expression of class II molecules, generally considered an indication of T cell activation. It is therefore possible that the physiology of T cell activation has unusual attributes in the dolphin. It is especially interesting that some land mammals, namely swine (ungulates) and dogs and cats (carnivores), also express class II molecules on peripheral blood T lymphocytes. Since ungulates and carnivores are thought to share a common distant ancestry with toothed whales, the evolutionary history may be more relevant than the environmental history in determining these unusual attributes.

Neuropeptide-Y innervation of the rat spleen: another potential immunomodulatory neuropeptide.

Neuropeptide-Y (NPY) is a 36 amino acid peptide that acts as a chemical messenger in the central and peripheral nervous systems. NPY often is found colocalized with the classical neurotransmitter norepinephrine (NE) and can potentiate the effects of this neurotransmitter postsynaptically in many systems. Using immunocytochemistry for NPY and specific lymphoid cell markers, we mapped the distribution of NPY-positive nerve fibers in the rat spleen. NPY-positive nerve fibers were present along the vasculature, trabeculae, and capsule, and also were found associated with specific lymphoid parenchymal compartments of the spleen, in close contact with lymphocytes and macrophages. These contacts were investigated further at the electron microscopic level. NPY-positive nerve terminals were found in close apposition with lymphocytes in the periarteriolar lymphatic sheath, and with lymphocytes and macrophages in the marginal zone. Previous studies have reported that postganglionic noradrenergic nerve fibers innervate specific lymphoid compartments of the rat spleen, with nerve terminals forming direct appositions with cells of the immune system. The possible colocalization of NPY and NE in these nerve fibers was investigated by chemical sympathectomy with 6-hydroxydopamine, followed by immunocytochemical labeling of NPY and tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine synthesis. Colocalization also was investigated by labeling for NPY with a fluorescent label, eluting the NPY, and staining for TH with diaminobenzidine as the label. These studies demonstrate that norepinephrine and NPY are colocalized in the postganglionic sympathetic nerve fibers of the rat spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas.

Given the importance of the cell surface recognition protein, CD4, in immune function, the cloning and characterization of CD4 at the molecular level from an odontocete cetacean, the white whale (Delphinapterus leucas), was carried out. Whale CD4 cDNA contains 2662 base pairs and translates into a protein containing 455 amino acids. Whale CD4 shares 64% and 51% identity with the human and mouse CD4 protein, respectively, and is organized in a similar manner. Unlike human and mouse, however, the cytoplasmic domain, which is highly conserved, contains amino acid substitutions unique to whale. Moreover, only one of the seven potential N-linked glycosylation sites present in whale is shared with human and mouse. Evolutionarily, the whale CD4 sequence is most similar to pig and structurally similar to dog and cat, in that all lack the cysteine pair in the V2 domain. These differences suggest that CD4 may have a different secondary structure in these species, which may affect binding of class II and subsequent T-cell activation, as well as binding of viral pathogens. Interestingly, as a group, species with these CD4 characteristics all have high constitutive expression of class II molecules on T lymphocytes, suggesting potential uniqueness in the interaction of CD4, class II molecules, and the immune response. Molecular characterization of CD4 in an aquatic mammal provides information on the CD4 molecule itself and may provide insight into adaptive evolutionary changes of the immune system.

The prevalence of osteopenia in pediatric renal allograft recipients varies with the method of analysis.

BACKGROUND: Pediatric renal allograft recipients often suffer from osteopenia and the potential for increased fractures. Although modern densitometers are widely available, their use in children is complicated by lack of optimal interpretive criteria. METHODS: We reviewed dual energy X-ray absorptiometry (DEXA) studies in 33 patients with functional renal allografts 4.4 +/- 3.6 years after transplantation. We interpreted our data using three previously described methods of assigning bone mineral density (BMD) Z scores. RESULTS: BMD was directly related to age, height, weight, body surface area, and pubertal status (p < 0.001). Using gender-mixed reference data matched by chronological age, the mean BMD Z score was -0.9 +/- 1.3 vs. 0.4 +/- 1.4 when matched by height-age (p < 0.001). Height-age adjustment particularly increased the BMD Z score of pubertal adolescents. In a subset of 22 patients, gender-matched reference data led to different results from the gender-mixed reference population (mean BMD Z score 0.0 +/- 1.7 vs. -0.8 +/- 1.4, p < 0.001). CONCLUSIONS: The perceived prevalence of osteopenia among pediatric kidney transplant recipients differs using analysis based on chronological age, height-age, or gender-matched reference data. Further studies are necessary to determine the clinical significance of measured bone density in this population.