Effects of polychlorinated biphenyls (PCB) on California sea lion (Zalophus californianus) lymphocyte functions upon in vitro exposure.

Polychorinated biphenyl (PCB) congeners are a cause for concern due to their persistence in the environment, their lipophilic properties that cause them to bio-accumulate in top predators, and their adverse effects on mammalian health. For example, the common urogenital carcinoma reported in California sea lions (Zalophus californianus) (CSL) is associated with high tissue levels of PCBs, but the mechanisms responsible for this association are unknown. This study investigated the effect of exposure to six PCB congeners and a congener mix at low and environmentally relevant concentrations on NK cell-like and T cell activity using in vitro assays on cryopreserved lymph node mononuclear cells isolated from dead CSL. Non dioxin-like congeners 153 and 180 increased lymphocyte proliferation at 5 and 10 ppm, while congener 138 decreased proliferation by up to 43% at 15 ppm. Dioxin-like PCBs 118 and 169 did not affect lymphocyte proliferation, while the effects of congener 105 depended on the mitogen concentration; these did not correlate with their predicted toxic equivalent factors. NK cell-like activity was affected only by the highest concentration of PCBs tested; it was increased by non-dioxin-like congeners 138 and 153, and decreased by dioxin-like congener 169. The PCB congener mix suggested that the effects of PCB congeners were not simply additive. Our results concur with effects of PCBs reported for other pinniped's lymphocytes and add further experimental support to the observation that dioxin-like PCBs are not the most toxic congeners for marine mammals, contrary to effects in other species. This is the first evidence of in vitro suppression of NK cell-like cytotoxicity by a dioxin-like congener in a pinniped. More importantly, the observed results suggest that PCBs can modulate the CSL immune system, increasing exposed individuals' susceptibility to viral and oncogenic challenges.

Prevalence and ultrastructure of sarcocystis in American woodcock harvested in Connecticut.

The range-wide population of American woodcock Scolopax minor has been in slow, steady decline since the late 1960s. The parasite load carried by woodcock and its possible role in the population decline has not been investigated since the early 1970s. A survey of parasites in American woodcock in Connecticut was undertaken in 2002: Sarcocystis spp. was found in 32 of 78 (42%) individuals examined. Elongate sarcocysts, 25 x 125 micro, containing numerous packed bradyzoites with distinct, tightly packed villar projections of the cyst wall, were found scattered throughout skeletal type and myocardial muscle. Sarcocystis spp. was also recorded during the earlier surveys and considered common, but was not examined with the use of electron microscopy. The present study includes the first ultrastructural description of Sarcocystis sp. in the skeletal muscle of woodcock and will serve as a basis for future comparisons in woodcock.

Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells'.

Paclitaxel is an antimicrotubule agent that induces mitotic block and apoptosis. We show for the first time that paclitaxel acts directly or mitochondria isolated from human cancer cells. In isolated yeast mito chondria, paclitaxel (15 microM) induced an 18% increase in the respiration rate, with no concomitant release of cytochrome c. In isolated neuroblas toma mitochondria, paclitaxel (10-100 microM) induced a 27-72% release o cytochrome c. Release was prevented by cyclosporin A, suggesting the involvement of the permeability transition pore. Doxorubicin did no induce cytochrome c release, whereas vinorelbine, another antimicrotu bule agent, did. Thus, antimicrotubule agents can directly affect mito chondria to induce apoptosis.

Phenotyping of beluga whale blood lymphocytes using monoclonal antibodies.

Widespread efforts are currently made to classify morphologically indistinguishable lymphocyte subpopulations in several species. In order to increase the knowledge in cetacean immunology, cross-reactivity of antibodies against bovine, human, ovine and mouse cell surface proteins was tested on beluga whale (Delphinapterus leucas) peripheral blood lymphocytes using flow cytometry. Anti-MHC class I and II as well as anti-CD2 reacted with virtually all peripheral blood lymphocytes. Anti-TCR gamma delta and anti-CD4 reacted with respectively 31% and 30% of peripheral blood lymphocytes. B lymphocytes were identified by an anti-surface IgM which was present on 6% of blood lymphocytes. Specificity of these antibodies was demonstrated by immunoprecipitation of beluga proteins with similar molecular weight to that of other species. These results could be useful for further immunotoxicological evaluation of highly versus mildly contaminated populations of belugas.

