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.

Occurrence of recreational water quality monitoring general fecal indicator bacteria and fecal source identification genetic markers in gray seal scat.

The number of gray seals (Halichoerus grypus) observed along the United States Northwest Atlantic region has been increasing for decades. These colonial animals often haul-out on beaches seasonally in numbers ranging from a few individuals to several thousands. While these larger aggregations are an important part of gray seal behavior, there is public concern that haul-outs could lead to large amounts of fecal waste in recreational areas, potentially resulting in beach closures. Yet, data to confirm whether these animals contribute to beach closures is lacking and minimal information is available on the occurrence of key water quality monitoring genetic markers in gray seal scat. This study evaluates the concentration of E. coli (EC23S857), enterococci (Entero1a), and fecal Bacteroidetes (GenBac3) as well as six fecal source identification genetic markers (HF183/BacR287, HumM2, CPQ_056, Rum2Bac, DG3, and GFD) measured by qPCR in 48 wild gray seal scat samples collected from two haul-out areas in Cape Cod (Massachusetts, U.S.A.). Findings indicate that FIB genetic markers are shed in gray seal scat at significantly different concentrations with the Entero1a genetic marker exhibiting the lowest average concentration (-0.73 log10 estimated mean copies per nanogram of DNA). In addition, systematic testing of scat samples demonstrated that qPCR assays targeting host-associated genetic markers indicative of human, ruminant, and canine fecal pollution sources remain highly specific in waters frequented by gray seals (>97 % specificity).

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.

Exploring fungal diversity in Antarctic wildlife: isolation and molecular identification of culturable fungi from penguins and pinnipeds.

AIMS: To survey the diversity of fungal species that may be cultured from Antarctic penguins and pinnipeds, and to test the in vitro susceptibility to triazole drugs of any medically important Aspergillus spp. isolates. METHODS: During an expedition to Argentinean Antarctic research stations at Potter Peninsula (South Shetland Islands) and Primavera Cape (Antarctic Peninsula) in February 2019, samples (n = 212) were collected from fur seals (Arctocephalus gazella), elephant seals (Mirounga leonine), leopard seals (Hydrurga leptonyx), Weddell seals (Leptonychotes weddellii) and crabeater seals (Lobodon carcinophaga) and gentoo penguins (Pygoscelis papua). Oral, nasal and rectal swabs and skin/hair brushings were collected from pinnipeds, and skin/feather brushings, cloacal swabs and moulted feathers from penguins. Samples were cultured on Sabouraud dextrose agar and/or potato dextrose agar plates and fungal isolates identified by morphological criteria followed by PCR amplification and DNA sequencing. Antifungal susceptibility of Aspergillus spp. isolates to triazoles was tested. RESULTS: Fungi from 21 genera were isolated from 121/212 (57.1%) samples obtained from pinnipeds and penguins. Among pinnipeds from Potter Peninsula (fur seals and elephant seals), the most frequent fungal species were Debaryomyces hansenii and Rhodotorula mucilaginosa, isolated from the oral, nasal and/or rectal mucosa, and Antarctomyces psychrotrophicus isolated from the skin/hair of all sampled individuals. Among pinnipeds from Primavera Cape (leopard seals, Weddell seals and crabeater seals), the most frequent fungal species were Naganishia adeliensis and Cryptococcus neoformans var. uniguttulatus, isolated from the nasal/oral mucosa of 4/33 (15.2%) and 5/33 (12.1%) animals, respectively. The most frequently isolated fungal species from gentoo penguins (Potter Peninsula), were Pseudogymnoascus pannorum and A. pyschrotrophicus, which both were isolated from skin/feathers of 7/15 (46.7%) birds, and Thelebolus microsporus, isolated from the cloacal mucosa and skin/feathers of 5/15 (33.3%) and 2/15 (13.3%) birds, respectively. Fungi that are potentially pathogenic to both humans and animals (Aspergillus fumigatus, Asp. flavus, Asp. versicolor, Candida parapsilosis and Microsporum canis) were isolated from 4/38 (10.5%), 1/38 (2.6%), 2/38 (5.3%), 4/38 (10.5%) and 2/38 (5.3%) sampled pinnipeds, respectively. Only non-azole-resistant isolates of Asp. fumigatus and Asp. flavus were identified. CONCLUSIONS: The fungal biodiversity in Antarctic pinnipeds and gentoo penguins was explored using standard mycological culture followed by PCR and DNA sequencing. The frequency of fungal carriage varied among animal species, sample type and location. This study constitutes an epidemiologic approach to monitoring of these marine animals for emerging fungal pathogens.

