Effects of ontogeny and oiling on the thermal function of southern sea otter (Enhydra lutris nereis) fur.

During the evolution of most marine mammals, fur as an insulator has been replaced with more buoyant, energy storing and streamlining blubber. By contrast, the sea otter (Enhydra lutris) relies on insulation from its dense, air-trapping pelage, which differs morphologically between natal and adult stages. In this study, we investigated the ontogenetic changes in thermal function of southern sea otter (Enhydra lutris nereis) pelts in air, in water, and when saturated with crude oil. Pelt thermal conductivity, thickness, and thermal resistance were measured for six age classes: neonate (<1 month), small pup (1-2 months), large pup (3-5 months), juvenile (6 months-1 year), subadult (1-3 years), and adult (4-9 years). Thermal conductivity was significantly higher for pelts in air than in water, with oiled pelts exhibiting the highest values (P < 0.001). Oiled pelts had the lowest thermal resistance, which suggests that regardless of age, all sea otters are vulnerable to the effects of oiling (P < 0.001). To scale up our laboratory findings, we used a volume-specific geometric model of conductive heat transfer for a simplified sea otter body, representing all tested age classes and treatments. Neonates, small pups, and large pups are more vulnerable to the effects of oiling compared with older age classes (P < 0.0001) due to a higher surface area-to-volume ratio. These results are consistent with the known thermal conductance values for adult sea otter pelts, yet this is the first time such thermal differences have been demonstrated in young otters. Overall, body size and age play a more important role in the thermal abilities of sea otters than previously thought.

Ontogenetic changes in southern sea otter (Enhydra lutris nereis) fur morphology.

Many animals exhibit morphological changes across ontogeny associated with adaptations to their environment. Sea otters (Enhydra lutris) have the densest fur of any animal, which is composed of guard hairs, intermediate hairs, and underhairs. Sea otters live in cold water environments, and their fur traps a layer of air to remain properly insulated, due to morphological adaptations that allow the hairs to trap air when submerged. When a sea otter is born, it has a natal pelage which it will eventually molt and replace with a pelt resembling the adult pelage. Past studies have investigated the morphology and hair density of adult sea otter fur, but these characteristics have not been measured for other age classes, including for the natal pelage. This study quantified ontogenetic changes in hair morphology of southern sea otter (E. lutris nereis) pelts. We measured guard hair length and circularity, shape of cuticular scales on guard hairs and underhairs, and overall hair density for sea otter pelts across six age classes: neonate (<1 month), small pup (1-2 months), large pup (3-5 months), juvenile (6 months-1 year), subadult (1-3 years), and adult (4-9 years). Neonate and small pup pelts had significantly longer guard hairs than older age classes. Natal pelage guard hairs were similarly shaped but smaller in diameter than adult guard hairs. Hairs of the natal pelage had similar cuticular scale patterns as adult hairs, indicating the importance of this structure for the function of the fur. Natal pelage had a lower hair density than the pelage of older age classes, with the adult pelage exhibiting the highest hair density. Overall, the morphological differences between natal and adult pelage in sea otters suggest functional differences that may make sea otter pups more vulnerable to heat loss.

Pathology, microbiology, and genetic diversity associated with Erysipelothrix rhusiopathiae and novel Erysipelothrix spp. infections in southern sea otters (Enhydra lutris nereis).

