INFLUENCE OF PATHOGENS, FISH-RELATED CHARACTERISTICS, AND ENVIRONMENTAL FACTORS ON THE DEVELOPMENT OF SKIN ULCERATIONS IN WILD COMMON DAB (LIMANDA LIMANDA) FROM THE NORTH SEA.

Environmental changes or stressors can result in the development of diseases. Through regular fish disease surveys in the Belgian part of the North Sea, attention was drawn to a sudden increase of skin ulceration prevalence between 2011 and 2014 in common dab (Limanda limanda). Information on prevalence, ulceration, bacteriology, fish-related (e.g., length, age, and sex) and (spatial and temporal) environmental factors, and fishing intensity were gathered. This detailed investigation was framed within a long-term monitoring program, executed every spring-autumn from 2000 to present. Ulcerations were observed in 1.3% of fish (n=3,999). Spatial and temporal differences were evident, and highest prevalence was found in summer. Vibrio was the dominant cultivated bacterial genus present in the lesions. Skin ulcerations appeared to be correlated with length and body condition of the fish, as well as with temperature and pH of the seawater and fishing vessel density. Our research suggested the involvement of multiple factors in the development of skin ulcerations in common dab and endorsed the effects of changing environment and human influence on the marine ecosystem through activities such as fishing.

Clinical, pathological, and laboratory diagnoses of diseases of harbour porpoises (Phocoena phocoena), live stranded on the Dutch and adjacent coasts from 2003 to 2016.

Harbour porpoises (Phocoena phocoena) in the North Sea live in an environment heavily impacted by humans, the consequences of which are a concern for their health. Autopsies carried out on stranded harbour porpoises provide an opportunity to assess health problems in this species. We performed 61 autopsies on live-stranded harbour porpoises, which died following admission to a rehabilitation centre between 2003 and 2016. The animals had stranded on the Dutch (n = 52) and adjacent coasts of Belgium (n = 2) and Germany (n = 7). We assigned probable causes for stranding based on clinical and pathological criteria. Cause of stranding was associated in the majority of cases with pathologies in multiple organs (n = 29) compared to animals with pathologies in a single organ (n = 18). Our results show that the three most probable causes of stranding were pneumonia (n = 35), separation of calves from their mother (n = 10), and aspergillosis (n = 9). Pneumonia as a consequence of pulmonary nematode infection occurred in 19 animals. Pneumonia was significantly associated with infection with Pseudalius inflexus, Halocercus sp., and Torynurus convolutus but not with Stenurus minor infection. Half of the bacterial pneumonias (6/12) could not be associated with nematode infection. Conclusions from this study are that aspergillosis is an important probable cause for stranding, while parasitic infection is not a necessary prerequisite for bacterial pneumonia, and approximately half of the animals (29/61) probably stranded due to multiple causes. An important implication of the observed high prevalence of aspergillosis is that these harbour porpoises suffered from reduced immunocompetence.

Dental anomalies and lesions in Eastern Atlantic harbor seals, Phoca vitulina vitulina (Carnivora, Phocidae), from the German North Sea.

Skulls of 1,901 Eastern Atlantic harbor seals (Phoca vitulina vitulina) were systematically studied for externally visible dental anomalies and lesions. The sample comprised 927 males and 974 female individuals, with age at death ranging between 1 week and 25 years. Most of the skulls originated from animals collected in 1988, when the population suffered from a mass mortality event caused by the phocine distemper virus (PDV). Mean age (± SD) of females (6.7 ± 6.4 years) was higher (p = 0.002) than that of males (5.9 ± 5.2 years). In 264 individuals, one or more teeth were missing either congenitally (n = 26 animals, 1.4%) or due to intravital loss (n = 238 animals, 12.5%). One male exhibited congenital absence of all teeth (anodontia). As this animal had been reported to be almost hairless, the condition was tentatively diagnosed as a case of hypohidrotic ectodermal dysplasia. Males were more frequently (p = 0.002) affected by intravital tooth loss (15.0%) than females (10.2%). Supernumerary teeth were found in 3.4% of the skulls, with females (4.7%) showing hyperodontia more frequently (p < 0.001) than males (1.9%). Fifty-nine individuals (3.1%; 28 males, 31 females, p = 0.84) exhibited abnormal tooth morphology. Tooth fractures were noted in 40 seals (2.1%), with males being more frequently affected than females (p = 0.017). Periapical lesions were diagnosed in 143 skulls, with a tendency (p = 0.05) for males (8.7%) to be more frequently affected than females (6.4%). Enamel hypoplasia was not observed in the study sample. Analyzing the occurrence of dental anomalies and lesions in wild mammals can substantially contribute to an assessment of population health and thereby broaden the basis for effective species conservation and informed management decisions.

