To investigate the variety and genetic characteristics of arboviruses epidemic in Yunnan province,Culicoides were collected in Shizong county and inoculated cells for virus isolation and identification.Whole genome sequence of the isolated virus was obtained by full-length cDNA amplification (FLAC) and next generation sequencing (NGS),then analyzed through sequence alignment and phylogenetic tree.The results showed that a virus strain (YNSZ043) causing cytopathic effect on C6/36 cells was isolated from collected Culicoides specimens.The genome of the isolated virus was composed with double-stranded segmented RNA,showing migration pattern of "2-4-3" by agarose gel electrophoresis.The viral particle of the isolated virus was about 70 to 80 nm in diameter exhibiting morphological feature of "finger ring",with fibrous protrusions on the surface.The genome size of the YNSZ043 was 20 683 bp in length with 12 gene segments ranging from 3 762 bp (Seg-1) to 861 bp (Seg-12),with nucleic acid sequence identities ranging from 64.8% to 99.6% and amino acid sequence identities ranging from 58.8% to 100%,compared with other Chinese BAV strains.On the phylogenetic tree,YNSZ043 clustered together with other Chinese BAV strains forming an independent Chinese evolutionary branch.YNSZ043 belonged to A2 genotype by phylogenetic analysis of Seg-12 which decided the genotype of BAV.Maximum nucleic and amino acid sequence identity of Seg-5/VP5 were observed between YNSZ043 and BAV strains isolated from Vietnam,which were 97.1% and 97.6%,respectively,indicated there might be Seg-5 reassortment between Vietnamese strains and YNSZ043 strain.The present study enriched the genome sequence of BAV,provided a foundation for further epidemiological studies of BAV in Yunnan.

In this study,a strain of probiotic Bacillus amyloliquefaciens B7 (B.amyloliquefaciens B7) was used as the research object,in order to improve its level of fermentation under liquid conditions,and to improve the optimum efficiency of spore fermentation.The method of determination of dipicolonic acid (DPA) concentration was proposed to replace the traditional plate spores counting method to explore the feasibility of this method in the optimum process of spore fermentation.The significant factors affecting spore production were first screening through the single-factor experiment.Firstly,the factors that had significant promoting effect on spore production of strain were screened out by single factor test.The optimum combination of peptone,corn powder,rice protein powder and Mn²⁺ was determined by orthogonal experiment.The result showed that the spore concentration was linearly dependent on the fluorescence intensity of DPA in the method,and the optimal medium for higher spore concentration was peptone 20 g/L,corn flour 10 g/L,rice protein powder 20 g/L,Mn²⁺ 1.0 mmol/L.After 48 h of fermentation,spore concentration reached 7.0×10⁹ CFU/mL.Compared with the LB medium and the industrial production medium used in practice,the spore concentration was increased by 3.3 and 2.2 times,respectively.This study provided a rapid method for the determination of spore concentration during fermentation of Bacillus,and a simpler medium with high spore yields was presented for industrial spore fermentation of B.amyloliquefaciens B7 based on the rapid quantification of spore concentration.

In mammals,follistatin (FST) is not only involved in reproductive regulation,but also significantly associated with muscle growth and fat deposition.In order to explore the biological function of this gene in fibroblasts,the second generation sequencing technique was used to sequence the transcriptome of pig fibroblasts with FST gene knock-down and screen the differentially expressed genes. GO functional annotation and pathway enrichment analysis were carried out using on-line biological software.The results showed that 370 genes were significantly up-regulated and 287 genes were significantly down-regulated after FST gene knockdown.There were significant changes in HOXA6,PARP14,ZBP1,NDUFB9,RAB13,FAM83D,THBS1 and BTF3 genes associated with cell proliferation and apoptosis,as well as disease-related genes ZNF354C,RNF213,IFIT1,CCL5 and VPS13C.51 GO terms were clustered for these differential genes.Biological regulation,cellular process,metabolic process and single-organism process were screened under biological process.Organelle,cell part and cell were screened under cellular component.Binding terms were screened under molecular function.These terms were all including more than 200 differential genes.12 significantly enriched pathways were identified,including 3 pathways related to cell proliferation,such as p53 signal pathway,cell cycle and ribosome biogenesis in eukaryotes,4 pathways were related to disease,such as Alzheimer's disease,Salmonella infection,Huntington's disease and non-small cell lung cancer,2 pathways were related to muscle contraction,such as vascular smooth muscle contraction,cardiac muscle contraction.In addition,there were ubiquitin mediated proteolysis,regulation of actin cytoskeleton and gap junction pathways.The results indicated that after FST gene knockdown in fibroblasts,genes and biological pathways related to cell proliferation,differentiation and some diseases were significantly enriched,suggesting that FST gene played an important role in wound repair process of fibroblasts.

Bone morphogenetic protein 15 (bone morphogenetic protein 15,BMP15) gene mutation had a great impact on sheep ovulation rate,litter size and fecundity.Therefore,this study was aimed to establish a rapid visual detection technology for B2 mutation of BMP15 gene.Combine the improved amplification refractory mutation system (ARMS) with the nucleic acid dye SYBR Green Ⅰ,and designed ARMS-specific primers for the B2 mutation of BMP15 gene,made the last base at the 3'end of the primer as A,meanwhile designed additional mismatches at the third base of the 3'end of the downstream primer to improve primer specificity.After ARMS PCR,added SYBR Green Ⅰ to the product,and detected the B2 mutation of BMP15 gene by visually observing the color change of the PCR tube.It was found through sequencing that the collected samples were all wild-type.Therefore,the B2 mutation of BMP15 gene template was constructed by overlap extension PCR.The sequencing results showed the B2 mutation of BMP15 gene template was successfully constructed.ARMS PCR results showed that ARMS-specific primers could only amplify mutant templates,could not amplify wild-type templates,and the amplification effect was good.After ARMS PCR,SYBR Green Ⅰ was added and it was found that the known wild-type template was consistent with the negative control and showed the original color of SYBR Green Ⅰ orange-yellow,while the known mutant template was bright green with obvious color changes.Using the established visual detection technology to detect 50 Small-tailed Han sheep genomic DNA (20 samples were added with the constructed B2 mutant template of BMP15 gene),and compare the number of the mutant sample detected visually with the number recorded when the sample was mixed.The results showed that this technology could accurately detect the B2 mutation of BMP15 gene,and the accuracy was as high as 100%.The constructed BMP15 gene B2 mutant template was serially diluted to detect the sensitivity of the visual detection system in this experiment.It was found that the sensitivity of the ARMS visual detection technology reached 0.006 ng/μL.Therefore,the technology established in this study could visually detect the B2 mutation of BMP15 gene in sheep with high accuracy,and it was expected to provide technical support for the breeding of high-fertility sheep.

The purpose of this study was to explore the relationship between the copy number of P28-ORF2 inserted into the genome of PCV2-Cap and the expression of PCV2-Cap protein when Swine pox virus vector was used to express PCV2-Cap protein.In this experiment,using Swine pox virus as vector,8 strains of recombinant Swine pox virus with different copy number (1-8 copies) of P28-ORF2 were constructed by double enzyme digestion and in-fusion cloning method.8 strains of recombinant Swine pox virus with different copy number (1-8 copies) were purified.The virion particles were observed by electron microscope and the results of Western blotting were judged based on the size of fluorescent spot and electron microscope.The fluorescent spot diameter of the 8 purified recombinant virus was about 317.41-384.96 μm,and there was no significant difference in the size of the fluorescent spots.The morphology of the recombinant Swine pox virus was the same as that of the parent virus.The results of Western blotting showed that the expression of recombinant protein with 1 copy of P28-ORF2 was the lowest,and the expression of PCV2-Cap increased with the increase of P28-ORF2 copy number.When 4 copies of P28-ORF2 were inserted,the expression of PCV2-Cap reached the peak,and then decreased.There was no significant difference in the fluorescent spot size of 8 strains of recombinant Swine pox virus.The insertion of different copy numbers of P28-ORF2 into the genome of Swine pox virus had no effect on the proliferation of recombinant virus in PK15 cells.The morphology of recombinant Swine pox virus particles did not change,indicating that the insertion of multiple copies of foreign genes did not affect the structure of Swine pox virus.The results showed that when Swine pox virus was used as vector to express foreign protein,single promoter initiation of multi copy foreign sequence could significantly increase the expression of foreign protein,and the foreign sequence inserted 4 copies was a suitable copy number.

