Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Comparative Immunopathology of Cetacean morbillivirus Infection in Free-Ranging Dolphins From Western Mediterranean, Northeast-Central, and Southwestern Atlantic.

Cetacean morbillivirus (CeMV; Paramyxoviridae) causes epizootic and interepizootic fatalities in odontocetes and mysticetes worldwide. Studies suggest there is different species-specific susceptibility to CeMV infection, with striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis) ranking among the most susceptible cetacean hosts. The pathogenesis of CeMV infection is not fully resolved. Since no previous studies have evaluated the organ-specific immunopathogenetic features of CeMV infection in tissues from infected dolphins, this study was aimed at characterizing and comparing immunophenotypic profiles of local immune responses in lymphoid organs (lymph nodes, spleen), lung and CNS in CeMV-molecularly (RT-PCR)-positive cetaceans from Western Mediterranean, Northeast-Central, and Southwestern Atlantic. Immunohistochemical (IHC) analyses targeted molecules of immunologic interest: caspase 3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFß, and PAX5. We detected consistent CeMV-associated inflammatory response patterns. Within CNS, inflammation was dominated by CD3+ (T cells), and CD20+ and PAX5+ (B cells) lymphocytes, accompanied by fewer Iba1+, CD68+, and lysozyme+ histiocytes, mainly in striped dolphins and bottlenose dolphins. Multicentric lymphoid depletion was characterized by reduced numbers of T cells and B cells, more pronounced in Guiana dolphins. Striped dolphins and bottlenose dolphins often had hyperplastic (regenerative) phenomena involving the aforementioned cell populations, particularly chronically infected animals. In the lung, there was mild to moderate increase in T cells, B cells, and histiocytes. Additionally, there was a generalized increased expression of caspase 3 in lymphoid, lung, and CNS tissues. Apoptosis, therefore, is believed to play a major role in generalized lymphoid depletion and likely overt immunosuppression during CeMV infection. No differences were detected regarding cytokine immunoreactivity in lymph nodes, spleen, and lung from infected and non-infected dolphins by semiquantitative analysis; however, there was striking immunoreactivity for IFNγ in the CNS of infected dolphins. These novel results set the basis for tissue-specific immunophenotypic responses during CeMV infection in three highly susceptible delphinid species. They also suggest a complex interplay between viral and host's immune factors, thereby contributing to gain valuable insights into similarities, and differences of CeMV infection's immunopathogenesis in relation to body tissues, CeMV strains, and cetacean hosts.

Insights Into Dolphins' Immunology: Immuno-Phenotypic Study on Mediterranean and Atlantic Stranded Cetaceans.

Immunology of marine mammals is a relatively understudied field and its monitoring plays an important role in the individual and group management of these animals, along with an increasing value as an environmental health indicator. This study was aimed at implementing the knowledge on the immune response in cetaceans stranded along the Italian coastline to provide a baseline useful for assessing the immune status of bottlenose (Tursiops truncatus) and striped (Stenella coeruleoalba) dolphins. In particular, since the Mediterranean Sea is considered a heavily polluted basin, a comparison with animals living in open waters such as the Atlantic Ocean was made. Formalin-fixed, paraffin-embedded spleen, thymus, and lymph node tissues from 16 animals stranded along Italian and 11 cetaceans from the Canary Island shores were sampled within 48 h from death. Information regarding stranding sites, gender, and age as well as virologic, microbiological, and parasitological investigations, and the cause and/or the death mechanism were also collected in order to carry out statistical analyses. Selected tissues were routinely stained with hematoxylin-eosin (H&E) and with immunohistochemical techniques (IHC). For IHC analysis, anti-human CD5 monoclonal mouse antibody to identify T lymphocytes, CD20 monoclonal mouse antibody for the identification of mature B lymphocytes and HLA-DR antigen (alpha-chain) monoclonal mouse antibody for the identification of the major histocompatibility complex type II were previously validated for both species by Western-blotting technique. T-test method applied to quantitative evaluation of IHC positive cells showed a significant relationship between the number of (expression) of CD20 stained lymphocytes and normal and hypoplastic lymph nodes, respectively. No other significant correlations were noticed. Analyses for organochlorines (OC) compounds were performed in animals (n°5) having frozen blubber tissue available. A simple linear regression was calculated to predict if the amount of OCs could influence the number of inflammatory cell subpopulations and a moderate negative correlation was found between the presence of high quantity of contaminants and the number of T lymphocytes. Future analysis should be aimed to understand the effect of the major immunomodulatory pathogens on sub-populations of B and T cells.

