Highly pathogenic avian influenza A virus (HPAIV) H5N1 infection in two European grey seals (Halichoerus grypus) with encephalitis.

ABSTRACTRecent reports documenting sporadic infections in carnivorous mammals worldwide with highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b have raised concerns about the potential risk of adaptation to sustained transmission in mammals, including humans. We report H5N1 clade 2.3.4.4b infection of two grey seals (Halichoerus grypus) from coastal waters of The Netherlands and Germany in December 2022 and February 2023, respectively. Histological and immunohistochemical investigations showed in both animals a non-suppurative and necrotising encephalitis with viral antigen restricted to the neuroparenchyma. Whole genome sequencing showed the presence of HPAIV H5N1 clade 2.3.4.4b strains in brain tissue, which were closely related to sympatric avian influenza viruses. Viral RNA was also detected in the lung of the seal from Germany by real-time quantitative PCR. No other organs tested positive. The mammalian adaptation PB2-E627K mutation was identified in approximately 40% of the virus population present in the brain tissue of the German seal. Retrospective screening for nucleoprotein-specific antibodies, of sera collected from 251 seals sampled in this region from 2020 to 2023, did not show evidence of influenza A virus-specific antibodies. Similarly, screening by reverse transcription PCR of tissues of 101 seals that had died along the Dutch coast in the period 2020-2021, did not show evidence of influenza virus infection. Collectively, these results indicate that individual seals are sporadically infected with HPAIV-H5N1 clade 2.3.4.4b, resulting in an encephalitis in the absence of a systemic infection, and with no evidence thus far of onward spread between seals.

Detection of Novel Poxvirus from Gray Seal (Halichoerus grypus), Germany.

We detected a novel poxvirus from a gray seal (Halichoerus grypus) from the North Sea, Germany. The juvenile animal showed pox-like lesions and deteriorating overall health condition and was finally euthanized. Histology, electron microscopy, sequencing, and PCR confirmed a previously undescribed poxvirus of the Chordopoxvirinae subfamily, tentatively named Wadden Sea poxvirus.

Usefulness of serum cardiac troponin I concentration as a marker of survival of harbor seal (Phoca vitulina) pups during rehabilitation.

OBJECTIVE To measure serum cardiac troponin I (cTnI) concentrations in orphaned harbor seal (Phoca vitulina) pups at various points during rehabilitation in a seal rescue center and determine whether cTnI concentration was associated with survival during rehabilitation and duration of rehabilitation. DESIGN Serial cross-sectional study. ANIMALS Fifty-five 2- to 9-day-old harbor seal pups. PROCEDURES Blood samples for serum cTnI concentration measurement, CBC, and serum biochemical analysis were obtained from seal pups at admission into a seal rescue center, after 2 weeks of rehabilitation at the center, and prior to release. Serum cTnI concentrations were compared between seals that did or did not survive rehabilitation. RESULTS Median serum cTnI concentration was highest at admission (0.03 ng/mL). After 2 weeks, the median value was 0.01 ng/mL; prior to release, it was 0.01 ng/mL. Seal pups that were found to have died during or after rehabilitation (n = 7) had a significantly higher median serum cTnI concentration at admission (0.06 ng/mL) than did seal pups that survived rehabilitation (and for which the postrelease fate was unknown; 48; 0.03 ng/mL). No correlation was identified between serum cTnI concentration and duration of rehabilitation. CONCLUSIONS AND CLINICAL RELEVANCE The results of this study suggested some degree of myocardial injury was present in most of the orphaned seal pups admitted for rehabilitation. Measurement of serum cTnI concentration in seal pups at admission might provide prognostic information about their likelihood of survival during or after rehabilitation.

Impacts of Underwater Noise on Marine Vertebrates: Project Introduction and First Results.

The project conducts application-oriented research on impacts of underwater noise on marine vertebrates in the North and Baltic Seas. In distinct subprojects, the hearing sensitivity of harbor porpoises and gray seals as well as the acoustic tolerance limit of harbor porpoises to impulsive noise from pile driving and stress reactions caused by anthropogenic noise is investigated. Animals are equipped with DTAGs capable of recording the actual surrounding noise field of free-swimming harbor porpoises and seals. Acoustic noise mapping including porpoise detectors in the Natura 2000 sites of the North and Baltic Seas will help to fully understand current noise impacts.

