Unraveling the gut microbiota of Mexican pinnipeds: the dominance of life histories over phylogeny.

Studying how phylogeny influences the composition and functions of microbiotas within animal hosts is essential for gaining insights into the connection between genetics, ecology, and health in the animal kingdom. However, due to limited comprehensive studies, this influence remains unclear for many wild mammals, including Mexican pinnipeds. We employed 16S rRNA gene deep-sequencing to investigate the impact of phylogeny on the gut microbiota of four pinniped species inhabiting Mexican shores: the Pacific harbor seal (Phoca vitulina richardii), the northern elephant seal (Mirounga angustirostris), the California sea lion (Zalophus californianus), and the Guadalupe fur seal (Arctocephalus philippii townsendi). Our results indicated that factors such as diets and shared life histories exerted more influence on microbiota composition than phylogeny alone. Notably, otariid species sharing similar life histories displayed greater microbiota similarity than phocids, which have distinct life histories and fewer microbiota similarities. Furthermore, harbor seals have more microbial similarities with the two otariid species than with elephant seals. Of particular concern, we observed a higher abundance of potentially pathogenic bacteria (e.g., Photobacterium damselae and Clostridium perfringens) in harbor seals and Guadalupe fur seals compared to other pinnipeds. This finding could pose health threats to these species and nearby human populations.IMPORTANCEPinnipeds in Mexico host microbial communities that remain understudied. While several factors can influence microbiota composition, the role of phylogenetic relationships among these pinnipeds remains unclear due to limited knowledge of the microbiota in certain species. This study aimed to fill this gap by characterizing the composition and function of the gut microbiota in the four pinniped species that occur in Mexico. Our analysis reveals that shared diets and life histories contribute to similarities in the composition of gut microbial communities. This study also highlights the potential differences in the metabolic capabilities and adaptations within the gut microbiota of pinnipeds. Understanding how phylogeny impacts microbial communities enhances our insights into the evolutionary dynamics of marine mammals.

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.

Survival and adaptation of Streptococcus phocae in host environments.

Marine mammals are sentinel species representing the "health" of our oceans on which we are dependent. There are many threats to marine mammals including infectious diseases that increase with climate change and pollution of the marine environment. Streptococcus phocae has frequently been isolated from diseased or dead marine mammals. However, its pathogenicity and contribution to disease in marine mammals is still unknown. As bacteria including (potential) pathogens has to deal with different host environments during colonization or infection, we investigated the survival of S. phocae in fresh porcine and phocid blood, in seawater and in the presence of macrophages and (epithelial) cells from harbor seals and pigs. Furthermore, we tested adherence on and invasion of different (marine) mammalian cells by S. phocae. Our results showed that S. phocae can survive in seawater for at least 11 and 28 days at 16°C and 4°C, respectively. It is able to grow in blood of harbor and grey seals, but not in porcine blood. Furthermore, S. phocae is adherent and invasive to cells from seals and pigs, while the portion of invasive cells was higher in seal derived cells. Macrophages of harbor seals were more efficient in killing S. phocae than porcine macrophages. Our results indicate that S. phocae has strategies enabling it to adapt to the marine environment and seal hosts.

Arcanobacterium buesumense sp. nov., isolated from an anal swab of a male harbour seal (Phoca vitulina).

A polyphasic taxonomic study was performed on an unidentified previously described Arcanobacterium-like Gram-positive strain 2701T isolated from an anal swab of a dead male harbour seal. Comparative 16S rRNA sequencing showed that the bacterium belonged to the genus Arcanobacterium in the family Arcanobacteriaceae. The genome sequence of the strain was obtained by Borowiak et al. [1]. The genome had a G+C content of 49 mol% and a total length of 1.94 Mb. The presence of the major menaquinone MK-9(H4) supported the affiliation of the isolate with the genus Arcanobacterium. The polar lipid profile consisted of diphosphatidylglycerol and an unidentified phospholipid as major components and two unidentified lipids, a further unidentified phospholipid, two unidentified phosphoglycolipids as well as phosphatidylglycerol. The major fatty acids were C16 : 0, C18 : 1 and C18 : 0. Biochemical and phylogenetic analyses clearly distinguished the isolate from other members of the genus Arcanobacterium and closely related other species. Based on these results, it is proposed that the unknown Arcanobacterium sp. strain 2701T should be classified as representing a novel species with the name Arcanobacterium buesumense sp. nov. The type strain is 2701T (=DSM 112952T=LMG 32446T).