Erysipelothrix spp., including E. rhusiopathiae, are zoonotic bacterial pathogens that can cause morbidity and mortality in mammals, fish, reptiles, birds, and humans. The southern sea otter (SSO; Enhydra lutris nereis) is a federally-listed threatened species for which infectious disease is a major cause of mortality. We estimated the frequency of detection of these opportunistic pathogens in dead SSOs, described pathology associated with Erysipelothrix infections in SSOs, characterized the genetic diversity and antimicrobial susceptibility of SSO isolates, and evaluated the virulence of two novel Erysipelothrix isolates from SSOs using an in vivo fish model. From 1998 to 2021 Erysipelothrix spp. were isolated from six of >500 necropsied SSOs. Erysipelothrix spp. were isolated in pure culture from three cases, while the other three were mixed cultures. Bacterial septicemia was a primary or contributing cause of death in five of the six cases. Other pathology observed included suppurative lymphadenopathy, fibrinosuppurative arteritis with thrombosis and infarction, bilateral uveitis and endophthalmitis, hypopyon, petechia and ecchymoses, mucosal infarction, and suppurative meningoencephalitis and ventriculitis. Short to long slender Gram-positive or Gram-variable bacterial rods were identified within lesions, alone or with other opportunistic bacteria. All six SSO isolates had the spaA genotype-four isolates clustered with spaA E. rhusiopathiae strains from various terrestrial and marine animal hosts. Two isolates did not cluster with any known Erysipelothrix spp.; whole genome sequencing revealed a novel Erysipelothrix species and a novel E. rhusiopathiae subspecies. We propose the names Erysipelothrix enhydrae sp. nov. and Erysipelothrix rhusiopathiae ohloneorum ssp. nov. respectively. The type strains are E. enhydrae UCD-4322-04 and E. rhusiopathiae ohloneorum UCD-4724-06, respectively. Experimental injection of tiger barbs (Puntigrus tetrazona) resulted in infection and mortality from the two novel Erysipelothrix spp. Antimicrobial susceptibility testing of Erysipelothrix isolates from SSOs shows similar susceptibility profiles to isolates from other terrestrial and aquatic animals. This is the first description of the pathology, microbial characteristics, and genetic diversity of Erysipelothrix isolates recovered from diseased SSOs. Methods presented here can facilitate case recognition, aid characterization of Erysipelothrix isolates, and illustrate assessment of virulence using fish models.

Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American Pacific coastal marine mammals.

Southern sea otters (SSO: Enhydra lutris nereis) are a federally-listed threatened subspecies found almost exclusively in California, USA. Despite their zoonotic potential and lack of host specificity, K. pneumoniae and Klebsiella spp. have largely unknown epizootiology in SSOs. Klebsiella pneumoniae is occasionally isolated at necropsy, but not from live SSOs. Hypermucoviscous (HMV) K. pneumoniae strains are confirmed pathogens of Pacific Basin pinnipeds, but have not been previously isolated from SSOs. We characterized the virulence profiles of K. pneumoniae isolates from necropsied SSOs, evaluated killing of marine mammal K. pneumoniae following in vitro exposure to California sea lion (CSL: Zalophanus californianus) whole blood and serum, and characterized lesion patterns associated with Klebsiella spp. infection in SSOs. Four of 15 SSO K. pneumoniae isolates were HMV and all were recovered from SSOs that stranded during 2005. Many K. pneumoniae infections were associated with moderate to severe pathology as a cause of death or sequela. All HMV infections were assessed as a primary cause of death or as a direct result of the primary cause of death. Klebsiella-infected SSOs exhibited bronchopneumonia, tracheobronchitis and/or pleuritis, enteritis, Profilicollis sp. acanthocephalan peritonitis, septic peritonitis, and septicemia. All SSO HMV isolates were capsular type K2, the serotype most associated with HMV infections in CSLs. Multiplex PCR revealed two distinct virulence gene profiles within HMV isolates and two within non-HMV isolates. In vitro experiments investigating CSL whole blood and serum killing of K. pneumoniae suggest that HMV isolates are more resistant to serum killing than non-HMV isolates.

Exposure to domoic acid is an ecological driver of cardiac disease in southern sea otters✰.