The present study was aimed to clone the complete CDS region of Yanbian cattle adipose differentiation-related protein PLIN2 gene,analyze the sequence and basic protein characteristics of PLIN2 gene CDS region through bioinformatics,and explore its presence in different tissues of Yanbian cattle.The 18-month-old castrated Yanbian cattle were selected as the research object.PLIN2 gene of Yanbian cattle was obtained by RT-PCR and gene cloning,and the homology comparison with other species was carried out and the phylogenetic tree was constructed.The PLIN2 encoded protein was predicted by bioinformatics methods the physicochemical properties,potential phosphorylation sites,glycosylation sites,disulfide bond analysis,signal peptides,subcellular localization and high-level structure of the protein,Real-time quantitative PCR was used to detect the expression level of PLIN2 gene in different tissues of Yanbian cattle.The results showed that the CDS of PLIN2 gene in Yanbian cattle was 1 353 bp long and encoded 450 amino acids.The homology comparison showed that Yanbian cattle PLIN2 gene had 100% homology with cattle,98.7% for buffalo,96.7% for sheep,97.1% for goat,85.5% for wild boar,86.3% for human,47.3% for mouse,and 49.1% for pheasant.The phylogenetic tree showed that Yanbian cattle had the closest genetic relationship with Yellow cattle and buffalo,and the farthest genetic relationship with pheasants.PLIN2 protein was an unstable protein with a certain degree of hydrophilicity.There were 61 potential phosphorylation sites,16 potential O-glycosylation sites,12 potential N-glycosylation sites,and 2 disulfide sites,there was no signal peptide,mainly distributed in the nucleus,a small amount in the cytoplasm and mitochondria.The high-level structure of PLIN2 protein predicted that the protein mainly contains a mixed protein composed of α-helix,extended chain,random coils and β-turns,which were connected by random coils,mainly α-helix.Real-time quantitative PCR results showed that PLIN2 gene was expressed the highest in small intestine tissue of Yanbian cattle,followed by longissimus dorsi muscle and kidney tissue.The results of this study could provide a reference for further research on the function of PLIN2 gene.

With the rapid development of biological products industry,the serum-free suspension culture technology of mammalian cells has been involved in more and more fields.This technology can expand the scale of mammalian cells,improve the production and quality of biological products,broaden the application field of mammalian cells,and promote industrialized production of mammalian cells.However,compared with the parent cells,the protein expression level and growth regulation pathway of the cells after suspension acclimation have changed to varying degrees,which affected the production performance of cell lines.In this regard,scholars at home and abroad have carried out a lot of research and made good progress.In this paper,the research status of mammalian cells serum-free suspension culture technology and the optimization strategy of clinical production process were descried,in order to provide references for the development of mammalian cells serum-free suspension culture technology.

This experiment was intended to explore the regulation mechanism of Gram-negative bacterium Escherichia coli (E.coli) and its surface molecules lipopolysaccharide (LPS) induced the pancreas regeneration protein Ⅲ gamma (RegⅢγ) expression.Firstly,different concentration E. coli (10⁹,10⁸,10⁷,10⁶,10⁵ and 10⁴ CFU/mL) and LPS (0.01,0.1,1,5,10,20,40 and 80 μg/mL) were used to induce intestinal epithelia cells of porcine (IPEC-JⅡ),and D_{490 nm}value was determined by MTT method to judge the effect of E. coli and LPS on IPEC-JⅡ cell viability.Then,different concentration E. coli (10⁷,10⁶ and 10⁵ CFU/mL) and LPS (0.01,0.1,1 and 5 μg/mL) were used to induce IPEC-JⅡ for 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of RegⅢγ.Finally,1 μg/mL LPS was used to treat IPEC-JⅡ for 24 h,and Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression and phosphorylation level of p65,p38,JNK and ERK.The results showed that except 0.01 μg/mL LPS did not inhibit IPEC-JⅡ cell viability,all the other concentration of E. coli and LPS inhibited IPEC-JⅡ cell viability,moreover,the cell viability of 10⁹ and 10⁸ CFU/mL E. coli and 10,20,40,80 μg/mL LPS group were extremely significantly decreased (P<0.01).Compared with control group,10⁷,10⁶ and 10⁵ CFU/mL E. coli could increase the expression of RegⅢγ in IPEC-JⅡ cells,and the RegⅢγ mRNA expression of 10⁵ CFU/mL E. coli group was extremely significantly higher than that of control group (P<0.01),the quantity of protein expression was significantly higher than control group (P<0.05).0.01,0.1,1 and 10 μg/mL LPS could increase the expression of RegⅢγ in IPEC-JⅡ cells,moreover,the RegⅢγ mRNA expression of 0.1 and 1 μg/mL LPS group was extremely significantly higher than that of control group (P<0.01),although RegⅢγ protein expression had increasing trend,but had no significant difference (P>0.05).Compared with control group,the mRNA expression of p65 and p38 of 1 μg/mL LPS group were extremely significantly increased (P<0.01),and the mRNA expressions of JNK and ERK were significantly increased (P<0.05).Meanwhile,the protein expression levels of p38 and JNK and the phosphorylation level of p65 were significantly increased (P<0.01), and the phosphorylation level of p65 was significantly increased (P<0.05).Both ERK protein and phosphorylation levels increased,but the difference were not significant (P>0.05).The above results showed that E. coli and LPS could induce RegⅢγ expression,1 μg/mL LPS increased the phosphorylation levels of p65,p38 and JNK proteins.

The purpose of this study was to establish the gingival mesenchymal stem cells (GMSCs) isolation and culture system in vitro,and to explore their biological characteristics,so as to provide seed cells for cell therapy and regenerative medicine.14 weeks of Simmental cattle gum was used to separate GMSCs digested by 0.1% type Ⅳ collagenase digestion,and then in vitro culture and cryopreservation were performed,cell viability was measured before and after cryopreservation,GMSCs growth curve was plotted by MTT assay.GMSCs specific markers (CD29,CD44,CD73,CD90,CD105,CD166 and STRO-1) were identified by immunofluorescence,flow cytometry and PCR,and their multidirectional differentiation potential was detected by adipogenic and osteogenic induction in vitro.The results showed that GMSCs of Simmental cattle had strong refraction and long spindle shape after adherent to the wall.The results of cell viability test before and after cryopreservation showed that the cell viability after cryopreservation was lower than that before cryopreservation,but remained above 85%,and the cell growth curve showed a typical "S" type.Immunofluorescence results showed that GMSCs of Simmental cattle expressed CD29,CD105 and STRO-1,but not CD31.PCR results showed that GMSCs of Simmental cattle expressed CD44,CD73,CD90 and CD166,but not CD34 and CD45.Flow cytometry results showed that CD73 (99.94%),CD90 (99.87%) and CD105 (99.90%) were highly expressed by Simental bovine GMSCs,while CD34 (1.16%) and CD45 (1.18%) were almost not expressed.After adipogenic differentiation,lipid droplets of different sizes were observed,which could be stained with oil red O,and the PCR results showed that they expressed the key lipid genes peroxidase proliferation factor activated receptor-γ (PPAR-γ) and lipoprotein enzyme (LPL).After the osteogenetic differentiation,mineralized nodules were displayed by Alizarin red staining,and PCR detection results showed that osteogenesis key genotype Collagen Ⅰ and osteopontin (OPN) gene were expressed.In conclusion,the GMSCs of Simmental cattle were successfully isolated in this study,and the isolation and culture system of GMSCs of Simmental was established in vitro.Moreover,the multidirectional differentiation potential was proved through adipogenic and osteogenic induction and differentiation,the results could provide references for studies on regenerative medicine and tooth repair.

