Emergence, Evolution, and Pathogenicity of Influenza A(H7N4) Virus in Shorebirds in China.

A 2-year surveillance study of influenza A viruses in migratory birds was conducted to understand the subsequent risk during the migratory seasons in Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China, a major stopover site on the East Asian-Australasian flyway. Overall, we isolated 27 influenza A viruses with multiple subtypes, including H3N8 (n = 2), H4N6 (n = 2), H4N7 (n = 2), H7N4 (n = 9), H7N7 (n = 1), H10N7 (n = 7), and H13N6 (n = 4). Particularly, a novel reassortant influenza A(H7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, posing a serious threat to public health. Here, we describe the genetic characterization and pathogenicity of the nine influenza A(H7N4) isolates. Phylogenetic analysis indicated that complex viral gene flow occurred among Asian countries. We also demonstrated a similar evolutionary trajectory of the surface genes of the A(H7N4) isolates and Jiangsu human-related A(H7N4) viruses. Our A(H7N4) isolates exhibited differing degrees of virulence in mice, suggesting a potential risk to other mammalian species, including humans. We revealed multiple mutations that might affect viral virulence in mice. Our report highlights the importance and need for the long-term surveillance of avian influenza virus in migratory birds combined with domestic poultry surveillance along migratory routes and flyways and, thereby, the development of measures to manage potential health threats. IMPORTANCE The H7 subtype avian influenza viruses, such as H7N2, H7N3, H7N4, H7N7, and H7N9, were documented as being capable of infecting humans, and the H7 subtype low pathogenicity avian influenza viruses are capable of mutating into highly pathogenic avian influenza; therefore, they pose a serious threat to public health. Here, we investigated the evolutionary history, molecular characteristics, and pathogenicity of shorebird-origin influenza A(H7N4) viruses, showing a similar evolutionary trajectory with Jiangsu human A(H7N4) viruses in HA and NA genes. Moreover, our isolates exhibited variable virulence (including moderate virulence) in mice, suggesting a potential risk to other mammalian species, including humans.

Historical records reveal the distinctive associations of human disturbance and extreme climate change with local extinction of mammals.

Accelerated anthropogenic impacts and climatic changes are widely considered to be responsible for unprecedented species extinction. However, determining their effects on extinction is challenging owing to the lack of long-term data with high spatial and temporal resolution. In this study, using historical occurrence records of 11 medium- to large-sized mammal species or groups of species in China from 905 BC to AD 2006, we quantified the distinctive associations of anthropogenic stressors (represented by cropland coverage and human population density) and climatic stressors (represented by air temperature) with the local extinction of these mammals. We found that both intensified human disturbances and extreme climate change were associated with the increased local extinction of the study mammals. In the cold phase (the premodern period of China), climate cooling was positively associated with increased local extinction, while in the warm phase (the modern period) global warming was associated with increased local extinction. Interactive effects between human disturbance and temperature change with the local extinction of elephants, rhinos, pandas, and water deer were found. Large-sized mammals, such as elephants, rhinos, and pandas, showed earlier and larger population declines than small-sized ones. The local extinction sensitivities of these mammals to the human population density and standardized temperature were estimated during 1700 to 2000. The quantitative evidence for anthropogenic and climatic associations with mammalian extinction provided insights into the driving processes of species extinction, which has important implications for biodiversity conservation under accelerating global changes.

Metagenomic analysis revealed the effects of goat milk feeding and breast feeding on the gut microbiome of Amur tiger cubs.

BACKGROUND: Ingredients in breast milk can help establish a healthy community of microorganisms in the infant gut, but no research exists regarding the effects of goat milk feeding and breast feeding on the gut microbiome of the Amur tiger, which is one of the most endangered species in the world. METHODS: In this study, we used whole-metagenome shotgun sequencing to analyze the effects of two different feeding patterns, goat milk feeding and breast feeding, on the composition and functional structures of gut microbiota in Amur tiger cubs. RESULTS: Goat milk-fed cubs have fewer beneficial bacteria and more pathogenic bacteria and a higher microbial diversity in their gut than breastfed cubs. A total of 15 genera showed statistically significant differences; the relative abundances of Streptomyces scabiei, Streptomyces avermitilis and Streptomyces davawensis were significantly decreased, whereas those of Niabella soli, Aeromonas media and Brochothrix thermosphacta were significantly increased in the goat milk-fed group compared with those in the breastfed group. At the functional level, carbohydrate metabolism, translation and replication and repair decreased, and amino acid metabolism, membrane transport and metabolism of cofactors and vitamins increased in the gut microbiota of goat milk-fed cubs compared with breastfed cubs. CONCLUSION: Our results indicate for the first time that the different milk feeding patterns of goat milk feeding and breast feeding can change the composition and functional structures of gut microbiota in Amur tiger cubs and that breastfed tiger cubs and goat milk-fed tiger cubs have distinct microbiotas in their guts.

