Multi-omic analysis reveals that Bacillus licheniformis enhances pekin ducks growth performance via lipid metabolism regulation.

Introduction: Bacillus licheniformis (B.licheniformis) was widely used in poultry feeds. However, it is still unclear about how B.licheniformis regulates the growth and development of Pekin ducks. Methods: The experiment was designed to clarify the effect and molecular mechanism of B. licheniformis on the lipid metabolism and developmental growth of Pekin ducks through multiomics analysis, including transcriptomic and metabolomic analyses. Results: The results showed that compared with the control group, the addition of 400 mg/kg B. licheniformis could significantly increase the body weight of Pekin ducks and the content of triglyceride (p < 0.05), at the same time, the addition of B. licheniformis could affect the lipid metabolism of liver in Pekin ducks, and the addition of 400 mg/kg B. licheniformis could significantly increase the content of lipoprotein lipase in liver of Pekin ducks. Transcriptomic analysis revealed that the addition of B. licheniformis primarily impacted fatty acid and glutathione, amino acid metabolism, fatty acid degradation, as well as biosynthesis and elongation of unsaturated fatty acids. Metabolomic analysis indicated that B. licheniformis primarily affected the regulation of glycerol phospholipids, fatty acids, and glycerol metabolites. Multiomics analysis demonstrated that the addition of B. licheniformis to the diet of Pekin ducks enhanced the regulation of enzymes involved in fat synthesis via the PPAR signaling pathway, actively participating in fat synthesis and fatty acid transport. Discussion: We found that B. licheniformis effectively influences fat content and lipid metabolism by modulating lipid metabolism-associated enzymes in the liver. Ultimately, this study contributes to our understanding of how B. licheniformis can improve the growth performance of Pekin ducks, particularly in terms of fat deposition, thereby providing a theoretical foundation for its practical application. Conclusion: B. licheniformis can increase the regulation of enzymes related to fat synthesis through PPAR signal pathway, and actively participate in liver fat synthesis and fatty acid transport, thus changing the lipid metabolism of Pekin ducks, mainly in the regulation of glycerol phospholipids, fatty acids and glycerol lipid metabolites.

Screening of Genes Related to Fat Deposition of Pekin Ducks Based on Transcriptome Analysis.

Subcutaneous fat deposition is an important index with which to evaluate meat-producing ducks, and affects their meat quality and feed conversion rate. Studying the differentially expressed genes in subcutaneous fat will help to comprehensively understand the potential mechanisms regulating fat deposition in ducks. In this study, 72 Nankou 1 Pekin Ducks and 72 Jingdian Pekin Ducks (half male and half female) at 42 days of age were selected for slaughter performance and transcriptome analysis. The results showed that the breast-muscle yield of Nankou 1 ducks was significantly higher than that of Jingdian ducks, but that the abdominal fat yield and subcutaneous fat yield were higher than that of Jingdian ducks. Thousands of DEGs, including many important genes involved in fat metabolism regulation, were detected by transcriptome. KEGG enrichment analysis showed that the DEGs were significantly enriched on pathways such as regulation of lipolysis in adipocytes, primary bile acid biosynthesis, and biosynthesis of unsaturated fatty acids. SCD, FGF7, LTBP1, PNPLA3, ADCY2, and ACOT8 were selected as candidate genes for regulating subcutaneous fat deposition. The results indicated that Nankou 1 had superior fat deposition ability compared to Jingdian ducks, and that the candidate genes regulated fat deposition by regulating fat synthesis and decomposition.

Application of Host-Depleted Nanopore Metagenomic Sequencing in the Clinical Detection of Pathogens in Pigs and Cats.

