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1.  Spatially Explicit Analysis of Genome-Wide SNPs Detects Subtle Population Structure in a Mobile Marine Mammal, the Harbor Porpoise 
PLoS ONE  2016;11(10):e0162792.
The population structure of the highly mobile marine mammal, the harbor porpoise (Phocoena phocoena), in the Atlantic shelf waters follows a pattern of significant isolation-by-distance. The population structure of harbor porpoises from the Baltic Sea, which is connected with the North Sea through a series of basins separated by shallow underwater ridges, however, is more complex. Here, we investigated the population differentiation of harbor porpoises in European Seas with a special focus on the Baltic Sea and adjacent waters, using a population genomics approach. We used 2872 single nucleotide polymorphisms (SNPs), derived from double digest restriction-site associated DNA sequencing (ddRAD-seq), as well as 13 microsatellite loci and mitochondrial haplotypes for the same set of individuals. Spatial principal components analysis (sPCA), and Bayesian clustering on a subset of SNPs suggest three main groupings at the level of all studied regions: the Black Sea, the North Atlantic, and the Baltic Sea. Furthermore, we observed a distinct separation of the North Sea harbor porpoises from the Baltic Sea populations, and identified splits between porpoise populations within the Baltic Sea. We observed a notable distinction between the Belt Sea and the Inner Baltic Sea sub-regions. Improved delineation of harbor porpoise population assignments for the Baltic based on genomic evidence is important for conservation management of this endangered cetacean in threatened habitats, particularly in the Baltic Sea proper. In addition, we show that SNPs outperform microsatellite markers and demonstrate the utility of RAD-tags from a relatively small, opportunistically sampled cetacean sample set for population diversity and divergence analysis.
PMCID: PMC5082642  PMID: 27783621
2.  Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe 
Journal of Virology  2016;90(9):4269-4277.
Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host.
IMPORTANCE Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.
PMCID: PMC4836327  PMID: 26819311
3.  Crop diversity loss as primary cause of grey partridge and common pheasant decline in Lower Saxony, Germany 
BMC Ecology  2016;16(1):39.
The grey partridge (Perdix perdix) and the common pheasant (Phasianus colchicus) are galliform birds typical of arable lands in Central Europe and exhibit a partly dramatic negative population trend. In order to understand general habitat preferences we modelled grey partridge and common pheasant densities over the entire range of Lower Saxony. Spatially explicit developments in bird densities were modelled using spatially explicit trends of crop cultivation. Pheasant and grey partridge densities counted annually by over 8000 hunting district holders over 10 years in a range of 3.7 Mio ha constitute a unique dataset (wildlife survey of Lower Saxony). Data on main landscape groups, functional groups of agricultural crops (consisting of 9.5 million fields compiled by the Integrated Administration and Control System) and landscape features were aggregated to 420 municipalities. To model linear 8 or 10 year population trends (for common pheasant and grey partridge respectively) we use rho correlation coefficients of densities, but also rho coefficients of agricultural crops.
All models confirm a dramatic decline in population densities. The habitat model for the grey partridge shows avoidance of municipalities with a high proportion of woodland and water areas, but a preference for areas with a high proportion of winter grains and high crop diversity. The trend model confirms these findings with a linear positive effect of diversity on grey partridge population development. Similarly, the pheasant avoids wooded areas but showed some preference for municipalities with open water. The effect of maize was found to be positive at medium densities, but negative at very high proportions. Winter grains, landscape features and high crop diversity are favorable. The positive effect of winter grains and higher crop diversity is also supported by the trend model.
The results show the strong importance of diverse crop cultivation. Most incentives favor the cultivation of specific crops, which results in large areas of monocultures. The results confirm the importance of sustainable agricultural policies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12898-016-0093-9) contains supplementary material, which is available to authorized users.
PMCID: PMC5016946  PMID: 27612946
Habitat model; Trend analysis; Grey partridge; Common pheasant; Citizen science; Diversity; Maize cultivation
4.  Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets 
PLoS ONE  2016;11(7):e0159625.
Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.
PMCID: PMC4957826  PMID: 27448168
5.  Seasonal Variation of Harbor Seal's Diet from the Wadden Sea in Relation to Prey Availability 
PLoS ONE  2016;11(5):e0155727.
