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Table of Content

20 January 2020, Volume 47 Issue 1
Biotechnology
Construction and Identification of Helicobacter hepaticus CdtB Gene Mutant Strain
ZHU Chen, CAO Shuyang, WU Zhihao, YIN Jun, ZHU Liqi, ZHANG Quan
2020, 47(1):  1-7.  doi:10.16431/j.cnki.1671-7236.2020.01.001
Abstract ( 269 )   PDF (2420KB) ( 90 )  
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The experiment was aimed to knock out the CdtB gene of Helicobacter hepaticus (H.hepaticus).A suicide plasmid for knocking out the CdtB gene of H.hepaticus ATCC 51449 strain was constructed using the pcDNA plasmid.According to the principle of homologous recombination,the plasmid was transferred into H.hepaticus by electroporation to construct a H.hepaticus CdtB gene mutant strain (ΔCdtB).The ΔCdtB mutant strain was identified by PCR and sequencing.Using biochemical test and calculating the growth curve to detect the difference between the ΔCdtB mutant strain and the wild type H.hepaticus.The results showed that the homologous recombinant knockout plasmid pcDNA-ΔCdtb was constructed by plasmid pcDNA,and the chloramphenicol positive transformant was obtained by subculture and screening after the transformation.The PCR product of the mutant strain was 964 bp,which was larger than that of the corresponding product 884 bp of wild type H.hepaticus. It was found that the urease test results of ΔCdtB mutant strain and wild type strain were all positive,and there was no significant difference in growth rate between them on the common blood agar plate,but the ΔCdtB mutant strain grew more on the blood agar plate containing chloramphenicol.In addition,the deletion strain was passed on and identified by PCR,and no reverse mutation was found.To sum up,the gene knockout plasmid pcDNA-ΔCdtb constructed in this study could knock out the target gene of H.hepatica,and provide an effective gene manipulation tool for gene function research and pathogenic factor discovery of H.hepatica.
Construction and Identification of Iron Responsive Regulator irr and rirA Genes Deletion Strain of Brucella melitensis M5-90 Vaccine Strain
ZHAO Xiaoli, WANG Benben, ZHANG Huan, YANG Yajun, YANG Ningning, WANG Zhen, YANG Qin, WANG Yong, CHEN Chuangfu, CAO Xudong
2020, 47(1):  8-17.  doi:10.16431/j.cnki.1671-7236.2020.01.002
Abstract ( 249 )   PDF (3469KB) ( 60 )  
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In order to explore the role of iron response regulator (irr) and rhizobial iron regulator (rirA) during Brucella melitensis infection,in this research,two mutants M5-90Δirr and M5-90ΔrirA were constructed through the kanamycin gene replacement method.These two mutants and their parental strain M5-90 were cultured for 36 h in the same condition,the growth ability were measured.1×108 CFU M5-90Δirr,M5-90ΔrirA and M5-90 were inoculated into the 1 mL Brucella broth supplemented with 1.5 mmol/L NaCl,pH 2.5,pH 11.5 and 10 mmol/L H2O2 to detect the growth ability of these two mutants.The adhesion,invasion and intracellular survival ability of these two mutants were evaluated in RAW264.7 cells infected with 1×106 CFU either M5-90Δirr,M5-90ΔrirA or M5-90.The results showed that under the same culture condition,the growth rates of two mutants M5-90Δirr and M5-90ΔrirA were significantly lower than that of their parental strain.The survival rates of M5-90Δirr and M5-90ΔrirA in high salt,strong acid and alkali environment were significantly lower than those in parental strains (P<0.05).The survival rates of these two mutants were significantly higher than the parental strain under hydrogen peroxide conditions (P<0.05).The invasion and adhesion capability of these two mutants inside the murine macrophages RAW264.7 were attenuated,especially at 45 min post infection,the invasion and adhesion capability of these two mutants were extremely significantly lower than M5-90 (P<0.01),however,24 h after infection,the ability of these two mutants to propagate in cells was enhanced compared with that of their parental strain.This was the first report that irr and rirA genes of B.melitensis not only associated with the virulence but also with the growth ability.To sum up,M5-90Δirr and M5-90ΔrirA were two promising Brucella vaccine candidates.
Cloning and Bioinformatics Analysis of oppD/F Gene of Mycoplasma bovis Isolate Strain in Xinjiang
LING Chen, HAO Chengwu, ZHANG Fei, HOU Feng, HE Sun
2020, 47(1):  18-27.  doi:10.16431/j.cnki.1671-7236.2020.01.003
Abstract ( 240 )   PDF (1937KB) ( 40 )  
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This study was aimed to clone and analyze the oppD/F gene of Mycoplasma bovis isolated strain in Xinjiang,explore the sequence characteristics of oppD/F gene and the structure and function of its encoded protein.One pair of specific primers was designed based on the sequence of oppD/F gene of PG45 strain (accession No.:AF130119.1) in GenBank,the sequence of oppD/F gene was amplified by PCR,and it was inserted into pMD19-T vector for sequencing.Its nucleotide sequence,amino acids sequence,the basic physicochemical properties,hydrophobicity,soluble,signal peptide,transmembrane,subcellular localization,phosphorylation site,glycosylation site,secondary structure,tertiary structure,function of oppD/F protein were predicted and analyzed by bioinformatics methods.The results showed that the sequence length of Mycoplasma bovis isolated strain oppD/F gene was 1 617 bp,the sequence and phylogenetic tree analysis indicated that it showed 100% homology with that of Mycoplasma bovis strain PG45 and Mycoplasma bovis isolate JF4278,its were closely relative which were in the same branch.The oppD/F protein encoded a protein composed of 539 amino acids,no signal peptide and transmembrane structure,it was a stable soluble hydrophilic protein.The oppD/F protein had a AAA super family domain,50 potential phosphorylation sites and 3 glycosylation sites.The secondary structure was the mixed,and the alpha helix were the most,accounting for 61.78%,followed by the random coil,accounting for 20.78%.Function prediction showed that the high odds predicted from signal transducer receptor,structural protein,ion channel,immune response and stress response in oppD/F protein.The results could provide a theoretical basis for further exploration of its function of oppD/F gene of Mycoplasma bovis isolated strain.
Cloning and Bioinformatics Analysis of S100A16 Gene in Sika Deer
CHEN Jiqian, XIA Yanling, GAO Yueyu, LIU Weishi
2020, 47(1):  28-36.  doi:10.16431/j.cnki.1671-7236.2020.01.004
Abstract ( 237 )   PDF (1531KB) ( 59 )  
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In order to explore the S100A16 gene sequence and biological characteristics of Sika deer (Cervus nippon),primers were designed according to the S100A16 gene sequence of cattle and sheep in GenBank database,and the cDNA of S100A16 gene in Sika deer was successfully obtained by RT-PCR and molecular cloning technology with the top tissue cDNA of sika deer antler as template.Bioinformatics analysis showed that the total length of CDS region of S100A16 gene in Sika deer was 312 bp,encoding 103 amino acids;the protein contained 11 phosphorylation sites,transmembrane domain,and no signal peptide,which was the stable protein that played a role in the cell;The protein only contained the EF-hand of S100 protein family in the C-terminal,which was composed of 12 amino acids,and the EF-hand at the N-terminal was composed of 15 amino acids.The second-order structure of S100A16 protein in Sika deer was mainly composed of alpha-helix and ranchorn coil;the third-order structure showed that the protein had two Ca2+ binding sites;the amino acid sequence of S100A16 protein in Sika deer had the highest homology with Eastern European red deer,which was 100%,and the phylogenetic tree was constructed with the amino acid sequence of S100A16 protein in some other species.S100A16 gene was relatively conservative in evolution and conformed to the characteristics of functional genes.The results provided the basis for further revealing the function and expression mechanism of S100A16 gene in Sika deer.
American Mink Ear Fibroblasts Transfected by Adenovirus Vector
LI Wen, HAN Yuping, ZHAO Xiangyuan, FAN Bingfeng, LI Xiaoxia, DIAO Yunfei, YANG Yifeng, XU Baozeng
2020, 47(1):  37-43.  doi:10.16431/j.cnki.1671-7236.2020.01.005
Abstract ( 207 )   PDF (3671KB) ( 39 )  
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The objective of this study was to investigate the feasibility and efficiency of transfection of adenovirus vector with enhanced green fluorescent protein (EGFP) into fibroblasts in the ear margin of mink.Firstly,ear fibroblasts of mink were isolated and cultured by tissue adherent method,and then the virus Ad-EGFP was packaged and the titer was detected by TCID50 method.Two groups of different concentration gradients were used to infect mink fibroblasts,multiplicity of infection(MOI):0/100/300/500/700/900 and 0/10/30/50/70/90.Transfection efficiency (%) was estimated by looking at the ratio of positive cells to the total number of cells under a microscope.The results showed that primary cells could be grown in the ear tissue of mink cultured by tissue adherent method for 11 d,and the fusion degree of passage cells could reach 80% in 3-4 d.The titer of the packaged Ad-EGFP virus was 1.99×1011 PFU/mL,the optimal MOI value of the infected mink fibroblasts was 30,and the instantaneous transfection efficiency was more than 90%.In conclusion,it was feasible to transfection mink fibroblasts with adenovirus vectors,which was an efficient transfection method and provided a basis for the preliminary validation test of mink genome editing.
