【Objective】 The aim of this study was to identify candidate genes and molecular markers affecting the contents of mineral elements in breast muscle of ducks by genome-wide association study (GWAS),and analyze the genetic mechanism of mineral element traits.【Method】 The F2 generation resource population of Pekin ducks×Anas platyrhynchos was constructed,and the whole genome was resequenced.The breast muscles of ducks were collected for 8 weeks and the contents of 7 major mineral elements were determined according to the national standard method.Combined with the resequencing data and mineral element phenotypic data,the genes related to element content were identified by whole gene association analysis.【Result】 The phenotypic heritability of the contents of seven mineral elements was P(0.007)＜Fe(0.100)＜Mg(0.120)＜Na(0.140)＜K(0.150)＜Ca(0.190)＜Zn(0.350).The results of correlation analysis showed that the correlation between Mg and K was the highest (r=0.62).There was a high positive correlation between P and Mg as well as K in duck breast,r were 0.43 and 0.56,respectively.A SNP locus significantly related to Zn content were identified by GWAS and reached a significant association (-log₁₀P-value=8.03) at 12 075 283 bp on chromosome 6. 39 SNPs highly correlated with the highest point SNP narrowed the candidate interval to 39 kb,in which there was only one gene SORCS3.Combined with gene functional annotation and transcriptome,SORCS3 was identified as a candidate gene for Zn content variation.The highest point of correlation between SNP and P content at the whole genome level was highly correlated with 260 SNPs(R²＞0.4),all of which were located at chromosome 4 between 17.1-18.6 Mb and contained 27 genes.Combined with correlation analysis,gene functional annotation and transcriptome,six candidate genes were highly related to P content were identified (TUSC3,MICU3,LOC101803915,MTMR7,SLC7A2 and ASAH1 genes).【Conclusion】 The candidate gene SORCS3 of Zn content was identified by GWAS,and combined with transcriptome,six candidate genes related to P content were identified.These results enriched the knowledge system of mineral nutrition of poultry meat,and provided an important theoretical basis for breeding excellent livestock and poultry products.

【Objective】 This study was aimed to explore the biological function of LIF gene in White muscovy duck,and clarify its expression pattern in hypothalamus,pituitary,ovary,oviduct enlargement and uterus.【Method】 In this study,the ovarian tissue of White muscovy duck was used as the research object,and the LIF gene sequence of White muscovy duck was cloned by rapid amplification of cDNA ends technology (RACE),and the similarity comparison and phylogenetic tree were constructed.The physicochemical properties and structure of LIF protein were analyzed by bioinformatics methods.The relative expression of LIF gene in the hypothalamus,pituitary,ovary,oviduct enlargement and uterus of White muscovy duck was detected by Real-time quantitative PCR.【Result】 The cloned LIF gene sequence of White muscovy duck was 1 820 bp,and the CDS region was 636 bp,encoding 211 amino acids.The results of nucleotide sequence similarity comparison showed that the sequence similarity of LIF gene between White muscovy duck and Aythya fuligula,Anas platyrhynchos,Oxyura jamaicensis,Cygnus atratus,Serinus canaria, Coturnix japonica,Gallus gallus,Nothoprocta perdicaria were 99.36%,99.21%,99.21%,98.11%,92.03%,90.68%,90.58%,87.60%,respectively.The analysis of phylogenetic tree showed that the genetic distance between White muscovy duck and Aythya fuligula was the closest,and the genetic distance between White muscovy duck and Gallus gallus was the farthest.The LIF protein in White muscovy duck was an alkaline hydrophilic stable protein with 16 phosphorylation sites,7 N-glycosylation sites and 1 signal peptide,which was mainly located in the extracellular and lysosome.The secondary structure of LIF protein was mainly composed of alpha helix and random coli,and the prediction result of protein tertiary structure was consistent with that of secondary structure.LIF protein in White muscovy duck might interact with IL6ST,CNTFR,IL6,OSMR,LIFR,IL6R,STAT3,CNTF,IFNGR1 and IFNGR2 proteins.The tissue expression showed that LIF gene was expressed in five tissues of White muscovy duck. LIF gene of White muscovy duck in the oviduct enlargement and ovarian tissue at nest stage was significantly higher than that at laying stage (P＜0.05).The expression of LIF gene in the oviduct enlargement was significantly higher than other tissues (P＜0.05) in two periods.【Conclusion】 The LIF gene in White muscovy duck was successfully cloned in the experiment,and its expression in the oviduct enlargement was significantly higher than that in other tissues,and the nesting stage was higher than that at laying stage.The results provided a theoretical basis for exploring the regulatory mechanism of ovarian development in White muscovy duck.

【Objective】 The purpose of this experiment was to clone and bioinformatically analyze the gene of the rate-limiting enzyme aralkylamine-N-acetyltransferase (AANAT) for N-acetyl-5-methoxytryptamine (MT) synthesis in American mink (Neovison vison),so as to provide a reference for biological functions in mink.【Method】 DNA was extracted from the caudal vein of mink,the sequence of AANAT gene was cloned by homologous recombination,the sequence of CDS region was analyzed,the amino acid sequence of AANAT gene was deduced for similarity alignment and phylogenetic tree construction,and the AANAT protein was analyzed by bioinformatics.【Result】 The sequence of AANAT gene in mink was about 1 631 bp,and the sequence of CDS region was 504 bp,which could encode 167 amino acids.The amino acid sequence similarity of AANAT gene in mink was 98.2% with Mustela putorius furo,and the phylogenetic tree analysis also showed that it was the closest relative to Mustela putorius furo.Bioinformatics analysis results showed that AANAT protein in mink was a hydrophobic protein without transmembrane domain and signal peptide,mainly distributed in the cytoplasm,and had 5 antigen-binding sites as a non-secreted protein.AANAT protein contained multiple post-translational modification sites,such as phosphorylation and glycosylation.AANAT protein contained a conserved domain of the N-acyltransferase superfamily,which was closely related to mammalian circadian rhythms.The secondary structure of AANAT protein was dominated by random coil,and the tertiary structure contained several conserved catalytic residues.Protein network interaction analysis results showed that AANAT protein could interact with multipleserotonin synthesis-related proteins.【Conclusion】 The sequence of AANAT gene in mink was successfully obtained.The structure and specific modification sites of AANAT protein might participate in the regulation of MT biosynthesis by localizing acetyl-CoA and 5-hydroxytryptamine.The reults provided a reference for research on the regulation mechanism of AANAT gene on embryonic diapause and improving fecundity in mink.

【Objective】 This study was aimed to better understand the molecular characteristics of complete genomes of waterfowl infected with H3 subtype Avian influenza virus (AIV) in the coast area of Guangxi.【Method】 Two strains of H3 subtype AIV named A/duck/Guangxi/S11359/2020 (H3N1) and A/duck/Guangxi/S11374/2020 (H3N8) were acquired by one-step RT-PCR amplification of genome segment,then conducted sequencing and evolutionary analysis further in this study.【Result】 The amino acid cleavage sites of HA protein of both H3 subtypes AIV were ³⁴⁰PEKQTR↓GLFG³⁴⁹ each,which was in accordance with the molecular characteristics of low pathogenic AIV,and the 226th and 228th amino acid sites correlated to receptor tropism were Q and T,indicating that were poultry receptor as possible.The BLAST analysis result showed that M and NP genes of H3N1 and PA gene of H3N8 with the highest similarity to H7 subtype AIV was 99.5%,98.7% and 98.9%,respectively,which suggested that genes recombination was occurred between H3 and H7 subtypes AIV.The genes of both H3 subtype AIV shared the highest similarity with that of waterfowl-origin H3,H4,H6,H7 and H9 subtypes AIV from Vietnam,Bangladesh and South Korea countries around China,which indicated that the isolates possessed the same evolutionary source.Genetic analysis indicated that all genes of both H3 subtype AIV isolates belonged to the Eurasian branch,the genetic distance of those was the closest to waterfowl-origin influenza virus strains,closer to canine and feline-origin strains,close to human and porcine-origin strains,but in addition to NS gene of H3N1 isolate which got the closest genetic distance to A/Aichi/2/1968 derived from Japanese earlier,suggesting that genes exchanged could occurred between them.Recombination sign of HA gene of H3N8 isolate was detected,the recombinant parent strains of that were A/duck/Beijing/40/04 and A/canine/Beijing/20121215-34/2012 strains,with the similarity was 93.1% and 97.1%,respectively.【Conclusion】 The source and genetic characteristics of genes of both sea duck-origin H3 subtype AIV from Guangxi was complicated and divergent.

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne virus of the genus Flavivirus in the family Flaviviridae.The disease caused by it is characterized by the decline of egg production of laying ducks and the damage of the nervous system of ducklings in clinical practice,which has caused significant economic losses to the domestic duck industry.Since its outbreak in Shanghai in 2010,DTMUV infection has gradually spread to major duck farms in Eastern,Southeastern,and Northern China.Several clinical diagnostic or laboratory tests for DTMUV have been established in China.Isolation and identification of DTMUV and immunohistochemistry are used to detect the presence of viral particles.Serological detection of DTMUV mainly includes indirect ELISA,competitive ELISA,and colloidal gold immunochromatographic strips,which can detect DTMUV-specific proteins or antibodies and is widely used for the field detection of DTMUV.The detection of DTMUV by conventional PCR,multiplex PCR and CRISPR techniques has enabled rapid clinical diagnosis of DTMUV and facilitated the epidemiological investigation and study of the pathogenesis of duck tambours virus disease.Currently,with the development and commercialization of some vaccines,the epidemic intensity of DTMUV infection in China has gradually changed from outbreaks to sporadic transmission.However,DTMUV has a wide host range,diverse transmission routes,and potential zoonotic potential.To identify and interrupt the spread of DTMUV at an early stage,there is an urgent need to establish rapid,accurate,safe,and effective detection methods and vaccines.The author summarizes the research progress of DTMUV detection methods and vaccines to provide references for the prevention of DTMUV infection.

