Effects of Polar Bear and Killer Whale Derived Contaminant Cocktails on Marine Mammal Immunity.

Most controlled toxicity studies use single chemical exposures that do not represent the real world situation of complex mixtures of known and unknown natural and anthropogenic substances. In the present study, complex contaminant cocktails derived from the blubber of polar bears (PB; Ursus maritimus) and killer whales (KW; Orcinus orca) were used for in vitro concentration-response experiments with PB, cetacean and seal spp. immune cells to evaluate the effect of realistic contaminant mixtures on various immune functions. Cytotoxic effects of the PB cocktail occurred at lower concentrations than the KW cocktail (1 vs 16 µg/mL), likely due to differences in contaminant profiles in the mixtures derived from the adipose of each species. Similarly, significant reduction of lymphocyte proliferation occurred at much lower exposures in the PB cocktail (EC50: 0.94 vs 6.06 µg/mL; P < 0.01), whereas the KW cocktail caused a much faster decline in proliferation (slope: 2.9 vs 1.7; P = 0.04). Only the KW cocktail modulated natural killer (NK) cell activity and neutrophil and monocyte phagocytosis in a concentration- and species-dependent manner. No clear sensitivity differences emerged when comparing cetaceans, seals and PB. Our results showing lower effect levels for complex mixtures relative to single compounds suggest that previous risk assessments underestimate the effects of real world contaminant exposure on immunity. Our results using blubber-derived contaminant cocktails add realism to in vitro exposure experiments and confirm the immunotoxic risk marine mammals face from exposure to complex mixtures of environmental contaminants.

Immunotoxic effects of environmental pollutants in marine mammals.

Due to their marine ecology and life-history, marine mammals accumulate some of the highest levels of environmental contaminants of all wildlife. Given the increasing prevalence and severity of diseases in marine wildlife, it is imperative to understand how pollutants affect the immune system and consequently disease susceptibility. Advancements and adaptations of analytical techniques have facilitated marine mammal immunotoxicology research. Field studies, captive-feeding experiments and in vitro laboratory studies with marine mammals have associated exposure to environmental pollutants, most notable polychlorinated biphenyls (PCBs), organochlorine pesticides and heavy metals, to alterations of both the innate and adaptive arms of immune systems, which include aspects of cellular and humoral immunity. For marine mammals, reported immunotoxicology endpoints fell into several major categories: immune tissue histopathology, haematology/circulating immune cell populations, functional immune assays (lymphocyte proliferation, phagocytosis, respiratory burst, and natural killer cell activity), immunoglobulin production, and cytokine gene expression. Lymphocyte proliferation is by far the most commonly used immune assay, with studies using different organic pollutants and metals predominantly reporting immunosuppressive effects despite the many differences in study design and animal life history. Using combined field and laboratory data, we determined effect threshold levels for suppression of lymphocyte proliferation to be between b0.001-10 ppm for PCBs, 0.002-1.3 ppm for Hg, 0.009-0.06 for MeHg, and 0.1-2.4 for cadmium in polar bears and several pinniped and cetacean species. Similarly, thresholds for suppression of phagocytosis were 0.6-1.4 and 0.08-1.9 ppm for PCBs and mercury, respectively. Although data are lacking for many important immune endpoints and mechanisms of specific immune alterations are not well understood, this review revealed a systemic suppression of immune function in marine mammals exposed to environmental contaminants. Exposure to immunotoxic contaminants may have significant population level consequences as a contributing factor to increasing anthropogenic stress in wildlife and infectious disease outbreaks.

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors.

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid--α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.

Evolution and survival of marine carnivores did not require a diversity of killer cell Ig-like receptors or Ly49 NK cell receptors.