Purification of functional T lymphocytes from splenocytes of the beluga whales (Delphinapterus leucas).

In an effort to gain knowledge on immune functions in beluga whales, Delphinapterus leucas, we have used two physical methods for the purification of T lymphocytes of spleen cells. Isolation by sheep red blood cells (SRBC) rosetting and by adherence on nylon wool columns were tested. SRBC-rosetting gave unreliable results in obtaining purified T cells. Therefore, the purification of T cells was done using nylon wool columns. Less than 3% of the IgM(+) B cells remained in effluent populations. In the later population, 45% gave positive staining with mouse anti-human CD4 allowing us to verify functionality of the cells. The study of calcium mobilization and tyrosine kinase activation, mediated by CD4 cross-linking permitted verification of the functionality of cells. We also showed that upon activation with mitogens, beluga T cells upregulate the density of MHC class II molecules on their surfaces. CD4 cross-linking with a specific antibody inhibited the proliferation response. Overall, the activation of beluga whales lymphocytes did not differ markedly from what is known in other species. This study can help in the groundwork for functional investigation of the beluga whale's immune system.

Possible mechanisms of action of environmental contaminants on St. Lawrence beluga whales (Delphinapterus leucas).

A small isolated population of beluga whales (Delphinapterus leucas) that are highly contaminated by pollutants, mostly of industrial origin, resides in the St. Lawrence estuary, Québec, Canada. Overhunting in the first half of the century was the probable cause for this population to dwindle from several thousand animals to the current estimate of 500. The failure of the population to recover might be due to contamination by organochlorine compounds, which are known to lead to reproductive failure and immunosuppression in domestic and laboratory animals and seals. Functional and morphological changes have been demonstrated in thyroid gland and adrenal cortex in many species exposed to organochlorinated compounds, including seals. Morphological lesions, although different, were also found in belugas. Functional evaluation of thyroid and adrenal glands of contaminated (St. Lawrence) versus much less contaminated (Arctic) belugas is currently under way. Necropsy of St. Lawrence belugas showed numerous severe and disseminated infections with rather mildly pathogenic bacteria, which suggests immunosuppression. Organochlorine compounds and other contaminants found in beluga whales cause immunosuppression in a variety of animal species including seals. Thirty-seven percent of all the tumors reported in cetaceans were observed in St. Lawrence beluga whales. This could be explained by two different mechanisms: high exposure to environmental carcinogens and suppression of immunosurveillance against tumors. Overall, St. Lawrence belugas might well represent the risk associated with long-term exposure to pollutants present in their environment and might be a good model to predict health problems that could emerge in highly exposed human populations over time.

Caulerpenyne from Caulerpa taxifolia has an antiproliferative activity on tumor cell line SK-N-SH and modifies the microtubule network.

Caulerpenyne, the major secondary metabolite synthesized by the green marine alga Caulerpa taxifolia, is cytotoxic against several cell lines. To identify possible targets of this toxin, we investigated the effect of caulerpenyne on the neuroblastoma SK-N-SH cell line. Caulerpenyne induced an inhibition of SK-N-SH cell proliferation with an IC50 of 10 +/- 2 microM after 2 hr of incubation. We observed no blockage in G2/M phase and an increase in cell death. On immunofluorescence microscopy, caulerpenyne affected the microtubule network in SK-N-SH cell line; we observed a loss of neurites and a compaction of the microtubule network at the cell periphery. In vitro, after 35 min of incubation, caulerpenyne inhibited the polymerization of pig brain purified tubulin or microtubule proteins, with an IC50 of 21 +/- 2 microM and 51 +/- 6 microM respectively. Analysis by electron microscopy indicated that caulerpenyne induced aggregation of tubulin, which may be responsible for inhibition of microtubule polymerization and bundling of residual microtubules.

Functional characterization of a swine CD4(+)/CD8(+) double positive lymphoblastoid T-cell line with a CD25(+)/CD45RA(-) phenotype generated in vitro with interleukin-2.