The Wadden Sea has an important role for marine mammals in terms of resting, nursing and foraging. Harbor seal is the most abundant marine mammal species in this area. The use of the food resources of the Wadden Sea by seals is not clear, and previous studies showed that this species can travel kilometers away from their haul-outs to forage in the North Sea. In this study, we analyzed the stable isotopes of vibrissae from 23 dead harbor seals found on the island of Sylt to investigate their diet. The predator´s carbon and nitrogen isotope compositions were compared to the compositions of different potential prey items from the Sylt-Rømø Bight and from the North Sea in order to study seasonal pattern in the diet and in the foraging location. In parallel, seasonal variation of abundance and biomass of the potential prey items from the Sylt-Rømø Bight were studied and compare to their contribution to the seal´s diet. The results revealed a change in the seal´s diet from pelagic sources in spring to a benthic based diet in summer, and an increasing use of the North Sea resources in fall and winter in accordance with the seasonal variation of the availability of prey in the Sylt-Rømø Bight.
PMCID: PMC4866785  PMID: 27176227
6.  Unique fur and skin structure in harbour seals (Phoca vitulina)—thermal insulation, drag reduction, or both? 
Journal of the Royal Society Interface  2015;12(104):20141206.
Vertebrate surface structures, including mammalian skin and hair structures, have undergone various modifications during evolution in accordance with functional specializations. Harbour seals rely on their vibrissal system for orientation and foraging. To maintain tactile sensitivity even at low temperatures, the vibrissal follicles are heated up intensely, which could cause severe heat loss to the environment. We analysed skin samples of different body parts of harbour seals, and expected to see higher hair densities at the vibrissal pads as a way to reduce heat loss. In addition to significantly higher hair densities around the vibrissae than on the rest of the body, we show a unique fur structure of hair bundles consisting of broad guard hairs along with hairs of a new type, smaller than guard hairs but broader than underhairs, which we defined as ‘intermediate hairs’. This fur composition has not been reported for any mammal so far and may serve for thermal insulation as well as drag reduction. Furthermore, we describe a scale-like skin structure that also presumably plays a role in drag reduction.
PMCID: PMC4345485  PMID: 25652462
guard hair; pilosebaceous unit; skin structure; intermediate hair; hydrodynamics; surface drag
7.  Lungworm seroprevalence in free-ranging harbour seals and molecular characterisation of marine mammal MSP 
Harbour seals (Phoca vitulina) are frequently infected with the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus. The infection is often accompanied by secondary bacterial infections and can cause severe bronchopneumonia and even death in affected animals. Hitherto, the detection of lungworm infections was based on post mortem investigations from animals collected within stranding networks and a valid detection method for live free-ranging harbour seals was not available. Recently, an ELISA was developed for detecting lungworm antibodies in harbour seal serum, using major sperm protein (MSP) of the bovine lungworm, Dictyocaulus viviparus as recombinant diagnostic antigen. To determine lungworm seroprevalence in free-ranging harbour seals, serum was taken from four different seal age groups (n = 313) resulting in an overall prevalence of 17.9% (18.9% of males, 16.7% of females). 0.7% of harbour seals up to six weeks of age were seropositive, as were 89% of seals between six weeks and six months, 53.6% between six and 18 months and 24.2% of seals over 18 months of age. In the 18 months and over age group, seropositive animals showed statistically significant reductions in body weight (P = 0.003) and length (P < 0.001). Sera from lungworm infected harbour seals in rehabilitation (n = 6) revealed that duration of antibody persistence may be similar to that of lungworm infected cattle, but further studies are needed to confirm this. Phylogenetic analyses of MSP sequences of different marine and terrestrial mammal parasitic nematodes revealed that lungworm MSP of the genus Dictyocaulus (superfamily Trichostrongyloidea) is more closely related to metastrongylid marine mammal lungworms than to trichostrongylid nematodes of terrestrial hosts.
Graphical abstract
•First study on lungworm seroprevalence in live free-ranging harbour seals.•Total seroprevalence was 17.9%, but age-dependent differences were observed.•Six weeks to six months old seals showed highest prevalences (89% positives).•Seropositive adult seals showed significantly reduced body weight and length.•Phylogenetic tree construction using MSP of marine and terrestrial mammal parasites.