The objective of this experiment was to analyze the differential proteomic between colostrum and mature milk of sheep,and provide further insight into the proteome characteristics during different lactation periods.6 Hu sheep with the same parity and similar due date were selected to collect colostrum on the 1st,3rd,7th day and mature milk on the 14th,28th,56th day postpartum.6 units of milk were mixed as a tested sample with equal volume at each time point,and defatted milk was prepared by removing the fat from the whole milk.The defatted milk of colostrum and mature milk were analyzed by two-dimensional electrophoresis (2-DE).The results showed that the content of milk protein was higher in colostrum,and decreased with the extension of lactation stage.According to the 2-DE map of milk protein on the 1st day,38 different protein spots were detected on the 3rd day,20 of which only existed on the 1st day and 1 only existed on the 3rd day,the other 17 protein spots showed differential expression.On the 7th day,35 different protein spots were detected,19 of which only existed on the 1st day and 1 only existed on the 7th day,the other 15 protein spots showed differential expression.On the 14th day,34 different protein spots were detected,11 of which only existed on the 1st day and 1 only existed on the 14th day,the other 22 protein spots showed differential expression.On the 28th day,38 different protein spots were detected,28 of which only existed on the 1st day and 1 only existed on the 28th day,the other 9 protein spots showed differential expression.On the 56th day,36 different protein spots were detected,26 of which only existed on the 1st day and 1 only existed on the 56th day,the other 9 protein spots showed differential expression.To sum up,a total of 44 differentially expressed protein spots were detected,8 of which only existed on the 1st day of postpartum,and these proteins were mainly involved in immunological activity.

To better develop and utilize local breed resources,the relevant indexes of Tibetan pigs' low-temperature adaptability was explored in this study and to provide theoretical basis for the study of Tibetan pig germplasm characteristics.20-day-old piglets were randomly selected (4 Tibetan pigs at -16 ℃ in winter and 4 Tibetan pigs at 30 ℃ in summer) in good body condition during lactation in a Tibetan pig farm in Yuzhong county,Gansu,and their body measurements and respiratory rate,back fat thickness,skin thickness,blood physiological,and biochemical indicators were measured.HE staining was used to observe the morphology of adipocytes in the groin,scapula,and back.Real-time fluorescent quantitative PCR technology was used to detect the relative expression of heat-related genes in different adipose tissues.The results showed that the respiratory rate of Tibetan pigs in the winter low temperature group was extremely significantly lower than that of the Tibetan pigs in the summer high temperature group (P<0.01).The thickness of back fat and the skin thickness of the neck,back and buttocks were extremely significantly higher than that of the high temperature Tibetan pigs (P<0.01).The number of platelets (PLT),platelet pressure (PCT),high-density lipoprotein cholesterol (HDL-C),glucose (GLU),and triglyceride (TG) contents of Tibetan pigs in the low temperature group were significantly higher than those of Tibetan pigs in the high temperature group (P<0.05).Mean platelet volume (MPV) and platelet distribution width (PDW) were significantly lower than of the high temperature group Tibetan pigs (P<0.05).The HE staining results showed that the size of fat cells in the groin and scapula of the low temperature group was different,and there were multi-cavity lipid droplets.Meanwhile,it was found that the relative expression of UCP3,PGC1α and Dio2 gene in the groin and scapula of the cold group was significantly higher among Tibetan pigs in high temperature group (P<0.05).In conclusion,environmental temperatures had a great influence on the physiological indexes and fat metabolism of Tibetan pigs,and the white fat browning stimulated by low temperature was one of the important factors for Tibetan pigs to resist cold.

Ruminants are important livestock in animal husbandry.It is of great significance to study the colonization process of rumen microorganisms in young ruminants,and take scientific early regulation measures by utilizing the colonization rules so as to improve the production efficiency and product quality of ruminants,and to maintain the high efficiency,health and sustainable development of animal husbandry.The colonization process of rumen microbes is accompanied by huge changes in the rumen development and diet of young ruminants.When young ruminants are breastfeeding,the rumen does not work due to the presence of the esophageal groove reflex,so the development is slow.At this time,only few functional microbes gradually colonized.As the age increases,young ruminants take in large amount of solid feed,the rumen develops rapidly and matures under the stimulation of solid feed.At this time,a large number of bacteria colonized,rumen fermentation is gradually active,the dominant microbial have changed greatly compared with the previous period,and the accumulation of rumen fermentation products further stimulated the development of rumen.Ruminants have experienced a physiological transition from non-rumination to rumination at the young stage.The stage before rumination is the period of the most sensitive and easy to intervene.It is possible to take artificial regulation measures for ruminant rumen microbes at the young stage to ensure the healthy and growth.In this paper,the early development of rumen,the types and sources of rumen microorganisms and their colonization in the rumen of young ruminants were reviewed.The important functions of rumen microorganisms in rumen digestion and metabolism,production performance and animal health were elucidated.The commonly used rumen microbial control techniques were also included,so as to provide a reference for the feeding management and nutrition regulation of young ruminants in the production system.

The aims of this experiment was to study the effects of Clostridium butyricum and sodium butyrate on egg production rate,freckles,dark spots and egg quality of laying hens in different breeds.320 270-day-old Langshan chickens,Barred Plymouth Rock or Beijing You chickens were randomly divided into 2 groups with 8 repeats in each group and 20 chickens in each repeat.The control groups(CON) were fed with basic diet and the experimental groups (EXP) were fed with diet added Clostridium butyricum (100 mg/kg) and sodium butyrate (500 mg/kg).The pre-feeding period was 3 days and the period of the experiment was 5 weeks.The results showed that:Compared with the control group,①The egg production rate of Langshan chicken,Barred Plymouth Rock and Beijing You chicken were increased by 12.5%,12.0% and 24.9% respectively (P<0.01).②The grade 3 rate of the freckles of the Langshan was decreased by 34.2% in the experimental group (P<0.05),there was no significant difference of dark spot rate of Langshan chicken,and no significant difference of freckle rates and dark spot rate in Barred Plymouth Rock and Beijing You chicken (P>0.05).③The egg weight was decreased by 1.7% (P<0.05),the egg shape index increased by 2.3% (P<0.05) of Langshan chicken in experimental group.The weight of the eggs of Barred Plymouth Rock increased by 1.5% (P<0.05).The protein height increased by 13.9% (P<0.05),and the Hastelloy unit increased by 4.7% (P<0.05) of Beijing You chicken in experimental group.In summary,adding Clostridium butyricum and sodium butyrate to the basic diets could increase the egg production rate of Langshan chicken,Barred Plymouth Rock and Beijing You chicken.It also had a certain effect on improving the freckles of Langshan chicken eggs,and could help to improve the egg quality of Barred Plymouth Rock and Beijing You chicken.