In-air evoked potential audiometry of grey seals (Halichoerus grypus) from the North and Baltic Seas.

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.

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.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Concentrations of selected essential and non-essential elements in blood of harbor seal (Phoca vitulina) pups of the German North Sea.

This study on harbor seal (Phoca vitulina) pups of the North Sea evaluated concentrations of 14 essential and non-essential elements (Al, As, Be, Ca, Cd, Cr, Fe, Mn, Mo, Ni, Pb, Se, Sn, and Zn) in whole blood samples. The essential elements are analyzed to give references for health status determinations of pups. The measurement of classic toxic metals, like Pb or Cd, and other elements that may be in toxic concentrations in blood, is important due to their influence on health, particularly on the immune system. Blood samples of six seal pups found on the German Wadden Sea coast of Schleswig-Holstein in 2004 and transported to the Seal Centre Friedrichskoog, Germany were collected. The blood sampling was performed three times, immediately after collection of the newborns, after 1.5 months, and after 2 months before their release back into the wild. Inductively coupled plasma mass spectrometry and total reflection X-ray fluorescence spectrometry were used to determine the element concentrations. We found higher concentrations of Al, As, Fe, Mo, and Zn in blood samples of newborns compared to samples collected later, probably due to transplacental and lactational transfer from mother to fetus. Furthermore, there is a high need for, in particular, Fe and Zn in the developing organism, which may cause reduced values after some month. In contrast, the concentrations of Be, Cd, Ca, Cr, Pb, Mn, Ni, Se, and Sn, which were low in newborns and increased during the study, may be due to the fish fed to infant pups. Compared to free-ranging adults, in pups, the concentrations of Al, Ni, and Pb were higher in contrast to lower concentrations of As, Mn, and Mo. This case study is the first report on element levels in harbor seal pups of the North Sea.

Blood metal levels and metal-influenced immune functions of harbour seals in captivity.

Immunological blood parameters and the effects of environmental pollutants on the immune system are important to assess the health status of seals. Animals living permanently in seal centres are useful for development and validation of diagnostic tools for free-ranging animals. In this study, parameters of cellular immunity as well as metal concentrations in blood and metal influence on cell proliferation of seven seals from a seal centre were investigated repeatedly using multi-element analysis and a lymphocyte proliferation assay. The metal concentrations, except for tin and chromium, were in general comparable to those of free-ranging animals of the North Sea. The unstimulated and mitogen-stimulated lymphocyte proliferation showed strong intra- and inter-individual variability, which reflected variability in activation of the immune status. Furthermore, both immunosuppressive and stimulative influences of metals on lymphocytes were found. Summarising, the methods used in this investigation provided useful information on these animals, and their application to free-ranging animals can be recommended.

Metal-induced impairment of the cellular immunity of newborn harbor seals (Phoca vitulina).

The cellular immunity of newborn harbor seals and the influence of pollutants are rarely investigated. This study evaluated the lymphocyte proliferation using a lymphocyte proliferation test (LTT) to understand the dynamics of immune response in seal pups of varying ages from the moment they arrived in a seal center after active beaching until their release into wildlife 3 months later after rehabilitation. Moreover, the effect of various metals (Ag, Al, Au, Be, Cd, Co, Cr, Cu, different Hg compounds, Mo, Ni, Pb, Pd, Pt, Sn, Ti) on lymphocyte proliferation in terms of immunosuppression and hypersensitivity was investigated. First, a strong lymphocyte proliferation in newborns as a reflection of relative immunocompetence was found. Second, different metal-induced influences on lymphocyte proliferation such as specific inhibition by Be, Cd, Hg, and Sn as well as stimulation induced by Mo and Ni were determined. For seals tested repeatedly, the suppressive effect was detected in newborns but not found in the same animals when they were older and had become immunologically competent. Summarizing, the lymphocyte proliferation used as a marker in this investigation provided useful immunological information on these developing animals, and its application for toxicological studies on pollutants can be recommended.