The resistance patterns and molecular characteristics of ESBL/AmpC-producing Escherichia coli from captive panda ecosystem in China.

Escherichia coli (E. coli) plays an important ecological role, and is a useful bioindicator to recognize the evolution of resistance in human, animal and environment. Recently, extended-spectrum ß-lactamases (ESBL) producing E.coli has posed a threat to public health. Generally, captive healthy giant pandas are not exposed to antibiotics; however, they still acquire antimicrobial resistant bacteria. In order to understand whether there is an exchange of resistance genes within the ecosystems of captive giant pandas, this study explored resistance characteristics of 330 commensal E. coli isolates from feces of giant pandas, the surroundings, and breeders. Isolates from different sources showed similar resistance phenotype, and ESBL/AmpC-producing isolates showed more profound resistance to antibiotics than non-ESBL/AmpC-producing isolates (P<0.05). Furthermore, the occurrence of broad-spectrum ß-lactamase related resistance genes and colistin resistance genes was detected, and isolates phylogenetic typing and multilocus sequence typing (MLST) were applied in this study. Seven different ß-lactamase resistance genes (blaCTX-M-55, blaCTX-M-15, blaCTX-M-27, blaCTX-M-65, blaTEM-1, blaOXA-1 and blaCMY) and mcr-1 were found in 68 ESBL/AmpC-producing isolates. blaCTX-M-55 (48.53 %) was found the most predominant resistance genes, followed by blaTEM-1 (19.12 %) and blaCTX-M-27 (16.18 %). Nonetheless, blaCTX-M-55 was commonly detected in the isolates from giant pandas (63.16 %), the surroundings (43.48 %), and breeders (33.33 %). However, there were no carbapenemase genes detected in this study. mcr-1 was harbored in only one isolate from giant panda. Forty-five tansconjugants were successfully obtained in the conjugation experiments. The presence of antimicrobial resistance and related resistance genes tested were observed in the transconjugants. The results indicated that 52.63 % of the isolates from giant panda 73.91 % of the isolates from surroundings, and 100 % of the isolates from breeders were phylogroup A. Total of 27 sequence types (ST) were recognized from the isolate by MLST and found that ST48 (19/68; 27.94 %) was the predominant ST type, especially in the isolates from giant pandas and the surroundings. In conclusion, commensal ESBL/AmpC-producing E. coli becomes a reservoir of ESBL resistance genes, which is a potential threaten to health of giant pandas. The interaction between giant pandas, surroundings and breeders contribute to development of resistant phenotypes and genotypes which might transfer across species or the surroundings easily; hence, strict monitoring based on a "One Health" approach is recommended.

Pseudomonas-associated bacteria play a key role in obtaining nutrition from bamboo for the giant panda (Ailuropoda melanoleuca).

Gut microbiota plays a vital role in obtaining nutrition from bamboo for giant pandas. However, low cellulase activity has been observed in the panda's gut. Besides, no specific pathway has been implicated in lignin digestion by gut microbiota of pandas. Therefore, the mechanism by which they obtain nutrients is still controversial. It is necessary to elucidate the precise pathways employed by gut microbiota of pandas to degrade lignin. Here, the metabolic pathways for lignin degradation in pandas were explored by comparing 209 metagenomic sequencing data from wild species with different feeding habits. Lignin degradation central pathways, including beta-ketoadipate and homogentisate pathway, were enriched in the gut of wild bamboo-eating pandas. The gut microbiome of wild bamboo-eating specialists was enriched with genes from pathways implicated in degrading ferulate and p-coumarate into acetyl-CoA and succinyl-CoA, which can potentially provide the raw materials for metabolism in pandas. Specifically, Pseudomonas, as the most dominant gut bacteria genus, was found to be the main bacteria to provide genes involved in lignin or lignin derivative degradation. Herein, three Pseudomonas-associated strains isolated from the feces of wild pandas showed the laccase, lignin peroxidase, and manganese peroxidase activity and extracellular lignin degradation ability in vitro. A potential mechanism for pandas to obtain nutrition from bamboo was proposed based on the results. This study provides novel insights into the adaptive evolution of pandas from the perspective of lignin metabolism. IMPORTANCE: Although giant pandas only feed on bamboo, the mechanism of lignin digestion in pandas is unclear. Here, the metabolic pathways for lignin degradation in wild pandas were explored by comparing gut metagenomic from species with different feeding habits. Results showed that lignin degradation central pathways, including beta-ketoadipate and homogentisate pathway, were enriched in the gut of wild bamboo-eating pandas. Genes from pathways involved in degrading ferulate and p-coumarate via beta-ketoadipate pathway were also enriched in bamboo-eating pandas. The final products of the above process, such as acetyl-CoA, can potentially provide the raw materials for metabolism in pandas. Specifically, Pseudomonas, as the most dominant gut bacteria genus, mainly provides genes involved in lignin degradation. Herein, Pseudomonas-associated strains isolated from the feces of pandas could degrade extracellular lignin. These findings suggest that gut microbiome of pandas is crucial in obtaining nutrition from lignin via Pseudomonas, as the main lignin-degrading bacteria.