The purpose of this experiment was to study the effect of adding cysteamine to the buffalo diet on the lactation performance,rumen fermentation parameters,antioxidant performance,and rumen microbial diversity of the buffalo.Twenty healthy Nili-Ravi buffaloes with similar body weight (615 kg±21 kg),parity (3~5 births) and milk production (6 kg/d±1 kg/d) were selected and divided into 4 groups with 5 buffalo in each group as a single-factor randomized block design.0 (control group),15,30 and 45 g/d cysteamine was added to the diet,respectively.The pre-feeding period was 1 week and the test period was 4 weeks.The results showed that:Compared with the control group,① Adding cysteamine had no significant effect on buffalo body surface temperature,rectal temperature and respiratory rate (P>0.05).② The addition of 30 and 45 g/d cysteamine in the diet significantly increased the DMI and the apparent digestibility of ADF (P<0.05).③ The addition of cysteamine in the diet could significantly increase the milk production of buffalo,4% milk fat corrected milk production,milk protein rate,total milk solids and the milk non-fat solids content (P<0.05).④ Dietary supplementation with 45 g/d cysteamine significantly increases the buffalo rumen MCP content (P<0.05).Dietary supplementation with cysteamine had no significant effect on the number of bacteria and fungi in rumen fluid of buffalo (P>0.05).Adding 15 g/d cysteamine significantly reduced the number of protozoa (P<0.05).When the dosage of cysteamine was 30 g/d,the number of rumen fluid protozoa was significantly increased (P<0.05).⑤ The addition of cysteamine in the diet significantly reduced the content of C14:1n5,C18:3n6 in milk and significantly increased the content of C18:1n9t,C19:0,C20:3n6 in milk (P<0.05).Adding 30 g/d cysteamine to the diet significantly reduced the content of C16:0,C18:0 and SFA in milk (P<0.05).Dietary supplementation of 30 and 45 g/d cysteamine significantly reduced the content of C20:4n6 (ARA) and UFA in milk (P<0.05).⑥ The buffalo serum T-AOC in cysteamine groups were significantly higher (P<0.05);MDA in the diet supplemented with 15 and 30 g/d cysteamine group was significantly lower than the control group (P<0.05);GSH-Px,total protein and globulin in the 30 and 45 g/d cysteamine group was significantly increased compared with the control group (P<0.05);Cor of the 15 and 45 g/d cysteamine group was significantly lower than that of the control group (P<0.05).The activity of AST in the 30 g/d cysteamine group was significantly increased compared with the control group (P<0.05).In conclusion,the addition of cysteamine in the diet could improve the lactation performance and antioxidant capacity of the buffalo in the mid-lactation period,and affect the rumen fermentation,milk fat fatty acid content and the number of rumen protozoan in the buffalo.Under the conditions of this experiment,the recommended amount of cysteamine added to the diet was 30 g/d.

The shortage of feed resources has always been the main factor restricting the development of animal husbandry industry in China.In long term,conventional feed can no longer maintain the sustainable development of China's animal husbandry,and the development of new feed resources is one of the important ways to solve the shortage of feed.Sunflower by-products have high nutritional value,wide source and low price,which is a new type of feed with great development value.Sunflower by-products include sunflower cake,sunflower disk,sunflower stalk and kernel shells.Sunflower cake is high in crude protein content,low fiber content,is a high-quality plant protein source,can improve the nutritional value of cow milk,promote the growth of beef cattle,improve the fermentation effect of sheep rumen.Sunflower disk is high in crude fat content,containing a large number of dietary fiber,can improve the milk yield and milk fat rate of cows,promote beef cattle weight gain,improve the concentration of rumen volatile fatty acids and the digestion of crude protein,crude fiber and neutral detergent fiber.Sunflower stalk is rich in nitrogen,phosphorus,potassium,calcium,magnesium and other mineral elements and a small amount of protein,can provide some constant elements and trace elements for cattle,improve the daily weight gain of sheep,reduce the feed to gain ratio.Kernel shells are mainly composed of cellulose and lignin,rich in biology active substances which can improve daily cow weight gain and beef feed conversion rate.The nutritional value of sunflower by-products including sunflower cake,sunflower disk,sunflower stalk and kernel shell,and their application in ruminant feed at present were introduced in detail,so as to provide theoretical basis for the development and utilization of sunflower by-products in the future.

In recent years,the improvement of pig muscle quality by changing muscle fiber properties and intramuscular fat content has become a hot research topic among scientists.The formation of muscle fiber property and the deposition of intramuscular fat are the result of the interaction of internal and external fact.In this paper,the genetic mechanism of muscle fiber property formation and intramuscular fat deposition in pigs was reviewed in order to find ways to control and improve pork muscle quality.The genetic mechanism of pig muscle fiber property formation and intramuscular fat deposition is very complex,regulated by the complex network of genes,transcription factors and signal pathways.The growth and development of pig muscle fibers are mainly regulated by myogenic determinant (MyoD) gene family and myostatin (MSTN) gene.AMPK/SIRT1/PGC-1α energy sensing network plays an important role in the transformation of pig muscle fiber types.The expression of genes related to fat deposition is closely related to intramuscular fat deposition.With the development of molecular biotechnology,many genes related to muscle fiber property formation and intramuscular fat deposition have been identified,which is helpful to reveal the genetic mechanism of muscle fiber property formation and intramuscular fat deposition in pigs.

In order to analyze the polymorphism of GHR gene in cattle,SNPs with significant effects on the growth traits of native cattle in Guizhou were selected.This study used 150 Guizhou local Yellow cattle (Guanling cattle,Sinan cattle,Weining cattle each 50) as the research object,and GHR as the candidate gene,design primers based on the exon sequence of the Yellow cattle GHR gene included in GenBank.The blood of Guizhou local Yellow cattle was collected,blood genomic DNA was extracted and a mixed pool was contructed.The SNPs and typing of GHR gene was verified by PCR amplification sequencing method.Using DNAStar and SPSS 19.0 software to conduct genetic analysis on the SNPs of GHR gene and the correlation analysis with the growth traits in Guizhou local Yellow cattle,looking for the difference in the growth performance of Guizhou local Yellow cattle with different genotypes.At the same time,bioinformatics was used to analyze the changes of mRNA secondary structure before and after GHR mutation,predictive analysis of the structure and function of GHR protein in Guizhou local Yellow cattle.The results showed that a total of 8 SNPs were detected of GHR gene in Guizhou local Yellow cattle,namely Exon9-C162T,Exon9-C201T,Exon9-G243C,Exon9-G383A,Exon9-A495T,Exon9-C622T,Exon9-C642T and Exon9-A650C.Among them,Sinan cattle had no Exon9-G243C.Analysis of genetic characteristics showed that except for Exon9-G243C locus deviated from Hardy-Weinberg equilibrium in Weining cattle,the remaining SNPs did not deviate from Hardy-Weinberg equilibrium.Correlation analysis showed that GHR gene mutation sites were not detected in Guanling cattle and Sinan cattle.However,in Weining cattle,the chest circumference of individuals with Exon9-G243C GC genotype was significantly lower than that of GG and CC genotypes (P<0.05),the obliquee body length and ischial width of individuals with Exon9-A650C AA genotype were significantly higher than those with CC genotype (P<0.05),while the chest depth was significantly lower than that of AC and CC genotypes (P<0.05).The remaining 6 SNPs were not significant (P>0.05).Bioinformatics analysis showed that before and after the mutation,except for Exon9-G383A mRNA secondary structure did not change,the other SNPs mRNA secondary structure changed,Exon9-G243C and Exon9-A650C would affect the changes of protein secondary structure,but the mutation did not affect the change of the tertiary structure of the protein.In addition,Guizhou local Yellow cattle GHR protein was a secreted protein with signal peptide cleavage sites and 6 N-glycosylation sites.It showed that GHR gene had a high degree of variation.The results showed that the two SNPs of GHR gene Exon9-G243C and Exon9-A650C had significant effect on the growth traits of Guizhou local Yellow cattle,and might be used as candidate molecular markers for the growth and development of Guizhou local Yellow cattle.

The purpose of this study was to understand the genetic variation of important economic traits and functional genes in experimental pig population using molecular breeding gene chip of "Zhongxin No.1",so as to provide effective information for selecting excellent breeding individuals.In this study,the effective mutation sites from the functional genes were selected as molecular genetic breeding markers.Those functional genes control the fat deposition,meat quality,growth,disease resistance and coat color phenotype of pigs.A total of 106 individuals inclding wild boars,Songliao Black sows and their hybrids generation were used as experimental animal model.The effective mutation sites on functional genes controlling different traits from experimental animals were statistically analyzed by pig molecular breeding chip "Zhongxin No.1".The results showed that the effective mutation sites for the functional genes controlling fat deposition traits (SCD,MYH4) in pigs were favorable genotypes (CC,TT) for intramuscular fat deposition.The effective mutation sites for the functional genes controlling meat quality trait (PHKG1,PRKAG3,RYR1) were the favorable genotypes (CC,GG,CC) except 21 pigs carrying allele T (heterozygous genotype CT) with effective mutation site of RYR1 functional gene which was unfavorable to meat quality traits.The proportion of homozygous genotype (GG) for disease-resistant favorable allele at the effective mutation site of disease-resistant trait functional gene MUC13 was higher than that of heterozygous (GA) and unfavorable allele homozygous genotype (AA).The detection of effective mutation sites for growth trait functional genes (HMGA1,VRTN,CCKAR) found that there was only one heterozygote,without individuals having TT genotype with the HMGA1 mutation site for body length increase in pigs.The frequency of VRTN effective mutation site T allele controlling rib number was high,which controlled the increase of the number of individual thoracic vertebrae.The effective mutation site for functional gene CCKAR were all C allele homozygous genotype that were beneficial to increase feed intake and daily weight gain.The effective mutation site for KIT functional gene controlling coat color phenotype traits were GG genotype in all tested animals,indicating that there were no gene mutation sites affecting white coat phenotype in the studied pigs.The above results were consistent the coat phenotype of experimental pigs.These results would provide useful information for pig breeding schemes.

