Immunogenicity and efficacy of recombinant subunit vaccines against phocid herpesvirus type 1

Phocid herpesvirus type 1 (PhHV-1) is an alpha-herpesvirus that causes significant morbidity and mortality among young and immunocompromised harbour seals (Phoca vitulina) and therefore represents a major problem for seal rehabilitation centres. Consequently, there is a need for a safe and effective PhHV-1 vaccine. We tested an ISCOM-based recombinant PhHV-1 gB vaccine alone (gB) or with the addition of recombinant PhHV-1 gD (gBD) for (i). immunogenicity and protective efficacy against feline herpesvirus (FHV) infection in cats and (ii). their immunogenicity in seals. The FHV-cat model was chosen based on the close antigenic relationship between PhHV-1 and FHV. Upon challenge, all vaccinated (gB and gBD) cats excreted significantly less FHV (P<0.01) and gBD vaccinated cats showed less weight loss (P=0.05) than the mock-vaccinated cats. However, adding gD to the gB vaccine did not result in significantly better protection. Based on these data, immunogenicity studies in seals under rehabilitation were performed with the gB vaccine only. To this end, gB vaccine was tested at two different doses (20 or 40 microg). PhHV-1 specific antibody titres and in vitro proliferative T cell responses were measured in all seals upon vaccination. No differences were observed in antibody titres between seals vaccinated with either 20 or 40 microgB, but the higher gB concentration did result in higher specific proliferative T cell responses (P<0.01). Based on the close antigenic relationship between PhHV-1 and FHV, the safety and efficacy data in the FHV-cat model, and the immunogenicity data in the vaccinated seals, the gB based vaccine is expected to be safe and effective in protecting against PhHV-1 related disease in harbour seals.     

Genome sequence based, comparative analysis of the fluorescent amplified fragment length polymorphisms (FAFLP) of tubercle bacilli from seals provides molecular evidence for a new species within the Mycobacterium tuberculosis complex.

Tuberculosis in seals is caused by a member of the Mycobacterium tuberculosis complex referred to as the 'seal bacillus'. Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was applied to isolates from four Australian and six Argentinean seals and compared with FAFLP pattern for standard strains belonging to the M. tuberculosis complex. The FAFLP profiles derived from EcoRI/MseI restricted fragments of blind coded DNA samples differentiated the seal bacillus from other members of the M. tuberculosis complex. According to the phylogenetic analysis performed using FAFLP data, seal bacilli appear to have diverged significantly from other members of the M. tuberculosis complex. We describe the suitability of a panel of 19 highly polymorphic markers for rapid identification and comparative genomic analyses of the seal bacillus strains. It is likely that these bacilli got separated from the M. tuberculosis lineage as a result of different insertion deletion events occurring on a genome wide scale. Our analysis reveals that the seal bacillus and M. bovis are genetically related and therefore, might have originated from a common ancestor. Our data additionally support the hypothesis that seal bacillus occupies a unique taxonomic position within the M. tuberculosis complex.     

Mercury in canadian seals

Summary The mercury concentration was determined in ringed and bearded seal claws, tissues from a harbour seal, tissues obtained from 6 gray seals of different sizes and ages, tissues from ten female harp seals and their pups, and a female gray seal and her fetus. It was suggested that the mercury concentration of the fur and claws of seals could be used as indicators of the degree of the seals' mercury contamination. Preliminary results indicate that mercury contamination in these species, from the same location, increases with size.The methylmercury concentration of seal pups was lower than that of their mothers. The fetus of the seal did not show a preference for mercury over that of the mother's tissues. It was suggested that seals may possess enzyme systems that demethylate methylmercury.     

A candidate phocid herpesvirus vaccine that provides protection against feline herpesvirus infection.