Investigating a whale mass mortality event.

In 2016, 30 sperm whales died having stranded in the southern North Sea. Researchers have now investigated what caused this. Georgina Mills reports.

Biodegradation in seawater of PAH and alkylphenols from produced water of a North Sea platform.

Operational planned discharges of produced water (PW) to the marine environment from offshore oil production installations, contain low concentrations of dispersed oil compounds, like polycyclic aromatic hydrocarbons (PAHs) and alkylated phenols (APs). Biotransformation in natural seawater (SW) of naphthalenes/PAHs and phenol/APs in field-collected PW from a North Sea platform was investigated in this biodegradation study. The PW was diluted in SW from a Norwegian fjord, and the biodegradation study was performed in slowly rotating carousels at 13 °C over a period of 62 days. Naphthalenes/PAHs and phenol/APs biotransformation was determined by first-order rate kinetics, after normalization against the recalcitrant biomarker 17α(H),21ß(H)-Hopane. The results from this study showed total biotransformation half-lives ranging from 10 to 19 days for groups of naphthalenes and PAHs, while half-lives for APs (C0- to C9-alkylated) were 10-14 days. Biotransformation half-lives of single compounds ranged from 8 to >100 days for naphthalenes and PAHs (median 16 days), and from 5 to 70 days (median 15 days) for phenols and APs. Four of the tested PAHs (chrysene, benzo(b)fluoranthene, benzo(e)pyrene, benzo(g,h,i)perylene) and one AP (4-tert-butylphenol) showed biotransformation half-lives >50 days. This is one of a few studies that has investigated the potential for biodegradation of PW in natural SW. Methods and data from this study may be used as a part of Risk Based Approaches (RBA) for assessments of environmental fate of PW released to the marine environment and as part of the persistence related to risk.

Osteoarthritis of the temporomandibular joint in the Eastern Atlantic harbour seal (Phoca vitulina vitulina) from the German North Sea: a study of the lesions seen in dry bone.

BACKGROUND: Pathological changes and resulting functional impairment of the temporomandibular joint (TMJ) can substantially affect physical condition, morbidity, and mortality of wildlife species. Analysis of TMJ disorders is therefore of interest for the characterization of the health status of populations of wild mammals. This paper, for the first time, analyses the prevalence of TMJ osteoarthritis (TMJ-OA) and the spectrum of osteoarthritic bone lesions of the TMJ in the Eastern Atlantic harbour seal (Phoca vitulina vitulina), applying a standardized scoring system. Dry skulls of 1872 individuals from the German North Sea, collected between 1961 and 1994, were examined for lesions consistent with a diagnosis of TMJ-OA. Of the skulls, 913 (48.8%) were from male, 959 (51.2%) from female seals, with age at death ranging from 2 weeks to 25 years. Possible associations of TMJ-OA with dental or periodontal disorders were also analysed. RESULTS: Lesions consistent with TMJ-OA were found in 963 (53.9%) of the 1787 juvenile/subadult (5 weeks to 5 years of age) and adult (> 5 years) specimens, the condition mostly (95.0% of affected individuals) occurring in a bilateral fashion. Males were affected more frequently than females (p < 0.001), while lesion severity tended to be higher in females (p < 0.05). Severity of TMJ-OA lesions was positively correlated with age (p < 0.001). Lesion severity was also weakly positively correlated with the number of fractured teeth (p < 0.05) and of intravitally lost teeth (p < 0.01), when controlling for age at death as a confounder. CONCLUSIONS: TMJ-OA is a common disorder in the Eastern Atlantic harbour seal. The more pronounced severity of the lesions in females compared to males is basically attributed to the higher average age of the female subsample. The causes underlying the high prevalence of TMJ-OA in the studied assemblage remain unknown. Most of the specimens (75.3%) analysed in the present study were found dead during the first phocine distemper virus epizootic in 1988. Therefore, it is assumed that, contrary to other museum collections, only little overrepresentation of pathological skeletal conditions is present in this death sample compared with the population from which it originated.

Parasites in the inner ear of harbour porpoise: cases from the North and Baltic Seas.