Harmful algal blooms produce toxins that bioaccumulate in the food web and adversely affect humans, animals, and entire marine ecosystems. Blooms of the diatom Pseudo-nitzschia can produce domoic acid (DA), a toxin that most commonly causes neurological disease in endothermic animals, with cardiovascular effects that were first recognized in southern sea otters. Over the last 20 years, DA toxicosis has caused significant morbidity and mortality in marine mammals and seabirds along the west coast of the USA. Identifying DA exposure has been limited to toxin detection in biological fluids using biochemical assays, yet measurement of systemic toxin levels is an unreliable indicator of exposure dose or timing. Furthermore, there is little information regarding repeated DA exposure in marine wildlife. Here, the association between long-term environmental DA exposure and fatal cardiac disease was investigated in a longitudinal study of 186 free-ranging sea otters in California from 2001 - 2017, highlighting the chronic health effects of a marine toxin. A novel Bayesian spatiotemporal approach was used to characterize environmental DA exposure by combining several DA surveillance datasets and integrating this with life history data from radio-tagged otters in a time-dependent survival model. In this study, a sea otter with high DA exposure had a 1.7-fold increased hazard of fatal cardiomyopathy compared to an otter with low exposure. Otters that consumed a high proportion of crab and clam had a 2.5- and 1.2-times greater hazard of death due to cardiomyopathy than otters that consumed low proportions. Increasing age is a well-established predictor of cardiac disease, but this study is the first to identify that DA exposure affects the risk of cardiomyopathy more substantially in prime-age adults than aged adults. A 4-year-old otter with high DA exposure had 2.3 times greater risk of fatal cardiomyopathy than an otter with low exposure, while a 10-year old otter with high DA exposure had just 1.2 times greater risk. High Toxoplasma gondii titers also increased the hazard of death due to heart disease 2.4-fold. Domoic acid exposure was most detrimental for prime-age adults, whose survival and reproduction are vital for population growth, suggesting that persistent DA exposure will likely impact long-term viability of this threatened species. These results offer insight into the pervasiveness of DA in the food web and raise awareness of under-recognized chronic health effects of DA for wildlife at a time when toxic blooms are on the rise.

Pathology and epidemiology of nasopulmonary acariasis (Halarachne sp.) in southern sea otters (Enhydra lutris nereis).

Halarachne sp. nasal mites infest harbor seals (Phoca vitulina) and southern sea otters (Enhydra lutris nereis) in California, but little is known about the pathophysiology of these infestations, or risk factors for exposure. To investigate these questions, a retrospective case-control study was performed using necropsy data from 70 mite-infested sea otters, and 144 non-infested controls. Case records for sea otters examined by pathologists from February 1999 through May 2015 were examined to assess risk factors for infestation, and lesions associated with nasopulmonary acariasis. Animals with a history of captive care within 10 days of death or carcass recovery were 3.2 times more likely to be infested with nasopulmonary mites than those with no history of recent rehabilitation. Sea otters stranding within 1 km of Elkhorn Slough in Monterey Bay were 4.9 times more likely to be infested with nasal mites than other areas; this site is characterized by high sea otter contact with sympatric harbor seals (a common host for Halarachne sp.), and a comparatively large population of rehabilitated and released sea otters. Aged adult otters were 9.4 times more likely to be infested than younger animals, and sea otters with nasopulmonary acariasis were 14.2 times more likely to have upper respiratory inflammation than un-infested animals. Additional findings in otters with nasopulmonary acariasis included lower respiratory tract bacterial infections, presence of medium-sized and/or fresh nose wounds at necropsy (indicators of recent face-to-face interaction between otters during copulation or fighting), and turbinate bone erosion. Our findings, although preliminary, suggest that captive rehabilitation and close contact with harbor seals could facilitate nasopulmonary mite transmission to sea otters. We also identified a high-risk zone for nasopulmonary acariasis in sea otters. We also provide preliminary data to suggest that nasopulmonary mite infestations can cause significant respiratory pathology in sea otters.

Mycoplasma enhydrae sp. nov. isolated from southern sea otters (Enhydra lutris nereis).