Reference gene catalog and metagenome-assembled genomes from the gut microbiome reveal the microbial composition, antibiotic resistome, and adaptability of a lignocellulose diet in the giant panda.

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet.

Occurrence and Molecular Characteristics of Microsporidia in Captive Red Pandas (Ailurus fulgens) in China.

Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidian pathogens with zoonotic potential that pose significant public health concerns. To ascertain the occurrence and genotypes of E. bieneusi and Encephalitozoon spp., we used nested PCR to amplify the internal transcribed spacer (ITS) gene and DNA sequencing to analyze 198 fecal samples from red pandas from 6 zoos in China. The total rate of microsporidial infection was 15.7% (31/198), with 12.1% (24/198), 1.0% (2/198), 2.0% (4/198) and 1.0% (2/198) for infection rate of E. bieneusi, Encephalitozoon cuniculi, Encephalitozoon intestinalis and Encephalitozoon hellem, respectively. One red panda was detected positive for a mixed infection (E. bieneusi and E. intestinalis). Red pandas living in semi-free conditions are more likely to be infected with microsporidia (χ2 = 6.212, df = 1, p < 0.05). Three known (SC02, D, and PL2) and one novel (SCR1) genotypes of E. bieneusi were found. Three genotypes of E. bieneusi (SC02, D, SCR1) were grouped into group 1 with public health importance, while genotype PL2 formed a separate clade associated with group 2. These findings suggest that red pandas may serve as a host reservoir for zoonotic microsporidia, potentially allowing transmission from red pandas to humans and other animals.

Gastrointestinal microbiome, resistance genes, and risk assessment of heavy metals in wild giant pandas.

The gastrointestinal microbiome (GM) of giant panda (GP) plays an important role in food utilization and health and is also an essential reservoir of resistance genes. Currently, little knowledge is available on the GM, acid resistance genes (AcRGs), antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs) in wild GPs. We sampled the gastrointestinal tract of a dead GP and explored the composition and function of GM and resistance genes through cryo-scanning electron microscopy, metagenomic sequencing, and genome-resolved metagenomics. The concentration of metals in the gastrointestinal lumen, feces, bamboo, and soil was measured by inductively coupled plasma mass spectrometry. Results showed that the composition of the microbiota varied in different gastrointestinal regions. Fecal microbiota was highly associated with small intestinal and colonic microbes. The lignocellulosic cross-linked structure of bamboo was destroyed in the stomach initially and destroying degree increased from stomach to anus. Reconstruction of metagenome-assembled-genomes confirmed that core GM, e.g., Streptococcus, Clostridium, Lactococcus, Leuconostoc, and Enterococcus, carried genes encoding the lignocellulose degradation enzyme. There were no significant differences of resistance genes between gastrointestinal and fecal samples, except MGEs. Multidrug and multi-metal resistance genes were predominant in all samples, while the transposase gene tnpA was the major type of MGE. Significant correlations were observed among the abundance of GM, resistance genes, and MGEs. Gastrointestinal and fecal mercury and chromium were the main metals influencing GM and resistance genes. The content of gastrointestinal and fecal metals was significantly associated with the presence of the same metals in bamboo, which could pose a threat to the health of wild GPs. This study characterized the gastrointestinal microbiome of wild GPs, providing new evidence for the role of the gastrointestinal microbiome in degrading lignocellulose from bamboo and highlighting the urgent need to monitor metal levels in soil and bamboo.