Peribullar sinuses of harbour porpoises Phocoena phocoena are parasitized with high prevalence by the nematode Stenurus minor. The effect of S. minor on the hearing ability of this species is still undetermined. Here, we review the occurrence of S. minor in the inner ear of harbour porpoises recovered from strandings in the North and Baltic Seas. In particular, we present the results from ears collected in German and Danish waters from 2002 to 2016 and from Dutch waters from 2010 to 2016. While the prevalence of S. minor in pterygoid and peribullar sinuses and tympanic cavity was high in harbour porpoises (66.67% in our cases), its prevalence in the cochlea was rare. Only 1 case out of 129 analysed by either histology, electron microscopy or immunofluorescence showed the presence of a nematode parasite morphologically consistent with S. minor at the most basal portion of the right cochlea. This individual also had severe haemorrhage along the right cochlear spiral, which was likely caused by ectopic S. minor migration. Although this animal might have had impaired hearing in the right ear, it was otherwise in good body condition with evidence of recent feeding. These findings highlight the need to study the effect of parasites on hearing, and other pathological changes that might impair appropriate processing of acoustic information.

Norovirus Infection in Harbor Porpoises.

A norovirus was detected in harbor porpoises, a previously unknown host for norovirus. This norovirus had low similarity to any known norovirus. Viral RNA was detected primarily in intestinal tissue, and specific serum antibodies were detected in 8 (24%) of 34 harbor porpoises from the North Sea.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe.

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health.

Toxoplasma gondii in stranded marine mammals from the North Sea and Eastern Atlantic Ocean: Findings and diagnostic difficulties.

The occurrence of the zoonotic protozoan parasite Toxoplasma gondii in marine mammals remains a poorly understood phenomenon. In this study, samples from 589 marine mammal species and 34 European otters (Lutra lutra), stranded on the coasts of Scotland, Belgium, France, The Netherlands and Germany, were tested for the presence of T. gondii. Brain samples were analysed by polymerase chain reaction (PCR) for detection of parasite DNA. Blood and muscle fluid samples were tested for specific antibodies using a modified agglutination test (MAT), a commercial multi-species enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA). Out of 193 animals tested by PCR, only two harbour porpoise (Phocoena phocoena) cerebrum samples, obtained from animals stranded on the Dutch coast, tested positive. The serological results showed a wide variation depending on the test used. Using a cut-off value of 1/40 dilution in MAT, 141 out of 292 animals (41%) were positive. Using IFA, 30 out of 244 tested samples (12%) were positive at a 1/50 dilution. The commercial ELISA yielded 7% positives with a cut-off of the sample-to-positive (S/P) ratio≥50; and 12% when the cut-off was set at S/P ratio≥20. The high number of positives in MAT may be an overestimation due to the high degree of haemolysis of the samples and/or the presence of lipids. The ELISA results could be an underestimation due to the use of a multispecies conjugate. Our results confirm the presence of T. gondii in marine mammals in The Netherlands and show exposure to the parasite in both the North Sea and the Eastern Atlantic Ocean. We also highlight the limitations of the tests used to diagnose T. gondii in stranded marine mammals.

Occurrence of microsporidians Glugea hertwigi and Pleistophora ladogensis, in smelt Osmerus eperlanus from two German rivers, North Sea coast.

Monthly samples of smelt Osmerus eperlanus (Linnaeus, 1758) were collected from July 1985 to May 1986, in the river Elbe (Germany), and examined for infections with microsporidians. Two microsporidians were found: Glugea hertwigi Weissenberg, 1911, infecting the digestive tract and Pleistophora ladogenis Voronin, 1978, infecting the skeletal musculature. G. hertwigi infection led to the formation of xenomas, whereas P. ladogensis was characterized by diffuse infections, with the production of macroscopic visible thread-like or oval-shaped infection foci. Development of G. hertwigi in the host cells showed characteristics typical of the genus Glugea. The ultrastructural development of P. ladogensis showed features typical of the genus Pleistophora, without evidence of the production of 2 types of spores. Host reaction consisted of inflammatory tissue surrounding some of the infection foci as well as phagocytosis of spores. G. hertwigi was only found in juvenile smelt (<10 cm in length), whereas P. ladogensis infected smelts from 6 to 26 cm in length. Prevalence increased with fish length to a maximum value of 9.6%. Seasonal fluctuations in prevalence of infection were also found, with the lowest value in the winter months (2.5% in January 1986) and the highest in summer (11.8% in July 1985). The differences in prevalence of infection with fish length and date of sampling were significant. Additionally, samples of smelt caught in April 1986 from the rivers Eider and Ems revealed infections with P. ladogensis in the first river system only.

Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates.

