To explore the structure and function of porcine connexin 43 (GJA1) protein,this study compared the coding sequences of GJA1 protein in 16 different species to analyze evolution and phylogeny relationship.The amino acid sequence information of porcine GJA1 protein such as physical and chemical properties,protein modification sites,transmembrane regions,subcellular localization,secondary and tertiary structures,polymorphic sites,functional regions and interaction proteins were analyzed by the related bioinformatic softwares and databases.Phylogenetic analysis results showed the variation of GJA1 protein did not reach saturation,the protein sequence displayed small genetic distance and high homology among 16 species.The porcine GJA1 protein had the greatest genetic relationship with that of equine,and the classification in the evolutionary tree also confirmed it.The protein structure analysis results indicated that the porcine GJA1 protein was composed of 20 kinds of amino acids with the molecular weight of 43.08 ku and theoretical isoelectric point of 8.88.It was predicted that the instability index of porcine GJA1 protein was 44.06,the lipid solubility index was 82.67,and its hydrophobic average coefficient was -0.240.There were 4 transmembrane regions,6 O-GlcNAc sites,38 phosphorylation sites and no signal peptide.The GJA1 protein was most likely exist in the plasma membrane and the Golgi apparatus,and its secondary and tertiary structures were mainly comprised of α-helix and random coil.The protein function analysis results demonstrated that the porcine GJA1 protein had 2 superfamily functional regions and 6 missense mutation sites in its CDS sequence.In addition,the porcine GJA1 protein interacted with ZO-1,Cx36 and many protein kinases.This study systematically revealed the structure and function of porcine GJA1 protein by bioinformatics analysis,which could provide a theoretical reference for further researches on the genetic regulatory mechanism of GJA1 protein in pigs.

This study was aimed to clone and analyze the CDS region of porcine glucose-regulated protein 78 (GRP78) gene using bioinformatics,and investigate the mRNA expression profile of GRP78 gene.Primers were designed according the predicted sequence of porcine GRP78 gene (accession No.:XM 001927795.6) in GenBank.The CDS region of porcine GRP78 gene was cloned by PCR amplification and sequencing.The physical and chemical property,transmembrane region,signal peptide,hydrophobicity,conserved domains,the secondary and tertiary structures were analyzed,and the phylogenetic tree of GRP78 gene was constructed by online prediction softwares.Finally,Real-time PCR was used to detect the tissue expression of porcine GRP78 gene.The results showed that the CDS of GRP78 gene was 1 965 bp,encoding 654 amino acids.Bioinformatics analysis showed that the molecular weight,theoretical electrical point (pI),half-life and the extinction coefficient of water solution at 280 nm of GRP78 were 72.3 ku,5.06,30 h,and 30 495,respectively.The peptide chain N-terminal of GRP78 was M (Met).The unstsble coefficient of GRP78 was 32.39,indicating that it belonged to the stable protein.The fat coefficient of GRP78 protein was 85.40,and the total average hydrophobic index was -0.496.GRP78 did not have transmembrane domain but it had one signal peptide,therefore,this protein belonged to the secreted protein.The secondary structure analysis of GRP78 showed that the percent of amino acids of α-helix,extended chain,β-turn and random coil were 40.06%,20.49%,8.10% and 31.35%,respectively.Homology analysis results showed that the nucleotide homology of GRP78 gene with human(NM_005347.4),mouse(NM_001163434.1),rat(NM_013083.2),goat (XM_005687138.3) and cattle (NM_001075148.1) were 93%,91%,90%,95% and 95%,whereas the amino acid homology were 99%,98%,98%,99% and 99%,respectively.These results indicated that GRP78 gene was highly conserved.Real-time PCR results showed that GRP78 gene was expressed in heart,liver,spleen,lung,kidney,small intestine,stomach,ovary,fallopian tube,breast,cerebellum,cerebrum and pituitary gland,and highly expressed in heart,spleen and lung.This study provided basic materials for further study of the biological function of GRP78 gene.

In order to study the molecular characteristics and biological function of NS1 gene of feline parvovirus (FPV),the NS1 gene of FPV-BJ 05 strain was amplified,cloned and sequenced.Nucleotide and amino acid homology analysis,signal peptide,transmembrane domain,B-cell preponderant epitope,phosphorylation site subcellular localization and protein structure and fuction,advanced structure of NS1 gene were predicted using bioinformatic softwares.The results showed that the length of NS1 gene was 2 007 bp,encoding 668 amino acids.The NS1 gene of FPV-BJ 05 shared 98.8% to 99.3% nucleotide homology with that of the other FPV strains,and the amino acid homology was 97.9% to 98.8%.Phylogenetic tree analysis showed that the NS1 protein of FPV-BJ 05 strain was classified as the same major branch with 3 FPV reference strains registered in GenBank,but it was a small independent branch due to amino acid mutation.The NS1 protein had neither a signal peptide nor a transmembrane domain,which was a non-secretory hydrophobic protein.The prediction results of B cell antigen parameters showed that NS1 protein had good flexibility,high antigenicity and surface accessibility,and it was predicted that the protein contained 13 dominant antigenic sites.62 phosphorylation sites,27 O-glycosylation sites and 2 N-glycosylation sites were found in NS1 protein.The result of subcellular localization showed that the probability of NS1 protein in the cytoplasm and nucleus was higher,which were 69.6% and 17.4% respectively.The prediction of secondary structure revealed that α-helix (39.37%),extended chain (15.42%),random coil (39.37%) and β-turn (5.84%),and the tertiary structure of NS1 protein was curved spiral structure.This study provided theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of FPV in Beijing.

This study was aimed to clone,prokaryotically express and purify toxA-N gene of Pasteurella multocida in goat,and analyze the expressed protein toxA-N by bioinformatics,which provided a reference for exploring the relevant characteristics of the Pasteurella multocida toxin gene in goat.The genome of the above-mentioned bacteria was used as a template,and the primer was designed with reference to the Pasteurella multocida HN06 toxA gene sequence published in GenBank(accession No.:CP003313.1),and the target fragment was amplified by PCR.The recombinant plasmid pET28a(+)-toxA-N was constructed and then transferred to E.coli BL21(DE3) competent cells.After inducted by IPTG,the expressed proteins were characterized by Coomassie blue staining,Western blotting,protein purification and bioinformatics softwares.The results showed that the 1 515 bp gene fragment was successfully amplified,and the two fragments of 5 369 and 1 515 bp were digested by BamH Ⅰ and Not Ⅰ,indicating that the recombinant plasmid pET28a(+)-toxA-N was successfully constructed.The Coomassie blue staining and Western blotting results showed that a size of about 60 ku toxA-N protein was successfully expressed by IPTG induction.Bioinformatics analysis results showed that toxA-N protein was an inclusion body with a molecular formula of C₂₆₃₅H₄₀₀₂N₆₆₄O₇₉₇S₁₇,the total number of atoms was 8 115,the extinction coefficient was 84 480,the instability index was 43.50,and the average hydrophilicity was -0.381.In the secondary structure of toxA-N protein,α-helix,β-turn,extended chain and random coil accounted for 53.47%,2.77%,11.28% and 32.48%,respectively,which were consistent with the prediction results of the tertiary structure.In summary,this study revealed the relevant properties of the Pasteurella multocida toxin by preliminary study of toxA-N gene,which was of great significance for disease prevention,diagnosis and treatment of livestock.