Metagenomic sequencing is a valuable tool for non-specifically detecting various microorganisms in samples, offering unique advantages for detecting emerging pathogens, fastidious or uncultivable pathogens, and mixed infections. It has recently been applied to clinically detect pathogenic microorganisms in animals; however, the high proportion of host genes, expensive sequencing equipment, and the complexity of sequencing and data analysis methods have limited its clinical utility. In this study, a combination of tissue homogenization and nuclease digestion was employed to remove host genes from pig and cat samples; DNA and RNA were then extracted and subjected to nonselective PCR amplification to simultaneously detect DNA and RNA pathogen genomes using R9.4.1 or R10.4.1 flow cells on the MinION platform. Real-time pathogen detection was conducted using EPI2M WIMP, and viral genome assembly was performed using NanoFilt, minimap2, samtools, and ivar. Pathogens in five clinical samples (serum, nasopharyngeal swab, feces, or ascites) from cats and four clinical samples (lung or small intestine tissue) from pigs were examined by metagenomic sequencing, and the results were consistent with those obtained by PCR and bacterial culture. Additionally, we detected four viruses and three bacteria that may be associated with diseases. A comparison of results before and after host gene removal in three samples showed a 9-50% reduction in host genes. We also compared the assembly efficiency of six virus genomes and found that data volumes ranging from 3.3 to 98.3 MB were sufficient to assemble >90% of the viral genomes. In summary, this study utilized optimized nanopore metagenomic sequencing and analysis methods to reduce host genes, decrease the required data volume for sequencing analysis, and enable real-time detection to determine when to stop sequencing. The streamlined sequencing and analysis process overcomes barriers to the veterinary clinical application of metagenomic sequencing and provides a reference for clinical implementation.

Dietary Saccharomyces cerevisiae improves intestinal flora structure and barrier function of Pekin ducks.

This study aimed at investigating the effects of dietary Saccharomyces cerevisiae (SC) on the intestinal flora structure and barrier function of Pekin duck. A total of 180 1-day-old Pekin ducks were randomly divided into 3 groups with 6 replicates in each group and 10 birds per replicate. The birds in the control group (CON) were fed the basal diet, and those in the experimental group were fed the basal diets supplemented with 600 mg/kg SC (LSC) and 1,200 mg/kg (HSC), respectively. The trial lasted for 42 d. Results showed that LSC and HSC treatments tended to improve the feed conversion efficiency during the trial. The ileum length of birds in the LSC and HSC groups was elevated. Additionally, with 600mg/kg SC supplemented, the mRNA levels of villin, claudin3, and MUC 2 in d21 were up-regulated, as well as the mRNA levels of villin, claudin3, occludin, i-FABP, ZO-1, and MUC 2 in d42. In addition, dietary SC supplementation improved the α-diversity of the bacteria in cecal chyme and tended to increase the abundance (RA) of Bacteroidetes (P = 0.071). Besides, the RA of Ruminococcaceae_UCG-014 was raised in the LSC group. Beyond that, the RA of Proteobacteria was descended with two levels of SC added. In conclusion, dietary Saccharomyces cerevisiae, particularly at 600 mg/kg level, improved the intestinal flora structure and barrier function of Pekin duck.

Effects of silybin supplementation on growth performance, serum indexes and liver transcriptome of Peking ducks.

As an emerging feed additive extracted from the traditional herb milk thistle, silybin has few applications and studies in Peking ducks. The aim of this study was to explore the practical significance of silymarin application in Peking ducks and to provide more theoretical support for the application of silymarin in livestock and poultry production. A total of 156 1-day-old healthy Peking ducks were randomly divided into four groups and supplemented with 0 mg/kg (control group), 400 mg/kg (S400), 800 mg/kg (S800) and 1,600 mg/kg (S1600) of silybin in the diets at day 14, to investigate the effects of silymarin on the growth, serum indexes and liver transcriptome of Peking ducks. The whole experiment lasted until day 42, and the sample collection was scheduled to take place in the morning. A substantial inprovement in average daily gain (ADG) and a decrease in feed conversion ratio (FCR) occurred in the S1600 group on days 14-28 compared to the control group (p < 0.05). The FCRs of other additive groups in the same period showed the same results. Supplementation of diets with silybin significantly increased serum IgA levels and when 1,600 mg/kg of silybin was given, levels of TNF-α and IL-6 were also significantly decreased (p < 0.05). In addition, we observed that the S1600 group had a significantly lower (p < 0.05) glutamine transaminase and an increased (p < 0.05) T-SOD level in the S400 group (p < 0.05). Liver transcriptome sequencing showed that 71 and 258 differentially expressed genes (DEGs) were identified in the S400 and S1600 groups, respectively, compared with the control group. DEGs related to cell composition and function, antigen processing and presentation were up-regulated, while DEGs related to insulin resistance and JAK-STAT were down-regulated. Conclusively, silybin can be used as a feed additive to improve the growth performance and health status of Peking ducks.

Multidrug-Resistant Klebsiella pneumoniae Complex From Clinical Dogs and Cats in China: Molecular Characteristics, Phylogroups, and Hypervirulence-Associated Determinants.

