【Objective】 The aim of this study was to compare single nucleotide polymorphism (SNP) and gene differences in regions with significant genomic differences between groups of ducks with different egg production using mixed pool whole-genome re-sequencing and selective clearance analysis technology, and screen and identify genetic variation sites and functional genes related to egg production, so as to provide a molecular basis for improving egg-laying performance.【Method】 In this study, two extreme phenotypes of Jinding ducks were selected according to individual egg production within 150 days after the opening:A high-egg-production group (CH) and a low-egg-production group (CL). SNP and related functional genes that located in regions with significant genomic differences between two groups were screened based on mixed pool whole-genome re-sequencing and selective clearance analysis technology.The screened SNP related to egg production were verified by PCR sequencing of each sample.GO function and KEGG pathway enrichment analysis were performed on the selected candidate genes to determine the most important biochemical metabolic pathway and signal transduction pathway involved by the candidate genes.And differences in egg production among different genotype of each SNP were analyzed.【Result】 192 071 438 and 229 836 820 clean reads were obtained in CL and CH groups, respectively.There were 1 368 significantly different SNP fragment regions, and 214 candidate genes were selected.These intervals and genes were mainly located on chromosome Z, mainly including KDM4C, LURAP1L, PTCH1, PRUNE2, TRPM3 and VPS13AD genes.The verification results showed that the re-sequencing results were accurate.GO function analysis showed that these selected genes were involved in three ontologies:Molecular function, cellular component and biological process.KEGG pathway enrichment analysis showed that these genes were mainly enriched in metabolic pathway, regulation of actin cytoskeleton, ribosome biogenesis in eukaryotes and other signal pathways.Multiple mutations, Z-28286537, Z-28286879, Z-28288421, Z-28434122, Z-28436368 in the candidate gene KDM4C, Z-30802227 in LURAP1L gene, Z-36500134, Z-36503668, Z-36534782, Z-36684262, Z-36710928, Z-36732487 in TRPM3 gene, Z-37498270, Z-37513004 in VPS13A gene, and Z-41510597 in PTCH1 gene were affected the ducks egg production.【Conclusion】 There were multiple SNPs and genes significantly related to egg production on chromosome Z in ducks. These results provided a molecular basis for improving egg-laying performance and accelerating the breeding process in ducks.

【Objective】 The aim of this study was to analyze the biological characteristics of solute carrier 30A(ZnT) gene in Yorkshire pigs, and detect the expression of ZnT gene in different tissues of pigs, so as to provide a scientific basis for the study on the regulation of zinc metabolism by ZnT gene family.【Method】 Various bioinformatics software were used to analyze the sequence of ZnT gene family (ZnT1-ZnT10), including motif, conserved domain, transmembrane domain, evolutionary tree and protein interaction.Real-time quantitative PCR was used to detect the expression differences of ZnT gene family in heart, liver, spleen, lung, kidney, ovary and breast in Yorkshire pigs.【Result】 ZnT1-ZnT10 genes were located on 8 chromosomes of pigs, the molecular weight were 41.61-85.30 ku, and the isoelectric points were 6.27(ZnT1)-9.30(ZnT6).In the ZnT gene family, they all belonged to stable proteins except for ZnT3, contained 6-layer transmembrane domains except for ZnT5 and ZnT9, and the sequence of motifs were the same except for ZnT6 and ZnT9.Cation-efflux was a common conservative domain of ZnT gene family.ZnT6 and ZnT2, ZnT3, ZnT7 and ZnT9 proteins had the highest interrelationship, ZnT1 and ZnT3 proteins had the highest interrelationship, and ZnT8 had a distant relationship with other family members.The sequences of ZnT gene family were relatively conserved during mammalian evolution.The 10 genes of ZnT gene family had higher expression in lung and lower expression in heart of Yorkshire pigs, except for ZnT5, ZnT7 and ZnT9 genes, the other members of ZnT gene family were almost not expressed in muscle.【Conclusion】 ZnT family belonged to transmembrane structural proteins, and the motifs were basically in the same order.Cation-efflux was a common conserved domain of ZnT gene family.Most members of ZnT gene family had the highest expression in lung, and the low expression in heart and muscle of Yorkshire pigs.

With the rapid development of genome editing tools, many significant breakthroughs have been achieved in numerous fields.Among the current gene editing tools, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-based system is the most widely used in researches.CRISPR/Cas9 and its derived systems, base editor (BE) and prime editor (PE), enable researchers to easily and efficiently accomplish desired modification in genomes, such as deletion, insertion and substitution.CRISPR/Cas9-based systems have great potential in producing animals with specific genetic characteristics.Sheep are economically important livestock which has great potential as ideal large animal models for genome engineering.CRISPR/Cas9-based systems have been utilized in sheep genome engineering for generating superior agricultural breeds, expression of therapeutic agents in mammary glands, and developing animal models for human disease and regenerative medicine research.Starting from the principle of CRISPR/Cas9-based systems, the author focused on the specific process of genome editing using CRISPR/Cas9-based systems, and summarized the application of CRISPR/Cas9-based systems in research and production of sheep.

【Objective】 The purpose of this study was to investigate the role of African swine fever virus (ASFV) capsid E120R protein in virus infection, and identify the host proteins that could interact with E120R protein.【Method】 The E120R gene was synthesized according to ASFV CADC_HN09 isolate.The bait plasmid pGBKT7-E120R, truncated plasmids pGBKT7-E120R-1 (amino acids 1 to 61) and pGBKT7-E120R-2 (amino acids 62 to 122) of E120R gene were constructed.After toxicity and auto-activation detected, yeast two-hybrid BMDMs cDNA library was applied to screen the host proteins with E120R protein.Then the host proteins sequences were aligned via NCBI database and verified by co-immunoprecipitation assay.The candidate host proteins were preliminarily analyzed by GO function and KEGG pathway analysis by webgenstal online analysis website, so as to determine the biological process and signal pathway of the screened host proteins.【Result】 The bait plasmid pGBKT7-E120R-2 was non-toxic and not auto-activation, so it was available for cDNA library screening.A total of 46 initial positive clones were screened from the yeast two-hybrid BMDMs cDNA library and verified.After sequence alignment in NCBI database, a total of 29 potential interacting proteins were obtained.Both poly(C) binding protein 2 (PCBP2) and interferon-stimulated gene product 15 (ISG15) were identified to interact with E120R protein by co-immunoprecipitation assay.GO function enrichment analysis showed that these host proteins could participate in metabolic process, biological regulation, response to stimulus and other biological processes.KEGG pathway enrichment analysis indicated that these host proteins could be involved in antigen processing and presentation, ferroptosis, necroptosis and other pathways.【Conclusion】 ASFV capsid protein E120R could interact with a variety of host proteins involved in the host immune response and cell death, which would provide an important theoretical basis for further research on the role of ASFV E120R protein in the process of virus infection.

【Objective】 This study was aimed to perform bioinformatics analysis, transcription kinetics, eukaryotic expression and subcellular localization of Orf virus (ORFV) ORFV114 protein.【Method】 DNAStar software was used to perform alignment analysis of ORFV114 gene.The online websites ExPASy, TMHMM-2.0, SignalP-5.0, SOPMA and SWISS-MODEL were used to predict the physical and chemical properties, hydrophobicity, the presence of transmembrane helices and signal peptide, the structure of ORFV114 protein.In the presence or absence of cytarabine (Arac), HeLa cells were harvested at various time post infection.RT-PCR was used to amplify ORFV114 gene to determine the dynamic transcriptional level of ORFV114.The ORFV114 gene was amplified by PCR and subcloned into eukaryotic expression vector pEGFP-N1 to construct the recombinant plasmid pEGFP-ORFV114.After restriction enzyme digestion and Sanger sequencing identification, the pEGFP-ORFV114 plasmid was transiently transfected into HEK293 cells with Lipofectamine 3000.The expression of ORFV114-EGFP fusion protein in HEK293 cells was detected by Western blotting.HeLa cells were transiently transfected with pEGFP-N1 plasmid and pEGFP-ORFV114 recombinant plasmid.After 24 h, the nucleus was stained with Hoechst 33342, and the subcellular localization of ORFV114 protein was observed under an inverted fluorescence microscope.【Result】 The ORFV114 gene of ORFV-JS strain was 1 041 bp, which was highly conserved among ORFV strains.The ORFV114 protein was 346 amino acids in length, with predicted molecular masses 38 ku.It was a weakly hydrophilic unstable protein without transmembrane region nor signal peptide.Its secondary structure was composed of random coil, alpha helix, extended chain and beta turn, which were 45.09%, 25.14%, 21.68% and 8.09%, respectively.Transcription of ORFV114 was detected as early as 2 h post infection, and the levels of expression increased during the infection cycle in the presence or absence of Arac.The results indicated that Arac did not inhibit the transcription of ORFV114, that was, ORFV114 belonged to an early gene of ORFV.The pEGFP-ORFV114 recombinant plasmid was successfully constructed and ORFV114-EGFP fusion protein with a molecular mass of 65 ku was successfully expressed in HEK293 cells.The ORFV114 protein mainly localized to the cytoplasm of HeLa cells, exhibiting a punctate distribution pattern in this compartment.【Conclusion】 In this study, the transcription kinetics, eukaryotic expression and subcellular localization of ORFV114 protein of ORFV were successfully performed, laying a foundation for further research on ORFV114 protein function and its interaction proteins.

【Objective】 The aim of this study was to prepare a polyclonal antibody against chicken ring finger protein 165 (RNF165) and perform bioinformatics analysis on RNF165 protein, in order to provide references for studying the biological function of chicken RNF165.【Method】 Using chicken embryo fibroblasts (DF-1) as the test material, total RNA was extracted from DF-1 and reverse transcribed to obtain cDNA. The RNF165 gene was amplified by PCR, and it was connected to the pET-28a (+) plasmid to construct the recombinant expression plasmid pET-28a-RNF165. The correctly sequenced recombinant expression plasmid was transformed into E. coli BL21 competent cells for induced expression, and the expressed fusion protein was purified and immunized 7-week-old BALB/c female mice according to the established immunization program. Seven days after the third immunization, the mouse serum was separated, and the specificity of mouse anti-RNF165 protein polyclonal antibody was detected by Western blotting, and the titer was determined by indirect ELISA. The physicochemical properties, signal peptide, phosphorylation site, subcellular localization, transmembrane structure, secondary structure and conserved domain of RNF165 protein were analyzed using online bioinformatics softwares.【Result】 The RNF165 gene with a size of 1 068 bp was successfully amplified. The prokaryotic expression vector pET-28a-RNF165 was successfully constructed, and the RNF165 protein of about 43 ku was induced. Western blotting results showed that the prepared mouse anti-RNF165 protein polyclonal antibody could specifically recognize the overexpressed RNF165 protein in DF-1 cells with a titer of 1:32 000. The results of bioinformatics analysis showed that RNF165 protein was composed of 350 amino acids, with a molecular weight of 39.88 ku, an isoelectric point of 7.13, and an instability index of 69.87. RNF165 protein was a hydrophilic protein without signal peptide and transmembrane domain, and it contained 29 phosphorylation sites. The secondary structures of RNF165 protein was mainly composed of random coil, alpha helix, extended chain and beta turn. The highest probability of RNF165 appeared in the nucleus was 47.8%. The amino acid residues 296-341 of the C-terminal of RNF165 protein were conserved domains of the RING-Ubox superfamily.【Conclusion】 The mouse anti-RNF165 polyclonal antibody that was prepared in this study had high reactivity and specificity. RNF165 protein was a hydrophilic protein and was mainly expressed in the nucleus. The results could provide materials for exploring the biological function of RNF165 protein.