Mycoplasma miroungirhinis sp. nov. and Mycoplasma miroungigenitalium sp. nov., isolated from northern elephant seals (Mirounga angustirostris), Mycoplasma phocoenae sp. nov., isolated from harbour porpoise (Phocoena phocoena), and Mycoplasma phocoeninasale sp. nov., isolated from harbour porpoise and California sea lions (Zalophus californianus).

Seven novel independent strains of Mycoplasma species were isolated from northern elephant seals (ES2806-NAST, ES2806-GENT, ES3157-GEN-MYC and ES3225-GEN-MYC), a harbour porpoise (C264-GENT and C264-NAST), and a California sea lion (CSL7498). These strains were phenotypically and genetically characterized and compared to the known Mycoplasma species. Four strains (C264-GENT, C264-NAST, CSL7498 and ES2806-NAST) hydrolysed arginine but not urea and did not produce acid from carbohydrates. Strains ES2806-GENT, ES3157-GEN-MYC and ES3225-GEN-MYC did not produced acid from carbohydrates and did not hydrolyse arginine or urea; hence, it is assumed that organic acids are used as the energy source for them. All were isolated and propagated in ambient air supplemented with 5±1 % CO2 at +35-37 °C using either SP4 or PPLO medium. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. The complete genomes were sequenced for all type strains. Average nucleotide and amino acid identity analyses showed that these novel strains were distant from the phylogenetically closely related Mycoplasma species. Based on these data, we propose four novel species of the genus Mycoplasma, for which the name Mycoplasma miroungirhinis sp. nov. is proposed with the type strain ES2806-NAST (=NCTC 14430T=DSM 110945T), Mycoplasma miroungigenitalium sp. nov. is proposed with the type strain ES2806-GENT (=NCTC 14429T=DSM 110944T) and representative strains ES3157-GEN-MYC and ES3225-GEN-MYC, Mycoplasma phocoenae sp. nov. is proposed with the type strain C264-GENT (=NCTC 14344T=DSM 110687T) and Mycoplasma phocoeninasale sp. nov. is proposed with the type strain C264-NAST (=NCTC 14343T=DSM 110688T) and representative strain CSL7498. The genome G+C contents are 24.06, 30.09, 28.49 and 29.05% and the complete genome sizes are 779 550, 815 486, 693 115, and 776 009 bp for strains ES2806-NAST, ES2806-GENT, C264-GENT and C264-NAST, respectively.

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.

Distinct gut microbiotas between southern elephant seals and Weddell seals of Antarctica.

The gut microbiome provides ecological information about host animals, but we still have limited knowledge of the gut microbiome, particularly for animals inhabiting remote locations, such as Antarctica. Here, we compared fecal microbiota between southern elephant seals (Mirounga leonina) and Weddell seals (Leptonychotes weddelli), that are top predatory marine mammals in the Antarctic ecosystem, using 16S rRNA amplicon sequencing and assessed the relationships of the gut microbial communities to functional profiles using gut metabolite analysis. The bacterial community did not differ significantly by host species or sex at the phylum level, but the distinction at the family level was obvious. The family Ruminococcaceae (Firmicutes) was more abundant in southern elephant seals than in Weddell seals, and the families Acidaminococcaceae (Firmicutes) and Pasteurellaceae (Gammaproteobacteria) were uniquely present in Weddell seals. The fecal bacterial community structure was distinctively clustered by host species, with only 6.7% of amplicon sequence variants (ASVs) shared between host species. This result implies that host phylogeny rather than other factors, such as diet or age, could be the major driver of fecal microbiotic diversification. Interestingly, there was no apparent sex effect on bacterial community structure in Weddell seals, but the effect of sex was pronounced in adult southern elephant seals mainly due to the prevalence of Edwardsiella sp., suggesting that extreme sexual dimorphism may modulate the gut microbiota of southern elephant seals. Unlike the clear distinction in the taxonomic composition of fecal bacterial communities, there were no discernible differences in the profiles of potential microbial functions and gut metabolites between host species or sexes, indicating that functional redundancy dominates the gut microbiota of seals surveyed in this study.