【Objective】 This study was aimed to investigate the effect of membrane permease ⅡC(EⅡC) gene in Listeria monocytogenes (LM) pathogenicity island 4 (LIPI-4) on the key virulence factor genes expression.【Method】 The EⅡC gene deletion strain LM928ΔEⅡC and complementary strain CLM928ΔEⅡC of isolate LM928 were constructed.The growth curve of each strain and the fermentation of different sugars were determined.The adhesion,invasion and intracellular proliferation of each strain to human brain microvascular endothelial cells (HCMEC/D3) were determined.The transcript levels of virulence genes of each strain were detected using Real-time quantitative PCR under BHI medium and HCMEC/D3.【Result】 LM928 EⅡC gene deletion strain LM928ΔEⅡC and complementary strain CLM928ΔEⅡC were successfully constructed.Compared with the LM928,the LM928ΔEⅡC strain showed consistent growth curves and fermentability to glucose,cellobiose,fructose,rhamnose,esculin and salicin.The invasive ability of LM928ΔEⅡC decreased extremely significantly (P＜0.01), but the adhesion ability was not change significantly (P＞0.05).The amount of intracellular proliferation of LM928ΔEⅡC was significantly lower than that of LM928 and CLM928ΔEⅡC from 2 to 4 h post-infection (P＜0.05),but it was significantly higher than that of LM928 and CLM928ΔEⅡC from 8 to 12 h post-infection (P＜0.05).Compared with LM928 and CLM928ΔEⅡC,in BHI medium,the transcriptional levels of hly,prfA,actA,plcB, inlA,inlB,inlC and mpl genes of LM928ΔEⅡC were extremely significantly down-regulated (P＜0.01),the transcriptional level of plcA gene was extremely significantly up-regulated (P＜0.01).While in HCMEC/D3 cell,the transcriptional levels of hly,prfA,inlB,inlC and mpl genes of LM928ΔEⅡC were significant or extremely significantly up-regulated (P<0.05 or P<0.01),the transcriptional levels of plcA and inlA genes were extremely significant down-regulated (P＜0.01),and there was no significant difference between the transcriptional levels of actA and plcB genes.【Conclusion】 EⅡC in LIPI-4 did not affect the growth and partial sugar fermentation of LM928,could regulate important invasion-related genes and virulence-related genes in LM,which laid a foundation for further exploration of the role of EⅡC in LM and LIPI-4.

【Objective】 This study was aimed to clone the cysteine dioxygenase type 1(CDO1) gene and conduct bioinformatics analysis,detect the expression of CDO1 gene in different tissues of Large White pigs,and locate the site of CDO1 in mammary gland,so as to provide the basis for further exploring the regulatory role of CDO1 gene in mammary gland development.【Method】 The full-length sequence of CDO1 gene CDS in Large White pigs was amplified by PCR.The CDO1 gene sequence was ligated with pMD18-T vector to transformed into E.coli DH5α competent cells.After PCR identification and sequencing of the amplified culture solution of positive bacteria,the sequence similarity among different species were compared and the phylogenetic tree was constructed.The bioinformatics analysis of CDO1 protein was carried out using online prediction software.The expression of CDO1 gene in different tissues of Large White pigs was detected by Real-time quantitative PCR,the localization of CDO1 protein in the mammary gland of Large White pigs was detected by immunohistochemistry.【Result】 The full-length sequence of CDO1 gene CDS in Large White pigs was 603 bp,encoding 200 amino acids.There was the highest similarity with the nucleotide sequence of CDO1 gene between Large White pigs and Sus scrofa.The phylogenetic tree results showed that Large White pigs and Sus scrofa were first clustered into a group and were closely related to Macaca mulatta.The molecular mass of CDO1 protein was 23.018 ku,the isoelectric point was 5.98,and the instability coefficient was 33.58 (＜40),which was a stable hydrophilic protein and located in the cytoplasm.CDO1 protein had glycosylation and phosphorylation sites.The secondary structure and tertiary structure model of CDO1 protein were consistent.Tissue expression analysis results showed that the expression of CDO1 gene in liver of Large White pigs was the highest,which was significantly higher than that in other tissues (P＜0.05),it was next expressed in mammary gland,longissimus dorsi muscle and heart,which were significantly higher than that in other tissues except for liver (P＜0.05),the expression of CDO1 gene in duodenum was the lowest.Immunohistochemical results showed that CDO1 was localized in somatic cells,epithelial cells and adipocytes of terminal bud (TEB) of mammary gland in sows.【Conclusion 】 The total length of CDO1 gene CDS in Large White pigs was 603 bp,encoding 200 amino acids,which was highly expressed in heart,liver and mammary gland.CDO1 protein was a stable hydrophilic protein,which was enriched in somatic cells,epithelial cells and adipocytes of TEB of mammary gland in sows.The results provided a reference for further exploring the effect of CDO1 protein on mammary gland development and lactation function of sows.

Lysine,as the first limiting amino acid of pig diet,is also the key to construct an ideal protein model.Lysine can improve the growth performance and nutrient digestibility,enhance body immunity,maintain intestinal health,affect protein synthesis in pigs.Lack or excess of lysine will have an impact on all aspects of pigs.Therefore,it is very important to explore the effect of lysine addition level and lysine requirement in the diet on pig performance and various indicators.Based on the relevant research on pig lysine in recent years at home and abroad,it was found that less than 15% of the recommended amount of lysine could induce inflammation.However,appropriate lysine could enhance the cellular immunity of piglets,and then affect the intestinal health of pigs,promote the proliferation of intestinal epithelial cells,enhance cell vitality,improve nutrient digestion of pigs,and promote skeletal muscle synthesis by regulating satellite cells.At the same time,the authors synthesized the lysine requirements of piglets,finishing pigs,boars and sows and low-protein diets,in order to provide scientific basis for the rational application of lysine in animal production.

【Objective】 This experiment was conducted to study the effects of dietary β-carotene supplementation on lactation performance,blood routine indexes and serum biochemical and immune indexes of dairy cows during the lactating peak period.【Method】 Twenty healthy Chinese Holstein dairy cows during the dry period with an average body weight of (550.00±32.71) kg and parities of 2-3 were randomly divided into 4 groups with 5 cows in each group.The cows in control group were fed a basal diet,the cows in experimental groups were fed the basal diet supplemented with 600,1 200 and 1 800 mg/d β-carotene,respectively.The experiment lasted for 150 days from 60 days before calving to 90 days after calving.During the experiment,the milk yield and milk composition of dairy cows were measured.Blood was collected from the tail vein to determine the blood routine indexes,and isolated serum was used to determine the indexes of biochemistry and immune at the end of the experiment.【Result】 Compared with the control group,dietary β-carotene supplementation had no significant effect on milk yield,milk fat rate,milk protein rate and lactose rate of dairy cows (P＞0.05).600,1 200 mg/d β-carotene supplementation significantly reduced somatic cells number of dairy cows (P＜0.05).Dietary β-carotene supplementation had no significant effect on blood white blood cell count (WBC),red blood cell count (RBC),platelet count (PLT),hemoglobin (HGB) and hematocrit (HCT) (P＞0.05).1 200 and 1 800 mg/d β-carotene supplementation significantly increased serum triglyceride (TG) content (P＜0.05),600,1 200 mg/d β-carotene supplementation significantly increased serum high density lipoprotein (HDL) content (P＜0.05).600 and 1 800 mg/d β-carotene supplementation significantly reduced the serum CD4⁺ content of dairy cows (P＜0.05),1 200 mg/d β-carotene supplementation significantly decreased the contents of serum CD8⁺,tumor necrosis factor-α (TNF-α),interleukin-1 β (IL-1β),interleukin-2 (IL-2) and interferon-γ (IFN-γ) contents,and significantly increased CD4⁺/CD8⁺ ratio in serum of dairy cows (P＜0.05).【Conclusion】 In conclusion,appropriate of β-carotene in diet could reduce somatic cell count in milk and inflammatory cytokines,and enhance cellular immune function of dairy cows during the lactating peak period,and 1 200 mg/d was the appropriate supplemental level.

【Objective】 The purpose of this experiment was to study the effects of guanidinoacetic acid (GAA) on the meat quality and abdominal fat deposition of broilers.【Method】 In this experiment,90 1-day-old Cobb broilers were selected and randomly divided into 3 groups,with 5 replicates and 6 broilers per replicate.The group Ⅰ (control group) was fed with basal diet,groups Ⅱ and Ⅲ were added with 1.2 and 3.6 g/kg GAA in basal diet,respectively.The feeding cycle was 42 days,at the end of experiment (42 days),the breast muscle,leg muscle,liver,abdominal fat and blood samples were collected to measure meat quality,pectoralmyopathies and abdominal fat deposition.【Result】 Compared with group Ⅰ,pH_{45 min} of pectoralis and leg muscle in groups Ⅱ and Ⅲ were extremely significantly or significantly increased (P＜0.01 or P＜0.05),the b^* value of pectoralis,a^* value and water loss rate of leg muscle in group Ⅱ were extremely significantly increased (P＜0.01),L^* value of leg muscle,the incidences of wooden breast and white striping in group Ⅱ were significantly decreased (P＜0.05),and the incidences of wooden breast in group Ⅲ was significantly increased (P＜0.05).The abdominal fat rate and abdominal adipocytes diameter in group Ⅱ was significantly lower than that in groups Ⅰ and Ⅲ (P＜0.05).The level of serum triglyceride (TG) in group Ⅱ was significantly higher than that in groups Ⅰ and Ⅲ (P＜0.05).【Conclusion】 Supplementation of 1.2 g/kg GAA in diet could improve the meat quality of broilers,reduce the occurrence of pectoral wooden breast and white striping in broilers, and significantly reduce abdominal fat deposition.

【Objective】 In order to improve the degradation rate of aflatoxin B₁ (AFB₁) in animal feed,artificial gastrointestinal fluid (AGIF) was used to make compound biological detoxifier for degrading AFB₁ effectively.【Method】 Firstly,the degradation rates of AFB₁ by four probiotics in AGIF were investigated,then the optimal proportion of 4 species of beneficial microbes was obtained by means of response surface methodology to make compound probiotics for degrading AFB₁.And then the compound probiotics was combined with different concentrations of AFB₁-degrading enzyme to screen the optimal portfolio of compound biological detoxifier for degrading AFB₁ effectively.Finally,effect of compound biological detoxifier on degrading AFB₁ was evaluated under the AGIF condition.【Result】 ①The AFB₁-degradation rates by four species of Bacillus subtilis, Lactobacillus casein,Enterococcus faecalis and Candida utilis were 20.59%,19.97%,28.75% and 25.67%,respectively.② The maximal AFB₁-degradation rate was 41.5% when the visible counts of four probiotics were 1.0×10⁵,1.0×10⁵,1.0×10⁷ and 1.0×10⁵ CFU/mL to make compound probiotics by response surface methodology,significantly higher than that of single probiotic alone (P＜0.05);③When compound probiotics was combined with 0.1% AFB₁-degradation enzyme to make compound biological detoxifier,AFB₁-degradation rate was increased to 55.28%,significantly higher than that of probiotics or AFB₁-degradation enzyme alone (P＜0.05).【Conclusion】 The combination of compound probiotics with mycotoxin-degradation enzyme (compound biological detoxifier) was able to degrade AFB₁effectively.It could provide reference for biological detoxification measures of AFB₁ in feed.