Vibrio vulnificus is a halophilic bacterium of coastal environments known for sporadically causing severe foodborne or wound infections. Global warming is expected to lead to a rising occurrence of V. vulnificus and an increasing incidence of human infections in Northern Europe. So far, infections in Germany were exclusively documented for the Baltic Sea coast, while no cases from the North Sea region have been reported. Regional variations in the prevalence of infections may be influenced by differences in the pathogenicity of V. vulnificus populations in both areas. This study aimed to compare the distribution of virulence-associated traits and genotypes among 101 V. vulnificus isolates from the Baltic Sea and North Sea in order to assess their pathogenicity potential. Furthermore, genetic relationships were examined by multilocus sequence typing (MLST). A high diversity of MLST sequences (74 sequence types) and differences regarding the presence of six potential pathogenicity markers were observed in the V. vulnificus populations of both areas. Strains with genotypes and markers associated with pathogenicity are not restricted to a particular geographic region. This indicates that lack of reported cases in the North Sea region is not caused by the absence of potentially pathogenic strains.

Anisakid nematodes in beaked redfish (Sebastes mentella) from three fishing grounds in the North Atlantic, with special notes on distribution in the fish musculature.

Parasitic anisakid nematodes commonly occur in the musculature and visceral organs of many fish species from the North Atlantic. In this respect, the presence of the third stage larvae of Anisakis spp. in the fish musculature may pose a potential consumer hazard due to the parasite's ability to cause anisakidosis. Thus, knowledge on the occurrence and distribution of these potentially zoonotic parasites in the commercially important North Atlantic fish species is crucial in order to evaluate and consequently prevent human infections. In the present study, 300 Sebastes mentella from three North Atlantic fishing grounds (Northern North Sea: Tampen; Barents Sea: off Bear Island; Irminger Sea: off SE Greenland) were examined for anisakid nematodes, with emphasis on occurrence and distribution in the musculature. Overall larval prevalence and mean intensity were significantly higher in redfish from Tampen (94%; 13.5±20.0) and Bear Island (94%; 14.5±19.4) than in fish from SE Greenland (75%; 6.0±5.8; p<0.01). The same trend was observed for larval infection in the musculature showing prevalence and mean intensities of 79%, 73%, and 55%, and 5.9±6.6, 5.8±6.5, and 3.2±2.4, in the musculature of redfish from Tampen, Bear Island, and Greenland, respectively. Conventional microscopy and rDNA ITS-gene sequencing of various subsamples of muscle-dwelling nematode larvae of redfish from every catching locality revealed the presence of two anisakid species; Anisakis simplex sensu stricto and the non-zoonotic Hysterothylacium aduncum. Since the larvae of H. aduncum typically occur in or on the viscera of fish, our findings of two specimens in the belly flaps of redfish were unusual. Additionally, more than 92% of the muscle-dwelling larvae occurred in the belly flaps, i.e. the hypaxial part of the musculature surrounding the visceral organs on either fish side. Thus, trimming the fillets of beaked redfish by removal of most of the belly flaps would significantly reduce the probability of anisakid nematode larvae to be present in the final product.

Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265) Brucella positive tissue samples were Brucella positive by culture and these were all confirmed by real-time polymerase chain reaction (real-time PCR) based on the insertion element 711 (IS711). In addition, two more Brucella-positive tissue samples from two animals collected in 2011 were identified using real-time PCR resulting in an overall Brucella prevalence of 6.3% (7/112 animals). Brucella spp. were obtained from lungs (n=3), pulmonary lymph node (n=3) and lungworms (n=2). Multi Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) typing based on the MLVA-16 showed that the Brucella isolates were B. ceti. Additional in silico Multi Locus Sequence typing (MLST) after whole genome sequencing of the 6 Brucella isolates confirmed B. ceti ST 23. According to the Brucella 2010 MLVA database, the isolated Brucella strains encountered were of five genotypes, in two distinct subclusters divided in two different time periods of harbour porpoises collection. This study is the first population based analyses for Brucella spp. infections in cetaceans stranded along the Dutch coast.

Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae.