Five Mycoplasma strains have been isolated from the oropharynx of southern sea otters (Enhydra lutris nereis) from the Central California Coast, USA. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma species. All five strains hydrolysed arginine but not urea, but did not produce acid from glucose, and all were isolated and propagated under anaerobic and aerobic atmospheric conditions at +35-37 ˚C using either SP4 or PPLO medium supplemented with arginine. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of the following genetic loci: 16S rRNA, 16S-23S rRNA ITS, rpoB, rpoC, polC, topIIA, tufB, arcA and smc. Complete 16S rRNA gene sequence analysis indicated that these strains were most closely related to M.ycoplasma phocicerebrale, and to M.ycoplasma arginini, M.ycoplasma gateae and M.ycoplasma canadense with nucleotide similarities of 99 and 98 %, respectively. Nucleotide analysis of other genetic loci revealed 73-91 % nucleotide similarity to the corresponding genes of the above closely related species. All five strains clustered into a distinct group on the 16S rRNA and rpoB phylogenetic trees. Serological testing via growth inhibition and metabolic inhibition tests employing antiserum to type strains of M. phocicerebrale, M. arginini, M. gateae and M. canadense failed to recognize these novel strains. Our results suggest that the strains isolated from southern sea otters represent a novel species of the genus Mycoplasma, for which the name Mycoplasma enhydrae sp. nov. is proposed; the type strain is 6243-11T (=DSM 106704T=ATCC TSD-140T).

Serologic Detection of Subtype-specific Antibodies to Influenza A Viruses in Southern Sea Otters (Enhydra lutris nereis).

There are approximately 3,000 southern sea otters (Enhydra lutris nereis) in the nearshore environment along the California coast, US, and the species is classified as Threatened under the Endangered Species Act. We tested sera from 661 necropsied southern sea otters sampled from 1997 to 2015 to determine overall exposure to influenza A viruses (IAVs) and to identify subtype-specific antibody responses. Using an enzyme-linked immunosorbent assay (ELISA), antibodies to IAV nucleoproteins were detected in 160 (24.2%) otters, with seropositive animals found in every year except 2008. When the ELISA-positive samples were tested by virus microneutralization, antibody responses were detected to avian-origin hemagglutinin subtypes H1, H3, H4, H5, H6, H7, H9, and H11. Strong antibody responses to pandemic H1N1 (pdmH1N1) were also detected, indicating that epizootic transmission of pdmH1N1 occurred among the southern sea otter population after the emergence of this human-origin virus in 2009. We conclude that southern sea otters are susceptible to infection with avian and human-origin IAV and that exposure to a wide array of subtypes likely occurs during a given otter's 10- to 15-yr life span. Important unanswered questions include what effect, if any, IAV infection has on sea otter health, and how these animals become infected in their nearshore environment.

ENDEMIC INFECTION OF STRANDED SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) WITH NOVEL PARVOVIRUS, POLYOMAVIRUS, AND ADENOVIRUS.

Over the past century, the southern sea otter (SSO; Enhydra lutris nereis) population has been slowly recovering from near extinction due to overharvest. The SSO is a threatened subspecies under federal law and a fully protected species under California law, US. Through a multiagency collaborative program, stranded animals are rehabilitated and released, while deceased animals are necropsied and tissues are cryopreserved to facilitate scientific study. Here, we processed archival tissues to enrich particle-associated viral nucleic acids, which we randomly amplified and deeply sequenced to identify viral genomes through sequence similarities. Anelloviruses and endogenous retroviral sequences made up over 50% of observed viral sequences. Polyomavirus, parvovirus, and adenovirus sequences made up most of the remaining reads. We characterized and phylogenetically analyzed the full genome of sea otter polyomavirus 1 and the complete coding sequence of sea otter parvovirus 1 and found that the closest known viruses infect primates and domestic pigs ( Sus scrofa domesticus), respectively. We tested archived tissues from 69 stranded SSO necropsied over 14 yr (2000-13) by PCR. Polyomavirus, parvovirus, and adenovirus infections were detected in 51, 61, and 29% of examined animals, respectively, with no significant increase in frequency over time, suggesting endemic infection. We found that 80% of tested SSO were infected with at least one of the three DNA viruses, whose tissue distribution we determined in 261 tissue samples. Parvovirus DNA was most frequently detected in mesenteric lymph node, polyomavirus DNA in spleen, and adenovirus DNA in multiple tissues (spleen, retropharyngeal and mesenteric lymph node, lung, and liver). This study describes the virome in tissues of a threatened species and shows that stranded SSO are frequently infected with multiple viruses, warranting future research to investigate associations between these infections and observed lesions.