【Objective】 The aim of this study was to express avrA protein of Salmonella Enteritidis which had biological activity and analyze its bioinformatics, so as to provide references for studying the influence of avrA protein of Salmonella Enteritidis on the immune function of porcine intestinal cells.【Method】 Primers were designed according to the complete gene sequence of avrA protein of Salmonella Enterititis in GenBank (gene ID:1254388), and avrA gene was amplified by PCR.After recombination and cloning of the recovered target fragment and expression vector PGEX-4T-1, the correctness of the recombinant expression plasmid was verified by restriction enzyme and gene sequencing.The verified recombinant expression plasmids were transformed into Escherichia coli Rosetta 2(DE3) competent cells, and the recombinant expression strains were induced to express under different conditions.The expression of avrA protein was detected by SDS-PAGE and Western blotting.The target gene sequences were analyzed by BLAST, and the amino acid composition of avrA protein was predicted by ExPASy-Translate Tool software.The transmembrane domain, secondary structure, tertiary structure and antigenicity of the target protein were predicted by bioinformatics softwares.【Result】 The total length of CDS region of avrA gene was 909 bp, encoding 302 animo acids.The recombinant expression plasmid PGEX-4T-1-avrA was successfully constructed by restriction enzyme and gene sequencing analysis.The results of SDS-PAGE and Western-blotting showed that avrA protein expressed by recombinant bacteria was soluble protein.The protein content of recombinant bacteria induced under 15 ℃ for 16 h was 10 mg/L and the protein solubility was 40%.Bioinformatics analysis showed that avrA protein was an extracellular protein, and amino acids 16-301 of avrA protein had a conserved domain from the superfamily YopJ serine/threonine acetyltransferase.In the secondary structure of avrA protein, random coil, alpha helix and extended chain were accounted for 43.71%, 39.40% and 16.89%, respectively.The predicted tertiary structure of avrA protein was consistent with the predicted secondary structure. AvrA protein had 9 B cell-binding epitopes at amino acid positions 5-40, 51-68, 80-92, 94, 101-109, 146-157, 160-168, 199-231, 236-298, respectively.【Conclusion】 avrA gene of Salmonella Enteritidis was successfully cloned, and the recombinant expression plasmid and expression bacteria were successfully constructed.The induced expressed avrA protein was soluble.AvrA protein was an extramembrane protein with 9 epitopes that could bind to B cells.The results of this study could provide references for the preparation of antibodies to detect Salmonella Enteritidis infection.

【Objective】 This study aimed to determine whether selenoprotein glutathione peroxidases 4 (GPX4) could regulate inflammatory responses affect in RAW264.7 macrophages upon lipopolysaccharide (LPS) stimulation and the underlying molecular mechanisms.【Method】 RAW264.7 macrophages were cultured in vitro.DMSO was used as the control, and 0.1-5.0 μmol/L GPX4 inhibitor FIN56 was used to treat RAW264.7 macrophages.The optimal concentration of GPX4 inhibitor FIN56 on RAW264.7 was determined by CCK-8 method and Western blotting.RAW264.7 was divided into 4 groups:Control group, RAW264.7 was treated with DMSO for 24 h;FIN56 group, RAW264.7 was treated with 0.5 μmol/L FIN56 and cultured for 24 h;DMSO-LPS group, RAW264.7 was treated with DMSO and then stimulated with 100 ng/mL LPS for 3 h;FIN56-LPS group, RAW264.7 was treated with FIN56 for 24 h then stimulated with 100 ng/mL LPS for 3 h.After cultured, the reactive oxygen species (ROS) level was detected by H₂DCFDA, the cellular malondialdehyde (MDA) level was detected by the kit method, the expression of IL-1β, IL-6, and TNF-α mRNA was analyzed by Real-time PCR and the levels of cytokines in supernatants were detected by ELISA.Toll-like receptor 4 (TLR4) relative protein expression levels was detected by Western blotting.【Result】 Compared with DMSO treatment, cell viability had no significantly varied (P>0.05), while GPX4 protein level was significantly downregulated by 0.5 μmol/L FIN56 pretreatment for 24 h on macrophages (P<0.05).Therefore 0.5 μmol/L FIN56 was used for subsequent assay.Compared with control group, both FIN56 and LPS significantly increased ROS production (P<0.05), while compared with DMSO-LPS group, FIN56 resulted in a remarkable increase in ROS production(P<0.05).Compared with control group, LPS could significantly increase the gene expression of IL-1β, IL-6, TNF-α and up-regulated IL-6 production in macrophages, and the phosphorylation levels of JNK and c-Jun (P<0.05).While FIN56 could significantly down-regulated LPS-induced IL-6 gene expression and production (P<0.05), and decreased JNK and c-Jun phosphorylation levels (P<0.05), but had no significant effect on the protein expression levels of p38 and Erk1/2(P>0.05).【Conclusion】 Treatment of GPX4 inhibitor significantly suppressed LPS induced-cinflammatory response via JNK1/3 and c-Jun phosphorylation in macrophages.The findings of the study would provide a basis for anti-inflammatory or cytoprotective therapy.

【Objective】 The aim of this experiment was to find a Bacillus coagulans strain with stable performance and strong environmental resistance, so as to play its role instead of antibiotics in actual production.【Method】 The fecal samples from 160-day old healthy Hyline Brown laying hens were collected for isolation and purification of bacteria, and morphological characteristics observation, physiological and biochemical identification, 16S rDNA sequencing analysis and identification of the isolated bacteria were carried out.The growth rule was determined by plotting the growth dynamic curve of the bacterium.Acid and bile salt resistance tests were used to analyze the resistance of the strain.The culture conditions of isolated strains were optimized by single factor experiment.【Result】 The isolated bacteria grew gray white colonies with rough and irregular surface on the ordinary culture medium, and were rod-shaped and spore terminal Gram-positive bacteria under microscope.Combined with biochemical experiments and 16S rDNA sequence analysis, the strain was identified as Bacillus coagulans, named NJ001.The growth dynamic curve showed that 7-24 h was the logarithmic growth period of the strain, and the number of viable number reached the peak at 24 h.The optimal culture time of Bacillus coagulans NJ001 was 24 h.The results of tolerance test showed that the survival rate was 59.5% at pH 3.0 for 2 h, and 87.6% at pH 4.0, showing strong resistance to acid.The survival rate was 54.0% after treatment with 0.3% bile salt for 2 h, and the survival rate was still more than 50% when the concentration of bile salt increased to 0.5%.The results of single factor experiment showed that the optimum culture temperature was 40 ℃, inoculation amount 2%, initial pH 7.0 and shaking speed was 220 r/min.Based on the above optimum cultivation conditions, the maximum viable count of the strain was 1.93×10⁸ CFU/mL.【Conclusion】 The isolated strain was Bacillus coagulans with strong acid and bile salt resistance, and its optimal growth conditions which was basically consistent with the gastrointestinal environment of animals were easy to achieve, so it had the potential of application as a microecological agent.

Threonine is the third limiting amino acid for poultry, which has important regulatory effects on growth and development, intestinal health, immune function and fat metabolism of Pekin ducks.Peking duck is a meat duck breed bred independently in China.It has the characteristics of fast growth and good fattening performance, and is the raw material for the famous "Peking Roast Duck".The author summarizes the research of threonine on the nutritional and physiological functions of Peking duck in recent years, including the effect of threonine on growth performance, slaughter performance, immune function, intestinal health and fat metabolism in different strains of Peking duck, and the mechanism of threonine regulation on lipid metabolism.Based on the research results of threonine requirement in recent years, the threonine requirements are evaluated with the quadratic curve break-line model and the straight-line-break line model, to meet daily weight gain, average daily feed intake, breast muscle rate, feed-to-weight ratio.It is recommended that the threonine requirement to meet the maximum daily weight gain and meat production of 1-21 day old Peking duck is 0.60%-0.67%.However, there is little research on the threonine requirement of Peking duck in the late growth stage, and further research is needed.In addition, it is necessary to further study the molecular mechanism of threonine regulating fat metabolism, intestinal health and immune function in Peking duck.

Silage is an indispensable part of animal feed.However, once the silage is opened and in contact with the air, inevitably causing aerobic deterioration to a certain extent.Aerobic deterioration of silage would cause great losses to feed industry and animal husbandry, and seriously restrict the development of animal husbandry.Therefore, it is necessary to study the measures to prevent aerobic deterioration.The authors had fully investigated the relevant studies at home and abroad on measures to prevent aerobic deterioration of silage, and on this basis, firstly, a systematic description on the causes, hazards, related microbial changes (mold and yeast) and their impact on the quality of silage were summarized.And then, physical method (the moisture of raw material, length and storage density), chemical method (effective components in the chemical agents as acid or salt), and biological fermentation method(with enzymes (such as cellulase and hemicellulase), microbial agents (lactic acid bacteria and other probiotic bacteria), and biological active substances) and other existing silage aerobic deterioration prevention methods were reviewed, of which, the research progress on lactic acid bacteria, especially heterofermentative lactic acid bacteria and their metabolites alone and combined with other methods in preventing aerobic deterioration was focused on described.Finally, a summary of the prevention of aerobic deterioration for important silage materials including corn, straw and forage were presented, as well as suggestions and perspectives of research priorities and preventive and control measures for aerobic deterioration in the future were offered.The aim of this paper was to provide a reference for solving the problem of aerobic deterioration of silage, establishing efficient measures to prevent aerobic deterioration, improving feed quality and even promoting the development of animal husbandry.