In order to investigate the prevalence and mutation of canine parvovirus (CPV) in Nanning,Guangxi,this study extracted the viral genomic DNA of 12 positive samples that were initially diagnosed as CPV.Using the extracted samples DNA as templates,the entire VP2 gene sequence was amplified and sequenced.In this experiment,the VP2 gene of all 12 positive strains were compared with the VP2 gene of 17 domestic and foreign CPV isolates registered in GenBank.The results showed that the VP2 gene fragments of 12 strains were successfully amplified with the size about 1 755 bp,and the homology between VP2 gene of 12 positive strains was between 99.2% and 100.0%,among which NN01 and NN07,NN02 and NN06 were the highest,accounting for 100.0%.The homology of VP2 gene between the positive sample strain and other domestic isolates was 97.6% to 100.0%,among which NN08,NN10 and NN04 were the highest with CPV-ZJ1579,all of which were 100.0%,belonging to CPV-2a subtype.The homology between positive sample strains and foreign representative strains was 98.1% to 99.8%.Phylogenetic tree analysis showed that among the 12 strains,3 strains were CPV-2a subtype,3 strains were CPV-2b subtype,and 6 strains were CPV-2c subtype.This was the first time that CPV-2c subtype CPV had been found in Nanning area since the isolation of CPV-2c subtype CPV in Guangxi in early 2018,which indicated that the epidemic of CPV-2c subtype CPV was slowly increasing in China.This experiment indicated that CPV-2a,CPV-2b and CPV-2c coexisted in Nanning area of Guangxi,but the proportion of CPV-2c subtype was larger than that of other areas,which also provided new prevention and control information for this area.In addition to paying attention to the traditional epidemic subtypes such as CPV-2a and CPV-2b,the prevention and control of CPV-2c subtype CPV should be paid more attention to in the actual prevention and control work of CPV.

In order to analyze the effect of listeriolysin S (LLS) llsB gene deletion on the biological characteristics of Listeria monocytogenes (LM),this study used homologous recombination to construct the llsB gene deletion strain LM90-ΔllsB,and the biological characteristics of growth characteristics,median lethal dose (LD₅₀) and organ-borne bacteria were studied in healthy Kunming male mice at 8 weeks old and weighing 40 g±5 g.The llsB gene deletion strain was successfully constructed,and the deletion strain had good genetic stability through continuous passage to 20 generations in vitro.Based on its growth curve examination,we found that the growth rate of the mutant strain was slightly higher than that of the parent strain.The results of mice infection test showed that the LD₅₀ of the parent strain and the deletion strain were 10^6.17 and 10^6.50 CFU,respectively.Compared with the parent strain,the amount of bacteria load of the deletion strain in the liver and spleen of the mice was extremely significantly decreased (P<0.01),the results showed that the infection ability of the mutant strain on mice was obviously weakened.No Listeria monocytogenes was detected in the brain.The results suggested that llsB gene might have direct or indirect regulatory effect on some biological characteristics of LM90,and it would provide a theoretical basis for further understanding of the pathogenic mechanism of LLS,prevention and control of listeriosis.

This study was aimed to screen and prepare a monoclonal antibody against chicken PD-1,and preliminarily study the immunological characteristics,binding activity of the monoclonal antibody and its blocking effect on the activation of the chicken PD-1/PD-L1 signaling pathway.The hybridism cell lines was obtained using hybridism technique,the immunological activity of antibody was identified by ELISA method,the biological activity of Ig antibody subtype identification kit and Western blotting.The combination of corresponding monoclonal antibody with the PBMCs was detected by indirect immunofluorescence technique and flow cytometry.Furthermore,the monoclonal antibody was used to treat chicken PBMC cells after IBDV infected for 7 days,and the expressions of IL-2,IL-6 and IFN-γ were detected by Real-time quantitative PCR.The results showed that a hybridism cell line with specific and stable secretion of chicken PD-1 monoclonal antibody was obtained and named PD-1-D05.The subtype of monoclonal antibody belonged to IgG1,and the titers of hybridism cell culture supernatant and ascites were 1:2¹¹ and 1:2.048×10⁵,respectively.ELISA and Western blotting results showed that PD-1-D05 monoclonal antibody reacted specifically with immunogens,no cross reaction was found between PD-1-D05 and pET-28a(+),Rosetta strain protein extract supernatant and unrelated proteins.The indirect immunofluorescence and flow cytometry results showed that the obtained PD-1-D05 monoclonal antibody could specifically combine with the chicken PBMC,the expression of IL-2 significantly increased after PBMC treatment with PD-1-D05 after 7 days of IBDV infection (P<0.05),the expression of IFN-γ significantly decreased (P<0.05),the expression of IL-6 decreased compared with IBDV attack group,but there was no statistical difference (P>0.05).The results suggested that it successfully screened and prepared a cell line that secretes the monoclonal antibody to the extracellular domain of chicken PD-1 and obtained a PD-1 monoclonal antibody with good immunological activity.This monoclonal antibody could specifically identify the PD-1 molecule and combine the PBMCs.Moreover,it could also effectively inhibit and restore immune regulation related cytokines caused by activation of PD-1/PD-L1 signaling pathways induced by IBDV infection,such as the abnormal expression of IL-2 and IFN-γ.

This study was aimed to explore the sequence characteristics of interleukin 10 (IL-10) gene and its relative expression of different tissues in goat.IL-10 gene of Jintang Black goat was amplified and cloned by PCR,the gene sequence was analyzed by bioinformatics softwares such as DNAMAN,DNAStar and ExPASy,then the expression of IL-10 gene in different tissues was detected by Real-time quantitative PCR.The results showed that the sequence of IL-10 gene was 379 bp,and the open reading frame was 276 bp,encoding 91 amino acid residues.The secondary structure of IL-10 protein contained alpha helix(76.92%),beta turn(2.20%) and random coil(20.88%).The tertiary structure model of IL-10 protein had the highest homology with human IL-10 (1ilk.1.A) gene (86.81%).In the phylogenetic tree,IL-10 gene in Jintang Black goat was closely related to Norwegian goat.Real-time quantitative PCR result showed that the expression level of IL-10 gene in lung of Jintang Black goat was significantly higher than that in spleen,heart,muscle and kidney (P<0.05),the expression of IL-10 gene in kidney was significantly higher than that in spleen,heart and muscle (P<0.05),the expressions of IL-10 gene in spleen,heart and muscle were lower,but there was no significant difference (P>0.05).It revealed that IL-10 gene was highly conserved,and it might be involved in regulating the immune response of goats.

This study was aimed to clone the flagellin FliC gene of Salmonella Enteritidis and analyze its genetic structure with bioinformatics.The genomic DNA of bacterium was extracted as a template,according to the published sequence of FliC gene in GenBank,and one pair of specific PCR primers was designed.FliC gene was cloned by PCR and linked into the cloning vector to be sequenced.The nucleotide sequence,amino acid sequence,homology and phylogenetic tree were analyzed by the bioinformatics softwares.The physicochemical properties,hydrophilicity,signal peptides,transmembrane domains,glycosylation sites,B cell epitopes,secondary structure and tertiary structure of the encoding protein were predicted by DNAStar software and online servers.The results showed that the gene of 1 518 bp was successfully cloned and encoded 505 amino acids.Homology analysis showed that FliC gene was relatively conserved,and this species had high homology with E.coli genus.The chemical formula of this protein was C₂₂₅₄H₃₇₀₁N₆₅₇O₈₀₃S₄,and the theoretical molecular mass was 52.981 ku.The theoretical isoelectric point was 4.91,and the instability index was 16.86,which was a stable hydrophilic protein.Structural analysis result showed that neither a signal peptide nor a transmembrane domain was found,however,a total of 8 N-linked glycosylation sites,13 O-linked glycosylation sites,60 phosphorylation sites,26 linear B cell piptopes and 5 T cell binding sites were predicted.Secondary structure analysis showed that α-helix,β-turn,extended chain and random coil accounted for 43.76%,3.76%,20.99% and 31.49%,respectively.In this study,FliC gene was cloned and analyzed successfully.The results laid a theoretical foundation for the further study of the role of flagella in the pathogenesis and the development of genetic engineering vaccines.

