Pathologic Findings and Trends in Mortality in the Beluga (Delphinapterus leucas) Population of the St Lawrence Estuary, Quebec, Canada, From 1983 to 2012.

An isolated population of beluga (Delphinapterus leucas) inhabits the St Lawrence Estuary, Quebec, Canada. This population has failed to recover despite the prohibition of hunting >30 years ago, suggesting the presence of other limiting factors. The authors summarize the reported causes of death and propose risk factors to explain the lack of recovery of this population. From 1983 to 2012, a total of 472 beluga were found stranded. Complete necropsies were carried out on 222 beluga, including 178 adults, 25 juveniles, and 19 newborn calves. Infectious diseases, the most prevalent cause of mortality in this population, accounted for the death of one-third of all beluga (32%). Verminous pneumonia was the cause of mortality of 13 juvenile beluga (52% of juvenile beluga). A total of 39 malignant neoplasms, diagnosed in 35 beluga, caused the death of 31 beluga (20% of beluga >19 years old). Median age at diagnosis of cancer was 48 years (range, 30-61 years). Dystocia and postpartum complications were the cause of death in 18 beluga, accounting for 19% of the females >19 years old examined. The occurrence of parturition-associated complications, as well as mortality of calves <1 year old, have increased recently in this population and may be the probable cause of the recent decrease in the size of this population. One of the hypotheses proposed to explain the unusually high occurrence of some of the pathologic conditions observed in this population is chronic exposure to environmental contaminants.

Preliminary evaluation on the use of homing pigeons as a biomonitor in urban areas.

This study evaluates the usefulness of homing pigeons as a biomonitor of polycyclic aromatic hydrocarbons (PAHs) in urban environments. The mean concentrations of total PAHs in liver and lung tissues were greater in pigeons from Beijing compared to pigeons from Chengdu, however, this difference was only statistically significant for PAH concentrations in liver tissue (P < 0.05). Similarly, the severity of anthracosis or pneumoconiosis in lung tissue and hepatitis in liver tissue was greater in pigeons from Beijing compared to pigeons from Chengdu. Low molecular weight PAHs dominated the contribution of individual PAHs in both tissues. Significant differences (P < 0.05) were observed for most low and moderate molecular weights PAHs in liver and for some low and high molecular weights PAHs in lung between the two cites. The profile patterns of individual PAHs were similar between lung tissue of pigeons and between local ambient airs in summer for both cities, whereas the profile patterns between liver tissue and pigeon food were less similar. These data suggest that homing pigeons may be of value as a biomonitor of environmental pollution in urban areas.

Lyme snap: A feasibility study of on-line declarations of erythema migrans in a rural area of France.

The incidence of Lyme borreliosis remains a matter of debate, but it can be estimated using the incidence of erythema migrans (EM), which is pathognomonic of the first phase. The aim of this prospective pilot study was to assess the feasibility of the on-line declaration of EM in rural areas where the incidence of Lyme borreliosis was previously estimated at 85 per 100,000 inhabitants per year. The study was limited to a rural area (Les Combrailles, Auvergne) of approximately 52,800 inhabitants and was preceded by an information campaign for the inhabitants and the healthcare professionals. Patients who sent a photo of the suspected EM by email or MMS message between April 2017 and April 2018 and who accepted to answer a questionnaire were included in the study. Two physicians then evaluated the quality of the photographs and the probability of EM. In parallel, the number of EM seen by physicians and pharmacists in the area over the given period was recorded. Out of the 113 emails and MMS messages received, 73 people were outside of the trial area or period and 9 did not complete the questionnaire. The photos of the remaining 31 people were analysed. The median age was 51.5 years old ([38-58] IQR) and 18 (58%) were women. Seven people (25%) stated that they did not have a smartphone and in 9 cases (29%) the photo was sent by a third party. The quality of the photos was considered very good in 22 (71%) cases, good in 7 (23%) cases, and average in 2 (6%) cases. The probability of EM was determined to be strong or possible in 12 (38%) cases, i.e. an estimated incidence of 22.7 per 100,000 inhabitants. Over the study period, 40 physicians and 20 pharmacists were contacted on a monthly basis. A median of 5 physicians [3;7] and 4 pharmacists [3 ;7] answered each month for a total of 18 and 36 declared EM respectively. The EM (strong probability/possible) collected by on-line declaration and those declared by healthcare professionals were all sent between April and October 2017. The total time spent on the information campaign and collection has been estimated at 265 h (divided between 10 people) for an overall cost of 10,669 Euros. The incidence of EM recorded by on-line self-declaration in our study seems to be lower than in previous studies, the under-reporting was probably linked to the low use of new technologies in the rural areas. Increasing the human resources and finances appears difficult to achieve in practice over a longer time period but the development of an application for the automatic recognition of EM could be one method for a more exhaustive collection in the long term and at lower cost.