Klebsiella pneumoniae complex is an increasingly important bacterial pathogen that is capable of causing severe organs and life-threatening disease. This study aimed to investigate the multidrug resistance, phylogroups, molecular characterization, and hypervirulence-associated determinants of the complex, which were isolated from clinical diseased dogs and cats. A total of 35 K. pneumoniae complex (2.3%; 95% confidence interval, 1.6-3.2) isolates were identified from 1,500 samples, all of which were collected randomly from veterinary hospitals in the 12 regions across China. Antimicrobial susceptibility testing showed that isolates were extremely resistant to amoxicillin-clavulanate (82.9%) and trimethoprim-sulfamethoxazole (77.1%). The rate of multidrug-resistant reached an astonishing 82.9% and found a carbapenemase-producing strain carrying IncX3-bla NDM-5 derived a cat from Zhejiang. The prevalence rates of extended-spectrum ß-lactamase gene bla CTX-M and plasmid-mediated quinolone resistance gene aac(6')Ib-cr were 51.4% and 45.7%, respectively. The resistance gene aph(3')-Ia of isolates from cats was more significantly (p < 0.05) prevalent than that from dogs. Likewise, K. pneumoniae complex harbored hypervirulence-associated genes ybt (11.4%), iuc (5.7%), and iroB (2.9%). Three (8.6%) of the 35 isolates were determined as hypermucoviscous by the string test. Lipopolysaccharide serotype O1v2 had the highest percentage of 25.7%, but capsular serotypes presented diversity distribution among the isolates. The core-genome phylogenetic tree demonstrated most of the isolates belonged to the KpI phylogroup (91.4%). Multilocus sequence typing analysis identified 25 different STs; ST15 and ST37 were the most abundant accounting for isolates, followed by ST307, ST656, ST1408, and ST4566. In addition, the prevalence of IncFIB-type plasmid for cat isolates was significantly higher (p < 0.05) than that for dogs. Sequences of IncX3 in bla NDM-5-positive strain contained regions showing >99% nucleotide sequence identity to the reference plasmid pNDM-MGR194 from the human.

Dietary Lactobacillus plantarum improves the growth performance and intestinal health of Pekin ducks.

Lactobacillus plantarum (LP) contributes to the intestinal health, whereas the study about the effects of LP on Pekin ducks is lacking. This study aimed to investigate the effects of LP on growth performance and intestinal health of Pekin ducks. A total of 180 one-day-old birds were randomly allotted to 3 treatments, and ducks were fed with a basal diet (Control) or basal diet supplemented with 400 (LP1) and 800 (LP2) mg/kg LP (5 × 109 CFU/g). The animal trial lasted for 42 d. Results showed that the LP1 and LP2 treatments improved growth performance (feed conversion) of ducks during the period of 1 to 42 d. At the end of 21 d, the decreased serum levels of interleukin (IL)-1ß, interferon (IFN)-γ as well as downregulated ileal mRNA expression of IL-1ß were observed in 2 doses of LP group. Meanwhile, the ileal mRNA levels of major histocompatibility complex (MHC)-II, IL-4, Claudin, Occludin were upregulated with 2 doses of LP supplemented. In addition, both LP treatments increased the relative abundance of Firmicutes and decreased Bacteroidetes wherein the relative abundance of Bacteroides fragilis was dropped parallelly. It is worth mentioning that markedly increased secretory immunoglobulin A content in ileal mucosa was observed in the LP1 group at d 21. At the end of the trial, the levels of serum complement 3 and ß-defense were elevated with 2 doses of LP treated. Additionally, the ileal mRNA expressions of MHC-II, lysozyme were upregulated, and the diversity of the flora was also improved in the LP1 and LP2 groups. In conclusion, dietary LP improved the growth performance and intestinal health of Pekin ducks, and 400 mg/kg LP seemed to work better.

Effect of Dietary Bacillus licheniformis Supplementation on Growth Performance and Microbiota Diversity of Pekin Ducks.

