Variations in antimicrobial resistance genes present in the rectal faeces of seals in Scottish and Liverpool Bay coastal waters.

Antibiotic resistance genes originating from human activity are considered important environmental pollutants. Wildlife species can act as sentinels for coastal environmental contamination and in this study we used qPCR array technology to investigate the variety and abundance of antimicrobial resistance genes (ARGs), mobile genetic elements (MGEs) and integrons circulating within seal populations both near to and far from large human populations located around the Scottish and northwest English coast. Rectal swabs were taken from 50 live grey seals and nine live harbour seals. Nucleic acids were stabilised upon collection, enabling extraction of sufficient quality and quantity DNA for downstream analysis. 78 ARG targets, including genes of clinical significance, four MGE targets and three integron targets were used to monitor genes within 22 sample pools. 30 ARGs were detected, as well as the integrons intl1 and intl2 and tnpA transposase. Four ß-lactam, nine tetracycline, two phenicol, one trimethoprim, three aminoglycoside and ten multidrug resistance genes were detected as well as mcr-1 which confers resistance to colistin, an important drug of last resort. No sulphonamide, vancomycin, macrolide, lincosamide or streptogramin B (MLSB) resistance genes were detected. Resistance genes were detected in all sites but the highest number of ARGs (n = 29) was detected in samples derived from grey seals on the Isle of May, Scotland during the breeding season, and these genes also had the highest average abundance in relation to the 16S rRNA gene. This pilot study demonstrates the effectiveness of a culture-independent workflow for global analysis of ARGs within the microbiota of live, free-ranging, wild animals from habitats close to and remote from human habitation, and highlights seals as a valuable indicator species for monitoring the presence, abundance and land-sea transference of resistance genes within and between ecosystems.

The first report of Listeria monocytogenes detected in pinnipeds.

The aim of this study was to describe the pathology in seals from which Listeria monocytogenes was isolated and investigate if the lesions' nature and severity were related to the phylogeny of isolates. L. monocytogenes was isolated from 13 of 50 (26%) dead grey seal (Halichoerus grypus) pups, six (12%) in systemic distribution, on the Isle of May, Scotland. Similar fatal L. monocytogenes-associated infections were found in a grey seal pup from Carnoustie, Scotland, and a juvenile harbour seal (Phoca vitulina) in the Netherlands. Whole genome sequencing of 15 of the L. monocytogenes isolates identified 13 multilocus sequence types belonging to the L. monocytogenes lineages I and II, but with scant phenotypic and genotypic antimicrobial resistance and limited variation in virulence factors. The phylogenetic diversity present suggests there are multiple sources of L. monocytogenes, even for seal pups born in the same colony and breeding season. This is the first description of L. monocytogenes isolated from, and detected in lesions in, pinnipeds and indicates that infection can be systemic and fatal. Therefore, listeriosis may be an emerging or overlooked disease in seals with infection originating from contamination of the marine environment.

Investigating the impacts of H2O2 treatment on gills of healthy Atlantic salmon reveals potential changes to mucus production with implications on immune activity.

Current treatment strategies for relevant infectious diseases in Atlantic salmon (Salmo salar L.) include the use of low salinity or freshwater bathing. However, often availability is restricted, and hydrogen peroxide (H2O2) is used as an alternative. The potential impacts of H2O2 on fish mucosal tissues, especially the gills therefore need to be considered. In this study the mucosal and immunological effects of H2O2 treatment on the gills of healthy Atlantic salmon were examined by gene expression (qPCR) and immunohistochemistry (IHC) investigating T-cell, B-cell, and mucin activity. Healthy fish were treated with H2O2 and sampled at different times: 4 h, 24 h and 14 days post-H2O2 treatment (dpt) (total n = 18) to investigate the effect of holding time and H2O2 treatment. Treatment with H2O2 resulted in up-regulation of markers for T-cell activity and anti-inflammatory response and down-regulation of mucin expression in the gills at 14 dpt compared to fish sampled prior to treatment (0h; n = 5 fish). These findings were supported by IHC analysis, which despite being highly variable between samples, showed an increase in the number of CD3+ T cells at 14 dpt in 50% of treated fish compared to pre-treatment fish. The results from this study suggest that H2O2 treatment does not immune compromise healthy Atlantic salmon after 14 dpt (i.e., post-recovery) but modulates gill immune activity and disrupts the mucus covering of the gills. However, further studies are required to determine whether the effects observed are related to H2O2 treatment in isolation or other variables such as holding time or environmental factors.

