Bacterial dysbiosis and epithelial status of the California sea lion (Zalophus californianus) in the Gulf of California.

Despite the high incidence of urogenital carcinoma (UGC) in California sea lions stranded along California, no UGC has been reported in other areas of their distribution; however, cell morphologies typical of premalignant states have been found. Risk factors for UGC include high of organochlorines and infection with a gammaherpesvirus, OtHV-1, but the importance of the bacteriome for epithelial status remains unknown. We characterized the genital bacteriome of adult female California sea lions along their distribution in the Gulf of California and examined whether the diversity and abundance of the bacteriome varied spatially, whether there were detectable differences in the bacteriome between healthy and altered epithelia, and whether the bacteriome was different in California sea lions infected with OtHV-1 or papillomavirus. We detected 2270 ASVs in the genital samples, of which 35 met the criteria for inclusion in the core bacteriome. Fusobacteriia and Clostridia were present in all samples, at high abundances, and Actinobacteria, Alphaproteobacteria, and Campylobacteria were also well-represented. Alpha diversity and abundance of the California sea lion genital bacteriome varied geographically. The abundance of bacterial ASVs varied depending on the genital epithelial status and inflammation, with differences driven by classes Fusobacteriia, Clostridia, Campylobacteria and Alphaproteobacteria. Alpha diversity and abundance were lowest in samples in which OtHV-1 was detected, and highest those with papillomavirus. Our study is the first investigation of how the bacteriome is related to epithelial status in a wild marine species prone to developing cancer.

Implications of Escherichia coli community diversity in free-ranging Australian pinniped pups.

Escherichia coli is a widely studied bacterium, commonly used as an indicator of faecal contamination. Investigations into the structure and diversity of E. coli in free-ranging wildlife species has been limited. The objective of this study was to characterise intra-individual and inter-species E. coli phylotype and B2 sub-type diversity in free-ranging Australian pinniped pups, to determine whether a single E. coli colony is representative of the phylotype and B2 sub-type diversity in these hosts. Faecal samples were collected from free-ranging Australian fur seal (Arctocephalus pusillus doriferus), Australian sea lion (Neophoca cinerea) and long-nosed fur seal (Arctocephalus forsteri) pups from three breeding colonies between 2018 and 2021. Faecal swabs from thirty randomly selected pups (n = 10 from each species) were cultured and ten E. coli colonies were selected from each culture based on morphology and separation between colonies on agar plates. Molecular screening techniques were utilised to assign isolates to phylotypes and B2 sub-types. There was no significant difference (p > 0.05) in either intra-individual or inter-species E. coli phylotype and B2 sub-type diversity. The B2 phylotype was the most dominant, with 78% of isolates (n = 234) assigned to this phylotype. Host factors (species, weight [kg] and standard length [cm]) did not significantly affect phylotype diversity. The absence of intra-individual and inter-species differences in E. coli diversity at a phylotype level suggests that a single E. coli colony could be used as an indicator of overall diversity of E. coli at a phylotype level in A. p. doriferus, N. cinerea and A. forsteri pups. These findings can be used to simplify and improve the efficiency of sampling protocols for ongoing monitoring of human-associated E. coli phylotypes in free-ranging pinniped populations.

ISOLATION, CHARACTERIZATION, AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA ISOLATED FROM SEA LION (ZALOPHUS CALIFORNIANUS) PUPS IN NORTHWESTERN MEXICO.

Bacterial infections have been documented in marine mammals for decades, and some are considered emerging pathogens with zoonotic potential. The aerobic oral (n=16) and rectal (n=17) bacterial microbiota and their antimicrobial resistance were characterized for 17 apparently healthy California sea lion pups (Zalophus californianus) captured with a hoop net in Farallon Island, Sinaloa, Mexico, in 2016. Bacteriologic cultures, Analytical Profile Index, and PCR were used to identify bacterial species. The Escherichia coli phylogenetic groups were identified by PCR, Salmonella serotypes were identified, and resistance to antibiotics was evaluated. Overall, 39 bacterial species were isolated, including E. coli and Salmonella spp. (35.9% each) and Pseudomonas aeruginosa (28.2%). For E. coli, UNKNOWN phylogroup was the most prevalent (57.7%), followed by the A phylogroup (37.1%). Most Salmonella serotypes were identified as Newport (92.8%); serotype Saintpaul was also identified (7.2%). Sea lions with bacterial co-colonization included 24.2%, from which two bacterial species were isolated, and 3% with three species. Overall, 59% of bacteria were resistant to at least one antibiotic tested, and 25.6% were extensively drug resistant. Bacteria were highly resistant to ampicillin and cefotaxime. This study demonstrates the importance of characterizing the microbiome of sea lions, and the potential effect of pathogens with antimicrobial resistance on wildlife conservation and public health.