Variations in gut microbiota and fecal metabolic phenotype associated with Fenbendazole and Ivermectin Tablets by 16S rRNA gene sequencing and LC/MS-based metabolomics in Amur tiger.

BACKGROUND: The Amur tiger is one of the most endangered species in the world, and the healthy population of captive Amur tigers assists the recovery of the wild population. Gut microbes have been shown to be important for human disease and health, but little research exists regarding the microbiome of Amur tigers in captivity. METHODS: In this study, we used an integrated approach of 16S rRNA gene sequencing combined with ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics to analyze the effects of Fenbendazole and Ivermectin Tablets on the gut microbiota and fecal metabolic phenotype of the Amur tiger. RESULTS: The relative abundances of the bacterial genera Collinsella, Clostridium XI and Megamonas were decreased, whereas those of Escherichia and Clostridium sensu stricto were increased in experimental Amur tigers compared with those in normal controls. Meanwhile, distinct changes in the fecal metabolic phenotype of the experimental Amur tigers were also found, including lower levels of acrylic acid, acetoacetate and catechol and higher amounts of 5,6-dihydrouracil, adenine hydrochloride hydrate and galactitol. Moreover, the differentially abundant gut microbes were substantially associated with the altered fecal metabolites, especially the bacteria in the Firmicutes and Actinomycetes, which were involved in the metabolism of 5,6-dihydrouracil, 6-phospho-d-gluconate and 1-methylnicotinamide. CONCLUSION: Our results indicate for the first time that Fenbendazole and Ivermectin Tablets not only disturb the gut microbiota at the abundance level but also alter the metabolic homeostasis of the Amur tiger.

Metagenomic analysis of captive Amur tiger faecal microbiome.

BACKGROUND: The gastrointestinal tracts of animals are home to large, complex communities of microbes. The compositions of these communities ultimately reflect the coevolution of microorganisms with their animal host and are influenced by the living environment, diet and immune status of the host. Gut microbes have been shown to be important for human disease and health, but little research exists in the gut microbiome of the Amur tiger, which is one of the most endangered species in the world. RESULTS: In this study, we present the use of whole-metagenome shotgun sequencing to analyze the composition and functional structures of the gut microbiota in captive Amur tigers. Our results showed a high abundance of four major phyla in captive Amur tigers, including Proteobacteria, Firmicutes, Actinobacteria and Fusobacteria. Moreover, at the genus level, Escherichia, Collinsella and Fusobacterium were most abundant in the captive Amur tiger fecal metagenome. At the species level, Escherichia coli, Fusobacterium ulcerans and Fusobacterium varium were the species with highest abundances in the captive Amur tiger gut microbiota. The primary functional categories of the Amur tiger faecal metagenome were associated mainly with Carbohydrate metabolism, Membrane transport and Amino acid metabolism based on the KEGG pathway database. The comparative metagenomic analyses showed that the captive Amur tiger fecal metagenome had a lower abundance of Spirochaetes, Cyanobacteria and Ascomycota than other animals, and the primary functional categories were primarily associated with carbohydrate metabolism subsystems, clustering-based subsystems and protein metabolism. CONCLUSIONS: We presented here for the first time the use of the shotgun metagenomic sequencing approach to study the composition and functional structures of the gut microbiota in captive Amur tiger.

Clues for two-step virion infectivity factor regulation by core binding factor beta.

Lentiviruses threaten human and animal health. Virion infectivity factor (Vif) is essential for the infectivity of most lentiviruses, except for the equine infectious anaemia virus (EIAV). Vif promotes viral infectivity by recruiting a Cullin-based E3 ligase to induce the degradation of a class of host restriction factors, named APOBEC3. Core binding factor beta (CBF-ß) is necessary for several primate lentiviral Vif functions, including HIV-1 Vif. Although much progress has been made in understanding the contribution of CBF-ß to Vif function, the precise mechanism has not yet been fully elucidated. In this study, we found that an interaction with CBF-ß altered the oligomerization and subcellular distribution pattern and increased the stability of two primate lentiviral Vifs, HIV-1 Vif and Macaca simian immunodeficiency virus (SIVmac) Vif. Moreover, using a CBF-ß loss-of-function mutant, we demonstrated that the interaction between CBF-ß and Vif was not sufficient for Vif assistance; a region including F68 in CBF-ß was also required for the stability and function of Vif. For the first time, this study separates the binding and regulating processes of CBF-ß when it is promoting Vif function, which further extends our understanding of the biochemical regulation of Vif by CBF-ß.

De novo transcriptomic analysis and development of EST-SSR markers in the Siberian tiger (Panthera tigris altaica).