The Pacific harbor seal gut microbiota in Mexico: Its relationship with diet and functional inferences.

Diet is a primary driver of the composition of gut microbiota and is considered one of the main routes of microbial colonization. Prey identification is fundamental for correlating the diet with the presence of particular microbial groups. The present study examined how diet influenced the composition and function of the gut microbiota of the Pacific harbor seal (Phoca vitulina richardii) in order to better understand the role of prey consumption in shaping its microbiota. This species is a good indicator of the quality of the local environment due to both its foraging and haul-out site fidelity. DNA was extracted from 20 fecal samples collected from five harbor seal colonies located in Baja California, Mexico. The V4 region of 16S rRNA gene was amplified and sequenced using the Illumina technology. Results showed that the gut microbiota of the harbor seals was dominated by the phyla Firmicutes (37%), Bacteroidetes (26%) and Fusobacteria (26%) and revealed significant differences in its composition among the colonies. Funtional analysis using the PICRUSt software suggests a high number of pathways involved in the basal metabolism, such as those for carbohydrates (22%) and amino acids (20%), and those related to the degradation of persistent environmental pollutants. In addition, a DNA metabarcoding analysis of the same samples, via the amplification and sequencing of the mtRNA 16S and rRNA 18S genes, was used to identify the prey consumed by harbor seals revealing the consumption of prey with mainly demersal habits. Functional redundancy in the seal gut microbiota was observed, irrespective of diet or location. Our results indicate that the frequency of occurrence of specific prey in the harbor seal diet plays an important role in shaping the composition of the gut microbiota of harbor seals by influencing the relative abundance of specific groups of gut microorganisms. A significant relationship was found among diet, gut microbiota composition and OTUs assigned to a particular metabolic pathway.

BRUCELLA PINNIPEDIALIS IN GREY SEALS ( HALICHOERUS GRYPUS) AND HARBOR SEALS ( PHOCA VITULINA) IN THE NETHERLANDS.

Brucellosis is a zoonotic disease with terrestrial or marine wildlife animals as potential reservoirs for the disease in livestock and human populations. The primary aim of this study was to assess the presence of Brucella pinnipedialis in marine mammals living along the Dutch coast and to observe a possible correlation between the presence of B. pinnipedialis and accompanying pathology found in infected animals. The overall prevalence of Brucella spp. antibodies in sera from healthy wild grey seals ( Halichoerus grypus; n=11) and harbor seals ( Phoca vitulina; n=40), collected between 2007 and 2013 ranged from 25% to 43%. Additionally, tissue samples of harbor seals collected along the Dutch shores between 2009 and 2012, were tested for the presence of Brucella spp. In total, 77% (30/39) seals were found to be positive for Brucella by IS 711 real-time PCR in one or more tissue samples, including pulmonary nematodes. Viable Brucella was cultured from 40% (12/30) real-time PCR-positive seals, and was isolated from liver, lung, pulmonary lymph node, pulmonary nematode, or spleen, but not from any PCR-negative seals. Tissue samples from lung and pulmonary lymph nodes were the main source of viable Brucella bacteria. All isolates were typed as B. pinnipedialis by multiple-locus variable number of tandem repeats analysis-16 clustering and matrix-assisted laser desorption ionization-time of flight mass spectrometry, and of sequence type ST25 by multilocus sequence typing analysis. No correlation was observed between Brucella infection and pathology. This report displays the isolation and identification of B. pinnipedialis in marine mammals in the Dutch part of the Atlantic Ocean.

Campylobacter blaseri sp. nov., isolated from common seals (Phoca vitulina).

During a study to assess the faecal microbiome of common seals (Phoca vitulina) in a Dutch seal rehabilitation centre, 16S rRNA gene sequences of an unknown Campylobacter taxon were identified. Campylobacter isolates, which differed from the established Campylobacter taxa, were cultured and their taxonomic position was determined by a polyphasic study based on ten isolates. The isolates were characterized by 16S rRNA and atpA gene sequence analyses and by conventional phenotypic testing. Based on the whole genome sequences, the average nucleotide identity and core genome phylogeny were determined. The isolates formed a separate phylogenetic clade, divergent from all other Campylobacter taxa and most closely related to Campylobacter corcagiensis, Campylobacter geochelonis and Campylobacter ureolyticus. The isolates can be distinguished phenotypically from all other Campylobacter taxa based on their lack of motility, growth at 25 °C and growth on MacConkey agar. This study shows that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter blaseri sp. nov. is proposed. The type strain for this novel species is 17S00004-5T (=LMG 30333T=CCUG 71276T).