Carriage of antibiotic resistant bacteria in endangered and declining Australian pinniped pups.

The rapid emergence of antimicrobial resistance (AMR) is a major concern for wildlife and ecosystem health globally. Genetic determinants of AMR have become indicators of anthropogenic pollution due to their greater association with humans and rarer presence in environments less affected by humans. The objective of this study was to determine the distribution and frequency of the class 1 integron, a genetic determinant of AMR, in both the faecal microbiome and in Escherichia coli isolated from neonates of three pinniped species. Australian sea lion (Neophoca cinerea), Australian fur seal (Arctocephalus pusillus doriferus) and long-nosed fur seal (Arctocephalus forsteri) pups from eight breeding colonies along the Southern Australian coast were sampled between 2016-2019. DNA from faecal samples (n = 309) and from E. coli (n = 795) isolated from 884 faecal samples were analysed for class 1 integrons using PCRs targeting the conserved integrase gene (intI) and the gene cassette array. Class 1 integrons were detected in A. p. doriferus and N. cinerea pups sampled at seven of the eight breeding colonies investigated in 4.85% of faecal samples (n = 15) and 4.52% of E. coli isolates (n = 36). Integrons were not detected in any A. forsteri samples. DNA sequencing of the class 1 integron gene cassette array identified diverse genes conferring resistance to four antibiotic classes. The relationship between class 1 integron carriage and the concentration of five trace elements and heavy metals was also investigated, finding no significant association. The results of this study add to the growing evidence of the extent to which antimicrobial resistant bacteria are polluting the marine environment. As AMR determinants are frequently associated with bacterial pathogens, their occurrence suggests that these pinniped species are vulnerable to potential health risks. The implications for individual and population health as a consequence of AMR carriage is a critical component of ongoing health investigations.

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.

Novel alphaherpesvirus in a wild South American sea lion (Otaria byronia) with pulmonary tuberculosis.

In 2017, an adult male South American sea lion (Otaria byronia), presenting emaciation and a cervical abscess, stranded alive in Florianópolis, southern Brazil. The animal was directed to a rehabilitation center, dying a few days later. On necropsy, the main gross findings were necrotizing lymphadenitis of the right prescapular lymph node and nodular bronchopneumonia. A novel alphaherpesvirus, tentatively named Otariid alphaherpesvirus 1, was amplified in several tissue samples. No histopathologic findings associated with viral infection were observed. Additionally, pulmonary tuberculosis by Mycobacterium pinnipedii was diagnosed by histopathological, immunohistochemical, and molecular techniques. Several bacteria were cultured from antemortem and postmortem samples, including Proteus mirabilis from the cervical abscess and cardiac blood, and Escherichia coli from the cervical abscess and pericardial effusion. Flavivirus, morbillivirus, and Apicomplexa were not detected by molecular techniques. Herein, we report a novel alphaherpesvirus in a pinniped species of the family Otariidae. Although previously described in Southern Hemisphere pinniped species, including South American sea lions, there is limited information regarding M. pinnipedii impact over this group. Further research is required to determine the associated pathogenesis of this novel herpesvirus, and prevalence of Otariid alphaherpesvirus 1 and M. pinnipedii in the reproductive colonies.

Streptococcus vicugnae sp. nov., isolated from faeces of alpacas (Vicugna pacos) and cattle (Bos taurus), Streptococcus zalophi sp. nov., and Streptococcus pacificus sp. nov., isolated from respiratory tract of California sea lions (Zalophus californianus).