Animal Nutrition and Feed Science
Optimization of the Fermentation Conditions of Recombinant B.subtilis WB600 Produced Keratinase
LIAO Chaoyong, ZHANG Tieying, LIU Junli, SUI Jingwei
2020, 47(1):  44-52.  doi:10.16431/j.cnki.1671-7236.2020.01.006
Abstract ( 293 )   PDF (827KB) ( 71 )  
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The aim of this experiment was to optimize the fermentation conditions of the constructed keratinase recombinant B.subtilis WB600 strain WB600-K,and obtain the optimal enzyme production conditions in order to increase the yield of recombinant keratinase;At the same time,the combination optimization between different types of carbon sources and different nitrogen sources was studied in order to obtain a low-cost recombinant fermentative medium formula.The results showed that when the fermentation D600 nm=1.0,and 0.5% xylose was added to induce fermentation,the expression of keratinase was best in the recombinant WB600-K (P<0.05).As a carbon source,sorbitol could significantly increase the activity of the recombinant strain WB600-K keratinase (P<0.05),but the cost was too high to be a source of mixed carbon sources.The combination of different carbon sources (glucose,corn flour,molasses and glycerin) could significantly increase the activity of keratinase (P<0.05).The combination of different kinds of nitrogen sources (peptone,yeast powder,soybean meal and urea) significantly increased the activity of keratinase less than single nitrogen source (peptone) (P<0.05).The WB600-K had the highest keratinase activity at 48 h after induction of fermentation (P<0.05).The overall experimental optimization results showed that,at the condition of corn flour 5.1 g/L,glucose 5.9 g/L,molasses 5.9 g/L,glycerol 3.0 g/L,peptone 20 g/L,NaCl 10 g/L,0.5% xylose was added when fermentation D600 nm=1.0,and 48 h fermentation,the activity of keratinase could be increased significantly (P<0.05),which was 56.9 U/mL and 49.74% higher than that in initial culture conditions.In summary,fermentation under optimized fermentation conditions could increase the activity of recombinant strain WB600-K keratinase,which laid a foundation for the industrial production of keratinase.
Effects of Cadmium-tolerant Lactobacillus on Cd Residue and Some Mineral Elements Accumulation in Hens' Tissues and Eggs
PU Junhua, CHEN Dawei, LU Junxian, ZHANG Xiaoyan, TANG Mengjun, KONG Lingwu, ZHOU Qian, LIU Yinyin, GAO Yushi
2020, 47(1):  53-62.  doi:10.16431/j.cnki.1671-7236.2020.01.007
Abstract ( 203 )   PDF (1007KB) ( 45 )  
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The purpose of this experiment was to find out the effects of cadmium-tolerant Lactobacillus on organ coefficient,Cd residue and the contents of Ca,Fe,Zn,Mn,Cu and Se in tissues and eggs of laying hens.The experiment was divided into 5 groups:Control group,fed basic diet;Cd group,basic diet+5 mg/kg Cd (added in the form of cadmium chloride);Lactobacillus group,basic diet+1×1010 CFU/kg cadmium-tolerant Lactobacillus;Cd+low dose Lactobacillus group,basic diet+5 mg/kg Cd+1×1010 CFU/kg cadmium-tolerant Lactobacillus;Cd+high dose Lactobacillus group,basic diet+5 mg/kg Cd+1×1011 CFU/kg cadmium-tolerant Lactobacillus.The test lasted for 6 weeks.At the end of the experiment,the blood of laying hens in each group were collected to measure the total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity;The visceral tissues and eggs of laying hens in each group were collected to calculate the organ coefficient,and the contents of Cd,Ca,Fe,Cu,Zn,Mn and Se in the tissues and eggs were measured.The results showed that compared with control group,Cd and cadmium-tolerant Lactobacillus groups significantly increased the total antioxidant capacity (P<0.05).Cd reduced spleen index to the lowest,which was significantly lower than spleen index of Cd with low dose or high dose cadmium-tolerant Lactobacillus (P<0.05),and compared with control group,cadmium-tolerant Lactobacillus significantly increased heart index (P<0.05).Cd residue in liver and kidney of laying hens was much higher than that in other tissues.Adding cadmium-tolerant Lactobacillus significantly reduced Cd residue in heart (P<0.05).Compared with control group,Cd significantly reduced the content of Ca in kidney,and Mn in spleen and yolk (P<0.05).Low dose cadmium-tolerant Lactobacillus significantly increased the Mn content in renal,Fe content in yolk and Cu content in kidney and spleen (P<0.05) compared with control group.The contents of Fe,Zn,Mn and Se in eggs were significantly increased by adding cadmium-tolerant Lactobacillus (P<0.05).The results indicated that adding 1×1010 CFU/kg of cadmium-tolerant Lactobacillus to the diets of laying hens could significantly improve the total antioxidant capacity of serum and the heart index of laying hens,significantly ameliorate Cd-induced spleen atrophy and reduce the Cd residue in the hearts of laying hens after Cd exposure,significantly increased the contents of Mn,Fe and Cu in kidney,spleen and yolk respectively,and significantly increased the contents of Fe,Zn,Mn and Se in eggs.
Genetics and Breeding
Genetic Diversity and Evolutionary Analysis of mtDNA D-loop Region in Jianchang Horse
ZHOU Fanli, HUANG Weiping, JIN Suyu, YANG Shizhong, HUANG Lin, RAO Kaiqing, ZHENG Yucai
2020, 47(1):  63-73.  doi:10.16431/j.cnki.1671-7236.2020.01.008
Abstract ( 223 )   PDF (1288KB) ( 56 )  
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In order to study the maternal genetic diversity and phylogenetic evolution of Jianchang horse using mitochondrial DNA (mtDNA),the genomic DNA was extracted from the blood of Jianchang horses (n=39),and the genetic diversity of mtDNA D-loop was assayed by PCR and direct sequencing.A 247 bp mtDNA D-loop hypervariable sequence was analyzed,the haplotypes and mutation sites of this region were assayed,and the haplotype diversity (Hd),nucleotide diversity (Pi) and average number of nucleotide differences (K) were calculated.The Neighbour-Joining (NJ) phylogenetic tree of 19 horse breeds including Jianchang horse was constructed,and the genetic distances among the breeds were calculated.The experiment obtained the PCR products with good resolution,and about 1 200 bp sequences were obtained by direct sequencing.The AT base content of the 247 bp mtDNA D-loop sequences from 39 samples (one of which lacked 1 bp) was 61.45%,with a characteristic of AT base pair enrichment region.26 unique haplotypes with 33 polymorphic sites were detected,and 4 of which were shared haplotypes,Hap7 and Hap1 were the dominant haplotypes.The Hd, Pi and K were 0.947,0.02399 and 5.901,respectively,indicating abundant maternal genetic diversity;The NJ phylogenetic tree demonstrated the presence of 6 major lineages (A,C,D,E,F and G),and about 50% were distributed in the A branch,indicating the complicated maternal origins of Jianchang horse.The genetic distance were the minimum (0.021) between Jianchang and Guanzhong horses,followed by Sanhe,Wenshan and Cheju horses (0.024),and the genetic distance with Jeju native horse was the maximum (0.032).In conclusion,the present study demonstrated that the mtDNA D-loop hypervariable region was rich in genetic diversity in Jianchang horse,showing multiple maternal origins,and the A branch had obvious advantages,which suggested that Jianchang,Guanzhong and Wenshan horses might have a common maternal origin.