A dual expressing (CD4(+)/CD8(+)) porcine lymphoblastoid T-cell line (pIL-2d) generated from peripheral blood mononuclear (MN) cells shown to be highly responsive to exogenous interleukin-2 (IL-2) was characterized. The swine MN cells were initially stimulated with concanavalin A (Con A), and sub-passaged using decreasing amounts of conditioned medium (CM), which was prepared from culture fluids of Con A activated porcine MN cells, until a steady growth was observed. The resulting pIL-2d cells require exogenous IL-2 from CM and are highly responsive to recombinant human IL-2 (rhIL-2). The pIL-2d cells exhibited a specific, dose-dependent proliferative response to stimulation with IL-2. The specificity of this proliferative response was confirmed to be IL-2 induced by its inhibition with an anti-swine IL-2 receptor (alpha-swIL-2R) monoclonal antibody (mAb). Furthermore, the pIL-2d cells are highly responsive to exogenous IL-2 contained in culture fluids derived from antigen-driven blastogenic tests performed with lymphocytes of vaccinated swine. This property makes the pIL-2d cells an ideal functional adjunct to immunochemical or molecular tests that are commonly used to measure total porcine IL-2. Interestingly, the phenotype of the pIL-2d cells after five or more passages was shown by flow cytometric analysis to be CD4(+)/CD8(+)/CD45RA(-)/CD25(+) and to remain unchanged thereafter. Although, the mechanism of selection and maintenance of the CD4(+)/CD8(+) DP cells developed here remains unclear, our data suggest that an oligoclonal or polyclonal expansion and maintenance of cells of this phenotype was mediated by exogenous IL-2.

Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry.

Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10,000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.

Immune functions in beluga whales (Delphinapterus leucas): evaluation of natural killer cell activity.

Natural killer (NK) activity, an important non-specific defense mechanism against viral infections and tumors, was demonstrated in beluga whales using two different methods: 51Cr release and flow cytometry. Using the 51Cr release assay, NK activity in belugas was shown to be higher against K-562 than against YAC-1 cell lines. Moreover, it was enhanced by the addition of human recombinant interleukin-2 with both cell lines. NK activity evaluated by flow cytometry in the peripheral blood of eight belugas increased when the effector:target cell (E:T) ratio increased, and averaged 13.9% +/- 3.8% (range 9.9% to 17.8%) at an E:T ratio of 100:1. While NK activity could be readily detected using both methods, the lack of radio-isotopes and related laboratory room make the flow cytometric method a viable and safe alternative. The evaluation of this function in cetaceans could lead to a better understanding of the early events that lead to viral epizootics in populations of marine mammals in different parts of the world, as well as to the high prevalence of neoplasms in St. Lawrence beluga whales.

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.

Effects of in vitro exposure of beluga whale leukocytes to selected organochlorines.

The effects of in vitro exposure to different organochlorines were evaluated on immune functions of beluga whale peripheral blood leukocytes and splenocytes. The effects of different concentrations of four different congeners of PCBs (138, 153, 180, and 169) as well as two DDT metabolites (p,p'-DDT and p,p'-DDE) were evaluated on phagocytosis and cell proliferation. The effects of dioxins and mixtures of organochlorines were also evaluated on cell proliferation. The different compounds tested had no marked effect on phagocytosis. PCB 138 and p,p'-DDT, but not PCB 153, PCB 180, PCB 169, and p,p'-DDE, reduced significantly the proliferative response of beluga splenocytes cultured either with or without phytohemagglutinin A (PHA). Proliferation of beluga splenocytes was not markedly affected by exposure to 5 ppm of PCB 138, 153, 180, and 169 separately. Exposure to a mixture of congeners 138, 153, and 180 (5 ppm each) significantly reduced splenocytes proliferation, but not the mixture of congeners 138, 153, 180, and 169 (5 ppm each). TCDD did not affect cell proliferation in our study. The reduced proliferation of beluga cells exposed in vitro to mixtures of organochlorines at concentrations in the range of those observed in tissues of St. Lawrence belugas might provide a basis to support the hypothesis that contaminants induce immunosuppression in these animals.

Mycobacterium marinum dermatitis and panniculitis with chronic pleuritis in a captive white whale (Delphinapterus leucas) with aortic rupture.