【Objective】 The aim of this study was to recombine and express laccase gene of Ganoderma lucidum L strain in Aspergillus niger (A.niger), and optimize its fermentation conditions, so as to obtain a genetic engineering strain with high yield of G.lucidum laccase.【Method】 Lac-L was cloned from G.lucidum L and optimized according to codon preference of A.niger.The promoter, signal peptide and terminator of A.niger glucoamylase (glaA) were selected as the expression source, and the pyrG gene of Aspergillus oryzae was selected as the screening marker.The laccase expression cassette was constructed and transformed into A.niger X1 (ΔpyrG) protoplast by polyethylene glycol-CaCl₂ method.PCR verification and enzyme activity determination of the transformants were performed to screen recombinant strains with high laccase production, and the carbon source, the ratio of inorganic and organic nitrogen sources, the concentration of Cu²⁺ and fermentation time of the solid-state fermentation medium for positive transformants were optimized.【Result】 Twenty-five recombinant laccase transformants were screened by uracil deficiency and 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt)(ABTS) medium, and the heterologous expression strain 6 of G.lucidum laccase was obtained by enzyme activity rescreening.The enzyme activity reached 3 532.6 U/kg after 72 h fermentation, while it was only 58.37 U/kg in the host strain.The optimal solid-state fermentation conditions for recombinant strain 6 were as follows:Wheat bran was used as the basic medium, adding 2% maltose, 2% (NH4)₂SO₄ and 0.25 g/L CuSO₄, and the laccase activity of the recombinant strain reached 6 255.47 U/kg after 60 h of fermentation.【Conclusion】 The heterologous expression of laccase gene Lac-L from G.lucidum L strain was successfully realized in A.niger.

Zinc is an essential micromineral for physiological processes such as the growth, development, and breeding in animals, and it ranks only second to iron micromineral according to micronutrient requirements.Zinc is widely distributed with tissue differences and is mainly absorbed in jejunum and ileum of animal.Both zinc-regulated transporter-like proteins (Zip) and zinc transporter (ZnT) of Zinc transport proteins, and metallothionein involve in zinc balance and metabolism in animal.Zinc is the component and the activity factor of many enzymes in animal, it on the one hand regulates cell processes as the constituent of enzyme, and on the other hand involves signal transduction as the signal molecule to regulate animal physiological functions of feed intake, redox, immune, metabolism, reproduction.At present, dietary zinc mainly includes inorganic, organic, and nano-zinc styles, which display different biological value.Dietary zinc is associated with physiological functions on feed intake, redox, immune, metabolism, reproduction of pigs, while zinc requirement varies with swine nutrient requirements during different growth stages.In addition, physiological regulation and applicative effects of dietary zinc display variations due to zinc levels and sources.The purpose of this article is to provide theoretical references for the deep excavation of microelement zinc, thereby promoting the healthy development of pig industry.

【Objective】 The aim of this study was to investigate the colonization ability of Clostridium butyricum in the digestive tract of broilers and the effects of Clostridium butyricum colonization on intestinal short-chain fatty acid content and microflora diversity of broilers.【Method】 90 one-day-old Abaric broilers with good health were randomly divided into blank group and Clostridium butyricum group with 3 replicates per group and 15 broilers per replicate. The blank group was fed with normal saline, while the Clostridium butyrate group was given fluorescently labeled Clostridium butyrate. At 3 h and 16 days of age, ileum epithelial cells were dissected and collected to observe the fluorescence. Then 30 one-day-old chicks with good health were randomly divided into control and experimental groups. The experimental group was fed the diet containing Clostridium butyricum from 1 to 6 days, followed by the basal diet, while the control group was fed the basal diet throughout the whole period, the period was 21 days. At 16 and 21 days of age, the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid and total short-chain fatty acid in ileum and cecum were determined. The difference analysis of microbial species, species abundance ratio, bacterial species and protein functional abundance were carried out.【Result】 There was fluorescence around the ileum epithelial cells in Clostridium butyricum group at 3 h and 16 days of age, indicating that Clostridium butyricum could colonize gastrointestinal ileum epithelial cells. The results of short-chain fatty acid content determination showed that compared with control group, the contents of acetic acid, propionic acid, isobutyric acid, iso-valeric acid and valeric acid in ileum of broilers at 16 days of age in experimental group were extremely significantly increased (P<0.01). The contents of propionic acid, iso-valeric acid and valeric acid in ileum of broilers at 21 days of age were extremely significantly increased (P<0.01), while the contents of acetic acid、isobutyric acid and total short-chain fatty acid were significantly increased (P<0.05). The contents of butyric acid and valeric acid in cecal of broilers at 16 days of age were extremely significantly increased (P<0.01), while the contents of iso-valeric acid and caproic acid were significantly increased (P<0.05). Compared with control group, the contents of butyric acid and isovaleric acid in cecum of broilers at 21 days of age were extremely significantly increased (P<0.01), while the contents of acetic acid, isobutyric acid, caproic acid and total short-chain fatty acid were significantly increased (P<0.05). The results of microbial species difference analysis at phylum level showed that Firmicutes at 16 days of age were the dominant flora with the highest abundance, and the number of microbial species was increased at 21 days of age samples, and Proteobacteria, Firmicutes and Bacteroidetes were the dominant bacterial groups with high abundance, and the dominant bacteria at 16 and 21 days of age experimental groups were higher than those in control group. The results of species abundance ratio analysis showed that compared with control group, the species abundance ratio of Anaerolineaceae at 16 days of age experimental group was significantly increased (P<0.05), while the proportions of 7 genera, such as Defluviicoccus, Comamonas, Enterobacter were significantly decreased (P<0.05). At 21 days of age in experimental group, the species abundance ratio of 6 genera, such as Ferribacterium, Trichococcus were increased significantly (P<0.05), while Proteobacteria, Plancyomycetes, and Armatimonadetes GP5 were significantly decreased (P<0.05). The results of genus difference analysis showed that the most important microorganisms in experimental group at 16 days of age were Blautia, while the most important microorganisms in control group were Clostridium and unclassified Lachnospiraceae. At 21 days of age, the most important microorganisms in experimental group were unclassified Bacteroidetes, Trichococcus, Lactobacillales and Lactococcus, while the most important microorganisms in control group were norank Acidobacteria GP4 and unclassified Comamonadaceae. The results of functional abundance analysis of flora proteins showed that the abundance of methyl-accepting chemotaxis protein, glutathione S-transferase and amino acidotransferase subunit A at 16 days of age experimental group were higher than those in control group, and the abundance of ECF subfamily RNA polymerases δ-70 factor and alanine at 21 days of age experimental group were higher than those in control group.【Conclusion】 Clostridium butyricum could colonize the ileum epithelial cells of broilers. Clostridium butyricum could increase the content of short-chain fatty acids, microbial species and beneficial bacteria genera in intestinal contents, and improve the abundance of species and functional proteins, so as to promote intestinal function.

There are complex microbiota in animal intestinal tract, and suitable microenvironment is beneficial to the colonization and growth of gut microbiota.Intestinal health is the basis for ensuring the health of animals, and it is also the current domestic and international research hot spot.As a beneficial microorganism for intestinal health, probiotics have great potential in improving animal intestinal health.Probiotics can inhibit the invasion of pathogenic microorganisms, and have a certain prevention and clearance effect on some viruses, but different strains have different effects.The author firstly summarized four protection modes of probiotics on intestinal health:Inhibiting pathogenic bacteria invasion and colonization, improving intestinal barrier function, maintaining intestinal healthy microbiota and improving immunity, and discussed the action modes of different strains.Secondly, the mechanism of probiotics antiviral was briefly described.Among them, probiotics regulate indirectly immune function is the main way of antiviral.Finally, the research progress on the application of probiotics in Rotavirus, Epidemic diarrhea virus and Infectious gastroenteritis virus in recent years were discussed, and the development prospect of probiotics was prospected, so as to provide certain reference for the research of probiotics in improving animal intestinal health and the development of products.

【Objective】 This study was aimed to analyze the influence of three fixed effects of parity, breeding season and breeding boar on gestational length in Yorkshire sows, estimate the genetic parameters of gestational length, and explore the relationship between gestational length and total number born, number born alive, total litter weight, number of stillbirth and number of mummy.【Method】 A total of 4 261 records with four parities (1-4) of 2 138 Yorkshire sows were collected, including gestational length, total number born, number born alive, total litter weight, number of stillbirth and number of mummy.A general linear model was used to analyze the effect of the fixed effects on gestational length in Yorkshire sows, and a multi-trait repeatability model was used to estimate the genetic parameters (heritability, repeatability, genetic correlation, phenotypic correlation) between gestational length and other reproductive traits.【Result】 The average gestational length of Yorkshire sows was 116.08 d, which was significant difference from 114 d according to the analysis of t test (P<0.05).The parity had a significant effect on gestational length, in which, the gestational length of the first pregnancy was significantly shorter than that of the second pregnancy (P<0.05).The gestational length was extremely significantly affected by breeding seasons (P<0.01).Sows bred in spring had the shortest gestational length, and sows bred in autumn had the longest gestational length, with a difference of 0.73 d (P<0.05).There was a significant difference in gestational length of sows after mating with different boars (P<0.05), and the maximum difference reached 3.05 d.The heritability of gestational length was 0.1603, which showed the negative phenotypic and genetic correlation with total number born, number born alive, total litter weight, number of stillbirth and number of mummy.【Conclusion】 The gestational length was affected by many factors and hereditary.Gestational length was significantly related to reproductive traits such as the total litter size of sows, and it could be used as an indirect index of swine production.Gestational length was of great significance to accurately calculate the prediction of the due date of sows and realize accurate feeding management.