In order to explore the effect of inbreeding on reproductive effects,this study selected Langshan chickens conserved in the National Chicken Genetic Resources as materials.Combining molecular markers and pedigre information,two groups of highly and low inbred chickens were established,and the reproductive performance data were recorded for both groups.Three chickens was selected as a material in each group,and ovarian tissues were taken for RNA-seq sequencing,and the functional annotation of different genes was carried out.The results showed that a total of 1 114 differential transcripts were obtained in the highly and low inbred groups,of which 783 genes were annotated,307 were up-regulated and 476 were down-regulated.GO and KEGG analysis showed that the differential genes were mainly enriched in biological processes such as ribosome biosynthesis,inflammatory response,reproduction,growth,immune system processes,and metabolic processes.Biological pathways including in folic acid biosynthesis,oocytes maturation and metabolism were also enriched by the differential genes.Functional analysis found that the differential genes (such as GGH,CPEB1,GNMT and PIWIL) were related to reproductive function.In addition,some genes related to immunity and stress (such as APOC3,HSP70,CD38 and LGMN) were also included.The results of this study were helpful for us to understand the genes and their regulatory mechanisms related to inbreeding depression of reproductive traits in Langshan chickens,and would provide a reference for the molecular mechanism of inbreeding depression of specific traits in poultry.

The entire development process of a follicle is precisely regulated,which includes primary follicles to mature follicles,ovulation and corpus luteum development.It is critical to produce large quantity of superior follicles for multiple births and rapid reproduction.It was found that through impacting the growth of oocyte and granulosa cells in sheep follicles,the associated signal pathways and transcription factors in turn regulate the growth and maturity of follicles.Further understanding of these signal pathways is useful for investigating the regulatory mechanism of follicular development.In addition,high efficiency of sheep breeding and reproduction can be achieved as soon as possible.Notch is a highly conservative signaling pathway that plays an important role in the development of follicles.The PI3K/AKT/mTOR signaling pathway is widely present in cells.Each member of this pathway is a signal transduction molecule and has a huge impact in the early stage of follicular development.Physical junctions such as gap junction (GJ) and transzonal projections (TZPs) play an important role in signal communication between cells.This article elaborated the regulatory function of Notch signaling pathway,PI3K/AKT/mTOR signaling pathway,gap junctions and transzonal projections in the development of sheep follicles,which would provide a reference for further exploring the regulatory mechanism of sheep follicle development.

In order to explore the polymorphism and tissue expression of porcine β-defensin 110 (PBD-110) gene,the CDS region and intron regions of PBD-110 gene were amplified in crossbred from wild boars,Min pigs and Large White pigs.Finally,the difference of tissue expression in liver and spleen of the three populations was explored by Real-time quantitative PCR method.The results showed that porcine PBD-110 gene CDS region and intron regions were amplified.Sequencing confirmed that there were no mutation sites in CDS region and genomic PCR product sequencing found that C314T mutation in intron 1.Polymorphism analysis showed that CC was the dominant genotype,and the genotype frequency were 0.750,0.781 and 0.516,respectively.Hardy-Weinberg equilibrium test also showed that the locus was in equilibrium and moderately or lowly polymorphic (0.25 < PIC < 0.5;PIC<0.25) in three populations.The results of expression profile analysis showed that PBD-110 gene was expressed in liver and spleen of three populations of pigs,and there were different expression pattern.PBD-110 gene might play an important role in innate immunity.

Calving interval is the time interval between calving,which can comprehensively reflect the reproductive performance of dairy cows,such as estrus,breeding,pregnancy and calving.Calving interval is an important index to measure the reproductive performance of dairy cows,and it is also an important factor affecting the milk yield and calving quantity of dairy cows,which has a direct impact on the economic benefits of dairy farming.The latest research showed that the suitable calving interval was correlated with the lactation ability of cattle.The higher the lactation ability,the longer the suitable calving interval should be.However,even though the milk yield of large-scale cattle farms in China has been greatly improved,most farms in China are still pursuing shorter calving intervals.Therefore,this paper summarized the factors affecting calving interval of Holstein dairy cows,especially high-yielding cows,and analyzed calving interval and economic benefits of dairy farms,in order to provide some references and theoretical basis for determining suitable calving interval for Holstein dairy cows farming in China.

The purpose of this study was to express the recombinant protein p49 via constructing the prokaryotic expression system of African swine fever virus (ASFV) B438L gene,then prepare the polyclonal antibody against p49 protein.B438L gene was synthesized according the gene sequence of ASFV Georgia 2007/1 (GenBank accession No.:FR682468.1),then inserted into pET-28a(+) vector to construct pET-28a-B438L recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,then induced by 1 mmol/L IPTG at 16 ℃ for 12 h,the recombinant protein expression form was identified by SDS-PAGE.MALDI-TOF/TOF was used to determine whether the recombinant protein was expressed correctly.The polyclonal antibody against p49 was prepared by immunizing mice with the purified recombinant protein p49,the antibody titer was detected by indirect ELISA,and the specificity was identified by indirect immunofluorescence assay (IFA) and Western blotting.The results showed the pET-28a-B438L was successfully constructed.Then the recombinant plasmid was transformed into E.coli BL21(DE3),and the recombinant protein was expressed mainly in the form of inclusion bodies,with mass at about 60 ku.The recombinant protein was further confirmed by tandem mass spectrometry.The indirect ELISA result showed that the titer of polyclonal antibodies was as high as 1:64 000.The results of IFA and Western blotting indicated that the polyclonal antibody could specifically recognize the recombinant protein p49.These results confirmed the recombinant protein p49 expressed via prokaryotic system had good immunogenicity,and the prepared polyclonal antibodies had high titer and specificity.This study provided technical support for further study of the biological structure and function of ASFV p49 protein for the further ASFV related diagnosis and vaccine development.

The purpose of the experiment was to establish a eukaryotic expression system for porcine colony-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4),and use the expressed two cytokines to induce porcine dendritic cells in vitro and test their cell function.Firstly,the eukaryotic expression vectors of GM-CSF and IL-4 were constructed and verified on HEK293 cells.Then,the two cytokines were used to induce porcine bone marrow-derived and blood-derived monocytes.Finally,the surface markers CD1a,SLA-Ⅱ and SWC3a of porcine dendritic cells were detected by fluorescence microscopy and flow cytometry respectively,and the morphological and immunological functions of dendritic cells were identified.The results showed that the two eukaryotic expression vectors successfully expressed GM-CSF and IL-4 in HEK293 cells.Morphological observation showed that monocytes induced by cytokines had aggregation after 3 days of culture,and typical dendritic processes could be observed at the 6th day.Flow cytometry showed that the expressed cytokines could successfully induce the transformation of porcine bone marrow-derived and blood-derived monocytes into dendritic cells,and the double positive rates of SWC3a⁺/SLA-Ⅱ⁺ and CD1a⁺/SLA-Ⅱ⁺ were significantly increased,and there was no difference from the commercial cytokine treatment group.The results of cell phagocytosis test showed that the dendritic cells induced by the expressed cytokines were immature dendritic cells with strong phagocytic ability.In this study,we successfully established a method for the expression of porcine recombinant protein GM-CSF and IL-4 in eukaryotic cells,and induced mononuclear dendritic cells from pig bone marrow and blood using two recombinant cytokines in vitro,which laid a foundation for further study on the role of porcine dendritic cells and various pathogenic microorganisms.