PMCID: PMC4781971  PMID: 26977405
Phoca vitulina; Lungworm infection; Otostrongylus circumlitus; Parafilaroides gymnurus; ELISA; Major sperm protein
8.  A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals 
Parasites & Vectors  2015;8:443.
Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well.
For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed.
Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100 % (95 % CI: 87.23-100 %) and a sensitivity of 97.83 % (95 % CI: 88.47-99.94 %). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies in animals positive for lungworm larvae at faecal examination.
The ELISA presented is a valuable method for detection of lungworm infections in live harbour and grey seals, providing a monitoring tool to reveal epidemiological dynamics of lungworm infections during health surveillance in free-ranging seals. Furthermore, ELISA results may aid institutions with harbour and grey seals under human care on decisions regarding anthelminthic treatment of individual animals.
PMCID: PMC4557280  PMID: 26329933
ELISA; Major sperm protein; Phoca vitulina; Halichoerus grypus; Lungworm infection; Verminous pneumonia; Metastrongyloidea; Otostrongylus circumlitus; Parafilaroides gymnurus
9.  Options for modulating intra-specific competition in colonial pinnipeds: the case of harbour seals (Phoca vitulina) in the Wadden Sea 
PeerJ  2015;3:e957.
Colonial pinnipeds may be subject to substantial consumptive competition because they are large, slow-moving central place foragers. We examined possible mechanisms for reducing this competition by examining the diving behaviour of harbour seals (Phoca vitulina) after equipping 34 seals (11 females, 23 males) foraging from three locations; Rømø, Denmark and Lorenzenplate and Helgoland, Germany, in the Wadden Sea area with time-depth recorders. Analysis of 319,021 dives revealed little between-colony variation but appreciable inter-sex differences, with males diving deeper than females, but for shorter periods. Males also had higher vertical descent rates. This result suggests that males may have higher overall swim speeds, which would increase higher oxygen consumption, and may explain the shorter dive durations compared to females. Intersex variation in swim speed alone is predicted to lead to fundamental differences in the time use of three-dimensional space, which may help reduce consumptive competition in harbour seals and other colonial pinnipeds.
PMCID: PMC4465952  PMID: 26082869
Pinniped; Dive behaviour; Sexual segregation; Dive duration; Swim speed; Harbour seal
10.  Avian Influenza A(H10N7) Virus–Associated Mass Deaths among Harbor Seals 
Emerging Infectious Diseases  2015;21(4):720-722.
PMCID: PMC4378483  PMID: 25811303
influenza A virus; harbor seals; H10N7; viruses; Germany; influenza; Phoca vitulina
11.  Phocine Distemper Virus: Current Knowledge and Future Directions 
Viruses  2014;6(12):5093-5134.
Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years.
PMCID: PMC4276944  PMID: 25533658
Morbillivirus; pinnipeds; sea otter; CD150/SLAM; phylogeny; pathology; epidemiology; immunity; vaccine
12.  Seasonal migrations of North Atlantic minke whales: novel insights from large-scale passive acoustic monitoring networks 
Movement Ecology  2014;2(1):24.
Little is known about migration patterns and seasonal distribution away from coastal summer feeding habitats of many pelagic baleen whales. Recently, large-scale passive acoustic monitoring networks have become available to explore migration patterns and identify critical habitats of these species. North Atlantic minke whales (Balaenoptera acutorostrata) perform seasonal migrations between high latitude summer feeding and low latitude winter breeding grounds. While the distribution and abundance of the species has been studied across their summer range, data on migration and winter habitat are virtually missing. Acoustic recordings, from 16 different sites from across the North Atlantic, were analyzed to examine the seasonal and geographic variation in minke whale pulse train occurrence, infer information about migration routes and timing, and to identify possible winter habitats.
Acoustic detections show that minke whales leave their winter grounds south of 30° N from March through early April. On their southward migration in autumn, minke whales leave waters north of 40° N from mid-October through early November. In the western North Atlantic spring migrants appear to track the warmer waters of the Gulf Stream along the continental shelf, while whales travel farther offshore in autumn. Abundant detections were found off the southeastern US and the Caribbean during winter. Minke whale pulse trains showed evidence of geographic variation, with longer pulse trains recorded south of 40° N. Very few pulse trains were recorded during summer in any of the datasets.