Basic immune response mechanisms in vertebrates against helminths are still poorly understood. Fish-nematode models may prove valuable for elucidation of this question. In this study we orally challenged rainbow trout (Oncorhynchus mykiss) with larvae of Anisakis simplex (Nematoda: Anisakidae) and subsequently investigated the expression of 18 immune relevant genes in spleen and liver 1, 4 and 8days post infection (d.p.i.). Gene expression data were analysed with regard to the infection status of the challenged rainbow trout at the time of necropsy; "worms rejected" (÷worms), "worms present" (+worms) and a combined group consisting of samples pooled from both previous groups (÷/+worms). No significant regulation of cytokine genes was recorded but fish which had rejected worms up-regulated the CD4 gene (6.1-fold change, 8d.p.i.) in liver. The gene encoding CD8 was significantly down-regulated 24h post challenge in livers in fish still carrying worms (2.7-fold change) but not in the worm-free group. The immunoglobulin gene IgM was significantly down-regulated (2.9-fold change, 8d.p.i.) in liver samples from the +worms group. Complement factor C3 and precerebellin genes were significantly up-regulated twofold in liver samples from infected fish 4d.p.i. Significant up-regulation of the acute-phase protein SAA was observed in all three groups and in both tissues. To our knowledge, this is the first study to describe the expression of immune genes in a fish host challenged with live nematode larvae.

Sealworm Pseudoterranova decipiens s.s. infection of European smelt Osmerus eperlanus in German coastal waters: ecological implications.

European smelt Osmerus eperlanus (n = 501) from the German Wadden Sea (North Sea) near the city of Cuxhaven were examined for their infestation with parasitic anisakid nematodes, especially with sealworms of the genus Pseudoterranova. The distribution of third-stage larvae (L3) in the musculature and viscera of the fish was analyzed. In total, we isolated 543 L3 from the hosts' body cavity and musculature. A subsample of 105 larvae were identified as the (sibling) species P. decipiens s.s. using direct sequencing of the highly variable ribosomal ITS1-5.8S-ITS2 genetic marker. The mean abundance was 1.1, the mean intensity was 2.3 P. decipiens s.s. and the prevalence was 47.3%. Total length and total weight, but not Fulton's condition factor (K), were significantly different in infected compared to uninfected smelt. No correlation was found between the total length of infected fish and the intensity of anisakid nematodes. The vast majority of P. decipiens s.s. was found in the musculature of the smelt. More than half (55.7%) of all nematodes were located in the 3 parts of the epaxial musculature, whereas 18.4 and 26.0% were found in the hypaxial musculature and the compartments of the tail muscles, respectively.

Hyperpigmentation in North Sea dab Limanda limanda. II. Macroscopic and microscopic characteristics and pathogen screening.

An increasing trend in the prevalence of hyperpigmentation in the common dab Limanda limanda from the North Sea prompted us to investigate the potential role of infectious agents as causes or contributing factors to the condition. Dab representing 3 severity grades of hyperpigmentation were sampled for virology, bacteriology, histopathology and ultrastructure assessments. No cytopathic effect was recorded during virology testing, and bacteriological results showed no differences between normal and hyperpigmented dab. Histopathological assessment showed that the most significant changes occurred in the dermis as a result of chromatophore hyperplasia, namely melanophores and iridophores, alongside loose melanin granules. Dermal lymphocytic infiltration occasionally expanding into the epidermis and the underlying musculature was more frequent in highly pigmented dab than in normal fish, suggesting an active immune response. Ultrastructure studies showed additional disruption of the epithelial layer, with loose melanin granules between cells and a number of single or aggregated melanocytes. Dab representing different grades of hyperpigmentation kept in the laboratory alongside normal fish for a monitoring period of 18 mo showed no changes in their pigment distribution pattern, nor occurrence of new pigment in the normal fish. The current investigation found no association of hyperpigmentation in the common dab with infectious agents; therefore, understanding the cause of the condition remains a challenge which can now more reliably focus on a non-infectious origin hypothesis.

First detection of piscine reovirus (PRV) in marine fish species.

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.

Sleep disturbances among offshore fleet workers: a questionnaire-based survey.

BACKGROUND. Shift work is related to fatigue and desynchronization with the external environment. This study investigates how 6-h shifts and 12-h shifts affects sleep and safety in workers onboard offshore supply vessels, and if any differences exist between the two working schedules. MATERIAL AND METHODS. A questionnaire study was carried out in the North Sea, Australia, Africa, South America, and the Far East, with 577 participants. The offshore fleet workers gave information on parameters related to sleep disturbances, causes of sleep disturbances, and safety. Regional differences in these parameters were also investigated. RESULTS. Workers on 6-hour shifts reported significantly more sleep problems than 12-hour shift workers did (p 〈 0.01). The 6-hour workers were more affected by noise (p 〈 0.01) and shift-work itself (p 〈 0.01). CONCLUSIONS. Those working 6-hour shifts suffer more from sleep disturbances than those on 12-hour shifts, but this is not reflected in the perception of safety within the individual. Noise and shift-work itself is more of a problem in the 12-hour workers. Differences in safety culture and work morale are likely to cause the differences between regions.