Microsporum gypseum Isolated from Ailuropoda melanoleuca Provokes Inflammation and Triggers Th17 Adaptive Immunity Response.

Microsporum gypseum causes dermatomycoses in giant pandas (Ailuropoda melanoleuca). This study aimed to investigate the immune response of M. gypseum following deep infection. The degree of damage to the heart, liver, spleen, lungs, and kidneys was evaluated using tissue fungal load, organ index, and histopathological methods. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detected the mRNA expression of receptors and cytokines in the lung, and immunofluorescence staining and flow cytometry, were used to assess immune cells in the lung. The results indicated that conidia mainly colonized the lungs and caused serious injury with M. gypseum infection. Furthermore, dectin-1, TLR-2, and TLR-4 played a role in recognizing M. gypseum cells. Numerous inflammatory cells, mainly macrophages, dendritic cells, polymorphonuclear neutrophils, and inflammatory cytokines (TGF-ß, TNF-α, IL-1ß, IL-6, IL-10, IL-12, and IL-23), were activated in the early stages of infection. With the high expression of IL-22, IL-17A, and IL-17F, the Th17 pathway exerted an adaptive immune response to M. gypseum infection. These results can potentially aid in the diagnosis and treatment of diseases caused by M. gypseum in giant pandas.

Blood transcriptome analysis revealed the immune changes and immunological adaptation of wildness training giant pandas.

The giant panda (Ailuropoda melanoleuca) is a global flagship species for biodiversity conservation. As the time for captive giant pandas to be released into the wild matures, wildness training is provided to allow adaptation to their natural environment. It is assumed that changes in the immune system would be integral in this adaptation from captive to wild, where many more pathogens would be encountered in their natural habitats. Therefore, this study aims to determine the expression changes of immune-related genes and their potential as immunoassay markers for adaptation monitoring in wildness training giant pandas, and then to understand the adaptation strategy of wildness training giant pandas to the wild environment, thereby improving the success rate of panda reintroduction. We obtained 300 differentially expressed genes (DEGs) by RNA-seq, with 239 up-regulated and 61 down-regulated DEGs in wildness training giant pandas compared to captive pandas. Functional enrichment analysis indicated that up-regulated DEGs were enriched in several immune-related terms and pathways. There were 21 immune-related DEGs, in which most of them were up-regulated in wildness training giant pandas, including several critical innate and cellular immune genes. IL1R2 was the most significantly up-regulated gene and is a signature of homeostasis within the immune system. In the protein-protein interaction (PPI) analysis, CXCL8, CXCL10, and CCL5 were identified as the hub immune genes. Our results suggested that wildness training giant pandas have stronger innate and cellular immunity than captive giant pandas, and we proposed that a gene set of CXCL8, CXCL10, CCL5, CD3D, NFKBIA, TBX21, IL12RB2, and IL1R2 may serve as potential immunoassay markers to monitor and assess the immune status of wildness training giant pandas. Our study offers the first insight into immune alterations of wildness training giant pandas, paving the way for monitoring and evaluating the immune status of giant pandas when reintroducing them into the wild.

Potential natural immunization against atherosclerosis in hibernating bears.

Brown bears (Ursus arctos) hibernate for 5-6 months during winter, but despite kidney insufficiency, dyslipidemia and inactivity they do not seem to develop atherosclerosis or cardiovascular disease (CVD). IgM antibodies against phosphorylcholine (anti-PC) and malondialdehyde (anti-MDA) are associated with less atherosclerosis, CVD and mortality in uremia in humans and have anti-inflammatory and other potentially protective properties. PC but not MDA is exposed on different types of microorganisms. We determine anti-PC and anti-MDA in brown bears in summer and winter. Paired serum samples from 12 free ranging Swedish brown bears were collected during hibernation in winter and during active state in summer and analyzed for IgM, IgG, IgG1/2 and IgA anti-PC and anti-MDA by enzyme linked immunosorbent assay (ELISA). When determined as arbitrary units (median set at 100 for summer samples), significantly raised levels were observed in winter for anti-PC subclasses and isotypes, and for IgA anti-PC the difference was striking; 100 IQR (85.9-107.9) vs 782.3, IQR (422.8-1586.0; p < 0.001). In contrast, subclasses and isotypes of anti-MDA were significantly lower in winter except IgA anti-MDA, which was not detectable. Anti-PCs are significantly raised during hibernation in brown bears; especially IgA anti-PC was strikingly high. In contrast, anti-MDA titers was decreased during hibernation. Our observation may represent natural immunization with microorganisms during a vulnerable period and could have therapeutic implications for prevention of atherosclerosis.

