Kristina Lehnert

Pathological findings in harbour seals (Phoca vitulina): 1996-2005.

Between 1996 and 2005 the carcasses of 355 harbour seals originating from the coast of Schleswig-Holstein, Germany, were investigated for pathological changes. The animals were collected before (n=280) and after (n=75) the second phocine distemper virus (PDV) epizootic in 2002. The seals were either found dead or were killed due to severe illness. Necropsy was performed in each case, in addition to histopathological, immunohistochemical, microbiological and parasitological examinations. Throughout the period of study, the respiratory and alimentary tracts were the organ systems most consistently affected by pathological change. The most common cause of death was bronchopneumonia caused by parasitic and/or bacterial infection of the lung. Less frequently identified changes included: trauma, gastroenteritis, uterine torsion or dystocia, polyarthritis/polymyositis, intestinal torsion, septicaemia, dermatitis, and keratitis. The most frequent causes of bronchopneumonia, gastroenteritis, polyarthritis, dermatitis and septicaemia were infections with alpha/beta-haemolytic streptococci, Escherichia coli and Clostridium perfringens. A number of changes were more frequently identified after 2002. These included the presence of parasites in the lung, stomach and intestine; bronchopneumonia, gastritis, enteritis, septicaemia and perinatal death. The increased prevalence of these changes may have been related to the preceding PDV epidemic.

Transmission of lungworms of harbour porpoises and harbour seals: molecular tools determine potential vertebrate intermediate hosts.

Harbour porpoises (Phocoena phocoena) and harbour seals (Phoca vitulina) from German waters are infected by six species of lungworms (Metastrongyloidea). These nematodes parasitise the respiratory tract, are pathogenic and often cause secondary bacterial infections. In spite of their clinical and epidemiological significance, the life cycle and biology of lungworms in the marine environment is still largely unknown. Regions of ribosomal DNA (ITS-2) of all lungworms parasitising harbour porpoises and harbour seals in German waters were sequenced to characterise and compare the different species. The phylogenetic relationship among the lungworm species was analysed by means of their ITS-2 nucleotide sequences and the species-specific traits of the ITS-2 were used to screen wild fish as possible intermediate hosts for larval lungworms. Molecular markers were developed to identify larval nematodes via in-situ hybridisation of tissues of harbour porpoise and harbour seal prey fish. Potential wild intermediate fish hosts from the North Sea were dissected and found to harbour larval nematodes. Histological examination and in-situ hybridisation of tissue samples from these fish showed lungworm larvae within the intestinal wall. Based on larval ITS-2 nucleotide sequences, larval nematodes were identified as Pseudalius inflexus and Parafilaroides gymnurus. Turbot (Psetta maxima) bred and raised in captivity were experimentally infected with live L1s of Otostrongylus circumlitus and ensheathed larvae were recovered from the gastrointestinal tract of turbot and identified using molecular tools. Our results show that fish intermediate hosts play a role in the transmission of metastrongyloid nematodes of harbour porpoises and harbour seals.

Evaluation of immune and stress status in harbour porpoises (Phocoena phocoena): can hormones and mRNA expression levels serve as indicators to assess stress?

BackgroundThe 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.ResultsBiomarker 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.ConclusionsThe 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.

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets

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.

Immune-relevant and new xenobiotic molecular biomarkers to assess anthropogenic stress in seals.

Harbour seals as top predators and indicators for ecosystem health are exposed to increasing pressure caused by anthropogenic activities in their marine environment. After their lactation period of about 24 days pups are weaned and left to hunt on their own. Little is known about the development of their immune system and a better understanding of anthropogenic impacts on the general health and immune system of harbour seal pups is needed. mRNA transcription of six immuno-relevant biomarkers was analysed in 13 abandoned harbour seal pups from the North Sea, fostered at the Seal Centre Friedrichskoog, Germany. RNAlater blood samples were taken at admission, day 22 and before release and analysed using RT-qPCR. Significant differences in HSP70, cytokine IL-2 and xenobiotic biomarkers AHR, ARNT and PPARα transcription were found between admission, during rehabilitation and before release. Highest levels at admission may result from dehydration, handling, transport and contaminant exposure via lactation. The significant decrease is linked to health improvement, feeding and adaptation. The increase before release is suspected to be due to infection pressure and contaminant exposure from feeding on fish. Molecular biomarkers are a sensitive tool to evaluate health and pollutant exposure and useful to serve as early warning indicators, monitoring and case-by-case tool for marine mammals in human care and the wild.

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.