Four novel independent strains of Streptococcus spp. were isolated from faeces of alpaca (SL1232T), cattle (KCJ4950), and from respiratory tract of wild California sea lions (CSL7508T, CSL7591T). The strains were indole-, oxidase- and catalase-negative, non-spore-forming, non-motile Gram-positive cocci in short and long chains, facultative anaerobes. The 16S rRNA gene of SL1232T and KCJ4950 shared 99.40-99.60% nucleotide similarity to strains of S. equinus, S. lutetiensis, S. infantarius, and the 16S rRNA gene of CSL7508T and CSL7591T demonstrated 98.72 and 98.92% similarity, respectively, to S. marimammalium. All other known Streptococcus species had the 16S rRNA gene sequence similarities of ≤95%. The genomes were sequenced for the novel strains. Average nucleotide identity (ANI) analysis for strains SL1232T and KCJ4950, showed the highest similarity to S. equinus, S. lutetiensis, and S. infantarius with 85.21, 87.17, 88.47, 85.54, 87.47 and 88.89%, respectively, and strains CSL7508T and CSL7591T to S. marimammalium with 87.16 and 83.97%, respectively. Results of ANI were confirmed by pairwise digital DNA-DNA hybridization and phylogeny, which also revealed that the strains belong to three novel species of the genus Streptococcus. Phenotypical features of the novel species were in congruence with closely related members of the genus Streptococcus and gave negative reactions with the tested Lancefield serological groups (A-D, F and G). MALDI-TOF mass spectrometry supported identification of the species. Based on these data, we propose three novel species of the genus Streptococcus, for which the name Streptococcus vicugnae sp. nov. is proposed with the type strain SL1232T (=NCTC 14341T=DSM 110741T=CCUG 74371T), Streptococcus zalophi sp. nov. is proposed with the type strain CSL7508T (=NCTC 14410T=DSM 110742T=CCUG 74374T) and Streptococcus pacificus sp. nov. is proposed with the type strain CSL7591T (=NCTC 14455T=DSM 111148T=CCUG 74655T). The genome G+C content is 36.89, 34.85, and 35.34 % and draft genome sizes are 1906993, 1581094 and 1656080 bp for strains SL1232T, CSL7508T, and CSL7591T, respectively.

CLASSIFICATION AND REGRESSION TREE ANALYSIS FOR PREDICTING PROGNOSIS IN WILDLIFE REHABILITATION: A CASE STUDY OF LEPTOSPIROSIS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

The spirochete bacterium Leptospira interrogans serovar Pomona is enzootic to California sea lions (CSL; Zalophus californianus) and causes periodic epizootics. Leptospirosis in CSL is associated with a high fatality rate in rehabilitation. Evidence-based tools for estimating prognosis and guiding early euthanasia of animals with a low probability of survival are critical to reducing the severity and duration of animal suffering. Classification and regression tree (CART) analysis of clinical data was used to predict survival outcomes of CSL with leptospirosis in rehabilitation. Classification tree outputs are binary decision trees that can be readily interpreted and applied by a clinician. Models were trained using data from cases treated from 2017 to 2018 at The Marine Mammal Center in Sausalito, CA, and tested against data from cases treated from 2010 to 2012. Two separate classification tree analyses were performed, one including and one excluding data from euthanized animals. When data from natural deaths and euthanasias were included in model-building, the best classification tree predicted outcomes correctly for 84.7% of cases based on four variables: appetite over the first 3 days in care, and blood urea nitrogen (BUN), creatinine, and sodium at admission. When only natural deaths were included, the best model predicted outcomes correctly for 87.6% of cases based on BUN and creatinine at admission. This study illustrates that CART analysis can be successfully applied to wildlife in rehabilitation to establish evidence-based euthanasia criteria with the goal of minimizing animal suffering. In the context of a large epizootic that challenges the limits of a facility's capacity for care, the models can assist in maximizing allocation of resources to those animals with the highest predicted probability of survival. This technique may be a useful tool for other diseases seen in wildlife rehabilitation.

A Novel Presentation of Tuberculosis with Intestinal Perforation in a Free-Ranging Australian Sea Lion (Neophoca cinerea).