Expression of Paracrine Effectors in Human Adipose-Derived Mesenchymal Stem Cells Treated With Plasma From Brown Bears (Ursus arctos).

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates for novel cell therapeutic applications. Hibernating brown bears sustain tissue integrity and function via unknown mechanisms, which might be plasma borne. We hypothesized that plasma from hibernating bears may increase the expression of favorable factors from human ADSCs. In an experimental study, ADSCs from patients with ischemic heart disease were treated with interventional media containing plasma from hibernating and active bears, respectively, and with control medium. Extracted RNA from the ADSCs was sequenced using next generation sequencing. Statistical analyses of differentially expressed genes were performed using fold change analysis, pathway analysis, and gene ontology. As a result, we found that genes associated with inflammation, such as IGF1, PGF, IL11, and TGFA, were downregulated by > 10-fold in ADSCs treated with winter plasma compared with control. Genes important for cardiovascular development, ADM, ANGPTL4, and APOL3, were upregulated in ADSCs when treated with winter plasma compared with summer plasma. ADSCs treated with bear plasma, regardless if it was from hibernating or active bears, showed downregulation of IGF1, PGF, IL11, INHBA, IER3, and HMOX1 compared with control, suggesting reduced cell growth and differentiation. This can be summarized in the conclusion that plasma from hibernating bears suppresses inflammatory genes and activates genes associated with cardiovascular development in human ADSCs. Identifying the involved regulator(s) holds therapeutic potential.

The immune and metabolic changes with age in giant panda blood by combined transcriptome and DNA methylation analysis.

Giant panda (Ailuropoda melanoleuca) is an endangered mammalian species. Exploring immune and metabolic changes that occur in giant pandas with age is important for their protection. In this study, we systematically investigated the physiological and biochemical indicators in blood, as well as the transcriptome, and methylation profiles of young, adult, and old giant pandas. The white blood cell (WBC), neutrophil (NEU) counts and hemoglobin (HGB) concentrations increased significantly with age (young to adult), and some indicators related to blood glucose and lipids also changed significantly with age. In the transcriptome analysis, differentially expressed genes (DEGs) were found in comparisons of the young and adult (257), adult and old (20), young and old (744) groups. Separation of the DEGs into eight profiles according to the expression trend using short time-series expression miner (STEM) software revealed that most DEGs were downregulated with age. Functional analysis showed that most DEGs were associated with disease and that these DEGs were also associated with the immune system and metabolism. Furthermore, gene methylation in giant pandas decreased globally with age, and the expression of CCNE1, CD79A, IL1R1, and TCF7 showed a highly negative correlation with their degree of methylation. These results indicate that the giant panda's immune function improves gradually with age (young to adult), and that changes in the methylation profile are involved in the effects of age on immune and metabolic functions. These results have important implications for the understanding and conservation of giant pandas.

Transcriptome analyses provide insights into maternal immune changes at several critical phases of giant panda reproduction.

Giant pandas (Ailuropoda melanoleuca) possess highly specialized reproductive characteristics, but the maternal immune changes during reproduction are largely unclear. Here, 20 blood transcriptomes were used to determine immune changes at four key phases of giant panda reproduction, and a total of 4640 differential expression genes were identified. During estrus, six immune-related genes (TLR4, IL1B, SYK, SPI1, CD80, and ITK) were identified as hub genes. The up-regulation of the TLR family genes (TLR4, TLR5, TLR6, and TLR8) and inflammatory response related genes (IL1B) may reflect innate immune enhancement and local tissue remodeling events, while the up-regulation of SYK and SPI1, and the down-regulation of CD80 and ITK suggested that the enhanced humoral immunity and inhibited cellular immunity of female giant pandas during estrus. During early pregnancy, antigen presentation related genes and proinflammatory cytokine (IL1B) were down-regulated. This may indicate that partial immune functions were suppressed in early pregnancy to achieve immune tolerance, including reducing inflammatory to protect embryos. By the late pregnancy, the antiviral related genes were up-regulated to strengthen defenses against external pathogen infection. KLRK1, which acts as a primary activation receptor for NK cells, was down regulated in estrus and pregnancy, suggesting that the activities of NK cells were inhibited, and KLRK1 may play a key role in the regulation the activities of pbNK cells during reproduction of giant pandas. Our results showed that there was no significant immune change in lactating females (post-natal 2 months) compared to anestrus females. This is the first time to observe the immune changes of giant panda during the breeding period and our data is expected to provide valuable resources for further studies on reproductive immunology of giant pandas.