This experiment was conducted to investigate the effects of different concentrations of Bacillus licheniformis (B. licheniformis) on growth performance and microbiota diversity of Pekin ducks. Three hundred 1-day-old healthy Pekin ducks were randomly divided into 5 groups with 6 replicates per group and 10 ducks per replicate. The five treatments supplemented with basal diets containing: either 0 (group CON), 200 (group LLB), 400 (group MLB), and 800 (group HLB) mg/kg B. licheniformis or 150 mg/kg aureomycin (group ANT) for 42 days, respectively, and were sacrificed and sampled in the morning of the 42nd day for detection of relevant indexes. The results showed as follows: The feed conversion ratio of the LLB group and MLB groups were lower than the CON group (P < 0.05). The body weight and average daily feed intake of the MLB group were significantly higher than that of the CON group and ANT group (P < 0.05). Compared with the CON group, the MLB group significantly increased the content of IgA (P < 0.05) and proinflammatory IL-6 were significantly decreased (P < 0.05), besides, the activity of SOD and T-AOC were also significantly increased in the MLB group (P < 0.05). The 16S rRNA analysis showed that B. licheniformis treatments had no effect (P > 0.05) on the alpha diversities of the intestine. The addition of B. licheniformis had a dynamic effect on the abundance of cecal microflora of Pekin ducks, and 1-21 d increased the diversity of microflora, while 21d-42 d decreased it. Compared with the CON group, the relative abundance of Epsilonbacteraeota in the MLB group was significantly increased on Day 21 (P < 0.05), and that of Tenericutes in the LLB group was significantly increased as well (P < 0.05). At 42 d, the relative abundance of Bacteroidetes in LLB, MBL, HBL, and ANT groups was significantly increased (P < 0.05). In addition, the addition of B. licheniformis increased the amount of SCAF-producing bacteria in the intestinal microbiota, such as Lachnospiraceae, Collinsella, Christensenellaceae, and Bilophila. The PICRUSt method was used to predict the intestinal microbiota function, and it was found that lipid transport and metabolism of intestinal microbiota in the MLB group were significantly affected. Overall, these results suggest diet supplemented with B. licheniformis improved growth performance, immune status, antioxidant capacity, and modulated intestinal microbiota in Pekin ducks. The optimal dietary supplement dose is 400 mg/kg.

Establishment of a New Cell Line of Canine Mammary Tumor CMT-1026.

Canine mammary tumors (CMTs) have histopathological, epidemiologic and clinical characteristics similar to those in humans and are known to be one of the best models for human breast cancer (HBC). This research aimed to describe a newly established canine cell line, CMT-1026. Tumor samples were collected from a female dog exhibiting clinical mammary neoplasm, and the adherent cells were cultured. Both the histology and immunohistochemistry (IHC) of tumor samples were estimated. Cell growth, ultrastructural, cytological and immunocytochemistry (ICC) features of CMT-1026 were examined. CMT-1026 cells were inoculated into 10 female BALB/c nude mice to evaluate oncogenicity and metastatic ability. Hematoxylin-eosin (H.E.) staining of the tumors revealed an epithelial morphology. Electron microscopy was used to detect histological and cytological of smears, and ultrathin sections showed that CMT-1026 cells were polygonal and characterized by atypia and high mitotic index in the tumor, with prominent nucleoli and multinucleated cells. IHC characterization of CMT-1026 indicated ER-, PR-, HER-2, p63+, CK5/6+, and α-SMA+ epithelial cells. ICC characterization of CMT-1026 showed high expression of Claudin-1, Delta-catenin, SOX-2, and KI-67. At 2 weeks after inoculation of the CMT-1026 cells, phyma was found in 100% of the mice. The xenograft cancers showed conservation of the original H.E. features of the female dog cancer. In conclusion, CMT-1026 may be a model of canine mammary cancer that can be used in research on the pathogenesis of both CMT and HBC.

Effect of Dietary Clostridium butyricum Supplementation on Growth Performance, Intestinal Barrier Function, Immune Function, and Microbiota Diversity of Pekin Ducks.