Seroprevalence for Brucella spp. in Baltic ringed seals (Phoca hispida) and East Greenland harp (Pagophilus groenlandicus) and hooded (Cystophora cristata) seals.

Zoonotic infections transmitted from marine mammals to humans in the Baltic and European Arctic are of unknown significance, despite given considerable potential for transmission due to local hunt. Here we present results of an initial screening for Brucella spp. in Arctic and Baltic seal species. Baltic ringed seals (Pusa hispida, n = 12) sampled in October 2015 and Greenland Sea harp seals (Pagophilus groenlandicus, n = 6) and hooded seals (Cystophora cristata, n = 3) sampled in March 2015 were serologically analysed for antibodies against Brucella spp. The serological analyses were performed using the Rose Bengal Test (RBT) followed by a confirmatory testing of RBT-positive samples by a competitive-enzyme linked immunosorbent assay (C-ELISA). Two of the Baltic ringed seals (a juvenile male and a juvenile female) were seropositive thus indicating previous exposure to a Brucella spp. The findings indicate that ringed seals in the Baltic ecosystem may be exposed to and possibly infected by Brucella spp. No seropositive individuals were detected among the Greenland harp and hooded seals. Although our initial screening shows a zoonotic hazard to Baltic locals, a more in-depth epidemiological investigation is needed in order to determine the human risk associated with this.

Arcanobacterium phocae infection in mink (Neovison vison), seals (Phoca vitulina, Halichoerus grypus) and otters (Lutra lutra).

BACKGROUND: Infectious skin disorders are not uncommon in mink. Such disorders are important as they have a negative impact on animal health and welfare as well as on the quality and value of the fur. This study presents the isolation of Arcanobacterium phocae from mink with severe skin lesions and other pathological conditions, and from wild seals and otters. RESULTS: In 2015, A. phocae was isolated for the first time in Denmark from outbreaks of dermatitis in mink farms. The outbreaks affected at least 12 farms. Originating from these 12 farms, 23 animals cultured positive for A. phocae. The main clinical findings were necrotizing pododermatitis or dermatitis located to other body sites, such as the lumbar and cervical regions. A. phocae could be isolated from skin lesions and in nine animals also from liver, spleen and lung, indicating a systemic spread. The bacterium was also, for the first time in Denmark, detected in dead seals (n = 9) (lungs, throat or wounds) and otters (n = 2) (throat and foot). CONCLUSIONS: An infectious skin disorder in mink associated with A. phocae has started to occur in Danish farmed mink. The origin of the infection has not been identified and it is still not clear what the pathogenesis or the port of entry for A. phocae infections are.

Identification of a novel host-specific IgG protease in Streptococcus phocae subsp. phocae.

Streptococcus (S.) phocae subsp. phocae causes bronchopneumonia and septicemia in a variety of marine mammals. Especially in harbor seals infected with phocine distemper virus it plays an important role as an opportunistic pathogen. This study was initiated by the detection of IgG cleavage products in Western blot analysis after incubation of bacterial supernatant with harbor seal serum. Hence, the objectives of this study were the identification and characterization of a secreted IgG cleaving protease in S. phocae subsp. phocae isolated from marine mammals. To further identify the responsible factor of IgG cleavage a protease inhibitor profile was generated. Inhibition of the IgG cleaving activity by iodoacetamide and Z-LVG-CHN2 indicated that a cysteine protease is involved. Moreover, an anti-IdeS antibody directed against the IgG endopeptidase IdeS of S. pyogenes showed cross reactivity with the putative IgG protease of S. phocae subsp. phocae. The IgG cleaving factor of S. phocae subsp. phocae was identified through an inverse PCR approach and designated IdeP (Immunoglobulin G degrading enzyme of S. phocae subsp. phocae) in analogy to the cysteine protease IdeS. Notably, recombinant (r) IdeP is a host and substrate specific protease as it cleaves IgG from grey and harbor seals but not IgG from harbor porpoises or non-marine mammals. The identification of IdeP represents the first description of a protein in S. phocae subsp. phocae involved in immune evasion. Furthermore, the fact that IdeP cleaves solely IgG of certain marine mammals reflects functional adaption of S. phocae subsp. phocae to grey and harbor seals as its main hosts.