Effects of environmental disinfection on microbial population and resistance genes: A case study of the microecology within a panda enclosure.

Widespread use of disinfectants raises concerns over their involvement in altering microbial communities and promoting antimicrobial resistance. This study explores the influence of disinfection protocols on microbial populations and resistance genes within an isolated enclosure environment and in the gut of giant pandas (GPs) held within. Samples of panda feces, air conditioning ducts, soil and bamboo were collected before and after disinfection. High-throughput sequencing characterized the microbial flora of GP gut and environmental microbes inside the artificial habitat. Microbial cultures showed that Escherichia coli (34.6%), Enterococcus (15.4%) and other pathogenic bacteria deposited in feces and the enclosure. Isolates exhibit a consistent resistance to disinfectant, with the greatest resistance shown to cyanuric acid, and the lowest to glutaraldehyde-dodecyl dimethyl ammonium bromide (GD-DDAB) and dodecyl dimethyl ammonium bromide (DDAB). The total number of the culturable bacteria in soil and bamboo were significantly diminished after disinfection but increased in the gut. After disinfection, the richness (Chao1 index) of environment samples increased significantly (P < 0.05), while the richness in gut decreased significantly (P < 0.05). Ten genera showed significant change in feces after disinfection. Metagenome sequencing showed that 126 types of virulence genes were present in feces before disinfection and 37 in soil. After disinfection, 110 virulence genes localized in feces and 53 in soil. Eleven virulence genes including ECP and T2SS increased in feces. A total of 182 antibiotic resistance genes (ARGs) subtypes, potentially conferring resistance to 20 classes of drugs, were detected in the soils and feces, with most belonging to efflux pump protein pathways. After disinfection, the number of resistance genes increased both in gut and soil, which suggests disinfection protocols increase the number of resistance pathways. Our study shows that the use of disinfectants helps to shape the microbial community of GPs and their habitat, and increases populations of resistant strain bacteria.

Protective Effects of Bacillus subtilis HH2 against Oral Enterotoxigenic Escherichia coli in Beagles.

This study evaluated the protective effect of Bacillus subtilis HH2 on beagles orally challenged with enterotoxigenic Escherichia coli (ETEC). We assessed the physiological parameters and the severity of diarrhea, as well as the changes in three serum immunoglobulins (IgG, IgA, and IgM), plasma diamine oxidase (DAO), D-lactate (D-LA), and the fecal microbiome. Feeding B. subtilis HH2 significantly reduced the severity of diarrhea after the ETEC challenge (p < 0.05) and increased serum levels of IgG, IgA, and IgM (p < 0.01). B. subtilis HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge (p < 0.05), but no significant changes were observed in D-LA (p > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with B. subtilis HH2 (p < 0.05), while B. subtilis HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles (p < 0.01). Moreover, 14 days of B. subtilis HH2 feeding reduced the relative abundance of Deinococcus-Thermus in feces. This study reveals that B. subtilis HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition.

Microbial species from multiple maternal body sites shape the developing giant panda (Ailuropoda melanoleuca) cub gut microbiome.

The gut microbiome of the giant panda (Ailuropoda melanoleuca) plays a vital role in nutrient acquisition from its specialized bamboo diet. Giant panda cubs harbour significantly different gut microbiota during their growth and development when feeding on milk before switching to bamboo. The fetal gut is sterile, and following birth, mother-to-infant microbial transmission has been implicated as a seeding source for the infant gut microbiota. Details of this transmission in giant pandas remain unclear. In this study, faecal samples were collected from seven panda mother-cub pairs when the cubs were 4-16 months old. Additional samples from the cubs' diet, soil and drinking water, and multiple body sites of the mothers were collected. Bacterial 16S rRNA gene sequencing and shotgun metagenomic sequencing were performed to determine the source and potential transmission routes of the cub gut microbiome. Source tracking analysis showed that maternal vagina, milk and faeces were the primary contributory sources of microbes, shaping the cub gut microbiome. Bacterial species from maternal faeces persisted the longest in the cub gut. Bacterial species in the diet contributed to the microbial community. Metagenomics analysis indicated that the predicted metabolic pathways of the gut microbiome also varied at different growth stages. Gut colonization with bacteria from various body sites of the mothers provides a foundational microbial community that is beneficial in fulfilling the evolving dietary needs of the cubs. This study suggests that mother-to-cub transmission is indispensable in shaping the gut microbiome of the developing panda cub.