Resistance to carbapenems and other antibiotics in Klebsiella pneumoniae found in seals indicates anthropogenic pollution.

BACKGROUND: The beta-lactamase enzyme OXA-48 has spread widely in recent years in Enterobacteriaceae associated with man, disseminated primarily on incompatibility group L/M plasmids. OXA-48 confers resistance to carbapenems, important antimicrobials for treating highly resistant bacterial infections in humans. This enzyme has rarely been detected in bacteria from animals. Furthermore, the use of carbapenem compounds is not permitted in food-producing animals in Europe and to our knowledge has not been reported in food-producing animals globally. METHODS: Bacterial isolates from lesions in stranded, free-living, juvenile common seals (Phoca vitulina) were identified. Antimicrobial susceptibility testing and whole genome sequencing analysis were used to characterise antimicrobial resistance genes carried by the bacteria. RESULTS: Here, we report the detection of Klebsiella pneumoniae subspecies pneumoniae carrying the bla OXA-48 gene on an incompatibility group L/M plasmid from an infection in a common seal. CONCLUSION: Evidence is accruing that marine mammals may be infected with bacteria originating from anthropogenic sources, such as human sewage, contaminating the environment.

Oceanivirga miroungae sp. nov., isolated from oral cavity of northern elephant seal (Mirounga angustirostris).

Two independent strains of a Leptotrichia species (ES3154-GLUT and ES2714_GLU) were isolated from the oral cavity of northern elephant seals (Mirounga angustirostris) that were admitted to The Marine Mammal Centre facilities in California, USA. The strains were isolated from oral swabs by cultivation in PPLO broth supplemented with serum, penicillin and colistin in anaerobic conditions. The strains were Gram-negative, pleomorphic, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile rods/coccobacilli in short chains. The 16S rRNA gene sequence of these strains shared 94.42 % nucleotide similarity with Oceanivirga salmonicida AVG 2115T but demonstrated ≤86.00-92.50 % nucleotide similarity to the 16S rRNA genes of other species of the family Leptotrichiaceae. The genome was sequenced for strain ES3154-GLUT. Average nucleotide identity values between strain ES3154-GLUT and 15 type strain genomes from the family Leptotrichiaceae ranged from 66.74 % vs. Sebaldella termitidis to 73.35 % vs. O. salmonicida. The whole genome phylogeny revealed that the novel species was most closely related to O. salmonicida AVG 2115T. This relationship was also confirmed by nucleotide similarity and multilocus phylogenetic analyses employing various housekeeping genes (partial 23S rRNA, rpoB, rpoC, rpoD, polC, adh, gyrA and gyrB genes). Chemotaxonomic and phenotypical features of strain ES3154-GLUT were in congruence with closely related members of the family Leptotrichiaceae, represented by similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis was capable to clearly discriminate strain ES3154-GLUT from all currently described taxa of the family Leptotrichiaceae. Based on these data, we propose a novel species of the genus Oceanivirga, for which the name Oceanivirga miroungae sp. nov. is proposed with the type strain ES3154-GLUT (=DSM 109740T=CCUG 73653T=ATCC TSD-189T=NCTC 14411T) and one representative strain ES2714_GLU. The G+C content is 26.82 %, genome size is 1 356 983 bp.