Identifying Global Patterns of Transshipment Behavior

Transshipment at sea, the offloading of catch from a fishing vessel to a refrigerated vessel far from port, can obscure the actual source of the catch, complicating sustainable fisheries management, and may allow illegally caught fish to enter the legitimate seafood market. Transshipment activities often occur in regions of unclear jurisdiction where policymakers or enforcement agencies may be slow to act against a challenge they cannot see. To address this limitation, we processed 32 billion Automatic Identification System (AIS) messages from ocean-going vessels from 2012 to the end of 2017 and identified and tracked 694 cargo vessels capable of transshipping at sea and transporting fish (referred to as transshipment vessels). We mapped 46,570 instances where these vessels loitered at sea long enough to receive a transshipment and 10,233 instances where we see a fishing vessel near a loitering transshipment vessel long enough to engage in transshipment. We found transshipment behaviors associated with regions and flag states exhibiting limited oversight; roughly 47% of the events occur on the high seas and 42% involve vessels flying flags of convenience. Transshipment behavior in the high seas is relatively common, with vessels responsible for 40% of the fishing in the high seas having at least one encounter with a transshipment vessel in this time period. Our analysis reveals that addressing the sustainability and human rights challenges (slavery, trafficking, bonded labor) associated with transshipment at sea will require a global perspective and transnational cooperation.

Long-Distance Travellers: Phylogeography of a Generalist Parasite, Pholeter gastrophilus, from Cetaceans.

We studied the phylogeography and historical demography of the most generalist digenean from cetaceans, Pholeter gastrophilus, exploring the effects of isolation by distance, ecological barriers and hosts’ dispersal ability on the population structure of this parasite. The ITS2 rDNA, and the mitochondrial COI and ND1 from 68 individual parasites were analysed. Worms were collected from seven oceanic and coastal cetacean species from the south western Atlantic (SWA), central eastern Atlantic, north eastern Atlantic (NEA), and Mediterranean Sea. Pholeter gastrophilus was considered a single lineage because reciprocal monophyly was not detected in the ML cladogram of all individuals, and sequence variability was <1% for mtDNA and 0% for ITS2. These results rule out a recent suggestion that P. gastrophilus would actually be a cryptic-species complex. The genetic cohesion of P. gastrophilus could rely on the extensive exploitation of wide-ranging and highly mobile cetaceans, with a putative secondary role, if any, of intermediate hosts. Unique haplotypes were detected in SWA and NEA, and an AMOVA revealed significant population structure associated to the genetic variation in these regions. The Equator possibly acts as a significant geographical barrier for cetacean movements, possibly limiting gene flow between northern and southern populations of P. gastrophilus. A partial Mantel tests revealed that the significant isolation of NEA populations resulted from geographic clustering. Apparently, the limited mobility of cetaceans used by P. gastrophilus as definitive hosts in this region, coupled with oceanographic barriers and a patchy distribution of potential intermediate hosts could contribute to significant ecological isolation of P. gastrophilus in NEA. Rather unexpectedly, no genetic differentiation was found in the Mediterranean samples of this parasite. Historical demographic analyses suggested a recent population expansion of P. gastrophilus in the Atlantic Ocean, perhaps linked to initial association and subsequent spreading in cetaceans.

Beached bachelors: An extensive study on the largest recorded sperm whale Physeter macrocephalus mortality event in the North Sea

Between the 8th January and the 25th February 2016, the largest sperm whale Physeter macrocephalus mortality event ever recorded in the North Sea occurred with 30 sperm whales stranding in five countries within six weeks. All sperm whales were immature males. Groups were stratified by size, with the smaller animals stranding in the Netherlands, and the largest in England. The majority (n = 27) of the stranded animals were necropsied and/or sampled, allowing for an international and comprehensive investigation into this mortality event. The animals were in fair to good nutritional condition and, aside from the pathologies caused by stranding, did not exhibit significant evidence of disease or trauma. Infectious agents were found, including various parasite species, several bacterial and fungal pathogens and a novel alphaherpesvirus. In nine of the sperm whales a variety of marine litter was found. However, none of these findings were considered to have been the primary cause of the stranding event. Potential anthropogenic and environmental factors that may have caused the sperm whales to enter the North Sea were assessed. Once sperm whales enter the North Sea and head south, the water becomes progressively shallower (<40 m), making this region a global hotspot for sperm whale strandings. We conclude that the reasons for sperm whales to enter the southern North Sea are the result of complex interactions of extrinsic environmental factors. As such, these large mortality events seldom have a single ultimate cause and it is only through multidisciplinary, collaborative approaches that potentially multifactorial large-scale stranding events can be effectively investigated.

Ecotoxicological Biomarkers and Accumulation of Contaminants in Pinnipeds

Abstract Pinnipeds are long-lived predators with amphibious lifestyles, often residing in remote environments. Environmental contaminants as well as other stressors due to anthropogenic impacts into their habitat are known to affect the immune and endocrine system in seals and to affect marine mammal health status and increase their susceptibility to infectious disease. Pinnipeds are under continuous pressure of anthropogenic activities such as fisheries, ship traffic, oil exploration, and chemical and noise pollution. This has prompted many studies over the past decades to evaluate the effects of cumulative stress on the health of these marine mammals. Different approaches have been used to assess the effects of contaminants on the health of marine mammals and better understand the impact on their physiology. To evaluate detrimental changes potentially caused by xenobiotics on wildlife health, parameters of biologic processes with prognostic or diagnostic explanatory power, so-called biomarkers need to be found. An overview about parameters used as markers to measure exposure and effects is given in the following chapter, combining long-standing, established methods with new techniques that are promising future tools.