Clostridium butyricum (C. butyricum) is increasingly being used to test the promotion of the gut health of animals. However, the modes of action for such applications for waterfowl remain unclear. Thus, we investigated whether or not intestinal barrier function, immune-related gene expression, and the diversity of the intestinal microbiota in Pekin ducks varied under C. butyricum supplementation. A total of 500 ducks were randomly assigned into five treatments supplemented with basal diets containing: either 0 (group Control), 200 (group CB200), 400 (group CB400) and 600 (group CB600) mg/kg C. butyricum or 150 mg/kg aureomycin (group A150) for 42 days. In comparison with the control group, C. butyricum supplementation enhanced the growth performance and intestinal villus height of Pekin ducks at 42 d. Serum immune indexes and fecal short-chain fatty acids (SCFAs) were all improved at both 21 d and 42 d after C. butyricum addition. The mRNA expression levels of Mucin2, Zonula occludens-1 (ZO-1), Caudin-3, and Occludin increased at 21 d and 42 d and the mRNA expression levels of IL-4 and IL-10 only increased at 42 d after C. butyricum addition. Dietary C. butyricum also resulted in an increase in the number of diversities of operational taxonomic units (OTUs), and an increase in the α-diversity of intestinal microbiota. The addition of C. butyricum altered the composition of the intestinal microbiota from 21 d to 42 d. The relative abundance of Firmicutes and Bacteroidetes showed little changes among groups; however, the relative abundance of Firmicutes/Bacteroidetes were found to have been significantly different between the 21 d and 42 d. C. butyricum administration improved the intestinal health of Pekin ducks by increasing the diversity of intestinal microbiota, enhancing the SCFAs contents, and strengthening the intestinal barrier function and immune systems. The optimal dietary supplementation dosage was recommended as 400 mg/kg in the diet.

Molecular Investigation of Klebsiella pneumoniae from Clinical Companion Animals in Beijing, China, 2017-2019.

This work is aimed to elucidate the prevalence and characteristics of antimicrobial resistance, virulence, and molecular typing in Klebsiella pneumoniae from clinical companion animals in Beijing, China. In total, 105 K. pneumoniae (2.0%) isolates were recovered from 5359 samples (dogs, n = 3356; cats, n = 2003). All tested isolates exhibited high resistance to amoxicillin-clavulanate (74.3%). Moreover, resistance rates in dog isolates (2.1%) were significantly higher than in cat isolates (0.9%); however, the rate of multidrug-resistance (MDR) was 57.1% and the MDR prevalence in cats was significantly higher than dogs. Whole-genome sequencing demonstrated plasmids IncX4 and IncFIA (HI1)/FII(K) carried mcr-1 (n = 1) and mcr-8 (n = 1), but blaOXA-181 (n = 1) and blaNDM-5 (n = 4) were harbored in IncX3-type plasmids, and the above genes were in different isolates. The most prevalent sequence types (STs) in companion animals were ST1 (n = 9) and ST37 (n = 9). Compared to National Center for Biotechnology Information (NCBI) data on human K. pneumoniae, resistance genes blaCTX-M and blaTEM were more prevalent in human isolates; however, aac(6')-Ib-cr and oqxAB showed a higher prevalence in companion animals. Hypermucoviscosity was reported in 9 (8.6%) isolates, whereas 64 isolates (61.0%) were hypervirulent K. pneumoniae (hvKP) via the Galleria mellonella. These findings validate the high risk of K. pneumonia and necessitate its relevant control in pet clinics.

Serum fibroblast growth factor 23 (FGF-23): associations with hyperphosphatemia and clinical staging of feline chronic kidney disease.

Fibroblast growth factor 23 (FGF-23) is an independent monitor of the progression of chronic kidney disease (CKD) in human medicine, and FGF-23 may have value as a biomarker in feline CKD. We evaluated the relationship between serum FGF-23 and CKD stages, and the effect of age on FGF-23 in normal cats. We measured FGF-23 and intact parathyroid hormone (iPTH) concentrations by ELISA, with intra- and inter-assay CVs ≤ 15%. The percentage recovery of FGF-23 and iPTH remained stable for up to 7 d in samples stored at -20°C and -80°C. We measured FGF-23 in 304 cats, among which 196 were diagnosed with CKD. The 108 clinically healthy cats were divided into 5 subgroups based on growth stage (0-2 y, 3-6 y, 7-10 y, 11-14 y, ≥ 15 y). No statistical difference was found in FGF-23 among age groups (p = 0.15) or by sex in healthy subjects. Using the International Renal Interest Society guideline, 34 cats were defined as CKD stage 1, 74 stage 2, 51 stage 3, and 37 stage 4. FGF-23 was higher in cats in all CKD stages than in controls. Higher serum phosphorus was observed in stage 3 (p = 0.04) and 4 (p < 0.01) compared to controls. iPTH increased as CKD progressed. Pearson analysis indicated a positive linear relationship between FGF-23 and iPTH (control: r = 0.70, p < 0.01; CKD: r = 0.46, p = 0.02). FGF-23 may be a useful biomarker of feline CKD and may precede hyperphosphatemia in advanced feline CKD.