Early sexual dimorphism in the developing gut microbiome of northern elephant seals.

The gut microbiome is an integral part of a species' ecology, but we know little about how host characteristics impact its development in wild populations. Here, we explored the role of such intrinsic factors in shaping the gut microbiome of northern elephant seals (Mirounga angustirostris) during a critical developmental window of 6 weeks after weaning, when the pups stay ashore without feeding. We found substantial sex differences in the early-life gut microbiome, even though males and females could not yet be distinguished morphologically. Sex and age both explained around 15% of the variation in gut microbial beta diversity, while microbial communities sampled from the same individual showed high levels of similarity across time, explaining another 40% of the variation. Only a small proportion of the variation in beta diversity was explained by health status, assessed by full blood counts, but clinically healthy individuals had a greater microbial alpha diversity than their clinically abnormal peers. Across the post-weaning period, the northern elephant seal gut microbiome was highly dynamic. We found evidence for several colonization and extinction events as well as a decline in Bacteroides and an increase in Prevotella, a pattern that has previously been associated with the transition from nursing to solid food. Lastly, we show that genetic relatedness was correlated with gut microbiome similarity in males but not females, again reflecting early sex differences. Our study represents a naturally diet-controlled and longitudinal investigation of how intrinsic factors shape the early gut microbiome in a species with extreme sex differences in morphology and life history.

Ureaplasma miroungigenitalium sp. nov. isolated from northern elephant seals (Mirounga angustirostris) and Ureaplasma zalophigenitalium sp. nov. isolated from California sea lions (Zalophus californianus).

Novel ureaplasma strains have been isolated from the genital tract of both sexes of northern elephant seals (Mirounga angustirostris; six strains) and California sea lions (Zalophus californianus; five strains) stranded along the Central California coast, USA. These strains were phenotypically and genetically characterized and compared to other seven known Ureaplasma species. All novel ureaplasma strains hydrolysed urea, but did not metabolize arginine, and all were isolated and propagated using PPLO medium supplemented with urea under aerobic, microaerophilic, and anaerobic atmospheric conditions at +35-37 °C. Transmission electron microscopy revealed typical mollicute cellular morphology. Molecular characterization included assessment of the following genetic loci: 16S rRNA, the 16S-23S ITS, 23S rRNA, rpoB, ftsH, tufB, rpoC, fusA and ureC. Complete 16S rRNA gene sequence analysis of these novel Ureaplasma species indicated that they were most closely related to each other with nucleotide identity 99.87 % and ≤93.08 % related to other known Ureaplasma species. The results of nucleotide analysis of the sequenced housekeeping genes revealed 71.68-93.02 % similarity to corresponding genes of other known Ureaplasma species. The multi-locus genetic characterization and the phylogenetic analysis of the 16S rRNA and rpoB genes of these Ureaplasma species clearly demonstrated their novelty and, reflecting their host specificites, the name Ureaplasma miroungigenitalium sp. nov. is proposed for the Ureaplasma species isolated from northern elephant seals, the type strain is ES2783-GENT (=DSM 24842T=ATCC BAA-2460T), and the name Ureaplasma zalophigenitalium sp. nov. is proposed for the Ureaplasma species isolated from California sea lions, the type strain is CSL7644-GENT (=DSM 24843T=ATCC BAA-2262T).

Pseudomonas leptonychotis sp. nov., isolated from Weddell seals in Antarctica.

