Lizabeth Bowen

Molecular characterization of expressed DQA and DQB genes in the California sea lion (Zalophus californianus)

Abstract. To date, there are no published MHC sequences from the California sea lion (Zalophus californianus), a thriving species that, by feeding high on the marine food web, could be a sentinel for disturbances in marine and coastal ecosystems. In this study, degenerate primers and RACE technology were used to amplify near-full-length (MhcZaca-DQB) and full-length (MhcZaca-DQA) expressed class II MHC gene products from the peripheral blood mononuclear cells of two California sea lions in rehabilitation. Five unique Zaca-DQA sequences and eight unique Zaca-DQB sequences, all encoding functional proteins, were identified in the two animals, indicating the presence of multiple DQ– loci in this species. An additional three Zaca-DQB sequences containing features compatible with pseudogenes or null alleles were also identified. Despite the identification of multiple DQA and DQB sequences, the degree of heterogeneity between them was extremely low. To confirm the limited degree of Zaca-DQ nucleotide variation between individuals, we used denaturing gradient gel electrophoresis to examine putative peptide binding region sequences from the peripheral blood leukocyte-derived RNAs of 19 wild-caught California sea lions from physically distinct populations. The pattern of Zaca-DQ sequence migration was identical between individuals and independent of geographical region. This apparent Zaca-DQ sequence identity between sea lions was confirmed by direct sequencing of individual bands. In combination, these findings raise important questions regarding immunogenetic diversity within this thriving species, and should prompt further research into the existence of a highly polymorphic sea lion class II MHC molecule with sequence features that support traditional peptide binding functions.

An immunogenetic basis for the high prevalence of urogenital cancer in a free-ranging population of California sea lions (Zalophus californianus).

In response to an unprecedented prevalence of cancer recently identified in free-ranging populations of California sea lions [(CSL) (Zalophus californianus], we examined the role of the immunologically important major histocompatibility (MHC) genes in this disease epidemic. Associations between MHC genes and cancer have been well established in humans, but have never before been investigated in wildlife. Using a previously developed technique employing sequence-specific primer-based PCR with intercalating dye technology, MHC genotypes were examined from 27 cancer-positive and 22 cancer-negative CSL stranded along the California coastline. Analyses elucidated an underlying immunogenetic component to the high prevalence of urogenital cancer in sea lions. Furthermore, these results demonstrate the functional relevance of CSL class II MHC by revealing a non-random nature of cancer susceptibility associated with the presence of specific genes.

Gene transcription in sea otters (Enhydra lutris); development of a diagnostic tool for sea otter and ecosystem health

Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real‐time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter (Enhydra lutris); sea otter‐specific qPCR primer sequences for the genes of interest are defined. We establish a ‘reference’ range of transcripts for each gene in a group of clinically healthy captive and free‐ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno‐modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell–cell adhesion. The cycle threshold (CT) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment.

Quantitation of leukocyte gene expression in cetaceans

Real-time quantitation of cytokine mRNA is a routine immunologic technique, especially fitting for use in those species for which monoclonal antibodies are not available. Quantitative gene expression assays were developed to assist in the immunologic assessment of three cetacean species including bottlenosed dolphins, Pacific white-sided dolphins and beluga whales. Nine cytokine genes (IL-2, -4, -10, -12, -13, -18, TNFalpha, TGFbeta and IFNgamma) and Cox-2 were selected for analysis. Most mitogen-induced mononuclear leukocyte responses were similar between the three cetacean species with either up- or down-regulation of cytokine genes. IL-10 expression was highly variable between species. No TH/1TH2 polarization was evident. Cytokine gene analysis has the potential to identify immune system perturbations induced by environmental insult as well as providing diagnostic tools for characterizing immune responses to environmental antigens and vaccines.

Differential Gene Expression Induced by Exposure of Captive Mink to Fuel Oil: A Model for the Sea Otter

Free-ranging sea otters are subject to hydrocarbon exposure from a variety of sources, both natural and anthropogenic. Effects of direct exposure to unrefined crude oil, such as that associated with the Exxon Valdez oil spill, are readily apparent. However, the impact of subtle but pathophysiologically relevant concentrations of crude oil on sea otters is difficult to assess. The present study was directed at developing a model for assessing the impact of low concentrations of fuel oil on sea otters. Quantitative PCR was used to identify differential gene expression in American mink that were exposed to low concentrations of bunker C fuel oil. A total of 23 genes, representing 10 different physiological systems, were analyzed for perturbation. Six genes with immunological relevance were differentially expressed in oil-fed mink. Interleukin-18 (IL-18), IL-10, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and complement cytolysis inhibitor (CLI) were down-regulated while IL-2 was up-regulated. Expression of two additional genes was affected; heat shock protein 70 (HSP70) was up-regulated and thyroid hormone receptor (THR) was down-regulated. While the significance of each perturbation is not immediately evident, we identified differential expression of genes that would be consistent with the presence of immune system-modifying and endocrine-disrupting compounds in fuel oil. Application of this approach to identify effects of petroleum contamination on sea otters should be possible following expansion of this mink model to identify a greater number of affected genes in peripheral blood leukocytes.