Epitope Mapping of PMab-241, a Lymphatic Endothelial Cell-Specific Anti-Bear Podoplanin Monoclonal Antibody.

Anti-bear podoplanin (bPDPN) monoclonal antibodies (mAbs), including PMab-247 and PMab-241, have been previously established. Although PMab-247 has shown positive immunostaining for lymphatic endothelial cells (LECs), type I alveolar cells of the lung, and podocytes of the kidney, PMab-241 stains LECs but does not react with lung type I alveolar cells. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and the PLAG-like domain (PLD). The binding epitope of PMab-247 was previously determined to include bPDPN residues Asp76, Arg78, Glu80, and Arg82. Among these, Glu80 and Arg82 are included in PLD of bPDPN. The purpose of this study is to determine the binding epitope of PMab-241 and to clarify the difference between these two anti-bPDPN mAbs. Analysis of bPDPN deletion mutants revealed that the N-terminus of the PMab-241 epitope exists between amino acids (aa) 75 and 80 of bPDPN. In addition, analysis of bPDPN point mutants demonstrated that the critical epitope of PMab-241 includes Thr75, Asp76, and Arg78 of bPDPN. The binding epitopes of PMab-241 and PMab-247 seem to overlap, but this slight difference may be sufficient to provide the specificity of PMab-241 to discriminate LECs from type I alveolar cells of the lung.

First demonstration of giant panda's immune response to canine distemper vaccine.

The Canine Distemper Virus (CDV) is a high fatal virus to the giant panda (Ailuropoda melanoleuca), where CDV vaccination is a key preventative measure in captive giant pandas. However, the immune response of giant pandas to CDV vaccination has been little studied. In this study, we investigated the blood transcriptome expression profiles of five giant panda cubs after three inoculations, 21 days apart. Blood samples were collected before vaccination (0 Day), and 24 h after each of the three inoculations; defined here as 1 Day, 21  Day, and 42  Day. Compared to 0 Day, we obtained 1262 differentially expressed genes (DEGs) during inoculations. GO and KEGG pathways enrichment analysis of these DEGs found 222 GO terms and 40 pathways. The maximum immune-related terms were enriched by DEGs from comparisons of 21  Day and 0 Day. In the PPI analysis, we identified RSAD2, IL18, ISG15 immune-related hub genes from 1 Day and 21 Day comparison. Compared to 0 Day, innate immune-related genes, TLR4 and TLR8, were up-regulated at 1 Day, and the expressions of IRF1, RSAD2, MX1, and OAS2 were highest at 21  Day. Of the adaptive immune-related genes, IL15, promoting T cell differentiation into CD8+T cells, was up-regulated after the first two inoculations, IL12ß, promoting T cell differentiation into memory cells, and IL10, promoting B cell proliferation and differentiation, were down-regulated during three inoculations. Our results indicated that the immune response of five giant panda cubs was strongest after the second inoculation, most likely protected against CDV infection through innate immunity and T cells, but did not produce enough memory cells to maintain long-term immunity after CDV vaccination.

PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Transcriptome analysis reveals immune-related gene expression changes with age in giant panda (Ailuropoda melanoleuca) blood.

The giant panda (Ailuropoda melanoleuca), an endangered species endemic to western China, has long been threatened with extinction that is exacerbated by highly contagious and fatal diseases. Aging is the most well-defined risk factor for diseases and is associated with a decline in immune function leading to increased susceptibility to infection and reduced response to vaccination. Therefore, this study aimed to determine which genes and pathways show differential expression with age in blood tissues. We obtained 210 differentially expressed genes by RNA-seq, including 146 up-regulated and 64 down-regulated genes in old pandas (18-21yrs) compared to young pandas (2-6yrs). We identified ISG15, STAT1, IRF7 and DDX58 as the hub genes in the protein-protein interaction network. All of these genes were up-regulated with age and played important roles in response to pathogen invasion. Functional enrichment analysis indicated that up-regulated genes were mainly involved in innate immune response, while the down-regulated genes were mainly related to B cell activation. These may suggest that the innate immunity is relatively well preserved to compensate for the decline in the adaptive immune function. In conclusion, our findings will provide a foundation for future studies on the molecular mechanisms underlying immune changes associated with ageing.