Genomic Regions Associated with Growth Traits in Nanyang Cattle Using Genome-wide Association Analysis
LYU Shijie, CHEN Fuying, ZHANG Zijing, SHI Qiaoting, XIN Xiaoling, CHU Qiuxia, LI Wenjun, BAI Zhonglin, WANG Eryao, XU Zhaoxue
2020, 47(1):  74-82.  doi:10.16431/j.cnki.1671-7236.2020.01.009
Abstract ( 253 )   PDF (3204KB) ( 102 )  
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The aim of this study was to screen and identify the genomic regions and candidate genes related to growth traits of Nanyang cattle,so as to better understand the genetic mechanism of growth traits.Blood samples of 71 Nanyang cattle were collected and used to extract genomic DNA.The specific-locus amplified fragment sequencing (SLAF-seq) technology was used to obtain genome-wide SNP markers and classify the genotypes of experimental individuals.A genome-wide association analysis (GWAS) was carried out for several growth traits consisting of body weight (BW),body height (BH),body length (BL),chest girth (CG),ischium width (IW) and body weight gain (BWG).BW,BH,BL,CG and IW of every individual were measured in every six months from birth to 36 months of age.BWG was calculated as the difference between subsequent body weight measurements with 6-month interval.Gene annotation was performed for the genes within the identified genomic regions to prioritize candidate genes.After filtering,141 755 SNPs remained for the GWAS.5 genomic regions (LOD ≥ 6.35) significantly associated with BW at 12 months of age(chromosome 8:17 320 634-17 347 720 bp),CG at 12 months of age (chromosome2:15 063 190-15 155 309 bp),BL at 24 months of age (chromosome 11:60 727 342-81 425 987 bp),IW at 36 months of age (chromosome 14:15 635 762-15 643 272 bp) and BWG between 12 and 18 months of age (chromosome 26:40 456 192-40 456 477 bp) were identified,respectively.8 genes (BMP10,IFT172,SDC1,TCF23,TRIM54,RAB1A,VPS54 and GDF7) on chromosome 11 were screened out by functional annotation of 186 genes in 5 genomic regions,which were related to bone growth,muscle development and growth regulation.It was suggested that they could be used as candidate genes for growth traits of cattle for further verification.
Effect of EGF on the Proliferation of Primary Cells of Sheep Endometrial Epithelium
WANG Lijuan, CHEN Chaolei, SU Yunze, LI Boyu, WANG Xiangguo, SHENG Xihui, GUO Yong, NI Hemin
2020, 47(1):  83-89.  doi:10.16431/j.cnki.1671-7236.2020.01.010
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In order to study the interaction mechanism between maternal and conceptus of sheep,an in vitro implantation model was established by establishing and improving the in vitro culture technology of primary sheep endometrial epithelial cells.Endometrial epithelial cells were isolated and cultured by tissue block method.The growth of endometrial epithelial cells was observed,and the effects of different EGF concentrations on the proliferation of endometrial epithelial cells were compared.The results showed that after 1-2 days of tissue culture,the endometrial epithelial cells migrated from the peri-tissue,and it took about 9-12 days to grow the 60 mm culture dish.After purification by differential digestion,the purity of the endometrial epithelial cells of F1 generation sheep could reach more than 90%,which indicated that the obtained cells could be used in subsequent experiments.F2 cells were cultured at different EGF concentrations (0,12.5,25,50,75 and 100 ng/mL) for 144 and 168 hours.The D450 nm values were extremely significant higher in 12.5,25 and 100 ng/mL EGF treatment group (P<0.01),and significantly higher in 50 ng/mL EGF treatment group (P<0.05) compared with control group at 144 h.At 168 h,the D450 nm values were significantly higher in 12.5 ng/mL EGF treatment group than those in control group (P<0.05).Therefore,at 12.5 ng/mL EGF concentration,the effect was the best.At 0 and 12.5 ng/mL EGF concentrations,F3 cells were cultured for different time (24,48,72,96,120,144,168,192 and 216 h).The D450 nm values were significantly different at 144 h (P<0.05),and extremely significant at 168 h (P<0.01).Consequently,the culture medium with 12.5 ng/mL EGF would obtain the endometrial epithelial cells more efficiently and rapidly.
Expression and Bioinformatics Analysis of Mastitis Resistance Related Genes in Xinjiang Brown Cattle
JIANG Hui, LIU Pan, LI Yuanyuan, LIU Kuiguo, HUANG Xixia, LIU Jiangwei, XU Lei, DU Jianwen, SANG Zhagen, YANG Xuemei, ZHAO Fanfan, WANG Dan
2020, 47(1):  90-97.  doi:10.16431/j.cnki.1671-7236.2020.01.011
Abstract ( 207 )   PDF (1258KB) ( 41 )  
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The aim of this study was to analyze the molecular biological functions of mastitis resistance related genes in Xinjiang Brown cattle,and lay a theoretical foundation for further development of mastitis resistance traits in Xinjiang Brown cattle by molecular marker-assisted breeding.Functional GO enrichment analysis and KEGG signaling pathway analysis were performed by DAVID 6.8 software for the genes related to mastitis resistance reported (20 genes in total) in previous studies and related literature.Using String 10.0 database for protein interaction network analysis,combined with bioinformatics analysis results and related literature research results,JAK2 and STAT5A genes were selected.Real-time PCR was used to detect the expression of JAK2 and STAT5A genes in blood of Xinjiang Brown cattle with different breast health.GO enrichment analysis showed that 20 genes mainly involved biological processes such as cell membrane and immune response regulation defense reaction;KEGG analysis found that genes were mainly involved in JAK-STAT signaling pathway,chemokine signaling pathway,ErbB signaling pathway,etc;PPI analysis showed that there were direct or indirect links between 8 genes in the 20 genes.Fluorescence quantitative test showed that the expression of JAK2 and STAT5A genes in blood of healthy Xinjiang Brown cattle was extremely significantly or significantly higher than that of mastitis Xinjiang Brown cattle(P<0.01;P<0.05).The JAK2 and STAT5A genes were screened by bioinformatics analysis.Furthermore,the intrinsic relationship between Xinjiang Brown cattle with different breast health and JAK2 and STAT5A gene expression was discussed at the molecular level.
Research Advance on Application of Chip Technology in Livestock and Poultry Breeding
MA Lina, LIU Yongjin, WANG Jin, ZHAO Zhengwei, MA Qing
2020, 47(1):  98-106.  doi:10.16431/j.cnki.1671-7236.2020.01.012
Abstract ( 365 )   PDF (1047KB) ( 149 )  
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There are abundant resources of livestock and poultry breeds in China,and there are many good trait genes.Unfortunately,these fine trait genes have not been fully utilized.Therefore,the development and utilization of genetics and resources at the genetic level is an important direction for improving economic traits of domestic animal genetics resources.The traditional pedigree selection method played a vital role in breeding while it also has shortcomings such as low accuracy and long breeding cycle.With the rapid development of molecular biology,advanced genome sequencing and gene typing technology have pushed the innovation of livestock and poultry breeding methods.The efficiency of genetic detection has been dramatically improved from the low-throughput,time-consuming restriction fragment polymorphism (RFLP) markers in the past to the single nucleotide polymorphism (SNP) with high-throughput and high density.Genotyping based on chip was widely used in research and breeding practice,becoming a new technology and hotspot in livestock and poultry breeding.In this review,the research and application of SNP chip technology in livestock and poultry breeding,its technical advantages,existing problems,challenges and application prospects are summarized.The paper showed that gene chip technology would become an important basic technology in molecular breeding of livestock and poultry,and play an important role in the rapid development of livestock and poultry breeding industry,promoting the genetics progress of livestock and poultry breeding in China,and enhancing the scientific and technological competiveness of livestock and poultry breeding industry in China.
Polymorphism of STAT3 Gene Exon 16 and Its Association Analysis with Meat Quality and Gene Expression in Jiangkou Luobo Pigs
ZHANG Ji, WU Xue, QIU Ganyuan, LIU Pengcheng, YANG Maolin, LIU Ruoyu
2020, 47(1):  107-115.  doi:10.16431/j.cnki.1671-7236.2020.01.013
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In order to study the relationship between signal transducers and activators of transcription 3 (STAT3) gene and meat quality traits in Jiangkou Luobo pigs,11 main meat quality indexes were determined in 105 Jiangkou Luobo pigs,and SNP locus were screened using mixed DNA pool sequencing.Direct sequencing and genotyping of individual samples were carried,the expression of STAT3 gene in longissimus dorsi muscle of individuals with different genotypes was detected.The correlation between SNP and meat quality and gene expression were analyzed by biostatistical software.The results showed that 1 SNP locus (Exon16-C+78T) was detected in exon 16 of STAT3 gene.Only CT and TT genotypes were found by single sample sequencing.Population genetic parameters analysis result showed that T was the dominant allele,and the polymorphism information content showed moderate polymorphism.χ2 test showed that Exon16-C+78T significantly deviated from Hardy-Weinberg equilibrium.The correlation analysis between genotypes and meat quality traits showed that the marbling score and cooked meat percentage of Jiangkou Luobo pigs with TT genotype were significantly higher than that of CT genotype (P<0.05),and the shearing force of TT genotype was significantly lower than that of CT genotype (P<0.05).The expression of STAT3 gene in longissimus dorsi muscle of Jiangkou Luobo pigs with TT genotype was significantly higher than that of CT genotype (P<0.05).The increase of STAT3 gene expression in Jiangkou Luobo pigs might have a positive effect on improving meat quality,TT genotype of STAT3 gene had higher expression level than CT genotype,and the meat quality traits were better.The results indicated that STAT3 gene could be used as a candidate gene for breeding of Jiangkou Luobo pigs,and Exon16-C+78T might be an important SNP locus affecting pork quality traits.