Using Blood Transcriptome Analysis to Determine the Changes in Immunity and Metabolism of Giant Pandas with Age.

A low reproductive rate coupled with human activities has endangered the giant panda, a species endemic to southwest China. Although giant pandas feed almost exclusively on bamboo, they retain carnivorous traits and suffer from carnivorous diseases. Additionally, their immune system is susceptible to aging, resulting in a reduced ability to respond to diseases. This study aimed to determine the genes and pathways expressed differentially with age in blood tissues. The differentially expressed genes in different age groups of giant pandas were identified by RNA-seq. The elderly giant pandas had many differentially expressed genes compared with the young group (3 years old), including 548 upregulated genes and 401 downregulated genes. Further, functional enrichment revealed that innate immune upregulation and adaptive immune downregulation were observed in the elderly giant pandas compared with the young giant pandas. Meanwhile, the immune genes in the elderly giant pandas changed considerably, including genes involved in innate immunity and adaptive immunity such as PLSCR1, CLEC7A, CCL5, CCR9, and EPAS1. Time series analysis found that giant pandas store glycogen by prioritizing fat metabolism at age 11, verifying changes in the immune system. The results reported in this study will provide a foundation for further research on disease prevention and the energy metabolism of giant pandas.

Lipidomics for Determining Giant Panda Responses in Serum and Feces Following Exposure to Different Amount of Bamboo Shoot Consumption: A First Step towards Lipidomic Atlas of Bamboo, Giant Panda Serum and Feces by Means of GC-MS and UHPLC-HRMS/MS.

Lipidic metabolites play essential roles in host physiological health and growth performance, serving as the major structural and signaling components of membranes, energy storage molecules, and steroid hormones. Bamboo, as wild giant pandas' exclusive diet, is the main determinant of giant pandas' lipidome, both as a direct source and through microbiota activity. Interestingly, the consumption of bamboo has attracted little attention from a lipidomic perspective. In the current study, we outline the lipidomic atlas of different parts of bamboo. By gas chromatography-mass spectrometry (GC-MS), we have been able to obtain the absolute quantification of 35 fatty acids pertaining to short chain fatty acids (8), medium chain fatty acids (6), long chain fatty acids (17), and very long chain fatty acids (4), while liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) allowed us to obtain the relative quantification of another 1638 lipids. Among the fatty acids quantified in absolute terms, eight showed significantly distinct concentrations among different bamboo parts. Subsequently, we investigated how the giant panda's serum and fecal lipidome adapt to the most important annual change in their diet, represented by the consumption of high amounts of bamboo shoots, typical of spring, the weight-gaining season. Five fatty acids were significantly altered in feces and two in serum, respectively, due to the different levels of bamboo shoot consumption. Furthermore, significant differences of the main bacteria strains were observed in feces between the two groups at the genus level, pertaining to Streptococcus, Leuconostoc, and Vagococcus. Correlations between giant panda fecal microbiome and lipidome were evaluated by Pearson correlation analysis. These findings suggest that a balanced diet, important for the overall lipidomic function and giant panda health, could be reached even in this remarkable case of a single food-based diet, by administering to the giant panda's combinations of different parts of bamboo, with specific lipidome profiles.

Dominant Components of the Giant Panda Seminal Plasma Metabolome, Characterized by 1H-NMR Spectroscopy.

As an assisted breeding technique, artificial insemination has become the main effective practical approach in the captive breeding programs of giant panda worldwide. The composition of seminal plasma plays an important role in the success of breeding. The present work is the first attempt to characterize, by proton magnetic resonance spectroscopy (1H-NMR), the metabolome of healthy giant panda seminal plasma. A total of 35 molecules were quantified, with the concentration of 2,3-butanediol being significantly different between individuals younger than 8 years and older than 13 years, and other distinct age-related trends were highlighted by a multivariate analysis. Isopropanol's concentration was significantly linked to estrus stages. Besides, the variations in the metabolome's profile during storage were also evaluated. This study may serve as a reference for further research wishing to shed light on the biological mechanisms affecting giant panda sperm's overall quality and may ultimately lead to novel approaches to giant panda artificial insemination.