Candida glabrata had gradually developed into the second largest pathogen in poultry Candida,which was usually a normal flora in the oral cavity,esophagus and gastrointestinal tract.When the immunity of the body decreased,it showed pathogenicity to the host,and with the increase of infection,once the strain entered the blood,it spread to the whole body,resulting in systemic candidiasis.Candida glabrata mainly infected chickens and pigeons.In clinic,the main symptoms was loss of appetite and emaciation,growth retardation,loose feathers and mental depression.Autopsy showed that white pseudomembrane or white round uplift ulcer was formed on the surface of crop mucosa,which was similar to carpet-like disease,commonly known as candidiasis.Candida glabrata existed in the form of yeast in the process of infection,and its mycelium morphology was not seen.During the invasion,Candida glabrata formed a biofilm to escape medical sterilization and disinfection,and used adhesin formed by adhesin family to absorb and invade the host.It could destroy host cells by hydrolyzing protease and other virulence factors,thus showing pathogenicity.The animal body used macrophage phagocytosis and oxidative stress to resist the infection of Candida glabrata.The bacteria detoxified actived oxides through the expression of catalase and superoxide dismutase detoxifying enzymes and the production of antioxidants glutathione and thioredoxin,thus survived immune attacks.In order to cope with the economic loss caused by Candida glabrata infection,the long-term and large-scale used of antifungal drugs led to the increasing of drug resistance,and Candida glabrata showed high drug resistance to first-line antifungal drugs,especially azoles.Researchers were increasingly inclined to use natural extracts to solve the problem of drug resistance of Candida glabrata.This paper reviewed the etiology,virulence factors and pathogenicity,epidemiology,antibiotics and detection techniques of Candida glabrata,in order to provide a theoretical basis for the development of effective antibiotics and more effective prevention and treatment of candidiasis in the future.

In order to provide guidance on the clinical treatment of pathogenic E. coli infection in morbid geese and to prevent and control pathogenic E. coli infection,in this study,spleen,liver and other tissues and organs of diarrhea geese and dead geese were collected aseptically for pathogen isolation and culture,staining observation,biochemical identification,pathogenicity test,partial gene sequencing,phylogenetic tree construction and drug sensitivity test.The results showed that off-white,round,raised,and neatly edged colonies grew on ordinary agar medium,and rose-red,smooth and round colonies grew on MacConkey agar medium.Gram negative bacteria were observed under microscope after Gram staining,the two ends were obtuse and medium in size,most of them were rod-shaped bacteria.The pathogenicity test showed that the isolated strain had high mortality to mice and goslings.The results of the phylogenetic tree showed that the isolated strain had the highest homology with the patient fecal isolate (GenBank accession No.:CP024992.1),reaching 99.5%.In vitro antibacterial test showed that the drug resistance of the strain was serious,and it had strong resistance to most antibiotics.Only ceftiofur had strong antibacterial effect on the isolated strain,and the disease was effectively controlled after rational drug use.This study provided a theoretical basis for the effective control of E.coli in geese,and had important value for the prevention and control of E.coli and other bacterial diseases in large-scale goose farms.

The purpose of this study was to investigate the clinical effect of Gongyingqiaolu powder on subclinical mastitis of dairy cow.In this experiment,78 cattle were selected,38 of which were sporadic and treated with Gongyingqiaolu powder,another 40 cattle were treated with concentrated contrast treatment and randomly divided into 2 groups with 20 cattle in each group,which were treated with Gongyingqiaolu powder and Gongying powder respectively.The California mastitis trial (CMT) method was used to detect the disease status of the udder after the treatment of Gongyingqiaolu powder and determined its therapeutic effect.Somatic cell count (SCC) method was used to detect the somatic cell number of milk after treatment with Gongyingqiaolu powder and Gongying powder and determined the therapeutic effect.The results showed that after 7 d of treatment with Gongyingqiaolu powder and Gongying powder,the number of affected udder was reduced from 75 to 28,and the treatment effect was very significant,with the cure rate reaching 62.67% and the effective rate reaching 90.67%.Compared with untreatment group,the number of somatic cells after 1 d of treatment with Gongyingqiaolu powder and Gongying powder was extremely significantly decreased (P<0.01),and the SCC of Gongyingqiaolu powder group decreased significantly than that of Gongying powder group.Compared with Gongying powder group,the SCC of Gongyingqiaolu powder group decreased significantly after 2 d of administration (P<0.05),and extremely significantly decreased after 3 d of administration (P<0.01).After 7 d of treatment,the cure rate of Gongyingqiaolu powder group reached 68.29%,while the cure rate of Gongying powder group only reached 20.00%,with a extremely significant difference (P<0.01).The effective rate of Gongyingqiaolu powder group was 97.56%,while the effective rate of Gongying powder group was 67.50%,the difference was extremely significant (P<0.01),indicating that the therapeutic effect of Gongyingqiaolu powder was extremely significantly better than that of Gongying powder (P<0.01).The above results indicated that Gongyingqiaolu powder could be used as an effective drug to treat subclinical cow mastitis and provided a reference for the development of cow mastitis drugs.

Parasitic diseases have a considerable socio-economic impact,zoonotic parasites bring a huge burden of disease to people and cause serious economic losses to the breeding industry.Therefore,the prevention and treatment of parasitic diseases is an urgent research topic.Parasites have many forms of immune evasion mechanisms.Inactivated vaccines,live attenuated vaccines,subunit vaccines,etc.have not achieved effective prevention of parasitic diseases.Many studies have shown that DNA vaccines are expected to be the prevention and effective treatment of parasitic diseases.DNA vaccine is a new type of vaccine which can simultaneously produce long-lasting durable humoral and cellular immune,and it provide protective immunity by expressing foreign proteins in the host.DNA vaccines are different from other subunit vaccines in that immunogens are synthesized in the host by cells that take up the DNA encoding the antigen.The synthesis of protein in the body can also carry out antigen processing,modification and presentation to the host's immune system,similar to the way of natural infection.This article discussed the immune mechanism,design principles,immune pathways,advantages and disadvantages of DNA vaccines and the research progress of parasite DNA vaccines in recent years.It was expected to theoretical reference for the control of parasites disease.

In order to establish and optimize the extraction process of polyphenols from Broussonetia papyrifera leaves,the ultrasonic assisted extraction method was used in this experiment.Taking the yield of polyphenols from Broussonetia papyrifera leaves as the index,the effects of the material to solvent ratio,ethanol concentration,ultrasonic time and ultrasonic temperature on the yield of polyphenols from Broussonetia papyrifera leaves were studied by single factor experiment.On the basis of single factor experiment,the experiment of 4 factors and 3 levels was designed by response surface methodology(RSM) to optimize the extraction process parameters of Broussonetia papyrifera leaf polyphenols,a regression model was established and response surface analysis was carried out to determine the optimal extraction process parameters,and three parallel validation were carried out.Taking vitamin C as the control,the antioxidant activity of Broussonetia papyrifera leaf polyphenols on DPPH and ABTS free radical scavenging rate was determined to evaluate in vitro antioxidant activity of Broussonetia papyrifera leaf polyphenols.The results showed that the order of the four factors on polyphenol yield was ethanol concentration (B)>the material to solvent ratio (A)>ultrasonic temperature(D)>ultrasonic time(C).RSM results showed that there was interaction between the two factors,but the interaction was not significant (P>0.05).When the material to solvent ratio was 70:1,ethanol concentration was 60%,ultrasonic time was 50 min,and ultrasonic temperature was 50 ℃,the yield of polyphenols from Broussonetia papyrifera leaves was 13.62 mg/g,which was close to the predicted value of RSM.In vitro antioxidant test showed that Broussonetia papyrifera leaf polyphenols could scavenge DPPH and ABTS free radicals,and could reach the level equivalent to vitamin C at high concentration.In conclusion,the extraction process of polyphenols from Broussonetia papyrifera leaves in this study was accurate and feasible.It was also proved that the polyphenols from Broussonetia papyrifera leaves had good antioxidant activity.The results could provide reference for the study of biological activity of Broussonetia papyrifera leaves and the development of medicinal value of Broussonetia papyrifera leaves.

