Immunology of whales and dolphins.

The increasing disease susceptibility in different whale and dolphin populations has led to speculation about a possible negative influence of environmental contaminants on the immune system and therefore on the health status of marine mammals. Despite current efforts in the immunology of marine mammals several aspects of immune functions in aquatic mammals remain unknown. However, assays for evaluating cellular immune responses, such as lymphocyte proliferation, respiratory burst as well as phagocytic and cytotoxic activity of leukocytes and humoral immune responses have been established for different cetacean species. Additionally, immunological and molecular techniques enable the detection and quantification of pro- and anti-inflammatory cytokines in lymphoid cells during inflammation or immune responses, respectively. Different T and B cell subsets as well as antigen-presenting cells can be detected by flow cytometry and immunohistochemistry. Despite great homologies between marine and terrestrial mammal lymphoid organs, some unique anatomical structures, particularly the complex lymphoepithelial laryngeal glands in cetaceans represent an adaptation to the marine environment. Additionally, physiological changes, such as age-related thymic atrophy and cystic degeneration of the "anal tonsil" of whales have to be taken into account when investigating these lymphoid structures. Systemic morbillivirus infections lead to fatalities in cetaceans associated with generalized lymphoid depletion. Similarly, chronic diseases and starvation are associated with a loss of functional lymphoid cells and decreased resistance against opportunistic infections. There is growing evidence for an immunotoxic effect of different environmental contaminants in whales and dolphins, as demonstrated in field studies. Furthermore, immunomodulatory properties of different persistent xenobiotics have been confirmed in cetacean lymphoid cells in vitro as well as in animal models in vivo. However, species-specific differences of the immune system and detoxification of xenobiotics between cetaceans and laboratory rodents have to be considered when interpreting these toxicological data for risk assessment in whales and dolphins.

First report of major histocompatibility complex class II loci from the Amazon pink river dolphin (genus Inia)

We report the first major histocompatibility complex (MHC) DQB1 sequences for the two species of pink river dolphins (Inia geoffrensis and Inia boliviensis) inhabiting the Amazon and Orinoco River basins. These sequences were found to be polymorphicwithin the Inia genus and showed shared homology with cetacean DQB-1 sequences, especially, those of the Monodontidae and Phocoenidae. On the other hand, these sequences were shown to be divergent from those described for other riverine dolphin species, such as Lipotes vexillifer, the Chinese river dolphin. Two main conclusions can be drawn from our results: 1) the Mhc DQB1 sequences seem to evolve more rapidly than other nuclear sequences in cetaceans, and 2) differential positive selective pressures acting on these genes cause concomitant divergent evolutionary histories that derive phylogenetic reconstructions that could be inconsistent with widely accepted intertaxa evolutionary relationships elucidated with other molecular markers subjected to a neutraldynamics.

Immunohistochemical detection of macrophages in the short-finned pilot whale (Globicephala macrorhynchus) and Risso's dolphin (Grampus griseus).

Macrophages play a central role in the immune system, but few markers are available for their detection in cetaceans. The purpose of the present study, therefore, was to examine the cross-reactivity for two cetacean species (short-finned pilot whale and Risso's dolphin) of four anti-human antibodies (SRA-E5, AM-3K, EBM11 and anti-human lysozyme). The distribution of SRA-E5- and AM-3K-positive cells was similar, both antibodies labelling (1) many resident macrophages in the spleen, lymph nodes, liver, lung, kidney, intestine and dermis, and (2) exudate macrophages in the hepatic interlobular septa. Anti-human lysozyme antibody also labelled both resident and exudate macrophages. However, double immunohistochemistry showed that the majority of AM-3K-positive cells in the spleen and liver were also labelled by SRA-E5; on the other hand, anti-human lysozyme-positive cells did not always correspond with AM-3K-positive cells. Cetacean tissues contained no EBM11-positive cells. The study demonstrated the potential values of SRA-E5, AM-3K and anti-human lysozyme antibody for cetacean macrophage studies.

Expression and functional characterization of killer whale (Orcinus orca) interleukin-6 (IL-6) and development of a competitive immunoassay.