Toxoplasmosis in captive dolphins (Tursiops truncatus) and walrus (Odobenus rosmarus).

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. Toxoplasma gondii infection was detected in captive marine mammals at a sea aquarium in Canada. Antibodies to T. gondii were found in all 7 bottlenose dolphins (Tursiops truncatus) tested. Two of these dolphins, as well as a walrus (Odobenus rosmarus) at the facility, died. Encephalitis and T. gondii tissue cysts were identified in histological sections of the brain of 1 dolphin (dolphin no. 1). Another dolphin (dolphin no. 2) had mild focal encephalitis without visible organisms, but viable T. gondii was isolated by bioassay in mice and cats from its brain and skeletal muscle; this strain was designated TgDoCA1. The PCR-RFLP typing using 11 markers (B1, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) identified a Type II strain. The DNA sequencing of B1 and SAG1 alleles amplified from TgDoCA1 and directly from the brains of dolphin no. 1 and the walrus showed archetypal alleles consistent with infection by a Type II strain. No unique polymorphisms were detected. This is apparently the first report of isolation of T. gondii from a marine mammal in Canada.

One-Year stable perovskite solar cells by 2D/3D interface engineering.

Despite the impressive photovoltaic performances with power conversion efficiency beyond 22%, perovskite solar cells are poorly stable under operation, failing by far the market requirements. Various technological approaches have been proposed to overcome the instability problem, which, while delivering appreciable incremental improvements, are still far from a market-proof solution. Here we show one-year stable perovskite devices by engineering an ultra-stable 2D/3D (HOOC(CH2)4NH3)2PbI4/CH3NH3PbI3 perovskite junction. The 2D/3D forms an exceptional gradually-organized multi-dimensional interface that yields up to 12.9% efficiency in a carbon-based architecture, and 14.6% in standard mesoporous solar cells. To demonstrate the up-scale potential of our technology, we fabricate 10 × 10 cm2 solar modules by a fully printable industrial-scale process, delivering 11.2% efficiency stable for >10,000 h with zero loss in performances measured under controlled standard conditions. This innovative stable and low-cost architecture will enable the timely commercialization of perovskite solar cells.

Evaluation of PCR and ELISA assays for screening clinical trial subjects for replication-competent retrovirus.

Gene delivery via murine-based recombinant retroviral vectors is currently widely used in gene therapy clinical trials. The vectors are engineered to be replication defective by replacing the structural and nonstructural genes of a cloned infectious retrovirus with a therapeutic gene of interest. The retroviral particles are currently generated in packaging cell lines, which supply all retroviral proteins in trans. Recombination between short homologous regions of the retroviral vector and packaging cell line elements can theoretically generate replication-competent retrovirus (RCR) and hence the Food and Drug Administration (FDA) requires the monitoring of clinical trial subjects for the presence of RCR. Sensitive polymerase chain reaction (PCR) assays have been used for the detection of murine leukemia virus (MLV) nucleotide sequences in peripheral blood mononuclear cells (PBMCs). A novel serological enzyme-linked immunosorbent assay (ELISA) for the detection of anti-MLV specific immunoglobulin (Ig) has been developed to be used as an alternative to the PCR assay. Both assays were used to monitor human immunodeficiency virus (HIV)-positive clinical trial subjects who had received multiple injections of HIV-IT (V), a retroviral vector encoding HIV-1 IIIBenv/rev. Western blot analysis and an in vitro vector neutralization assay were used to characterize further a subset of serum samples tested by ELISA. Results show no evidence of RCR infection in clinical trial subjects. PCR and ELISA assays are discussed in terms of their advantages and limitations as routine screening assays for RCR. The PCR assay is our current choice for monitoring clinical trial subjects receiving direct administration of vector, and the ELISA is our choice for those receiving ex vivo treatment regimens.