Novel urease-negative Helicobacter sp. 'H. enhydrae sp. nov.' isolated from inflamed gastric tissue of southern sea otters.

A total of 31 sea otters Enhydra lutris nereis found dead or moribund (and then euthanized) were necropsied in California, USA. Stomach biopsies were collected and transected with equal portions frozen or placed in formalin and analyzed histologically and screened for Helicobacter spp. in gastric tissue. Helicobacter spp. were isolated from 9 sea otters (29%); 58% (18 of 31) animals were positive for helicobacter by PCR. The Helicobacter sp. was catalase- and oxidase-positive and urease-negative. By electron microscopy, the Helicobacter sp. had lateral and polar sheathed flagella and had a slightly curved rod morphology. 16S and 23S rRNA sequence analyses of all 'H. enhydrae' isolates had similar sequences, which clustered as a novel Helicobacter sp. closely related to H. mustelae (96-97%). The genome sequence of isolate MIT 01-6242 was assembled into a single ~1.6 Mb long contig with a 40.8% G+C content. The annotated genome contained 1699 protein-coding sequences and 43 RNAs, including 65 genes homologous to known Helicobacter spp. and Campylobacter spp. virulence factors. Histological changes in the gastric tissues extended from mild cystic degeneration of gastric glands to severe mucosal erosions and ulcers. Silver stains of infected tissues demonstrated slightly curved bacterial rods at the periphery of the gastric ulcers and on the epithelial surface of glands. The underlying mucosa and submucosa were infiltrated by low numbers of neutrophils, macrophages, and lymphocytes, with occasional lymphoid aggregates and well-defined lymphoid follicles. This is the second novel Helicobacter sp., which we have named 'H. enhydrae', isolated from inflamed stomachs of mustelids, the first being H. mustelae from a ferret.

THE HIGH COST OF MOTHERHOOD: END-LACTATION SYNDROME IN SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) ON THE CENTRAL CALIFORNIA COAST, USA.

Sea otters ( Enhydra lutris ) have exceptionally high energetic requirements, which nearly double during lactation and pup care. Thus, females are extremely vulnerable to caloric insufficiency. Despite a number of compensatory strategies, the metabolic challenge of reproduction culminates in numerous maternal deaths annually. Massive depletion of energy reserves results in a case presentation that we define as end-lactation syndrome (ELS), characterized by moderate to severe emaciation not attributable to a concurrent, independent disease process in females dying during late pup care or postweaning. We compiled detailed data for 108 adult female southern sea otters ( Enhydra lutris nereis) examined postmortem that stranded in California, US, 2005-12, and assessed pathology, reproductive status, and the location and timing of stranding. We introduce simple, grossly apparent, standardized physical criteria to assess reproductive stage for female sea otters. We also describe ELS, examine associated risk factors, and highlight female life history strategies that likely optimize reproduction and survival. Our data suggest that females can reset both the timing and energetic demands of reproduction through fetal loss, pup abandonment, or early weaning as part of specific physiologic checkpoints during each reproductive cycle. Females appear to preload nutritionally during delayed implantation and gestation to increase fitness and reproductive success. We found that ELS was a major cause of death, affecting 56% of enrolled adult females. Peak ELS prevalence occurred in late spring, possibly reflecting the population trend toward fall/winter pupping. Increasing age and number of pregnancies were associated with a higher risk of ELS. Although the proportion of ELS females was highest in areas with dense sea otter populations, cases were recovered throughout the range, suggesting that death from ELS is associated with, but not solely caused by, population resource limitation.