A comparative study of the fecal microbiota of gray seal pups and yearlings - a marine mammal sentinel species.

Gray seals (Halichoerus grypus) can act as sentinel species reflecting the condition of the environment they inhabit. Our previous research identified strains of pathogenic Campylobacter and Salmonella, originating from both human and agricultural animal hosts, on rectal swabs from live gray seal (H. grypus) pups and yearlings on the Isle of May, Scotland, UK. We examined rectal swabs from the same pup (n = 90) and yearling (n = 19) gray seals to gain further understanding into the effects of age-related changes (pup vs. yearling) and three different natal terrestrial habitats on seal pup fecal microbiota. DNA was extracted from a subset of rectal swabs (pups n = 23, yearlings n = 9) using an optimized procedure, and the V4 region of the 16S ribosomal RNA gene was sequenced to identify each individual's microbiota. Diversity in pup samples was lower (3.92 ± 0.19) than yearlings (4.66 ± 0.39) although not significant at the p = 0.05 level (p = 0.062) but differences in the composition of the microbiota were (p < 0.001). Similarly, differences between the composition of the microbiota from pups from three different terrestrial habitats (Pilgrim's Haven [PH], Rona Rocks [RR], and Tarbet Slope [TS]) were highly significant (p < 0.001). Pairwise tests showed significant differences between all three habitats: PH versus TS (p = 0.019), PH versus RR (p = 0.042) and TS versus RR (p = 0.020). This preliminary study suggests a general trend, that seal microbiomes are modified by both age and, in pups, different terrestrial habitats. Furthermore, knowledge of the microbiota species present has the potential to be used in determining the environmental quality index.

Presence of DNA from Chlamydia-like organisms in the nasal cavities of grey seal pups (Halichoerus grypus) and three different substrates present in a breeding colony.

BACKGROUND: Chlamydia-like organisms (CLO) have been found to be present in many environmental niches, including human sewage and agricultural run-off, as well as in a number of aquatic species worldwide. Therefore, monitoring their presence in sentinel wildlife species may be useful in assessing the wider health of marine food webs in response to habitat loss, pollution and disease. We used nasal swabs from live (n = 42) and dead (n = 50) pre-weaned grey seal pups and samples of differing natal substrates (n = 8) from an off-shore island devoid of livestock and permanent human habitation to determine if CLO DNA is present in these mammals and to identify possible sources. RESULTS: We recovered CLO DNA from 32/92 (34.7%) nasal swabs from both live (n = 17) and dead (n = 15) seal pups that clustered most closely with currently recognised species belonging to three chlamydial families: Parachlamydiaceae (n = 22), Rhabdochlamydiaceae (n = 6), and Simkaniaceae (n = 3). All DNA positive sediment samples (n = 7) clustered with the Rhabdochlamydiaceae. No difference was found in rates of recovery of CLO DNA in live versus dead pups suggesting the organisms are commensal but their potential as opportunistic secondary pathogens could not be determined. CONCLUSION: This is the first report of CLO DNA being found in marine mammals. This identification warrants further investigation in other seal populations around the coast of the UK and in other areas of the world to determine if this finding is unique or more common than shown by this data. Further investigation would also be warranted to determine if they are present as purely commensal organisms or whether they could also be opportunistic pathogens in seals, as well as to investigate possible sources of origin, including whether they originated as a result of anthropogenic impacts, including human waste and agricultural run-off.

Novel Dermatitis and Relative Viral Nucleic Acid Tissue Loads in a Fin Whale (Balaenoptera physalus) with Systemic Cetacean Morbillivirus Infection.