Immunohistochemical investigation of the cross-reactivity of selected cell markers in formalin-fixed, paraffin-embedded lymphoid tissues of Franciscana (Pontoporia blainvillei).

A considerable amount of knowledge on natural and anthropogenic pathologic conditions affecting different cetacean species has been gained over the last decades. Nonetheless, the immunopathological bases for most of these processes have been poorly documented or remain unknown. Comparative immunopathological investigations in these species are precluded by the limited number of specific antibodies, most of which are not commercially available, and the reduced spectrum of validated and/or cross-reactive ones. To partially fill in this gap of knowledge, a set of commercially available primary antibodies were tested for cross-reactivity against leukocytes and cytokines in formalin-fixed, paraffin-embedded (FFPE) lymphoid tissues (lymph nodes, spleen and thymus) of three bycaught, apparently healthy and fresh Franciscanas (Pontoporia blainvillei) using immunohistochemistry. On the basis of similar region specificity within the lymphoid organs, cellular morphology and staining pattern with human control tissues, 13/19 primary antibodies (caspase 3, CD3, CD57, CD68, FoxP3, HLA-DRα, IFNγ, IgG, IL4, IL10, Lysozyme, TGFß and PAX-5) exhibited satisfactory cross-reactivity. Our results expand the spectrum of suitable cross-reactive primary antibodies in FFPE cetacean tissues. Further comparative immunopathological studies focused on infectious diseases and ecotoxicology may benefit from establishment of baseline expression of immunologically relevant molecules in various cetaceans species.

Bridging immunogenetics and immunoproteomics: Model positional scanning library analysis for Major Histocompatibility Complex class II DQ in Tursiops truncatus.

The Major Histocompatibility Complex (MHC) is a critical element in mounting an effective immune response in vertebrates against invading pathogens. Studies of MHC in wildlife populations have typically focused on assessing diversity within the peptide binding regions (PBR) of the MHC class II (MHC II) family, especially the DQ receptor genes. Such metrics of diversity, however, are of limited use to health risk assessment since functional analyses (where changes in the PBR are correlated to recognition/pathologies of known pathogen proteins), are difficult to conduct in wildlife species. Here we describe a means to predict the binding preferences of MHC proteins: We have developed a model positional scanning library analysis (MPSLA) by harnessing the power of mixture based combinatorial libraries to probe the peptide landscapes of distinct MHC II DQ proteins. The algorithm provided by NNAlign was employed to predict the binding affinities of sets of peptides generated for DQ proteins. These binding affinities were then used to retroactively construct a model Positional Scanning Library screen. To test the utility of the approach, a model screen was compared to physical combinatorial screens for human MHC II DP. Model library screens were generated for DQ proteins derived from sequence data from bottlenose dolphins from the Indian River Lagoon (IRL) and the Atlantic coast of Florida, and compared to screens of DQ proteins from Genbank for dolphin and three other cetaceans. To explore the peptide binding landscape for DQ proteins from the IRL, combinations of the amino acids identified as active were compiled into peptide sequence lists that were used to mine databases for representation in known proteins. The frequency of which peptide sequences predicted to bind the MHC protein are found in proteins from pathogens associated with marine mammals was found to be significant (p values <0.0001). Through this analysis, genetic variation in MHC (classes I and II) can now be associated with the binding repertoires of the expressed MHC proteins and subsequently used to identify target pathogens. This approach may be eventually applied to evaluate individual population and species risk for outbreaks of emerging diseases.

The environment as a driver of immune and endocrine responses in dolphins (Tursiops truncatus).

Immune and endocrine responses play a critical role in allowing animals to adjust to environmental perturbations. We measured immune and endocrine related markers in multiple samples from individuals from two managed-care care dolphin groups (n = 82 samples from 17 dolphins and single samples collected from two wild dolphin populations: Indian River Lagoon, (IRL) FL (n = 26); and Charleston, (CHS) SC (n = 19). The immune systems of wild dolphins were more upregulated than those of managed-care-dolphins as shown by higher concentrations of IgG and increases in lysozyme, NK cell function, pathogen antibody titers and leukocyte cytokine transcript levels. Collectively, managed-care care dolphins had significantly lower levels of transcripts encoding pro-inflammatory cytokine TNF, anti-viral MX1 and INFα and regulatory IL-10. IL-2Rα and CD69, markers of lymphocyte activation, were both lower in managed-care care dolphins. IL-4, a cytokine associated with TH2 activity, was lower in managed-care care dolphins compared to the free-ranging dolphins. Differences in immune parameters appear to reflect the environmental conditions under which these four dolphin populations live which vary widely in temperature, nutrition, veterinary care, pathogen/contaminant exposures, etc. Many of the differences found were consistent with reduced pathogenic antigenic stimulation in managed-care care dolphins compared to wild dolphins. Managed-care care dolphins had relatively low TH2 lymphocyte activity and fewer circulating eosinophils compared to wild dolphins. Both of these immunologic parameters are associated with exposure to helminth parasites which is uncommon in managed-care care dolphins. Less consistent trends were observed in a suite of hormones but significant differences were found for cortisol, ACTH, total T4, free T3, and epinephrine. While the underlying mechanisms are likely multiple and complex, the marked differences observed in the immune and endocrine systems of wild and managed-care care dolphins appear to be shaped by their environment.