Effect of miR-93 on GH Secretion in Pituitary Cells of Yanbian Yellow Cattle
LOU Angang, CUI Changyan, XIANG Siyu, JIN Taihua, CHEN Yingying, ZHANG Rui, GUAN Lizeng
2020, 47(1):  116-123.  doi:10.16431/j.cnki.1671-7236.2020.01.014
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In order to analyze the effect of miRNAs on growth hormone (GH) secretion in pituitary cells of Yanbian Yellow cattle,the differently expressed miR-93 in the blood of Yanbian Yellow cattle and Hanyan cattle was selected to analyze the effects of miR-93 on GH secretion in pituitary cells of Yanbian Yellow cattle.The primary culture of Yanbian Yellow cattle pituitary cells was carried out,and mimics (miR-93-mi group),mimics reference substance (NC control group),inhibitor (miR-93-in group) and inhibitor reference substance (iNC group) of miR-93 were transfected into the established pituitary primary cells.After 48 h,the cells were collected and the total RNA and protein were extracted.The target genes of miR-93 were predicted by targetscan and RNA hybrid analysis software,and the target relationship of miR-93 was validated by double luciferase reporter gene system.The GH mRNA transcription and protein expression were detected by Real-time PCR and Western blotting,respectively.The results showed that miR-93 targeted 3'UTR of growth hormone releasing hormone receptor (GHRHR);The transcription and protein expression of GH in pituitary cells of Yanbian Yellow cattle in miR-93-mi group were extremely significantly lower than those of NC control group (P<0.01);The mRNA transcription and protein expression of GHRHR in miR-93-mi group were extremely significantly lower than those of NC control group (P<0.01),while the protein expression of GHRHR in miR-93-in group were significantly higher than those of iNC group (P<0.05).The above results demonstrated that miR-93 could regulate GH secretion in pituitary cells by regulating the expression of GHRHR gene and further regulated the growth and development of Yanbian Yellow cattle.
Effects of Resveratrol on Plasma Membrane,DNA Integrity, and Thermal Tolerance of Frozen Goat Sperm
LYU Chunrong, Allai Larbi, LI Junxi, HONG Qionghua, QUAN Guobo
2020, 47(1):  124-130.  doi:10.16431/j.cnki.1671-7236.2020.01.015
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The aim of this study was to investigate the effects of resveratrol on plasma membrane,DNA integrity and thermal tolerance of frozen goat sperm.Semen was collected from 8 Yunshang Black goats using artificial vagina.Semen was diluted with the Optidyl extender supplemented with resveratrol at 0,0.1,1,10 and 20 μmol/L.Then the diluted semen was floaded into IMV plastic straw.The semen frozen without resveratrol was used as control group.After equilibration at 5 ℃ for 4 h,the straws were pre-frozen in liquid nitrogen vapor for 10 min,followed by storage in liquid nitrogen for 30 d.After thawing in 37 ℃ water bath,the plasma membrane integrity and temperature tolerance were measured by low osmolality tolerance test and sperm chromatin diffusion method was used to detect DNA fragmentation rate.The results showed that the rate of sperm with curled tail in 10 μmol/L resveratrol freezing group was significantly higher than that in other groups (P<0.05),but there was no significant difference in other groups (P>0.05).The DNA fragmentation rate of 10 μmol/L resveratrol freezing group was extremely significantly higher than that of fresh sperm (P<0.01),and was extremely significantly lower than that in non-resveratrol control group (P<0.01).The results of thermal tolerance test showed that the sperm tail-bending rate was the highest when sperm frozen with 10 μmol/L resveratrol were equilibrated in 37 ℃ water bath for 1 h,and there was extremely significant difference between 10 μmol/L resveratrol freezing group and the other freezing groups (P<0.01).The rate of sperm with curled tail decreased gradually with prolonging of the incubation time.After incubation for 4 h,there was no significant difference in the rate of sperm with curled tail between 10 μmol/L resveratrol freezing group and control group (P>0.05).The rate of sperm with intact plasma membrane in 10 μmol/L resveratrol freezing group was significantly higher than that in control group (P<0.05).In conclusion,resveratrol could improve the plasma membrane status,DNA integrity and thermal tolerance of frozen goat semen.The optimally functional concentration of resveratrol was 10 μmol/L.However,whether resveratrol could improve the effect of artificial insemination of frozen goat semen remains to be studied.
Expression of Porcine DHRS4 Gene in Skeletal Muscle and Its Association with Growth Traits
ZHANG Leixia, YANG Yanzhao, LI Wentong, LIU Ying, ZHOU Rong, YANG Yalan, LI Kui
2020, 47(1):  131-139.  doi:10.16431/j.cnki.1671-7236.2020.01.016
Abstract ( 270 )   PDF (1136KB) ( 47 )  
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In order to investigate the expression of porcine DHRS4 gene in skeletal muscle and its association with growth traits,this study analyzed the expression of DHRS4 gene in different types of skeletal muscles in Landrace pigs at 30,180 and 300 d.The single nucleotide polymorphism (SNP) of DHRS4 gene in Lansi White pig resource population were identified and genotyped by the Sequenom genotyping platform.The association of the SNP in DHRS4 gene was analyzed with growth traits,including feed gain ratio,average daily gain and average backfat thickness.The results showed that DHRS4 gene was expressed in breast,leg and dorsal muscles of Landrace pigs at 30,180 and 300 d.In breast and leg muscles,the expression of DHRS4 was significantly or extremely significantly higher at 30 d than that at 180 and 300 d (P<0.05;P<0.01),but there was no significant difference of dorsal muscles at different developmental stages (P>0.05).In addition,this study found 3 SNPs of DHRS4 gene in Lansi White pig resource population:rs334250151,rs342446613 and rs326982309.The association analysis results showed that rs334250151 loci was significantly associated with feed gain ratio,and the feed gain ratio of AA genotype individuals was significantly lower than that of TT genotype individuals (P<0.05).This results reveals that DHRS4 gene might be involved in porcine skeletal muscle development,and rs334250151 loci could be used as a new molecular marker for genetic improvement of growth traits in pig.
Research Progress on Application of CRISPR/Cas9 System in Poultry
ZHANG Lei, ZHANG Haibo, ZHANG Jingqi, ZHANG Yang, CHANG Guobin, CHEN Guohong, WANG Jian
2020, 47(1):  140-147.  doi:10.16431/j.cnki.1671-7236.2020.01.017
Abstract ( 242 )   PDF (864KB) ( 143 )  
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The CRISPR/Cas system is an adaptive immune system of bacteria and archaebacteria,which includes clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas).Based on CRISPR/Cas9 (CRISPR-associated protein 9,Cas9) system,sgRNA (single guide RNA) could be designed and genome editing could be operated in specific location.As agricultural economic animals,poultry could produce eggs and meat quickly and efficiently.It's an important source of protein,and could be used as the valuable biological model for vertebrate development.However,as the oviparous animals,the fertilized eggs of poultry are closely bound to the surface of the yolk,and the unfertilized eggs are difficult to extract.So it's very hard to operate the specific genetic modification on poultry eggs,which prevents the research progress of functional genes and transgenic poultry.CRISPR/Cas9 system has the advantages of high targeting,easy operation and with wide range of application,and has greatly promoted the research progress on poultry and soon become one of the most effective tools in poultry science.This paper reviews the progress of CRISPR/Cas9 research on poultry cells,growth and development,gonadal differentiation,diseases,embryo editing and transgenic poultry preparation,which would provide theoretical basis and practical reference for the future research and application of CRISPR/Cas in poultry.