BACTERIAL MICROBIOTA IN HARBOR SEALS (PHOCA VITULINA) FROM THE NORTH SEA OF SCHLESWIG-HOLSTEIN, GERMANY, AROUND THE TIME OF MORBILLIVIRUS AND INFLUENZA EPIDEMICS.

We present microbiologic findings in harbor seal (phoca; Phoca vitulina ) carcasses collected from the North Sea of Schleswig-Holstein, Germany, 1996-2014, and interpret results in relation to potential variations caused by phocine distemper virus and influenza A virus mass mortalities. We conducted microbiologic investigations on 2,124 tissue samples from lung, liver, kidney, spleen, intestine, and mesenteric lymph nodes from 549 dead harbor seals of the German North Sea. A large variety of bacteria, including potentially pathogenic species such as Bordetella bronchiseptica , Brucella spp., Clostridium perfringens , Escherichia coli , Erysipelothrix rhusiopathiae , ß-hemolytic streptococci, and Staphylococcus aureus , were isolated. These bacteria were associated with bronchopneumonia, gastroenteritis, hepatitis, polyarthritis, nephritis, myositis, myocarditis, and septicemia. Bordetella bronchiseptica and Streptococcus equi subsp. zooepidemicus were significantly associated with the seal die-offs from phocine distemper in 2002 and influenza in 2014. Many bacteria were detected in tissues of dead harbor seals, of which E. coli , ß-hemolytic streptococci, and Brucella spp. might be responsible for pathologic changes. Zoonotic bacteria such as Brucella spp. and E. rhusiopathiae are frequently isolated from harbor seals. Brucella spp. was less and Vibrio spp. more frequently found in summer. Erysipelothrix rhusiopathiae showed an almost regular 4-yr oscillating trend. We found C. perfringens less frequently and E. coli more frequently in harbor seals from St. Peter-Ording. Because zoonotic bacteria are regularly found, handling of dead and live harbor seal specimens should be conducted carefully to prevent transmission to humans. Further investigations are needed to understand microbiota changes in relation to increasing seal populations, reintroduction of rehabilitated seals to the wild, and increasing pressure from anthropogenic activities.

Comparative analysis of the fecal bacterial community of five harbor seals (Phoca vitulina).

The gut microbiota has many beneficial effects on host metabolism and health, and its composition is determined by numerous factors. It is also assumed that there was a co-evolution of mammals and the bacteria inhabiting their gut. Current knowledge of the mammalian gut microbiota mainly derives from studies on humans and terrestrial animals, whereas those on marine mammals are sparse. However, they could provide additional information on influencing factors, such as the role of diet and co-evolution with the host. In this study, we investigated and compared the bacterial diversity in the feces of five male harbor seals (Phoca vitulina). Because this small population included two half-brother pairs, each sharing a common father, it allowed an evaluation of the impact of host relatedness or genetic similarity on the gut microbial community. Fresh feces obtained from the seals by an enema were analyzed by fluorescence in situ hybridization and amplicon sequencing of 16S rRNA genes. The results showed that the bacterial communities in the seals' feces mainly consisted of the phyla Firmicutes (19-43%), Bacteroidetes (22-36%), Fusobacteria (18-32%), and Proteobacteria (5-17%) . Twenty-one bacterial members present in the fecal samples of the five seals contributed an average relative abundance of 93.7 + 8.7% of the total fecal microbial community. Contrary to all expectations based on previous studies a comparison of the fecal community between individual seals showed a higher similarity between unrelated than related individuals.

First isolation of Schizophyllum commune in a harbor seal (Phoca vitulina).