We detail a novel presentation of tuberculosis associated with intestinal perforation in an endangered Australian sea lion (Neophoca cinerea) from South Australian waters and confirm the presence of this disease in the region of highest pup production. In February 2017, a 3-yr-old juvenile male died shortly after hauling out at the Kingscote beach on Kangaroo Island. On postmortem examination, we found a mid-jejunal intestinal perforation and partial obstruction (from a strangulating fibrous and granulomatous mesenteric mass), a marked multicentric abdominal fibrosing granulomatous lymphadenitis, and a large volume serosanguinous peritoneal effusion. Acid-fast bacteria were detected postmortem in cytologic preparations of the mesenteric lymph node and in histologic sections of jejunum and the encircling mass. Mycobacterial infection was confirmed by positive culture after 3 wk. Molecular typing using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing with 12-locus analysis identified Mycobacterium pinnipedii. This case highlights the need for vigilance of zoonotic disease risk when handling pinnipeds, including in the absence of specific respiratory signs or grossly apparent pulmonary pathology. Increased serologic population surveillance is recommended to assess the species' risk from this and other endemic diseases, especially given its endangered status.

Molecular and genome characterization of colistin-resistant Escherichia coli isolates from wild sea lions (Zalophus californianus).

Using molecular and whole-genome sequencing tools, we investigated colistin-resistant Escherichia coli isolates from wild sea lions. Two unrelated E. coli colistin-resistant isolates, ST8259 and ST4218, were identified, both belonging to the B2 phylogroup and different serotypes. Polymorphisms in PmrA, PmrB, and PhoQ proteins were identified, and the role of PmrB and PhoQ in contributing to colistin resistance was determined by complementation assays. However, the mutations characterized in the present study are not involved in colistin resistance, which have been described in E. coli isolates from clinical settings. Therefore, the acquired mutations in pmrB and phoQ genes in resistance to colistin in bacteria related to marine environment animals are different. This work contributes to the surveillance and characterization of colistin resistance in Escherichia coli obtained from animals from aquatic environments.

Linking longitudinal and cross-sectional biomarker data to understand host-pathogen dynamics: Leptospira in California sea lions (Zalophus californianus) as a case study.

Confronted with the challenge of understanding population-level processes, disease ecologists and epidemiologists often simplify quantitative data into distinct physiological states (e.g. susceptible, exposed, infected, recovered). However, data defining these states often fall along a spectrum rather than into clear categories. Hence, the host-pathogen relationship is more accurately defined using quantitative data, often integrating multiple diagnostic measures, just as clinicians do to assess their patients. We use quantitative data on a major neglected tropical disease (Leptospira interrogans) in California sea lions (Zalophus californianus) to improve individual-level and population-level understanding of this Leptospira reservoir system. We create a "host-pathogen space" by mapping multiple biomarkers of infection (e.g. serum antibodies, pathogen DNA) and disease state (e.g. serum chemistry values) from 13 longitudinally sampled, severely ill individuals to characterize changes in these values through time. Data from these individuals describe a clear, unidirectional trajectory of disease and recovery within this host-pathogen space. Remarkably, this trajectory also captures the broad patterns in larger cross-sectional datasets of 1456 wild sea lions in all states of health but sampled only once. Our framework enables us to determine an individual's location in their time-course since initial infection, and to visualize the full range of clinical states and antibody responses induced by pathogen exposure. We identify predictive relationships between biomarkers and outcomes such as survival and pathogen shedding, and use these to impute values for missing data, thus increasing the size of the useable dataset. Mapping the host-pathogen space using quantitative biomarker data enables more nuanced understanding of an individual's time course of infection, duration of immunity, and probability of being infectious. Such maps also make efficient use of limited data for rare or poorly understood diseases, by providing a means to rapidly assess the range and extent of potential clinical and immunological profiles. These approaches yield benefits for clinicians needing to triage patients, prevent transmission, and assess immunity, and for disease ecologists or epidemiologists working to develop appropriate risk management strategies to reduce transmission risk on a population scale (e.g. model parameterization using more accurate estimates of duration of immunity and infectiousness) and to assess health impacts on a population scale.