Effects of Astaxanthin on Mouse Implantation Embryos Development and Gene Expression
ALAFAT·Amulati, ZHAO Xi, NURIBIYAMU·Maimaitituoheti, AERMAN·Haire, LIU GuoShi, ABULIZI·Wusiman
2020, 47(1):  148-155.  doi:10.16431/j.cnki.1671-7236.2020.01.018
Abstract ( 309 )   PDF (2633KB) ( 66 )  
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In present study,the effects of astaxanthin (AX) supplement in in vitro culture (IVC) medium on mouse embryo development and gene expression were determined.The female ICR strain mice (body weight 23-25 g) were super ovulated.The oocytes obtained from mouse oviduct were fertilized in vitro (IVF).The zygotes were divided randomly into 5 media suppled with different concentration of astaxanthin (0,5,10,25 and 50 μmol/L) for culture in vitro.Cultured for 24,96 h,the cleavage rate and blastocyst rate were analyzed.At 96 h,the cell number of blastocysts in each group was extracted.The total RNA of blastocysts in each group was abstracted,and relative expression amounts of TGF-β and Bcl-2 genes were determined by Real-time PCR.The results indicated that 10 μmol/L astaxanthin supplement could improve the in vitro development environment of mouse embryos and improve significantly embryo cleavage rate and blastocyst rate (P<0.05),increase cell number of blastocyst significantly (P<0.05),and increase expression amount of TGF-β gene extremely significantly (P<0.01).TGF-β and Bcl-2 genes were likely to be involved in the regulation of embryonic development.These results demonstrated that supplement of 10 μmol/L astaxanthin was beneficial to mouse embryo development in vitro,and could improve the developmental environment of mouse embryo.
Research Progress on Genomic Information for Livestock Improvement
ZHOU Xirong, YU Lijuan, ZHANG Yuhua, MAERZIYA·Yasen, JIANG Xiaomei, LAZHATI·Ainiwaer, ZHANG Yanhua
2020, 47(1):  156-163.  doi:10.16431/j.cnki.1671-7236.2020.01.019
Abstract ( 266 )   PDF (861KB) ( 54 )  
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In order to meet the rapid growth of demand,we must speed up the development of livestock and poultry industry and minimize the impact on the environment.Improving the genetic characteristics of livestock and poultry is expected to promote the solution of this problem.Since the 21st century,the molecular breeding technology with genome selection as the core has provided opportunities.Early and accurate selection can be realized using this technology,which can greatly shorten the generation interval,accelerate the progress of population genetics,and significantly reduce the breeding cost.Although in some breeds,such as dairy cows,genome selection has been successful,and the population has also made great genetic progress,but still can not meet the rapid growth needs.Therefore,it is necessary to find ways to further accelerate genetic progress.The results showed that adding the known functional gene information of target traits to SNP marker data could improve the accuracy of gene breeding value prediction and accelerate the genetic progress.While mining more genomic information,developing more optimized analysis methods can be more conducive to the realization of the goal.This paper summarizes the available genomic data of major livestock and poultry species,including cattle,sheep,goats,pigs and chickens,and how these data can help identify genetic markers and genes that affect important traits,so as to further improve the accuracy of genomic selection.
Preventive Veterinary Medicine
Genetic Variation Analysis of gE and gB Genes of Pseudorabies Virus in Jiangxi Province During 2013 to 2018
GAO Yingxue, JIANG Xinhua, ZHOU Hetian, LUO Feng, YANG Danfeng, WAN Wenzhong, DENG Shunzhou
2020, 47(1):  164-173.  doi:10.16431/j.cnki.1671-7236.2020.01.020
Abstract ( 273 )   PDF (2465KB) ( 51 )  
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In order to grasp the molecular epidemiology and genetic variation of pseudorabies virus (PRV) in Jiangxi province,this study used the established PCR method in the laboratory to amplify gE and gB genes of the collected PRV positive tissue samples from Nanchang,Yichun,Ganzhou,Ji'an,Jiujiang,Shangrao,Fuzhou and Xinyu in Jiangxi province during 2013 to 2018,and the PCR products were sequenced and analyzed.The results showed that a total of 64 PRV gE gene sequences and 12 PRV gB gene sequences were obtained.The phylogenetic tree of gE gene showed that 64 strains of PRV belonged to one large branch,and they were closely related to Asian strains and domestic isolates in recent years,all of which were GⅠ type,but had far-reaching relationship with GⅡ type European and American classic strains;The nucleotide and amino acid homology bewteen Jiangxi strain gE gene and 19 reference strains were 97.2% to 100% and 94.6% to 100%,respectively;The nucleotide and amino acid homologies between gB gene and 11 reference strains were 98.2% to 100% and 96.5% to 100%,respectively;There were base deletions,insertion and site mutations exist in gB gene of Jiangxi strain compared with Bartha-K61 vaccine strain.This study confirmed the prevalence and variation of PRV in Jiangxi province from the perspective of molecular epidemiology,and provided theoretical basis for scientific prevention and control of PRV in Jiangxi province.
Epidemiological Investigation of Major Porcine Viral Diseases in Guangxi During 2018
SHI Kaichuang, YIN Yanwen, WANG Xiaode, XIE Shouyu, SHI Yongsheng, LIU Hongmei, LU Wenjun, QU Sujie, FENG Shuping, SU Yanqiong
2020, 47(1):  174-181.  doi:10.16431/j.cnki.1671-7236.2020.01.021
Abstract ( 243 )   PDF (751KB) ( 81 )  
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To investigate the epidemiology of major porcine viral diseases in Guangxi during 2018,clinical tissue samples were detected by multiplex Real-time RT-PCR for classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV),multiplex Real-time PCR for porcine pseudorabies virus (PRV),porcine circovirus (PCV) type 1 (PCV1),PCV2 and PCV3.Clinical diarrhea samples were detected by multiplex RT-PCR for porcine epidemic diarrhea virus (PEDV),porcine deltacoronavirus (PDCoV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV).The results showed that of 694 tissue samples detected,the positive rates of CSFV,PRRSV,HP-PRRSV,PRV,PCV1,PCV2,PCV3 were 11.10%,18.88%,7.20%,5.19%,2.45%,67.00% and 5.76%,respectively,of which co-infection rates of two pathogens and three pathogens were 41.21% and 4.32% respectively,and PRRSV and PCV2 co-infection rate was the highest.792 diarrhea samples (intestinal contents and rectal swabs) were detected,the positive rates of PEDV,PDCoV,TGEV and PRoV were 9.72%,5.81%,1.77% and 6.31%,respectively,of which co-infection rate of two pathogens was 5.30%,and PEDV and PRoV co-infection rate was the highest.The results indicated that several major viral diseases were still widespread distribution in swine herds from Guangxi,and mixed infections were common.It should be further imposed for epidemic surveillance,prevention and control of these diseases in the future.
Isolation,Identification and ERIC-PCR Genotyping of E.coli O157:H7 from Xinjiang Cattle
SU Zhanqiang, ZHANG Ling, LIU Luyao, WANG Dong, MA Xiaoyu, AIKEDAI·TULUHONG, ZHANG Yi, TONG Panpan
2020, 47(1):  182-189.  doi:10.16431/j.cnki.1671-7236.2020.01.022
Abstract ( 187 )   PDF (4267KB) ( 34 )  
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In order to understand the pollution status and genetic diversity of bovine Escherichia coli O157:H7 in Xinjiang,to explore the genetic relationship of isolates from different areas,and to provide experimental basis for controlling the transmission of bovine E.coli O157:H7 in cattle,in this experiment,the samples were added to EC broth (37 ℃,180 r/min),then inoculated on SMAC plate and cultured overnight in 37 ℃ incubator for about 18 h.Then,white or colorless single colonies on SMAC were inoculated the MUG medium,and cultured at 37 ℃ for 18 h.The non-fluorescent samples were inoculated on the SMAC plate,and cultured at 37 ℃ for about 18 h.The white or colorless single colonies were selected the next day for PCR identification.The positive strains with rfbE and fliC gene bands were identified.ERIC-PCR fingerprinting was used to analyze the homology among the positive strains.ERIC-PCR results showed that there were 3 groups of strains with 100% similarity.The strains isolated from the Yili region were the most diverse,with 6 types,followed by Urumqi with 4 types.The most diverse source of strains was in cluster D.It could be traced by ERIC-PCR typing.ERIC-PCR was able to distinguish the isolates of specific sampling points or species.It could demonstrate that there were some similar ERIC characteristics among strains from different sources and they were clustered in the same cluster.The E.coli O157:H7 strain selected in this study had extensive genetic diversity,which was very sensitive to detect bacterial differences among different species.Therefore,ERIC-PCR could be used as an effective tool for routine monitoring and identification of E.coli O157:H7.