To date, Schizophyllum commune infection has been identified in only humans and dogs. A 7-year-old female harbor seal (Phoca vitulina) died after exhibiting corneal opacity, anorexia, and labored respiration. At necropsy, phthisis of the left eyeball was detected, and multiple nodular lesions were observed in the thoracic and abdominal regions, especially in the lung, heart, and lymph nodes. Histopathologically, numerous hyphae were seen in granulomatous lesions in the eyes, lung, heart, and lymph nodules. An isolate on potato dextrose agar from the eyes, lung, and sputum yielded a rapidly growing white woolly mycelia with basidiocarps (fruiting bodies) at 37°C. A suitable temperature for mycelial growth was obtained at 25°C, although sustained growth also occurred at 37°C. The fungal isolate, KH-JPN15-011, had distinctive features including hyphae bearing spicules and clamp connections, which were consistent with the characteristics of basidiomycete fungus. The sequence of the internal transcribed spacer region of nuclear ribosomal DNA showed 99.67% (617 bp) similarity with those of S. commune Phylogenetic analysis showed that the present isolate is most closely related to the samples from the Old World. This is the first report of a fatal disease caused by S. commune in exotic animals. Previously reported human and canine infections have not included granulomatous endophthalmitis and myocarditis. After considering these and previous findings, there is a possibility that S. commune from the Old World may include numerous highly pathogenic strains.

Brain Meta-Transcriptomics from Harbor Seals to Infer the Role of the Microbiome and Virome in a Stranding Event.

Marine diseases are becoming more frequent, and tools for identifying pathogens and disease reservoirs are needed to help prevent and mitigate epizootics. Meta-transcriptomics provides insights into disease etiology by cataloguing and comparing sequences from suspected pathogens. This method is a powerful approach to simultaneously evaluate both the viral and bacterial communities, but few studies have applied this technique in marine systems. In 2009 seven harbor seals, Phoca vitulina, stranded along the California coast from a similar brain disease of unknown cause of death (UCD). We evaluated the differences between the virome and microbiome of UCDs and harbor seals with known causes of death. Here we determined that UCD stranded animals had no viruses in their brain tissue. However, in the bacterial community, we identified Burkholderia and Coxiella burnetii as important pathogens associated with this stranding event. Burkholderia were 100% prevalent and ~2.8 log2 fold more abundant in the UCD animals. Further, while C. burnetii was found in only 35.7% of all samples, it was highly abundant (~94% of the total microbial community) in a single individual. In this harbor seal, C. burnetii showed high transcription rates of invading and translation genes, implicating it in the pathogenesis of this animal. Based on these data we propose that Burkholderia taxa and C. burnetii are potentially important opportunistic neurotropic pathogens in UCD stranded harbor seals.

Arcanobacterium pinnipediorum sp. nov., isolated from a harbour seal.

A polyphasic taxonomic study was performed on an unidentified Arcanobacterium-like, Gram-stain-positive bacterium, strain 2710T, isolated from a harbour seal. Comparative 16S rRNA gene sequence analysis showed that this bacterial strain belonged to the genus Arcanobacterium and was related most closely to the type strains of Arcanobacterium phocae (98.4 % similarity) and Arcanobacterium phocisimile (97.5 %). 16S rRNA gene sequence similarities to the type strains of other Arcanobacterium species were between 95.3 and 96.9 %. DNA-DNA hybridization values between strain 2710T and A. phocae DSM 10002T and A. phocisimile LMG 27073T were 4.7 % (reciprocal 56 %) and 23 % (reciprocal 7.7 %), respectively. The presence of the major menaquinone MK-9(H4) and a polar lipid profile with the major compounds diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside supported the affiliation of strain 2710T to the genus Arcanobacterium. The major fatty acids were C16:0, C18:1ω9c, C18:0 and C18:2ω6,9c/anteiso-C18:0. The peptidoglycan structure was of cross-linkage type A5α (l-Lys-l-Lys-d-Glu). Physiological and biochemical tests clearly distinguished the isolate from other members of the genus Arcanobacterium. Based on these tests, it is proposed that this unknown bacterium should be classified as a novel species of the genus Arcanobacterium, with the name Arcanobacterium pinnipediorum sp. nov. The type strain is 2710T ( = DSM 28752T = LMG 28298T).

Otariodibacter oris and Bisgaardia genomospecies 1 isolated from infections in pinnipeds.