This study highlights the feasibility of using acoustic monitoring networks to explore migration patterns of pelagic marine mammals. Results confirm the presence of minke whales off the southeastern US and the Caribbean during winter months. The absence of pulse train detections during summer suggests either that minke whales switch their vocal behaviour at this time of year, are absent from available recording sites or that variation in signal structure influenced automated detection. Alternatively, if pulse trains are produced in a reproductive context by males, these data may indicate their absence from the selected recording sites. Evidence of geographic variation in pulse train duration suggests different behavioural functions or use of these calls at different latitudes.
Electronic supplementary material
The online version of this article (doi:10.1186/s40462-014-0024-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4337769  PMID: 25709833
Passive acoustic monitoring (PAM); Minke whales; Balaenoptera acutorostrata; Migration; Pulse trains; Seasonality; Geographic variation
13.  A Systematic Health Assessment of Indian Ocean Bottlenose (Tursiops aduncus) and Indo-Pacific Humpback (Sousa plumbea) Dolphins Incidentally Caught in Shark Nets off the KwaZulu-Natal Coast, South Africa 
PLoS ONE  2014;9(9):e107038.
Coastal dolphins are regarded as indicators of changes in coastal marine ecosystem health that could impact humans utilizing the marine environment for food or recreation. Necropsy and histology examinations were performed on 35 Indian Ocean bottlenose dolphins (Tursiops aduncus) and five Indo-Pacific humpback dolphins (Sousa plumbea) incidentally caught in shark nets off the KwaZulu-Natal coast, South Africa, between 2010 and 2012. Parasitic lesions included pneumonia (85%), abdominal and thoracic serositis (75%), gastroenteritis (70%), hepatitis (62%), and endometritis (42%). Parasitic species identified were Halocercus sp. (lung), Crassicauda sp. (skeletal muscle) and Xenobalanus globicipitis (skin). Additional findings included bronchiolar epithelial mineralisation (83%), splenic filamentous tags (45%), non-suppurative meningoencephalitis (39%), and myocardial fibrosis (26%). No immunohistochemically positive reaction was present in lesions suggestive of dolphin morbillivirus, Toxoplasma gondii and Brucella spp. The first confirmed cases of lobomycosis and sarcocystosis in South African dolphins were documented. Most lesions were mild, and all animals were considered to be in good nutritional condition, based on blubber thickness and muscle mass. Apparent temporal changes in parasitic disease prevalence may indicate a change in the host/parasite interface. This study provided valuable baseline information on conditions affecting coastal dolphin populations in South Africa and, to our knowledge, constitutes the first reported systematic health assessment in incidentally caught dolphins in the Southern Hemisphere. Further research on temporal disease trends as well as disease pathophysiology and anthropogenic factors affecting these populations is needed.
PMCID: PMC4159300  PMID: 25203143
14.  Mysterious bio-duck sound attributed to the Antarctic minke whale (Balaenoptera bonaerensis) 
Biology Letters  2014;10(4):20140175.
For decades, the bio-duck sound has been recorded in the Southern Ocean, but the animal producing it has remained a mystery. Heard mainly during austral winter in the Southern Ocean, this ubiquitous sound has been recorded in Antarctic waters and contemporaneously off the Australian west coast. Here, we present conclusive evidence that the bio-duck sound is produced by Antarctic minke whales (Balaenoptera bonaerensis). We analysed data from multi-sensor acoustic recording tags that included intense bio-duck sounds as well as singular downsweeps that have previously been attributed to this species. This finding allows the interpretation of a wealth of long-term acoustic recordings for this previously acoustically concealed species, which will improve our understanding of the distribution, abundance and behaviour of Antarctic minke whales. This is critical information for a species that inhabits a difficult to access sea-ice environment that is changing rapidly in some regions and has been the subject of contentious lethal sampling efforts and ongoing international legal action.
PMCID: PMC4013705  PMID: 24759372
Balaenoptera bonaerensis; bio-duck; Antarctic minke whale; Southern Ocean; acoustic monitoring
15.  In-Air Evoked Potential Audiometry of Grey Seals (Halichoerus grypus) from the North and Baltic Seas 
PLoS ONE  2014;9(3):e90824.