Phocid herpesvirus type 1 (PhHV-1) causes significant morbidity and mortality among young and immunocompromised harbour seals. Therefore, the availability of an effective PhHV-1 vaccine would be of importance for orphanages and seal rehabilitation centres. Since possibilities to test PhHV-1 candidate vaccines in the target species are limited, a suitable animal model is needed. Given the close genetic and antigenic relationships between PhHV-1 and feline herpesvirus (FHV), the FHV cat system could be considered to test candidate PhHV-1 vaccines. Here we have tested a PhHV-1 based ISCOM vaccine for its protective efficacy against FHV infection in cats. To this end, three groups of cats were vaccinated thrice with ISCOM adjuvanted PhHV-1, FHV, and mock vaccines, respectively. One month after the last vaccination, all cats were challenged with a virulent FHV strain. All PhHV-1 and FHV vaccinated cats were protected from developing severe disease and excreted significantly less FHV than the mock vaccinated cats.     

Coplanar polychlorinated biphenyl congener levels and patterns and the identification of separate populations of harbor seals (Phoca vitulina) in Denmark

Blubber samples from 21 harbor seals (Phoca vitulina), found dead on Danish beaches during the morbilli virus epidemic in 1988, have been analyzed for toxic coplanar chlorinated biphenyl (CB) congeners. The 21 samples consist of seven samples from 1–2-year-old animals (both males and females) from each of three geographical separate locations: The Limfjord, the Kattegat, and the Wadden Sea.The level of the CBs, defined as the sum of all the measured CB congeners, were highest in the samples from the Wadden Sea and lowest in the Limfjord samples. The quotient between highest level and lowest level was 4.Toxic equivalency factors given by Safe (1990) was used for calculation of the contribution to dioxin-like toxicity from the coplanar CB congeners, and the result was compared to literature data on polychlorinated dibenzo-p-dioxins and -dibenzofurans in harbor seal. CB congeners CB-118, CB-156, CB-157, and CB-105 were major contributors to TEQ in all samples.The patterns of polychlorinated biphenyl congeners in harbor seals from the three locations were compared by principal component analysis. The two first principal components could separate the samples in groups corresponding to each of the three geographical locations. The geographical and biological variations were estimated from the data. It was shown, that the geographical variation was the largest. Possible reasons behind the geographical variation in CB congener patterns are discussed.     

Chromosome survey of seals in the Baltic Sea in 1988–1992

The populations of grey seals (Halichoerus grypus) and ringed seals (Phoca hispida botnica) in the Baltic Sea have decreased very much, especially during the last decades. Environmental pollution has been seen as an important cause. As top predators, the seals accumulate pollutants from their environment. High concentrations of chlorinated hydrocarbons and heavy metals have been found in seals from the Baltic Sea. In the present study the possible mutagenic effects of environmental pollutants on the seals have been studied by screening cultured lymphocytes for chromosome aberrations.Blood samples were taken from grey seal pups before weaning in March–April, and from adult ringed seals at the end of April during 1988–1992. Blood cells were cultured for 48 h for screening for chromosome aberrations, and for 61 h with bromodeoxyuridine for the study of sister chromatid exchange (SCE).The types of aberrations found were chromosome and chromatid breaks, gaps, and fragmentations. More complicated rearrangements were rare. Blood samples from a total of 47 grey seal pups and from ten adult ringed seals were analysed. The mean frequency of cells with chromosome aberrations from the grey seals was 5.7% (SD ±5.3). The median was 4, because the distribution was skewed with some animals having a considerably higher frequency of aberrations than the average. Some cells with several aberrations and fragmentation of the chromosomes were observed among the lymphocytes from these animals. The frequencies of SCEs were not markedly higher than those normally found in humans. The frequencies of aberrations found in the adult ringed seals were lower than those found in the grey seal pups. The chromosome aberrations are probably caused by chemical pollutants.     