We document the first associations of two recently described species of Pasteurellaceae bacteria with lesions in wild pinnipeds in rehabilitation. Samples were collected from nine lesions in four California sea lions (Zalophus californianus) and two Pacific harbor seals (Phoca vitulina) during necropsy or admission examinations at a rehabilitation facility in northern California. Seven Pasteurellaceae isolates were identified using phenotypic tests and partial rpoB gene sequencing. Six strains of Otariodibacter oris were isolated from California sea lions. Otariodibacter oris was isolated in pure culture from four abscesses, an affected lymph node, and a bone lesion consistent with osteomyelitis. Otariodibacter oris was also cultured with Arcanobacterium phocae and ß-hemolytic streptococci. A pure culture of Bisgaardia genomospecies 1 was obtained from an abscess in a harbor seal. This is the first time, to our knowledge, that O. oris has been associated with infection. Isolation of these bacteria in pure culture from abscesses and osteomyelitis strongly indicates a pathogenic potential of this organism. Likewise, the isolation of Bisgaardia genomospecies 1 in pure culture from an abscess in a harbor seal implies causality.

Infection due to Mycobacterium avium subsp. avium in a free-ranging common seal (Phoca vitulina) in Scotland.

We describe the first reported case of mycobacterial infection in a free-ranging pinniped in the Northern Hemisphere. Acid-fast bacteria were demonstrated histologically in the liver of an adult female common seal (Phoca vitulina), and Mycobacterium avium subsp. avium was cultured from the liver.

Quantifying sequence proportions in a DNA-based diet study using Ion Torrent amplicon sequencing: which counts count?

A goal of many environmental DNA barcoding studies is to infer quantitative information about relative abundances of different taxa based on sequence read proportions generated by high-throughput sequencing. However, potential biases associated with this approach are only beginning to be examined. We sequenced DNA amplified from faeces (scats) of captive harbour seals (Phoca vitulina) to investigate whether sequence counts could be used to quantify the seals' diet. Seals were fed fish in fixed proportions, a chordate-specific mitochondrial 16S marker was amplified from scat DNA and amplicons sequenced using an Ion Torrent PGM™. For a given set of bioinformatic parameters, there was generally low variability between scat samples in proportions of prey species sequences recovered. However, proportions varied substantially depending on sequencing direction, level of quality filtering (due to differences in sequence quality between species) and minimum read length considered. Short primer tags used to identify individual samples also influenced species proportions. In addition, there were complex interactions between factors; for example, the effect of quality filtering was influenced by the primer tag and sequencing direction. Resequencing of a subset of samples revealed some, but not all, biases were consistent between runs. Less stringent data filtering (based on quality scores or read length) generally produced more consistent proportional data, but overall proportions of sequences were very different than dietary mass proportions, indicating additional technical or biological biases are present. Our findings highlight that quantitative interpretations of sequence proportions generated via high-throughput sequencing will require careful experimental design and thoughtful data analysis.

Dynamics of Vibrio with virulence genes detected in Pacific harbor seals (Phoca vitulina richardii) off California: implications for marine mammal health.

Given their coastal site fidelity and opportunistic foraging behavior, harbor seals (Phoca vitulina) may serve as sentinels for coastal ecosystem health. Seals using urbanized coastal habitat can acquire enteric bacteria, including Vibrio that may affect their health. To understand Vibrio dynamics in seals, demographic and environmental factors were tested for predicting potentially virulent Vibrio in free-ranging and stranded Pacific harbor seals (Phoca vitulina richardii) off California. Vibrio prevalence did not vary with season and was greater in free-ranging seals (29 %, n = 319) compared with stranded seals (17 %, n = 189). Of the factors tested, location, turbidity, and/or salinity best predicted Vibrio prevalence in free-ranging seals. The relationship of environmental factors with Vibrio prevalence differed by location and may be related to oceanographic or terrestrial contributions to water quality. Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae were observed in seals, with V. cholerae found almost exclusively in stranded pups and yearlings. Additionally, virulence genes (trh and tdh) were detected in V. parahaemolyticus isolates. Vibrio cholerae isolates lacked targeted virulence genes, but were hemolytic. Three out of four stranded pups with V. parahaemolyticus (trh+ and/or tdh+) died in rehabilitation, but the role of Vibrio in causing mortality is unclear, and Vibrio expression of virulence genes should be investigated. Considering that humans share the environment and food resources with seals, potentially virulent Vibrio observed in seals also may be of concern to human health.

A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals.

Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.