In-air anthropogenic sound has the potential to affect grey seal (Halichoerus grypus) behaviour and interfere with acoustic communication. In this study, a new method was used to deliver acoustic signals to grey seals as part of an in-air hearing assessment. Using in-ear headphones with adapted ear inserts allowed for the measurement of auditory brainstem responses (ABR) on sedated grey seals exposed to 5-cycle (2-1-2) tone pips. Thresholds were measured at 10 frequencies between 1–20 kHz. Measurements were made using subcutaneous electrodes on wild seals from the Baltic and North Seas. Thresholds were determined by both visual and statistical approaches (single point F-test) and good agreement was obtained between the results using both methods. The mean auditory thresholds were ≤40 dB re 20 µPa peak equivalent sound pressure level (peSPL) between 4–20 kHz and showed similar patterns to in-air behavioural hearing tests of other phocid seals between 3 and 20 kHz. Below 3 kHz, a steep reduction in hearing sensitivity was observed, which differed from the rate of decline in sensitivity obtained in behavioural studies on other phocids. Differences in the rate of decline may reflect influence of the ear inserts on the ability to reliably transmit lower frequencies or interference from the structure of the distal end of the ear canal.
PMCID: PMC3954592  PMID: 24632891
16.  Further Studies on Arcanobacterium phocisimile: a Novel Species of Genus Arcanobacterium 
Journal of veterinary medicine  2014;2014:923592.
Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698T isolated from a vaginal swab of a harbour seal and four additional A. phocisimile strains also isolated from four harbour seals could reliably be identified by phenotypic properties, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and by sequencing the genomic targets 16S rDNA and 16S-23S rDNA intergenic spacer region and the genes rpoB and gap. The A. phocisimile strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of A. phocisimile remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approach might help to identify A. phocisimile in future.
PMCID: PMC4590858  PMID: 26464945
17.  Analysis of blood gases, serum fat and serum protein: a new approach to estimate survival chances of stranded Harbor seal (Phoca vitulina) pups from the German North Sea 
Facing numerous challenges, such as illness, storms or human disturbance, some harbor seal (Phoca vitulina) pups lose contact to their dams and are found abandoned along the North Sea coast. In Schleswig-Holstein, pups with the prospect of surviving rehabilitation are admitted to the Seal Center Friedrichskoog. Despite elaborate clinical health assessments on admission, including differential hematology, in 2010, 17% of 108 admitted pups did not survive the first 20 days. The death rate during the years 2006 and 2009 varied between 9 and 19%. To broaden the spectrum of variables which could be predictive for survival, blood gas and serum analyses were performed for 99 pups using venous blood. Variables included total CO2, pH, partial CO2, HCO3–, base excess and anion gap as well as glucose, urea nitrogen, sodium, potassium and chloride. Moreover, total serum protein and fat (triglyceride) concentrations were measured for all pups on admission.
Repeated measurements of 12 randomly selected individuals revealed a significant (p = 0.002) positive influence of time in rehabilitation on triglyceride concentrations. This trend probably shows the improvement of the pups’ nutritional status as a consequence of the shift from milk replacer formula to fish. No such positive influence was detected for total protein concentrations though. Hematologic values, including blood gases, were not predictive for survival.
For the first time blood gas values are reported in this study for a large sample size (N = 99) of seal pups (regardless of their health status). The ranges and medians calculated from the data can serve as a stepping stone towards the establishment of reference values for neonate harbor seals. However, future investigations on the development of blood gases in harbor seals with different health conditions and ages over time are necessary to allow for a better understanding of acid–base regulation in harbor seals.
PMCID: PMC3930289  PMID: 24490584
Blood gas analysis; Serum fat (triglycerides); Serum protein; Harbor seal pups; Rehabilitation; Phoca vitulina
19.  Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress? 
The harbour porpoise is exposed to increasing pressure caused by anthropogenic activities in its marine environment. Numerous offshore wind farms are planned or under construction in the North and Baltic Seas, which will increase underwater noise during both construction and operation. A better understanding of how anthropogenic impacts affect the behaviour, health, endocrinology, immunology and physiology of the animals is thus needed. The present study compares levels of stress hormones and mRNA expression of cytokines and acute-phase proteins in blood samples of harbour porpoises exposed to different levels of stress during handling, in rehabilitation or permanent human care.