To investigate the antioxidation and immune activity of Oxytropis kansuensis flavonoids,the antioxidant activities of Oxytropis kansuensis flavonoids were determined by DPPH,superoxide anion and hydroxyl free radical scavenging activities,and the immunomodulatory of Oxytropis kansuensis flavonoids were studied by measuring the effects of different concentrations of Oxytropis kansuensis flavonoids on the proliferation of mouse spleen lymphocytes,the concentrations of IL-2 and IgG.The results showed that the highest scavenging rate of DPPH free radicals of Oxytropis kansuensis flavonoids was 32.23% which was slightly higher than vitamin E (28.55%) and lower than vitamin C (97.05%).The highest scavenging rate of superoxide anion free radicals of Oxytropis kansuensis flavonoids was 80.03% which was higher than vitamin E (69.70%) and lower than vitamin C (98.69%).The highest clearance rate of hydroxyl free radicals of Oxytropis kansuensis flavonoids was 30.39% which was lower than that of vitamin C (98.98%) and slightly lower than that of vitamin E (32.57%).In the spleen lymphocyte proliferation assay on mice,the relative proliferation rate of spleen lymphocytes increased with the increase concentration of Oxytropis kansuensis flavonoids.When the concentration of Oxytropis kansuensis flavonoids was 200 μg/mL,the spleen lymphocyte proliferation rate was 23.40%.The IL-2 concentration in 200 μg/mL Oxytropis kansuensis flavonoids group was extremely significantly higher than that of control group (P<0.01),and there was no significant difference between 50,100 μg/mL Oxytropis kansuensis flavonoids groups and control group (P>0.05);The IgG antibody concentration of Oxytropis kansuensis flavonoids groups were extremely significantly higher than that of control group (P<0.01),50 μg/mL Oxytropis kansuensis flavonoids group was extremely significantly higher than that of 100 and 200 μg/mL Oxytropis kansuensis flavonoids groups (P<0.01),and 100 μg/mL Oxytropis kansuensis flavonoids group was extremely significantly higher than 200 μg/mL Oxytropis kansuensis flavonoids group (P<0.01).In conclusion,the Oxytropis kansuensis flavonoids had good antioxidant and immune activity.

To explore the effect of linoleic acid on the synthesis of fatty acids of Butyrivibrio fibrisolvens in ruminant animals,different levels of linoleic acid medium were used to conduct an anaerobic culture test with Butyrivibrio fibrisolvens.Linoleic acid was added to the culture medium to make its concentrations in test groups 1 (control group),2,3 and 4 were 0,2.5,5.0 and 7.5 mg/100 mL when D_{600 nm} value of bacteria reached 1.0.The bacteria fluid was collected and tested for 35 kinds of fatty acids after cultured for 24 h.The results showed that there were no significant difference in the contents of C8:0,C11:0,C14:0,C15:0,C17:0,C17:1,C18:1(n-9)C,C18:2(n-6)C,C18:3(n-6),C20:0,C20:2,C20:3(n-3),C20:3(n-6),C20:5(n-3),C21:0,C22:0,C22:2,C22:1(n-9)and C22:6(n-3) with the increase of linoleic acid (P>0.05);The amount of C10:0,C12:0,C16:0,C18:0,C20:4(n-6),C23:0 and C24:1 were significantly decreased response to the linoleic acid addition (P<0.05).The contents of C15:1 and C18:3(n-3) were significantly increased in 2.5 mg/100 mL linoleic acid group (P<0.05),the amount of C14:1 in control group was the highest and that would increase with the linoleic acid increase.The amount of C24:0 in 7.5 mg/100 mL linoleic acid group was the highest.In conclusion,linoleic acid had a certain effect on the synthesis of fatty acids of Butyrivibrio fibrisolvens,it could inhibit the synthesis of C10:0,C12:0,C16:0,C18:0,C23:0,C14:1 and C20:4(n-6),increase the synthesis of C15:1,C16:1 and C18:3(n-3) which would decrease when the linoleic acid content exceed a certain value.

H₂S is a colorless and odorous gas,it is not only a toxic and harmful substance in livestock houses,but also is the third gas signal molecule after NO and CO.H₂S can be produced through decompositing and reducting of sulfur-containing organic compounds and sulfates by Salmonella,Helicobacter pylori,etc.in the gastrointestinal tract,and also through catalyzing sulfur-containing amino acid such as L-cysteine by cystathionine-β-synthase (CBS),cystathionine-γ-lyase (CSE),cysteine transferase (CAT) and 3-mercapto-pyruvate thiotransferase (3-MST).H₂S has a variety of physiological and pathological effects.It can inhibit mitochondrial respiration and blocks apoptosis through blocking cytochrome C (at high content),while also down-regulates anti-apoptotic genes such as BCL-2 and NF-κB to promote apoptosis.It can inhibit apoptosis by regulating Caspase3 (at low content),and also can play a thiolation reaction with NF-κB p65 to promote the expression of downstream anti-apoptotic genes and play an anti-apoptotic effect.High concentration of H₂S could up-regulate the expression of NF-κB,P38 and ERKE1/2 genes to promote inflammatory responses,low concentrations of H₂S could exert anti-inflammatory effects through inhibiting NF-κB signaling pathway.Summarizing the formation of H₂S gas and its dual roles in apoptosis and inflammatory response,this paper provides a reference for the understanding of the physiopathological mechanism of H₂S in the process of disease development.

In this study,the effects of recombinant Enterococcus faecium expressing porcine lactoferrin peptide (pNZ8112-PLFcin/Ef) on growth performance and its protection activity aganist enterotoxigenic Escherichia coli (ETEC) of weaned piglets were examined.36 healthy weaned piglets of 28 days old with similar body weight were selected and randomly divided into 3 groups with 3 replicates per group and 4 piglets per replicate.The piglets in recombinant Enterococcus faecium group was fed the basal diet supplemented with 6×10¹² CFU/kg pNZ8112-PLFcin/Ef,that in pNZ8112/Ef group was fed basal diet supplemented with 6×10¹² CFU/kg pNZ8112/Ef,while that in the medium group was fed basal diet supplemented with the same volume of GM17 medium.The experiment lasted for 26 d.The results showed that compared with medium group,the average daily gain of weaned piglets in the recombinant Enterococcus faecium group was extremely significantly increased (P<0.01),the F/G was significantly decreased (P<0.05).And the occurrence of piglet diarrhea was reduced to a certain extent.The evenness and diversity indexes of intestinal flora in weaned piglets were decreased.In order to further explore the antibacterial activity of pNZ8112-PLFcin/Ef,the protective effect of ETEC infection was explored in this study.After continuous feeding for 21 days,one piglet randomly selected from each replicate and fed with ETEC.The results showed that compared with the medium group,the levels of IL-2 and IgG in serum,sIgA in intestinal mucus and spleen index of recombinant Enterococcus faecium group were significantly increased (P<0.05),but there was no significant difference in thymus index and the length and weight of small intestinal (P>0.05).In conclusion,the studies had shown that recombinant Enterococcus faecium expressing porcine lactoferrin could promote the growth of piglets and had protective effect of ETEC infection.

Plant extracts (PE) are single or mixed substances obtained from plants through physical or chemical methods.They contain polyphenols,alkaloids,polysaccharides,essential oils and other active substances.Their biological functions are diverse:Regulating the activities of antioxidant enzymes,reducing peroxide,inhibiting or eliminating free radicals,etc;Achieving immune responses by regulating the activation of NF-κB,ERK,JNK and expression of related transcription factors;Reducing inflammatory cytokines and related gene expression to alleviate inflammation;Have antibacterial effect and are not easy to produce drug resistance;Achieving apoptosis and anticancer effect by changing the cell cycle of cancer cells;Having significant inhibitory effects on parasites,such as plasmodium,filariosis and Trypanosoma cruzi.Plant extracts are widely used in piglets and have good application results:Improving the growth performance of piglets,such as pineapple stem,star anise extracts;Promoting the digestion and absorption of piglets to improve the metabolism of nutrients;Reducing aminotransferase and AKP activity to relieve stress;Increasing the activities of antioxidant enzymes and reducing the level of peroxide in piglets;Increasing the content of cytokines and immunoglobulin of blood in piglets,and enhancing their immune functions;Regulating intestinal flora,improving intestinal mucosal morphology,and reducing piglet diarrhea.As a kind of green and healthy additive,the widespread use of plant extracts can greatly reduce the use of antibiotics.It is believed that,with the development of biotechnology,plant extracts will play more important role in the healthy breeding.