Application of Bcteriophage to Prevent and Control Bacterial Infection in Livestock and Poultry
WEI Bingdong, CONG Cong, LI Shuying, LI Jibin, YU Wei, YANG Huan, XU Yongping, WANG Xin
2020, 47(1):  190-200.  doi:10.16431/j.cnki.1671-7236.2020.01.023
Abstract ( 255 )   PDF (1802KB) ( 76 )  
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In recent years,it has resulted in significant misuse and overuse of antibiotics contributing to the emergence of antibiotic resistant,drug residue and food safty.Especially,the emergence of multi-resistant and pan resistant bacteria has threated the human health.Therefore,it is necessary to find a safe and effective antibiotic substitute to ensure animal-derived food,improve animal health and promote social development.Bacteriophage are defined as viruses that infect bacteria,fungus and actinomycetes.Compared with traditional antibiotics,bacteriophage has many advantages for therapeutic purpose,including existence wildly,high specificity,safety,low cost,and become the weapon with human fighting bacterial infections.The early history of phage therapy has been controlled by bacterial infection.However,the emergence of antibiotic and chemical antibacterial agent,phage research and therapy was largted to medical history in the western countries.Until 1980s,the emergence of multidrug resisitant bacteria has led researcher to re-consider phage therapy.With the development of modern biotechnology,new investigations were focused on the use of phage therapy in the treatment of human infections as well as in crop,veterinary and food safety.The authors reviewed the biological characteristics of phage,advantages and disadvantages of phage therapy,prevention E.coil,Salmonella and Campylobacter infections in livestock and poultry,and provided a theoretical reference for the further applications in animal production.
Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody
KONG Zimeng, JIANG Bo, CAI Yunhong, HE Houjun, LI Yongqing, JIN Huan
2020, 47(1):  201-208.  doi:10.16431/j.cnki.1671-7236.2020.01.024
Abstract ( 200 )   PDF (2482KB) ( 44 )  
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In order to identify chicken complement receptor 2 (ChCR2) gene,we designed specific primers and amplified ChCR2 gene by RT-PCR.The prokaryotic expression vectors pGEX-6P-1-ChCR2-△TM and pET-32a-ChCR2-△TM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM were constructed by homologous arm ligation.The two prokaryotic plasmids were transferred into E.coli expression system to express GST-ChCR2-△TM and His-ChCR2-△TM proteins.The polyclonal antibody was prepared by immunizing BALB/c mice with GST-ChCR2-△TM protein.The antibody titer was detected by ELISA.Indirect immunofluorescence test and Western blotting were used to identify the specific binding of antigen and antibody.The results showed that a ChCR2 gene with a size of 1 113 bp was amplified.The prokaryotic expression vectors pGEX-6P-1-ChCR2-ΔTM and pET-32a-ChCR2-ΔTM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM with the transmembrane region removed were successfully constructed.The GST-ChCR2-△TM protein was soluble at low temperature (16 ℃),while His-ChCR2-△TM protein was in the form of inclusion body.The serum titer of BALB/c mice immunized with GST-ChCR2-△TM protein was 1:512 000 detected by ELISA.The results of indirect immunofluorescence assay and Western blotting showed that the polyclonal antibody against GST-ChCR2-△TM protein could specifically bind to ChCR2 protein.The results of this study provided a reference for further identification of ChCR2 gene and related immunological research of chicken and even poultry.
Sequencing and Genetic Variation Analysis of the Whole Gene of Epidemic Strain of PEDV in Pengzhou,Sichuan Province
XIANG Meng, ZHANG Huanrong, REN Yupeng, ZHANG Keke
2020, 47(1):  209-220.  doi:10.16431/j.cnki.1671-7236.2020.01.025
Abstract ( 203 )   PDF (3215KB) ( 63 )  
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In order to study the genomic characteristics and genetic variation of porcine epidemic diarrhea virus (PEDV) strains infected by a pig farm immunized with PEDV vaccine in Pengzhou,Sichuan province,RT-PCR was used to screen the positive samples of PEDV,named SCXM strain.The whole genome sequence was cloned and sequenced to obtain the whole genome sequence,and the main structural genes were analyzed for genetic variation and phylogenetic analysis.The results showed that the genetic variation of SCXM strain was mainly concentrated in S gene,the homologies were 90.4%-99.2% compared with 71 strains and 93.8% compared with CV777.Compared with common vaccine strains in China,amino acid mutations were found in 5 linear epitopes (P1,S1P2,S1P3,SS5 and SS6) and 1 neutralizing epitope (SID).The analysis of genetic evolution showed that SCXM strain belonged to the G2b subtype of PEDV,which was the same branch with HBXY3 strain in Hubei province and Vietnam strain.In addition,the analysis of ORF3,M and N genes showed that there were a certain number of amino acid mutations in SCXM compared with the vaccine strain,but the degree of variation was relatively small,and did not cause the change of antigenicity prediction value.The results showed that PEDV SCXM strain was a new variant strain,and its S gene was mutated to a large extent.The mutation and antigenicity change of multiple amino acids in S gene might be one of the reasons for the failure of vaccine immunization in pig farms.This study enriched the genetic data of PEDV,discussed the genetic variation and evolution characteristics of PEDV of SCXM strain,and laid a foundation for the molecular evolution of PEDV.
Identification and Complete Genomic Analysis of a Newcastle Disease Virus from a Laying Hen
LI Jiajia, SONG Jianling
2020, 47(1):  221-228.  doi:10.16431/j.cnki.1671-7236.2020.01.026
Abstract ( 213 )   PDF (1237KB) ( 138 )  
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In order to study the molecular epidemic characteristics and pathogenicity of Newcastle disease virus (NDV) in Yunnan province,the pathogen was isolated from the tissue samples of an laying hens farm in Yunnan province.The isolates were proved to be NDV by hemagglutination,hemagglutination inhibition test and RT-PCR detection.The virulence of the virus was determined by the minimal lethal dose of (MLD),50% egg infectious does of (EID50),the mean death time of (MDT),the pathigenicity index of (ICPI),and the pathogenicity index (IVPI) of intravenous inoculation.The results showed that the EID50 was 10-4.4,MLD was 10-4,MDT was 97.5 h,ICPI was 0 and ICPI was 0,which showed that the isolates were attenuated strains.High throughput sequencing and genetic evolution analysis showed that the genomic length of the isolated strain was 15 198 bp,and there were 6 structural genes from 3'end to 5'end,which were 1-48 nucleotides interval between NP-P-M-F-HN-L genes.The genetic evolution analysis of the whole genome,F gene and HN gene showed that the isolated strain belonged to Class Ⅰ branch.The full length ORF of F gene was 1 662 bp,encoding 553 amino acids,and the amino acid sequence of F protein cracking site was 112-ERQERL-117,which consistent with the characteristics of NDV weak virus cleavage site.The ORF of HN gene was 1 851 bp,encoding 616 amino acids.There was no amino acid sequence variation at 234-NRKSCS-239 site in the conservative region of HN gene.There were six glycosylation sites in F protein and six potential glycosylation sites in HN protein,but there were missing and more glycosylation sites compared with LaSota.The cysteine residues of F protein were 76,199,338,347,362,370,394,399,401,424,514,523 amino acids,the neutral antigen sites of F protein were mutated at 72,75,79 and 170 amino acids,the amino acids of HN protein receptor binding site and NA active site were 174R,401E,416R,526Y,no mutation occurred.In this study,the whole gene sequence characteristics of Newcastle disease Class Ⅰ strain in laying hens in Yunnan Province were reported for the first time,which laid a foundation for the study of Newcastle disease virus Class Ⅰ strain.
Basic Veterinary Medicine
Main Pathogenic Bacteria Causing Subclinical Mastitis in Yak and the Distribution of Antibiotic Resistance and Virulence Genes
WANG Fei, WANG Cailian, SONG Shuzhen, LANG Xia
2020, 47(1):  229-239.  doi:10.16431/j.cnki.1671-7236.2020.01.027
Abstract ( 227 )   PDF (1061KB) ( 23 )  
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To investigate the main pathogenic bacteria causing subclinical mastitis (SCM) in yak and the distribution of antibiotic resistance genes and virulence genes,yak milk samples were collected from the summer pasture in Gannan Tibetan Autonomous Prefecture,Gansu.The Lanzhou mastitis test (CMT) was carried out to grade SCM.16S rDNA was used to identify the main pathogenic bacteria isolated from SCM samples,antibiotic resistance was determined by Kirby-Bauer method,and related antibiotic resistance and virulence genes were detected by PCR.The results showed that a total of 324 SCM samples were found,with the detection rate of 14.43%;The main pathogenic bacteria were Staphylococcus,Escherichia and Enterococcus;Staphylococcus were highly resistant to penicillin and tetracycline with the rate of 59.57% and 47.52% respectively,43.40% and 20.75% E.coli were resistant to tetracycline and ampicillin respectively,as well as 25.00% and 16.67% E.faecalis were resistant to tetracycline and erythromycin respectively;12 strains of methicillin-resistant S.aureus (MRSA) were detected in 59 strains of penicillin-resistant S.aureus,inclouding 7 strains of mecA MRSA and 5 strains of mecC MRSA;For tetracycline-resistant S.aureus,efflux pumps genes tetK and tetA had the highest carrying rate (85.45%,56.36%) and ribosomal protection gene tetM had the lowest frequency (34.55%);Among virulence genes,the detection rates of clfA,clfB,fib and coa genes were higher than others with the ratio of 87.64%,84.27%,83.15%,82.02%,respectively.In conclusion,the main pathogenic bacteria causing SCM in yaks were S.aureus and E.coli which were both highly resistant to penicillins and tetracyclines,and the main virulence factors of S.aureus isolates were clumping factors and coagulase.