Whole blood transcriptome profiling identifies candidate genes associated with alopecia in male giant pandas (Ailuropoda melanoleuca).

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is a threatened species endemic to China. Alopecia, characterized by thinning and broken hair, mostly occurs in breeding males. Alopecia significantly affects the health and public image of the giant panda and the cause of alopecia is unclear. RESULTS: Here, we researched gene expression profiles of four alopecia giant pandas and seven healthy giant pandas. All pandas were approximately ten years old and their blood samples collected during the breeding season. A total of 458 up-regulated DEGs and 211 down-regulated DEGs were identified. KEGG pathway enrichment identified that upregulated genes were enriched in the Notch signaling pathway and downregulated genes were enriched in ribosome, oxidative phosphorylation, and thermogenesis pathways. We obtained 28 hair growth-related DEGs, and identified three hub genes NOTCH1, SMAD3, and TGFB1 in PPI analysis. Five hair growth-related signaling pathways were identified with abnormal expression, these were Notch, Wnt, TGF-ß, Mapk, and PI3K-Akt. The overexpression of NOTCH1 delays inner root sheath differentiation and results in hair shaft abnormalities. The delayed hair regression was associated with a significant decrease in the expression levels of TGFB1. CONCLUSIONS: Our data confirmed the abnormal expression of several hair-related genes and pathways and identified alopecia candidate genes in the giant panda. Results of this study provide theoretical basis for the establishment of prevention and treatment strategies for giant pandas with alopecia.

Antimicrobial resistance of Escherichia coli, Enterobacter spp., Klebsiella pneumoniae and Enterococcus spp. isolated from the feces of giant panda.

BACKGROUND: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae and Enterococcus spp., common gut bacteria in giant pandas, include opportunistic pathogens. The giant panda is an endangered species, classified as vulnerable by the World Wildlife Foundation. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from giant pandas is vital not only for their protection but also for public health. RESULTS: A total of 166 E. coli, 68 Enterobacter spp., 116 K. pneumoniae and 117 Enterococcus spp. isolates were collected from fecal samples of 166 giant pandas. In the antimicrobial susceptibility tests, 144 E. coli isolates, 66 Enterobacter spp. isolates, 110 K. pneumoniae isolates and 43 Enterococcus spp. isolates were resistant to at least one antimicrobial. The resistant isolates carried antimicrobial resistance genes (ARGs), including sul3, blaTEM, blaSHV and tetA. The differences in the prevalence of the bla types implied that the genetic basis for ß-lactam resistance among the E. coli, Enterobacter spp. and K. pneumoniae isolates was different. The strain K. pneumoniae K85 that was resistant to sixteen antimicrobials was selected for whole genome sequencing. The genome contained Col440I, IncFIBK and IncFIIK plasmids and altogether 258 ARGs were predicted in the genome; 179 of the predicted ARGs were efflux pump genes. The genetic environment of the ß-lactamase genes blaCTX-M-3 and blaTEM-1 in the K. pneumoniae K85 genome was relatively similar to those in other sequenced K. pneumoniae genomes. In comparing the giant panda age groups, the differences in the resistance rates among E. coli, K. pneumoniae and Enterobacter spp. isolates suggested that the infections in giant pandas of different age should be treated differently. CONCLUSIONS: Antimicrobial resistance was prevalent in the bacterial isolates from the giant pandas, implying that the gut bacteria may pose serious health risks for captive giant pandas. The resistance genes in the genome of K. pneumoniae K85 were associated with insertion sequences and integron-integrase genes, implying a potential for the further spread of the antimicrobial resistance.

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.

Comparative transcriptome analyses of immune responses to LPS in peripheral blood mononuclear cells from the giant panda, human, mouse, and monkey.