In this experiment,suitable calcium requirements in Jinghong 1 commercial laying hens were studied through feeding experiments to provide certain basic data for the formulation of nutritional standards in Jinghong 1 commercial laying hens.Single-factor test design was used for the experiment.360 Jinghong 1 layers at age of 52 weeks were randomly divided into 4 treatments with 6 replicates per treatment and 15 layers per replicate.Corn-soybean-phytasebasic diets were fed in this experiment.Dietary calcium levels were 2.91%,3.94%,4.38%and 4.98%,respectively.And the trial period was 6 weeks.The number of laying eggs,egg weight and the number of unqualified eggs were recorded every day during the experiment,and the feed intake was counted by repetition every week.At the end of the experiment,3 eggs were taken from each replicate to measure egg quality.The results showed that:① Different calcium levels had no significant difference on egg production rate,average egg weight,daily egg production,average daily feed intake and feed-egg ratio (P>0.05).But feed-egg ratio and the average daily feed intake in the 3.94% group were lower than those in other groups,and the broken egg rate in the 3.94% treatment group was significantly lower than that in the 4.38% and 4.98% groups (P<0.05).②Different calcium level diets had significant effect on eggshell thickness (P<0.05).The 4.38% group was significantly higher than the 2.91% and 4.98% groups (P<0.05),and the 3.94% group was significantly higher than the 4.98% group (P<0.05).The eggshell intensity of 3.94% group was the highest,and the eggshell ratio in this group was the most suitable,but there was no significant difference among all the treatments (P>0.05).③Through establishing a regression relationship between eggshell thickness and dietary calcium levels,the optimal dietary calcium level was estimated to be 3.84%.Based on the results of this experiment,under the condition of feeding corn-soybean-phytasebasic diets,3.84% to 3.94% calcium level diets could improve egg shell thickness,increase egg weight and reduce broken egg rate in late period of Jinghong 1 laying hens,and were beneficial to the production performance.

There are a large number of microorganisms that inhabit in the gut of mammals animal.The gut microbiome are closely related to host digestion,absorption and immunization.The metagenomics of intestinal microbiota has received significant attention from researchers.With the application of molecular biologic technology in the field of the intestinal microbiome,especially metagenomic sequencing of the next-generation sequencing technology,progress has been made in the study of the intestinal microbiome.Metagenomics can be used to study intestinal microbiome diversity and its relationship to health and disease.Moreover,functional metagenomics can identify novel functional genes,and determine interactions and co-evolution between microbiota and host.This review will introduce the recent advances and successful application in mammalian gastrointestinal microbes and the limitations of metagenomics,and the prospects of their application in the researches of gut microbiota were discussed,so as to further understanding the importance of intestinal microbiota in intestinal function of mammals.

This experiment was conducted to investigate the effects of Astragalus polysaccharides,Clostridium butyricum and their combination on growth performance,serum biochemical indices of ducklings.A total of 600 one-day-old healthy Shaoxing male ducklings with the similar body weight were randomly arranged into 5 groups with 6 replicates per group and 20 ducklings per replicate.The ducklings in the group Ⅰ (control group) were fed with a basal diet,and that in groups Ⅱ,Ⅲ,Ⅳ and Ⅴ were fed with the basal diets supplemented with 40 mg/kg of bacitracin (antibiotic group),800 mg/kg Astragalus polysaccharide,250 mg/kg Clostridium butyricum and 800 mg/kg Astragalus polysaccharide+250 mg/kg Clostridium butyricum,respectively.The experiment lasted for 28 d.The results showed as follows:①At 1 to 14 days of age,there were no significant differences of ADG,ADFI and F/G between the control group and the test groups (P>0.05).At 15 to 21 days of age,compared with groups Ⅰ and Ⅱ,the ADG of groups Ⅲ,Ⅳ and V was significantly increased (P<0.05),and F/G was significantly reduced (P<0.05).At 22 to 28 days of age,the ADG of groups Ⅲ,Ⅳ and V was significantly increased than that in group Ⅰ (P<0.05),the ADFI of group V was significantly higher than other groups (P<0.05),while F/G of groups Ⅲ,Ⅳ and V was significantly reduced than that of groups Ⅰ and Ⅱ (P<0.05).At 1 to 28 days of age,compared with groups Ⅰ and Ⅱ,the ADG of groups Ⅲ,Ⅳ and Ⅴ was significantly increased (P<0.05),and F/G was significantly decreased (P<0.05).② At 28 days of age,compared with group Ⅰ,the serum T3 content in group Ⅴ,T4 and GH contents in groups Ⅲ and Ⅴ were significantly increased (P<0.05).There was no significant difference of CORT content among groups (P>0.05).The IL-2 content in groups Ⅳ and Ⅴ were significantly improved compared with groups Ⅰ and Ⅱ (P<0.05).The IFN-γ content of the groups Ⅲ and Ⅴ was significantly higher than other group (P<0.05).In conclusion,the addition of Astragalus polysaccharides and Clostridium butyricum in diets could improve the growth performance and serum biochemical indexes of ducklings,and their combined effect was better than the single one.

This experiment was conducted to study the effects of feeding melatonin (MLT) on the breeding ewes in the off-season and feeding letrozole (LE) and N-methyl-D-aspartate (NMDA) on the basis of MLT,and to provide reference for the development and utilization of regulating ewes estrus drugs.Forty health multiparous Suffolk ewes with the age of (3.0±0.5) years old and the average weight of 76.23 kg±12.25 kg were randomly divided into 4 groups,control group and test groups Ⅰ,Ⅱ and Ⅲ,10 in each group.Control group:Basal diet;Test groupⅠ:Basal diet+ MLT 10 mg/d;Test groupⅡ:Basal diet+MLT 10 mg/d+LE 7 mg/d;Test group Ⅲ:Basal diet+MLT 10 mg/d+LE 7 mg/d+NMDA 300 mg/d.The results showed that,compared with the control group,the levels of FSH,E₂,E₃ and DHT in ewes plasma extremely significantly increased (P<0.01),and the level of T significantly decreased (P<0.05) in test group Ⅰ;Plasma MLT and LH levels were extremely significantly increased (P<0.01) and the FSH level was significantly increased (P<0.05) in test groupⅡ;In test group Ⅲ,the levels of MLT and GnRH in the plasma of the ewe were significantly increased (P<0.05).Compared with test groupⅠ,the MLT in the plasma of test group Ⅱ was significantly increased (P<0.05),and the LH and T levels were extremely significantly increased (P<0.01).Compared with test groups Ⅰ and Ⅱ,the FSH level was extremely significantly lower in test group Ⅲ (P<0.01).In summary,MLT and LE had a promoting effect on the release of reproductive-related hormones in ewes,and the combination of the two could achieve the effect of regulating the hormone levels related to reproductive growth of ewes;The combined application of MLT,LE and NMDA had a poor effect on the regulation of reproductive hormones in ewes.

This experiment was conducted to evaluate the quality of ramie silage and explore the effects of different crude protein (CP) levels of concentrate supplement on growth performance and serum biochemical parameters of Simmental cattle when ramie silage was used as the main roughage.In this study,the mixture ratio of ramie and corn straw at 1:1 was designed and bundled together for ensiling.Eighteen healthy Simmental beef cattle with similar body weight (324.83±30.27) kg and age were assigned randomly to 3 groups with 6 replicates per group and 1 cattle per replicate.Beef cattle were fed concentrate supplement containing 18.0% CP (high CP level,group Ⅰ),16.0% CP (middle CP level,group Ⅱ) and 14.0% CP (low CP,group Ⅲ),respectively.The adjustment period was 10 d,and formal experiment period was 60 d.The results showed that,in mixed ensiling experiment,the pH of ramie and maize straw mixed silage was 4.24.The content of NH₃-N was increased significantly (P<0.05),while there were no significant differences for the contents of DM,OM,CP,NDF,ADF,Ca and TP (P>0.05) before and after silage and the fermentation quality grade of silage ramie was excellence.The results of feeding experiment showed that the ADG of Simmental cattle in group Ⅰ was the highest,which was 1.49 kg/d,and that in groups Ⅱ and Ⅲ were reduced by 14.09% and 11.41% compared with group Ⅰ (P>0.05).Simultaneously,no significant difference in DMI and F/G of Simmental cattle were detected among three treatments (P>0.05).Moreover,the contents of ALP,AST,ALT/AST,TP,ALB,GLB,A/G,TCHO,TG,HDL and LDL in serum were not significantly changed with different CP levels of concentrate supplement (P>0.05).Only the content of ALT in group Ⅰ was significantly increased compared with group Ⅲ (P<0.05),but not significantly higher than group Ⅱ (P>0.05).Overall,the nutritive value of ramie was higher,and ramie and maize straw mixed ensiling could produce better ramie silage.Moreover,concentrate supplement with high protein level (18%) might improve growth performance of beef cattle when ramie silage was used as the only roughage.