【Objective】 The aim of this study was to investigate the effects of selenium on lead-induced apoptosis in mouse Leydig cells in vitro.【Method】 Mouse Leydig cells were divided into control,selenium,lead and selenium＋lead groups.2 μmol/L sodium selenite was added into the cell culture medium of selenium group,40 μmol/L lead acetate was added into the cell culture medium of lead group,2 μmol/L sodium selenite and 40 μmol/L lead acetate were added into the cell culture medium of selenium＋lead group.The cells were cultured for 24 h in vitro.Cell proliferation was detected by MTT assay.The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in cells were detected by biochemical analysis.Reactive oxygen species (ROS) level was measured by chemical fluorescence.Real time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of cysteinyl aspartate specific proteinase 3 (Caspase3),cysteinyl aspartate specific proteinase 8 (Caspase8),B-cell lymphoma-2 (Bcl-2) and Bcl2-associated X (Bax).【Result】 Compared with control group, cell proliferation was significantly inhibited in lead group (P＜0.05),the content of MDA and ROS level were significantly increased (P＜0.05),and the activities of SOD and GSH-Px were significantly decreased (P＜0.05).At the same time,lead significantly up-regulated the mRNA and protein levels of Caspase3,Caspase8 and Bax (P＜0.05),but down-regulated the mRNA and protein levels of Bcl-2 (P＜0.05).The proliferative activity of Leydig cells in selenium group was significantly increased than that in control group (P＜0.05).Compared with lead group,the content of MDA and ROS level in the cells of the selenium＋lead group were significantly decreased,and the activities of SOD and GSH-Px were significantly increased,which were between control and lead groups (P＜0.05).The mRNA and protein expressions of Caspase3,Caspase8,and Bax in selenium＋lead group were significantly lower than those in lead group (P＜0.05),while Bcl-2 showed an opposite trend (P＜0.05).【Conclusion】 Lead could inhibit the proliferative activity of mouse Leydig cells in vitro and induce oxidative stress and apoptosis.Supplementing appropriate amount of selenium could promote cell proliferation,alleviate lead-induced cell oxidative stress and apoptosis by increasing the activity of antioxidant enzymes.

【Objective】 This study was aimed to investigate the polymorphism of the gonadotropin-inhibitory hormone (GnIH) gene in pigeons,and screen the single nucleotide polymorphism (SNP) that significantly affect egg-laying traits in female pigeons for early selection of high-yielding female pigeons.【Method】 A total of 78 pairs of White King pigeons was selected as the research objects,and primers were designed according to the mRNA sequence of GnIH gene in pigeons (GenBank accession No.:MG589639.1) obtained by earlier cloning,and GnIH gene was detected by PCR amplification and direct sequencing for SNP and genotyping.The linkage imbalance analysis was conducted on GnIH gene SNP using Haploview software,and the correlation between the polymorphism of GnIH gene and egg-laying trait in White King pigeons was analyzed by SPSS 22.0 software.【Result】 Seven SNPs were detected in GnIH gene of White King pigeons,which were located in exon 1 (c.59 C＞T and c.72 T＞A),exon 2 (c.398 G＞C) and 3'-UTR (c.768 C＞T,c.860 G＞A,c.909 A＞G and c.961 T＞C),respectively.Expect c.59 C＞T deviated from Hardy-Weinberg equilibrium (P＜0.05),all other SNPs were in Hardy-Weinberg equilibrium (P＞0.05).c.768 C＞T was completely linked to c.961 T＞C (D’=1 and r²=1),c.860 G＞A was strongly linked to c.768 C＞T and c.961 T＞C (D’=1 and r²=0.69),and other SNPs were weakly linked.c.398 G＞C mutation caused changes in amino acid properties and mRNA secondary structure,which was significantly correlated with annual egg production and body weight at the first egg of female pigeons (P＜0.05).【Conclusion】 The SNP on exon 2 of GnIH gene had certain genetic effects on the laying traits of pigeons,which could be used as candidate molecular marker for the egg-laying performance of female pigeons.

Bone mineral density is an important index of bone strength,which is widely used in the evaluation of the bone health of human and animals.In recent years,the problem of animal limb and hoof has brought a certain degree of economic loss to the breeding industry,especially the pig industry.The determination of bone mineral density level will be helpful to establish the early warning standard of animal elimination in farm,timely treatment or elimination of animals with limb and hoof disease,reduce the breeding cost,and improve the production efficiency of farm.Since bone mineral density levels are predictive of hoof and limb problems,in vivo measurement of animal bone mineral density can greatly improve animal performance and longevity.The bone mineral density of mammals is affected by parity,pregnancy,lactation,and hormone.In vivo measurement of bone mineral density of mammals can timely reflect the bone health status of animals,so as to improve the bone mineral density of animals and the bone health status of animals.The author reviewed the research progress on bone mineral density at home and abroad in recent years,including several common methods of bone mineral density measurement in vivo,such as dual-energy X-ray absorption,quantitative computed tomography,quantitative ultrasound,etc.,the relationship between bone mineral density and bone health,and the relationship between bone mineral density and parity,pregnancy and lactation,hormone,etc.,so as to establish an early warning standard for the treatment or elimination of animal with hoof disease,and maximally improve the production performance and utilization life of animals.

【Objective】 The purpose of this study was to explore the maturation characteristics of raccoon dog oocytes,and to provide a basis for the in vitro maturation culture of raccoon dog oocytes and to select high-quality mature oocytes,so as to improve the in vitro fertilization efficiency of raccoon dog.【Method】 Fresh ovaries from 1 to 3 years old female raccoon dogs at reproductive stage were collected to prepare light and electron microscope samples to observe the changes of organelles during oocyte maturation of raccoon dog. X-ray microanalysis was used to explore the relationship between the changes of chemical elements in plasma membrane and maturation of oocyte.【Result】 The maturation and development of raccoon dog oocytes could be divided into 8 stages according to the layer number of granulosa cells,the diameter of follicles,the size of oocytes and the changes of the zona pellucida.Raccoon dog oocytes were surrounded by granulosa cells in the early developmental stage (stages Ⅰ to Ⅳ),with the gradual appearance of the zona pellucida and the follicular cavity,the chromatin in the germinal vesicles became more and more compact.With the maturation and development of oocytes (stages Ⅴ to Ⅶ),cortical granules and microvilli appeared and the number of organelles such as Golgi complex,mitochondria increased significantly and they gradually migrated to the cortex,while mitochondria and lipid droplets experienced morphologic variation.When the oocyte grew mature (stage Ⅷ),the Golgi complex and the rough endoplasmic reticulum disappeared,and the cortical granules were arranged under the plasmalemma.After ovulation,the nucleolus became compact.The contents of sodium,chloride and sulfur in oocyte plasma membrane decreased with the increase of follicular cavity.The contents of sodium,chlorine and sulfur in oocyte plasma membrane in stage Ⅴ were significantly higher than those in stage Ⅶ (P＜0.05),and the relative content of calcium was significantly lower than that of stage Ⅵ and Ⅶ oocytes (P＜0.05).Potassium showed a low-high-low variation,and the content of potassium in oocyte plasma membrane in stage Ⅵ was significantly higher than that of stage Ⅴ and Ⅵ oocytes (P＜0.05).【Conclusion】 After the raccoon dog oocytes developed to stage Ⅴ,the type and number of organelles increased,the plasma membrane chemical elements changed,and oocyte membrane potential was depolarized,and the oocytes began to enter the mature stage.At this time,the oocytes had the ability to mature in vitro.

【Objective】 The aim of this study was to analyze the genetic diversity of forkhead box A2 (FOXA2) gene exon 3 polymorphism in local pig breeds and its correlation with reproductive traits in Songliao Black pigs.【Method】 The single nucleotide polymorphisms (SNPs) (genotype frequency,gene frequency,genetic homozygosity,genetic heterozygosity,number of effective alleles,polymorphic information content) of FOXA2 gene exon 3 were screened by direct sequencing in ear tissues of 90 healthy Songliao Black pigs,and the correlation between SNP of FOXA2 gene eoxn 3 and reproductive performances (total number of litter,number born alive,number of nipples,birth weight,3-week weight,weaning weight and weaned piglets) were analyzed using SPSS 26.0 software.【Result】 The results obtained that SNPs sites (C1337A,C1343G,C1626T)were found of FOXA2 gene exon 3 in Songliao Black pig.Genotypes of CC,CA and AA were contained in C1337A,genotypes of CC,CG and GG were contained in C1343G,and genotypes of CC,CT and TT were contained in C1626T.The chi-square fitness test showed that SNPs (C1337A,C1626T) were in Hardy-Weinberg equilibrium (P＞0.05),C1343G was not in Hardy-Weinberg equilibrium (P<0.05).The calculation of polymorphism information content (PIC) showed that the genetic heterozygosities of C1337A and C1626T were in moderate polymorphism (0.25＜PIC＜0.50),the genetic heterozygosity of C1343G was in the highly polymorphism (PIC＞0.50).The results of association analysis showed that the total number born of CC genotype with C1337A locus was significantly higher than that of CA genotype (P＜0.05),the 3-week weight and weaning weight of piglets born by AA genotype individuals were extremely significantly higher than that of CC and CA genotype (P＜0.01);The 3-week weight and weaning weight of piglets born by CC genotype individuals with C1626T locus were significantly or extremely significantly higher than that of CT and TT genotype (P＜0.05 or P＜0.01),the number of nipples of CC genotype was significantly higher than that of CT genotype (P＜0.05).【Conclusion】 There were three SNPs sites (C1337A,C1343G,C1626T) in exon 3 of FOXA2 gene,which had a significant correlation with the total number born,3-week weight,weaning weight and number of nipples of Songliao Black pig.

【Objective】 The purpose of this study was to explore the haplotypes diversity,genetic differentiation and phylogenetic relationships of Aedes albopictus in different populations in Huaihua district,Hunan province.【Method】 Thirty Aedes albopictus were collected from Huaihua district,Hunan province.The partial sequences of the ribosomal 18S rRNA and cytochrome c oxidase subunit 1 (cox1) gene from Aedes albopictus were amplified using PCR method.The variation sites on both genes sequences were analyzed with ClustalX and the haplotypes diversity,genetic differentiation and phylogenetic relationships of Aedes albopictus were studied by Dnasp 5.0,Network 4.6,Arlequin version 3.0,PhyML 3.0 and Mega 5.0,respectively.【Result】 The results showed that the 18S rRNA and cox1 gene sequences of all samples was 489 and 1 004 bp,respcetively.A total of 30 mutation loci were found in the 18S rRNA sequences of Aedes albopictus,including 17 haplotypes (A1 to A17,Hd=0.9149),among which haplotype A5 was the oldest haplotype.The gene differentiation coefficient (Gst),genetic differentiation coefficient (Fst) and gene flow (Nm) of 18S rRNA gene of Aedes albopictus were 0.05614,0.24001 and 0.792,respectively.There were 2 mutation loci and 3 haplotypes (B1 to b3,Hd=0.1908) in the cox1 gene sequence of Aedes albopictus.Haplotype B1 was the oldest haplotype.The Gst,Fst and Nm of cox1 gene of Aedes albopictus were 0.05000,0.04762 and 4.999,respectively.Among the phylogenetic tree,30 Aedes albopictus from Huaihua were located in a branch of two developmental trees.【Conclusion】 The results indicated that there was a certain degree of genetic variation and haplotype polymorphism among the individuals and populations of Aedes albopictus from Huaihua district,Hunan province.However,the haplotypes and individuals had the close genetic relationships,and the frequent genetic exchanges occurred between the individuals of the two populations,whithout genetic differentiation.

