Integrated analysis of blood mRNAs and microRNAs reveals immune changes with age in the Yangtze finless porpoise (Neophocaena asiaeorientalis).

Populations of Yangtze finless porpoises (YFPs) have rapidly declined in recent decades, raising the specter of extinction. In order to protect YFPs, a greater understanding of their biology is needed, including studying how their immune functioning changes with age. Here, we systematically studied the hematologic and biochemical parameters, as well as mRNAs and miRNAs profiles of old, adult, and young YFPs. The lymphocyte (LYMPH), neutrophils (NEUT) and eosinophils (EOS) counts in old YFPs were lower than those in young or adult YFPs. When comparing old to adult YFPs, the latter showed higher expression of genes associated with the innate and adaptive immune systems, including complement components, major histocompatibility complex, interleukins, TNF receptors, and chemokines/cytokines. When comparing old to young YFPs, the most striking difference was in higher toll-like receptor signaling in the latter. When comparing adult to young YFPs, the former exhibited higher expression of genes related to adaptive immunity and the FoxO signaling pathway, but lower expression of genes associated with the PI3K-Akt signaling pathway. Negative miRNA-mRNA interactions were predicted in comparisons of the old and adult (326), old and young (316), adult and young (211) groups. Overall, these results delineate a progression from early innate immune function dominance to adaptive immune function enhancement (young to adult) and deterioration (adult to old), and the changes in miRNAs profile correlate with the effects of age on immune functions. This study is the first to observe the changes of immune function of Yangtze finless porpoise with age using transcriptome method, and the study's findings are of great significance for protecting this endangered species.

Organization and characteristics of the major histocompatibility complex class II region in the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis).

Little is known about the major histocompatibility complex (MHC) in the genome of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) (YFP) or other cetaceans. In this study, a high-quality YFP bacterial artificial chromosome (BAC) library was constructed. We then determined the organization and characterization of YFP MHC class II region by screening the BAC library, followed by sequencing and assembly of positive BAC clones. The YFP MHC class II region consists of two segregated contigs (218,725 bp and 328,435 bp respectively) that include only eight expressed MHC class II genes, three pseudo MHC genes and twelve non-MHC genes. The YFP has fewer MHC class II genes than ruminants, showing locus reduction in DRB, DQA, DQB, and loss of DY. In addition, phylogenic and evolutionary analyses indicated that the DRB, DQA and DQB genes might have undergone birth-and-death evolution, whereas the DQB gene might have evolved under positive selection in cetaceans. These findings provide an essential foundation for future work, such as estimating MHC genetic variation in the YFP or other cetaceans. This work is the first report on the MHC class II region in cetaceans and offers valuable information for understanding the evolution of MHC genome in cetaceans.

High similarity at three MHC loci between the baiji and finless porpoise: trans-species or convergent evolution?

Two DRA alleles and six MHC-I alleles were identified from a group of 15 baiji (Lipotes vexillifer), the most threatened cetacean in the world. Little sequence variation was detected at the DRA locus but extensive variation at the MHC-I locus. In combination with data at the DQB locus previously reported, three MHC loci exon 2 of the baiji all revealed striking similarity with those of the finless porpoise. Especially, some identical alleles shared by both species at the MHC-I and DQB loci suggested the convergent evolution as a consequence of common adaptive solutions to similar environmental pressures in the Yangtze River. As for DRA locus, the identity alleles were shared not only by baiji and finless porpoise but by some other cetacean species of the families Phocoenidae and Delphinidae, suggesting trans-species evolution on this gene.

Sequence variability at three MHC loci of finless porpoises (Neophocaena phocaenoides).

Major histocompatibility complex (MHC) class II DQB and DRA genes and class I gene of finless porpoises (Neophocaena phocaenoides) were investigated by single-strand conformation polymorphism and sequence analysis. The DRA, DQB, and MHC-I loci each contained 5, 14, and 34 unique sequences, respectively, and considerable sequence variation was found at the MHC-I and DQB loci. Gene duplication was manifested as three to five distinct sequences at each of the DQB and MHC-I loci from some individuals, and these sequences at each of the two loci separately clustered into four groups (cluster A, B, C, and D) based on the phylogenetic trees. Phylogenetic reconstruction revealed a trans-species pattern of evolution. Relatively high rates of non-synonymous (dN) vs synonymous (dS) substitution in the peptide-binding region (PBR) suggested balancing selection for maintaining polymorphisms at the MHC-I and DQB loci. In contrast, one single locus with little sequence variation was detected in the DRA gene, and no non-synonymous substitutions in the PBR indicated no balancing selection on this gene.

Relationships between polychlorinated biphenyls and health status in harbor porpoises (Phocoena phocoena) stranded in the United Kingdom.

To investigate possible relationships between polychlorinated biphenyl (PCB) exposure and infectious disease mortality in harbor porpoises (Phocoena phocoena) in United Kingdom waters, summed blubber concentrations of 25 chlorobiphenyl congeners (sigma25CB) in healthy harbor porpoises that died of acute physical trauma (mainly by-catch; n = 175) were compared with sigma25CB values in animals that died of infectious disease (n = 82). The infectious disease group had significantly greater sigma25CB values (mean, 27.6 mg/kg lipid) than the physical trauma group (mean, 13.6 mg/kg lipid; p < 0.001). This association occurred independently of other potentially confounding variables, including age, sex, two indices of nutritional status, season, region, and year found. Total blubber PCB levels (as Aroclor 1254) were also calculated, enabling direct comparison with a proposed threshold for adverse health effects (including immunosuppression) in marine mammals of 17 mg/kg lipid. In porpoises with total PCB levels greater than 17 mg/kg lipid (n = 154), total PCB levels were significantly higher in the infectious disease group compared to the physical trauma group (p < 0.001). This association was no longer significant in porpoises with total PCB levels of less than 17 mg/kg lipid (n = 103; p > 0.55). These findings are consistent with a causal (immunotoxic) relationship between PCB exposure and infectious disease mortality, and they provide a framework for future quantitative risk-assessment analyses of porpoise populations of known size and PCB exposure.