Gram-negative bacteria are major pathogens that can cause illnesses in giant pandas. Lipopolysaccharides (LPS), components of Gram-negative bacteria, can activate immune responses in mammals (i.e., humans and mice) through recognition by toll-like receptors (TLRs). However, the giant pandas' immune response to LPS stimulation and the differences between the giant panda and other mammals are not fully known. In this study, we administrated peripheral blood mononuclear cells (PBMCs) from giant pandas, humans, C57BL/6 mice, and rhesus monkeys by LPS treatment at 6 h followed by RNA sequencing (RNA-seq), respectively, with control of non-stimulation. KEGG analyses of differentially expressed genes (DEGs) pathways indicated that LPS could activate the classic signaling pathway of NF-κB in PBMCs from those four tested species. Thus, similar to the other three species, NF-κB is an LPS-responsive regulator of innate immune responses in giant pandas. Furthermore, the expression patterns of adapter genes, inflammatory cytokine genes, chemokines, interferon genes, cytokine genes related to cell growth and development, costimulatory molecules, Th1/Th2 cytokine genes, Th17 cytokine genes, Th9, and Th22 cytokine genes were compared among giant pandas and three other species. Our data indicated that in addition to the similar expression patterns of certain genes among giant pandas and other species, the unique expression pattern response to LPS in giant pandas was also discovered. Furthermore, Th9, Th17, and Th22 cells might be involved in the response to LPS in giant pandas at this tested time point. This study reveals that LPS-induced immune responses have different sensitivities and response timelines in giant pandas compared with other mammals. This study facilitates further understanding of the role of the TLR signaling pathway and the immune system in giant pandas, which might be helpful for disease prevention and protection.

The Fecal and Serum Metabolomics of Giant Pandas Based on Untargeted Metabolomics.

Little is comprehensively known or understood about giant panda fecal and serum metabolites, which could serve as important indicators of the physiological metabolism of giant pandas. Therefore, we determined the contents of fecal and serum metabolites of giant pandas based on an untargeted metabolome. Four hundred and 955 metabolites were detected in the feces and serum of giant panda, respectively. Glycerophospholipid and choline metabolism were the main metabolic pathways in feces and serum. A significant correlation between the gut microbiota and fecal metabolites was found (P < 0.01). Fecal metabolites were not greatly affected by the age or gender of giant pandas, but serum metabolites were significantly affected by age and gender. The majority of different metabolites caused by age were higher in serum of younger giant pandas, including fatty acids, lipids, metabolites of bile acids, and intermediate products of vitamin D3. The majority of different metabolites caused by gender included fatty acids, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). A separate feeding diet should be considered according to different ages and genders of giant panda. Therefore, our results could provide helpful suggestions to further protect captive giant pandas.

Diversity and Composition of Gut Bacterial Community in Giant Panda with Anorexia.

The giant panda (GP) is the most precious animal in China. Gastrointestinal tract disease, especially associated with dysbiosis of gut microbiota, is the leading cause of death in GPs. Here, we performed 16S rRNA high-throughput sequencing to investigate the gut microbiota of GPs having symptoms of anorexia. Results showed that gut microbiota of GP with anorexia had lower richness (Chao1 index) than the healthy GP. However, no significant differences in alpha diversity were observed. There is a significance in the microbial structure between anorexia and healthy GPs. The abundance of phylum Firmicutes (99.23% ± 7.1%), unidentified genus Clostridiales (24.75% ± 2.5%), was significantly higher in the subadult anorexia group (P < 0.01), and that of the unidentified genus Clostridiales (4.53% ± 1.2%) was also significantly higher in the adult anorexia group (P < 0.01). Weissella and Streptococcus were found to be decreased in both anorexia groups. The decreased abundance of Weissella (0.02% ± 0.0%, 0.08% ± 0.0%) and Streptococcus (73.89% ± 4.3%, 91.15% ± 7.6%) and increase in Clostridium may cause symptoms of anorexia in giant pandas. The correlation analysis indicated that there is a symbiotic relationship among Streptococcus, Leuconostoc, Weissella, and Bacillus which are classified as probiotics (r > 0.6, P < 0.05). Importantly, a negative correlation has been found between Streptococcus and unidentified_Clostridium in two groups (r > 0.6, P < 0.05). Our results suggested that Streptococcus might be used as probiotics to control the growth of Clostridium causing the anorexia.

Immune profiles of male giant panda (Ailuropoda melanoleuca) during the breeding season.

