Transcriptional Profiles of California Sea Lion Peripheral NK and CD+8 T Cells Reflect Ecological Regionalization and Infection by Oncogenic Viruses

The California sea lion is one of the few wild mammals prone to develop cancer, particularly urogenital carcinoma (UGC), whose prevalence is currently estimated at 25% of dead adult sea lions stranded along the California coastline. Genetic factors, viruses and organochlorines have been identified as factors that increase the risk of occurrence of this pathology. Given that no cases of UGC have as yet been reported for the species along its distribution in Mexican waters, the potential relevance of contaminants for the development of urogenital carcinoma is highlighted even more as blubber levels of organochlorines are more than two orders of magnitude lower in the Gulf of California and Mexican Pacific than in California. In vitro studies have shown that organochlorines can modulate anti-viral and tumor-surveillance activities of NK and cytotoxic T-cells of marine mammals, but little is known about the activity of these effectors in live, free-living sea lions. Here, we examine leukocyte transcriptional profiles of free-ranging adult California sea lions for eight genes (Eomes, Granzyme B, Perforin, Ly49, STAT1, Tbx21, GATA3, and FoxP3) selected for their key role in anti-viral and tumor-surveillance, and investigate patterns of transcription that could be indicative of differences in ecological variables and exposure to two oncogenic viruses: sea lion type one gammaherpesvirus (OtHV-1) and sea lion papillomavirus type 1 (ZcPV-1) and systemic inflammation. We observed regional differences in the expression of genes related to Th1 responses and immune modulation, and detected clear patterns of differential regulation of gene expression in sea lions infected by genital papillomavirus compared to those infected by genital gammaherpesvirus or for simultaneous infections, similar to what is known about herpesvirus and papillomavirus infections in humans. Our study is a first approach to profile the transcriptional patterns of key immune effectors of free-ranging California sea lions and their association with ecological regions and oncogenic viruses. The observed results add insight to our understanding of immune competence of marine mammals, and may help elucidate the marked difference in the number of cases of urogenital carcinoma in sea lions from US waters and other areas of their distribution

Phylogeographic analysis reveals association of tick-borne pathogen, Anaplasma marginale, MSP1a sequences with ecological traits affecting tick vector performance

<p>Abstract</p> <p>Background</p> <p>The tick-borne pathogen <it>Anaplasma marginale</it>, which is endemic worldwide, is the type species of the genus <it>Anaplasma </it>(Rickettsiales: Anaplasmataceae). <it>Rhipicephalus </it>(<it>Boophilus</it>) <it>microplus </it>is the most important tick vector of <it>A. marginale </it>in tropical and subtropical regions of the world. Despite extensive characterization of the genetic diversity in <it>A. marginale </it>geographic strains using major surface protein sequences, little is known about the biogeography and evolution of <it>A. marginale </it>and other <it>Anaplasma </it>species. For <it>A. marginale</it>, MSP1a was shown to be involved in vector-pathogen and host-pathogen interactions and to have evolved under positive selection pressure. The MSP1a of <it>A. marginale </it>strains differs in molecular weight because of a variable number of tandem 23-31 amino acid repeats and has proven to be a stable marker of strain identity. While phylogenetic studies of MSP1a repeat sequences have shown evidence of <it>A. marginale</it>-tick co-evolution, these studies have not provided phylogeographic information on a global scale because of the high level of MSP1a genetic diversity among geographic strains.</p> <p>Results</p> <p>In this study we showed that the phylogeography of <it>A. marginale </it>MSP1a sequences is associated with world ecological regions (ecoregions) resulting in different evolutionary pressures and thence MSP1a sequences. The results demonstrated that the MSP1a first (R1) and last (RL) repeats and microsatellite sequences were associated with world ecoregion clusters with specific and different environmental envelopes. The evolution of R1 repeat sequences was found to be under positive selection. It is hypothesized that the driving environmental factors regulating tick populations could act on the selection of different <it>A. marginale </it>MSP1a sequence lineages, associated to each ecoregion.</p> <p>Conclusion</p> <p>The results reported herein provided the first evidence that the evolution of <it>A. marginale </it>was linked to ecological traits affecting tick vector performance. These results suggested that some <it>A. marginale </it>strains have evolved under conditions that support pathogen biological transmission by <it>R. microplus</it>, under different ecological traits which affect performance of <it>R. microplus </it>populations. The evolution of other <it>A. marginale </it>strains may be linked to transmission by other tick species or to mechanical transmission in regions where <it>R. microplus </it>is currently eradicated. The information derived from this study is fundamental toward understanding the evolution of other vector-borne pathogens.</p