【Objective】 The purpose of this study was to explore the relationship between insertion/deletion (InDels) mutation of KAP gene family keratin-associated protein 9.2 (KAP9.2) gene and growth traits of Inner Mongolia cashmere goat, in order to provide a theoretical basis for molecular breeding of goats.【Method】 A total of 606 female Inner Mongolia cashmere goats were selected as the research objects, the ear tissue was collected to extract DNA, and their body weight and body size, such as body height, length and chest circumference were measured.The InDel mutation of KAP9.2 gene was detected by agarose gel electrophoresis and direct sequencing technology, and genetic diversity indicators were detected.The association between mutation locus and growth traits was analyzed by SPSS 23.0 software.【Result】 There was a 30 bp InDelmutation in the exon region of the KAP9.2 gene in Inner Mongolia cashmere goats, which produced three genotypes of II, ID and DD.The polymorphism information content (PIC) showed that the locus was moderately polymorphic (0.25<PIC<0.50) in the breeding goat population, low polymorphism (PIC<0.25) in the adult goat population, and both were in Hardy-Weinberg equilibrium (P>0.05).In the whole population, the locus was low polymorphism (PIC<0.25), but deviated from Hardy-Weinberg equilibrium (P<0.05).The results of association analysis showed that in the breeding goat population, the 30 bp InDel mutation had significant effect on body length and height at hip cross (P<0.05), and had extremely significant effect on body height, chest depth and chest width (P<0.01).In the adult goat population, this mutation had extremely significant effect on cannon circumference, chest depth and chest width (P<0.01).Further analysis found that this mutation had significant effect on body length and height at hip cross (P<0.05), and had extremely significant effect on body height, chest depth and chest width (P<0.01) in the whole population.【Conclusion】 The InDel mutation of KAP9.2 gene had significant or extremely significant effect on many growth traits of Alashan-type Inner Mongolia cashmere goat, and could be used as a molecular marker for the breeding of excellent traits of Inner Mongolia cashmere goat.

【Objective】 This study aimed to explore the polymorphism of methionine sulfoxide reductase B3 (MSRB3) gene and its association with antler weight trait.【Method】 8 sika deer with high and 8 sika deer with low antler yield were selected, and the gene mutation sites were detected by direct DNA sequencing. The genetic variation of MSRB3 gene in 314 24-month-old sika deer was genotyped by MassARRAY^® SNP typing technology, and the association analysis and haplotype analysis were carried out combined with the data of antler weight traits of sika deer.【Result】 A total of 6 SNPs were found in MSRB3 gene of sika deer, of which 2 SNPs were located in the exon region, and the mutations did not cause amino acid changes, belonging to synonymous mutations, and the other 4 SNPs were located in the intron region.The typing results showed that 4 samples were not successfully typed, and the remaining 310 samples were subjected to follow-up analysis.The observed heterozygosity of each locus was basically consistent with the expected heterozygosity.The heterozygosity of g.44455582 T>C, g.44455759 C>T, g.44414424 T>C, g.44350306 T>C and g.44340836 G>A were higher, which showed moderately polymorphic (0.25<PIC<0.5).And g.44340734 G>C locus had low heterozygosity, which showed low polymorphism (PIC<0.25).The results of chi-square test showed that g.44455759 C>T deviated from Hardy-Weinberg equilibrium (P<0.05), and the other 5 loci were in Hardy-Weinberg equilibrium (P>0.05).The results of association analysis between the different genotypes of 6 SNPs and antler weight of sika deer showed that the antler weight of TT genotype with g.44455582 T>C locus was extremely significantly higher than that of CC and TC genotypes (P<0.01).The haplotype analysis results showed that g.44340734 G>C and g.44350306 T>C, g.44455582 T>C and g.44455759 C>T were strongly linked, each producing 3 haplotypes.The antler weight of haplotype TC in Block 2 was significantly higher than that of haplotype CT (P<0.05).【Conclusion】 MSRB3 gene was closely related to the antler weight traits of sika deer, g.44455582 T>C locus and haplotype TC could be used as molecular markers for screening high-yield sika deer.

Rabbits are stimulated ovulating animals and require intercourse to stimulate the release of gonadotropin-releasing hormone (GnRH), which then triggers an increase in luteinizing hormone (LH), leading eventually to ovulation of mature oocytes, but the mechanism of ovulation in rabbits is not fully understood.In artificial insemination of rabbits, intramuscular or intravaginal administration of GnRH and its analogues is used to induce ovulation due to the lack of stimulation by intercourse.But these compounds usually have detrimental effects, so a new approach is needed to replace hormonal stimulation.β-nerve growth factor (β-NGF) is one of the most promising substances recently identified to play a role in female reproductive physiology.The widespread presence of β-NGF and its receptors in the reproductive system of rabbits, and has an important physiological role. At the same time, β-NGF, a protein that is itself present in the semen of animals, is likely to be a natural ovulation-promoting factor in rabbits.Therefore, the use of β-NGF may be able to reduce the use of hormones or other drugs.In this regard, in order to provide ideas and theoretical basis for studying the mechanism of ovulation stimulation in rabbits and to promote the application of β-NGF in assisted reproduction techniques in rabbits, the authors briefly describes the role of β-NGF in promoting ovulation in rabbits and the current status of research, mainly from the expression of β-NGF and its receptors in the reproductive system of rabbits, the species specificity of the β-NGF system in inducing ovulation in rabbits, and the mechanism of action of β-NGF in inducing ovulation in rabbits.

【Objective】 The purpose of this experiment was to analyze the molecular characteristics and differentiation process of main cell types in the morphogenesis of the secondary hair follicles in Erdos fine wool sheep during the embryonic stage.【Method】 HE staining was performed on skin samples from the lateral part (posterior edge of scapula) of 87 fetal age of 3 Ordos fine wool sheep to identify the development period of hair follicles.Single-cell transcriptome sequence (scRNA-Seq) was performed on some skin samples after mixing.Cell clusters were analyzed by t-distributed stochastic Neigh or embedding (tSNE), and dermal lineage cells and epidermal lineage cells were identified by Col1a1 and Krt15 genes, respectively.The marker genes of different skin cells were used for cell type analysis.The sequence data were analyzed to explore the expression of differential genes in the process of differentiation.The function of the gene was further verified by GO function enrichment analysis.【Result】 HE staining results showed that Ordos fine wool sheep were in the secondary hair follicle induction stage at 87 days of gestational age. 10 603 cells and 18 704 genes were obtained by scRNA-Seq in skin cell samples from the body side of fine wool sheep at 87 days of gestational age could be used for downstream analysis.Based on tSNE analysis, 15 cell clusters were found in scRNA-Seq data.The identification of Col1a1 and Krt15 marker genes showed that dermal and epidermal cells were highly heterogeneous.The differentiation trajectories and dynamic gene expression profiles of dermal/epidermal cell lineage in the process of hair follicle morphogenesis were constructed by quasi-sequential analysis, during the differentiation of dermal lineate cells from fibroblast (Fb) to mature dermal condensates (DC), several different stage marker genes FST recombinant protein (Fst), inhibin subunit βα(Inhba) and transcriptional repressor GATA binding 1(Trps1) were specifically expressed on the quasisequential track, and Wnt, Noggin, bone morphogenetic protein (BMP) and other signaling pathways related to hair follicle morphogenesis were enriched.During the differentiation of epidermal lineage cells, the marker genes Krt10, HOXC13 and SHH of matrix and interfollicle epidermis (IFE) were specifically expressed in both branches of the quasi-sequential trajectory of epidermal lineage cells, and the pathways related to cell proliferation and cell adhesion were enriched.【Conclusion】 The results showed that dermal lineage cells differentiated from Fb to DC during the induction of the secondary hair follicles in fine wool sheep.Epidermal lineage cells were in the stage of proliferation and differentiation of epidermal cells between matrix and hair follicle.The results of this study could enrich people's understanding of the morphogenesis of the secondary hair follicles in fine wool sheep, and provide strong theoretical and technical support for the breeding of Ordos fine wool sheep.

【Objective】 The objective of this study was to clone and bioinformatic analysis of myostatin (MSTN) gene in Tibetan sheep, and investigate its expression in different tissues of Tibetan sheep, so as to provide a referencce for exploring the biological function of MSTN gene in Tibetan sheep.【Method】 Using cDNA of longissimus dorsi muscle of Tibetan sheep as template, the complete CDS region of MSTN gene of Tibetan sheep was cloned and sequenced, and the sequencing results were spliced by SeqMan program, and the assembled sequence was analyzed and identified by BLAST online procedure.Similarity comparison, phylogenetic tree construction and bioformztics analysis were carried out by bioinformatics softwares, and the expression of MSTN gene in different tissues of Tibetan sheep was detected by Real-time quantitative PCR.【Result】 The CDS length of MSTN gene in Tibetan sheep was 1 128 bp and encode 375 amino acids.The amino acid sequence similarity of MSTN gene in Tibetan sheep were 100.0%, 93.4%, 93.4%, 95.2%, 94.4%, 94.2%, 94.4%, 93.1%, 87.8% and 87.5% with Ovis aries, Bos mutus, Bos taurus, Sus scrofa, Macaca mulatta, Homo sapiens, Pan troglodytes, Canis lupus familiaris, Gallus gallus and Maylandia zebra.The results of phylogenetic tree analysis showed that Tibetan sheep was the closest to Ovis aries and the furthest to Maylandia zebra and Gallus gallus. MSTN protein of Tibetan sheep was a hydrophilic secretory protein, and had instability, without transmembrane structure, contains 1 signal peptide, has 31 potential phosphorylation sites and 2 N-glycosylation modification sites, it was mainly distributed in mitochondria and cytoplasm.The secondary structure of MSTN protein was consistent with the prediction of tertiary structure, mainly random coil, followed by α-helix, extended chain and β-turn, which was a mixed protein.Real-time quantitative PCR results showed that MSTN gene was expressed in hypothalamus, pituitary, heart, liver, spleen, lungs, kidneys, rumen, duodenum, triceps brachio, semitendinosus, quadriceps femoris and longissimus dorsi, and the MSTN gene was expressed in different tissues of Tibetan sheep, and the expression of MSTN gene in triceps brachii and semitendinosus muscle was significantly higher than that in lung, hypothalamus, heart, liver, duodenum, rumen and kidney(P<0.05).【Conclusion】 MSTN gene was successfully cloned from Tibetan sheep, and its expression in muscle was higher than that in visceral tissue, which laid a foundation for further study of the regulatory mechanism of MSTN gene on muscle growth and development in Tibetan sheep.

Intramuscular fat (IMF) is a key indicator of pork quality, which can affect the tenderness, shear force, juiciness and flavor. microRNA (miRNA) is a class of short non-coding RNA with a length of about 22 nt, which plays an important role in various physiological processes such as embryonic development, adipogenic differentiation, muscle fiber formation, neuroregulation and immune response.More and more studies have shown that miRNA plays an important regulatory role in porcine IMF deposition and is an important regulator of lipid metabolism.By summarizing relevant studies at home and abroad on the regulation of porcine IMF deposition by miRNA, the authors found that miR-34a, miR-125a-5p, miR-32-5p could regulates porcine IMF deposition by targeting Krüppel-like factors (KLF) family members, miR-130a regulates porcine IMF deposition by targeting peroxisometer-activated receptor(PPAR) family members, miR-34a, miR-17-5p and miR-125A-5p can target other family members, such as acyl-CoA synthetase long chain family member 4(ACSL4) and Nuclear receptor coactivator 3 (NCOA3) regulate porcine IMF deposition.However, the specific mechanism of miRNA regulating porcine IMF is not fully understood and needs to be further explored.By sorting out the miRNAs that have been confirmed to be related to porcine IMF deposition, the authors sorted out the target genes and main pathways of related miRNAs, in order to provide convenience for screening IMF related miRNAs, provide new ideas and strategies for improving meat quality, and provide reference for elucidating the mechanism of miRNA in porcine lipid metabolism.