A 16-year-old female white whale, Delphinapterus leucas, died after nearly 18 months of chronic lymphopenia and pyogranulomatous dermatitis. Necropsy revealed rupture of the aorta with hemorrhage into the cranial mediastinum and between fascial planes of the ventral neck musculature. Multiple foci of ulcerative dermatitis and panniculitis were present across the thorax and abdomen and surrounded the genital folds. In addition, there was a chronic proliferative pleuritis with over 20 liters of histiocytic exudate in the thoracic cavity. Acid-fast bacteria consistent with Mycobacterium sp. were identified in sections of skin lesions and in cytospins of pleural exudate. Cultures of pleura and 1 skin lesion collected at necropsy yielded sparse growth of an acid-fast bacillus with colony characteristics and morphology consistent with Mycobacterium marinum. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed the presence of M. marinum DNA in samples of skin. This is the first documented occurrence of mycobacteriosis in a white whale and is a unique presentation of mycobacterial dermatitis and panniculitis with chronic pleuritis in a cetacean. The improved PCR-RFLP protocol utilized in this case unifies techniques from several protocols to differentiate between species of Nocardia and rapidly growing mycobacteria clinically relevant to aquatic animals.

Pathology and toxicology of beluga whales from the St. Lawrence Estuary, Quebec, Canada. Past, present and future.

An indigenous population of 450-500 beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence Estuary has been exposed chronically for more than 50 years to a complex mixture of industrial pollutants including organochlorinated compounds (OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals. From 1983 to 1990, we have necropsied 45 well preserved carcasses out of a total of 120 beluga whales reported dead over this period. Of these 45 animals, nine were affected by 10 malignant neoplasms. Fifteen animals (33%) were affected by pneumonia. Milk production was compromised in eight of 17 mature females (41%), by inflammatory changes (seven animals) and cancer (one animal) which affected the mammary glands. Opportunistic bacteria were found in pure culture, and/or in significant amounts in at least two organs in 20 belugas (44%). The concentrations of both total PCBs and highly chlorinated PCB congeners were much higher in St. Lawrence animals than in Arctic beluga whales. OC-induced immunosuppression has been repeatedly demonstrated in a wide variety of animal species. Therefore, it is probable that the immune functions of St. Lawrence beluga whales are impaired. Benzo[a]pyrene adducts were detected in 10 of the 11 St. Lawrence beluga whales of which tissues (six livers, 10/11 brains) were analyzed by a method based on HPLC. No such adducts were found in four Arctic animals. Since benzo[alpha]pyrene is one of the most potent chemical carcinogens known to man, these compounds might be responsible for some of the cancers observed in that population. Overall, our findings contrast vividly with those of others who found that cancers are exceedingly rare in free-ranging odontocete populations and that the major causes for mortalities in these populations are bacteria, parasites, and trauma.

Non-neoplastic lesions in beluga whales (Delphinapterus leucas) and other marine mammals from the St Lawrence Estuary.

In a 3-year (1988-1990) pathological study, 24 carcasses of beluga whales from the St Lawrence Estuary, Québec, Canada, showed numerous severe lesions, many of which had never been reported in cetaceans. The most common lesions were found in the digestive tract (21 animals) and consisted mainly of periodontitis and of erosions and ulcers in the oesophagus and the first two gastric compartments. Pneumonia, usually of parasitic origin, was also a common finding (12 animals). The adrenal glands often contained nodules (five animals) or cysts (seven animals), and mastitis was observed in five females. Overall, the incidence of degenerative, infectious, hyperplastic or necrotic lesions, in addition to numerous neoplasms described in another paper, was considerably higher than that found in marine mammals elsewhere or in other species of marine mammal from the same waters.

Immune function of bovine leukocytes after in vitro exposure to selected heavy metals.