Heightened Immune System Function in Polar Bears Using Terrestrial Habitats.

Climate change is altering the distribution of some wildlife species while warming temperatures are facilitating the northward expansion of pathogens, potentially increasing disease risk. Melting of Arctic sea ice is increasingly causing polar bears (Ursus maritimus) of the southern Beaufort Sea (SBS) to spend summer on land, where they may encounter novel pathogens. Here, we tested whether SBS polar bears on shore during summer exhibited greater immune system activity than bears remaining on the sea ice. In addition, we tested whether the type of immune response correlated with body condition, because adaptive responses (slowly developing defenses against specific pathogens) often require less energy than innate responses (rapid defenses not based on pathogen identity). After accounting for body condition, we found that polar bears on shore exhibited higher total white blood cell counts, neutrophils, and monocytes than bears on the ice, suggesting more infections. Lymphocytes, eosinophils, basophils, and globulins did not differ. C-reactive protein, an indicator of inflammation, also did not differ between habitats. Body condition was associated with variables indicative of both innate and adaptive immunity, suggesting that neither response was uniquely limited by energy resources. Our data indicate that as more polar bears spend longer periods of time on shore, they may experience more infections. We encourage continued health monitoring of this species and studies of the long-term fitness consequences from disease.

Immune function in arctic mammals: Natural killer (NK) cell-like activity in polar bear, muskox and reindeer.

Natural killer (NK) cells are a vital part of the rapid and non-specific immune defense against invading pathogens and tumor cells. This study evaluated NK cell-like activity by flow cytometry for the first time in three ecologically and culturally important Arctic mammal species: polar bear (Ursus maritimus), muskox (Ovibos moschatus) and reindeer (Rangifer tarandus). NK cell-like activity for all three species was most effective against the mouse lymphoma cell line YAC-1, compared to the human leukemia cell line K562; NK cell response displayed the characteristic increase in cytotoxic activity when the effector:target cell ratio increased. Comparing NK activity between fresh and cryopreserved mouse lymphocytes revealed little to no difference in function, highlighting the applicability of cryopreserving cells in field studies. The evaluation of this important innate immune function in Arctic mammals can contribute to future population health assessments, especially as pollution-induced suppression of immune function may increase infectious disease susceptibility.

Effects of Polar Bear and Killer Whale Derived Contaminant Cocktails on Marine Mammal Immunity.

Most controlled toxicity studies use single chemical exposures that do not represent the real world situation of complex mixtures of known and unknown natural and anthropogenic substances. In the present study, complex contaminant cocktails derived from the blubber of polar bears (PB; Ursus maritimus) and killer whales (KW; Orcinus orca) were used for in vitro concentration-response experiments with PB, cetacean and seal spp. immune cells to evaluate the effect of realistic contaminant mixtures on various immune functions. Cytotoxic effects of the PB cocktail occurred at lower concentrations than the KW cocktail (1 vs 16 µg/mL), likely due to differences in contaminant profiles in the mixtures derived from the adipose of each species. Similarly, significant reduction of lymphocyte proliferation occurred at much lower exposures in the PB cocktail (EC50: 0.94 vs 6.06 µg/mL; P < 0.01), whereas the KW cocktail caused a much faster decline in proliferation (slope: 2.9 vs 1.7; P = 0.04). Only the KW cocktail modulated natural killer (NK) cell activity and neutrophil and monocyte phagocytosis in a concentration- and species-dependent manner. No clear sensitivity differences emerged when comparing cetaceans, seals and PB. Our results showing lower effect levels for complex mixtures relative to single compounds suggest that previous risk assessments underestimate the effects of real world contaminant exposure on immunity. Our results using blubber-derived contaminant cocktails add realism to in vitro exposure experiments and confirm the immunotoxic risk marine mammals face from exposure to complex mixtures of environmental contaminants.

Demographic and temporal variations in immunity and condition of polar bears (Ursus maritimus) from the southern Beaufort Sea.