【Objective】 The aim of this study was to explore the effects of adding soybean lecithin instead of 10% egg yolk to the frozen diluent on the cryopreservation of semen of sika deer, in order to provide references for the improvement of the artificial insemination system of sika deer.【Method】 The semen of sika deer was collected by electrical stimulation, and 1%, 2%, 3%, 4% and 5% soybean lecithin was added to the frozen diluent to replace 10% egg yolk as the experimental group, and 20% egg yolk was added as control group, respectively.Cryopreservation of semen in each group was performed.After 5 days, the semen was thawed, and the sperm motility, plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity, and survival time of each group were detected after thawing, and soybean lecithin of suitable concentration was screened.Healthy female sika deer aged 4-5 years were selected, intramuscularly injected with 300 IU PMSG and 0.4 mg cloprostol sodium for simultaneous estrus treatment, and artificial insemination was performed with frozen semen in the 20% egg yolk group and the selected soybean lecithin group at 20 h after estrus, 30 days after insemination, B-ultrasound detector was used to detect the pregnancy, and count the pregnancy rate.【Result】 Compared with control group, the sperm motility, forward motility, fast-forward motility, motility, plasma membrane integrity rate, acrosome integrity rate and mitochondrial activity were significantly improved in 1% soybean lecithin group after freezing and thawing (P<0.05).With the increase of the concentration of soybean lecithin in the diluent, the sperm motility, forward motility, fast forward motility, motility, plasma membrane integrity rate, acrosome integrity rate and mitochondrial activity showed a downward trend, and sperm survival time also decreased with the increasing concentration.The pregnancy rate of sika deer in 1% soybean lecithin group was 61.11% after artificial insemination of frozen-thawed sperm, which was higher than that of control group, 2% and 3% soybean lecithin groups, but the difference was not significant (P>0.05).【Conclusion】 Adding 1% soybean lecithin to replace 10% egg yolk in the frozen diluent of sika deer sperm could effectively improve the quality of the frozen and thawed semen of sika deer, and provide a theoretical basis for further screening of new frozen diluents of sika deer semen.

The follicle is fundamental for female mammals to exert their reproductive capacity, and its development is a dynamic process that mainly involves the formation of primordial follicles, the recruitment of follicles, the selection of dominant follicles, the ovulation of mature follicles and the luteinization of follicles after ovulation.Follicle development is regulated by complex physiological activities, such as the endocrine system, autophagy, apoptosis, and so on.Aautophagy is an evolutionarily conserved stress-responsive process that forms autophagosomes by enveloping intracellular materials and delivering them to lysosomes for degradation to help cells maintain the intracellular material metabolic balance, which plays an important role in the process of follicle development, on the one hand it can alleviate the follicle damage caused by stress by degrading or recycling damaged proteins or harmful metabolites; On the other hand, it can cause follicular atresia by generating a large number of autophagosomes for excessive degradation of organelles.The regulation of follicular development by autophagy requires the involvement of multiple canonical signaling pathways, such as PI3K-Akt-mTOR, MAPK-ULK1, ERK1/2 and Sirt1-FOXO1-Atg7, which have been shown to regulate the physiological activity of follicle cells by promoting or inhibiting autophagy through independent actions or interactions under the stimulation of stresses, such as hormones, oxidative stress, and cell starvation.Different levels of autophagy are currently known to have different effects on the survival of follicle cells, but there are still relatively few studies on the level of autophagy that determines whether a cell can survive.In addition, studies on the regulation of follicular development by autophagy have focused on granulosa cells, while the role of oocyte maturation and follicular membrane cells has been less well reported.In this review, the authors briefly describe the role of autophagy in the formation of ovarian reserve, the development of growing follicles, the formation and regression of corpus luteum and follicular atresia.In addition, the effects of autophagy induced by chemicals and stress on follicular development are analyzed, which will provide a comprehensive understanding of the regulation of autophagy in follicular development.

【Objective】 This study was aimed to construct a genotype Ⅶ Newcastle disease virus (NDV) chimeric vaccine, and access its immune efficacy.【Method】 The F and HN protein ectodomains of recombinant plasmid pT7-Y2 of the Avian metaavulavirus-2 (AMAV-2) Y2 strain were replaced with the corresponding F and HN protein ectodomains of genotype Ⅶ NDV HB0901 strain by utilizing reverse genetic technology.Then F protein cleavage site of HB0901 strain was transformed into that of lentogenic LaSota strain, and the chimeric virus was recovered and named rY2-FHNR.The growth characteristics, pathogenicity and genetic stability of rY2-FHNR strain were evaluated, then 2-week-old SPF chickens were immunized with rY2-FHNR to determine the immunogenicity and cross-reactivity between immune serum and Y2 strain.The immune serum was identified by indirect NDV NP ELISA to verify its diagnosis effect.【Result】 Chimeric recombinant virus strain rY2-FHNR was constructed successfully.The biological characteristics results showed that the proliferation titer and pathogenicity in chicken embryos of rY2-FHNR strain were avirulent, while the chimeric virus was genetic stable when serially passaged in chicken embryos.Immunization with rY2-FHNR stimulated antibodies against NDV, and the immune serum had no cross-reactivity with rY2 strain.Through indirect NDV NP ELISA could distinguish infected animals from vaccinated animals.【Conclusion】 This study developed a genotype Ⅶ NDV chimeric vaccine candidate, which could be used as chimeric vaccine, provided technological support for the monitoring, prevention and eradication of genotype Ⅶ NDV.

【Objective】 This study was aimed to study the antiviral effects of silver nanoparticles (AgNPs) on Porcine reproductive and respiratory syndrome virus (PRRSV) in vitro, and preliminarily analyze its mechanism, so as to provide a new idea for the prevention and control of PRRSV.【Method】 The safe concentration of AgNPs for subsequent experiments was selected from 0.1875, 0.375, 0.75, 1.5, 3, 6 and 12 μg/mL by CCK-8 kit which was used to evaluate the cytotoxicity of AgNPs to Marc145 cells.The antiviral effects of AgNPs on PRRSV were evaluated by microscopic observation, indirect immunofluorescence assay, virus titer determination and Real-time quantitative RT-PCR.Direct inactivation effect of AgNPs on PRRSV was evaluated by indirect immunofluorescence assay and Real-time quantitative RT-PCR.The effects of AgNPs on the adhesion and invasion of different multiplicity of infection (MOI) of PRRSV (0.0001 to 0.1) to Marc145 cells were analyzed by Real-time quantitative RT-PCR.The effects of AgNPs on PRRSV proliferation were analyzed by Real-time quantitative PCR after Marc145 cells were treated with AgNPs at the indicated time points that infected with PRRSV for 3, 6, 12, 18 and 24 h.【Result】 The maximum safe concentration of AgNPs to Marc145 cells was 1.5 μg/mL.There were both a certain inhibitory and inactivation effect of 0.375, 0.75 and 1.5 μg/mL AgNPs on PRRSV.The adhesion and invasion of different MOI strains to Marc145 cells was inhibited by AgNPs, and the proliferation of PRRSV was inhibited by adding AgNPs at 3, 6, 12, 18 and 24 h.【Conclusion】 AgNPs was anti-PRRSV, and the resistance was exerted through direct inactivation of PRRSV and inhibition of adhesion, invasion and proliferation of virus.

【Objective】 Ring finger protein 185 (RNF185) is a member of Ring family E3 ubiquitin ligases.This study was aimed to explore the effect of chicken-derived RNF185 on the replication of Avian leukemia virus subgroup A (ALV-A) in vitro, and study the effect of chicken-derived RNF185 on the pathogenicity and mediated tumorigenic mechanism of ALV-A.【Method】 Referring to the sequence of RNF185 gene in chicken (accession No.:NP_001007841) published in GenBank, RNF185 overexpression primers and RNF185 single-stranded shRNA were designed, respectively.The RNF185 gene and double-stranded shRNA were synthesized by PCR and cloned into eukaryotic expression vectors psi-flag and interference vector pLKO.1-GFP, respectively.The constructed recombinant plasmids were verified by PCR, double restriction digestion and sequencing, and the CCK-8 kit was used to detect the effect of the recombinant plasmid on the viability of DF-1 cells.10⁴ tissue culture infective dose 50% (TCID₅₀) ALV-A (rHB2015012) virus solution was inoculated into DF-1 cells after RNF185 overexpression and interference, respectively, the cells were harvested at 3, 4 and 5 d after inoculation, and the viral load of ALV-A was detected by Western blotting.【Result】 The RNF185 gene was successfully amplified by PCR and the single-stranded shRNA was annealed to form double-stranded shRNA.The recombinant plasmids psi-flag-RNF185, pLKO.1-RNF185-1 and pLKO.1-RNF185-2 were successfully constructed.Western blotting results showed that the constructed overexpression and interference plasmid could stably express and interfere with RNF185 in DF-1 cells.The CCK-8 results indicated that overexpression and interference of RNF185 did not affect the activity of DF-1 cells.Overexpression of RNF185 could promote the replication of ALV-A in DF-1 cells, and interfere with the expression of RNF185 could inhibit the replication of ALV-A in DF-1 cells.【Conclusion】 This study constructed an overexpression plasmid and an interference plasmid that could highly express and interfere with RNF185 in DF-1 cells.RNF185 had no significant effect on the proliferation of DF-1 cells, confirming that RNF185 could promote the replication of ALV-A (rHB2015012) in vitro.