Molecular identification of Cryptosporidium spp. in pet snakes in Beijing, China.

Few reports of Cryptosporidium spp. in snakes in China have been published. To determine the infection rate and document the presence of Cryptosporidium in pet snakes using molecular methods, 273 fecal samples were collected from eight species of pet snakes from 13 pet households in Beijing, China, and were examined by PCR amplification of the small subunit ribosomal RNA gene. Cryptosporidium was detected from 17 of 273 (6.2%) samples, and nine out of 13 households tested positive for Cryptosporidium with a range of 3.3 to 33.3% among households showing significant difference (p < 0.01). The infection rate of Cryptosporidium for females and males was 6.5% (13/201) and 5.6% (4/72), respectively, showing no significant difference (p > 0.05). Six out of eight pet snake species tested positive for Cryptosporidium with a range of 4.2 to 9.1% among species, showing no significant difference (p > 0.05). Two Cryptosporidium species were identified: Cryptosporidium serpentis in 10 samples and Cryptosporidium varanii in seven samples. No zoonotic Cryptosporidium species occur in our study populations.

Antimicrobial resistance of bacterial pathogens isolated from canine urinary tract infections.

Urinary tract infections (UTIs), many of which are caused by bacterial pathogens, are some of the most common infections in dogs. To effectively treat UTIs, it is important to identify the predominant bacterial pathogens and their susceptibility to antimicrobial agents. In this study, we collected 326 samples from cases with UTIs or other urinary system diseases at the China Agricultural University Veterinary Teaching Hospital, Beijing, from 2016-2018. In total, 129 non-duplicate bacterial isolates were recovered from 103 clinical samples. The proportion of positive female samples was higher than that of males. The predominant Gram-negative bacteria were Escherichia coli and Klebsiella spp., while Staphylococcus spp. were the predominant Gram-positive bacteria. Broth microdilution-based antimicrobial susceptibility testing showed that 39 % of E. coli and 51.5 % of Staphylococcus spp. isolates were multidrug-resistant. Specifically, E. coli isolates showed high rates of resistance to ampicillin (40.5 %), ceftazidime (59.5 %), and florfenicol (42.9 %), but limited resistance to amikacin (2.38 %), meropenem (7.14 %), and polymyxin E (7.14 %). In comparison, Staphylococcus spp. showed high rates of resistance to erythromycin (60.6 %), trimethoprim-sulfamethoxazole (54.6 %), and penicillin (45. 5 %), but low resistance rates to vancomycin (6.06 %) and nitrofurantoin (6.06 %). Pulsed-field gel electrophoresis (PFGE)-based typing identified 31 PFGE patterns among the 43 E. coli isolates. These results suggested that multiple bacterial strains, many of which are multidrug-resistant, can cause UTIs in dogs. Thus, basic antimicrobial susceptibility tests should be performed to provide guidance for the selection of first-line clinical therapeutics, and to help prevent the occurrence and spread of induced antimicrobial resistance.

Protective effects of L-arginine on the intestinal epithelial barrier under heat stress conditions in rats and IEC-6 cell line.

Heat stress (HS) and the associated restricted blood flow to the intestine have been proven to destroy intestinal integrity. Considering the beneficial properties of L-arginine on gut function, we investigated the protective effects of L-arginine on the intestine under HS conditions. In vivo, the serum cortisol level and the rectal temperature increased in response to HS. Under HS, the intestinal damage showed obvious morphological changes. Furthermore, HS decreased the mRNA and protein expression levels of Nurr1, ZO-1, occludin, claudin-6 and E-cadherin, increased the mRNA expression of NF-κB and IL-1ß, and increased the protein expression of cleaved caspase-3. In contrast, L-arginine supplementation maintained intestinal integrity and increased the villus/crypt ratio. L-arginine also suppressed the expression of inflammation-related genes and the protein expression of cleaved caspase-3, whereas it upregulated the mRNA and protein expression of tight junction proteins and LC3B protein expression. In vitro, L-arginine attenuated HS-induced apoptosis as demonstrated by flow cytometry and decreased cleaved caspase-3 protein expression. L-arginine induced autophagy, which was demonstrated by decreased expression of p62 and p-mTOR/mTOR, and increased expression of LC3B. The protein expression levels of TJ proteins also enhanced by L-arginine in IEC-6 cells. Taken together, these results suggest that L-arginine can alleviate intestinal damage and protect the intestinal integrity by suppressing local inflammation response, promoting the production of TJs and facilitating autophagy under HS conditions.