【Objective】 The purpose of this experiment was to study the polymorphism of myogenin (MyoG) gene and its relationship with body size and meat quality traits in White feather King pigeons,and provide reference for the breeding of molecular markers in this breed.【Method】 A total of 108 12-week-old White feather King pigeons were selected as the research objects,PCR and direct sequencing were used to screen the SNP of MyoG gene in White feather King pigeons,RNAfold was used to predict the mRNA secondary structure before and after MyoG gene mutation,and SPSS 26.0 software was used to analyze the correlation between SNP of MyoG gene and body size and meat quality traits of White feather King pigeons.【Result】 Four SNPs were detected in exon 3 of MyoG gene in White feather King pigeons:g.590075 C＞T,g.590084 G＞A,g.590102 G＞A and g.590114 C＞T,all of which were synonymous mutations and resulted in three genotypes,among them,g.590075 C＞T belonged to low polymorphism,the other 3 SNPs showed moderate polymorphism.χ² results showed that g.590084 G＞A and g.590114 C＞T were in Hardy-Weinberg equilibrium (P＞0.05),and there was strong linkage disequilibrium between the two SNPs.The mutations of g.590084 G＞A and g.590102 G＞A resulted in the change of secondary structure and free energy of MyoG gene mRNA sequence.The combination of 4 SNPs produced 4 haplotypes and 9 diplotypes.The results of correlation analysis showed that g.590075 C＞T was extremely significantly associated with keel length in White feather King pigeons (P＜0.01);g.590102 G＞A had an extremely significant effect on body length (P＜0.01).The body length of H3H3 (CCAAAACC) individuals were superior to the other diplotypes,and the keel length and chest width of H4H4 (TTGGGGCC) individuals were superior to the other diplotypes.g.590084 G＞A and g.590114 C＞T were extremely significantly or significantly associated with pectoral muscle shear force (P＜0.01 or P＜0.05).The diplotype H3H3 (CCAAAACC) individuals were better than the other diplotypes in pH and flesh color of chest muscle,while in the shear force and water loss rate,the diplotype H2H4 (CTGAGGCT) and H4H4 (TTGGGGCC) performed better.【Conclusion】 MyoG gene had significant effect on some body size and meat quality traits in White feather King pigeons,which could be used as a candidate gene for breeding new lines in White feather King pigeons.

【Objective】 By obtaining and comparing the partial sequence of mtDNA D-loop hypervariable region Ⅰ of Sujiang pig,the germplasm resource characteristics and genetic diversity of Sujiang pig population were revealed.【Method】 Ear tissues of 100 parturient sows from the core group of Sujiang pigs were collected and DNA were extracted.And gene fragments of mtDNA D-loop hypervariable region Ⅰ of sows in the core group of Sujiang pigs were amplified and sequenced.The NCBI online BLAST was used to compare and calibrate the sequencing and reference sequences,and homologous sequences were found to determine the length and location of the sequence of high variable region Ⅰ of mtDNA D-loop.DnaSP v.5.10.1 software was used to calculate the number of nucleotide polymorphic sites and mutation sites in the population and the haplotype diversity (Hd),nucleotide polymorphism (Pi),average nucleotide difference (K) and other parameters were analyzed.The genetic distance of the sample was calculated based on Kimura’s 2-prarmetermodel using Mega 7.0 software and the genetic distance between different haplotypes of Sujiang pig mtDNA D-loop region sequences were obtained.The phylogenetic tree was constructed by using Neighbor-Joining (NJ) on the mtDNA D-loop hypervariable region Ⅰ sequences of 4 haplotypes of Sujiang pig,17 Chinese pig breeds and European domestic pigs (accession No.:AF034253.1).【Result】 The highly variable region Ⅰ of mtDNA D-loop sequence of 100 Sujiang pigs was amplified and sequenced,and the effective sequence with a length of 429 bp was obtained,in which the content of AT was 63.1%,and the content of GC was 36.9%,neutral test Tajima’s D value was -1.80517 (P＜0.05).It was confirmed that Sujiang pig population significantly deviated from neutral test.A total of 19 mutation sites were detected in the sequencing column,and 4 haplotypes were defined,Hd was 0.578,Pi was 0.0032, and the average genetic distance between the four haplotypes in the population ranged from 0.004 to 0.031.Phylogenetic tree results showed that the joining group were divided into two groups:Domestic and foreign.The four haplotypes of Sujiang pig were clustered in the domestic branch,among which haplotype H2 and Erhuface pig were clustered in the same group,and haplotype H4 was clustered in the same group with Jiangquhai and Wannanhua pig.【Conclusion】 The proportion of polymorphic sites in the high variation region Ⅰ of mtDNA D-loop in Sujiang pig population was high,Hd was high,and Pi was low.The genetic distance of dominant haplotypes in Sujiang pig population was relatively close,and the proportion of dominant haplotypes was high,which reflected that there were fewer maternal sources in Sujiang pig population.

Exosomes are nanoscale and homogeneous membranous vesicles that widely exist in various body fluid environments and some tissues,which are mainly composed of various biological components such as proteins,lipids,mRNA,miRNA and lncRNA.In recent years,researchers have isolated exosomes from the follicular fluid of different animals.In addition,it has been scientifically confirmed that follicular fluid exosomes are involved in a series of reproductive processes,such as proliferation of follicular granulosa cells,egg formation and development,animal fertilization,embryo development,and male animal reproductive function,which provides new ideas for further research on the regulation of follicular fluid exosomes on mammalian reproductive function.Therefore,the author described the latest progress in the isolation and identification of exosomes from follicular fluid,their main components and the molecular mechanisms regulating mammalian reproductive function,which provided a reference for a comprehensive understanding of the structure and function of exosomes,germinal apoptosis and mechanism of action.At the same time,it provided relevant basic information for exploring animal reproductive theory and improving animal reproductive technology using exosomes and applying it to practical production.

【Objective】 This study was aimed to investigate the immunogenicity of OmpW and TbpA proteins of swine Pasteurella multocida and explore their potential as vaccine candidate antigens.【Method】 The similarity of amino acid sequences of OmpW and TbpA proteins in different serotypes of swine Pasteurella multocida were compared by NCBI.Further bioinformatics analysis of OmpW and TbpA proteins were carried out.Based on this,the recombinant expression strains pET28a-OmpW-BL21 and pET28a-TbpA-BL21 were successfully constructed,which were purified and mixed with aluminum hydroxide adjuvant to prepare subunit vaccine.After immunizing mice,the immunogenicity of OmpW and TbpA proteins was evaluated using swine Pasteurella multocida GX-PmD4 strain to challenge.【Result】 The results of similarity analysis indicated that OmpW and TbpA proteins were highly conserved in different serotypes of swine Pasteurella multocida.SignalP 6.0 Server and TMHMM Server v.2.0 softwares predicted that OmpW protein had signal peptide and transmembrane region,while TbpA protein only had signal peptide without transmembrane region.ABCpred software analysis showed that OmpW and TbpA proteins had 14 and 22 B cell antigen epitopes,respectively.SOPMA and SWISS-MODEL softwares further predicted the protein structure of OmpW and TbpA,which showed that the alpha helix,beta turn,extended strand and random coil accounted for 16.18%,5.39%,36.76%,41.67%,and 40.42%,5.69%,16.77%,37.13%,respectively.The recombinant OmpW and TbpA proteins obtained by prokaryotic expression technique had strong antigenicity.Compared with control group,the serum specific antibody levels of OmpW and TbpA in serum were extremely significantly increased on 14 and 28 d after immunizing mice (P<0.01).The survival rate of mice in OmpW and TbpA groups was 50.0% and 37.5%,respectively.【Conclusion】 In this study,the OmpW and TbpA proteins of swine Pasteurella multocida were successfully expressed, the recombinant OmpW protein had strong immunogenicity and could be used as an effective candidate antigen for subunit vaccine against swine Pasteurella multocida.

【Objective】 The purpose of this study was to clone the heme oxygenase-1 (HO-1) gene in Dezhou donkey,carry out its prokaryotic expression and prepare polyclonal antibody,and detect the expression of HO-1 gene in different tissues of Dezhou donkey,so as to provide materials for functional study of HO-1 gene.【Method】 The specific primers were designed according to HO-1 gene of horse (GenBank accession No.:XM_023631135.1).The HO-1 gene coding sequences (CDS) were amplified in cDNA following RNA extracted from donkey primary alveolar macrophages (DPAMs),the sequences obtained by sequencing were compared with other species for similarity,and the phylogenetic tree was constructed.The encoding protein of HO-1 gene was analyzed by bioinformatics software online.HO-1 gene was cloned into pCold-SUMO vector,the recombinant HO-1 protein was purified by Ni-chelating affinity chromatography,and immunized Zealand rabbits to prepare polyclonal antibodies.The specificity of polyclonal antibody and maximum dilution binding to antigen were identified by ELISA and Western blotting.The expression of HO-1 protein in 4 tissues of Dezhou donkey were detected by immunohistochemical assay.【Result】 The CDS sequence of HO-1 gene in Dezhou donkey was 873 bp in total length,and coding for 290 amino acids.The amino acid sequence of HO-1 gene exhibited the highest similarity and the closest genetic relationship with Equss caballus,and showed the lowest similarity and the farthest genetic relationship with Gallus gallus.The HO-1 protein in Dezhou donkey was an instable and hydrophilic protein without signal peptide,but with one transmembrane domain,its molecular mass was 33.04 ku,it contained alpha helix in the secondary structure.ELISA and Western blotting results showed that the maximum dilution of anti-HO-1 polyclonal antibody binding to HO-1 was 1∶102 400,and the host HO-1 protein could be recognized by the polyclonal antibody.The immunohistochemical assay results showed that HO-1 protein was effectively expressed in 4 tissues of Dezhou donkey.【Conclusion】 The recombinant HO-1 protein in Dezhou donkey and specific polyclonal antibody were obtained successfully,these data would provide reference for the role of HO-1 gene in equids infected with pathogens.

【Objective】 The aim of this study was to prokaryotically express VP2 protein of Luoyang Feline panleukopenia virus (FPV) isolates and prepare polyclonal antibodies from mice.【Method】 A pair of specific primers were designed according to the published FPV VP2 gene sequence (GenBank accession No.:EU018143.1),DNA was extracted from the feces of 7 cats suspected of FPV infection in Luoyang area,and VP2 gene was identified by PCR and sequenced.The genetic evolution of VP2 gene was analyzed by Mega 11.0 software.The online software was used to predict the structure characteristics of VP2 protein and find the dominant epitopes.The VP2 dominant antigen (sVP2) was cloned into pET-32a(+) vector,the recombinant plasmid pET-32a-sVP2 was constructed and transformed into E.coli Rosetta (DE3) competent cell for induction and optimal expression.The recombinant protein was identified by SDS-PAGE and Western blotting.BABL/c mice were immunized with pET-32a-sVP2 purified by nickel column affinity chromatography to prepare polyclonal antibody,and the titer of the antibody was detected by ELISA method.【Result】 A FPV strain was isolated and named LY-1.Genetic evolution analysis showed that this strain belonged to FPV-G2 subgroup,which was the closest relative to Beijing strain (GenBank accession No.:MT270581).Bioinformatics analysis showed that VP2 was a stable intramembrane protein with no signal peptide and transmembrane region,and there were abundant B cell epitopes (1-800 bp,sVP2) at the N-terminus.The recombinant plasmid pET-32a-sVP2 was successfully constructed and expressed.SDS-PAGE showed that the molecular mass of the recombinant protein was about 50 ku,mainly in the form of inclusion bodies,the optimal protein expression was induced by 1 mmol/L IPTG at 30 ℃ for 6 h;Western blotting results showed that the recombinant protein could react specifically with mouse anti-His monoclonal antibody.ELISA results showed that the antibody titer of the recombinant protein was 1∶128 000.The antibody could recognize the specific reaction between sVP2 and prokaryotic expression of VP2 protein.【Conclusion】 FPV sVP2 fragment was cloned and polyclonal antibody was prepared,which provided a reference for further study of the role of VP2 in FPV replication and pathogenesis and establishing a diagnostic method for FPV.