Organochlorine concentrations in the Saimaa ringed seal (Phoca hispida saimensis) from Lake Haukivesi,Finland, 1981 to 2000, and in its diet today

Organochlorine and mercury (Hg) pollution has been one possible reason for a decline in the size of the population of the Saimaa ringed seal (Phoca hispida saimensis) in Lake Haukivesi, Finland. In this study, we analyzed changes in the concentrations of organochlorine compounds (OCls) and extractable organic halogen (EOX) in the blubber of Saimaa ringed seals that died from 1981 to 2000. In addition, the present concentrations and biomagnification factors of OCls and Hg from the main prey fish of ringed seal and pike (Esox lucius) were determined. Concentrations of pollutants in bream (Abramis brama) also were analyzed. The decline in the OCl concentrations in blubber was, on average, 25% during the two decades since polychlorinated biphenyls and DDT compounds were banned. Today, concentrations in blubber of Saimaa ringed seals are similar of those in ringed seals from the Arctic. Correspondingly in fish, the OCl and Hg concentrations were, on average, similar to those found in other studies on Finnish and Arctic areas. The low diet concentrations and metabolism of OCls also reflect the lower age correlation of OCls in the male ringed seals in the 1990s than in the 1980s. Biomagnification was considerably higher from fish to the ringed seal than from prey fish to pike because of higher feeding rate of ringed seals and differences in the metabolisms of these animals.     

Canine distemper virus ISCOMs induce protection in harbour seals (Phoca vitulina) against phocid distemper but still allow subsequent infection with phocid distemper virus-1.

A candidate canine distemper virus (CDV) ISCOM vaccine has been shown to be effective in protecting harbour seals (Phoca vitulina) from phocid distemper in 1988. However, of the 35 harbour seals receiving this vaccine upon admission to a seal rehabilitation and research centre (Pieterburen, The Netherlands) in 1989, six developed mild inflammatory symptoms of the respiratory tract. Phocid distemper virus-1 (PDV-1) could be isolated from three of these animals. This indicates that the vaccine affords protection from phocid distemper, but may still allow PDV-1 infection of the respiratory tract. Contacts with non-vaccinated seals should then be prevented until no more virus is excreted. It is speculated that this PDV-1 infection of the respiratory tract in CDV-ISCOM vaccinated seals is followed by a lifelong immunity.     

Persistent Organochlorine Contaminants and Enantiomeric Signatures of Chiral Pollutants in Ringed Seals (Phoca hispida) Collected on the East and West Side of the Northwater Polynya, Canadian Arctic

To examine the influence of diet and age on organochlorine contaminant (OC) concentrations in two closely related ringed seal (Phoca hispida) populations enantiomeric fractions (EFs) of chiral contaminants and stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) were measured along with OCs in ringed seals collected from the east and west side of the Northwater Polynya. Seals from these two locations were feeding at the same trophic level based on δ¹⁵N values in muscle but had slightly different sources of carbon based on δ¹³C measurements in muscle. After removing the influence of age, sex, and blubber thickness, OC concentrations did not vary between ringed seals from the east and west side of the polynya. ΣPCB, ΣDDT, and Σchlordane were found to increase with age for both male and female seals. The inclusion of older (>20 years) female seals, which may have a reduced reproductive effort, may influence the relationships in females. Stable isotopes failed to describe OC concentrations in ringed seals suggesting that diet was not a major factor in variation of OC concentrations within this ringed seal population. Cis- and trans-chlordane, oxychlordane, and heptachlor epoxide were all nonracemic in the ringed seal blubber but did not vary with age, sex, or collection site. α-HCH appeared racemic (enantiomeric fraction = 0.50 ± 0.01) in the seals, although this EF is different than those previously observed in their prey species, and was found to vary significantly with age. EF values in the ringed seals varied considerably from other Arctic marine mammals and seabirds, providing addition evidence that the type(s) and characteristic(s) of the enzymes involved in biotransformation of chiral OCs vary between these organisms. Received: 11 April 2001/Accepted: 27 June 2001     

Phocine distemper outbreak, The Netherlands, 2002

During the 2002 phocine distemper epidemic, 2,284 seals, primarily harbor seals (Phoca vitulina), were found stranded along the Dutch coast. Stranding pattern varied with age, sex, state of decomposition, wind, and location. Cumulative proportion of deaths (54%) was comparable to that in the first reported epidemic in 1988.     