Interleukin-6 (IL-6) is a cytokine that can reach detectable systemic levels and is a major inducer of the acute phase response. As such, clinical assays to identify this cytokine in mammalian sera are of diagnostic value. A 558 base-pair (bp) fragment of killer whale IL-6 was cloned and expressed as a 21 kDa protein in Escherichia coli. Biological activity of the recombinant killer whale IL-6 (rkwIL-6) was demonstrated using the IL-6-dependent B9 mouse hybridoma cell line; acute phase sera from a killer whale and supernatants from lipopolysaccharide (LPS)-stimulated killer whale peripheral blood mononuclear cells (PBMCs) also supported the proliferation of the B9 hybridoma. Rat anti-mouse IL-6 receptor antibody effectively blocked biological activity of all three sources of IL-6. Polyclonal antisera, specific for the recombinant protein, were obtained by successive immunization of a rabbit with rkwIL-6. The polyclonal antibody was capable of neutralizing the biological activity of both recombinant and native kwIL-6. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal rabbit anti-rkwIL-6 and the recombinant protein; sensitivity of the assay was in the range of 1 ng/ml. The ELISA was subsequently used to identify the presence of native IL-6 in acute phase sera of two species of delphinidae, a killer whale and a bottlenose dolphin. The application of quantitative cytokine assays as diagnostic tools for monitoring cetacean health are becoming feasible as many animals are now being trained for fluke presentation, making blood collection a routine procedure.

Molecular cloning and functional characterization of bottlenose dolphin (Tursiops truncatus) tumor necrosis factor alpha.

Bottlenose dolphin tumor necrosis factor alpha (doTNF-alpha) cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the nucleic and deduced amino acid sequences were determined. The sequence of the cDNA clones shows that doTNF-alpha has an open reading frame of 699bp encoding 233 amino acids. The nucleic acid sequence of doTNF-alpha indicates 90, 88, 87, and 79% similarity with the cattle, pig, human, and mouse TNF-alpha gene, respectively. Based on the analysis of human and mouse TNF-alpha molecules, doTNF-alpha is processed to a mature protein with 157 amino acids. The 233 amino acids precursor has a hydrophobic region that could serve as a transmembrane domain. The recombinant doTNF-alpha expressed in Escherichia coli as a glutathione S-transferase fusion protein reacted with anti-human TNF-alpha antibody and exerted cytotoxity to the TNF-alpha sensitive murine cell line L929.

Molecular cloning and functional expression of bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist.

The bottle-nosed dolphin (Tursiops truncatus) interleukin-1 receptor antagonist IL-1ra cDNA was cloned from mitogen-stimulated peripheral blood mononuclear cells (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequence of this cDNA showed that dolphin IL-1ra clones contained open reading frames encoding 177 amino acids. Comparison of the deduced amino acids showed that dolphin IL-1ra sequence shared 87.6, 77.9, 77.4, 77.4, 76.4, and 75.8% similarity with the bovine, rabbit, equine, human, mouse, and rat IL-1ra sequences, respectively. Recombinant glutathione S-transferase (GST) dolphin IL-1ra produced in Escherichia coli (E. coli) was purified. This protein suppressed the cytostatic activity of dolphin IL-1beta on A375S2 cells, indicating that the dolphin IL-1ra cDNA obtained in the present study encodes biologically active dolphin IL-1ra.

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.

A monoclonal antibody, DL10, which recognizes a sugar moiety of MHC class I antigens expressed on NK cells, NK+ T cells, and granulocytes in humans.

One mAb, DL10, was established from mice injected with dolphin lymphocytes. In addition to its reactivity against all dolphin lymphocytes, it reacted with some human leukocytes, including NK cells, NK+ T cells, and granulocytes. When its reactivity was examined in various animals, bovine, ovine, and equine leukocytes were DL10+. Murine, rat, and canine leukocytes were DL10-. Although the reactivity of DL10+ was similar to those of CD56 and CD57 antigens in humans, the actual molecules it recognized were different. Thus, all reactivity of DL10 disappeared after treatment of cells with glycopeptidase or after culture of cells with tunicamycin. Furthermore, the immunoprecipitation method revealed that DL10 indirectly recognized the heavy chain (45kD) of MHC class I antigen in humans and animals. Considering data from analysis of the N-terminal amino acid sequence of the DL10 molecule and the HLA typing of reactive cells, DL10 recognized a sugar moiety of some monomorphic MHC antigens and polymorphic MHC antigens such as HLA-B60 and -B61. If the donors are HLA-B60- and -B61 (> 80% in Japan and > 95% in the United States), DL10 would appear to be a very useful agent for the detection of pan-NK+ T cells.

Regression of cetacean tattoo lesions concurrent with conversion of precipitin antibody against a poxvirus.

Tattoo lesions linked to the cetacean poxvirus of bottlenose dolphins regressed without treatment. Two types of regression were observed: (1) The tattoo lesions became raised and blanched, then disappeared along with sloughing skin. (2) When an incision was made through a tattoo lesion, the tattoo disappeared in a zone around the incision. Poxviruses removed from the raised, blanched skin lesions and from typical tattoo lesions were reacted with dolphin serums and examined by immunoelectron microscopy. Antibody was not detected against either of these poxvirus preparations when the dolphins had only typical tattoo lesions. However, after the raised, blanched lesions appeared, serums obtained during the acute or convalescent stages were positive for the poxvirus separated from the lesions. Regression of the typical tattoo lesions was concurrent with antibody conversion.