Table of Content

20 February 2019, Volume 46 Issue 2
Cloning and Expression Analysis in Reproductive Axis of Yak FSHR Gene
XIA Yi, WANG Qin, HE Xiangdong, CHEN Ying, JIGE Moti, ZI Xiangdong
2019, 46(2):  329-337.  doi:10.16431/j.cnki.1671-7236.2019.02.001
Abstract ( 195 )   PDF (1688KB) ( 158 )  
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The aim of this study was to investigate the potential role of follicle stimulating hormone receptor (FSHR) in regulating yak reproduction by clarifying its CDS sequence and expression characteristics in the reproductive axis of yak (Bos grunniens).The hypothalamus,anterior pituitary,ovary,oviduct and uterus of yak and cattle (Bos taurus) were collected during the follicular phase.The yak FSHR gene was amplified and cloned by RT-PCR,and its mRNA expression levels in different tissues was detected by Real-time PCR.The results showed that the FSHR coding region of the yak was 2 088 bp,encoding 695 amino acids.The formula of FSHR protein was C6378H10670N2088O2637S576,the molecular weight of FSHR protein was 177 263.85 u,and the theoretical isoelectric point was 4.88.Yak FSHR amino acids shared high homology with cattle,sheep,goat and wild boars (91.50% to 99.38%).The FSHR protein was an acid labile hydrophobin protein with 8 transmembrane structures and signal peptides;The secondary structure consisted of extended chain (15.54%),α-helix (42.30%),β-turn (1.44%) and random coil (40.72%).The phylogenetic tree showed the genetic relationship was the closest between yak and cattle.The Real-time PCR results showed that FSHR gene expressed in all tissues examined.Its expression in yak uterus was significantly or extremely significantly higher than other tissues (except ovary)(P<0.05;P<0.01),while it was significantly of extremely significantly higher in ovary of cattle than other tissues (P<0.05;P<0.01).Its expression level in cattle ovary was extremely significantly higher than yak (P<0.01).These data indicated that FSHR gene was relatively conservative in the course of animal evolution,and its lower expression in yak ovary possibly affected yak reproduction.

Cloning of ENaCα Subunit Gene from Alax League Bactrian Camel and Effect of Hormone on the ENaCα Expression of Renal Cortex Cells
ZHU Chunxiao, SUN Rui, ZHANG Yanru, ZHANG Dong, LIU Chunxia, MENG Fanhua
2019, 46(2):  338-344.  doi:10.16431/j.cnki.1671-7236.2019.02.002
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This study was aimed to explore the effect of sodium channel in epithelial cells of Alax League Bactrian camel.PCR primers were designed using the ENaCα subunit gene fragment which was provided by Shenzhen Huada company.Total RNA was extracted from the renal cortex of Alax League Bactrian camel,and cDNA was obtained by reverse transcription synthesis,then the renal cortex ENaCα subunit gene fragments were amplified by PCR.The cloning plasmid pMD19-T-ENaCα was constructed,the recombinant plasmid was identified by double enzyme digestion and squencing.The renal cortex of Alax League Bactrian camel was collected,after the cells were cultured in the P3 generation,different concentrations of aldosterone and angiotensin Ⅱ were added,the expression of ENaCα subunit gene was detected by Real-time quantitative PCR to determine the sensitivity of ENaCα changes in two hormone concentrations.The results showed that the ENaCα subunit gene was successfully cloned with the length of 2.2 kb,double digestion with Sma Ⅰ and Pst Ⅰ gave 2.2 and 2.7 kb bands,the recombinant plasmid pMD19-T-ENaCα was successfully constructed.Real-time quantitative PCR results confirmed that the expression levels of ENaCα treated with 1×10-9 mol/L aldosterone and angiotensin Ⅱ were significantly higher than that of control group (P<0.05);The expression levels of ENaCα treated with 1×10-6,1×10-7 and 1×10-8 mol/L aldosterone and angiotensin Ⅱ were significantly lower than that of control group (P<0.05).The results provided the experimental evidence for revealing the physiological mechanism of water and salt metabolism in Bactrian camel.