【Objective】 This study was aimed to explore the biological and genome characteristics of Escherichia coli (E.coli) phage vB_EcoP_GN07,and provide biological materials for the development of phage cocktail formulations against pathogenic E.coli disease.【Method】 A strain of E.coli lytic phage vB_EcoP_GN07 was isolated from a chicken farm in Nanning,Guangxi using the double-layer plate method.Transmission electron microscope observation,host spectrum determination,pH and temperature stability evaluation,the optimal multiplicity of infection (MOI) determination,one-step growth curve determination and whole genome sequencing were used to understand the morphology,biological characteristics and genome-wide characteristics of the phage.【Result】 Transmission electron microscopy showed vB_EcoP_GN07 had a head diameter of about 45 nm±5 nm and tail length of about 25 nm,belonging to the Podoviridae.which had strong host specificity.The phage titer could be kept stable at 4 to 60 ℃,and the phage activity could be maintained at pH 3.0-11.0.The optimal MOI of the phage was 10^-2.The one-step growth curve showed that the incubation period of the phage was 60 min,and the burst size was 560 PFU/cell.Sequencing results showed that it belonged to double stranded DNA,with a length of 70 120 bp and a GC content of 42.8%.The phage vB_EcoP_GN07 had 81 open reading frames,of which 23 were known functional proteins.BLASTN comparison showed that the phage vB_EcoP_GN07 had low similarity with other phages in NCBI database,conformed to the characteristics of N4-like phage,and lacked virulence genes and drug resistance genes associated with antibiotic resistance,toxins and virulence factors.【Conclusion】 An E.coli phage vB_ EcoP_ GN07 was successfully isolated in this study,which had strong cracking ability,host specificity and acid and alkali resistance.vB_ EcoP_ GN07 was less similar to other phages,and determined to be a new N4-like phage.This results provided a theoretical basis for the development of new fungicides with application E.coli phage vB_EcoP_GN07.

【Objective】 The aim of this study was to screen the inhibin α subunit (INHα)-specific nanobody (VHH) gene from the lymphocyte genome of Xingjiang Bactrian camel, and prepare a novel inhibin immune preparation,so as to indirectly increase the blood follicle-stimulating hormone (FSH) level and ovulation rate and lambing rate in animal.【Method】 INHα protein was induced by pET32a-INHA prokaryotic expression vector and purified.The purified INHα protein was renatured with urea solution and then immunized Xinjiang Bactrian camels.The VHH amino acid difference database was established by nested PCR amplification of lymphocyte VHH gene in whole blood before and after immunization and high-throughput sequencing.The pre-immune serum,the post-immune serum and the INHα-specific antibodies screened from the post-immunization serum were analyzed by liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) to obtain mass spectrometry data.After performing protein database search and data analysis on mass spectrometry data,a peptide library and protein library of INHα-specific antibodies and post-immunization-specific antibodies were established.By aligning the VHH amino acid difference database with the peptide library of INHα-specific antibody and post-immunization-specific antibody,respectively,the INHα-specific nanobody gene was screened out.Finally,the screened VHH genes were analyzed by sequence alignment,three-dimensional structure prediction and protein-protein simulation docking.【Result】 INHα protein was successfully expressed and purified,and the antibody titer of Xinjiang Bactrian camel immunized with INHα protein reached 1∶1 024 000.The VHH gene was successfully cloned from the lymphocytes,and the specific antibody against INHα was screened from the immunized serum.Finally,5 INHα-specific nanobody genes were screened by high-throughput sequencing,mass spectrometry analysis and data processing.The amino acid sequence alignment of the 5 strains of VHH showed that the overall similarity was 70.98%,and the length of the complementarity determining region 1 (CDR1) ranged from 10 to 13,the length of CDR2 ranges from 12 to 13,the length of CDR3 ranges from 12 to 22,and CDR3 had the largest amino acid sequence difference among the three complementarity determining regions.Phylogenetic tree showed that the selected 5 amino acid sequences had rich diversity.The results of protein-protein docking simulation showed that the other 4 VHH strains could pair with INHα except for VHH-267,and the main forces were hydrophobic interaction and hydrogen bonding.【Conclusion】 In this study,5 strains of INHα-specific VHH genes were firstly screened from the lymphocyte genome of Xinjiang Bactrian camel by high-throughput sequencing technology combined with mass spectrometry.This study would provide theoretical guidance and technical support for improving the level of FSH and ovulation rate in animals,and had certain reference value for the development of reproductive immunology.

【Objective】 The purpose of this experiment was to explore the risk of contamination of yak meat caused by Listeria monocytogenes in yak slaughterhouses.【Method】 Listeria monocytogenes was isolated and identified in 50 sewage samples of yak slaughterhouse by means of isolation culture,morphological observation,biochemical test and molecular method.The serotype and virulence genes of the isolate were identified and detected by PCR,and D_{600 nm}value of the isolate was determined under different growth conditions.The virulence of the isolate was determined by cell adhesion and invasion tests and mouse median lethal dose (LD₅₀).【Result】 One Gram-positive short-rod-shaped bacterium was isolated from 50 sewage samples.The isolates showed bluish-green colonies on Listeria chromogenic medium,and were Gram-positive short rod-shaped bacteria with blunt round ends,which was suspected to be Listeria monocytogenes.The results of biochemical tests showed that the isolates could hydrolyze aestin and ferment glucose,maltose and rhamnose.MR,VP,kinetic and catalase tests were positive,while mannitol and xylose fermentation tests were negative.Phylogenetic tree showed that the isolates had a high homology with Listeria monocytogenes EDG-e,the sequence similarity was 98%,and the serotype was identified as 1/2a.Virulence gene test results showed that the isolates carried inlB,inlC,inlJ,actA,plcA,prfA,mpl,hly,inlA and SigmaB virulence genes.The stress resistance test showed that the growth rate of the isolated strain was extremely significantly higher than that of the standard strain ATCC 19111 in extreme environment such as low temperature,strong acid,strong alkali,high salt,ethanol stress and oxidation (P＜0.01).The cell test showed that the adhesion rate and invasion rate of RAW246.7 cells were 8.3% and 2.5%,and the adhesion and invasion ability of RAW246.7 cells were extremely significantly higher than that of standard strain ATCC 19111 (P＜0.01).The adhesion rate and invasion rate of Caco-2 cells were 6.2% and 1.5%,respectively.The adhesion and invasion ability of Caco-2 cells were extremely significantly higher than those of standard strains ATCC 19111 (P＜0.01).Animal tests showed that the LD₅₀ of the isolated strain in mice was 10^4.7 CFU.【Conclusion】 The serotype of Listeria monocytogenes isolated in this study was 1/2a,which was the main serotype causing human listeriosis,it had strong anti-stress ability and belonged to strong strain.This study provided molecular biological basis for monitoring Listeria monocytogenes in pastoral areas and theoretical basis for ensuring food safety of yak meat.

【Objective】 The purpose of this study was to explore the function of RNA helicase DEAD-box family protein DDX1 in porcine ileal epithelial cells (IPI-2I) infected by Porcine epidemic diarrhea virus (PEDV),and lay a foundation for revealing the biological function of DDX1.【Method】 According to the sequence of porcine DDX1 gene in GenBank (accession No.XM_021087822.1),specific primers were designed.The full length of DDX1 gene was amplified by PCR and ligated into pMD19-T cloning vector.After successful identification by double enzyme digestion,the DDX1 gene was ligated into pcDNA3.1 expression vector.After the transformation of the product,the positive colonies were screened.IPI-2I cells were cultured,and the cells that had been passaged twice were spread in a six-hole plate for follow-up experiment.The recombinant plasmids of the positive colonies were transfected into the six-well plate for 24 h.The protein was collected,and the transfection efficiency of DDX1 was detected by Western blotting.IPI-2I cells transfected with pcDNA3.1-DDX1 or PBS (control) were infected with PEDV with a multiple of infection (MOI) of 1.0 to establish a cell model of PEDV infection.Total protein and RNA were collected 12 h after PEDV infection.The expression of PEDV N protein at cell level was detected by indirect immunofluorescence assay,the expression of PEDV N gene mRNA was detected by Real-time quantitative PCR,and the expression of PEDV N protein was detected by Western blotting.Bioinformatics software was used to predict the phosphorylation site of DDX1 peotein,and immunoprecipitation test was used to detect the phosphorylation level of DDX1 after PEDV infection in IPI-2I cells for 12 h.【Result】 The target DDX1 gene band of 2 223 bp was successfully obtained by PCR amplification.The results of double digestion identification showed that pMD19-T-DDX1 cloning vector and pcDNA3.1-DDX1 eukaryotic expression vector were successfully constructed.The expression of DDX1 protein in the cells transfected with pcDNA3.1-DDX1 was extremely significantly higher than that in PBS group (P＜0.01),indicating that DDX1 transfection was successful.The results of indirect immunofluorescence assay showed that compared with PBS group,there were a large number of green fluorescence signals in PEDV infection group,indicating that the IPI-2I cell model infected by PEDV was successfully constructed.In IPI-2I cells transfected with pcDNA3.1-DDX1 recombinant plasmid,compared with PBS group,the fluorescence intensity of PEDV N protein significantly decreased,the mRNA and protein expression of PEDV N gene were extremely significantly decreased (P＜0.01).Bioinformatics analysis showed that DDX1 protein had multiple phosphorylation sites,including 17 serine,12 threonine and 6 tyrosine.The results of immunoprecipitation test showed that the level of DDX1 phosphorylation in PEDV infected group was extremely significantly higher than that in uninfected group (P＜0.01).【Conclusion】 In this study,the eukaryotic expression vector of pcDNA3.1-DDX1 was successfully constructed.It was found that 12 h after PEDV infection,DDX1 inhibited the replication of PEDV and increased the phosphorylation level,indicating that DDX1 protein might inhibit the replication of PEDV through phosphorylation during PEDV infection.