L-Arginine alleviates heat stress-induced intestinal epithelial barrier damage by promoting expression of tight junction proteins via the AMPK pathway.

Heat stress (HS) and secondary restricted blood flow to the intestines cause dysfunction of the intestinal epithelial barrier. Tight junctions (TJs) are essential to maintain intestinal integrity. L-Arginine has beneficial effects on gut functions. However, the underlying mechanisms remain largely unknown. This study tested the hypothesis that L-arginine regulates the TJ network by activating AMP-activated protein kinase (AMPK) signaling, which in turn improves intestinal barrier functions under HS. IEC-6 cells and rat small intestines were used as experiment models of heat stress. AICAR and dorsomorphin were used to activate and inhibit the AMPK pathway, respectively. Cell proliferation, apoptosis, differential gene expression and KEGG pathway analysis, intestinal paracellular permeability, intestinal morphology, and expression of HSP and TJ proteins, and p-AMPK were determined. L-Arginine promoted cell proliferation and reduced apoptosis after heat exposure at an optimal concentration of 5 mmol. Transcriptome sequencing analysis revealed that differentially expressed genes associated with the HSP family and TJs were elevated by L-arginine. According to KEGG pathway analysis, L-arginine activated the AMPK signaling pathway. In vivo, intestinal damage resulted in obvious morphological changes as well as apoptosis with TUNEL and caspase-3 staining under HS and dorsomorphin treatments. Furthermore, HS and dorsomorphin increased the serum D-lactate concentration, diamine oxidase activity, and mRNA expression level of MLCK (P < 0.05). In contrast, L-arginine and AICAR treatments reduced intestinal injury, maintained intestinal permeability, and increased the villus/crypt ratio under hyperthermia. L-Arginine had the same effect as AICAR both in vitro and in vivo, namely increasing p-AMPK protein expression. L-Arginine and AICAR also upregulated the mRNA expression level of HSP70 and HSP90, and downregulated mRNA expression of MLCK (P < 0.05). The protein expression levels of TJ proteins ZO-1 and claudin-1 were suppressed by heat stroke and dorsomorphin, but enhanced by L-arginine and AICAR. Our findings indicate that activation of AMPK signaling by L-arginine is associated with improved intestinal mucosal barrier functions by enhancing the expression of TJs in rat small intestines and IEC-6 cells during HS.

A Discovery of Relevant Hepatoprotective Effects and Underlying Mechanisms of Dietary Clostridium butyricum Against Corticosterone-Induced Liver Injury in Pekin Ducks.

Clostridium butyricum (C. butyricum) can attenuate oxidative stress, inflammation, and hepatic fatty deposition in poultry, however, the underlying mechanisms for this in Pekin ducks remain unclear. This study evaluated these hepatoprotective effects and the underlying mechanisms in a corticosterone (CORT)-induced liver injury model in Pekin ducks fed a C. butyricum intervention diet. A total of 500 Pekin ducks were randomly divided into five groups: one group (CON group) was only provided with a basal diet, three groups were provided a basal diet with 200 mg/kg (LCB group), 400 mg/kg (MCB group), or 600 mg/kg (HCB group) C. butyricum, respectively, and one group was provided a basal diet with 150 mg/kg aureomycin (ANT group) for 42 d. At 37 days-old, all ducks received daily intraperitoneal injections of CORT for five days to establish a liver injury model. C. butyricum intervention alleviated liver injury by decreasing the liver organ indices, hepatic steatosis and hepatocyte necrosis, and improving liver function, antioxidant capacity, and inflammatory factors. Hepatic RNA-seq revealed 365 differentially expressed genes (DEGs) between the MCB and CON groups, with 229 up- and 136 down-regulated DEGs in the MCB group. Between the MCB and ANT groups, 407 DEGs were identified, including 299 up- and 108 down-regulated genes in MCB group. Some DEGs in the MCB group related to oxidative stress and inflammatory responses such as Sod3, Tlr2a/b, and Il10, which were up-regulated, while Apoa1, Cyp7a1, Acsl1/5, Fasn, Ppar-γ, and Scd, which are involved in lipid metabolism, were down-regulated, indicating that these genes were responsive to dietary C. butyricum for the alleviation of corticosterone-induced hepatic injury. Toll-like receptor signaling, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, peroxisome proliferator-activated receptor (PPAR) signaling pathway, adipocytokine and glycerophospholipid metabolism signaling pathway were significantly enriched in the MCB group. These findings indicate that C. butyricum intervention can protect Pekin ducks from corticosterone-induced liver injury by the modulation of immunoregulatory- and lipid metabolism-related genes and pathways.