A taxonomic study was carried out on four Gram-stain-negative strains P5773T, P6169, P4708 and P6245, isolated from anus or mouth samples of Weddell seals at James Ross Island, Antarctica. The results of initial 16S rRNA gene sequence analysis showed that all four strains formed a group placed in the genus Pseudomonas and found Pseudomonas guineae and Pseudomonas peli to be their closest neighbours with 99.9 and 99.2 % sequence similarity, respectively. Sequence analysis of rpoD, rpoB and gyrB housekeeping genes confirmed the highest similarity of isolates to P. peli (rpoD) and to P. guineae (rpoB and gyrB). The average nucleotide identity value below 86 %, as calculated from the whole-genome sequence data, showed the low genomic relatedness of P5773T to its phylogenetic neighbours. The complete genome of strain P5773T was 4.4 Mb long and contained genes encoding proteins with biotechnological potential. The major fatty acids of the seal isolates were summed feature 8 (C18 : 1 ω7c), summed feature 3 (C16 : 1 ω 7 c/C16  : 1 ω6c) and C16:0. The major respiratory quinone was Q9. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Putrescine and spermidine are predominant in the polyamine pattern. Further characterization performed using repetitive sequence-based PCR fingerprinting and MALDI-TOF MS analysis showed that the studied isolates formed a coherent cluster separated from the remaining Pseudomonas species and confirmed that they represent a novel species within the genus Pseudomonas, for which the name Pseudomonas leptonychotis sp. nov. is suggested. The type strain is P5773T (=CCM 8849T=LMG 30618T).

Forensic microbiology reveals that Neisseria animaloris infections in harbour porpoises follow traumatic injuries by grey seals.

Neisseria animaloris is considered to be a commensal of the canine and feline oral cavities. It is able to cause systemic infections in animals as well as humans, usually after a biting trauma has occurred. We recovered N. animaloris from chronically inflamed bite wounds on pectoral fins and tailstocks, from lungs and other internal organs of eight harbour porpoises. Gross and histopathological evidence suggest that fatal disseminated N. animaloris infections had occurred due to traumatic injury from grey seals. We therefore conclude that these porpoises survived a grey seal predatory attack, with the bite lesions representing the subsequent portal of entry for bacteria to infect the animals causing abscesses in multiple tissues, and eventually death. We demonstrate that forensic microbiology provides a useful tool for linking a perpetrator to its victim. Moreover, N. animaloris should be added to the list of potential zoonotic bacteria following interactions with seals, as the finding of systemic transfer to the lungs and other tissues of the harbour porpoises may suggest a potential to do likewise in humans.

Detection of serum antibodies to Brucella in Russian aquatic mammals.

A serologic survey of Brucella infection was performed in Caspian seals (Pusa caspica, n=71), Baikal seals (P. sibirica, n=7), ringed seals (P. hispida hispida, n=6), and beluga whales (Delphinapterus leucas, n=4) inhabiting Russian waters, by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis as antigens. The sera of 4 Caspian seals (4%) tested positive for B. abortus. The same sera samples demonstrated weaker yet detectable affinity for B. canis antigens. Several discrete bands against B. abortus and B. canis antigens were detected on Western blot analysis of the ELISA-positive seal sera; the bands against B. canis were weaker than those against B. abortus. The sera of 3 beluga whales (75%) were positive for B. abortus antigens but showed no binding to B. canis antigens in the ELISA. The positive whale sera showed a strong band appearance only against B. abortus antigens in the Western blot analysis. Many detected bands were discrete, while some of them had a smeared appearance. The present results indicate that Brucella infection occurred in Caspian seals and beluga whales inhabiting Russian waters, and that the Brucella strains infecting the seals and the whales were antigenetically distinct.

Vibrio vulnificus in aquariums is a novel threat to marine mammals and public health.

Vibrio vulnificus is a Gram-negative, curved, obligate halophilic marine bacterium that exclusively exists in coastal seawaters. Previous studies revealed that V. vulnificus is one of the most dangerous foodborne zoonotic pathogens for human beings. However, it remains unknown whether marine mammals can be infected by V. vulnificus. In May 2016, a captive spotted seal (Phoca largha) died due to septicemia induced by V. vulnificus. Upon post-mortem examination, V. vulnificus was isolated, identified, and named as BJ-PH01. Further analysis showed that BJ-PH01 belongs to biotype 1 and the Clinical genotype. Furthermore, we performed an epidemiological investigation of V. vulnificus in six aquariums in northern China. As a result, V. vulnificus was successfully isolated from all investigated aquariums. The positive rates ranged from 20% to 100% in each investigated aquarium. During the investigation, 12 strains of V. vulnificus were isolated, and all 12 isolates were classified into biotype 1. Eleven of the 12 isolates belonged to the Clinical genotype, and one isolate belonged to the Environmental genotype. All 12 isolated V. vulnificus strains showed limited antibiotic resistance. Overall, our work demonstrated that V. vulnificus is frequently distributed in aquariums, thus constituting a threat to captive marine mammals and to public health.