Anti-vector immunoglobulin induced by retroviral vectors.

Replication-incompetent retroviruses have been employed as gene therapy vectors in experimental settings for more than a decade. More recently, these vectors have been tested in the clinic as immunotherapeutic agents and anticancer agents. One potential problem with the use of such vectors is the possible development of immune responses directed against the vector particles themselves. Here, we examine immunoglobulin (Ig) responses specific for retroviral vectors derived from murine leukemia virus (MLV). Anti-MLV Ig is seen following intramuscular (i.m.) administration of retroviral vectors in mice, and in nonhuman primates; as expected, these responses are dependent upon the vector dose delivered. Furthermore, serum from vector-treated animals is capable of partially neutralizing vector-mediated transduction of target cells in an in vitro assay. Nevertheless, even in the presence of significant levels of anti-vector Ig in vivo, i.m. administration of retroviral vector is still capable of driving both Ig and cytotoxic T lymphocyte (CTL) responses specific for vector-encoded gene products. This work suggests that although retroviral vectors may readily induce immune responses directed against the vector particles themselves, such responses will not significantly affect the efficiency of these vectors in an immunotherapeutic protocol.

Plasmid pFCE4: a new system of Escherichia coli expression-modification vectors.

Two versatile expression-modification vectors were obtained by inserting the origin of replication (ori) of phage f1 into the expression vector pOTS. The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4+ and pFCE4-) and excrete it into the medium as virus-like particles following infection with phage f1. These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations. The human IFN alpha-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Expression of the human apolipoprotein AI gene fused to the E. coli gene for beta-galactosidase.

The human apoAI gene was expressed in E. coli by in-frame fusion to a modified beta-galactosidase gene present in plasmid pUR291. The fused beta-galactosidase-apoAI gene product was expressed at a high level and was recognized by an anti-human apoAI antiserum. Besides the fused protein, at least one degradation product having an Mr similar to that of beta-galactosidase was present in high amounts in bacterial extracts. These results and those of a pulse-chase experiment indicate that degradation took place only in the apoAI moiety of the chimeric protein.

Phase I trial of retroviral vector-mediated interferon (IFN)-gamma gene transfer into autologous tumor cells in patients with metastatic melanoma.

The purpose of this study was to determine the safety of treating melanoma patients with retroviral vector-mediated interferon (IFN)-gamma gene-transduced autologous tumor cells. We designed a phase I study, in which irradiated, autologous, transduced melanoma cells expressing the IFN-gamma gene were injected subcutaneously every 2 weeks with escalating cell doses for six injections. Tumor tissue was harvested from 58 patients with metastatic melanoma. Twelve patients had sufficient expansion of autologous tumor (0.56-160 x 10(7) cells) and adequate IFN-gamma expression after gene transduction (2-79,000 U/10(6) cells/24 hours) for injections. Five patients received injections. No toxicity was attributed to the IFN-gamma retroviral vector in the patients injected. One of the injected patients remains disease-free after 13 injections, following the surgical removal of brain, adrenal, and lung metastases. We found that injections of autologous tumor cells transduced by IFN-gamma gene were well tolerated. However, the ability to develop primary autologous melanoma cell lines was limited, and only a minority of patients were injected.

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation.

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

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.

Neoplastic and nonneoplastic hepatic changes in lake whitefish (Coregonus clupeaformis) from the St. Lawrence River, Quebec, Canada.