H3N2 canine influenza virus and Enterococcus faecalis coinfection in dogs in China.

BACKGROUND: In May 2017, 17 dogs in a German Shepherd breeding kennel in northern China developed respiratory clinical signs. The owner treated the dogs with an intravenous injection of Shuang-Huang-lian, a traditional Chinese medicine, and azithromycin. The respiratory signs improved 3 days post-treatment, however, cysts were observed in the necks of eight dogs, and three of them died in the following 2 days. CASE PRESENTATION: Quantitative real-time PCR was used to detect canine influenza virus (CIV). All of the dogs in this kennel were positive and the remaining 14 dogs had seroconverted. Two of the dogs were taken to the China Agricultural University Veterinary Teaching Hospital for further examination. Two strains of influenza virus (A/canine/Beijing/0512-133/2017 and A/canine/Beijing/0512-137/2017) isolated from the nasal swabs of these dogs were sequenced and identified as avian-origin H3N2 CIV. For the two dogs admitted to the hospital, hematology showed mild inflammation and radiograph results indicated pneumonia. Cyst fluid was plated for bacterial culture and bacterial 16 s rRNA gene PCR was performed, followed by Sanger sequencing. The results indicated an Enterococcus faecalis infection. Antimicrobial susceptibility tests were performed and dogs were treated with enrofloxacin. All 14 remaining dogs recovered within 16 days. CONCLUSIONS: Coinfection of H3N2 CIV and Enterococcus faecalis was detected in dogs, which has not been reported previously. Our results highlight that CIV infection might promote the secondary infection of opportunistic bacteria and cause more severe and complicated clinical outcomes.

Increasing Prevalence of ESBL-Producing Multidrug Resistance Escherichia coli From Diseased Pets in Beijing, China From 2012 to 2017.

We investigated antimicrobial resistance trends and characteristics of ESBL-producing Escherichia coli isolates from pets and whether this correlates with antibiotic usage in the clinic. Clinical samples containing E. coli from diseased cats and dogs were screened for antibiotic sensitivity and associated genotypic features. We identified 127 E. coli isolates from 1886 samples from dogs (n = 1565) and cats (n = 321) with the majority from urinary tract infections (n = 108, 85%). High rates of resistance were observed for ß-lactams and fluoroquinolones and resistance to > 3 antibiotic classes (MDR) increased from 67% in 2012 to 75% in 2017 (P < 0.0001). This was especially true for strains resistant to 6-9 antibiotics that increased from 26.67 to 60.71%. Increased rates in ß-lactam use for clinical treatment accompanied these increasing resistance rates. Accordingly, the most frequently encountered subtypes were bla CTX-M (n = 44, 34.65%), bla CTX-M-65 (n = 19) and bla CTX-M-15 (n = 18) and qnrB (n = 119, 93.70%). The bla CTX-M-isolates possessed 36 unique pulsed field electrophoretic types (PFGEs) and 28 different sequence types (STs) in ST405 (7, 15.9%), ST131 (3, 6.8%), ST73, ST101, ST372, and ST827 (2, 4.5% each) were the most prevalent. This data demonstrated a high level of diversity for the bla CTX-M-positive E. coli isolates. Additionally, bla NDM-5 was detected in three isolates (n = 3, 2.36%), comprised of two ST101 and one ST405 isolates, and mcr-1 was also observed in three colistin-resistant E. coli with three different STs (ST6316, ST405, and ST46). Our study demonstrates an increasing trend in MDR and ESBL-producing E. coli and this correlated with ß-lactam antibiotic usage for treatment of these animals. This data indicates that there is significant risk for the spread of resistant bacteria from pets to humans and antibiotic use for pets should be more strictly regulated.

Canine Influenza Virus A(H3N2) Clade with Antigenic Variation, China, 2016-2017.

During 2012-2017, we collected throat swabs from dogs in China to characterize canine influenza virus (CIV) A(H3N2) isolates. A new antigenically and genetically distinct CIV H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and replaced previously circulating strains. This clade probably poses a risk for zoonotic infection.