Harbor seals (Phoca vitulina) in British Columbia, Canada, and Washington State, USA, reveal a combination of local and global polychlorinated biphenyl, dioxin, and furan signals

The harbor seal (Phoca vitulina) can serve as a useful indicator of food web contamination by persistent organic pollutants (POPs) because of its high trophic level, wide distribution in temperate coastal waters of the Northern Hemisphere, and relative ease of capture. In 1996 through 1997, we live-captured 60 harbor seal pups from three regions, spanning remote (Queen Charlotte Strait, BC, Canada), moderately industrialized (Strait of Georgia, BC, Canada), and heavily industrialized (Puget Sound, WA, USA) marine basins straddling the Canada-United States border. Biopsy samples of blubber were taken and analyzed for congener-specific polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) by using high-resolution gas chromatography-high-resolution mass spectrometry. Harbor seals in Puget Sound were heavily contaminated with PCBs, whereas seals from the Strait of Georgia had relatively high concentrations of PCDDs and PCDFs. Pattern evaluation and principal components analysis suggested that proximity to sources influenced the mixture to which seals were exposed, with those inhabiting more remote areas being exposed to lighter PCB congeners (those with lower Henry's law constant and K(ow)) that disperse more readily through atmospheric and other processes. Total toxic equivalents to 2,3,7,8-tetrachlorodibenzo-p-dioxin for the PCBs, PCDDs, and PCDFs suggest that Puget Sound seals are at greatest risk for adverse health effects, and that PCBs represent the class of dioxinlike contaminants of greatest concern at all sites.     

Polychlorinated biphenyls and organochlorine pesticides in Baltic and Atlantic gray seal (Halichoerus grypus) pups

Organochlorine pollutants (OCs), such as polychlorinated biphenyls (PCBs), DDT, chlordanes (CHLs), hexachlorobenzene (HCB) and hexachlorocyclohexanes (HCHs) were determined in blubber biopsies from free-ranging Baltic and Atlantic gray seal (Halichoerus grypus) pups. Well-fed pups from the Baltic Sea had concentrations of DDT, PCBs, and HCHs that were 2 to 10 times higher than in corresponding pups from populations in the Atlantic Ocean. The OC pattern in the Baltic seals differed significantly from that of their Atlantic relatives, reflecting the predominance of regional point source inputs into the Baltic Sea and long-range atmospheric inputs into the Atlantic Ocean. The differences in the pattern of the compounds also indicated an enhanced metabolism of the more metabolizable compounds in the more contaminated Baltic seals. Surprisingly, the proportions of the high chlorinated and low-volatile PCB congeners (> 6 Cl atoms) were comparable or lower in the Baltic pups as compared to the Atlantic pups. This difference might be due to Baltic seals occupying a lower trophic level than Atlantic seals and/or to the eutrophication situation in the Baltic Sea, which causes sedimentation of these PCB congeners. Significantly higher OC concentrations were found in starved and/or abandoned Baltic pups as compared to well-fed pups. The most contaminated Baltic seal pups in the present study had PCB concentrations that are comparable or higher than those reported to impair the immune systems and vitamin A dynamics in phocids.     

Bone-related effects of contaminants in seals may be associated with vitamin D and thyroid hormones

The high levels of polychlorinated biphenyls (PCBs) and DDT in gray seal (Halichoerus grypus) and ringed seal (Phoca hispida botnica) in the Baltic Sea have been associated with pathological disruptions, including bone lesions and reproductive failures. The underlying environmental and toxicological mechanisms leading to these pathological changes are not yet fully understood. The present study investigated the relationship between the individual contaminant load and bone- and thyroid-related effects in adult gray seals (n=30) and ringed seals (n=46) in the highly contaminated Baltic Sea and in reference areas (Sable Island, Canada, and Svalbard, Norway). In the gray seals, multivariate and correlation analyses revealed a clear relationship between circulating 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D), calcium, phosphate, and thyroid hormone (TH) levels and hepatic PCB and DDT load, which suggests contaminant-mediated disruption of the bone and thyroid homeostasis. Contaminants may depress 1,25(OH)(2)D levels or lead to hyperthyroidism, which may cause bone resorption. In the ringed seals, associations between circulating 1,25(OH)(2)D, THs, and hepatic contaminants were less prominent. These results suggest that bone lesions observed in the Baltic gray seals may be associated with contaminant-mediated vitamin D and thyroid disruption.     