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).

Pup mortality in New Zealand sea lions (Phocarctos hookeri) at Enderby Island, Auckland Islands, 2013-18.

New Zealand sea lions (Phocarctos hookeri) are an endemic and endangered species. Pup mortality at Enderby Island (50.5°S, 166.28°E) in the New Zealand sub-Antarctic has been well studied, with subsequent investigations yielding more intricate detail of the causes of mortality, as new diagnostic methods become available. Klebsiella pneumoniae was first reported in 2001-02 at this site, causing a pup mortality epizootic and is now known to be present at several colonies. This bacterium is a common mucosal commensal of humans and animals, however the agent found in pups at necropsy is a hypervirulent strain, readily recognised in microbial culture as being hypermucoviscous. Infection causes septicaemia with a common syndrome of subsequent meningitis and polyarthritis. This investigation uses histopathology and microbiology, with new modalities such as matrix assisted laser desorption/ionisation-time of flight mass spectrometry to show that Klebsiella septicaemia could have historically been, and continues to be, the most important cause of pup mortality, but has been previously underrepresented due to the often cryptic presentation and sometimes peracute course of disease. Hypermucoviscous K. pneumoniae should be considered a serious threat to pup survival in the species, causing on average 60.2% of pup deaths annually at Enderby Island between 2013 and 2018, with likely more continuing mortality following pup dispersal and the cessation of the summer monitoring season. Less common causes of death included starvation (14.8%), trauma/asphyxiation (9.9%) and other infections (7%). This study forms the basis for further evaluation of risk factors for pup mortality in the species, with a view to developing active mitigation.

Genome sequencing of Mycobacterium pinnipedii strains: genetic characterization and evidence of superinfection in a South American sea lion (Otaria flavescens).

BACKGROUND: Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis Complex (MTBC), is capable of infecting several host species, including humans. Recently, ancient DNA from this organism was recovered from pre-Columbian mummies of Peru, sparking debate over the origin and frequency of tuberculosis in the Americas prior to European colonization. RESULTS: We present the first comparative genomic study of this bacterial species, starting from the genome sequencing of two M. pinnipedii isolates (MP1 and MP2) obtained from different organs of a stranded South American sea lion. Our results indicate that MP1 and MP2 differ by 113 SNPs (single nucleotide polymorphisms) and 46 indels, constituting the first report of a mixed-strain infection in a sea lion. SNP annotation analyses indicate that genes of the VapBC family, a toxin-antitoxin system, and genes related to cell wall remodeling are under evolutionary pressure for protein sequence change in these strains. OrthoMCL analysis with seven modern isolates of M. pinnipedii shows that these strains have highly similar proteomes. Gene variations were only marginally associated with hypothetical proteins and PE/PPE (proline-glutamate and proline-proline-glutamate, respectively) gene families. We also detected large deletions in ancient and modern M. pinnipedii strains, including a few occurring only in modern strains, indicating a process of genome reduction occurring over the past one thousand years. Our phylogenomic analyses suggest the existence of two modern clusters of M. pinnipedii associated with geographic location, and possibly host species, and one basal node associated with the ancient M. pinnipedii strains. Previously described MiD3 and MiD4 deletions may have occurred independently, twice, over the evolutionary course of the MTBC. CONCLUSION: The presence of superinfection (i.e. mixed-strain infection) in this sea lion suggests that M. pinnipedii is highly endemic in this population. Mycobacterium pinnipedii proteomes of the studied isolates showed a high degree of conservation, despite being under genomic decay when compared to M. tuberculosis. This finding indicates that further genomes need to be sequenced and analyzed to increase the chances of finding variably present genes among strains or that M. pinnipedii genome remodeling occurred prior to bacterial speciation.

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.

Comparative ecology of Escherichia coli in endangered Australian sea lion (Neophoca cinerea) pups.