Free-ranging harbour porpoises, incidentally caught in pound nets in Denmark, were compared to harbour porpoises in rehabilitation at SOS Dolfijn in Harderwijk, the Netherlands, and individuals permanently kept in human care in the Dolfinarium Harderwijk and Fjord & Belt Kerteminde, Denmark. Blood samples were investigated for catecholamines, adrenaline, noradrenaline and dopamine, as well as for adrenocorticotropic hormone (ACTH), cortisol, metanephrine and normetanephrine. mRNA expression levels of relevant cell mediators (cytokines IL-10 and TNFα, acute-phase proteins haptoglobin and C-reactive protein and the heat shock protein HSP70) were measured using real-time PCR.
Biomarker expression levels varied between free-ranging animals and porpoises in human care. Hormone and cytokine ranges showed correlations to each other and to the health status of investigated harbour porpoises. Hormone concentrations were higher in free-ranging harbour porpoises than in animals in human care. Adrenaline can be used as a parameter for the initial reaction to acute stress situations; noradrenaline, dopamine, ACTH and cortisol are more likely indicators for the following minutes of acute stress. There is evidence for different correlations between production of normetanephrine, metanephrine, cortisol and the expression of IL-10, HSP70 and haptoglobin.
The expression patterns of the selected molecular biomarkers of the immune system are promising to reflect the health and immune status of the harbour porpoise under different levels of stress.
PMCID: PMC3734172  PMID: 23866055
Harbour porpoise; Stress hormones; Cytokines; Anthropogenic impact; Offshore wind farms; Underwater noise
20.  The Stranding Anomaly as Population Indicator: The Case of Harbour Porpoise Phocoena phocoena in North-Western Europe 
PLoS ONE  2013;8(4):e62180.
Ecological indicators for monitoring strategies are expected to combine three major characteristics: ecological significance, statistical credibility, and cost-effectiveness. Strategies based on stranding networks rank highly in cost-effectiveness, but their ecological significance and statistical credibility are disputed. Our present goal is to improve the value of stranding data as population indicator as part of monitoring strategies by constructing the spatial and temporal null hypothesis for strandings. The null hypothesis is defined as: small cetacean distribution and mortality are uniform in space and constant in time. We used a drift model to map stranding probabilities and predict stranding patterns of cetacean carcasses under H0 across the North Sea, the Channel and the Bay of Biscay, for the period 1990–2009. As the most common cetacean occurring in this area, we chose the harbour porpoise Phocoena phocoena for our modelling. The difference between these strandings expected under H0 and observed strandings is defined as the stranding anomaly. It constituted the stranding data series corrected for drift conditions. Seasonal decomposition of stranding anomaly suggested that drift conditions did not explain observed seasonal variations of porpoise strandings. Long-term stranding anomalies increased first in the southern North Sea, the Channel and Bay of Biscay coasts, and finally the eastern North Sea. The hypothesis of changes in porpoise distribution was consistent with local visual surveys, mostly SCANS surveys (1994 and 2005). This new indicator could be applied to cetacean populations across the world and more widely to marine megafauna.
PMCID: PMC3632559  PMID: 23614031
21.  Possible Causes of a Harbour Porpoise Mass Stranding in Danish Waters in 2005 
PLoS ONE  2013;8(2):e55553.