Cetacean morbilliviruses (CeMVs) are significant causes of mortality in many cetacean species in epizootics and smaller outbreaks. Despite the prominence of skin lesions in seals and terrestrial animals, including humans, affected by other morbilliviruses, they have not been reported in CeMV-infected cetaceans. Here we report CeMV-associated skin lesions in a fin whale (Balaenoptera physalus) with subacute, systemic CeMV infection that live-stranded in Scotland, UK. Grossly, the skin was sloughing in large sheets, presumed due to autolysis, but histological examination showed syncytia, below the dermoepidermal junction, that were strongly immunopositive for morbillivirus antigen, as were syncytia in other organs. By polymerase chain reaction (PCR), the relative load of CeMV-specific RNA was largest in the liver and urinary bladder, even in formalin-fixed, paraffin-wax embedded samples. Levels were low in skin and only detectable in frozen samples. Genetic comparison of the CeMV revealed close alignment with isolates from fin whales from the North Atlantic Ocean and Mediterranean Sea, but that it was distinct from the porpoise CeMV clade. These findings show skin samples can be used to diagnose CeMV infection in cetaceans, highlighting the potential of ante-mortem sampling for monitoring disease in current populations and assessment of changes in host and pathogen genetics.

Novel atypical Aeromonas salmonicida bath challenge model for juvenile ballan wrasse (Labrus bergylta, Ascanius).

Atypical Aeromonas salmonicida (aAs) is currently one of the most routinely recovered bacterial pathogens isolated during disease outbreaks in farmed cleaner fish, ballan wrasse (Labrus bergylta, Ascanius). Vibrionaceae family bacteria have also been isolated from ballan wrasse in Scotland. This study determined the infectivity, pathogenicity and virulence of aAs and Vibrionaceae isolates in juvenile farmed ballan wrasse (n = 50; approx. 2 g) using a bath challenge, and fish were monitored for a period of 16 days. Atypical As caused significant mortalities in contrast to Vibrionaceae isolates. Notably, differential virulence was observed between two aAs vapA type V strains at similar challenge doses. Diseased fish exhibited a systemic infection where aAs was detected in all analysed tissues (liver, spleen and kidney) by PCR and qPCR. Macroscopically, moribund and survivor fish exhibited hepatomegaly and splenomegaly. In moribund and surviving fish, histopathology showed granulomatous hepatitis with eosinophilic granular cells surrounding bacterial colonies and endocarditis along with splenic histiocytosis. This is the first report of a successful aAs bath challenge model for juvenile ballan wrasse which provides an important tool for future studies on vaccine efficacy and immunocompetence.

Intrasarcoplasmic Polyglucosan Inclusions in Heart and Skeletal Muscles of Long-Finned Pilot Whales (Globicephala melas) may be Age-Related.

Polysaccharide storage myopathies have been described in several animal species and are characterized by periodic acid-Schiff (PAS)-positive, diastase-resistant intrasarcoplasmic inclusions in myocytes. Skeletal and cardiac muscle samples from a subset of a single pod of stranded long-finned pilot whales (Globicephala melas) were evaluated by light and transmission electron microscopy. Twelve individuals demonstrated sporadic basophilic packets of PAS-positive, diastase-resistant complex polysaccharide material, either centrally or peripherally, in skeletal and cardiac myocytes. Few microscopic myopathic changes were found but included focal inflammation and internalized nuclei. Ultrastructurally, the inclusions consisted of loosely arranged, tangled filaments and were not membrane-bound, which is consistent with polyglucosan inclusions. Within skeletal muscle, the number of inclusions had a marginal statistically significant (P = 0.0536) correlation with length, as a proxy for age, suggesting that such inclusions in skeletal muscles may be age-related, although the cause remains unclear.

Campylobacter pinnipediorum subsp. caledonicus and C. pinnipediorum subsp. pinnipediorum recovered from abscesses in pinnipeds.

Campylobacter pinnipediorum was described recently for isolates recovered from pinnipeds. The novel species was further split into 2 subspecies based on host and geography, with C. pinnipediorum subsp. pinnipediorum recovered from otariid seals in California (USA) and C. pinnipediorum subsp. caledonicus recovered from phocid seals in Scotland. We report details of the infections of 7 pinnipeds from which C. pinnipediorum was isolated: C. pinnipediorum subsp. caledonicus was isolated from 2 harbour seals Phoca vitulina and a single grey seal Halichoerus grypus, and C. pinnipediorum subsp. pinnipediorum was isolated from California sea lions Zalophus californianus. Six of the isolates were recovered from samples collected at post-mortem investigation. In 2 of the Scottish seals and in 3 of the California seals, C. pinnipediorum was the sole bacterial isolate recovered from abscesses present and suggests they may have resulted from conspecific or intraspecific bite wounds.