【Objective】 This study was aimed to identify the pathogenic bacteria and its characteristics that caused diarrhea and death of porcupine in a porcupine farm in Longyan city, Fujian province, and provide a reference for scientific prevention and control of the disease and rational drug use.【Method】 The pathogens of diarrhea-dead porcupines were isolated, and the isolates were identified by combining morphological characteristics, physiological and biochemical tests, 16S rRNA gene amplification and phylogenetic grouping.The pathogenicity and drug resistance of the isolates were studied by virulence gene detection, animal pathogenicity test and drug sensitivity test.【Result】 A strain of Escherichia coli was isolated from liver of the morbid and dead porcupine, and named as Fj/Porcupine2018.The sequence homology between the isolate and 22 reference strains from different sources of 16S rRNA was 97.4% to 99.8%.Among them, the sequence homology with the avian isolate (accession No.:MN022583) and the human isolate (accession No.:MW881377) were as high as 99.8%.The phylogenetic grouping confirmed that the isolate belonged to B1 group.The virulence gene test results showed that that in addition to the enteroinvasive Escherichia coli virulence gene EinV, 6 virulence-related genes of papC, iroC, Afa, luxs, stx2f and ompA were also detected.The results of animal pathogenicity tests showed that the isolate had strong virulence to the challenged mice, and all the mice died within 31 h after the challenge, and obvious pathological damage was caused to liver, spleen, lung, intestine and other organs and tissues of the dead mice.In the drug susceptibility test, the isolate showed different degrees of resistance to 7 classes of 11 drugs, including macrolides, tetracyclines, quinolones, sulfonamides, cephalosporins, aminoglycosides and penicillins, the drug resistance rate was 25% to 100%, and it was only sensitive to ceftriaxone and ceftriaxone.【Conclusion】 In this study, a pathogenic Escherichia coli strain of diarrhea-dead porcupines was isolated and obtained.Through a series of biological characteristics studies, it was confirmed that the isolate was a group B1 enteroinvasive Escherichia coli with strong pathogenicity and multi-drug resistance, which provided a certain reference for the prevention and control of colibacillosis in wild animals such as porcupine.

【Objective】 The purpose of this study was to explore the structure and function of S protein of Porcine epidemic diarrhea virus (PEDV), so as to provide a theoretical basis for promoting the diagnosis of PEDV infection and vaccine development.【Method】 The classic CV777 strain of PEDV was used as the template, the S and the S1 genes fragments were amplified by PCR.The PCR amplification products were cloned into pET-30a (+) prokaryotic expression vector and pFLAG-CMV-3 eukaryotic expression vector respectively, and the prokaryotic expression plasmid pET-30a-PEDV-S and eukaryotic expression plasmid pFLAG-CMV-3-PEDV-S1 were constructed. pET-30a-pEDV-S was transformed into E.coli BL21(DE3) competent cells, and expressed PEDV-S recombinant protein induced by IPTG.The recombinant protein was purified by denaturation and renaturation, and then detected by Western blotting.The recombinant protein PEDV-S was immunized in C57BL mice to prepare polyclonal antibodies.The obtained polyclonal antibody specificity was detected by indirect immunofluorescence assay (IFA) and polyclonal antibody titer was detected by indirect ELISA. pFLAG-CMV-3-PEDV-S1 was transfected into HEK293T cells, and the prepared polyclonal antibody was used as the primary antibody to detect the antigenicity of PEDV-S1 protein and the reactivity of polyclonal antibody by IFA.【Result】 The PEDV S and S1 genes were successfully cloned, and a prokaryotic expression vector capable of expressing recombinant protein PEDV-S and a eukaryotic expression vector capable of efficiently expressing S1 protein in HEK293T cells were constructed.The highest expression of PEDV-S recombinant protein was obtained when IPTG was 0.5 mmol/L and induced at 16 ℃ for 8 h, mainly in the form of inclusion bodies.Western blotting showed that the recombinant protein of PEDV-S was successfully expressed.ELISA results showed that the titer of the polyclonal antibody was 1:3 280 500.IFA results showed that the polyclonal antibody had good specificity and could specifically recognize PEDV-S and PEDV-S1 proteins.【Conclusion】 In this study, PEDV-S recombinant protein and S1 protein were obtained, and polyclonal antibody against PEDV S protein was successfully prepared, which provided conditions for studying the structure and function of S protein and laid a foundation for revealing the pathogenic mechanism of PEDV.

Long non-coding RNA (lncRNA) is an RNA with nucleic acid length greater than 200 nt, which does not encode or has limited coding ability.In the early days, it has been considered as genetic dark matter, but with the progress of molecular biology technology, more and more lncRNAs have been discovered.Studies have shown that lncRNA, as a key regulator of gene expression, can affect gene expression in histone modification, transcriptional regulation and post transcriptional regulation, and participate in almost all cell biological processes.Apicomplexan parasite is an obligate intracellular parasite with complex invasion mechanism.The research on the interaction between Apicomplexan parasites and their host has become the basis for the development of new anti-insect drugs.In recent years, it has been found that lncRNA, as a new type of regulatory factor, is widely involved in the interaction between Apicomplexan parasites and host cells, including infection immunity, development and differentiation, signal transduction and other cellular biological processes, which can not only help the host resist the invasion of parasites, but also help the parasites proliferate and develop in the cell, showing an important regulatory role in the occurrence and development of diseases.By summarizing the biological classification of lncRNA and its main functions in cell physiological processes, and combining the relevant studies of lncRNA in Apicomplexan parasites, which is mainly represented by Toxoplasma gondii, Plasmodium and Cryptosporidium in recent years, the author system arranged the mechanism of lncRNA in host immune response, parasite development and differentiation, epigenetic regulation and other aspects, in order to study the pathogenesis of Apicomplexan parasites, and provide new ideas and references for the research and development of efficient prevention and control technology.

【Objective】 This study was aimed to understand the situation of cattle infected with Chuzan virus (CHUV) in the southwest frontier area.【Method】 BHK21 cells were used to isolate virus from the blood samples of healthy cattle collected in Jinghong city and Jiangcheng county, Yunnan province.The samples with cytopathy were identified by morphology, genomic banding and molecular biology.The sequences of S7 and S2 genes were determined and compared.【Result】 Four samples with cytopathy were found.The virus particles were spherical with a diameter of about 50 nm by electron microscopy.The results of agarosegel electrophoresis showed that the genome of the four newly isolated viruses was 10 segments and the band was "3-3-4", which was similar to the strain of CHUV SZ187 isolated from Shizong in Yunnan province in 2012.The nucleotide similarity of the S7 and S2 genes of the four viruses were 98.7%-99.7%, 97.9%-99.1%.The sequence of S2 fragment had the highest homology with SZ187 and GHN-GS-16 strains isolated in China.The nucleotide and amino acid sequence of S7 gene had the highest homology with ON-1/E/18, ON-3/E/17 in Japan and SZ187, GHN-GS-16 in China.The results of genetic evolution analysis of S7 and S2 genes showed that the four newly isolated strains were the closest relatives.The S7 gene sequences of the four strains were in the same branch as the known PALV strains isolated from China and Japan, indicating that the four strains were PALV and formed a cluster, which had a certain regional character.The four strains were further confirmed to be CHUV, because the sequence of S2 gene of the four strains was on the same branch as the isolated CHUV from China.【Conclusion】 This study first reported the isolation of CHUV from cattle in the border area of Yunnan province.It provided an important reference and a powerful basis for the epidemiology of CHUV in China and the prevention and control of epidemic disease risk in border areas.

【Objective】 The objective of this study was to investigate the protein components of Haemaphysalis flava egg and their roles in embryonic development, so as to lay a foundation for the screening vaccine antigen of anti-tick reproduction.【Method】 The egg proteins of Haemaphysalis flava incubated for 0, 7, 14 and 21 days were extracted and digested by the FASP method.The unique peptides were separated and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.The midgut transcriptome library of Haemaphysalis flava was searched to identify proteins and annotated with NCBI.The iBAQ and LFQ of each protein were calculated by Maxquant, and the abundance of each protein at different incubation time was analyzed.【Result】 1 044 unique peptides were detected and 291 proteins were identified, 112 of which were high confidence (unique peptides ≥ 2), only 39 existing relevant literatures. The 39 proteins had been studied belong to six groups, including enzyme, protease inhibitor, transporter, heat shock protein, cytoskeleton protein, and unknown protein.Enzymes, protease inhibitors, transporters, and heat shock proteins all coexisted with family proteins.Quantitative analysis showed that the highest abundance was Contig14782, followed by Contig6575 in fresh eggs of Haemaphysalis flava.By the end of incubation, among the 20 highly abundant proteins, only Contig2242 was significantly increased (P<0.05), 14 were significantly decreased (P<0.05), and 5 were not significantly changed.GO functional enrichment analysis showed that the high confidence proteins identified in this work were mainly localized in the extracellular space, which was involved in immunoregulatory processes, and the molecular functions were correlated with protein binding, and RNA binding.【Conclusion】 Haemaphysalis flava egg protein composition was complex and numerous, including enzyme, protease inhibitor, transporter, heat shock protein, cytoskeleton protein, and unknown protein, of which enzyme was the most one.During incubation, most enzymes and protease inhibitors decreased significantly.

【Objective】 The aim of this experiment was to investigate the prevalence of the main viral pathogens of Angus calves diarrhea in Kashgar, Xinjiang and the genome-wide genetic relationship of Bovine norovirus (BNoV).【Method】 RT-PCR was used to detect four viral pathogens of Bovine rotavirus (BRV), Bovine coronavirus (BCoV), Bovine viral diarrhoea virus (BVDV) and BNoV in 363 calf diarrhea fecal samples collected from 4 cattle farms in Kashgar in different seasons in 2021, and the amplified BNoV gene sequences were analyzed.【Result】 The positive rates of BCoV and BNoV were 36.09% (131/363) and 25.07% (91/363), respectively.In mixed infection, BCoV+BNoV was the most, with a positive rate of 44.00% (22/50).Four fields in Kashgar area were tested.BCoV and BNoV were mainly detected in field A, with a detection rate of 43.52% (47/108) and 32.41% (35/108), respectively.BCoV was mainly detected in field B, with a detection rate of 52.21% (71/136).The pathogen detection rates in fields C and D were generally low.The viruses detected the most in autumn and winter were BNoV and BCoV, with detection rates of 66.67% (50/75) and 77.59% (90/116), respectively.The analysis of the whole gene sequence of BNoV showed that BNoV strain Bo/XJ-KS/01/CHN (accession No.:ON076888) detected in this experiment belonged to BNoV GⅢ.2, and was in the same branch of the evolutionary tree as CN/HB-SJZ-2 and CH/HB/BD/2019 strains in China, and had the highest nucleotide similarities with CN/HB-SJZ-2 strains, which was 93.70%.The nucleotide similarity with Jena/1999/UK strain isolated from Britain were the lowest, which was 72.49%. Further comparison and analysis of three open reading frames (ORFs) showed that the nucleotide and amino acid similarities between the whole genome sequence of BNoV and the domestic reference strain CN/HB-SJZ-2 were mainly the highest in ORF1 region, with 94.24% and 98.69%, respectively.The nucleotide and amino acid similarities with the foreign reference strain Jena/1999/UK was the lowest in ORF3 region, which was 63.59% and 64.57%, respectively.The comparison with domestic and foreign strains showed that there was no gene recombination.【Conclusion】 The epidemic situation of calf diarrhea in Southern Xinjiang was different due to different seasons and fields.The infection rate of viral pathogens was mainly high in autumn and winter, and single BCoV, BRV and BNoV were the main infections.The whole gene sequence of BNoV type GⅢ.2 Bo/XJ-KS/01/CHN determined in this study had the closest relationship with the reference strain CN/HB-SJZ-2 in China, and had no gene recombination phenomenon compared with the whole genome reference sequence at home and abroad.