OBJECTIVE: To study effects of in vitro exposure of bovine leukocytes to mercury, cadmium, and lead on phagocytosis, natural killer cell activity, and lymphocyte proliferation. SAMPLE POPULATION: Leukocytes from 6 nonpregnant Holstein heifers. PROCEDURE: Leukocytes were exposed in vitro to the aforementioned metals, and leukocyte functions were assessed. RESULTS: Phagocytosis was suppressed by 10(-5) to 10(-7) M CdCl2 and by 10(-5) and 10(-6) M HgCl2, but not 10(-7) M HgCl2 nor 10(-4) to 10(-6) M PbCl2. Spontaneous and concanavalin A- or phytohemagglutinin-stimulated proliferation of metal-treated bovine blood mononuclear cells was not significantly different from that of nontreated control cells, except for enhanced spontaneous proliferation in response to 10(-5) M HgCl2. When proliferation was expressed as a stimulation index, a dose-dependent increase of spontaneous proliferation was observed in response to exposure to HgCl2 and PbCl2. Compared with response to 10(-6) or 10(-7) M CdCl2, reduction of mitogen-induced and spontaneous proliferation was observed on exposure to 10(-5) M CdCl2. Natural killer cell activity against YAC-1 target cells, evaluated by flow cytometry, was decreased only in cells exposed to 10 M HgCl2. CONCLUSION AND CLINICAL RELEVANCE: Bovine leukocytes are susceptible to the immunomodulatory effects of in vitro exposure to heavy metals at concentrations equal to or higher than those at which similar effects are seen for leukocytes from most other animal species for which data are available for comparison. Exception is phagocytosis, which is severely affected by low concentrations of CdCl2 and HgCl2 in cattle. Reduction of defense mechanisms on exposure to metals could lead to increased susceptibility to potential pathogens.

True hermaphroditism in a St. Lawrence beluga whale (Delphinapterus leucas).

A hermaphrodite beluga whale (Delphinapterus leucas) was found in the St. Lawrence Estuary, Québec, Canada. This animal had two testicles, two separate ovaries, and the complete ducts of each sex; cervix, vagina and vulva were absent. Mature spermatozoa were found in the lumen of seminiferous tubules in the testicles, and numerous involuted corpora lutea were recognized in the ovaries. This represents the first case of true hermaphroditism in a cetacean, and is the fourth hermaphrodite mammal with two testicles and two separate ovaries.

Uterine adenocarcinoma with abdominal carcinomatosis in a beluga whale.

A case of uterine adenocarcinoma is reported in a 26-yr-old, free-ranging beluga whale (Delphinapterus leucas) from the St. Lawrence estuary (Quebec, Canada). This neoplasm appeared as a segmental stenotic thickening of the left uterine horn composed of well differentiated, but disorganized and infiltrative, glandular structures surrounded by an extensive scirrhous stroma. Abdominal carcinomatosis was observed on the mesosalpinx and on the serosal aspect of the gastric compartments. This is the first report of a malignancy originating in the uterus of a cetacean.

Adrenal hyperplastic and degenerative changes in beluga whales.

Thirty stranded beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary (Quebec, Canada) population and five animals from the Hudson Bay aboriginal hunt (North-west Territories, Canada) were examined. Twenty one animals from the St. Lawrence Estuary had mild to severe adrenal lesions and four whales from the Hudson Bay population were affected by minimal adrenal changes. Cortical hyperplasia was observed in 24 adult beluga whales all from the St. Lawrence Estuary. Bilateral cortical cysts and cellular vacuolar degeneration were observed in the adrenal glands of 19 beluga whales from both populations. The cysts, filled with a cortisol-rich liquid, were present in both sexes. Beluga whales with adrenal cysts were significantly older than animals without cysts, and the severity of the lesions increased with age. Nodular hyperplasia of the medulla was observed in seven of the beluga whales, all from the St. Lawrence Estuary population. All lesions could be part of a normal aging process. The adrenocortical lesions might be due to stress or adrenocorticolytic xenobiotics, while the medullary hyperplasia might be caused by hypoxia or exposure to estrogenic xenobiotics.

Bone fluoride concentrations in beluga whales from Canada.

Beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary have been reported to have dental and bone abnormalities. To determine whether these lesions could be caused by high exposure to fluorides, we measured bone fluoride levels in eight beluga whales stranded on the shores of the St. Lawrence Estuary (Quebec, Canada), and in nine beluga whales killed by Inuit hunters in the Hudson Bay (North Western Territories, Canada). In both groups, fluoride concentrations were higher than those found in terrestrial mammals intoxicated by fluorides. Unexpectedly, fluoride concentration was significantly higher in beluga whales from the Hudson Bay (mean +/- SD: 10.365 +/- 1.098 ppm) than in beluga whales from the St. Lawrence Estuary (4.539 +/- 875 ppm) and was positively correlated with age in the latter population. Differences in diet might explain the differences in fluoride concentrations found between these two populations.