Hookworm infection, anaemia and genetic variability of the New Zealand sea lion

Hookworms are intestinal blood-feeding nematodes that parasitize and cause high levels of mortality in a wide range of mammals, including otariid pinnipeds. Recently, an empirical study showed that inbreeding (assessed by individual measures of multi-locus heterozygosity) is associated with hookworm-related mortality of California sea lions. If inbreeding increases susceptibility to hookworms, effects would expectedly be stronger in small, fragmented populations. We tested this assumption in the New Zealand sea lion, a threatened otariid that has low levels of genetic variability and high hookworm infection rates. Using a panel of 22 microsatellites, we found that average allelic diversity (5.9) and mean heterozygosity (0.72) were higher than expected for a small population with restricted breeding, and we found no evidence of an association between genetic variability and hookworm resistance. However, similar to what was observed for the California sea lion, homozygosity at a single locus explained the occurrence of anaemia and thrombocytopenia in hookworm-infected pups (generalized linear model, F = 11.81, p < 0.001) and the effect was apparently driven by a particular allele (odds ratio = 34.95%; CI: 7.12–162.41; p < 0.00001). Our study offers further evidence that these haematophagus parasites exert selective pressure on otariid blood-clotting processes

Persistent contaminants and herpesvirus OtHV1 are positively associated with cancer in wild California Sea Lions (Zalophus californianus)

This work was funded by the Geoffrey Hughes Fellowship, the National Institutes of Health (Fogarty International Center) and National Science Foundation joint program for the Ecology of Infectious Disease, the National Marine Fisheries Service Marine Mammal Heath and Stranding Program, and the Natural Environment Research Council grant number NE/R015007/.The prevalence of cancer in wild California sea lions (Zalophus californianus) is one of the highest amongst mammals, with 18–23% of adult animals examined post-mortem over the past 40 years having urogenital carcinoma. To date, organochlorines, genotype and infection with Otarine herpesvirus-1 (OtHV-1) have been identified in separate studies using distinct animals as associated with this carcinoma. Multi-year studies using large sample sizes to investigate the relative importance of multiple factors on marine mammal health are rare due to logistical and ethical challenges. The objective of this study was to use a case control approach with samples from 394 animals collected over 20 years in a multifactorial analysis to explore the relative importance of distinct factors identified to date as associated with sea lion cancer in the likelihood of sea lion carcinoma. Stepwise regression indicated that the best model to explain carcinoma occurrence included herpesvirus status, contaminant exposure, and blubber depth, but not genotype at a single microsatellite locus, PV11. The odds of carcinoma was 43.57 times higher in sea lions infected with OtHV-1 (95% CI 14.61, 129.96, p <0.001), and 1.48 times higher for every unit increase in the loge[contaminant concentrations], ng g–1 (an approximate tripling of concentration), in their blubber (95% CI 1.11, 1.97, p <0.007), after controlling for the effect of blubber depth. These findings demonstrate the importance of contaminant exposure combined with OtHV1 infection, in the potential for cancer occurrence in wild sea lions.Publisher PDFPeer reviewe

Context-dependent associations between heterozygosity and immune variation in a wild carnivore