Isolation,Identification and Detection of Drug Resistance and Drug Resistance Gene of Pulmonary Pathogenic Escherichia coli from Calves
GU Xiaoxiao, WU Qin, TAO Qiaoxiaoci, MA Xue, LI Jie, ZHOU Xia, HAN Mengli, HUANG Xin, WU Tongzhong, ZHANG Xingxing, ZHONG Fagang
2020, 47(1):  240-248.  doi:10.16431/j.cnki.1671-7236.2020.01.028
Abstract ( 231 )   PDF (2178KB) ( 49 )  
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The purpose of this study was to identify the bacterial pathogens that cause the death of calves due to respiratory diseases in a cattle farm in Shihezi,Xinjiang,and to analyze the phylogenetic classification,drug resistance phenotype and drug resistance gene of the isolated pathogens.The routine isolation of bacteria combined with automatic biochemical analysis system was used to isolate and identify the isolates.The paper and PCR methods were used to analyze the systematic grouping,drug sensitive phenotype and drug resistance genes of the isolates.Six strains of pathogenic E.coli were isolated and identified from the lung,liver and lymph nodes of infected calves.All of the six strains were multidrug resistant.The drug resistance rates of penicillin,amoxicillin,cefazolin,cefradine,streptomycin,gentamicin,florfenicol,teicoplanin,clindamycin,meropenem,tetracycline and tobramycin were 100%.The carrying rates of aminoglycoside resistance genes aacC2,β-lactam resistance genes blaCTX-M and blaTEM,tetracycline resistance gene tetA,quinolone resistance gene gyrA,and sulfonamide resistance gene sul2 were 66.67%,83.3%,66.7%,100%,100% and 100%,respectively.E.coli was the bacterial pathogen causing respiratory tract infections in dead calves in a cattle farm in Shihezi,Xinjiang.It showed strong multi-drug resistance,and the isolates carried more abundant drug resistance genes.
Research Progress on Detection of Small Molecule Chemical Residues by Single-chain Antibody
HOU Ren, HUANG Lingli, TAO Yanfei, XIE Changqing, LIU Zhenli, PENG Dapeng
2020, 47(1):  249-257.  doi:10.16431/j.cnki.1671-7236.2020.01.029
Abstract ( 237 )   PDF (1095KB) ( 30 )  
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Excessive residues of small molecular chemical substances such as veterinary drugs,pesticides and biological toxins in food,feed or water cause a certain degree of harm to human and animal health and the balance of ecological environment.Therefore,it is necessary to establish a rapid detection method for the residue of small molecules.Immunoassay based on specific binding of antigen and antibody is a common method for residue detection.The key of this technique is to prepare antibodies that can be used to detect specific antigens.With the development of antibody structure analysis and recombinant DNA technology,single-chain antibody provide a new technique for the detection of small molecule chemical residues.Single-chain antibody has the advantages of small molecular weight,low immunogenicity,strong plasticity,and mass production,and have broad development space.In recent years,the detection of small molecule chemical residues by enzyme-linked immunosorbent assay based on single-chain antibody has become the most commonly used analytical tool.The single-chain antibody screening strategy,expression strategy,mutation strategy and fusion protein with alkaline phosphatase further improve antibody yield,sensitivity,broad spectrum and simplify the immunoassay method,thus promoting single-chain antibody immunoassay method for the detection of small molecule chemical residues.In this paper,recent advances on the application of single-chain antibody-based immunoassay methods in small molecule chemical substances such as veterinary drugs,pesticides and biotoxins are reviewed,aiming at providing reference for detection technology of small molecule chemical residue.
Isolation,Identification and Probiotic Characteristics of a Clostridium butyricum
WANG Qian, LI Haihua, DOU Caixia, LIU Lili, QIAO Jiayun
2020, 47(1):  258-266.  doi:10.16431/j.cnki.1671-7236.2020.01.030
Abstract ( 271 )   PDF (4764KB) ( 75 )  
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This study explored the probiotic characteristics of Clostridium butyricum isolates and aimed to provide a theoretical basis for their application in piglet diets.Clostridium butyricum was isolated by routine methods and cultured in pure culture.The isolates were identified by biochemical detection and 16S rRNA gene sequencing;The viable cell count method and the Oxford cup method were used to study the inhibitory effect of the culture supernatant of the isolate on the three pathogenic bacteria,the adhesion of the isolated pig intestinal epithelial cells (IPEC-J2) and its inhibitory effect on the adherent cells of pathogenic bacteria;The effect of the isolate on the growth of IPEC-J2 was studied by cell counting method;The cytokine level in the culture supernatant was determined by ELISA after treatment of the isolate.The results showed that a strain of Clostridium butyricum was successfully isolated in this study.Compared with the control group,the antibacterial effect of Clostridium butyricum culture supernatant on the three pathogenic bacteria was significant (P<0.05).Clostridium butyricum could adhere to IPEC-J2,and the optimal multiplicity of infection(MOI) was 50,and the optimal treatment time was 3 h;Clostridium butyricum had inhibitory effect on three pathogenic adhesion IPEC-J2 cells (P<0.05).When the MOI of Clostridium butyricum was 1,10 and 50,the cell growth was normal and the morphology was intact.When MOI was 100,the growth of IPEC-J2 was significantly inhibited,and some cell death occurred at the same time.There was no difference in cytokine production levels when MOI was 1,and cytokine production levels were significantly increased at MOI of 10,50,and 100 (P<0.05).In conclusion,a Clostridium butyricum strain was successfully isolated in this study,which has good probiotic characteristics.The results provided a theoretical basis for the application of the isolated strain in production.
Comparative Study on Pharmacokinetics of Long-acting Transdermal Agents of Ivermectin and Common Injection in Rabbits
LIU Yingying, YUE Zhiguang, WU Ningpeng, LIU Zhenzhen, YAN Yaqun, ZHANG Sumei, JIAN Fuchun, ZHANG Longxian, WANG Rongjun, NING Changshen
2020, 47(1):  267-274.  doi:10.16431/j.cnki.1671-7236.2020.01.031
Abstract ( 320 )   PDF (1054KB) ( 27 )  
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To compare the drug metabolism and efficacy time of ivermectin long-acting transdermal preparation and ordinary ivermectin injection,the study prepared the long-acting transdermal preparation with ivermectin content of 0.5%,1.0% and 1.5% respectively.The pharmacokinetics of ivermectin long-acting transdermal preparation and common ivermectin injection (1.0%) in rabbits with different dosage and the same volume were detected by HPLC,and the data were analyzed by PKSolver pharmacokinetic processing software.The results showed that the t1/2ka of 0.5%,1.0%,1.5% ivermectin long-acting transdermal agent and 1.0% injection were 0.81,0.52,1.02 and 0.12 d,respectively;The Tmax were 1.55,0.97,1.62 and 0.42 d,respectively;The Cmax were 47.36,72.02,115.30,99.53 ng/mL,respectively;The t1/2ke were 3.61,5.92,5.59 and 1.79 d;The MRT were 5.27,7.37,5.13 and 2.16 d,respectively;The AUC were 1 488.70,3 081.98,3 161.20 and 480.00 ng·d/mL,respectively.The effective concentration of long-acting transdermal agent of ivermectin was maintained for 35 d in vivo and 9 d in general injection.The effect of long-acting transdermal agents of ivermectin was confirmed,which could be further studied.
Acute Toxicity and Hypoglycemic Activity of Total Flavonoids from Melastoma dodecandrum Lour.
WENG Jingyu, LIANG Liqing, ZHOU Jingkai, LIU Yuting, LI Li
2020, 47(1):  275-281.  doi:10.16431/j.cnki.1671-7236.2020.01.032
Abstract ( 238 )   PDF (922KB) ( 30 )  
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This experiment was conducted to explore the acute toxicity and hypoglycemic activity in diabetic mice of total flavonoids from Melastoma dodecandrum Lour. (MDF).The mice were intragastrically administered with MDF solution (5.6 mg/(g·BW)) with the maximum gastric volume (40 μL/(g·BW)),solution was administered twice for 24 h.The state of mice in 14 d was recorded.At the end of the test,the levels of aspartate aminotransferase (AST),alanine aminotransferase (ALT),creatinine (Cre) and urea nitrogen (BUN) were measured.The organ index was calculated by weighting the heart,liver,spleen,lung and kidney.The diabetic mouse model was established by intraperitoneal injection of 0.160 mg/(g·BW) streptozotocin (STZ).The mouse were divided into blank group,model group,metformin group (0.6 mg/(g·BW)),MDF high dose,medium dose and low dose groups (1.2,0.8,0.53 mg/(g·BW)).After 21 days of continuous administration,fasting blood glucose (FBG),body mass,feed intake,water intake and 24 h urine protein content of each group of mice were measured.The results showed that the maximum daily dose of MDF was 11.2 mg/(g·BW)).Compared with control group,there was no significant difference in body mass,organ index,and serum AST,ALT,Cre and BUN of the mouse after administration (P>0.05).In the hypoglycemic activity test,compared with the model group,the FBG,water and feed intake of MDF-treated group were significantly or extremely significantly reduced (P<0.05;P<0.01).The above results confirmed that the MDF was safe and non-toxic,it could effectively alleviate the symptoms and decrease blood glucose in diabetic mice.