Ly49 lectin-like receptors and killer cell Ig-like receptors (KIR) are structurally unrelated cell surface glycoproteins that evolved independently to function as diverse NK cell receptors for MHC class I molecules. Comparison of primates and various domesticated animals has shown that species have either a diverse Ly49 or KIR gene family, but not both. In four pinniped species of wild marine carnivore, three seals and one sea lion, we find that Ly49 and KIR are each represented by single, orthologous genes that exhibit little polymorphism and are transcribed to express cell surface protein. Pinnipeds are therefore species in which neither Ly49 nor KIR are polygenic, but retain the ancestral single-copy state. Whereas pinniped Ly49 has been subject to purifying selection, we find evidence for positive selection on KIR3DL during pinniped evolution. This selection, which focused on the D0 domain and the stem, points to the functionality of the KIR and most likely led to the sea lion's loss of D0. In contrast to the dynamic and rapid evolution of the KIR and Ly49 genes in other species, the pinniped KIR and Ly49 have been remarkably stable during the >33 million years since the last common ancestor of seals and sea lions. These results demonstrate that long-term survival of placental mammal species need not require a diverse system of either Ly49 or KIR NK cell receptors.

Antibodies to marine caliciviruses in the Steller sea lion (Eumetopias jubatus Schreber).

Sera from 145 Steller sea lions (76 adults, three subadults, 37 pups, and 29 fetuses) were tested for neutralizing antibodies to nine marine calicivirus serotypes. Antibodies were found to San Miguel sea lion virus (SMSV) types 1, 5, 6, 7, 8, 10 and 13, and to Tillamook (bovine) calicivirus, but no antibodies were found to the walrus calicivirus. Titers (microtiter neutralization assay) ranged from 1:20 to 1:320, with many positive reactions at the higher dilutions (greater than or equal to 1:80). Antibodies to SMSV's 5 and 10 were most common among animals sampled in Alaskan waters, while antibodies to SMSV-6 were most common among pups from the southern Oregon coast. These data provide evidence that Steller sea lions, like their California sea lion (Zalophus c. californianus Lesson) counterparts, have experienced widespread exposure to multiple serotypes of marine caliciviruses.

Prevalence and distribution of serum neutralizing antibodies to Tillamook (bovine) calicivirus in selected populations of marine mammals.

Neutralizing antibodies to Tillamook calicivirus (TCV) were found in sera collected from California sea lions (Zalophus c. californianus Lesson) in 1983 and 1984 and in sera collected from Steller sea lions (Eumetopias jubatus Schreber) in 1976 and 1985. The combined prevalence of antibodies for these two species was 10/228 = 4.38%. Titers ranged from 1:20 (five animals), to 1:40 (four animals), to 1:80 (one animal) by standard microtiter neutralization assay. The seropositive pinnipeds were dispersed widely along the margins of the eastern Pacific rim, from the Bering Sea to the Santa Barbara Channel. Antibodies to TCV were not found in sera collected from northern fur seals (Callorhinus ursinus L.), Pacific walruses (Odobenus rosmarus divergens Illiger), seals of the family Phocidae, or several cetacean species. Tillamook calicivirus was isolated originally in 1981 from dairy calves in Oregon; the finding of neutralizing antibodies in two widely distributed species of sea lions suggests the possibility of a marine origin for this agent.

Prevalence of vesicular exanthema of swine antibodies among feral mammals associated with the southern California coastal zones.

Serum-neutralizing antibodies to both vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) were found in a number of animal species having an association with the southern California coastal zones. California sea lions (Zalophus californianus) had antibodies to 9 VESV types (A48, C52, D53, E54, F54, G55, I55, J56, and K56). Fur seals (Callorhinus ursinus) and elephant seal pups (Mirounga angustirostris) were tested for antibodies to 6 VESV types and all were negative. California gray whales (Eschrichtius robustus) were tested for antibodies to 9 VESV types, and sperm whale (Physeter catodon), finback whale (Balaenoptera physalus), and sei whale (Balaenoptera borealis) were tested for 6 VESV types. Among the last 4 species, antibodies were present for each VESV type except C52, and all species were positive for antibodies to 2 or more VESV types. Feral swine from both Santa Cruz Island and Santa Catalina Island were tested and antibodies were present for 8 of 9 VESV types and all SMSV types except SMSV-4. One donkey from San Miguel Island was positive for VESV I55 and 2 were positive for SMSV-2.