PREVALENCE, PATHOLOGY, AND RISK FACTORS ASSOCIATED WITH STREPTOCOCCUS PHOCAE INFECTION IN SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS), 2004-10.

Recent studies have implicated beta-hemolytic streptococci as opportunistic pathogens of marine mammals, including southern sea otters (Enhydra lutris nereis), but little is known about their prevalence or pathophysiology. Herein, we focus on risk factors for sea otter infection by a single beta-hemolytic streptococcal species, Streptococcus phocae. Streptococcus phocae was first identified as a marine mammal pathogen in 1994, and the first report in southern sea otters was in 2009. Its broad host range encompasses fish, pinnipeds, cetaceans, and mustelids, with S. phocae now recognized as an important pathogen of marine species worldwide. We assessed risk factors and lesion patterns for S. phocae infection in southern sea otters. Using archival necropsy data, S. phocae prevalence was 40.5% in fresh dead otters examined 2004-10. Skin trauma of any type was identified as a significant risk factor for S. phocae infection. The risk of infection was similar regardless of the cause and relative severity of skin trauma, including mating or fight wounds, shark bite, and anthropogenic trauma. Streptococcus phocae-infected sea otters were also more likely to present with abscesses or bacterial septicemia. Our findings highlight the importance of S. phocae as an opportunistic pathogen of sea otters and suggest that the most likely portal of entry is damaged skin. Even tiny skin breaks appear to facilitate bacterial colonization, invasion, abscess formation, and systemic spread. Our data provide important insights for management and care of marine species.

Oral papillomatosis caused by Enhydra lutris papillomavirus 1 (ElPV-1) in southern sea otters (Enhydra lutris nereis) in California, USA.

The southern sea otter (Enhydra lutris nereis) is a threatened marine sentinel. During postmortem investigations of stranded sea otters from 2004 to 2013 in California, US, papillomas were detected in the oral cavity of at least seven otters via necropsy and histopathology. Next-generation sequencing of viral particles purified from a single papilloma revealed a novel papillomavirus, Enhydra lutris papillomavirus 1 (ElPV-1). The genome of ElPV-1 was obtained, representing the first fully sequenced viral genome from southern sea otters. Phylogenetic analysis of the entire L1 gene, as well as a concatenated protein identities plot of all papillomaviral genes revealed that ElPV-1 is a λ-papillomavirus, related to a raccoon papillomavirus (Procyon lotor papillomavirus type 1) and a canine oral papillomavirus. Immunohistochemical staining, using a cross-reactive bovine papillomavirus antibody, suggested that ElPV-1 is present in intranuclear inclusions and intracytoplasmic keratin granules. Virus-infected cells were scattered throughout the stratum granulosum and stratum spinosum of the gingival and buccal papillomas. Using ElPV-1-specific PCR, we confirmed viral DNA in oral papillomas from all seven stranded sea otters, with identical L1 sequences. This virus is associated with the development of oral papillomatosis in southern sea otters.

Pelodera strongyloides infection in Pacific harbor seals (Phoca vitulina richardii) from California.

Skin biopsies were collected from free-ranging harbor seals (Phoca vitulina richardii) from central California (n = 53). Microscopic examination of hematoxylin and eosin-stained tissue sections revealed the presence of tightly coiled nematode larvae within the ostia of numerous hair follicles of four seals. Parasites were characterized by paired lateral alae, platymyarian musculature, and an indistinct, uninucleate digestive tract. Mild chronic superficial dermatitis and perifolliculitis were evident microscopically in association with the intrafollicular parasites. Histomorphologic features of the larvae and their presence within hair follicles are consistent with previous reports of the facultative nematode parasite Pelodera strongyloides. This is the first published report of P. strongyloides infection in any marine mammal. This parasite may be acquired by marine mammals through close contact with soil or decaying organic material and should be considered as a potential differential diagnosis for dermatitis in marine mammals that use terrestrial resting sites.