Progress in PK-PD Research on the Combination of Veterinary Antibacterial Drugs
YUAN Yuanyuan, HUANG Lingli, HAO Haihong, LIU Zhenli, XIE Changqing, YUAN Zonghui, PENG Dapeng
2020, 47(1):  282-289.  doi:10.16431/j.cnki.1671-7236.2020.01.033
Abstract ( 256 )   PDF (881KB) ( 70 )  
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With the rapid development of the breeding industry,the application of veterinary antibacterial drugs is more and more extensive.However,due to the unreasonable selection and abuse of antimicrobial drugs,the resistance rate of bacteria to antimicrobial drugs is gradually increasing,and almost all bacteria have acquired resistance genes.At present,bacterial resistance has become a global problem,and clinically,the cause of the disease is complex,often manifested as a variety of pathogens secondary infection and mixed infection,single drug is often difficult to effectively control the disease,seriously endangering the development of aquaculture.The combination of antibiotics can improve the therapeutic effect of drugs,alleviate or reduce adverse reactions,reduce the incidence of bacterial resistance.For cases of mixed infection or unable to carry out bacteriological diagnosis,the combination of antibiotics can also expand the scope of antibacterial.The combination model of PK and PD can effectively integrate the relationship among drugs,organism and pathogenic bacteria,and provide a reasonable medication plan for clinical use.Therefore,by introducing the advantages and problems of the combination of drugs,the classification of PK/PD and the optimization and guidance of PK/PD on the combination of drugs,this paper summarizes the research progress of PK-PD on the combination of veterinary drugs and antibacterial drugs at present,in order to promote the rational use of veterinary clinical antibacterial drugs.
Clinical Veterinary Medicine
Effect of Subclinical Ketosis on Reproductive Performance and Blood Clinical Pathology of Dairy Cows During Early Lactation
ZHANG Feng, BAI Yunlong, Song Yuxi, SUN Shuhan, XIA Cheng, XU Chuang, ZHANG Hongyou
2020, 47(1):  290-295.  doi:10.16431/j.cnki.1671-7236.2020.01.034
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The aim of this study was to explore the relationship between perinatal subclinical ketosis,reproductive performance and follicular development in early lactation,and to detect changes in blood biochemical of the dairy cows.60 cows from a large intensive dairy farm in Heilongjiang province were assigned to subclinical ketosis group (SCK) and control health group (C) according to ketone bodies levels postpartum.According to the estrus condition within 50 d in milk,16 cows and 14 cows of the SCK group was further divided into the estrus group (SCKE) and the anestrus group (SCKA),respectively.The cows in group C was also divided into estrus group (CE,25 cows) and anestrus group (CA,5 cows).All groups of cows were tracked to record reproductive performance data and follicular development status at 50 d postpartum by rectal examination and B-ultrasound.Results showed that the first estrus days of SCKE cows were 10 d longer than the CE cows (P<0.05),and the difference of diameter of follicles was more than 4 mm (P<0.01).The incidence of uterine incompleteness in SCKA group was significantly higher than that in CE group (P<0.05).The plasma insulin-like growth factor-1 (IGF-1) level was significantly decreased (P<0.05) and the milk yield was extremely significantly increased in SCK group (P<0.01).Compared with estrus cows,the content of triglyceride (TG) in plasma of anestrus cows was increased significantly (P<0.05);The contents of glucose (Glu),insulin (Ins),estradiol (E2) and progesterone (P4) decreased significantly or extremely significantly (P<0.05;P<0.01).In conclusion,abnormal energy metabolism and reproductive hormone in cows with subclinical ketosis are the risk factor for reproductive and follicles development disorder postpartum in dairy cows,which will lead to poor fertility in dairy cows.
Products Quality and Safety
Study on the Contents of Trace Elements and the Influencing Factors in Buffalo Milk in Guangxi
YU Xiaonan, LIU Hongyan, GUO xiaoqing, ZHAO Qingyu, LI Fadi, ZHANG Junmin
2020, 47(1):  296-305.  doi:10.16431/j.cnki.1671-7236.2020.01.035
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Buffalo milk was an important milk source in the South of China.In order to study the contents of trace elements in buffalo milk in Guangxi and the influence of different factors such as region,breed,lactation,60 samples of buffalo milk with 2 breeds in 3 lactation periods were collected from 3 buffalo farms in Xingning and Yongning district in Nanning and Lingshan district in Qinzhou,Guangxi province.The samples were digested by microwave digestion method.12 trace elements (Cr,Mn,Co,Cu,As,Se,Cs,Ba,Mo,Pb,Cd and Ni) were analyzed by inductively coupled plasma mass spectrometry method (ICP-MS).Correlation among 12 trace element contents was analyzed using Pearson correlation analysis.Differences in trace element contents between buffalo milk in different regions,breeds and lactation stages were compared by one-way analysis of variance.Multivariate analysis of variance was used to analyze the effect of interaction on trace elemental contents.The results showed that the spiked recovery of 12 trace elements in buffalo milk was 84.94% to 120.91%.The contents of Mn,Se,Cs and Ni in buffalo milk were significantly different among different regions (P<0.05).The contents of Mn and Cs in Lingshan were higher than those in other regions.The contents of Se and Ni in Xingning were higher than those in other region.The contents of Se and Ni in buffalo milk were significantly different between different breeds (P<0.05).The contents of Se and Ni in Nile buffalo were higher than that in Mora buffalo.The contents of Mn,Co,Cu,Se and Mo were significantly different among different lactation periods (P<0.05).The contents of Co,Cu and Se in colostrum were the highest among three lactation periods.The content of Mn in mature milk was higher than that in other lactation periods.The content of Mo in late milk was the highest among three lactation periods.The interaction between region and breed had no significant effect on the contents of trace elements in buffalo milk.
Environmental Safety
Comparative Study on Spring Environmental Indices and Lamb's Health Status of Two Intensive Sheep Farms in South and North of Xinjiang
JIAO Weina, LI Zhuocheng, LIU Hongjia, YANG Huiguo, HE Zonglin, YAO Gang, WANG Jinquan
2020, 47(1):  306-314.  doi:10.16431/j.cnki.1671-7236.2020.01.036
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The purpose of this study was to understand the spring changes of the environment of intensive mutton sheep sheds in Xinjiang and the correlation between the environmental changes and lamb health.Environmental indices including the illumination,temperature,humidity,noise,concentrations of CO2,NH3,CH4,PM10 and TSP were measured consecutively for 15 days in two typical sheep farms which were located in Wensu county (WS) in Southern Xinjiang and Mulei county (ML) in Northern Xinjiang.24 h changes of the above indices were measured in 5 consecutive days,then which were compared between the two sheep farms.At the same time,the clinical observation of lamb health and disease was carried out in two sheep farms.The results showed that the average noise in ML sheep farm was extremely higher than that in WS (P<0.01).The average concentration of NH3,CO2,TSP and PM10 in WS sheep farm was extremely higher than that in ML (P<0.01);24 h change of the above parameters showed that the temperature in the two farms about 15,24 h in one day was over the limit of NY/T 388-1999,respectively.The main diseases of lambs in the spring were diarrhea,pneumonia and paralysis,and the cough rate of the lambs in WS was higher than that in ML (P<0.05).The paralytic rate of lambs in ML farm was higher than that in WS (P<0.05).In conclusion,the environmental indices in two sheep farms were not exceeded to the limit of NY/T 388-1999 except the temperature.The results showed that the environment of two sheep farms in spring was more suitable for the growth and development of sheep.However,the temperature of the sheep house needed to be controlled to ensure the welfare and health of the animals.The lambs in two sheep farms were in good health in spring,but it was necessary to strengthen the feeding management of newborn and weak lambs in colostrums and supplementary feeding of fattening lambs in ML sheep farm.This study laid a certain foundation for the environment standard of meat sheep farm in Xinjiang,which also provided a scientific basis for the feeding management,animal health and welfare and the improvement of production level for Xinjiang sheep farms.