The Siberian tiger, Panthera tigris altaica, is an endangered species, and much more work is needed to protect this species, which is still vulnerable to extinction. Conservation efforts may be supported by the genetic assessment of wild populations, for which highly specific microsatellite markers are required. However, only a limited amount of genetic sequence data is available for this species. To identify the genes involved in the lung transcriptome and to develop additional simple sequence repeat (SSR) markers for the Siberian tiger, we used high-throughput RNA-Seq to characterize the Siberian tiger transcriptome in lung tissue (designated 'PTA-lung') and a pooled tissue sample (designated 'PTA'). Approximately 47.5 % (33,187/69,836) of the lung transcriptome was annotated in four public databases (Nr, Swiss-Prot, KEGG, and COG). The annotated genes formed a potential pool for gene identification in the tiger. An analysis of the genes differentially expressed in the PTA lung, and PTA samples revealed that the tiger may have suffered a series of diseases before death. In total, 1062 non-redundant SSRs were identified in the Siberian tiger transcriptome. Forty-three primer pairs were randomly selected for amplification reactions, and 26 of the 43 pairs were also used to evaluate the levels of genetic polymorphism. Fourteen primer pairs (32.56 %) amplified products that were polymorphic in size in P. tigris altaica. In conclusion, the transcriptome sequences will provide a valuable genomic resource for genetic research, and these new SSR markers comprise a reasonable number of loci for the genetic analysis of wild and captive populations of P. tigris altaica.

Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

Multiple lysines combined in HIV-1 Vif determines the responsiveness to CBF-ß.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type-1 (HIV-1) is critical for HIV-1 infectivity. CBF-ß is required for HIV-1 Vif function, as it increases the steady-state level of the HIV-1 Vif protein to promote host restriction factor APOBEC3 degradation. However, the precise mechanism by which CBF-ß promotes HIV-1 Vif levels remains unclear. In the present study, we provided evidences that CBF-ß promoted steady-state levels of HIV-1 Vif by inhibiting the degradation of HIV-1 Vif through the proteasome pathway. Our results reveal a new mechanism by which a cellular protein supports viral infectivity by inhibiting viral protein degradation.

Core-binding factor subunit beta is not required for non-primate lentiviral Vif-mediated APOBEC3 degradation.

Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-ß) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-ß is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-ß. In addition, CBF-ß did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-ß silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. Importance: The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-ß was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-ß knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-ß to degrade APOBEC3. CBF-ß did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vif-CBF-ß interaction.

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities.

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (-) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB- oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

Phylogenetic and pathogenic analysis of a novel H6N2 avian influenza virus isolated from a green peafowl in a wildlife park.

H6 subtype avian influenza virus, which has been circulating among different species, causes considerable concern for both veterinary medicine and public health. We isolated a strain of H6N2 avian influenza virus from healthy green peafowl (Pavo muticus) in Qinghuangdao Wildlife Park in Hebei Province, China, in 2012. A phylogenetic analysis indicated that the isolated H6N2 strain had the same gene constellation as southern China strains, which were predominantly isolated from waterfowl distributed in Shantou, Guangxi, and Hunan in 2001-2010. The isolate showed no and low pathogenicity in chickens and ducks, respectively. However, it replicated efficiently in the lungs and turbinate of infected mice, resulting in thickened alveolar septa and moderate interstitial pneumonia. This finding raises concerns that the H6N2 subtype maybe evolve into a novel endemic avian influenza virus. Therefore, periodical surveillance of avian influenza viruses must be undertaken to monitor the advent of novel viruses.

Construction and analysis of Siberian tiger bacterial artificial chromosome library with approximately 6.5-fold genome equivalent coverage.

Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

Two new species of Hemielimaea Brunner von Wattenwyl from China.

Two new species Hemielimaea (Hemielimaea) paracari sp. nov. and H. (H.) parva sp. nov. from southwestern China are described. Characteristics of the stridulatory file on underside of male left tegmen, male stridulatory area on left and right tegmen, and abdominal apex of male are provided. Important and necessary illustrations of the new species are presented. Materials come from the following two depositories: Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS), and China Agricultural University, Beijing, China (CAU).

Comparative analysis of the intestinal microbiota of black-necked cranes (Grus nigricollis) in different wintering areas.