Hepatic microsomal cytochrome p450s and chlorinated hydrocarbons in largha and ribbon seals from Hokkaido, Japan: differential response of seal species to Ah receptor agonist exposure

From 16 largha seals (Phoca largha) and 15 ribbon seals (Phoca fasciata) in the coastal waters of Hokkaido, Japan, blubber chlorinated hydrocarbon (CHC) levels and hepatic cytochrome P450 (CYP) catalytic activities and their immunochemically detected protein content levels were measured. Concentrations of DDTs (2,2-bis(4-chlorophenyl)-1,1-dichloroethylene,p,p'-DDE; 2,2-bis(4-chlorophenyl)-1,1-dichloroethane, p,p'-DDD; dichlorodiphenyltrichloroethane, p,p'-DDT), polychlorinated biphenyl congeners (PCBs), and chlordane compounds (oxychlordane, chlordanes, and nonachlors) in both species were in the range of 290 to 5,300, 420 to 4,000, and 130 to 1,500 ng/g lipid weight, respectively. Aryl hydrocarbon receptor (AhR) agonists, non-ortho (IUPAC 77 and 126) and mono-ortho (IUPAC 105, 118, and 156) coplanar PCB congeners, were also detected, and the 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) toxic equivalents (TEQs) were 4.9 to 120 pg TEQ/g lipid weight. Cross-reactive proteins with polyclonal antibodies against rat CYP1A1 and CYP3A2 were notably detected in seal liver microsomes. Interestingly, a polyclonal antibody against rat CYP2B1 recognized proteins only at trace levels. In largha seals, both levels of alkoxyresorufin- (methoxy-, ethoxy-, pentoxy-, and benzyloxyresorufin) O-dealkylase (AROD) activities and proteins detected by polyclonal antibodies against rat CYP1A1 were significantly correlated with the concentrations of individual coplanar PCB congeners, total TEQs, and total PCBs. Threshold concentrations for TEQs in blubber of the largha seal to induce hepatic CYP1A protein and EROD activity were estimated to be 8.5 and 19 pg TEQ/g fat weight, respectively. In ribbon seals, similar correlations were not detected, although the TEQ levels were not significantly lower than those in largha seals. These results suggest that AROD activity and CYP1A1 protein in the liver of the largha seal could be a biomarker for the exposure to AhR agonists such as coplanar PCB congeners. This study also indicates differences in AhR-mediated responses to the CHC exposures between largha and ribbon seals.     

Mercury and major essential elements in seals,penguins, and other representative fauna of the Antarctic

Concentrations of total Hg and major essential elements, Ca, Mg, Na, and K, were measured in the muscle, liver, and kidney of three species of seals, crabeater seal (Lobodon carcinophagus), leopard seal (Hydrurga leptonyx), and Weddell seal (Leptonychotes weddelli) collected in the Antarctic. The muscle and liver of three species of penguins, i.e., gentoo penguin (Pygoscelis papua), Adelie penguin (P. adeliae), chinstrap penguin (P. antarctica), and other representative fauna were also analyzed for the elements. Distinct inter-tissue differences in the metal concentration were observed; liver had the greatest concentrations of Hg, kidney showed maximum concentration of Ca and Na, while muscle was characterized by the greatest content of Mg and K. Inter-specimen differentiation of concentrations among the same species was distinctly visualized for Hg but not for the major essential elements. The Hg concentration in the seals analyzed are in keeping with those reported previously by other authors. Such comparison for Ca, Mg, Na, and K was impossible because of the lack of available literature data for their concentrations in marine seals. Numerous significant correlations were observed between concentrations of the several metals analyzed. There was no correlation between Hg, which is a non-essential element and both Zn and Cd which are essential and non-essential elements, respectively in the liver or kidney of seals. However, there were significant correlations between concentration of Zn and the sum of molar concentrations of Hg + Cd in kidney (r=0.82) and liver (r=0.76). The results suggest that several control mechanisms operate to maintain physiologically required levels that decreases any effect of heavy metal toxicants such as Hg and Cd.     