【Objective】 This study was aimed to develop an immunological diagnostic reagent for Canine rotavirus (CRV), prepare and identify specific monoclonal antibodies (MAbs) against CRV, and establish colloidal gold immunochromatography for detection of CRV.【Method】 Female BALB/c mice were immunized with clinical CRV strain SL006, and cell fusion was performed using hybridoma cell method.Hybridoma cells that secreted CRV MAb were screened by indirect immunofluorescence assay (IFA).Then ascites were prepared and purified by affinity chromatography.The monoclonal antibodies obtained was identified and the colloidal gold strip detection method was established through optimization.The sensitivity, specificity, repeatability and application of the strip were evaluated respectively.【Result】 Four hybridoma cells were obtained, named 2C12, 4A5, 1H3 and 5F3, and the IFA titers were 1:6 400, 1:12 800, 1:1 600 and 1:3 200, respectively.Their heavy chains belonged to IgG2b, IgG2a, IgG1, IgG2a, and all light chains were kappa. The 4A5 was coated on a porous nitrocellulose membrane as the detection line, the goat anti-mouse IgG antibody was coated as the control line, and the MAb 2C12 was conjugated with colloid gold, respectively.The detection limit of the strip was 10^4.2 TCID₅₀/mL.Other viruses of canine origin, Canine parvovirus (CPV), Canine parainfluenza virus (CPIV), Canine adenovirus type Ⅰ (CAV-Ⅰ), CAV-Ⅱ, Canine distemper virus (CDV), CRV negative anal swabs and negative feces were negative.Intra-assay and inter-assay repeatability test results were consistent.47 samples were detected by the colloidal gold strip and RT-PCR, and the coincidence rate was 93.6%.【Conclusion】 The strip had high sensitivity and specificity, good repeatability, and high coincidence rate with RT-PCR method, providing an effective method for rapid clinical diagnosis of CRV.

【Objective】 The purpose of this experiment was to study the application effects of small-cell fully-closed partition hoggery on pig epidemic control, purification and vaccine reduction.【Method】 All negative, all positive and partial positive groups of Porcine circovirus type 2 (PCV2) serum antibody were set up in this experiment.6 same-aged nursing pigs were selected in each group and fed as mode A and B for 140 days.Mode A was fed in small-cell fully-closed partition hoggery for 140 days, and mode B was first fed in small-cell fully-closed partition hoggery for 70 days and then fed in common opened hoggery for 70 days.All pigs were immunized with Classical swine fever virus (CSFV) and Pseudorabies virus (PRV) vaccine only once.The serum antibody levels and positive rates of PCV2, Porcine reproductive and respiratory syndrome virus (PRRSV), CSFV, PRV-gB and PRV-gE were detected on the 0, 70, 140 feeding day.【Result】 The results showed that compare with the 0 day, the PCV2 antibody level of all groups and serum antibody positive rate of all positive and partial positive groups in modle A decreased significantly (P<0.05) on the 70th and 140th day.In mode B, compare with the 0 day, the PCV2 antibody level were extremely significantly decreased in all positive group (P<0.01) and maintained in all negative and partial positive groups on the 70th day, and were extremely significantly increased in all groups on the 140th day (P<0.01).The serum antibody positive rate of PCV2 in mode B decreased greatly in the first 70 days and increased greatly to 100% in the last 70 days.Compare with the 0 day, the antibody level and serum antibody positive rate of CSFV were increased significantly (P<0.05) in mode A and B in the first 70 days.Compare with the 70 day, the antibody level of CSFV increased slightly in all negative and partial positive groups, and the serum antibody positive rate maintained 100% in all negative group and all positive group of mode A in the last 70 days.In the last 70 days, compare with the 70 day, the antibody level of CSFV decreased significantly (P<0.05) and approached the level of day 0 in all negative group and all positive group of mode B.In the first 70 days, the antibody level and serum antibody positive rate of PRV-gB decreased in some groups, and decreased in all groups in the last 70 days in mode A and B.In the whole experimental period, the antibody levels of PRRSV and PRV-gE were at a low level, and the serum antibody positive rates were 0.【Conclusion】 The small-cell fully-closed partition hoggery could significantly block the transmission of PCV2, purify PCV2, enhance the CSFV vaccine immunity protection rate, and reduce vaccine cost and was a feasible guarantee equipment to enhance the disease prevention ability of pig breeding and has broad application prospects.

【Objective】 The purpose of this study was to explore the mechanism of modified Wumei powder in the treatment of porcine transmissible gastroenteritis (TGE) by network pharmacology.【Method】 The active components and action targets of modified Wumei powder were obtained using Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) and Swiss TargetPrediction, and the related targets of TGE were obtained using the Public Comparative Toxicology Genomics Database (CTD).The protein interaction (PPI) network diagram of the intersection targets of modified Wumei powder and TGE was drawn using STRING database.The GO function and KEGG pathway enrichment analysis of the intersection targets were performed by DAVID, so as to explore the main action mechanism of modified Wumei powder in the treatment of TGE.【Result】 A total of 125 active components such as quercetin, kaempferol and formononetin and 58 action targets such as JUN, TNF and STAT3 were screened from modified Wumei powder.GO functional enrichment analysis showed that a total of 138 items of biological process were obtained, including immunoreaction, generation of vascular endothelial factors, and positive regulation of pri-miRNA transcription by the RNA polymerase Ⅱ promoter.Cellular component was 13 items, involving extracellular region, extracellular space, membrane raft, etc.Molecular function was 32 items, involving cytokine activity, growth factor activity, and interleukin 2 (IL2) receptor activity.Enrichment analysis of KEGG pathway revealed that a total of 135 signaling pathways were identified, including IL17 signaling pathway, Th17 cell differentiation, TNF signaling pathway, and HIF-1 signaling pathway, which were the main pathways of modified Wumei powder in the treatment of TGE.【Conclusion】 The results above indicated that modified Wumei powder mainly regulated JUN, TNF and STAT3 targets by multiple active components such as quercetin, kaempferol and formononetin, and treated TGE by participating in IL17 signaling pathway and Th17 cell differentiation.

【Objective】 The aim of this experiment was to investigate the protective effect of puerarin (PU) on acetaminophen (APAP)-induced acute liver injury in mice, and provide a new idea for the clinical prevention and treatment of liver injury caused by APAP.【Method】 Forty 4-week-old SPF-grade male Kunming mice weighing (20±2) g were randomly divided into four groups:Normal (CON), model (APAP), puerarin low-dose (L-PU), and puerarin high-dose (H-PU) groups, 10 mice in each group.Mice were kept normally in CON group, and mice in L-PU and H-PU groups were gavaged with 50 and 100 mg/kg puerarin, respectively, once daily for 7 days.After the last administration, the mice in APAP, L-PU and H-PU groups were fasted for 16 h and gavaged with 200 mg/kg acetaminophen once.Continue fasting for 12 h, blood was collected from the orbit after anesthesia, and the mice were killed after cervical dislocation.The liver was quickly collected, and the liver morphology was observed and the liver index was calculated.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured to evaluate the liver injury.The liver catalase (CAT) activity and total antioxidant capacity (T-AOC) were determined to evaluate the liver oxidative stress.The levels of glutathione S-transferase (GST) and cytochrome P450 family 2 subfamily E member 1 (cytochrome P450 2E1, CYP2E1) were measured to analyze the metabolism of APAP in liver.The apoptosis-related protein cysteine protease caspase-3 and caspase-9 levels were determine to evaluate the apoptosis of liver cells.The pathological sections of the liver were made and the histological changes of the liver were observed after HE staining.【Result】 Compared with CON group, the liver in APAP group was hard with obvious bleeding, the liver tissue structure was severely damaged, the hepatic cords were disordered, the structure of the liver lobules was not clear, and there was massive necrosis of hepatocytes, the liver index and ALT and AST levels in serum were significantly increased (P<0.05), the activity of CAT, T-AOC and GST level were significantly decreased (P<0.05), and the contents of apoptotic proteins caspase-3, caspase-9 and CYP2E1 were significantly increased (P<0.05).Compared with APAP group, the liver morphology and tissue structure of L-PU and H-PU groups were basically normal, and the effect of H-PU group was more significant.In L-PU and H-PU groups, the liver index and the activities of AST and ALT in serum were significantly decreased (P<0.05), CAT activity and T-AOC in liver were significantly increased (P<0.05), and the level of caspase-9 was significantly decreased (P<0.05).In addition, L-PU and H-PU groups increased the level of GST and significantly decreased the level of CYP2E1 (P<0.05).【Conclusion】 The significant protective effect of puerarin on APAP-induced liver injury in mice might be related to the antioxidant, anti-apoptotic, and accelerated APAP detoxification effects of puerarin.