【Objective】 The purpose of this experiment was to prepare ferritin nanoparticles carrying the epitope of Porcine epidemic diarrhea virus (PEDV) antigen and evaluate their immunogenicity,so as to provide a new candidate subunit vaccine for the prevention and control of PEDV.【Method】 The COE and S1D regions of PEDV fibrin S were designed as the tandem epitope S1CD,and the codon was optimized and synthesized.The recombinant plasmid pET28a-S1CD expressing S1CD protein alone and the recombinant plasmid pET28a-S1CD-Ferritin fusion expression S1CD protein and ferritin subunit were constructed,and then induced in E.coli and purified.SDS-PAGE and Western blotting were used to verify the expression of S1CD and S1CD-Ferritin proteins in E.coli.The morphology and structure of S1CD and S1CD-Ferritin proteins were observed by transmission electron microscope.The mice were immunized with purified S1CD-Ferritin protein,and S1CD protein and PBS were used as controls.Indirect ELISA was used to detect the level of S-specific antibody and the content of interferon-γ (IFN-γ).The level of neutralizing antibody was determined by neutralization test,and the immunogenicity of recombinant protein in mice was analyzed.【Result】 The recombinant plasmids pET28a-S1CD and pET28a-S1CD-Ferritin were successfully constructed.The result of SDS-PAGE showed that the recombinant protein S1CD and S1CD-Ferritin were efficiently expressed,with the size of about 22 and 39 ku,respectively,and both existed in the form of inclusion bodies.Western blotting results showed that the purified proteins could react specifically with the PEDV positive serum,indicating that the recombinant proteins S1CD and S1CD-Ferritin expressed by prokaryotic expression system had good reactivity.The result of transmission electron microscope showed that the purified S1CD-Ferritin protein could self-assemble into uniform nanoparticles with the diameter of 12-20 nm,indicating that S1CD-Ferritin protein had been successfully assembled into nanoparticles.Serums antibody test results showed that 7 days after the first immunization,the titer of PEDV specific antibody in S1CD-Ferritin group was significantly higher than that in S1CD group (P＜0.05).28 days after the first immunization,the level of neutralizing antibody and the content of IFN-γ were significantly higher than that in S1CD group (P＜0.05).【Conclusion】 The PEDV epitope fused with ferritin was highly expressed in E.coli,and could self-assemble into nanoparticles,and effectively enhance the immunogenicity of the protein antigens,which broadened the research ideas for studying new PEDV subunit vaccine.

【Objective】 The purpose of this study was to design and synthesize antigen epitopes for the receptor binding domain (RBD) of Porcine deltacoronavirus (PDCoV),preparation and identification polyclonal antibodies.【Method】 In order to specifically detect PDCoV RBD antigen,bioinformatics techniques were applied to predict its potential antigenic epitopes.Match the epitope amino acid sequence with 15 other strains of delta Coronavirus and 14 other Porcine coronavirus strains were compared for similarity. The selected dominant epitope was coupled with the carrier protein keyhole limpet hemocyanin (KLH) to synthesize polypeptides and immunize mice to prepare specific antibody against PDCoV RBD.The PDCoV RBD expressed in different systems and S protein expressed in PDCoV,TGEV and PEDV -infected ST cells were identified by Western blotting,and the antibody potency was determined by indirect ELISA.【Result】 After sequence alignment,bioinformatics technology predicted that the synthetic epitope antigens had very low sequence similarity with other 15 delta Coronaviruses (0-14.28%) and other 14 Porcine coronaviruses (0-7.14%),with good conservation.And the Western blotting results showed that after 1∶500,1∶1 000 and 1∶2 000 dilutions all the prepared peptide antibodies at fold ratio dilution could specifically recognize the PDCoV RBD protein expressed by Expi293F transfected with pcDNA3.1-PDR-Strep,the PDCoV RBD protein expressed by Baculovirus infected SF9 cells.The PDCoV S protein expressed in ST cells infected with PDCoV could be detected after 1∶1 000 dilution,while the expression of specific protein was not detected in cells infected with TGEV and PEDV.ELISA detection of peptide-specific antibodies in antisera could reach a potency of 1∶12 800.【Conclusion】 PDCoV RBD protein antigenic epitopes were successfully predicted using bioinformatics techniques.The antibodies obtained after immunization had good specificity.

Macrophage-expressed gene 1 (Mpeg1) protein is a pore-forming protein belonging to the membrane attack complex/perforin (MACPF) superfamily.It is widely found in animals and is considered to be the oldest MACPF pore-forming protein in existence.Mpeg1 is involved in host innate immunity by killing bacteria,viruses and parasites.In antimicrobial immunity,cells with high expression of Mpeg1 can resist variety of pathogens such as intracellular bacteria,extracellular bacteria,acid-resistant bacteria and drug-resistant bacteria.In the host,the spread and infection of pathogens is limited and the development of chronic progressive diseases is inhibited indirectly by this broad-spectrum bactericidal activity.In anti-parasitic immunity,Mpeg1 can directly kill Cryptocaryon irritans,Miamiensis avidus and other protozoa.In antiviral immunity,cytokines induce the expression of Mpeg1 to resist viral infection.The studies in recent years have shown that Mpeg1 plays an important role in inflammatory response,antigen presentation and initial diagnosis of some tumor diseases.The molecular mechanism of Mpeg1 in pathogen clearance:The induced expression of Mpeg1 is redistributed in cells through ubiquitination and finally co-localized with pathogens in lysosomes,the acidic environment triggers Mpeg1 to form pores on the surface of bacteria,various antimicrobial effectors enter pathogens to participate in pathogen clearance.The discovery,pathogen clearance and other immune-related functions,and its molecular mechanisms were reviewed in this article,in order to provide reference for the further research and application of Mpeg1.

Bovine viral diarrhea(BVD) is an infectious disease caused by Bovine viral diarrhea virus (BVDV) infection.BVD will lead to the decrease of animal performance and economic losses in global cattle industry.The clinical symptoms and pathological changes of BVD are similar to other bovine-derived diarrhea virus diseases.Therefore,it is necessary to select a fast and efficient BVDV diagnosis method according to different conditions,then take immediate measures to block BVDV transmission between cattle,between PI cattle and healthy cattle,so as to reduce the economic losses caused by BVDV to the cattle industry.There are many methods for BVDV detection,mainly include virus isolation and identification,Real-time quantitative RT-PCR,RT-PCR,loop-mediated isothermal amplification (LAMP),recombinase polymerase amplification (RPA),colloidal gold immunodetection technology (GICT),enzyme-linked immunosorbent assay (ELISA),antigen capture ELISA (AC-ELISA),indirect immunofluorescence (IFA),etc.In recent years,some new technologies and optimal methods have been applied to the detection of BVDV,such as antibody liquid chip technology,CRISPR-LwCas13a,dual nano RT-PCR method,all of which have the advantages of strong specificity and high sensitivity.The author summarizes research progress of BVDV detection methods in two aspects of antigen detection and antibody detection,and compares the advantages and disadvantages of each detection method.It will be convenient for veterinary research institutions and departments at all levels to select appropriate and efficient BVDV detection methods according to the actual situation,so as to provide a reference for the epidemiological investigation and prevention of BVD in China.

【Objective】 This study was aimed to investigate the effects of Clostridium butyrate (C.butyricum) and sodium butyrate on semen quality and testicular tissue of mice stimulated by high fat diet,so as to provide experimental basis and theoretical support for the treatment of male reproductive diseases.【Method】 In experiment 1,32 8-week-old C57BL/6 male mice were randomly divided into 4 groups.The mice in control group (NC) were fed a basal diet, in high fat diet group (HFD), C. butyricum treatment group (HFD+C.butyricum) and sodium butyrate treatment group (HFD+Sodium butyrate) were fed high fat diet containing 60% fat.HFD+C.butyricum group was gavaged with 1×10⁸ CFU/kg C.butyricum,while HFD+Sodium butyrate group was gavaged with 300 mg/kg sodium butyrate,once a day.In experiment 2,16 8-week-old C57BL/6 male mice were randomly divided into two groups.The normal diet of control group was supplemented with normal saline,and the experiment group was supplemented with C.butyricum solution 1×10⁸CFU/kg by gavage,once a day.After 12 weeks,the animals were sacrificed for sampling.The semen quality was detected by CASA,and the pathological changes of testicular tissue were observed by HE staining.The total antioxidant capacity (T-AOC),the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px),the content of malondialdehyde (MDA) in testicular tissue and serum testosterone (T), and the activities of alkaline phosphatase (AKP),acid phosphatase (ACP),lactate dehydrogenase (LDH) and gamma glutamyltransferase (γ-GT) in testicular tissue were detected by corresponding kits.【Result】 In experiment 1,compared with high-fat group,the body weight of mice in C.butyricum treatment group was extremely significantly decreased (P＜0.01),the sperm motility,the number of A+B grade sperm,sperm linear motility and the velocity of average path of mice were significantly increased (P＜0.05),and the straight-line velocity of mice was extremely significantly increased (P＜0.01).The activity of AKP in testicular tissue of mice was significantly increased (P＜0.05).Compared with high-fat group,the body weight of mice in sodium butyrate treatment group was extremely significantly decreased (P＜0.01),the testis index,sperm linear motility,sperm forward motility,straight-line velocity and swing of sperm were significantly increased (P＜0.05),and the sperm motility,the number of A+B grade sperm,the velocity of average path and the activity of γ-GT in testicular tissue were extremely significantly increased (P＜0.01).In addition,sodium butyrate significantly increased the content of serum testosterone and the activity of ACP (P＜0.05),and significantly decreased the content of MDA in testicular tissue of mice (P＜0.05).The pathological section of testicular tissue showed that there was no significant difference in testicular tissue morphology of mice in each group.In experiment 2,compared with control group,the testicular coefficient of mice treated with C.butyricum were extremely significantly increased (P＜0.01),and the level of serum testosterone was significantly increased (P＜0.05),but there were no significant change in semen quality and antioxidant indexes of testicular tissue (P＞0.05).【Conclusion】 C.butyricum and sodium butyrate could significantly increase the content of serum testosterone in mice,improve the antioxidant capacity of testicular tissue,improve the quality and quantity of sperm,protect the structure of testicular tissue,and effectively protect the reproductive system injury caused by obesity in male mice.