As part of a survey of fish diseases, lake whitefish (Coregonus clupeaformis) were collected in fall 1995 from the St. Lawrence River 15 km upstream of Quebec City, Quebec, Canada, to assess the prevalence of liver lesions. A total of 141 fish were captured and necropsied, and three standard sections of liver were taken for histological examination. Prevalences of altered hepatocyte foci, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma were 0.7%, 2.1%, 0.7%, and 2.1%, respectively. Thus, the overall prevalence of liver neoplasia was 4.9% (7/141). Hepatic tumors were only observed in fish 7 years old or older. Fish age was significantly and positively correlated with the index assessing the number and size of macrophage aggregates (p<0.001; rs = 0.16). Hepatocyte vacuolation, anisokaryosis, lymphocytic infiltration, and bile duct hyperplasia were also observed but were not related to the age, length, sex, or condition factor of the fish. These results represent the first report on a series of hepatic tumors in a wild salmonid species.

Single-tube heminested PCR coupled with 'touchdown' PCR for the analysis of the walleye dermal sarcoma virus env gene.

The nucleotide sequences of geographically distant isolates of a newly characterized retrovirus of fish, the walleye dermal sarcoma virus (WDSV) were compared. Primers were designed based on the nucleotide sequence of a single molecular clone isolated from a tumor collected from a fish in New York state, USA. Conventional polymerase chain reaction (PCR) did not allow the satisfactory amplification of WDSV isolates from a different geographic region (Quebec, Canada), probably owing to the high genetic variability of retroviruses. To allow the successful amplification of Quebec WDSV isolates, heminested PCR and 'touchdown' PCR were combined in a highly sensitive, short and simple protocol taking place in a single tube, thus minimizing cross contamination. This technique allowed the first unambiguous detection of a fish retrovirus in Canada and the phylogenetic analysis of various geographic isolates of this virus. Other potential applications for this technique include PCR amplification using degenerate primers, and amplification of templates with some expected degree of genetic variability.

Anatomy of the heart of the beluga whale (Delphinapterus leucas).

The heart of the beluga whale (Delphinapterus leucas) is described from the dissection of seven specimens. As in most whales the heart is characterized by a transverse broadness and a flatness of the ventricles from one surface to the other and by an apex formed by both ventricles. Heart size parameters are used for comparison with parameters of other marine and land mammals. The heart width index (heart height/heart circumference) averages 31.3 in comparison to 28.7 for the Weddell seal and 39.0 for the felids. The right ventricle is relatively long and narrow with a mean length index (TS/heart height) of 64.7 and a mean breadth index (TP/heart height) of 38.7. These heart parameters are discussed in functional terms.

Arterial and venous vasculature of the heart of the beluga whale (Delphinapterus leucas).

The arteries and veins of the heart of the beluga whale (Delphinapterus leucas) are described from the dissection of nine specimens. The arterial distribution is composed of the basic mammalian pattern of two major vessels, the left and right coronary arteries, which supply the cardiac tissue. The venous drainage is provided by three major systems which are the great, middle, and small cardiac veins. The vascular characteristics of the heart of the beluga whale are the marked sinuosity of both coronary arteries and their main branches, the numerous large interarterial anastomoses between major vessels, and the duplication of vessels in parallel branches. These characteristics are discussed in functional terms and correlated with the diving ability of the species.

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 mitogen-induced blastic transformation of lymphocytes from peripheral blood, spleen and thymus.

A quantitative assay was developed to evaluate mitogen-induced lymphoblastic transformation in beluga whales (Delphinapterus leucas) using peripheral blood mononuclear cells, splenocytes and thymocytes. Optimal concentrations of four different mitogens (Con-A, PHA, LPS and PWM) were determined with the use of standard curves. Addition of human recombinant IL-2 (rhIL-2) after 48 h in culture with the different mitogens suggests that Con-A, PHA and PWM, but not LPS, stimulate T cells in belugas, as they do in other animal species. The addition of 2-mercaptoethanol did not enhance significantly the proliferation of cells stimulated by Con-A, PHA and LPS, while it did with the cells stimulated by PWM and those cultured without mitogen. The proliferative response of cells was suppressed when the culture medium was supplemented by beluga serum instead of fetal calf serum. This assay will be useful to assess the status of the immune functions in different populations of beluga whales as well for further in vitro immunotoxicological experiments.