【Objective】 The purpose of this experiment was to study the carriage and drug resistance of Escherichia coli (E.coli) high pathogenicity island (HPI) related genes in artificially bred Tupaia belangeri and to provide some ideas for the breeding and management of Tupaia belangeri and the prevention and treatment of colibacillosis.【Method】 129 samples of Tupaia belangeri anal wipe were collected aseptically.The bacteria were isolated and identified by McConkey medium, LB agar medium, Gram staining and biochemical test.HPI-related genes were detected by PCR.HPI-positive strains were screened after PCR identification.The similarity analysis and phylogenetic tree construction of the main structural genes irp2 and fyuA of the isolates were performed.Disk diffusion method was used for drug sensitivity test of the isolates.【Result】 A total of 123 strains of E.coli were isolated from 129 Tupaia belangeri anal wipe samples, and 95.35% (123/129) were isolated.The results of Gram staining showed that the isolates were microscopically examined as stubby rubrum.The biochemical identification results showed that the isolates were positive for lactose, glucose, maltose, peptone, and mannitol biochemical reactions, and negative for hydrogen sulfide and urease biochemical reactions, all of which conformed to the characteristics of E.coli.The electrophoresis results of PCR products showed that the detection rate of HPI-related genes was 88.62% (109/123), the carrying rates of irp2 and fyuA genes were 34.15% (42/123) and 47.97% (59/123), respectively.The results of sequence analysis of the main structural genes showed that the irp2 and fyuA genes were more than 98.6% and 98.9% similar to the sequences published irp2 and fyuA gene nucleotide sequences in GenBank, respectively, and the phylogenetic tree results showed that the isolates were closely related to Yersinia spp.The results of drug resistance analysis showed that the isolated strains were sensitive to amikacin and florfenicol, and resistant to amoxicillin and oxacillin, the drug resistance rates were 87.80% and 81.30% respectively.【Conclusion】 HPI related genes were widely distributed in E.coli of Tupaia belangeri, which further confirmed that HPI could be horizontally transferred, and the inheritance of the main structural genes irp2 and fyuA were highly conserved.

【Objective】 This study was aimed to analyze the presence of Bacillus cereus on eggs in different environments and the basic characteristics of these isolated strains.【Method】 9 kinds fresh eggs from different farms and supermarkets were collected.Isolation and identification, genotyping, virulence gene screening and drug resistance analysis of Bacillus cereus carried on the surface of eggs were carried out.Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for preliminary identification of isolated strains.The strains were identified, genotyped and screened for virulence genes and resistance genes after whole genome sequencing.The resistance of the strains to 12 antibiotics including ceftriaxone, erythromycin, vancomycin, etc., was tested by the microbroth method.【Result】 After the preliminary identification by MALDI-TOF MS and genomic strain identification, it was determined that 6 strains isolated from 3 kinds of unsterilized fresh eggs in the farms were all Bacillus cereus. The sequence typing (ST) of 6 strains were various, and two new ST types appeared.Non hemolytic enterotoxin gene nheB, diarrheal type virulence factor gene HlyⅡ and immune system invader gene inhA were detected in all 6 strains of Bacillus cereus.The carrying rates of nheA and nheC genes were both 83.3%, and the carrying rates of hblA, hblC and hblD genes which encoded hemolysin BL (HBL) were all 66.7%.The carrying rates of cytK and hlyⅢ genes were 33.3% and 50.0%, respectively.While the enterotoxin genes BceT and entFM and cereulide gene ces were not detected.All of 6 strains were sensitive to gentamicin, tetracycline, chloramphenicol, rifampicin, linezolid and ciprofloxacin, and they were resistant to ampicillin, ceftriaxone and tiamulin.The mediations to erythromycin and clindamycin were 83.3% and 100%, respectively, and 33.3% of strains were resistant to vancomycin.All 6 strains carried the fosfomycin resistance genes FosB and vanRSYZ, part of the vancomycin resistance gene cluster vanA.5 strains carried the macrolide drug resistance gene mphL, and some strains carried the β-lactam drug resistance gene or nucleoside drug resistance gene.【Conclusion】 In this experiment, Bacillus cereus strains were successfully isolated from the shells of unsterilized eggs in the farms.It was found that the isolated strains carried a variety of virulence genes and had multiple drug resistance, with potential pathogenicity and public safety risks.

【Objective】 The aim of this study was to explore the effect of mangiferin (Man) on the inflammatory injury of duck embryo hepatocytes (DEHCs) induced by Duck hepatitis A virus-1 (DHAV-1).【Method】 The primary DEHCs were isolated from the liver tissues of 14-day-old SPF duck embryos, and cultured for 48 h with different concentrations of mangiferin (0, 5, 10, 20, 40, 80 and 160 μmol/L).Then, the safe concentration range of mangiferin was detected by CCK-8 method.The toxicity of mangiferin to DEHCs was determined by detecting the activity of lactate dehydrogenase (LDH) in the supernatant of DEHCs cultured with safe concentration of mangiferin for 12, 24 and 48 h.The DEHCs were divided into control group (Mock), model group (Model), mangiferin groups (Man10, Man20 and Man40) and ribavirin group (Rib), with three replicates in each group, and cells in all groups were cultured with serum starvation for 12 h.After that, Mock group was added with DMEM medium containing 10% FBS, Model, Man10, Man20, Man40 and Rib groups were challenged with DHAV-1 (MOI=1.0) for 2 h, and then Model group was replaced with DMEM medium containing 10% FBS, the culture medium of Man10, Man20, Man40 groups was supplemented with 10, 20 and 40 μmol/L mangiferin, respectively, and 1 μmol/L ribavirin was added to Rib group.After 48 h, cells in each group were collected, and the content of malondialdehyde (MDA) and the activities of catalase (CAT), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), total nitric oxide synthase (T-NOS) and glutathione peroxidase (GSH-Px) were detected by colorimetric method.The distribution of DHAV-1 in DEHCs cells in each group was detected by immunofluorescence (IF).Expression of NLRP3/Pro-Caspase1/IL-1β pathway proteins were detected by Western blotting method.The fresh whole blood of 2-day-old SPF ducks was extracted, and the peripheral blood mononuclear cells (duPBMCs) of the ducks were isolated.The grouping and processing of duPBMCs were the same as DEHCs.ELISA assay was used to detect the content of IL-8 and IL-1β in the supernatant of duPBMCs.【Result】 Compared with 0 μmol/L mangiferin group, the cell proliferation ability of 80 and 160 μmol/L mangiferin was significantly reduced (P<0.05), so 5-40 μmol/L mangiferin was a safe administration concentration for DEHCs.The activity of LDH in the supernatant of cells treated with 20 and 40 μmol/L mangiferin for 48 h was significantly lower than that of cells treated with 12 and 24 h (P<0.05), the activity of LDH in the supernatant of cells treated with 20 and 40 μmol/L mangiferin for 12 and 24 h was significantly higher than that treated with 5 and 10 μmol/L mangiferin (P<0.05), there was no significant difference in LDH activity in the supernatant of cells treated with mangiferin at different concentrations for 48 h (P>0.05), so 48 h was the optimum treatment time.Compared with the Mock group, the activities of CAT, T-AOC, SOD and GSH-Px in DEHCs of the Model group were significantly decreased (P<0.05), the content of MDA, the activity of T-NOS, DHAV-1 copy number, DHAV-1 positive rate, the expression of NLRP3 in DEHCs and the secretion levels of IL-8 and IL-1β in duPBMCs were significantly increased (P<0.05).Compared with Model group, the activities of CAT, T-AOC, SOD and GSH-Px in DEHCs of Man10, Man20 and Man40 groups were significantly increased (P<0.05), the content of MDA, the activity of T-NOS, DHAV-1 copy number, DHAV-1 positive rate, the expression of NLRP3 in DEHCs and the secretion levels of IL-8 and IL-1β in duPBMCs were significantly decreased (P<0.05).【Conclusion】 Mangiferin could resist the inflammatory damage caused by DHAV-1 infection of DEHCs by increasing the antioxidant capacity, reducing the replication level of DHAV-1, inhibiting the activation of NLRP3 pathway and reducing the release of pro-inflammatory cytokines.

【Objective】 This study was aimed to screen the phage-resistant strains, and analyze the mutation sites of the receptor gene, in order to provide theoretical basis for revealing the mechanism of phage resistance mutation of Salmonella cholerae.【Method】 Salmonella cholerae CICC 21501 and its lytic phage vB_SenS_S528 (phage S528) were used as the research objects, the spontaneous phage-resistant strains were screened by fluctuation experiment, and the colony morphology was observed.The sensitivity to phage, adsorption characteristics, growth curve and sensitivity to temperature and pH were measured.The whole genome resequencing and PCR were used to verify the mutation site of the potential receptor gene.【Result】 A mutant strain resistant to phage S528 named B6-2 was successfully obtained.The strain B6-2 displayed rough edge of colony different from wild-type strain.Compared with wild bacteria, the growth curves showed no significant difference, but showed sensitivity to pH and temperature tolerance at 40 and 50 ℃. The adsorption rate of phage S528 to the phage-resistant strain was reduced by 60% (93% to host strain).The whole genome resequencing and comparative analysis showed that there were 3 mutation sites of the phage-resistant strain B6-2, 2 sites of PROKKA_04510 gene and 1 site of rfbC gene, respectively.The PCR product electrophoresis and sequencing results of the mutant gene fragment showed that 2 sites of PROKKA_04510 gene were not mutated, but the actual mutation site was located at 350 bp of rfbC gene, and the base was mutated from C to T.【Conclusion】 A phage-resistant strain B6-2 related to receptor changes was screened and it might prevent phage adsorption through base mutation in rfbC gene.

【Objective】 The purpose of this study was to study the antiviral effect of Tamoxifen on the infection of porcine Pseudorabies virus (PRV) in PK15 cell model.【Method】 Taking PK15 cells as the model, the effect of Tamoxifen on cell viability was detected by CCK-8 cell counting method.The effects of Tamoxifen on cell cycle and apoptosis were detected by ANNEXIN V-FITC/PI apoptosis kit.CytoFLEX flow cytometry and fluorescence microscope were used to detect the difference of virus proliferation in Tamoxifen treated cells infected with PRV-GFP.The changes of PRV gB gene mRNA and protein expression in Tamoxifen treated cells infected with PRV-QXX were detected by Real-time quantitative PCR and Western blotting, respectively.Virus titer assay was used to detect the inhibition of Tamoxifen treated cells infected with PRV-QXX.【Result】 Tamoxifen had no effect on cell viability at the concentration of 6 μmol/L.When the concentration of Tamoxifen was below 6 μmol/L, compared with control group, Tamoxifen treatment group had no significant effect on cell cycle and apoptosis(P>0.05).At the same time point, the proliferation rate of PRV-GFP in PK15 cells in Tamoxifen treatment group was extremely significantly lower than that in control group(P<0.01).Real-time quantitative PCR showed that Tamoxifen extremely significantly inhibited the mRNA expression of PRV gB gene in PK15 cells (P<0.01).The results of Western blotting showed that Tamoxifen significantly or extremely significantly inhibited the expression of PRV gB protein in the treatment groups with different concentrations at the same time point (P<0.05 or P<0.01).The results of virus titer showed that the replication rate of PRX-QXX progeny virus in Tamoxifen treatment group was significantly or extremely significantly lower than that in control group at the same time point (P<0.05 or P<0.01).【Conclusion】 Tamoxifen could significantly inhibit the proliferation of PRV in PK15 cells.