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is a threatened endemic Chinese species and a flagship species of national and global conservation concern. Life history theory proposes that reproduction and immunity can be mutually constraining and interrelated. Knowledge of immunity changes of male giant pandas during the breeding season is limited. RESULTS: Here, we researched peripheral blood gene expression profiles associated with immunity. Thirteen captive giant pandas, ranging from 9 to 11 years old, were divided into two groups based on their reproductive status. We identified 318 up-regulated DEGs and 43 down-regulated DEGs, which were enriched in 87 GO terms and 6 KEGG pathways. Additionally, we obtained 45 immune-related genes with altered expression, mostly up-regulated, and identified four hub genes HSPA4, SUGT1, SOD1, and IL1B in PPI analysis. These 45 genes were related to pattern recognition receptors, autophagy, peroxisome, proteasome, natural killer cell, antigen processing and presentation. SUGT1 and IL1B were related to pattern recognition receptors. HSP90AA1 was the most up-regulated gene and is a member of heat shock protein 90 family. HSP90 contributes to the translocation of extracellular antigen. KLRD1 encodes CD94, whose complex is an inhibitor of the cytotoxic activity of NK cells, was down-regulated. IGIP, which has the capability of inducing IgA production by B cells, was down-regulated, suggesting low concentration of IgA in male giant pandas. Our results suggest that most immune-related genes were up-regulated and more related to innate immune than adaptive immune. CONCLUSIONS: Our results indicated that breeding male giant pandas presented an immunoenhancement in innate immunity, enhanced antigen presentation and processing in cellular immunity compared to non-breeding males. The humoral immunity of male giant pandas may show a tendency to decrease during the breeding season. This study will provide a foundation for further studies of immunity and reproduction in male giant pandas.

Metagenomic analysis revealed a wide distribution of antibiotic resistance genes and biosynthesis of antibiotics in the gut of giant pandas.

BACKGROUND: The gut microbiome is essential for the host's health and serves as an essential reservoir of antibiotic resistance genes (ARGs). We investigated the effects of different factors, including the dietary shifts and age, on the functional characteristics of the giant panda's gut microbiome (GPs) through shotgun metagenome sequencing. We explored the association between gut bacterial genera and ARGs within the gut based on network analysis. RESULTS: Fecal samples (n=60) from captive juvenile, adult, and geriatric GPs were processed, and variations were identified in the gut microbiome according to different ages, the abundance of novel ARGs and the biosynthesis of antibiotics. Among 667 ARGs identified, nine from the top ten ARGs had a higher abundance in juveniles. For 102 ARGs against bacteria, a co-occurrence pattern revealed a positive association for predominant ARGs with Streptococcus. A comparative KEGG pathways analysis revealed an abundant biosynthesis of antibiotics among three different groups of GPs, where it was more significantly observed in the juvenile group. A co-occurrence pattern further revealed a positive association for the top ten ARGs, biosynthesis of antibiotics, and metabolic pathways. CONCLUSION: Gut of GPs serve as a reservoir for novel ARGs and biosynthesis of antibiotics. Dietary changes and age may influence the gut microbiome's functional characteristics; however, it needs further studies to ascertain the study outcomes.

Diet, habitat environment and lifestyle conversion affect the gut microbiomes of giant pandas.

Gut microbiota (GM) are important for the health of giant pandas (GPs), in addition to the utilization of bamboo in their diets. However, it is not fully understood how diet, habitat environment and lifestyle contribute to the composition of GM in GP. Consequently, we evaluated how dietary changes, habitat environment conversions and lifestyle shifts influence the GM of GPs using high-throughput sequencing and genome-resolved metagenomics. The GM of GPs were more similar when their hosts exhibited the same diet. High fiber diets significantly increased the diversity and decreased the richness of gut bacterial communities alone or interacted with the age factor (p < 0.05). The abundances of Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium significantly increased during diet conversion process (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA > 4). Reconstruction of 60 metagenome-assembled-genomes (MAGs) indicated that these bacteria were likely responsible for bamboo digestion via gene complements involved in cellulose, hemicellulose, and lignin degradation. While habitat environment may play a more important role in shaping the GM of GP, lifestyle can also greatly affect bacterial communities. The GM structure in reintroduced GPs notably converged to that of wild pandas. Importantly, the main bacterial genera of wild GPs could aid in lignin degradation, while those of reintroduced GPs were related to cellulose and hemicellulose digestion. Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium may contribute to lignocellulose digestion in GP. The results revealed that diet conversion, habitat environment and lifestyle could remarkably influence the GM of GP. In addition, results suggested that increasing the ability of lignin degradation with GM may aid to change the GM of reintroduced pandas to resemble those of wild pandas.