• Determining the immune status of Steller sea lions (Eumetopias jubatus): an environmental agents of disease perspective

      Kennedy, Stephanie Nichole; O'Hara, Todd; Ferrante, Andrea; Kuhn, Thomas; Trainor, Thomas; Rea, Lorrie (2019-05)
      The integrity of the immune system is paramount for preserving overall health for many organisms. Investigating environmental and physiological factors that may be associated with alterations of the immune status in non-traditional sentinel species, like the Steller sea lion (SSL), is a prominent undertaking in eco-immunology research. Changes to immune homeostasis likely impacts the health and survival of SSLs. Recent studies have reported that mercury concentrations in hair in 24 to 36% of newborn SSLs of the Western Aleutian Islands (WAI) exceed thresholds (>30 ppm) for potential adverse effects. Many of these individuals were from WAI rookeries that have historically experienced significant population declines with some slow to recover. Retrospective, and ongoing, analyses of mercury in lanugo coats (natal hair) from young pups of the WAI demonstrate in utero exposure to relatively high levels of mercury during late gestation. Therefore, this dissertation focuses on the notion that dietary acquired mercury could potentially alter immune response in SSLs, especially young pups, and may contribute to the lack of recovery from population declines. In order to gain an understanding of the potential for mercury to adversely affect the immune response of SSLs, selected aspects of immunity were measured (blood cell counts, haptoglobin, immunoglobulins, and cytokines) and investigated within the context of body condition, age, mercury exposure and regional population dynamics. In Chapter Two, the acute phase response protein, haptoglobin, was found to vary significantly with age and region. Individual SSL pups with greater concentrations of mercury had lower predicted concentrations of haptoglobin. In Chapter Three, a colorimetric protein A enzyme-linked immunosorbent assay was modified for enhancing accurate measurement of immunoglobulin concentrations in SSL serum. This improved methodology was then used in Chapter Four for comparing immunoglobulins in young developing SSL pups and dams as a measure of maternal investment of immunity among different rookeries. Lastly, Luminex multiplex technologies were employed for quantifying cell-signaling proteins (cytokines) in SSL serum to compare associations among rookery pups in Chapter Five. Although mercury concentrations in some individuals exceed adverse effects thresholds that are defined for other mammals, no statistically significant associations were found between immunoglobulins and cytokines relative to mercury concentrations in young developing pups. These thesis chapters provide a powerful baseline and improved methods for ongoing and future assessments of haptoglobin, immunoglobulins, and cytokines (combined with traditional hematologic measures) observed in young developing SSL pups in regions experiencing population decline when compared with rookeries with stable or increasing pup production. Some of these findings, especially for haptoglobin, are indicative of alterations in immune status in young SSL pups born to dams from different natal rookeries with higher mercury exposure. Understanding the cause of the differences in the immune status of young SSLs will require additional assessments of the maternal-fetal interface of immunity and other factors like nutrition, metabolic status, and infectious disease that may shape neonatal immunity leading to the regional differences observed.
    • Dietary effects on protein turnover in three pinniped species, Eumetopias jubatus, Phoca vitulina, and Leptonychotes weddellii

      Inglis, Susan D.; Castellini, Michael; Atkinson, Shannon; Barboza, Perry; Carpenter, James; Rea, Lorrie (2019-05)
      The role of dietary protein in pinniped (seal and sea lion) nutrition is poorly understood. Although these marine mammals derive the majority of their daily energetic needs from lipid, lipids cannot supply essential amino acids which have to come from protein fractions of the diet. Protein regulation is vital for cellular maintenance, molt, fasting metabolism, exercise and development. Proteins are composed of linked amino acids (AA), and net protein turnover is the balance between protein synthesis from component AA, and degradation back to AA. Protein regulation is influenced by dietary intake and quality, as well as physiological and metabolic requirements. In this work, pinniped diet quality was assessed through comparisons of amino acid profiles between maternal milk, blood serum, and seasonal prey of wild juvenile Steller sea lions (Eumetopias jubatus) in Southcentral Alaska. Both Pacific herring (Clupei pallasi) and walleye pollock (Gadus chalcogramma) showed similar patterns to milk in essential and branched chain amino acid content. Serum amino acid profiles suggest the juvenile sea lions were not in protein deficit at the time of capture. Protein metabolism in the blood and urine was assessed through turnover studies using amino acid tracers. The turnover kinetics of ¹⁵N-labelled glycine in the blood amino acid and protein pool, red blood cells, and urine urea were measured in wild adult female Weddell seals (Leptonychotes weddellii) in the Antarctic. Labelled glycine moved quickly into serum protein and red blood cells (1-2 hours) and urinary urea (2-4 hours). The turnover rates in the blood amino acid and urine urea pools demonstrated a reduced turnover rate associated with molting. Lastly, whole body protein turnover experiments using a single bolus ¹⁵N-labelled glycine tracer method with endproduct collection of blood, feces and urine were conducted on 2 Cohort groups of captive Alaskan harbor seals over 2 years. Season was found to have the greatest effect on whole body protein turnover, which increased during the winter and decreased in the summer molt. Conversely, protein intake decreased during the winter and increased in the summer molt. This pattern corresponded with an increase in mass and protein synthesis in the winter, while mass decreased and protein degradation rates increased in molting seals. Weaning also influenced the patterns with reduced protein turnover in newly weaned animals that had recently transitioned from milk to a fish diet. This project presents results on whole body protein turnover rates in nonfasting pinnipeds and reveals that protein turnover is strongly regulated by developmental and seasonal physiological and metabolic demands.
    • One health toxicology: expanding perspectives and methods to assess environmental contaminants