MHC gene configuration variation in geographically disparate populations of California sea lions (Zalophus californianus)

Major histocompatibility complex (MHC) class II DRB genotypes were examined in two geographically isolated populations of California sea lions (Zalophus californianus) (Gulf of California and California coastal Pacific Ocean). Genomic DNA from 227 California sea lions was examined using eight sequence‐specific primer (SSP) pairs flanking the putative peptide‐binding site. A total of 40 different Zaca‐DRB genotype configurations were identified among the 227 individuals. Using SSP‐PCR, significant differences were found between coastal California and Gulf of California Zalophus populations in numbers of DRB sequences per individual and configuration of sequences within individuals. Additionally, unique local patterns of MHC diversity were identified among the Midriff Island animals. These population differences are consistent with either ecologically distinct patterns of selection pressures and/or geographical isolation. The consequences of these partitioned MHC configurations at the population level are as yet unknown; however, the worldwide increase in emerging marine diseases lends urgency to their examination.

Cellular immune responses in cetaceans immunized with a porcine erysipelas vaccine

Clinical erysipelas represents a significant health problem in managed cetacean species. Vaccination was suspended in many oceanariums in the past due to losses associated with vaccine-induced hypersensitivities which were deemed to be a greater threat than clinical erysipelas. A perceived shift in clinical presentation of erysipelas from a chronic dermatologic form to an acute systemic form in dolphins sparked interest in re-initiating vaccination with improved subunit vaccines of Erysipelothrix rhusiopathiae. This manuscript describes the development and application of in vitro correlates of immunity (T(H)1, T(H)2 and T(REG)) in Tursiops truncatus induced by immunization with a commercial porcine 65 kDa subunit E. rhusiopathiae vaccine. Variable degrees of pre-existing T cell memory were identified prior to vaccination. Vaccine-induced IFN gamma responses were consistent with a T(H)1 response and associated with elimination of erysipelas in all vaccinated animals. Comparative analysis between six-month and 12-month vaccination booster regimes demonstrated maintenance of superior memory in the six-month group; however, anamnestic responses induced by booster were only identified in the 12-month group. To our knowledge, this is the first study to develop and apply advanced immunologic analyses for assessing vaccine efficacy in captive or free-ranging wildlife.

Differential expression of immune response genes in Steller sea lions (Eumetopias jubatus): An indicator of ecosystem health?

Characterization of the polygenic and polymorphic features of the Steller sea lion major histocompatibility complex (MHC) provides an ideal window for evaluating immunologic vigor of the population and identifying emergence of new genotypes that reflect ecosystem pressures. MHC genotyping can be used to measure the potential immunologic vigor of a population. However, since ecosystem-induced changes to MHC genotype can be slow to emerge, measurement of differential expression of these genes can potentially provide real-time evidence of immunologic perturbations. MHC DRB genes were cloned and sequenced using peripheral blood mononuclear leukocytes derived from 10 Steller sea lions from Southeast Alaska, Prince William Sound, and the Aleutian Islands. Nine unique DRB gene sequences were represented in each of 10 animals. MHC DRB gene expression was measured in a subset of six sea lions. Although DRB in genomic DNA was identical in all individuals, relative levels of expressed DRB mRNA was highly variable. Selective suppression of MHC DRB genes could be indicative of geographically disparate environmental pressures, thereby serving as an immediate and sensitive indicator of population and ecosystem health.

Ectoparasites of the Island Fox on Santa Cruz Island

The ectoparasite fauna for island foxes (Urocyon littoralis) on Santa Cruz Island (California, USA) in April (wet season) and September (dry season) 1998 was evaluated. Three taxa of ectoparasites were identified. These were fleas (Pulex irritans), lice (Neotrichodectes mephitidis), and ticks (Ixodes pacificus). Ectoparasite abundances varied seasonally. Typical of insular endemic species, island foxes may be especially vulnerable to the introduction of novel disease organisms and their vectors.

Hematology and Serum Chemistry of the Island Fox on Santa Cruz Island

Serum and hematologic biochemistry values for island foxes (Urocyon littoralis) on Santa Cruz Island (California, USA) in April (wet season) and September (dry season) 1998 were evaluated. Serum chemistry of island foxes generally varied seasonally; 10 (40%) of the 25 serum characteristics were higher in the wet season, and three (12%) of the 25 serum characteristics were higher in the dry season. No hematologic parameters varied between seasons, although some measures varied between sexes. Blood analytes also varied with age; fox pups had higher values than adults for one hematologic and four serum parameters, whereas adult foxes had higher values for five hematologic characteristics. The information on blood chemistry provides baseline data useful in the monitoring of this threatened insular endemic carnivore.