An unprecedented 85 harbour porpoises stranded freshly dead along approximately 100 km of Danish coastline from 7–15 April, 2005. This total is considerably above the mean weekly stranding rate for the whole of Denmark, both for any time of year, 1.23 animals/week (ranging from 0 to 20 during 2003–2008, excluding April 2005), and specifically in April, 0.65 animals/week (0 to 4, same period). Bycatch was established as the cause of death for most of the individuals through typical indications of fisheries interactions, including net markings in the skin and around the flippers, and loss of tail flukes. Local fishermen confirmed unusually large porpoise bycatch in nets set for lumpfish (Cyclopterus lumpus) and the strandings were attributed to an early lumpfish season. However, lumpfish catches for 2005 were not unusual in terms of season onset, peak or total catch, when compared to 2003–2008. Consequently, human activity was combined with environmental factors and the variation in Danish fisheries landings (determined through a principal component analysis) in a two-part statistical model to assess the correlation of these factors with both the presence of fresh strandings and the numbers of strandings on the Danish west coast. The final statistical model (which was forward selected using Akaike information criterion; AIC) indicated that naval presence is correlated with higher rates of porpoise strandings, particularly in combination with certain fisheries, although it is not correlated with the actual presence of strandings. Military vessels from various countries were confirmed in the area from the 7th April, en route to the largest naval exercise in Danish waters to date (Loyal Mariner 2005, 11–28 April). Although sonar usage cannot be confirmed, it is likely that ships were testing various equipment prior to the main exercise. Thus naval activity cannot be ruled out as a possible contributing factor.
PMCID: PMC3584061  PMID: 23460787
22.  Health status of seabirds and coastal birds found at the German North Sea coast 
Systematic pathological investigations to assess the health status of seabirds and coastal birds in Germany were performed. The investigation was conducted to obtain data on possible causes of decline in seabird and coastal bird populations.
48 individuals of 11 different species of seabirds and coastal birds were collected by the stranding network along the entire German North Sea coast from 1997 to 2008, including mainly waders such as Eurasian oystercatchers (Haematopus ostralegus) and red knots (Calidris canutus) as well as seabirds such as northern fulmars (Fulmaris glacialis) and common scoters (Melanitta nigra).
For most birds (n = 31) found dead along the shore no obvious cause of death was evident, while 17 individuals were killed by collisions with lighthouses.
Overall, the nutritional status of the investigated birds was very poor, and the body mass in most cases was significantly lower compared to masses of living birds caught during the same periods of the year. This is partly linked to chronic parasitic or bacterial infections in different organs or to septicaemia. In some cases infections with zoonotic tuberculosis caused by Mycobacterium spp. were found. Avian influenza was not found in any of the collected birds.
The presented data contribute to the evaluation of the health status of birds in the German North Sea. Moreover, they present an important tool for the assessment of potential pathogens with an impact on the health status of seabirds and coastal birds.
PMCID: PMC3441360  PMID: 22812640
Seabirds; Coastal birds; Pathology; North Sea; German waters
23.  Mercury immune toxicity in harbour seals: links to in vitro toxicity 
Environmental Health  2008;7:52.
Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes).
Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 μM). The expression of cytokines (IL-2, IL-4 and TGF-β) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 μM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 μM (n = 3).
The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 μM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-β mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 μM). Proteomics showed some variation in the protein expression profile (e.g. vimentin).
Our results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.
PMCID: PMC2600635  PMID: 18959786
24.  Distemper in a Dolphin 
Emerging Infectious Diseases  2007;13(12):1959-1961.
PMCID: PMC2876748  PMID: 18258062
Dolphin; distemper; morbillivirus; encephalitis; letter
25.  Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters 
BMC Biology  2007;5:30.
Understanding the role of seascape in shaping genetic and demographic population structure is highly challenging for marine pelagic species such as cetaceans for which there is generally little evidence of what could effectively restrict their dispersal. In the present work, we applied a combination of recent individual-based landscape genetic approaches to investigate the population genetic structure of a highly mobile extensive range cetacean, the harbour porpoise in the eastern North Atlantic, with regards to oceanographic characteristics that could constrain its dispersal.
Analyses of 10 microsatellite loci for 752 individuals revealed that most of the sampled range in the eastern North Atlantic behaves as a 'continuous' population that widely extends over thousands of kilometres with significant isolation by distance (IBD). However, strong barriers to gene flow were detected in the south-eastern part of the range. These barriers coincided with profound changes in environmental characteristics and isolated, on a relatively small scale, porpoises from Iberian waters and on a larger scale porpoises from the Black Sea.
The presence of these barriers to gene flow that coincide with profound changes in oceanographic features, together with the spatial variation in IBD strength, provide for the first time strong evidence that physical processes have a major impact on the demographic and genetic structure of a cetacean. This genetic pattern further suggests habitat-related fragmentation of the porpoise range that is likely to intensify with predicted surface ocean warming.
PMCID: PMC1971045  PMID: 17651495

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