The aim of this study was to evaluate the nutritional value of different degrees heat-treated of corn dried distillers grains and solubles (DDGS) using the principle and method of Cornell Net Carbohydrate Protein System (CNCPS),and to explore the correlation of CNCPS components and its ruminal degradability and intestinal digestibility to furosine which was Maillard reaction intermediate.This study was respected to provide new methods and ideas for the evaluation of feed nutritional value.The wet DDGS were dried at different temperatures (110,120,130℃) for different times (0.5,1.0,1.5 h),the protein components and their ruminal degradability and intestinal digestibility were estimated by conventional chemical analysis combined with CNCPS and AMTS-Cattle database,and the furosine content was determined by high performance liquid chromatography.Then the correlation between ruminal degradability and intestinal digestibility of the CNCPS components and furosine content were researched and the corresponding regression equations were established.The results showed that:①There were significant differences in furosine content,protein components,ruminal degradable,ruminal non-degradable and intestinal digestibility of protein components among DDGS treated with different degrees of heat (P<0.05).② There were significant correlation between furosine content and PA2,PB1,PB2 and PC components,RDPB2,RUPB2,RUPC and TRUP components,and IDGPB2 components (P<0.05).And the predictive equations could be established by regression.In summary,there were significant correlations between the protein components and furosine content of DDGS with different degrees of heat treatment,and the furosine content could be used to estimate the rumen degradability and intestinal digestibility of protein components in DDGS treated with different degrees of heat.

The innate immune response of poultry plays a key role in protecting the host from viral infections.Retinoic acid inducible gene-Ⅰ (RIG-Ⅰ) is used as a class of cytoplasmic internal recognition receptor of viral double-stranded RNA which is closely related to the natural immune response.It can monitor viral RNA in the cytoplasm by RNA ligand binding to pathogen-associated molecular patterns,and that process can activate RIG-Ⅰ and downstream mitochondrial antiviral signaling protein (MAVS),eventually leading to the activation of interferon regulatory factor (IRF3/7) and NF-κB,induce the production of type Ⅰ interferon and other immune cytokines,and then the cells make a corresponding anti-viral natural immune response.However,due to the lack of RIG-Ⅰ gene in chickens,the current researches are most to transfer RIG-Ⅰ gene from duck or geese source to chicken fibroblasts (DF-1) to study whether RIG-Ⅰ gene has immune function in chicken infected avian viruses.This article describes the expression of RIG-Ⅰ in poultry and its mediated antiviral innate immune signaling pathway,outlines the research status of antiviral effects of RIG-Ⅰ in poultry to provide references in the study of infection and immune system of poultry and the development of new anti-virus vaccines and immune adjuvants.

This study was aimed to analyze the genetic variance in each breed and the genetic correlation among three rabbit breeds including Fujian Yellow rabbit,New Zealand rabbit and Japanese White rabbit.In this experiment,all blood samples were took from 25 rabbits of each breed,DNA from the blood was extracted by DNA extract kit.The target sequence was got using 15 microsatellite loci,fluorescent PCR and capillary electrophoresis.The genetic diversity of three rabbit breeds was discussed by calculating alleles,effective alleles,heterozygosity,polymorphic information content and genetic distance.The results showed that a total of 110 alleles were detected at 15 microsatellite loci in three rabbit breeds,and the average number of allele was 7.3.The number of alleles of Fujian Yellow rabbit,New Zealand rabbit and Japanese White rabbit were 95,95 and 88,respectively.The number of microsatellite loci with high polymorphism variability in three rabbit breeds were below:Fujian Yellow rabbit was 11,New Zealand rabbit was 12,Japanese White rabbit was 11,so the selected 15 microsatellite loci had rich polymorphic information in three rabbit breeds.The result of Hardy-Weinberg equilibrium test showed that 8 microsatellite loci deviated from the Hardy-Weinberg equilibrium in Fujian Yellow rabbit and New Zealand rabbit,and 6 microsatellite loci in Japanese White rabbit.Nei's genetic distance analysis showed that the genetic distances between Fujian Yellow rabbit and New Zealand rabbit,Fujian Yellow rabbit and Japanese White rabbit,New Zealand rabbit and Japanese White rabbit were 0.1761,0.2347 and 0.0432,respectively.The smaller genetic distance lead to the higher similarity and the closer genetic relationship.In a word,the genetic relationship between New Zealand rabbit and Japanese White rabbit was the most close in three rabbit breeds,and the genetic relationship between Fujian Yellow rabbit and Japanese White rabbit was the most distant in three rabbit breeds.

This study was aimed to investigate the polymorphism of adiponectin (ADIPOQ) gene exon 2 and its correlation on body weights and body sizes in Shanxi White pig.A total of 392 pigs from Landrace pig,Large White pig,Duroc pig,Shanxi White pig,Shanxi Black pig and Mashen pig populations were used to detect the polymorphism of ADIPOQ gene exon 2 by PCR-SSCP,and the association of the SNPs on body weights and body sizes in Shanxi White pig was analyzed by GLM.The results showed that there was a missense mutation of G→A at 89 bp of ADIPOQ gene exon 2,which resulted in the change of amino acid from valine (Val) to isoleucine (Ile).There were three genotypes (AA,AB and BB) and two alleles (A and B) at this site.There was only BB genotype in Duroc pig,and the advantageous genotype was also BB genotype in Landrace pig,Large White pig,Shanxi White pig and Shanxi Black pig,while AA genotype frequency in Mashen pig was greater than AB and BB genotypes.In the imported pig breeds such as Landrace pig,Large White pig and Duroc pig,the frequency of the dominant allele B were 0.96,0.96,and 1.00,respectively.In Chinese domestic Mashen pig,the frequency of allele A (0.52) was little greater than that of allele B (0.48).The frequency of allele B in Shanxi White pig and Shanxi Black pig were 0.76 and 0.78,respectively.The genotype distribution among Mashen pig,Shanxi White pig and Shanxi Black pig was not significant(P>0.05),there was no significant difference between Duroc pig and Landrace pig or Duroc pig and Large White pig (P>0.05),however,the difference of genotype distribution between Landrace pig and Large White pig was significant (P<0.05).Comparing to the imported breeds with Chinese domestic Mashen pig and the composite populations of Shanxi White pig and Shanxi Black pig,the genotype distribution difference of each other was extremely significant (P<0.01).The effect of the SNP in exon 2 of ADIPOQ gene on weaning weight was significant,the weaning weight at 28-day-old of BB genotype individuals was significantly greater than those of AA and AB genotypes(P<0.05),and there was no significant difference between AA and AB genotypes(P>0.05).The effect of this polymorphic site on other traits was not significant,which indicated that it mainly played role at early stage of pig growth and development.

Molecular markers are genetic markers based on nucleotide sequence variation in individuals,and they are direct responses to genetic diversity at the DNA level.Molecular marker technology is an efficient and accurate technical method for polymorphism analysis at the DNA level,it has been widely used in sheep breeding.Molecular marker technology can not only locate gene in sheep,but also analyze the genetic structure of sheep population.Additionally,molecular marker technology plays an important role in sheep breeding through marker-assisted breeding.This article briefly introduced the basic principles,advantages and disadvantages of molecular marker technology based on Southern hybridization,PCR,simple sequence repeat (SSR),and single nucleotide polymorphism (SNP).The application of these molecular markers in marker-assisted selection (MAS) among sheep's traits such as body size,slaughter,and reproduction reveals the importance of molecular marker technology for the selection and assortative mating of sheep and improving its economic value in actual production.Finally,the future application of molecular marker technology is prospected based on the application of molecular marker technology in sheep breeding.