Presence of Helicobacter and Campylobacter species in faecal samples from zoo mammals.

Helicobacter and Campylobacter species (spp.) colonize the gastrointestinal tract of various domesticated animals. Apart from their pathogenic significance in animals, several species are of zoonotic importance as well. For most non-domesticated animal spp., however, little is known on the presence and importance of these agents. Therefore, we investigated the presence of Helicobacter and Campylobacter spp. in marine and terrestrial zoo mammals. Faecal samples of various marine and terrestrial zoo mammals were collected from 6 different zoos in Belgium. These samples were tested for the presence of Helicobacter and Campylobacter spp. by isolation and direct demonstration of DNA using genus-specific PCRs, followed by sequencing of the obtained amplicons. Helicobacter spp. were detected in 12 and Campylobacter spp. in 5 of the 22 faecal samples from marine mammals. In 4 of 5 dolphins, H. cetorum was demonstrated, a well-known pathogen associated with gastritis and gastric ulceration in marine mammals. C. insulaenigrae was isolated from 4 of 6 sea lions and from 1 of 11 seals. To our knowledge, this is the first description of the presence of C. insulaenigrae on the European mainland. Helicobacter spp. were detected in 5 and Campylobacter spp. (mainly C. jejuni subsp. jejuni and C. coli) in 9 of the 26 faecal samples from terrestrial mammals. Potential novel enterohepatic Helicobacter spp. were detected in both marine and terrestrial zoo mammals. For the first time, the presence of several known and unknown Campylobacter and Helicobacter spp. was demonstrated in the gastrointestinal tract of various marine and terrestrial zoo mammals. Further investigation is needed on the pathogenic and zoonotic importance of these species.

HYPERMUCOVISCOUS KLEBSIELLA PNEUMONIAE ISOLATES FROM STRANDED AND WILD-CAUGHT MARINE MAMMALS OF THE US PACIFIC COAST: PREVALENCE, PHENOTYPE, AND GENOTYPE.

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 102 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.

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.

Frequency of exposure of endangered Caspian seals to Canine distemper virus, Leptospira interrogans, and Toxoplasma gondii.

Canine distemper virus (CDV), Leptospira interrogans, and Toxoplasma gondii are potentially lethal pathogens associated with decline in marine mammal populations. The Caspian Sea is home for the endangered Caspian seal (Pusa caspica). In the late 1990s and early 2000s, CDV caused a series of mortality events involving at least several thousand Caspian seals. To assess current infection status in Caspian seals, we surveyed for antibodies to three pathogens with potential to cause mortality in marine mammals. During 2015-2017, we tested serum samples from 36, apparently healthy, Caspian seals, accidentally caught in fishing nets in the Caspian Sea off Northern Iran, for antibodies to CDV, L. interrogans, and T. gondii, by virus neutralization, microscopic agglutination, and modified agglutination, respectively. Twelve (33%), 6 (17%), and 30 (83%) samples were positive for CDV, L. interrogans and T. gondii antibodies, respectively. The highest titers of CDV, L. interrogans, and T. gondii antibodies were 16, 400, and 50, respectively. Frequencies of antibody to these pathogens were higher in seals >1 year old compared to seals <1 year old. Two serovars of L. interrogans (Pomona and Canicola) were detected. Our results suggest a need for additional studies to clarify the impact of these pathogens on Caspian seal population decline and the improvement of management programs, including systematic screening to detect and protect the remaining population from disease outbreaks.

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.