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 polymorphic within 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 neutral dynamics.

Immunostimulatory oligodeoxynucleotides induce dolphin neutrophil NADPH-oxidase activation in a CpG-independent but phosphorothioate backbone-dependent manner.

Immunocytes, which include antigen-presenting cells, B cells, natural killer cells and neutrophils, can be stimulated directly or indirectly with bacterial DNA and synthetic oligodeoxynucleotides (ODNs) with different structures and sequences. In the present study, we investigated the effect of synthetic ODNs on the respiratory burst of dolphin neutrophils using a chemiluminescence assay. Phosphorothioate (PS)-ODNs dose-dependently induced the respiratory burst, while phosphodiester (PO)-ODNs did not, regardless of CpG-content. The PS-ODN-induced activity was completely abolished by the flavoprotein inhibitor diphenyleneiodonium, which indicates that the NADPH-oxidase is activated by PS-ODNs. These results reveal that PS-ODNs induce dolphin neutrophil NADPH-oxidase activation in a CpG motif-independent but phosphorothioate-dependent manner.

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage.

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.

Decreased lymphocyte responses in free-ranging bottlenose dolphins (Tursiops truncatus) are associated with increased concentrations of PCBs and DDT in peripheral blood.

Since 1987, large-scale mortalities of dolphins have been reported along the Atlantic coast of North America, in the Gulf of Mexico, and in the Mediterranean Sea. Autopsied bottlenose dolphins, Tursiops truncatus, which were collected from the large-scale mortality along the Atlantic coast in 1987 to 1988, exhibited opportunistic infections indicative of immune dysfunction. Further, these animals had high levels of chlorinated hydrocarbons, such as PCBs and DDT, that can suppress immune functions. The purpose of this study was to determine whether there is a relationship between chemical contaminant exposure and immune response in free-ranging dolphins. In June of 1991, peripheral blood was obtained from members of a bottlenose dolphin population that resides along the west coast of Florida. Peripheral blood lymphocyte responses to Concanavalin A (Con A) and phytohemagglutinin (PHA) were determined in vitro and compared by regression analysis with contaminant concentrations in whole blood from a small subset of these animals (n = 5). These data indicate that a reduced immune response in these bottlenose dolphins was correlated with increasing whole blood concentrations of several contaminants. Specifically, inverse correlations were found between Con A-induced lymphocyte proliferation and tetrachlorinated to octachlorinated biphenyls (r2 values ranged from 0.70 to 0.87). Con A-induced lymphocyte responses also correlated inversely with p,p'DDT (r2 values of 0.73 and 0.79); o.p'-DDE (r2 values of 0.93 and 0.96); and p,p'-DDE (r2 values of 0.73 and 0.81).

Distribution of MHC II (+) cells in skin of the Atlantic bottlenose dolphin (Tursiops truncatus): an initial investigation of dolphin dendritic cells.

The skin is an important tissue of the immune system; however, little is known about immune cells in dolphin skin, and very few cetacean-specific immunoreagents are available for investigative purposes. Therefore, in this study immunohistochemistry techniques were used with species-specific and non-species-specific antibodies to characterize immune cells, primarily focusing on Langerhans cells, in skin from the Atlantic bottlenose dolphin (Tursiops truncatus). An antibody to human major histocompatibility complex (MHC) class II molecules labeled cells with a dendritic-like morphology. The immunophenotype, morphology, and distribution of some of these cells are consistent with those of Langerhans cells. The cells were predominantly found in dermal papillae, primarily along the epidermal-dermal junction. Thus, the location of these cells was somewhat different from that in terrestrial mammals. Other MHC II (+) cells of varying morphology were observed deeper in the dermis, with a perivascular concentration, and had characteristics of macrophages and dermal dendritic cells. There was no immunostaining with cetacean-specific CD2 or CD21. In diseased skin, a subjective increase of MHC II (+) cells, most notably in the superficial skin layers, was associated with an ulcerative dermatitis. A few CD2 (+) cells were also present. Differences between dolphins and terrestrial mammals in terms of morphology, mechanisms of response to insult and repair, and environmental challenges may explain the modified distribution of MHC II (+) cells in dolphin skin. An elucidation of the immune cells in cetacean skin will contribute to our understanding of the evolution of functional adaptations to various environments, facilitate diagnosis of skin diseases, and define the potential for intradermal administration of vaccines and other immunotherapeutics.