Follicle-stimulating hormone receptor (FSHR) gene,as a major gene affecting animal fertility,had been extensively and continuously studied in different species.The authors summarized and analyzed the structure of the goat FSHR gene and the corresponding protein's structure,action mechanism,expression scope and regularity,as well as the genetic effect of single nucleotide polymorphisms of the gene,and found that the research on the biological information and gene effects of goat FSHR gene had been carried out in depth,and the recent studies in signal transduction confirmed that FSHR gene was the key factor in the process of formation and development of ovarian follicles.Correlation analysis with reproductive performance of different populations suggested that some single nucleotide mutation sites of FSHR gene could be used as marker-assisted selection,but the mutation effects in different goat populations need to be further verified.

To study the genetic mutation and evolution of gB and gE genes of porcine pesudorabies virus (PRV),the FS-2015 wild strain PRV was purified in the tissue disease by plaque assay method (three times),the whole gene fragments of gB and gE genes were amplified,and PCR products were sequenced and analyzed in this study.The results showed that the homology of nucleotide sequence of gB and gE genes of PRV FS-2015 strain with other PRV strains from GeneBank were 97.0% to 100.0% and 97.5% to 99.7%,the homology of amino acid sequence were 96.4% to 100.0% and 95.3% to 99.7%,respectively.The amino acid variation site analysis results showed that gB and gE genes of FS-2015 strain had site mutations and deletions.Phylogenetic tree analysis results showed that the PRV FS-2015 stain with GY,ZJ01,HB1201,HN1201,JS2012,BJ-YT and BP stains had closer relationship,while that with Kaplan,Becker,NIA3,Kolchis,Bartha and Yangsan strains had a distant relationship.The results of PRV FS-2015 strains and classical strains at home and abroad,and the currently domestic variant strains suggested that PRV FS-2015 strain belonged to the variant popular in recent years,which could provide data for the molecular epidemiological investigation,the prevention and control of pseudorabies and the selection of vaccine strains in Guangdong province.

To understand the etiology situation of bovine respiratory disease complex (BRDC) in Guangxi,117 samples with respiratory diseases during 2016 to 2017 were diagnosed by RT-PCR/PCR method and bacterial isolation and identification method.And the antibiotic susceptibility test of the main isolates were carried out.The results showed that detection rates of Klebsiella pneumoniae,Mycoplasma bovis,Trueperella pyogenes,Pasteurella multocida,IBRV and Mannheimia haemolytica were 41.0%(48/117),28.2%(33/117),20.5%(24/117),15.4%(18/117),12.8%(15/117) and 5.1%(6/117) detected by RT-PCR/PCR method,while the detection rates of Klebsiella pneumoniae,Escherichia coli,Mycoplasma bovis,Trueperella pyogenes,Pasteurella multocida and Mannheimia haemolytica by bactorial isolation and identification method were 41.0%(48/117),33.3%(39/117),17.1%(20/117),7.7%(9/117),2.6%(3/117) and 2.6%(3/117),indicating the former had the better sensibility,and BPIV3,BRSV and BVDV had not been detected.The results of antibiotic susceptibility tests showed that resistance rates of 20 Mycoplasma bovis strains against β-lactams,sulfonamides and peptides were from 90.0% to 100.0%,and they were sensitive to quinolones,gentamicin,amikacin and kanamycin (resistance rates were 10.0% to 25.0%).30 Escherichia coli strains and 30 Klebsiella pneumoniae strains were sensitive to cefotaxime,ceftriaxone and ceftazidime (13.3% to 20.0%,10.0% to 30.0%,respectively),and they were resistant to other 15 kinds of drugs (50.0% to 100.0%,40.0% to 100.0%,respectively).The results indicated that mixed infection was dominant in bovine respiratory disease in Guangxi caused by Mycoplasma bovis,Escherichia coli and Klebsiella pneumoniae which had developed different degrees of drug resistance.

The aim of this study was to isolate the rabbit hemorrhagic disease virus (RHDV) strain from Zhenjiang and analyze genetic evolution and variation,as well as prokaryotic expression recombinant VP60 protein with good reactionogenicity.By means of excluding bacterial infection,hemagglutination (HA) and hemagglutination inhibition (HI) test,animal challenge test and virus passage,LD₅₀ determination,the pathogen was isolated and identified from the liver tissue sample of infected dead animals in a rabbit farm located in Zhenjiang city,Jiangsu province.The virus VP60 gene was obtained by RT-PCR,and the nucleotide and amino acid sequences were analyzed to study its genetic evolution.The low temperature inducible and Sumo-tag fused prokaryotic expression vector was constructed via recombining the VP60 gene into a low temperature induction vector pCold-Sumo,and the recombinant protein was expressed induced with IPTG at 15℃ overnight,and the reactivity was identified thereafter.The results showed that the RHDV ZJ2015 strain was isolated,and the virus could strongly agglutinate human type O erythrocytes,the HA titer reached to 11log2,and this activity could be blocked by the RHDV (AV33) antiserum.The LD₅₀ of this strain reached to 10^-6.38/mL,indicating the virus was highly lethal to rabbit.The size of the specific PCR product was about 1 740 bp,and the strain was classified as the antigen genetic variant (RHDVa) of RHDV1 according to the phylogenetic tree based on the nucleotide sequences.The nucleotide sequence homologies compared to RHDV1 and RHDV2 were 89.4% to 97.6% and 81.1% to 81.5%,and the amino acid sequence homologies were 93.8% to 98.3% and 87.4% to 87.6%,respectively.The recombinant VP60 fused with the Sumo-tag was successfully expressed in E.coli Rosetta (DE3) cells,the molecular weight was about 74 ku via SDS-PAGE,and the recombinant protein had a good reactivity with anti-serum via Western blotting analysis.This study laid the foundation for the epidemiological study of rabbit hemorrhagic disease and the development of recombinant vaccines and diagnostic reagents.

In order to establish an indirect ELISA method for detecting African swine fever virus (ASFV) antibody in serum,p30 gene of ASFV was expressed in prokaryotic system,the expression and immunogenicity of recombinant protein were identified by SDS-PAGE and Western blotting,respectively.Subsequently,the purified recombinant protein was used as a coating antigen,and a method for the detection of ASFV antibodies in serum was established condition optimization,specificity test,sensitivity test and repetitive test.The results showed that the ASFV p30 gene was successfully cloned into the prokaryotic expression vector pET-32a(+),and the recombinant plasmid pET-32a-p30 was obtained.The recombinant plasmid was transformed into E.coli BL21 (DE3) for expression,and the recombinant protein was obtained with 42 ku,it was mainly in the form of inclusion bodies.Western blotting results showed that the purified protein had good immunogenicity.An indirect ELISA method for detecting ASFV antibody was established by using purified P30 recombinant protein as coating antigen.The indirect ELISA method was optimized by square matrix test,and the optimal coating concentration of antigen was determined to be 1.2 μg/mL.The optimal dilution factor of the serum to be tested was 1:100,the optimal blocking solution was 1% BSA,and the optimal dilution of the enzyme-labeled antibody was 1:4 000.The established ASFV indirect ELISA method had a cut-off value of 0.322.The method showed high specificity,only specifically reacted with ASFV-positive serum,and had no cross-reaction with classical swine fever virus,porcine reproductive and respiratory syndrome virus,foot-and-mouth disease virus,pseudorabies virus,porcine circovirus type 2 and porcine epidemic diarrhea virus positive serum.The sensitivity of the method for detecting ASFV positive serum could reach 1:1 600;The intra-assay repeatability and the inter-assay repeatability coefficient of variation were both <10%.The established indirect ELISA method had good specificity,sensitivity and reproducibility,and could be initially applied to the detection of ASFV antibodies.