Following the Veterinary Pharmacopoeia of the People's Republic of China and The Compendium of Technical Guidelines for Research on Veterinary Drugs,the appearance,viscosity,pH,moisture,density,endotoxin,content and releated-substances tests were carried out on eprinomectin (EPR) extended-release injection agent to establish the quality standard and compare the pharmacological equivalence with the reference formulation.Content and related substances were determined on ZORBAX Eclipse Plus C18 column (2.1 mm×50 mm,1.8 μm),mobile phase:formic acid:water:acetonitrile (0.04 mL:40 mL:60 mL);Flow rate:0.4 mL/min;Detection wavelength:245 nm;Column temperature:35 ℃;Injection volume:2 μL.The pharmacological equivalence of the agent (f2 value) was calculated by comparing the release degree under the conditions of PBS buffer of 0.5% SDS at 37 ℃ and the rotation speed of 50 r/min.After testing,the appearance of EPR sustained-release injection agent was clear and transparent,the average relative viscosity was 46.45 mPa·s,the average pH was 6.88,the preparation did not contain water,the density was 1.13 g/cm³,the endotoxin met the limit of less than 0.1 EU/mL,the content was greater than the labeled value of 5%,the amount of the substance was less than 5%,the test results were all in line with the injectable preparation requirements.After 31 d of release under the same conditions,both self-research and the original EPR sustained-release injection agent release were more than 80%,and the f2 value of self-research and the original was more than 50.The test results of self-researched EPR sustained-release injection agent were all in accordance with the standard for injectable agents,and the self-researched and original EPR sustained-release injection agent were pharmacologically equivalent.

The study was aimed to isolate,cultivate,identify and purify the prevalent strains of Mycoplasma gallisepticum in Tai'an,Shandong,and to screen sensitive traditional Chinese medicines against the isolates in order to provide materials for further research.The methods of liquid culture and solid culture were used to isolate,culture and purify the Mycoplasma gallisepticum strain from the tissue samples of diseased chickens,the isolate was preliminarily identified by Swiss staining and Giemsa staining,serological methods and molecular biological methods.On this basis,the micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the eight Chinese medicines against the isolated strains,in order to screen the Chinese medicines sensitive to the isolated strains.The results showed that after the isolated strains were proliferated in the liquid medium of Mycoplasma gallisepticum,the color of the medium changed from red to yellow and was translucent,after cultured in the solid medium,it showed a typical ‘fried egg’-like colony,which were consistent with the culture characteristics of Mycoplasma gallisepticum.The Swiss staining and Giemsa staining results indicated that the morphology of the bacteria was consistent with the characteristics of Mycoplasma gallisepticum.The serological identification results showed that the isolate had agglutinated with the positive serum of Mycoplasma gallisepticum.The molecular biology identification results showed the matching degree of the amplified nucleic acid sequence with Mycoplasma gallisepticum was as high as 99%.The result of MIC showed that the antibacterial activity of traditional Chinese medicines Coptis chinensis and Corcagium were sensitive to the isolated Mycoplasma gallisepticum,the MIC were ≤0.98 and 0.39 mg/mL,respectively.While the traditional Chinese medicine Fagopyrum dibotrys (D.Don) Hara and Houttuynia aucuparia were moderately sensitive to Mycoplasma gallisepticum,with MIC ≥125 mg/mL.Isatidis Radix,Nova cortices album,Angelica sinensis and Et persequebantur Josue were not sensitive to Mycoplasma gallisepticum.In conclusion,a Mycoplasma gallisepticum strain was successfully isolated in this study,and screened out four kinds of sensitive traditional Chinese medicines to this strain,which were Coptis chinesis,Corcagium,Fagopyrum dibotrys (D.Don) Hara and Houttuynia aucuparia,respectively,it provided a reference basis for further study of traditional Chinese medicine prescriptions of prevention and treatment of Mycoplasma gallisepticum.

The overuse and misuse of antibiotics contributed to the accumulative emergence of antibiotics resistance,which has been deemed as a major threat to the public health.Among the existing antibiotics,carbapenems are often considered as the last escort against infection caused by the multi-drug resistant (MDR) bacteria. The emergence of carbapenem-resistant bacteria poses a great threat to public health safety.The production of carbapenemases is the main mechanism for those carbapenem-resistant (CR) Enterobacteriaceae,consequently the detections of carbapenemases is of importance in the diagnosis of CR bacterial infection.The detection of carbapenemases mainly depend on phenotypic methods and molecular methods.Phenotypic detections enjoy the benefit of easy-handling,cost-efficiency,and the high feasibility in clinical cases.However,these methods are also less-effctive and tedious.Molecular detections and the newly-developed technologies enable the specific and sensitive detection in a rapid way,yet these methods always call for high expense and professional equipment.Some technologies are still to be developed in the field of carbapenemase detection.Currently,different detection methods are established and refined for particular purposes,and none of them is able to be employed to meet all demands.In practice,the time-consumption,ease of handling and the cost have to be comprehensively considered to select a suitable method which fits the purpose most.In this review,the different detection methods for carbapenemases are summarized and compared from the aspects specificity and sensitivity,operability,time consumption and the cost,providing an integrated view of the current progresses and advances the detection methods for carbapenemases.

This experiment was aimed to study the drug resistance and pathogenicity of the duck isolate.Through isolation and identification of bacteria,drug sensitivity test,PCR detection of drug resistance genes and virulence genes,and animal pathogenicity test,a bacterial strain was successfully isolated.The purified bacteria had two forms of Gram staining,some looked like short rod-shaped,some were as long and thin as hair,and Gram-negative,the isolated strain was named as GZGY2020.The biochemical reaction showed that the isolated bacteria had obvious motility,M-R test and V-P test were positive,and the biochemical characteristics were consistent with Proteus mirabilis.The 16S rDNA gene phylogenetic tree showed that the isolate was in the same branch as Proteus mirabilis.The results of drug sensitivity test showed that the isolate was resistant to multiple drugs,it was resistant to 16 drugs including carbenicillin,cephalexin,and oxacillin among 18 drugs,and the drug resistance rate was as high as 88.9%,and only sensitive to ceftazidime and ciprofloxacin.PCR results showed that it contained sulfonamides resistance genes sul1 and sul2 and β-lactam resistance gene VIM,and 9 virulence genes including hpmA,rsmA,atfA,ucaA,ureC,zapA,pmfA,fliL and mrpA.The results of animal regression test showed that the isolated bacteria had strong pathogenicity to ducklings,1×10⁸ CFU/mL bacterial solution could kill all ducklings within 1 day.In terms of drug sensitivity,the strain GZGY2020 isolated from dead ducks was different from Proteus mirabilis previously reported,which indicated that the strain might mutate and lead to increased virulence.However,only one of the 10 virulence genes detected had no corresponding bands,indicating that Proteus mirabilis was an important pathogen causing the death of the duck,which should be paid more attention.