Liver colour scoring index, carotenoids and lipid content assessment as a proxy for lumpfish (Cyclopterus lumpus L.) health and welfare condition.

Ensuring lumpfish health and welfare in salmon farms is vital to reduce the high mortality rates reported and to guarantee a high delousing efficiency. Recent observations of farmed lumpfish livers have shown colours ranging from pale (colours 1 and 2), through bright orange (colours 3 and 4), to dark reddish-brown (colours 5 and 6), some of which may be related to welfare condition. To characterize the status of lumpfish deployed in four Faroese salmon farms, several welfare indicators were assessed: a weight-length relationship, scoring of external physical damage, and after dissection, stomach content and liver colour scoring. Liver samples were weighed, stored and analysed for lipid content, lipid classes, total pigments, fatty acid profile and histopathology to explain the differences between the mentioned liver colours. Bright orange livers, liver colours 3 and 4, were related to increased levels of carotenoid pigments rather than levels of lipids and appear to reflect good fish welfare. However, dark reddish-brown colours, liver colours 5 and 6, were associated with very low levels of triacyl glycerides in the liver, indicating use of lipid reserves and poor welfare condition. Histopathology confirmed that the dark reddish-brown livers, liver colours 5 and 6, formed a distinct group. Thus, liver colour was shown to be a good welfare indicator and should be monitored in farms.

Changes in distribution, morphology and ultrastructure of chloride cell in Atlantic salmon during an AGD infection.

Amoebic gill disease (AGD) is emerging as one of the most significant health challenges affecting farmed Atlantic salmon in the marine environment. It is caused by the amphizoic amoeba Neoparamoeba perurans, with infestation of gills causing severe hyperplastic lesions, compromising overall gill integrity and function. This study used histology, transmission electron microscopy (TEM), immunohistochemistry and transcript expression to relate AGD-associated pathological changes to changes in the morphology and distribution of chloride cells (CCs) in the gills of Atlantic salmon (Salmo salar L.) showing the progression of an AGD infection. A marked reduction in numbers of immunolabelled CCs was detected, and a changing pattern in distribution and morphology was closely linked with the level of basal epithelial hyperplasia in the gill. In addition, acute degenerative ultrastructural changes to CCs at the lesion site were observed with TEM. These findings were supported by the early-onset downregulation of Na+ /K+ -ATPase transcript expression. This study provides supportive evidence that histological AGD lesion assessment was a good qualitative tool for AGD scoring and corresponded well with qPCR genomic Paramoeba perurans quantification. Ultrastructural changes induced in salmon CCs as a result of AGD are reported here for the first time.

Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.).

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non-aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non-aqueous-based fixatives; qualitative and semi-quantitative analysis revealed a greater preservation of the gill mucus using the non-aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid-Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn-fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus-embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.

Streptococcus canis Are a Single Population Infecting Multiple Animal Hosts Despite the Diversity of the Universally Present M-Like Protein SCM.

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence.

Widespread neonatal infection with phocid herpesvirus 1 in free-ranging and stranded grey seals Halichoerus grypus.

Phocid herpesvirus 1 (PhHV-1) is known to infect grey seals Halichoerus grypus but little is known about its pathogenicity or true prevalence in this species. To investigate the prevalence of and risk factors associated with PHV-1 infection, nasal swabs were collected from grey seal pups and yearlings on the Isle of May, a well-studied grey seal breeding colony, and from stranded grey seal pups submitted to a rehabilitation centre. PhHV-1 nucleic acids were detected in nasal swabs from 58% (52/90) of live free-ranging grey seal pups, 62% (18/29) of live stranded grey seal pups and 28% (5/18) of live free-ranging yearlings, suggesting recrudescence in the latter. Location within the colony, pup body mass and stranding were determined to be risk factors for shedding PhHV-1 in live seal pups with a significantly higher prevalence of PhHV-1 in pups born on the tidal boulder beach when compared to other sites; a significantly positive correlation of PhHV-1 shedding and pup body mass and a higher prevalence in stranded grey seal pups compared to their free-ranging conspecifics. The prevalence of PhHV1 in dead pups on the Isle of May was 56% (27/48) with a positive PhHV-1 PCR status significantly associated with hepatic necrosis (p = 0.01), thymic atrophy (p < 0.001) and buccal ulceration (p = 0.027). Results indicate that PhHV1 was widespread in the pups in the Isle of May grey seal breeding colony.