In order to understand the genetic evolution and pathogenicity of fowl adenovirus (FAdV) in Shandong province,we conducted two henneries suspected outbroke inclusion body hepatitis and pericardial effusion syndrome of pathogenetic materials (liver and spleen) by PCR identification,preliminary considered they were two FAdV virus strains.The positive pathogenetic materials were grinded by the extract of inoculate LMH cells,then we subculture the strain viruses.The CPE observation,PCR analysis,TCID₅₀ calculation,chicken embryos pathogenicity test,SPF chicken regression analysis,and the phylogenetic analysis of hexon gene were performed in this study.We successfully isolated two FAdV virus strains,and found that the CPE could be found in the first generation,and the PCR analysis showed the bands (500 bp) were in the target locations.The F5 TCID₅₀ was 10^-7.75/0.1 mL for the first strain and 10^-7.60/0.1 mL for the second strain,and they both had strong pathogenicity to 7 days old SPF chicken embryo.When the 21 days old SPF chickens were infected by these two strains,the death rates were 80% and 15%,respectively.The nucleotide homology analysis of hexon gene showed that the first isolate had the highest homology with FAdV-4 (99.57%),and the second isolate had the highest homology with FAdV-8b (99.18%).Both of these two strains were the group Ⅰ viruses,which were different from the hemorrhagic enteritis of turkeys viruses (in group Ⅱ) and the egg drop syndrome viruses (in group Ⅲ).The first isolate was type C in the genotype,type 4 in the serotype,and was named as FAdV-SDC4;While the second isolate was type E in the genotype,type 8b in the serotype genotype,and was named as FAdV-SDE8b.Our study indicated that both of FAdV-SDC4 and FAdV-SDE8b strains were the epidemic strains in China in recent years,and our analysis laid the basis for the management to inclusion body hepatitis and pericardial effusion syndrome,and could contribute to the vaccine research to these diseases.

This study was aimed to investigate the reproductive characteristics of swine Japanese encephalitis virus (SA14-14-2 strains) in Vero cell and the immunogenicity of its inactivated vaccine.The swine Japanese encephalitis virus was cultured in Vero cell.The proliferation technology of swine Japanese encephalitis virus in Vero cell line was studied by the selection and optimization of the following seven culture conditions,including Vero cell incubation time,the amount of inoculated virus,adsorption time,adsorption temperature,pH of maintenance fluid,maintain culturing temperature and culture time.After one time of freezing and thawing,the virus titer was determined by means of viral plaque number determination.The inactivated vaccine of swine Japanese encephalitis virus was prepared by reproducing a batch of viruses under optimized conditions and inactivating it by means of inactivation of β-propiolactone and mixed with bidirectional adjuvant.Two immunizations (interval 14 d) were given to piglets with negative antibody to encephalitis,and the serum was collected before (0 d) and after 7,14,21,28,35 and 42 d for the first immunization,and serum neutralization antibodies were detected.We attacked piglets using the encephalitis P3 strain at the 28th after two immunizations,and the plasma was collected before (0 d) and after 1,2,3,5,7 and 9 d;14 d after the attack,piglets were dissected and brain tissues were collected,and detecting the swine Japanese encephalitis virus in plasma and brain tissue.The results showed that the cells were cultured in a cell culture flask,and the Vero cells were cultured for 48 h for virus inoculation,the inoculum size was 1 000 PFU/mL,the virus adsorption temperature was 37℃,the adsorption time was 90 min,and pH was 7.6 to 8.8 after adsorption.The maintenance solution was continued to be cultured at a temperature of 35℃.After 96 h of culture,the venom was harvested and frozen and thawed to obtain a higher titer.The serum antibody level of the inactivated vaccine prepared by immunization of piglets increased rapidly.No Japanese encephalitis virus was detected in plasma or brain tissue.The test piglets of immunized group could resist strong poisoning and obtain an effective immune protection.This results provided a basis for the production of swine encephalitis vaccine.

To study the acute toxicity of Chinese herbal medicine compound additive for invigorating qi-blood,liver and kidney,the intragastric administration was adopted in this study,40 SPF Kunming mice were divided into four groups:Control group,low dose group,middle dose group and high dose group,and supplemented with the concentration 0.86 g/mL of Chinese herbal medicine compound additive.The suspension was administered intragastrically at a volume of 0.4 mL/10 g per gavage,twice within 24 h in high dose group (0.4 mL each time) and middle dose group (0.3 mL each time) for 7 d,the control group was given intragastrically 0.4 mL distilled water twice within 24 h for 7 d,and the low dose group was given 0.2 mL,once within 24 h for 7 d.The general condition and death of mice were observed daily.At end of the test,blood was collected from the eyeballs of mice to detect physiological and biochemical indicators.The mice were anatomized,and the organ index was calculated by weighing the heart,liver,spleen,lung and kidney,and pathological sections were made to observe the pathological changes.The results showed that there was no abnormality in mice of experimental groups.Compared with the blank control group,the body weight of mice in middle dose group and high dose group was extremely significantly increased (P<0.01).The liver index and spleen index of the high dose group were extremely significantly higher than other groups (P<0.01).The total number of white blood cells of mice in Chinese herbal medicine compound additive groups were extremely significantly increased (P<0.01),but it was still within the normal range.The blood creatinine was significantly decreased in Chinese herbal medicine compound additive groups (P<0.05),and the urea nitrogen of middle dose group was significantly increased (P<0.05);There was no significant difference in other biochemical indexes among all groups (P>0.05).The results of pathological tissue sections showed that there was no pathological changes in liver,spleen and kidney in all groups.In conclusion,Chinese herbal medicine of invigorating qi-blood,liver and kidney played a significant role in promoting protein metabolism which could significantly improve the body immunity and kidney function.The maximum dose (34.4 g/kg,crude drug) was 92.7 times as much as the amount of clinical adult (0.371 g/kg) daily,proving that the drug was non-toxic and safe.

Moxidectin (MOX) is derived from derivatization of nemadectin produced by Streptomyces (Cyanneogrisens noncyanogenus),which has good insect repellent activity and is safe for mammals,and it is mainly used in animal husbandry.Moxidectin has better fat-soluble and water-soluble than other avermectin drugs,and has the highest distribution in adipose tissues of animals.Moxidectin binds to GABA,and neural membrane potential hyperpolarization is caused by massive influx of Cl^-,resulting in the death due to the insect's body paralysis.Differences in the route of administration and the physiological condition of animals would affect the peak concentration and depletion half-lives.Moxidectin has good insect repellent activity in animals such as cattle,sheep and dogs.The application range of moxidectin gradually expands from mammals to birds,and the control objects gradually extend from nematodes to mite and several kinds of germs.The research direction extends from anthelmintic activity to the treatment of human diseases.With the increase of the application range of moxidectin and the prolongation of time,the drug resistance began to appear,and the rate of drug resistance could be reduced by the combination of moxidectin and other agents.Exploring the wider role of moxidectin requires the efforts of researchers in different fields.

To explore the effects of sulfated polysaccharide from algae (SPA) and the compound extracts of Chinese medicine (CECM) on H9N2 subtype avian influenza virus (AIV),SPA and CECM were applied to the 10-day-old SPF chicken embryos inoculated with H9N2 subtype AIV in 3 different ways.The chicken embryo allantoic fluid was collected aseptically,the blood coagulation titer was measured,and the prevention,treatment and direct inactivation of the two kinds of liquids to H9N2 subtype AIV were tested.The results showed that the maximum safe concentrations of SPA and CECM were 50 and 200 mg/mL,respectively.Under different dosing methods,different concentrations of SPA and CECM could extremely significantly reduce the blood coagulation of H9N2 subtype AIV in chicken embryo allantoic fluid (P<0.01).The hemocoagulation titer of the direct inactivation group decreased the most,the preventive group and the therapeutic group followed.In the preventive group,the anti-viral effect of CECM high dose group (200 mg/mL) was significantly better than that of SPA dose groups (12.5,25,50 mg/mL) and CECM low and medium dose groups (50,100 mg/mL) (P<0.05);SPA high-dose group (50 mg/mL) and CECM medium-and high-dose groups (100,200 mg/mL) were significantly better than SPA low group (12.5 mg/mL) (P<0.05);The direct inactivation group,SPA and CECM both had good antiviral effects,and the antiviral effects of CECM dose groups were extremely significantly different from those of SPA dose groups (P<0.01).The results showed that SPA and CECM had almost no toxic side effects on 10 days old chicken embryos;SPA and CECM could significantly inhibit the proliferation of H9N2 subtype AIV in chicken embryo allantoic fluid,and the antiviral effect of direct inactivation group was the best.The strength of its anti-H9N2 subtype AIV was closely related to the drug concentration,medication and viral infection time sequence.