The dissemination of human-associated bacteria into the marine environment has the potential to expose wildlife populations to atypical microbes that can alter the composition of the gut microbiome or act as pathogens. The objective of the study was to determine whether endangered Australian sea lion (Neophoca cinerea) pups from two South Australian colonies, Seal Bay, Kangaroo Island and Dangerous Reef, Spencer Gulf, have been colonised by human-associated Escherichia coli. Faecal samples (n = 111) were collected to isolate E. coli, and molecular screening was applied to assign E. coli isolates (n = 94) to phylotypes and detect class 1 integrons; mobile genetic elements that confer resistance to antimicrobial agents. E. coli phylotype distribution and frequency differed significantly between colonies with phylotypes B2 and D being the most abundant at Seal Bay, Kangaroo Island (55% and 7%) and Dangerous Reef, Spencer Gulf (36% and 49%), respectively. This study reports the first case of antimicrobial resistant E. coli in free-ranging Australian sea lions through the identification of class 1 integrons from an individual pup at Seal Bay. A significant relationship between phylotype and total white cell count (WCC) was identified, with significantly higher WCC seen in pups with human-associated phylotypes at Dangerous Reef. The difference in phylotype distribution and presence of human-associated E. coli suggests that proximity to human populations can influence sea lion gut microbiota. The identification of antimicrobial resistance in a free-ranging pinniped population provides crucial information concerning anthropogenic influences in the marine environment.

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.

Neisseria zalophi sp. nov., isolated from oral cavity of California sea lions (Zalophus californianus).

Three independent strains of Neisseria sp. were isolated from the oral cavity of California sea lions (Zalophus californianus) that were admitted to The Marine Mammal Center facilities in California, USA. The strains were isolated from oral swabs by cultivation on Trypticase Soy agar with 5% sheep blood under aerobic conditions. The 16S rRNA gene sequence of these three strains shared 99% similarity, but demonstrated only 97-98% nucleotide similarity to the phylogenetically closest relatives such as N. canis, N. zoodegmatis, N. animaloris, and N. dumasiana. These three strains also shared 99% sequence similarity of their rplF, rpoB, and gyrB gene sequences. Based on the biochemical tests alone (i.e., without genetic analysis of housekeeping genes), it is difficult to discriminate this novel species from N. canis; however, it can be easily discriminated from all phylogenetically closely related species using the sequencing analysis of its housekeeping genes (e.g., rplF, rpoB, or gyrB genes). Thus, genetic testing is indispensable for accurate identification of this species in a routine laboratory practice. The species is an obligate aerobe and able to grow in Mueller-Hinton broth supplemented with 6% NaCl, but the phylogenetically closely related species (N. canis, N. zoodegmatis, N. animaloris, and N. dumasiana) were not. Based on these phenotypic and genotypic characteristics and phylogenetic data, we conclude that these new strains represent a novel species of the genus Neisseria, for which the name Neisseria zalophi sp. nov. is proposed. The type strain is CSL 7565T (= ATCC BAA2455T = DSM 102031T).

MHC class II DRB diversity predicts antigen recognition and is associated with disease severity in California sea lions naturally infected with Leptospira interrogans.

We examined the associations between California sea lion MHC class II DRB (Zaca-DRB) configuration and diversity, and leptospirosis. As Zaca-DRB gene sequences are involved with antigen presentation of bacteria and other extracellular pathogens, we predicted that they would play a role in determining responses to these pathogenic spirochaetes. Specifically, we investigated whether Zaca-DRB diversity (number of genes) and configuration (presence of specific genes) explained differences in disease severity, and whether higher levels of Zaca-DRB diversity predicted the number of specific Leptospira interrogans serovars that a sea lion's serum would react against. We found that serum from diseased sea lions with more Zaca-DRB loci reacted against a wider array of serovars. Specific Zaca-DRB loci were linked to reactions with particular serovars. Interestingly, sea lions with clinical manifestation of leptospirosis that had higher numbers of Zaca-DRB loci were less likely to recover from disease than those with lower diversity, and those that harboured Zaca-DRB.C or -G were 4.5 to 5.3 times more likely to die from leptospirosis, regardless of the infective serovars. We propose that for leptospirosis, a disadvantage of having a wider range of antigen presentation might be increased disease severity due to immunopathology. Ours is the first study to examine the importance of Zaca-DRB diversity for antigen detection and disease severity following natural exposure to infective leptospires.

Characterisation of North American Brucella isolates from marine mammals.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.