【Objective】 The purpose of the experiment was to prepare a new antibacterial peptide-Shengtaicin post-medicated bath solution,and characterize its effectiveness and safety.【Method】 The optimal formulation of Shengtaicin post-medicated bath solution was screened by one-way effect test and L₉(3)³ orthogonal test.In vitro bactericidal test was used to characterize the bactericidal efficacy of the post-medicated bath solution.The safety was verified by hemolysis test,cytotoxicity test,skin irritation test in single-administered mice,skin irritation test in multiple-administered mice and skin sensitization test in mice.【Result】 The orthogonal test results showed that,the film solution was configured with 7% film-forming agent PVA1788 as the substrate,and glycerol had the greatest experimental impact,followed by Tween 80.The optimal formulation of the Shengtaicin post-medicated bath solution was as follow:The optimal addition volume fraction of the plasticizer glycerol was 4%,the optimal addition volume fraction of the surfactant Tween 80 was 1%,and the optimal addition mass percentage concentration of the thickener pullulan polysaccharide was 0.4%.More than 99.999% of the tested Staphylococcus aureus,Staphylococcus epidermidis,Streptococcus agalactiae and Streptococcus dysgalactiae were killed within 30 min.The bactericidal effect was comparable to that of povidone iodine.In addition,the hemolytic and cytotoxic properties of Shengtaicin post-medicated bath solution were lower than those of the post-bath solution of povidone-iodine.The results of single and multiple skin irritation test and skin sensitization test in mice showed that Shengtaicin post-medicated bath solution was non-irritating and non-sensitizing to the skin of mice.【Conclusion】 The antimicrobial peptide Shengtaicin post-medicated bath solution was a validity,green and safe bath,which could effectively prevent the occurrence of mastitis in cows,and it would ensure the quality and safety of raw milk and promote the healthy development of the dairy industry.

【Objective】 Based on network pharmacology and molecular docking technology,this paper explored the role of chebulagic acid in preventing and treating cow endometritis,and clarified its potential mechanism.【Method】 The action targets of chebulagic acid were collected and predict through SwissTargetPrediction and PharmMapper databases,and standardized and de-duplicated through UniProt database.Cow endometritis related genes were acquire from OMIM and DRUGBANK databases and de-duplicated.Venn map was drawn to obtain the intersection target of chebulagic acid and cow endometritis.The protein-protein interaction (PPI) network was constructed using STRING database,and the GO function and KEGG pathway enrichment analysis were performed using DAVID database.The molecular docking technology was used to dock the key pathway targets obtained after enrichment analysis of KEGG pathway to verify the accuracy of network pharmacological analysis results.【Result】 333 potential target genes of chebulagic acid and 342 target genes related to cow endometritis were obtained by searching and screening multiple databases,with a total of 18 overlapping targets.PPI results showed that chebulagic acid might act on 18 key targets,such as prostaglandin G/H synthase 1(PTGS1),PTGS2,arachidonate 12-lipoxygenase (ALOX12),ALOX15,etc.. The results of GO founction and KEGG pathway enrichment analysis showed that chebulagic acid could prevent and treat cow endometritis through lipoxygenase pathway,arachidonic acid metabolism pathway,estrogen signal pathway,etc.Molecular docking results showed that chebulagic acid had good connectivity with the key targets obtained,which could play a role in preventing and treating cow endometritis through multiple targets,proving that the network pharmacological prediction results were accurate.【Conclusion】 Chebulagic acid might play a role in preventing and treating cow endometritis by combining with PTGS1,PTGS2,ALOX12 and cellular tumor antigen p53 (TP53) in the lipoxygenase pathway and arachidonic acid metabolism process.

【Objective】 The aim of the study was to explore the effect of dimethyl alpha-ketoglutarate (DMKG) on immunomodulatory effect of canine adipose-derived mesenchymal stem cells (cAD-MSCs).【Method】 In this experiment,DMKG medium with different concentrations was used to explore the effects of DMKG on the proliferation of cAD-MSCs.After cAD-MSCs were pretreated with 1 mmol/L DMKG,the levels of inflammatory cytokines of tumor nerosis factor-α (TNF-α),interleakin-1β (IL-1β),IL-6,IL-10 and transforming growth factor-β (TGF-β) in culture supernatant were determined by ELISA.RAW264.7 cells were cultured in medium containing 100,250,500,1 000 and 2 000 ng/mL lipopolysaccharide (LPS),respectively.After 24 h,the survival rate of cells was detected by CCK-8 assay to screen the optimal concentration of LPS was selected to establish RAW264.7 inflammation model.The cells were divided into blank control group,LPS group,MSCs group and DMKG-MSCs group,the cAD-MSCs pretreated with DMKG were co-cultured with RAW264.7 cells under 250 ng/mL LPS condition for 24 h,and the proliferation activity of the cells was detected by CCK-8 assay,the cell migration capacity was detected by Transwell assay,the content of NO in culture supernatant was determined by Griess assay,the mRNA expression levels of iNOs,IL-1β,IL-6,IL-10 and Arg-1 genes were detected by Real-time quantitative PCR,the surface markers CD80⁺ and CD86⁺ of M1 macrophages were detected by flow cytometry.【Result】 Compared with MSCs group,the secretion of TGF-β and IL-10 in culture supernatant after DMKG pretreatment of MSCs was significantly increased (P＜0.05),while the secretion of IL-1β and IL-6 were significantly inhibited (P＜0.05),and the secretion of TNF-α was not significantly different(P＞0.05).The proliferation activity of RAW264.7 cells reached the highest when LPS concentration was 250 ng/mL.Compared with LPS group,MSCs and DMKG pretreated cAD-MSCs extremely significantly inhibited the proliferation activity and migration ability of RAW264.7 cells (P＜0.01),inhibited NO release and down-regulated the mRNA expression level of iNOs (P＜0.01).In addition,mRNA expression levels of IL-1β (P＜0.01) and IL-6 in RAW264.7 cells were down-regulated,IL-10 was up-regulated (P＜0.05 or P＜0.01),the mRNA expression level of M2 macrophage surface marker Arg-1 was extremely significantly up-regulated (P＜0.01),and the expressions of surface markers CD80⁺ and CD86⁺ in M1 macrophages were extremely significantly decreased (P＜0.01).Compared with MSCs group,DMKG pretreated cAD-MSCs inhibited macrophage activation better.【Conclusion】 DMKG could effectively enhance the immunomodulatory effect of cAD-MSCs and increased expression of trophic factor TGF-β and anti-inflammatory cytokine IL-10,and the decreased expression of proinflammatory cytokines IL-1β and IL-6,inhibit the activation of RAW264.7 cells under the condition of LPS stimulation,and reduce the cellular inflammatory response.

【Objective】 This study was aimed to investigate the inter-group distribution,drug resistance characteristics and genetic relationships of carbapem-resistant Escherichia coli (CREC) strains from different poultry,so as to provide technical support for blocking the potential hazards of CREC.【Method】 A total of 1 131 cloacal swabs were collected from broilers,laying hens and waterfowls in Jiaodong area,and CREC strains were identified by selective isolation and culture,mass spectrometry identification,PCR,broth microdilution,multi-locus sequence typing (MLST) and whole genome sequencing (WGS).【Result】 A total of 364 bla_NDM gene positive CREC strains were isolated with a separation rate of 32.18% and the overall proportion of positive farms was 76.32%.The positive rates at both farm and individual levels of broilers were the highest (93.33% and 55.56% respectively).All the strains from three types of poultry were multi-drug resistant.83.65% of the isolates were resistant to 8 or more classes of drugs simultaneously,and the resistance rate to most of the tested drugs was more than 80%.The multi-drug resistance in broilers was the most serious,followed by waterfowl and laying hens.45 whole-genome sequenced CREC strains carried 52 drug resistance genes in 12 classes within 4 kinds of bla_NDM variants (mainly bla_NDM-5,77.78%).The detection rates of specific drug resistant gene of CREC originated from three types of poultry were different.A total of 46 ST types were detected,with a diversity ratio of 44.23%.There were 82-71 307 SNPs among the CREC strains from three types of poultry,and the dominant ST types of different poultry were quite different.【Conclusion】 CREC that carrying bla_NDM was widely prevalent in different poultry farms,especially in broiler farms,and the bla_NDM-5subtype was dominant.They were generally resistant to a variety of antimicrobial agents and carried a large number of drug-resistant genes,mainly plasmid transmission.CREC strains from three types of poultry showed diverse distribution,and the genetic relationship among most strains was quite far.It was suggested that surveillance and risk factors of CREC among different animals should be strengthened.

【Objective】 In order to reasonably prevent and control Salmonella Enteritidis (S.Enteritidis) infection,this experiment was conducted on the infection of S.Enteritidis from avian in four regions of Sichuan.【Method】 Bacterial isolation and identification was carried out by bacterial isolation and culture,biochemical test and molecular method.The sensitivity of isolated bacteria to 14 antibacterial drugs was further detected by K-B method,and drug resistance genes,virulence genes and pathogenicity of isolated bacteria were studied.【Result】 The results of bacterial isolation and culture showed that the isolated bacteria conformed to the culture characteristics of Salmonella,the microscopic examination was Gram-negative short bacilli,and the biochemical identification results were consistent with the biochemical characteristics of Salmonella.The PCR amplification results of Salmonella specific virulence target gene invA and S. Enteritidis specific virulence target gene sdfI showed that the target bands of 571 and 304 bp were obtained,which were consistent with the expected size.It was confirmed that 10 strains of S.Enteritidis were isolated,with a total isolation rate of 2.8% (10/361).The results of drug sensitivity test showed that the isolated bacteria had a high resistance rate to aminoglycosides,sulfonamides and peptides.Among aminoglycosides,the drug resistance rates to gentamicin and streptomycin were 50% (5/10) and 70% (7/10), respectively.Among sulfonamides,the drug resistance rates to compound sulfamethoxazole and sulfamethoxazole were 40% (4/10) and 100% (10/10) respectively.Among polypeptide drugs,the resistance rate to polymyxin B was 90% (9/10).The isolated bacteria were sensitive to quinolones,and moderately sensitive to cephalosporins β-lactamides and tetracyclines.Multiple drug resistance was common in isolated bacteria.Tetracycline resistance genes tetB (40.0%) and tetM (90%),aminoglycoside resistance genes aadA1 (80%) and aph(3')-Ⅱa (90%),β-lactam resistance gene bla_TEM-1 (100%),sulfonamide resistance gene sul3 (100.0%) were detected in 14 drug resistance genes.Among the six virulence related genes,the detection rates of ssaQ (SPI-2),mgtC (SPI-3),spi4D (SPI-4),pipA (SPI-5),invA (SPI-1) and spvB were 60%,80%,70%,60%,100% and 80%,respectively.The results of the pathogenicity test showed that the mice died after 24 h of intragastric administration of isolated bacterial solution.Blood stasis,hemorrhage,cell degeneration,etc.were observed in the organs and pathological sections,and the villi in the intestinal segment were broken and shed.【Conclusion】 In this study,10 strains of S.Enteritidis from avian were successfully isolated,and the isolated bacteria had strong drug resistance and multiple drug resistance,and the detection rate of virulence genes of isolated bacteria was high and the pathogenicity was strong,which could provide reference for the prevention and control and clinical treatment of S.Enteritidis.