      Harley, John Robinson; O'Hara, Todd; Dunlap, Kriya; Duffy, Lawrence; Rea, Lorrie (2017-12)
      The discipline of One Health is founded on the principal that environmental health, animal health, and human health are interconnected. Although the field has been largely focused on zoonotic diseases, examining concepts such as toxicology under a One Health lens can offer a more holistic and preventative approach to research and implementation and, in particular, how fish-based diets may be involved with One Health outcomes. Here we present three general case studies that demonstrate new approaches to investigating One Health toxicology. In Chapter One we show how Arctic canids can be used as environmental sentinels for human health. We discuss three separate canid studies; in the first we find that Arctic foxes can act as sentinels of Arctic contaminants due to their foraging plasticity, in the second we examine the use of fish-fed sled dogs as a model for the effects of a fish-based diet on contaminants exposure and gene transcription, and in the third we develop the sled dog as a model for particulate matter air pollution in the Fairbanks North Star Borough. In Chapter Two we utilize the Steller sea lion, a nonmodel organism, as a sentinel for the effects of fish-based diet mercury exposure induced whole-genome changes in gene transcription (RNA-Seq). Using newly developed informatics tools we assemble a de novo transcriptome and examine large scale changes in gene expression related to mercury exposure and other One Health uses. This approach is extremely adaptable and has the potential to be applied across numerous non-model organisms and contaminants. We also applied a microbial mining algorithm to our RNA-Seq data and found evidence for a hemotropic Mycoplasma spp. in one of our samples. In Chapter Three we examine sources of mercury exposure for pregnant women from La Paz, Baja California Sur, Mexico. We found mercury concentrations to be generally low among the examined fish species and staple foods. While typical dietary assessments rely on recall surveys and questionnaires, we found that examining chemical biomarkers of diet including stable isotopes of carbon and nitrogen are critical in dietary risk assessment. Taken together these three investigations offer valuable lessons and techniques which can be applied to the field of One Health toxicology; especially to those fish diet based systems.
    • Steller sea lion (Eumetopias jubatus) strandings and the role of pathogens in reproductive failure

      Esquible, Janessa A.; Atkinson, Shannon; Burek-Huntington, Kathy; Cox, Keith; Tamone, Sherry (2018-08)
      Steller sea lions (SSL, Eumetopias jubatus) have faced severe population fluctuations over the last five decades with a myriad of possibilities affecting their SSL population including disease, malnutrition, predation, climate change, entanglement in marine debris, and other factors. This thesis examined the effects that anthropogenic factors and disease may play in SSL strandings and reproductive failure. The goal of this study was to characterize long-term seasonality and spatial trends in SSL strandings and to investigate the role Brucella spp., Coxiella burnetti. Chlamydophila spp. and morbilliviruses may play in reproductive failure including spontaneous abortion and premature parturition. In Chapter 1, we utilized stranding data (n=1507) collected in Alaska, Oregon, and Washington from 1990-2015. We assessed temporal trends by identifying seasonality patterns across all years, analyzing sex, age class, body length, and characterizing signs of human interaction including factors contributing to mortality. Clear seasonality trends were evident, with the greatest number of reported strandings occurring during the spring and summer. Gunshot wounds and fishery interactions accounted for a large proportion (46%) of human interaction cases in strandings. Adult males were the most frequently stranded sex and age class in the Alaska and West Coast Regions. This study attempted to quantify efforts to monitor strandings and determined that the apparent increase in strandings following 2000 was likely due to increased stranding response effort resulting from increased federal grant awards. We encourage conducting further spatial analyses of strandings in addition to continued stranding surveillance monitoring with attempts to improve stranding response time. In Chapter 2 of my thesis, we analyzed archived lung, skin lesion and placenta tissues for the pathogens of interest in SSL fetuses (n=18) and neonatal pups (n=2) collected from 1998 2015 in Alaska. Associated pathological findings and morphometric data were examined to identify signs of pathology or abnormalities in all cases. Marine mammal Brucella was detected in the lung tissue of three cases. This is the first documented detection of Brucella in SSL by PCR methods. Phocine distemper virus was also detected in the skin lesion of two cases and in the placenta of one case, in which the cases with skin lesions exhibited abnormal pathology that included vesiculoulcerative dermatitis. Currently, there is very little available information on the significance of Brucella spp. and morbilliviruses in marine mammal populations inhabiting Alaskan waters. Therefore, this study demonstrates the clear need to continue disease surveillance programs and further investigate the role disease may play in SSL reproductive health, and more generally on cohort population stability.