First isolation of Brucella pinnipedialis and detection of Brucella antibodies from bearded seals Erignathus barbatus.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

Pathological and phylogenetic characterization of Amphibiothecum sp. infection in an isolated amphibian (Lissotriton helveticus) population on the island of Rum (Scotland).

Outbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infected L. helveticus in France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction from Amphibiocystidium spp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that the L. helveticus-infecting pathogen represents a single, novel species; Amphibiothecum meredithae.

Salmonella infection in grey seals (Halichoerus grypus), a marine mammal sentinel species: pathogenicity and molecular typing of Salmonella strains compared with human and livestock isolates.

Microbial pollution of the marine environment through land-sea transfer of human and livestock pathogens is of concern. Salmonella was isolated from rectal swabs of free-ranging and stranded grey seal pups (21.1%; 37/175) and compared with strains from the same serovars isolated from human clinical cases, livestock, wild mammals and birds in Scotland, UK to characterize possible transmission routes using pulsed-field gel electrophoresis and multi-locus variable number of tandem repeat analyses. A higher prevalence of Salmonella was found in pups exposed to seawater, suggesting that this may represent a source of this pathogen. Salmonella Bovismorbificans was the most common isolate (18.3% pups; 32/175) and was indistinguishable from isolates found in Scottish cattle. Salmonella Typhimurium was infrequent (2.3% pups; 4/175), mostly similar to isolates found in garden birds and, in one case, identical to a highly multidrug resistant strain isolated from a human child. Salmonella Haifa was rare (1.1% pups; 2/175), but isolates were indistinguishable from that of a human clinical isolate. These results suggest that S. Bovismorbificans may circulate between grey seal and cattle populations and that both S. Typhimurium and S. Haifa isolates are shared with humans, raising concerns of microbial marine pollution.

Isolation of Brucella ceti from a Long-finned Pilot Whale (Globicephala melas) and a Sowerby's Beaked Whale (Mesoploden bidens).

Brucella ceti is an emerging zoonotic pathogen that has been recovered from several species of cetaceans in the world's oceans over the past 20 yr. We report the recovery of B. ceti from a Sowerby's beaked whale (Mesoploden bidens) and a long-finned pilot whale (Globicehala melas). Recovery from the testis of a long-finned pilot whale provides further evidence of potential for B. ceti infection to impact the reproductive success of cetaceans, many of which are threatened species. The addition of another two cetacean species to the growing number from which B. ceti has been recovered also further emphasizes the concern for human infections with this organism.

Evidence of land-sea transfer of the zoonotic pathogen Campylobacter to a wildlife marine sentinel species.

Environmental pollution often accompanies the expansion and urbanization of human populations where sewage and wastewaters commonly have an impact on the marine environments. Here, we explored the potential for faecal bacterial pathogens, of anthropic origin, to spread to marine wildlife in coastal areas. The common zoonotic bacterium Campylobacter was isolated from grey seals (Halichoerus grypus), an important sentinel species for environmental pollution, and compared to isolates from wild birds, agricultural sources and clinical samples to characterize possible transmission routes. Campylobacter jejuni was present in half of all grey seal pups sampled (24/50 dead and 46/90 live pups) in the breeding colony on the Isle of May (Scotland), where it was frequently associated with histological evidence of disease. Returning yearling animals (19/19) were negative for C. jejuni suggesting clearance of infection while away from the localized colony infection source. The genomes of 90 isolates from seals were sequenced and characterized using a whole-genome multilocus sequence typing (MLST) approach and compared to 192 published genomes from multiple sources using population genetic approaches and a probabilistic genetic attribution model to infer the source of infection from MLST data. The strong genotype-host association has enabled the application of source attribution models in epidemiological studies of human campylobacteriosis, and here assignment analyses consistently grouped seal isolates with those from human clinical samples. These findings are consistent with either a common infection source or direct transmission of human campylobacter to grey seals, raising concerns about the spread of human pathogens to wildlife marine sentinel species in coastal areas.