【Objective】 The aim of this study was to understand the effect of Xiaochengqi powder on the intestinal microbiota of sows with constipation and investigate the mechanism of Xiaochengqi powder to alleviate constipation.【Method】 A total of 27 bi-crossbreeding sows (Yorkshire×Landrace,3-5 fetal periods) were selected and divided into healthy control group,constipation group and experimental group according to fecal conditions,each group had 3 replicates and each replicate had 3 pigs.The pigs in healthy control group and pigs in constipation group were given normal diets,and experimental group were added Xiaochengqi powder to the diet (60 g/(head·d)) once daily for three consecutive days.The fresh fecal samples of each group were collected on the fourth day,and the total DNA of the microbial genome was extracted.The microbial community diversity of the samples was detected using Illumina MiSeq sequencing technology.【Result】 The microbial flora in the feces of constipation group was significantly changed,and the Simpson index was significantly different from healthy control group and experimental group (P＜0.05).At the phylum level,there was a significant decrease in Firmicutes and Bacteroidetes (P＜0.05) and extremely significant increase in Proteobacteria (P＜0.01) in the gut microbiome of the sows in constipation group compared to healthy control group.Compared with constipation group,Bacteroidetes were significantly increased (P＜0.05),and Firmicutes had on increating tendency (P>0.05), and Proteobacteria were extremely significantly reduced (P＜0.01) in experimental group.At the genus level,Lactobacillus and Prevotella were extremely significantly decreased (P＜0.01),Clostridium_ⅪVa,Ruminococcaceae and Lachnospiraceae were significantly decreased (P＜0.05),while Acinetobacter and Escherichia_Shigella were extremely significantly increased(P＜0.01),Clostridium_sensu_stricto and Romboutsia were both significantly increased (P＜0.05) in the intestinal microbiome of sows in constipation group compared to healthy control group.In experimental group,there was an extremely significant increase in Prevotella (P＜0.01),a significant increase in Lachnospiraceae (P＜0.05),and an extremely significant decrease in Acinetobacter compared to constipation group (P＜0.01).Acetic acid,butyric acid,and propionic acid concentrations in the stools of sows in experimental group were all be considerably increased compared to constipation group (P＜0.05).【Conclusion】 Xiaochengqi powder might enhance the intestinal microbiome structure of sows with constipation,increase the amount of short-chain fatty acids (SCFAs) in the intestine,and accomplish a new balance of intestinal microorganisms.

【Objective】 It was planned to isolate a strain of phages with good bacteriostatic effect and wide adaptability for Escherichia coli,which provided a basis for the development of new Escherichia coli control methods.【Method】 Taking Escherichia coli H11 as the host bacteria,a strain of Escherichia coli phage was isolated and purified from sewage samples by double-layer agar plate method.The morphology of the phage was observed under electron microscope,and the titer,host spectrum,optimal multiplicity of infection(MOI),one-step growth curve,pH stability and temperature sensitivity of the phage were determined.The whole phage genome was analyzed and sequenced,and the inhibitory effect of the phage on H11 in liquid medium was explored.【Result】 A lytic Escherichia coli phage was successfully isolated and named EP_H11(accession number:OP688485).The results of double-layer agar culture method showed that the phage EP_H11 could form plaques with uniform size,transparent bright spots in the middle and halo around,and the phage cleavage titer could reach 10⁹-10¹⁰ PFU/mL.Electron microscopy showed that the phage EP_H11 had a regular polyhedral head with a diameter of about 85 nm,a tail with a diameter of about 23 nm and a length of about 109 nm.Using Escherichia coli H11 as host bacteria,the optimal MOI of phage EP_H11 was 0.1.The one-step growth curve showed that the incubation period was 40 min and the outbreak period was 80 min.The amount of cleavage was about 200 PFU/cell.The phage could survive for at least 2 h in the environment of pH 4.0-10.0,and could remain stable for 90 min at 10-60 ℃.Combined with transmission electron microscopy and whole genome analysis,the phage EP_H11 belonged to the Caudovirales,Siphoviridae family,Myoviridae genus.The total length of the phage genome was 111 373 bp and the GC content was 44.49%.According to the results of phage linear comparison,Escherichia coli phage EP_H11 and phage Escherichia phage vB_EcoM_IME392 had the highest homology.Phylogenetic tree analysis showed that the phage EP_H11 and Escherichia phage vB_EcoM_IME392 (Myoviridae genus) were located in the same branch,belonging to the order of Caudovirales and the Siphoviridae family.BLAST sequence analysis showed that there were 153 CDSs in genome of EP_H11,28 of which were predicted to encode functional proteins.In vitro bacteriostasis results showed that within 8 h,EP_H11 showed good bacteriostatic effect when MOI was 10. 【Conclusion】 In this study,a bacteriophage with strong lytic ability,wide adaptability and good bacteriostatic effect in vitro was isolated.The bacteriophage had the potential to be further developed as an antimicrobial agent for Escherichia coli.

【Objective】 The purpose of this study was to investigate the prevalence virulence and drug resistance of salmonella strains isolated from Yellow chicken in a large-scale hatchery.【Method】 The yolk swabs of weak chicks were collected and inoculated with selective medium for purifying and isolating bacteria.The isolated strains were identificated by Gram staining,microscopic examination and PCR amplification were carried out according to the Salmonella specific gene invA.Kirby-Bauer disk diffusion method was used for drug sensitivity test and amplify drug resistance genes to investigate and analyze the bacterial resistance of clinical isolates.The pathogenicity of the isolates was tested by SPF Kunming mice and the related virulence genes were amplified by PCR. 【Result】 The results showed that five isolated strains (JSS-Q3, JSS-Q4, JSS-Q5, JSS-Q8 and ZJS-C1-11) were suspected to be Salmonella.Colorless transparent colonies were formed in Eosin-Methylene blue medium,and purple transparent colonies were formed in Salmonella chromogenic medium,which were detected as short rod-shaped Gram-negative bacteria.Salmonella specific gene (invA) was positive and 99% similarity with Salmonella and the specific gene (IpaJ) amplification of Salmonella Gallinarum was positive,so the five isolated strains were all Salmonella Gallinarum.The results of virulence gene amplification showed that a total of fifteen virulence genes were detected,of which eleven were 100%.Drug resistance analysis showed that five isolated strains had strong resistance and were only sensitive to some antimicrobial agents.The isolates were sensitive to cefepime,flufenicol and enrofloxacin.In addition,JSS-Q5 and JSS-Q8 were sensitive to polymyxins B and neomycin,respectively,JSS-Q3 was sensitive to ceftazme and macromycin,and ZJS-C1-11 was sensitive to cefaclor,macromycin and polymyxins B.Some strains carried bla_SHV,bla_TEM,tet(A),tet(B) and other drug-resistant genes.Pathogenicity analysis showed that JSS-Q8 and ZJS-C1-11 strains were highly virulent and lethal to mice.【Conclusion】 The Salmonella isolates from Yellow chicken hatchery involved in this study were Salmonella Gallinarum.All of them showed multiple drug resistance and some of them were highly pathogenic,which might be one of the important reasons affecting the hatchery hatching rate and weak chick rate.The results of this study provided certain experimental basis for the detection,diagnosis and treatment of salmonella.

【Objective】 This study was aimed to explore the therapeutic effect of Sini decoction on piglet diarrhea and the influence of growth performance,blood physiological and biochemical indicators and intestinal flora.【Method】 One hundred and twenty 3-day-old diarrhea piglets with similar body weight were randomly divided into Chinese medicine group and Western medicine group,with 60 piglets in each group, and 60 healthy piglets of the same group were seleted as blank control group.The piglets in Chinese medicine group were fed with 5 g modified Sini decoction compound Chinese medicine solution every day for 7 days,while those in Western medicine group were injected intramuscularly with 0.1 mg/kg BW of gentamicin once a day for 4 days,blank control group was not treated with drugs.The mental status,food intake,diarrhea and average daily weight gain were recorded during the treatment period,and the blood physiological and biochemical indexes and intestinal microflora were determined before and after treatment.【Result】 After 5 days of treatment,the cure rate in traditional Chinese medicine group was 97%,which was significantly higher than that in Western medicine group(87%) (P＜0.05),and no death occurred.After the treatment,the average daily gain of piglets in traditional Chinese medicine group was significantly higher than that in Western medicine and blank control groups (P＜0.05).The results of blood routine test showed that the amount of white blood cell (WBC),lymphocyte (LYM),mononuclear cell (MONO),neutrophil (GRAN) and hematocrit (HCT) in traditional Chinese medicine group were significantly higher than those in Western medicine group (P＜0.05).There was no significant difference in other blood routine indicators between groups (P＞0.05).The blood biochemical results showed that the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in traditional Chinese medicine group were significantly lower than those in Western medicine group (P＜0.05),the creatinine (CRE) content was significantly higher than that of Western medicine group (P＜0.05),there was no significant difference between traditional Chinese medicine and blank control groups (P＞0.05).High-throughput sequencing of intestinal flora showed that, compared with Western medicine group, the Simpson and Shannon indexes of intestinal flora in traditional Chinese medicine group were significantly higher (P＜0.05), and the abundance of Firmicutes and Bacteroides, Lactobacillus and Clostridium were increased,the abundance of Proteobacteria and Fusobacteria, E.coli,Bacillus and Enterococcus were decreased.【Conclusion】 Modified Sini decoction could effectively treat diarrhea piglets,and the effect was better than Western medicine group,and could effectively improve the growth performance of diarrhea piglets,promote the body damage repair,adjust the structure of intestinal flora.

【Objective】 The purpose of this study was to determine the cause of death of the Tupaia belangeri chinensis,and to find out the pathogenicity and drug resistance of isolated strain.【Method】 In this study,the pathogenic bacteria were isolated from the heart blood,liver,spleen,lung and kidney of diseased and dying Tupaia belangeri chinensis by the conventional plate scribing method.The strain was identified by morphological observation,biochemical test and identification of 16S rRNA gene.And drug sensitivity test and pathogenicity test were carried out.【Result】 A suspicious bacterial strain was isolated from the liver and lung of Tupaia belangeri chinensis,which died of illness.The isolate showed a single colony of light orange on McConkey medium,which was detected as Gram-negative short bacillus by microscopy.The biochemical test results showed that the isolated bacteria could decompose glucose,did not ferment lactose,produce gas,produce hydrogen sulfide,etc.,which conformed to the biochemical characteristics of Salmonella.Genetic evolution analysis showed that the isolate was closely related to the Salmonella CP082916 strain from broiler chickens,and the nucleotide similarity was 99%.The results of drug sensitivity test showed that the isolate was sensitive to chloramphenicol,gentamicin,neomycin,norfloxacin and other drugs,not sensitive to clindamycin and vancomycin.The results of pathogenicity test showed that the isolate had strong pathogenicity to Tupaia belangeri chinensis and adult mice,and the median lethal dose (LD₅₀) to mice was 1.6×10⁶ CFU.Histopathological observation showed that the alveolar septum of the lung of Tupaia belangeri chinensis and mice widened and inflammatory cells infiltrated in the septum.The arrangement of liver cells was disordered,with a large number of necrotic cells disintegrating,sinus space being deformed or reduced,blood stasis in the central vein,and inflammatory cells infiltrating around,some spleen cells were disordered.【Conclusion】 Salmonella was the key cause of the disease of the laboratory Tupaia belangeri chinensis and the isolated was sensitive to chloramphenicol,gentamicin and neomycin and had strong pathogenicity.