Induction of cross-protection in mice against dolphin Erysipelothrix rhusiopathiae isolates with a swine commercial vaccine.

Erysipelothrix rhusiopathiae is well known to cause disease in dolphins. This disease occurs either in an peracute way, leading to mortality even before clinical signs are observed or in a sub-acute way, characterized by rhomboidal skin lesions, that can be treated with penicillin or its derivatives. Commercial swine vaccines, containing inactivated serotype 2 strains, are currently used for vaccination but it is not known whether these vaccines induce protection against E. rhusiopathiae isolates from dolphins. In the present study, it was demonstrated in a mouse model that vaccination with a commercial swine vaccine (Eurovac Ery, Eurovet, Belgium) containing inactivated serotype 2 E. rhusiopathiae strains induced protection against challenge with three E. rhusiopathiae isolates from dolphins. The duration of the protection varied, depending on the challenging isolate, between 8 and >23 weeks. There was however no positive correlation between the amount of antibodies at the moment of challenge and the observed protection. In conclusion, vaccination trials in mice indicate that commercial serotype 2 swine Erysipelothrix vaccines induce protection against erysipelas caused by dolphin pathogenic isolates.

Characterization of F21.A, a monoclonal antibody that recognize a leucocyte surface antigen for killer whale homologue to beta-2 integrin.

The specificity of F21.A, a monoclonal antibody raised against bottlenose dolphin leucocytes, was characterized in killer whale on the basis of immunoprecipitation of a protein of 94 kDa, as well as flow cytometric analysis. While minimally expressed on resting cells, F21.A labeled a homologue to beta-2 integrin in 89-97% of PMA-activated neutrophils, 53-66% of activated monocytes, and activated B cells but not T cells. Activation of neutrophils reached its maximum 10 min after PMA stimulation. F21.A did not label intracellular stores as did both cross-reacting anti-canine CD11b and CD18, suggesting that an activation-induced conformational change would expose a neoepitope recognized by F21.A. F21.A labeling was largely inhibited by pre-incubation with plasma, suggesting a binding site closely related to that for fibrinogen. In vitro phagocytosis and respiratory burst were almost fully inhibited upon pre-incubation with F21.A, demonstrating its functional importance. This antibody is foreseen as a possible valuable diagnostic and research tool in cetacean immunology.

Cross-reactivity of human and bovine antibodies in striped dolphin paraffin wax-embedded tissues.

This study evaluates the cross-reactivity of seven anti-human and one anti-bovine antibodies in formalin-fixed, paraffin-embedded tissue samples of liver and mesenteric lymph nodes of 13 striped dolphins (Stenella coeruleoalba). Four antibodies (CD3, IgG, lysozyme and S100 protein) reacted with striped dolphin lymph nodes in a similar pattern to that observed in the species of origin. The anti-human MHC class II mAb reacted strongly with macrophages and dendritic-like cells of striped dolphins, whereas a small number of lymphocytes were labelled with this antibody. These antibodies were used to study the immunophenotype of the inflammatory infiltrated in non-specific chronic reactive hepatitis (eight cases) and chronic parasite cholangitis (two cases) and normal liver (three cases) of striped dolphins. Non-specific chronic reactive hepatitis was composed of inflammatory infiltration of CD3+ T lymphocytes and IgG+ plasma cells in portal spaces and hepatic sinusoids. Lymphonodular aggregates observed in chronic parasitic cholangitis showed a cellular distribution similar to that found in lymph node cortex, including the presence of S100+ and MHC class II+ dendritic-like cells in lymphoid follicles and interfollicular areas. This result suggests that those inflammatory infiltrates are highly organised to enhance antigen presentation to B and T cells.

Monoclonal antibodies to lymphocyte surface antigens for cetacean homologues to CD2, CD19 and CD21.