Development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction.

Impairment of immune function is suggested to play a contributing role for the increasing incidence of infectious diseases in the harbor porpoise (Phocoena phocoena) of the North and Baltic Seas. Both, lymphocyte-transformation-assay of peripheral blood lymphocytes (PBMC) and detection of cytokine expression are important tools for the characterization of the cellular immune response. To evaluate optimal parameters for the lymphocyte-transformation-assay isolated blood lymphocytes from four healthy harbor porpoises were stimulated with different concentrations of concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Cell proliferation was measured photometrically after 72 h using 5-bromo-deoxyuridine-assay and stimulation indices were calculated. The expression of pro- and anti-inflammatory cytokines such as interleukin (IL)-2, IL-4, IL-6, IL-10, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha was investigated in control and mitogen-stimulated lymphocytes using reverse-transcription polymerase chain reaction (RT-PCR). Primers for IL-2, IL-4 and IL-6 were selected from published cDNA-sequences of other cetaceans. Established canine and human primers were taken for the detection of TNF-alpha, TGF-beta, IL-10 and the house keeping transcript glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Specificity of the amplicon was confirmed by DNA sequence analysis and comparison with nucleotide sequences of other marine and terrestrial mammals. Con A and PHA represented the most powerful mitogens for harbor porpoise lymphoid cells at concentrations of 5 and 2 microg/ml, respectively, while PWM induced a comparatively low maximum proliferation at a concentration of 2 microg/ml. GAPDH was amplified in non-stimulated and all mitogen-stimulated cells. With the exception of IL-10 none of the other cytokines were detected in non-stimulated cells. Transcription of IL-4, IL-6, IL-10, TNF-alpha and TGF-beta-mRNA was observed after incubation with all the three phytomitogens, whereas IL-2 was only detected in Con A and PWM treated cells. Lymphocyte-transformation-assay and RT-PCR for detection of cytokines will allow to investigate possible impaired immune function in the harbor porpoise in future studies.

Immunogenicity and efficacy of recombinant subunit vaccines against phocid herpesvirus type 1.

Phocid herpesvirus type 1 (PhHV-1) is an alpha-herpesvirus that causes significant morbidity and mortality among young and immunocompromised harbour seals (Phoca vitulina) and therefore represents a major problem for seal rehabilitation centres. Consequently, there is a need for a safe and effective PhHV-1 vaccine. We tested an ISCOM-based recombinant PhHV-1 gB vaccine alone (gB) or with the addition of recombinant PhHV-1 gD (gBD) for (i). immunogenicity and protective efficacy against feline herpesvirus (FHV) infection in cats and (ii). their immunogenicity in seals. The FHV-cat model was chosen based on the close antigenic relationship between PhHV-1 and FHV. Upon challenge, all vaccinated (gB and gBD) cats excreted significantly less FHV (P<0.01) and gBD vaccinated cats showed less weight loss (P=0.05) than the mock-vaccinated cats. However, adding gD to the gB vaccine did not result in significantly better protection. Based on these data, immunogenicity studies in seals under rehabilitation were performed with the gB vaccine only. To this end, gB vaccine was tested at two different doses (20 or 40 microg). PhHV-1 specific antibody titres and in vitro proliferative T cell responses were measured in all seals upon vaccination. No differences were observed in antibody titres between seals vaccinated with either 20 or 40 microgB, but the higher gB concentration did result in higher specific proliferative T cell responses (P<0.01). Based on the close antigenic relationship between PhHV-1 and FHV, the safety and efficacy data in the FHV-cat model, and the immunogenicity data in the vaccinated seals, the gB based vaccine is expected to be safe and effective in protecting against PhHV-1 related disease in harbour seals.

Immunohistochemical investigation of the cross-reactivity of selected cell markers from various species for characterization of lymphatic tissues in the harbour porpoise (Phocoena phocoena).

To facilitate a detailed investigation of cetacean lymphoid organs, 13 canine-, six bovine-, one equine-, one human- and four killer whale-specific monoclonal antibodies (mAbs) directed against cell surface antigens of the haematopoietic system (including CD2, CD4, CD8, CD45R, MHC class II, granulocyte, thrombocyte, pan-T cell and B-cell antigen), as well as a mAb and a polyclonal antibody (pAb) directed against the -peptide of the human CD3 complex, were tested for immunohistochemical cross-reactivity on frozen or formalin-fixed, paraffin wax-embedded lymphatic tissues of harbour porpoises. Eight of 26 mAbs and the pAb showed a specific reaction with harbour porpoise cells. Lymphocytes in T-cell compartments were labelled by the mAb and the pAb directed against the CD3 complex and by two killer whale mAbs specific for CD2 antigen. CD45R, labelled by a killer whale-specific mAb, was strongly expressed on B and weakly on T cells. MHC class II antigen, recognized by killer whale- and bovine-specific mAbs, was expressed on B and T cells. A canine MHC class II-specific mAb recognized an epitope on the surface of antigen-presenting cells and B lymphocytes. An anti-equine-pan-leucocyte marker labelled the majority of cells in B- and T-cell compartments. Thus, with leucocyte antigen markers from various species, it is now possible to determine the phenotype of lymphocytes in normal and diseased lymphoid organs of harbour porpoises.