Background: A multitude of correlations between heterozygosity and fitness proxies associated with disease have been reported from wild populations, but the genetic basis of these associations is unresolved. We used a longitudinal dataset on wild Galapagos sea lions (Zalophus wollebaeki) to develop a relatively new perspective on this problem, by testing for associations between heterozygosity and immune variation across age classes and between ecological contexts. Results: Homozygosity by locus was negatively correlated with serum immunoglobulin G production in pups (0-3 months of age), suggesting that reduced genetic diversity has a detrimental influence on the early development of immune defence in the Galapagos sea lion. In addition, homozygosity by locus was positively correlated with total circulating leukocyte concentration in juveniles (6-24 months of age), but only in a colony subject to the anthropogenic environmental impacts of development, pollution and introduced species, which suggests that reduced genetic diversity influences mature immune system activity in circumstances of high antigen exposure. Conclusions: These findings demonstrate the environmental context-dependency of the phenotypic expression of immune variation, which is implicit in the theory of ecoimmunology, but which has been rarely demonstrated in the wild. They also indicate that heterozygosity may be linked to the maintenance of heterogeneity in mammalian immune system development and response to infection, adding to the body of evidence on the nature of the mechanistic link between heterozygosity and fitness

Immune activity, body condition and human-associated environmental impacts in a wild marine mammal

PMB was funded by the Natural Environment Research Council (www.nerc.ac.uk), the Universities Federation for Animal Welfare (www.ufaw.org.uk) and the Sea Mammal Research Unit (www.smru.st-and.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.Within individuals, immunity may compete with other life history traits for resources, such as energy and protein, and the damage caused by immunopathology can sometimes outweigh the protective benefits that immune responses confer. However, our understanding of the costs of immunity in the wild and how they relate to the myriad energetic demands on free-ranging organisms is limited. The endangered Galapagos sea lion (Zalophus wollebaeki) is threatened simultaneously by disease from domestic animals and rapid changes in food availability driven by unpredictable environmental variation. We made use of this unique ecology to investigate the relationship between changes in immune activity and changes in body condition. We found that during the first three months of life, changes in antibody concentration were negatively correlated with changes in mass per unit length, skinfold thickness and serum albumin concentration, but only in a sea lion colony exposed to anthropogenic environmental impacts. It has previously been shown that changes in antibody concentration during early Galapagos sea lion development were higher in a colony exposed to anthropogenic environmental impacts than in a control colony. This study allows for the possibility that these relatively large changes in antibody concentration are associated with negative impacts on fitness through an effect on body condition. Our findings suggest that energy availability and the degree of plasticity in immune investment may influence disease risk in natural populations synergistically, through a trade-off between investment in immunity and resistance to starvation. The relative benefits of such investments may change quickly and unpredictably, which allows for the possibility that individuals fine-tune their investment strategies in response to changes in environmental conditions. In addition, our results suggest that anthropogenic environmental impacts may impose subtle energetic costs on individuals, which could contribute to population declines, especially in times of energy shortage.PostprintPeer reviewe

A novel non-invasive tool for disease surveillance of large free-ranging whales and its relevance to conservation programs

The numbers of potentially pathogenic microorganisms that have been isolated from stranded cetaceans in the last three decades underscore the urgent need for methods of detection of microorganisms that might cause significant disease and increase the likelihood of population declines. We have designed and implemented two non-invasive techniques for the collection of exhaled breath condensate (blow) from free-ranging whales and demonstrated their suitability for the detection of respiratory bacteria. We successfully collected 22 individual blow samples from eight cetacean species. Using well-established molecular techniques we detected three bacterial genera (Haemophilus, Streptococcus and Staphylococcus). Haemophilus spp. was detected in fin whale Balaenoptera physalus, sperm whale Physeter macrocephalus, humpback whale Megaptera novaeangliae and gray whale Eschrichtius robustus blows, while unidentified β-hemolytic streptococci and Staphylococcus aureus were detected in gray whale and blue whale Balaenoptera musculus blows. The detection limit of the test was determined as 1 CFU mL−1. None of the identified bacteria were found in environmental (control) samples, suggesting that their presence in the blows was genuine and not due to inadvertent contamination. While the population-level relevance of these bacteria is as yet unclear and it is possible that they are commensal microorganisms, S. aureus has been identified previously as a high-risk pathogen to cetacean health, and streptococci have increasingly been associated with cetacean mortality events. We suggest that future cetacean monitoring programs of vulnerable or threatened species include blow sampling as a means to determine the prevalence of the respiratory bacteria in the populations and monitor spatiotemporal fluctuations as indicators of changes in cetacean health

Data from: 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