Molecular identification and drug susceptibility of a strain pathogenic bacteria isolated from a yak suffered from bleeding from a farm of Naqu city,Tibet,were analyzed,in order to provide treatment bases for hemorrhagic disease of yaks in Tibet.The suspected strains were obtained by bacterial isolation and purification from lungs and livers of dead yaks,then the suspected strains were screened out by morphological observation and Columbia blood plate test,and then physiological and biochemical tests were done,16S rDNA sequencing and homology comparison test on the obtained suspected strains,and then obtained sensitive drugs of the suspected pathogenic strains by drug sensitivity tests,finally verified treatment effect of the sensitive drugs by animal regression test.The results showed that 6 suspected strains were obtained by separation and purification of the lungs and livers of dead yaks,and a strain of hemolytic Gram-positive cocci S-4 was screened out by morphological observation test and Columbia blood plate test,and the S-4 strain was identified as Staphylococcus epidermidis by the physiological and biochemical identification test,16S rDNA sequencing,homology comparison test.The drug sensitivity test showed that the S-4 strain was sensitive to enrofloxacin,neomycin,polymyxin B,kanamycin,and ciprofloxacin.And it was medium sensitive to florfenicol and doxycycline.And it was tolerant to streptomycin,erythromycin,tetracycline,penicillin and cephalexin.The animal regression tests showed that the strain was pathogenic and enrofloxacin,neomycin,and kanamycin,the 3 drugs had good treatment effects,while polymyxin B and ciprofloxacin had poor treatment effects.The results showed that this experiment obtained a pathogenic Staphylococcus epidermidis from yaks,the pathogenic bacteria could be treated with enrofloxacin,neomycin,and kanamycin during the breeding.

The purpose of this experiment was to explore the correlation between polymorphism of inducible nitric oxide synthase (iNOS) gene and brucellosis in Kazakh sheep.The serum of 231 Kazakh sheep were tested for serology by brucellosis using RBPT method.Refer to the sheep iNOS gene sequence in GenBank,design primers for its exons 6,7,8,PCR-SSCP and DNA sequencing technology were used to detect the polymorphism of iNOS gene in 231 Kazakh sheep,and analyze the correlation between their SNPs and the susceptibility to brucellosis.The detection results showed that 67 Kazakh sheep were positive for Brucella infection,and the positive detection rate was 29.00%.No polymorphic sites were detected on the exons 6 and 8 fragments of iNOS gene in Kazakh sheep,F7-T18054C and F7-C18084T were detected on the exon 7 fragment,and three genotypes (TC,TT and CC) were detected on the F7-T18054C polymorphic site,the dominant allele and genotype were C and CT genotype,and the allele frequency and genotype frequency were 0.660 and 0.446,respectively.Two genotypes (CT and CC) were detected on the F7-C18084T polymorphic site.The dominant allele and genotype were C and CC,the allele frequency and genotype frequency were 0.946 and 0.892,respectively.F7-C18084T belonged to low polymorphism (PIC<0.25),F7-T18054C belonged to moderate polymorphism (0.25 < PIC < 0.5).The correlation analysis results showed that the correlation between F7-T18054C and F7-C18084T polymorphic sites and brucellosis susceptibility was not significant (P>0.05).The results showed that F7-T18054C and F7-C18084T of iNOS in Kazakh sheep didn't correlate with the susceptibility to brucellosis.

To investigate the characteristics and antimicrobial resistance of a dominant bacterium isolated from the liver of a dead raccoon in a zoo in Sichuan province.In this study,Gram staining,biochemical identification,16S rRNA sequence determination,pathogenicity test,antimicrobial susceptibility test and resistance gene amplification test were used to conduct morphological,biochemical identification and study on pathogenicity and drug resistance of the isolated strain.The result showed that the isolated strain showed a translucent colony with smooth surface,round protrusion,neat edge and needle tip size on LB medium.Blue-purple cocci could be observed with Gram staining under oil microscope.The isolate was 99.5% similar to Enterococcus faecalis whose GenBank accession No. was KY003107.1,and the morphological and biochemical characteristics were consistent with those of Enterococcus faecalis.The pathogenicity test showed that the bacterium could cause hepatocytes swelling and degeneration,and had certain pathogenicity.Antimicrobial susceptibility test and resistance gene amplification test showed that the strain was resistant to 14 common antibiotics,cefoxitin,cefoperazone,ceftriaxone,cefepime,cefotaxime,ampicillin,kanamycin,streptomycin,netimicin,butanicin,azithromycin,erythromycin,doxycycline and tetracycline,and was only moderately sensitive to vancomycin and gentamicin.Tetracycline resistance gene tetC was positive,tetM and tetA gene were negative.Aminoglycoside resistance gene aac(6')/aph(2″) was positive,aph(3')-Ⅲ and ant(6)-Ⅰ were negative.The ermB of macrolide resistance genes was positive and ermA and ermC were negative.Vancomycin resistance gene vanA,vanB and β-lactam resistance gene TEM were all negative.In this study,the Enterococcus faecalis isolated from raccoons showed high drug resistance and pathogenicity,which could be used as a reference for the selection of drugs for clinical Enterococcus faecalis infection,and a reasonable infection prevention and treatment scheme could be formulated.

The trace detection of pesticides,veterinary drugs,heavy metals,biotoxins and other residual substances is of great significance in the fields of medical diagnosis,food safety and environmental monitoring.The immunoassays based on antigen-antibody specific recognition have advantages of strong specificity,high sensitivity and good stability,simple and quick operation,etc.,but how to obtain specific antibodies against small molecular compounds is the key to develop immunoassays.In this paper,the hybridoma technology for the preparation of monoclonal antibody of small molecular compounds was firstly introduced.Important factors affecting the production of monoclonal antibody of small molecular compounds in hybridoma technology was then briefly analyzed.The applications of monoclonal antibody in veterinary medicine,such as veterinary drug residues detection,drug development and environmental protection,were reviewed.Finally,the development trend of the monoclonal antibody production and application was prospected.This review provided guidance for the preparation and application of the monoclonal antibodies of small molecular compound.

The present study investigated the effect of FEA on inflammatory cytokines secretion in PRV induced RAW264.7 cells.Briefly,CCK-8 method was used to detect the activity of cells in vitro,in order to screen the safe concentrations of FEA on RAW264.7 cells.Then,the groups were assigned as blank control group,PRV positive group,rutin group and five FEA groups.The RAW264.7 cells(except for blank control group)were incubated with PRV for 1.5 h,then cultured with different concentrations of rutin or FEA for 4,8,12 and 24 h,respectively.CCK-8 method was used to detect the activity of PRV induced cells for screen three concentrations of FEA (high,medium and low doses)and then ELISA method were used to detect the secretion levels of TNF-α,IL-1β,IL-6,IL-10,MCP-1 and IFN-γ,in order to evaluate the optimal time and concentration of FEA for anti-inflammatory effect.The results showed as follows:①The safe concentration of FEA was ranged from 12.5 to 200 μg/mL.②In vitro,25-100 μg/mL of FEA could significantly improve the proliferation activity of PRV-induced RAW264.7 cell.③The levels of inflammatory cytokines were increased in PRV induced RAW264.7 cells,treatment of FEA for 8-12 h markedly decreased the levels of TNF-α,IL-1β,IL-6 and MCP-1 (P<0.05),increased the level of IFN-γ when compared with PRV group (P<0.05),and the IL-10 level was also regulated by FEA to a certain extent.Moreover,the anti-inflammatory effect was much better post FEA treatment for 8 h.In conlusion,FEA could regulate the secretion levels of inflammatory cytokines in PRV-induced RAW264.7 cell and exert anti-inflammatory effects in vitro.The results provided reference for further research on the molecular mechanism for antiviral drug.

The objective of this study was to report one case of the feline pseudomycetoma infected by Microsporum canis,from clinical symptom,diagnostic process to treatment strategies,which might strengthen awareness to the disease and provide potential insights for veterinary clinicians.Samples from the tail were taken for cytological examination,fungal culture and identification with MALDI-TOF MS.A large number of inflammatory cells,fungal spores and hyphae were observed in the cytology smear which indicated granulomatous inflammation.Based on the above results,fungal culture was performed.Staining and microscopic examination revealed more than six separated macroconidia in the cultured strains,which was finally identified as Microsporum canis by MALDI-TOF MS.In conclusion,the case was diagnosed as feline dermatophytic pseudomycetoma caused by Microsporum canis.Treatment for this case included shampoos and oral antifungal agents.In the early stage,the tail lesion was slightly improved and continued to deteriorate in the later stage.The patient died 7 months later with unknown reason.It suggested that veterinarians should pay attention when they encounter similar cases and use cytological examination,fungal culture and strain identification for diagnosis.Surgical removal in conjunction with medical therapy could improve the survival rate of the patient in the early stage of this disease.