Assessing the health and condition of animals in their natural environment can be problematic. Many physiological metrics, including immunity, are highly influenced by specific context and recent events to which researchers may be unaware. Thus, using a multifaceted physiological approach and a context-specific analysis encompassing multiple time scales can be highly informative. Ecoimmunological tools in particular can provide important indications to the health of animals in the wild. We collected blood and hair samples from free-ranging polar bears (Ursus maritimus) in the southern Beaufort Sea and examined the influence of sex, age, and reproductive status on metrics of immunity, stress, and body condition during 2013-2015. We examined metrics of innate immunity (bactericidal ability and lysis) and stress (hair cortisol, reactive oxygen species, and oxidative barrier), in relation to indices of body condition considered to be short term (urea to creatinine ratio; UC ratio) and long term (storage energy and body mass index). We found the factors of sex, age, and reproductive status of the bear were critical for interpreting different physiological metrics. Additionally, the metrics of body condition were important predictors for stress indicators. Finally, many of these metrics differed between years, illustrating the need to examine populations on a longer time scale. Taken together, this study demonstrates the complex relationship between multiple facets of physiology and how interpretation requires us to examine individuals within a specific context.

Cloning and characterization of giant panda (Ailuropoda melanoleuca) IL-18 binding protein.

The giant panda (Ailuropoda melanoleuca) is an endangered species. Interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses by inducing IFN-γ. IL-18 has been implicated in the pathogenesis of various diseases. IL-18 binding protein (IL-18BP) is an intrinsic inhibitor of IL-18 that possesses higher affinity to IL-18. In this study, we cloned and characterized IL-18BP in giant panda (AmIL-18BP) from the spleen. The amino acid sequence of giant panda IL-18BP ORF shared about 65% identities with other species. To evaluate the effects of AmIL-18BP on the immune responses, we expressed the recombinant AmIL-18BP in Escherichia coli BL21 (DE3).The fusing protein PET-AmIL-18BP was purified by nickel affinity column chromatography. The biological function of purified PET-AmIL-18BP was determined on mice splenocyte by qRT-PCR. The results showed that AmIL-18BP was functional and could significantly reduce IFN-γ production in murine splenocytes. These results will facilitate the study of protecting giant panda on etiology and immunology.

Immunotoxic effects of environmental pollutants in marine mammals.

Due to their marine ecology and life-history, marine mammals accumulate some of the highest levels of environmental contaminants of all wildlife. Given the increasing prevalence and severity of diseases in marine wildlife, it is imperative to understand how pollutants affect the immune system and consequently disease susceptibility. Advancements and adaptations of analytical techniques have facilitated marine mammal immunotoxicology research. Field studies, captive-feeding experiments and in vitro laboratory studies with marine mammals have associated exposure to environmental pollutants, most notable polychlorinated biphenyls (PCBs), organochlorine pesticides and heavy metals, to alterations of both the innate and adaptive arms of immune systems, which include aspects of cellular and humoral immunity. For marine mammals, reported immunotoxicology endpoints fell into several major categories: immune tissue histopathology, haematology/circulating immune cell populations, functional immune assays (lymphocyte proliferation, phagocytosis, respiratory burst, and natural killer cell activity), immunoglobulin production, and cytokine gene expression. Lymphocyte proliferation is by far the most commonly used immune assay, with studies using different organic pollutants and metals predominantly reporting immunosuppressive effects despite the many differences in study design and animal life history. Using combined field and laboratory data, we determined effect threshold levels for suppression of lymphocyte proliferation to be between b0.001-10 ppm for PCBs, 0.002-1.3 ppm for Hg, 0.009-0.06 for MeHg, and 0.1-2.4 for cadmium in polar bears and several pinniped and cetacean species. Similarly, thresholds for suppression of phagocytosis were 0.6-1.4 and 0.08-1.9 ppm for PCBs and mercury, respectively. Although data are lacking for many important immune endpoints and mechanisms of specific immune alterations are not well understood, this review revealed a systemic suppression of immune function in marine mammals exposed to environmental contaminants. Exposure to immunotoxic contaminants may have significant population level consequences as a contributing factor to increasing anthropogenic stress in wildlife and infectious disease outbreaks.

ANTIDOG IgG SECONDARY ANTIBODY SUCCESSFULLY DETECTS IgG IN A VARIETY OF AQUATIC MAMMALS.

Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter ( Enhydra lutris ), polar bear ( Ursus maritimus ), grey seal ( Halichoerus grypus ), harbor seal ( Phoca vitulina ), northern elephant seal ( Mirounga angustirostris ), California sea lion ( Zalophus californianus ), Pacific walrus ( Odobenus rosmarus ) and one freshwater mammal: Asian small-clawed otter ( Aonyx cinerea ). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

Molecular cloning, characterization, and bioactivity analysis of interleukin 18 in giant panda (Ailuropoda melanoleuca).