Fecal microbiota is essential for host health because it increases digestive effectiveness. The crane species Grus nigricollis (G. nigricollis) is considered to be near threatened. The fecal microbial composition of crane is less understood, particularly in the Tibet, China. This study was performed to investigate the differences in fecal microbial composition and diversity of crane in different wintering areas using third-generation single-molecule real-time sequencing technology in the Tibet, China. According to the findings, 20 samples were used to generate 936 bacterial amplicon sequence variants (ASVs) and 1,800 fungal ASVs, only 4 bacterial ASVs and 20 fungal ASVs were shared in four distinct locations. Firmicutes were the dominant bacterial phylum in all samples, and Ascomycota and Basidiomycota were the dominant fungal phylum. At the genus level, Lactobacillus was the dominant genus in Linzhi City (LZ), Shannan City (SN), and Lasa City (LS), whereas Megamonas was the dominant genus in Rikaze City (RKZ). Naganishia and Mycosphaerella were the dominant fungal genera in SN and RKZ. Mycosphaerella and Tausonia were the dominant fungal genera in LZ. Naganishia and Fusarium were the dominant fungal genera in LS. And the fecal microbial composition varied between the four groups, as shown by the underweighted pair-group method with arithmetic means and principal coordinates analysis. This study offers a theoretical basis for understanding the fecal microbial composition of crane.

Hemorrhagic enterocolitis and death in two felines (Panthera tigris altaica and Panthera leo) associated with Clostridium perfringens type A.

Severe hemorrhagic enterocolitis was observed in a Siberian tiger (Panthera tigris altaica) and a lion (Panthera leo). Both animals developed acute depression, anorexia, and bloody diarrhea several days before death. Small and large intestines were diffusely congested, edematous, necrotic, and filled with hemorrhagic fluid, and mesenteric lymph nodes were enlarged and congested. Pure and abundant growth of gram-positive bacilli was obtained in culture under anaerobic conditions from the livers of both felines. Identification of highly virulent Clostridium perfringens Type A was based on pathologic lesions, hemolytic patterns, morphologic structure, and polymerase chain reaction. Animal inoculation assays indicated that C. perfringens Type A played an important role in the pathogenesis of both felines.

Correlation and Influence of Seasonal Variation of Diet with Gut Microbiota Diversity and Metabolism Profile of Chipmunk.

Tamias Sibiricus is the only member of the genus Tamias, a significant and vigorous seed distributor and vital food for their predators. No information is known about the strict diet, gut microbiota structure, and metabolism profile of chipmunks and how they diversify seasonally. The above factors, as well as flexibility toward seasonal shifts, are critical in defining its growth rates, health, survivorship, and population stability. This study explored the diet, gut microbiota composition, and chipmunk metabolism. Additionally, the influence of different seasons was also investigated by using next-generation sequencing. Results revealed that seasons strongly affected a diet: streptophyte accounted for 37% in spring, which was lower than in summer (34.3%) and autumn (31.4%). Further, Ascomycota was observed at 43.8% in spring, which reduced to 36.6% in summer and the lowest (31.3%) in autumn. Whereas, nematodes showed maximum abundance from spring (15.8%) to summer (20.6%) and autumn (24.1%). These results signify the insectivorous nature of the chipmunk in summer and autumn. While herbivorous and fungivorous nature in spring. The DNA analysis revealed that chipmunk mainly feeds on fungi, including Aspergillus and Penicillium genus. Similar to diet composition, the microbiome also exhibited highly significant dissimilarity (p < 0.001, R = 0.235) between spring/autumn and spring/summer seasons. Proteobacteria (35.45%), Firmicutes (26.7%), and Bacteroidetes (23.59%) were shown to be the better discriminators as they contributed the most to causing differences between seasons. Moreover, PICRUSt showed that the assimilation of nutrients were also varied seasonally. The abundance of carbohydrates, lipids, nucleotides, xenobiotics, energy, terpenoids, and polyketides metabolism was higher in spring than in other seasons. Our study illustrates that seasonal reconstruction in the chipmunk diet has a significant role in shaping temporal variations in gut microbial community structure and metabolism profile.

Population Density and Driving Factors of North China Leopards in Tie Qiao Shan Nature Reserve.

The North China leopard (Panthera pardus japonesis) is a rare leopard subspecies distributed only in China. In this study, we conducted camera-trap surveys of a North China Leopard population in Tie Qiao Shan Nature Reserve, Shanxi Province, China. We estimated population abundance and density distribution, and explored the effects of distribution of different prey populations, habitat, and anthropogenic factors on the spatial distribution of North China leopard density. Our results suggested that the North China leopard density was 4.23 individuals/100 km2, and that 17.98 individuals might live within the study area. The population density of the North China leopard increased with the distribution of wild boars, and, on the contrary, decreased with the distribution of roe deer. We found that habitat environmental factors and anthropogenic interference also significantly affected the population density and spatial distribution of the North China leopard. These insights informed us that in order to protect this predator, which is only distributed in China, we should adopt a comprehensive customized adaptive landscape protection strategy.

[Identification and production of monoclonal antibody of Siberian tiger's immunoglobulin].

OBJECTIVE: To purify immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig,which can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger. METHODS: The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. RESULTS: We obtained three mouse hybridoma clones that produced mAbs against Ig of Siberian Tiger. The derived McAbs could recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also could be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine efficacy against the infectious disease on the Siberian Tiger. CONCLUSION: Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.