Jeffrey L. Stott

Morbillivirus infection in stranded common dolphins from the pacific ocean

From August 1995 to August 1997, six of 18 common dolphins (Delphinus delphis) that stranded along beaches of southern California (USA) tested antibody positive for dolphin morbillivirus (DMV). Titers ascertained by virus neutralization ranged from 1:50 to 1:910 while those determined by ELISA ranged from 1:80 to 1:195. The first individual to strand survived and was released back into the Pacific Ocean 14 mo later. Histopathologic examination of tissues from the other five dolphins did not reveal lesions characteristic of morbilliviral disease; however, morbilliviral RNA was detected in three of the five by reverse transcriptase-polymerase chain reaction testing. This is the first report of morbilliviral infection in any marine mammal species in the northern hemisphere of the Pacific Ocean. These data indicate that DMV, or a closely related morbillivirus, is present in the Pacific Ocean and infection of common dolphins may not be associated with morbillivirus disease.

Otarine herpesvirus-1: a novel gammaherpesvirus associated with urogenital carcinoma in California sea lions (Zalophus californianus).

The incidence of neoplasia in California sea lions (CSLs) is considered to be unusually high. Electron microscopic examination of some of these urogenital tumours revealed the presence of virions with typical herpes-like structure. While current attempts to cultivate this virus have not been successful, molecular studies employing DNA extracted from tumour tissues allowed both the classification of the agent and its identification in tumours and archived tissue samples. Two genome fragments generated using degenerate primers in PCR demonstrated highest identities with other mammalian gammaherpesviruses. Phylogenetic analysis showed that this novel virus, tentatively designated Otarine herpesvirus-1 (OtHV-1), grouped with members of the gammaherpesvirus subfamily and was distinct from PHV-2, a previously described pinniped gammaherpesvirus. An OtHV-1 specific PCR was established and used to investigate the presence of this virus in CSL tissues. PCR of DNA isolated from animals with these tumours, demonstrated that this virus was present in 100% (16/16) of tumours. Furthermore, DNA extracted from archived brain and muscle tissues was also positive in 29% (4/14) and 50% (7/14) of cases examined. This preliminary study provides evidence to support the hypothesis that the presence of this novel gammaherpesvirus is a factor in the development of urogenital carcinoma in CSLs.

Association of a mutation in the ryanodine receptor 1 gene with equine malignant hyperthermia

Equine malignant hyperthermia (MH) has been suspected but never genetically confirmed. In this study, we investigated whether mutations in a candidate gene, RyR1, were associated with MH in two clinically affected horses. RyR1 gene sequences revealed polymorphisms in exons 15, 17, and 46 in WTRyR1 and MHRyR1 horses with one derived amino acid change in MHRyR1 exon 46, R2454G. The MHRyR1 horses were genetically heterozygous for this mutation, but presented an MH phenotype with halothane challenge. Skeletal sarcoplasmic reticulum from a R2454G heterozygote collected during a fulminant MH episode showed significantly higher affinity and density of [3H]ryanodine‐binding sites compared to WTRyR1, but no differences in Ca2+, Mg2+, and caffeine modulation. In conclusion, an autosomal missense mutation in RyR1 is associated with MH in the horse, providing a screening test for susceptible individuals. [3H]ryanodine‐binding analysis suggests that long‐lasting changes in RyR1 conformation persists in vitro after the triggering event. Muscle Nerve 30: 356–365, 2004

INFECTIOUS DISEASE MONITORING OF THE ENDANGERED HAWAIIAN MONK SEAL

As part of conservation efforts between 1997 and 2001, more than 25% (332 animals) of the endangered Hawaiian monk seal (Monachus schauinslandi) population was sampled in the northwestern Hawaiian Islands. Serum samples were tested for antibodies to viruses, bacteria, and parasites known to cause morbidity and mortality in other marine mammal species. Antibodies were found to phocine herpesvirus-1 by using an enzyme-linked immunosorbent assay, but seropositive results were not confirmed by virus neutralization test. Antibodies to Leptospira bratislava, L. hardjo, L. icterohaemorrhagiae, and L. pomona were detected in seals from several sites with the microagglutination test. Antibodies to Brucella spp. were detected using 10 conventional serologic tests, but because of inconsistencies in test results and laboratories used, and the lack of validation by culture, the Brucella serology should be interpreted with caution. Antibodies to B. canis were not detected by card test. Chlamydophila abortus antibodies were detected by complement fixation (CF) test, and prevalence increased significantly as a function of age; the low sensitivity and specificity associated with the CF make interpretation of results difficult. Antibodies to Toxoplasma gondii and Dirofilaria immitis were rarely found. There was no serologic evidence of exposure to four morbilliviruses, influenza A virus, canine adenovirus, caliciviruses, or other selected viruses. Continuous surveillance provides a means to detect the introduction or emergence of these or other infectious diseases, but it is dependent on the development or improvement of diagnostic tools. Continued and improved surveillance are both needed as part of future conservation efforts of Hawaiian monk seals.

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.

Bovine cytokine expression during different phases of bovine leukemia virus infection

The potential role of aberrant cytokine production in the pathogenesis of bovine leukemia virus (BLV) was studied by analyzing cytokine mRNA expression in pokeweed-stimulated PBMLs of cows in different phases of disease progression. To analyze the mRNA, a semi-quantitative RT-PCR assay was developed. The RT-PCR assay was developed for detection of IL-2, -4, -6, -10, -12, IFN-gamma and actin using cDNA derived from phorbol-stimulated peripheral blood mononuclear leukocytes. Using a PCR specific for BLV tax, agar gel immunodiffusion and white blood cell counts, BLV-negative, BLV-positive aleukemic (AL), and BLV-positive persistently lymphocytotic (PL) cattle were identified. Peripheral blood lymphocytes cultured in vitro for 24 h in pokeweed mitogen were analyzed for cytokine production using the RT-PCR assay. Consistently elevated levels of IL-2 and IL-12 in AL and PL cattle in pokeweed mitogen-stimulated cells was detected, while IFN-gamma was elevated in the AL but not the PL cattle.

Monoclonal antibodies to lymphocyte surface antigens for cetacean homologues to CD2, CD19 and CD21

CD2 is a pan-T cell marker, while CD19 and CD21 are important molecules in signal transduction of B lymphocytes. CD19 and CD21 are both present on mature B cells, while CD19 is also present in developing B cells and plasma cells. Monoclonal antibodies (mAbs) against cetacean lymphocyte putative homologues to CD2 (two different antibodies), CD19 and CD21 were characterized. The proteins immunoprecipitated were as follows: F21.I (putative anti-CD2), 43 and 59kDa; F21.B (putative anti-CD19), 83 and 127kDa; F21.F (putative anti-CD21), 144kDa. The second putative anti-CD2 (F21.C) selectively inhibited the binding of F21.I. Both the putative anti-CD2 (T cell markers) stained T-cell zones on lymph node sections, while both the B cell markers (putative CD19 and CD21) stained B-cell zones. F21.B and F21.F were absent from thymus single cell suspension but labeled 63 and 65% mesenteric lymph node lymphocytes, respectively, while both F21.C and F21.F were present on 100% thymocytes and fewer lymph node lymphocytes. B and T cell markers were mutually exclusive on double labeling using flow cytometry. These mAbs are foreseen as possible valuable diagnostic and research tools to assess immune functions of captive and wild cetaceans.

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.