Customized design and 3D printing of face seal for an N95 filtering facepiece respirator

Filtering Facepiece Respirator (FFR) is the most common respirator users in the health care environment utilize for personal protection from outside particles. Comfort and fit are important while wearing an FFR. This paper proposes a novel technology to produce customized face seal design for improving the wearing comfort and fit of FFR wearers. In order to customize the design of face seals, we scanned the faces of three subjects using three-dimensional (3D) laser scanning method. A customized face seal for a 3M 8210 N95 FFR for each headform was designed using reverse engineering technique. Next, the face seal prototypes were fabricated with Acrylonitrile Butadiene Styrene (ABS) plastic using the 3D printing method. A force sensing system based on Arduino Uno R3 was developed, and the force sensor of this system was inserted between the FFR and headform to measure contact pressure. Experimental results showed that the newly designed FFR face seal provided the subjects with an improved contact pressure.     

Patterns of chlorinated biphenyl congeners in harbor seals (Phoca vitulina) and in their food: Statistical analysis

Patterns of chlorinated biphenyl (CB) congeners have been compared in two groups of samples, namely blood samples from harbor seals (Phoca vitulina) and muscle tissue samples in the fish with which the seals were fed. The data originate from a Dutch controlled feeding study, performed in 1981 and 1983. The seals were living in captivity in two separate groups, and the fish samples were plaice (Pleuronectes platessa) from the Dutch part of the Wadden sea and mackerel (Scomber scombrus) from the Atlantic ocean. The levels of CB congeners were different in the two types of fish.The CB congener patterns of these four matrices were compared by analysis of principal components, and were found to be different in the two groups of harbor seals, which were fed the different types of fish. The corresponding CB congener patterns of the two groups of fish were also different.There was no indication for a relatively larger impact of biotransformation due to induction of isoforms of the cytochrome P450-system at the higher absolute CB congener levels in the Wadden sea group of seals compared to the Atlantic group of seals. The differentiation between the CB congener patterns in the two groups of seals in the Dutch study can be ascribed solely to different CB congener patterns in their food.The difference between CB congener patterns in the seals and in their diet can be explained by the structure-related biotransformation of the CB congeners in the harbor seal.     

Phocine distemper virus in seals, east coast, United States, 2006

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.     

外周静脉留置针不同封管液封管效果的研究

目的 比较外周静脉留置针两种封管液的封管效果。以选择更有效的封管液封管。减少临床上由于封管液选用不当而致堵管情况的发生，避免由于堵管而不得不再次穿刺给患者带来的痛苦。方法 将68例患者按随机抽样的方法分为对照组和实验组，每组均34例，对照组用0．9％生理盐水5ml封管。实验组用肝素盐水（50u／m1）5ml封管，两组均采用正压封管方法，进行留置效果比较。结果 实验组用肝素盐水（50u／ml）5ml封管。有效率为93．10％。对照组用0.9％生理盐水5ml封管，有效率为64．93％。经统计学处理χ^2=20．16．P〈0．01两组封管方法有显著性差异。肝素盐水封管优于生理盐水。结论 对外周静脉留置针而言．主张用肝素盐水封管。     

Phocine distemper in German seals, 2002

Approximately 21,700 seals died during a morbillivirus epidemic in northwestern Europe in 2002. Phocine distemper virus 1 was isolated from seals in German waters. The sequence of the P gene showed 97% identity with the Dutch virus isolated in 1988. There was 100% identity with the Dutch isolate from 2002 and a single nucleotide mismatch with the Danish isolate.