CD2 is a pan-T cell marker, while CD19 and CD21 are important molecules in signal transduction of B lymphocytes. CD19 and CD21 are both present on mature B cells, while CD19 is also present in developing B cells and plasma cells. Monoclonal antibodies (mAbs) against cetacean lymphocyte putative homologues to CD2 (two different antibodies), CD19 and CD21 were characterized. The proteins immunoprecipitated were as follows: F21.I (putative anti-CD2), 43 and 59kDa; F21.B (putative anti-CD19), 83 and 127kDa; F21.F (putative anti-CD21), 144kDa. The second putative anti-CD2 (F21.C) selectively inhibited the binding of F21.I. Both the putative anti-CD2 (T cell markers) stained T-cell zones on lymph node sections, while both the B cell markers (putative CD19 and CD21) stained B-cell zones. F21.B and F21.F were absent from thymus single cell suspension but labeled 63 and 65% mesenteric lymph node lymphocytes, respectively, while both F21.C and F21.F were present on 100% thymocytes and fewer lymph node lymphocytes. B and T cell markers were mutually exclusive on double labeling using flow cytometry. These mAbs are foreseen as possible valuable diagnostic and research tools to assess immune functions of captive and wild cetaceans.

Measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion.

Killer whales and sea otters maintained in captivity are the subjects of routine health monitoring programs, and interest in immunologic studies in sea otters has been rising recently in response to potential impacts from infectious disease and environmental pollution on the threatened southern sea otter population. Development of species-specific reagents for immunologic studies in these two marine mammals is currently in its infancy. In this study, killer whale and sea otter immunoglobulin-specific polyclonal antibodies were generated, and used to develop tests for serum Ig concentration in the killer whale (Orcinus orca) and the southern (Enhydra lutris nereis) and northern sea otter (Enhydra lutris lutris). Killer whale serum IgG was purified using caprylic acid/ammonium sulfate precipitation. Sea otter plasma IgG was purified using protein-A-agarose. Polyclonal anti-Ig antisera were produced in rabbits, and specificity confirmed by immunoelectrophoresis. Radial immunodiffusion was used to measure Ig concentration in serum or plasma samples derived from 21 captive killer whales, 18 wild and 4 captive southern sea otters and 15 wild and 4 captive northern sea otters grouped by age. Mean killer whale serum Ig concentration (+/-95% confidence interval) ranged from 15.04 +/- 3.97 g/l for animals aged 0-5 years to 26.65 +/- 9.8 g/l for animals aged >10 years. Mean sea otter serum Ig concentration (+/-95% confidence interval) ranged from 28.39 +/- 11.00 g/l for southern sub-adults to 32.76 +/- 11.58 g/l for southern adults. No significant difference in serum Ig concentration was found between southern and northern sea otters. Serum Ig concentrations in two northern sea otter pups were low compared to those of adult sea otters. The two serum Ig quantitation assays produced were highly specific and reproducible and will be useful additions to the limited number of tests available for immune function in these marine mammal species.

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.

Specific non-coplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure.

Contaminant-induced immunosuppression by organochlorines (OC), particularly polychlorinated biphenyls (PCBs), has been suspected as a cofactor in the deaths of thousands of marine mammals. One important innate defense mechanism is phagocytosis, the ability of cells to ingest extracellular macromolecules. The present study was aimed at characterizing the immunomodulatory potential of representative OCs on phagocytosis in bottlenose dolphins and beluga whales. The ability of peripheral blood leukocytes to engulf fluorescent microspheres was evaluated using flow cytometry. The immunomodulatory effects of three non-coplanar PCB congeners, 138, 153, and 180, one coplanar PCB, 169, and 2,3,7,8-TCDD and all possible mixtures (26) were tested upon in vitro exposure. In both species, all mixtures containing at least two non-coplanar PCBs significantly reduced both neutrophil and monocyte phagocytosis, with effects more marked in dolphins than in belugas. Coplanar OCs, on their own or when added to non-coplanar congeners, did not further modulate phagocytosis, suggesting an Ah receptor-independent mechanism. Concentration-response experiments with individual congeners further demonstrated a non-coplanar PCB-induced suppression of phagocytosis, while coplanar congeners produced no consistent effects. Our results suggest simple additive interactions of chemicals in a mixture. However, calculation of toxic equivalency (TEQs) failed to predict the experimentally induced immunomodulatory effects of OCs on dolphin and beluga phagocytosis, confirming the Ah receptor-independent nature of the effects on phagocytosis. Overall, our results suggest that non-AhR mechanisms may explain one facet of immunotoxicity (phagocytosis), something that is not captured using the TEQ approach. This is the first report demonstrating the immunomodulatory effects of OCs on dolphin and beluga phagocytosis, and the first overall demonstration of immunomodulatory effects on phagocytosis mediated specifically by non-coplanar PCBs.