To screen some effective disinfectants against coccidian,oocysts of Eimeria tenella had been suspended and maintained in diluted 16 common disinfectants and 1 anti-coccidial drug (250 mg/L toltrazuril) for 2 or 4 h,and then sporulated in 2.5% potassium dichromate solution at 28℃ or inoculated into coccidian-free chickens at 14 days of age.Disinfection efficacy of these disinfectants against both unsporulated and sporulated oocysts were evaluated through oocyst sporulation rates,inhibitory rates of sporulation,OPG values,cecal lesion score and blood stool level in this study.The results of inhibitory efficacy tests of disinfectants and anti-coccidial drugs against both unsporulated and sporulated oocysts showed that 8% ammonia hydroxide could 100% inhibit the unsporulated and sporulated oocysts.250 mg/L toltrazuril,3% formaldehyde,4% phenol,0.5% peroxyacetic acid,5% NaOH and 5% NaOH+10% NaCl solutions showed partial inhibitory activities.Although inhibitory rates of sporulation of these six disinfectants were more than 10%,the inhibitory efficacy of them against sporulated oocysts were not significant.However,the remaining 10 disinfectants didn't have significant inhibitory activities against oocysts.The optimization results of inhibitory conditions of ammonia hydroxide showed that 1% to 8% ammonia hydroxide mixed with unsporulated oocysts for more than 1 h were able to 100% inhibit the activities of oocysts.In addition,the anti-coccidial efficacy of 2% and 4% ammonia hydroxide could not be interfered by animal feces.In conclusion,almost all common disinfectants and anti-coccidial drugs fail to effectively inhibit the activities of coccidian oocysts,while ammonia hydroxide was a highly effective disinfectant.These findings would provide important references for development of novel effective coccidian disinfectants and control of coccidiosis in livestock.

Exosome is a nanoscale extracellular vesicle produced by fusion of intracellular multivesicular bodies with cell membranes.They are widely distributed in serum,urine,saliva and other biological fluids.As an important transfer vector for intercellular communication and genetic material,exosomes can directly stimulate target cells through receptor-mediated interactions or through the transfer of various biologically active molecules such as cell membrane receptors,proteins,mRNAs and miRNAs,thereby exerts its biological functions.The article mainly reviews the functions of proteins and miRNAs in exosomes and the clinical application of exosomes.The functions of proteins and miRNAs in exosomes include inducing immunity,presenting antigens,and participating in intercellular signaling under physiological conditions;Changing the tumor microenvironment under pathological conditions,promoting cancer cell proliferation,invasion,accelerating angiogenesis,and promoting cancer development;Certain viruses can package and integrate their components into exosomes to achieve intercellular transmission,or hijack exosomes for immune evasion.Exosome in the clinical should mainly introduce its biological markers for early diagnosis of various cancers such as lung cancer,breast cancer,pancreatic cancer and colorectal cancer,as well as high stability,high transport efficiency and strong targeting drug carrier is used for treatment.

In order to screen the Mongolian medicine which had good anti-bacterial effect on pathogenic Escherichia coli O8 (E.coli O8) and Staphylococcus aureus (S.aureus) in vitro,Oxford cup method was used to determine the inhibition zone diameter,microdilution borth method combine plate method was adopted to investigate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Mongolian medicine,and the fractional inhibitory concent ration index (FICI) of the sensitive Mongolian medicine against E.coli O8 and S.aureus was determined by chessboard method.The results showed that the inhibition zone diameter of six Mongolian medicines to S.aureus,such as Cortex Phellodendri,Lonicera japonica,Munronia henryi Harms,Schisandra chinensis,Herba Taraxaci and Cortex Fraxini,was 10.22 to 22.44 mm and MIC was 15.6 to 250 mg/mL.The inhibition zone diameter of four Mongolian medicines to E.coli O8,such as Schisandra chinensis,Lonicera japonica,Munronia henryi Harms and Cortex Fraxini,was 11.29 to 26.41 mm and MIC was 15.6 to 500 mg/mL.The FICI of Cortex Phellodendri combined Munronia henryi Harms to S.aureus was 0.36,while that of Munronia henryi Harms combined with Sanguisorba officinalis L.to E.coli O8 was 0.48.The comprehensive bacteriostatic effects of the Cortex Phellodendri,Lonicera japonica,Munronia henryi Harms,Schisandra chinensis,Herba Taraxaci and Cortex Fraxini were extremely sensitive or highly sensitive to S.aureus.The comprehensive antibacterial effect of Schisandra chinensis,Lonicera japonica,Munronia henryi Harms and Cortex Fraxini were extremely sensitive or highly sensitive to E.coli O8.Cortex Phellodendri,Munronia henryi Harms and Cortex Fraxini were the first the choice in the group of inhibiting S.aureus,Munronia henryi Harms and Sanguisorba officinalis L. were the first the choice in the group of inhibiting E.coli O8.

To study the inflammatory response of germ-free rats with deep second-degree scald injury,a deep second-degree scald injury model of germ-free rats which were infected with Pseudomonas aeruginosa and a deep second-degree scald injury model of SPF rats were established,then the similarities and differences between the three groups in terms of healing process,inflammatory response,and pathological changes were compared,and the effect of inflammatory reaction of deep second-degree scald injury sterile rats were explored.Deep second degree scald wound was performed in 20 6-week-old female germ-free rats at the temperature of 94℃,8 s in a clean bench.And then the scald germ-free rats were randomly divided into two groups,one group was infected with Pseudomonas aeruginosa with a concentration of 1×10⁵ CFU/mL in the wound,the other group was not infected and the wound kept clean.SPF scald Wistar rats were set up as control group under the same conditions.The scab escharosis time,dislocation time and healing time of the three groups were observed,and serum samples were taken to detect the changes of inflammatory cytokines such as TNF-α,IL-1α and GM-CSF before burned (0 h) and 24 h,3 d and 7 d after burned.The results showed that the Pseudomonas aeruginosa infection rats had a long period of scab escharosis time (P<0.05) and the decrustation and healing time was significantly short compared with SPF rat and germ-free rats (P<0.05).When the burned rats were infected with Pseudomonas aeruginosa for 24 h,lymphocytes were observed in the subcutaneous tissues and there were chronic inflammatory reactions.At the 3rd day,there was a purulent infection in the deep dermis,and a large number of inflammatory cells were seen around the striated muscle.On the 7th day,there was slight blood vessel dilatation and congestion,and purulent infection was relieved.The skin of germ-free scald rats and SPF burned rats had mild inflammatory reactions within one week after scald.The serum level of TNF-α,GM-CSF and IL-1α in three groups of rats were significantly increased after burned (P<0.05);At 24 h,the level of IL-1α in the germ-free scald rats and the scald rats infected with Pseudomonas aeruginosa were significantly higher than the SPF rats (P<0.05),while the GM-CSF content in germ-free scald rats was significantly higher than that in the scald rats infected with Pseudomonas aeruginosa and SPF rats (P<0.05).At the 3rd day,the IL-1α content in germ-free scald rats and the scald rats infected with Pseudomonas aeruginosa were significantly higher than the SPF rats (P<0.05),and the GM-CSF content in germ-free scald rats was significantly higher than that in the scald rats infected with Pseudomonas aeruginosa (P<0.05).In conclusion,germ-free scald rats reacted fiercely,and serum inflammatory factors increased rapidly,while the inflammatory response of SPF rats was slower to the deep second degree scald.Pseudomonas aeruginosa infected deep second-degree scald sterile rats had a shorter decrustation time and healing time.