Interleukin 18 (IL-18), as a member of IL-1 superfamily, is an important pleiotropic cytokine that modulates Th1 immune responses. In this report, we cloned and identified a homolog of IL-18 in giant panda (Ailuropoda melanoleuca) (designated as AmIL-18) from peripheral blood mononuclear cells stimulated with lipopolysaccharide. The open readin g frame of AmIL-18 cDNA is 579 bp encoding a deduced protein of 192 amino acids. AmIL-18 gDNA fragments contained 5 exons and 4 introns. The amino acid sequence of AmIL-18 shared 23.9 to 87.0% identity with other species. To evaluate the effects of AmIL-18 on the immune response, we expressed the recombinant AmIL-18 in Escherichia coli BL21 (DE3). The fusion protein PET-AmIL-18 was purified by nickel affinity column chromatography and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The biological function of purified PET-AmIL-18 was determined on mouse splenocytes by quantitative real-time polymerase chain reaction. INF-γ and other cytokines were increased when stimulated by PET-AmIL-18, particularly when combined with recombinant human interleukin 12, while a Th2-type cytokine, interleukin-4, was strikingly suppressed. These results will provide information for the potential use of recombinant proteins to manipulate the immune response in giant pandas and facilitate the study to protect this treasured species.

No evidence for the effect of MHC on male mating success in the brown bear.

Mate choice is thought to contribute to the maintenance of the spectacularly high polymorphism of the Major Histocompatibility Complex (MHC) genes, along with balancing selection from parasites, but the relative contribution of the former mechanism is debated. Here, we investigated the association between male MHC genotype and mating success in the brown bear. We analysed fragments of sequences coding for the peptide-binding region of the highly polymorphic MHC class I and class II DRB genes, while controlling for genome-wide effects using a panel of 18 microsatellite markers. Male mating success did not depend on the number of alleles shared with the female or amino-acid distance between potential mates at either locus. Furthermore, we found no indication of female mating preferences for MHC similarity being contingent on the number of alleles the females carried. Finally, we found no significant association between the number of MHC alleles a male carried and his mating success. Thus, our results provided no support for the role of mate choice in shaping MHC polymorphism in the brown bear.

MHC class II DQB diversity in the Japanese black bear, Ursus thibetanus japonicus.

BACKGROUND: The major histocompatibility complex (MHC) genes are one of the most important genetic systems in the vertebrate immune response. The diversity of MHC genes may directly influence the survival of individuals against infectious disease. However, there has been no investigation of MHC diversity in the Asiatic black bear (Ursus thibetanus). Here, we analyzed 270-bp nucleotide sequences of the entire exon 2 region of the MHC DQB gene by using 188 samples from the Japanese black bear (Ursus thibetanus japonicus) from 12 local populations. RESULTS: Among 185 of 188 samples, we identified 44 MHC variants that encoded 31 different amino acid sequences (allotypes) and one putative pseudogene. The phylogenetic analysis suggests that MHC variants detected from the Japanese black bear are derived from the DQB locus. One of the 31 DQB allotypes, Urth-DQB*01, was found to be common to all local populations. Moreover, this allotype was shared between the black bear on the Asian continent and the Japanese black bear, suggesting that Urth-DQB*01 might have been maintained in the ancestral black bear population for at least 300,000 years. Our findings, from calculating the ratio of non-synonymous to synonymous substitutions, indicate that balancing selection has maintained genetic variation of peptide-binding residues at the DQB locus of the Japanese black bear. From examination of genotype frequencies among local populations, we observed a considerably lower level of observed heterozygosity than expected. CONCLUSIONS: The low level of observed heterozygosity suggests that genetic drift reduced DQB diversity in the Japanese black bear due to a bottleneck event at the population or species level. The decline of DQB diversity might have been accelerated by the loss of rare variants that have been maintained by negative frequency-dependent selection. Nevertheless, DQB diversity of the black bear appears to be relatively high compared with some other endangered mammalian species. This result suggests that the Japanese black bears may also retain more potential resistance against pathogens than other endangered mammalian species. To prevent further decline of potential resistance against pathogens, a conservation policy for the Japanese black bear should be designed to maintain MHC rare variants in each local population.