Cloning and Bioinformatics Analysis of Prolactin Receptor Gene of Mink
WANG Liying, ZHANG Yufei, CAO Manyuan, ZHAO Weigang, XU Baozeng
2019, 46(2):  345-353.  doi:10.16431/j.cnki.1671-7236.2019.02.003
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The aim of this study was to clone the full-length coding sequence of prolactin receptor (PRLR) gene from mink ovary tissues and analyze its biological characteristics to lay a foundation for studying its structure and function.A pair of primers was designed based on the predicted mRNA sequence of the dog PRLR gene in GenBank (accession No.:XM_025436332.1),and the ovary tissue samples of sexually mature healthy mink were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA,and PRLR gene was amplified by PCR using cDNA as template.The PCR products then were inserted into the pEASY-Blunt Simple Vector.Bioinformatics analysis was carried out after positive clone sequencing.The results showed that the coding region of PRLR gene of mink was 1 878 bp in length and encoded 625 amino acids.The homology of nucleotide sequence of PRLR gene between mink and Enhydra lutris kenyoni was the highest which reached 97.7%.The phylogenetic tree analysis also showed the closest relationship with Enhydra lutris kenyoni.The bioinformatics analysis of the PRLR protein of mink ovary showed that it contained a signal peptide,an extracellular region,a transmembrane region and an intramembranous region.It was a single transmembrane protein,and the D1 domain at the N-terminal contained two pairs of conserved disulfide bonds (Cys36-Cys46 and Cys75-Cys86),the D2 domain contained a conserved WS-motif (WS-E-WS).The PRLR protein of mink had a similar structure to other PRLR proteins.In conclusion,the study provided basic experimental data and theoretical basis for the further study of the structure and function of PRLR protein.

Physiology and Biochemistry
Effect of miR-425-5p on Proliferation and Differentiation of 3T3-L1 Preadipocyte
YANG Qiong, GU Hao, DU Jingjing, LIU Jinyuan, ZHANG Shunhua, ZHU Li
2019, 46(2):  354-364.  doi:10.16431/j.cnki.1671-7236.2019.02.004
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In order to explore the effect of miR-425-5p on the proliferation and differentiation of 3T3-L1 preadipocyte,the expression of miR-425-5p in tissues and cells were measured by Real-time PCR,the effect of miR-425-5p on the proliferation and differentiation of preadipocyte were measured by CCK8,EdU,oil red O staining and triglyceride detection methods,respectively.The target genes of miR-425-5p regulation preadipocyte differentiation were predicted and identified using bioinformatics softwares and dual luciferase reporter assays,respectively.The results showed that miR-425-5p was expressed at lower levels in adipose tissues of obese mice,and dynamically expressed during preadipocyte proliferation and differentiation.Compared with negative control,miR-425-5p overexpression promoted preadipocyte proliferation,inhibited marker genes related to adipocyte differentiation such as PPARγ,C/EBPα and FAS genes,and reduced lipid droplets and triglyceride accumulation.Inhibition of miR-425-5p expression inhibited the proliferation and prevented the differentiation of preadipocyte.Further analysis found that overexpression or inhibition of miR-425-5p decreased or increased IGF1 gene expression in preadipocyte differentiation,respectively.Compared with negative control,miR-425-5p overexpression inhibited the fluorescence activity of IGF1 gene 3'-UTR,whereas mutation of the miR-425-5p binding site in IGF1 gene 3'-UTR completely abolished this response.This results indicated that miR-425-5p might promote 3T3-L1 preadipocyte proliferation,whereas negatively regulates its differentiation by targeting IGF1.

Expression and Purification of Trx1 and Trx2 Proteins and Evaluation of Their Antioxidative Stress
YU Wenlan, HU Lianmei, LIANG Bai, WU Fuwang, YANG Fan, GUO Jianying, TANG Zhaoxin
2019, 46(2):  365-372.  doi:10.16431/j.cnki.1671-7236.2019.02.005
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The aim of this experiment was to express and purify Trx1 and Trx2 proteins and evaluate their antioxidant properties.The constructed recombinant plasmid GgTrx1-pET28a(+) and GgTrx2-nsp-pET28a(+) were transformed into E.coli for expression under induction of IPTG and purified by Ni-NAT chromatography.The recombinant proteins GgTrx1 and GgTrx2 activity were determined by insulin assay.The protective effect of GgTrx1 and GgTrx2 on oxidative stress induced by hydrogen peroxide (H2O2) in BRL-3A cell line was also evaluated.The results showed that the two recombinant proteins GgTrx1 and GgTrx2 were successfully obtained,and the optimal induction conditions were 37℃,0.4 mmol/L IPTG induction for 4 h and 37℃,1.0 mmol/L IPTG for 4 h,respectively.The recombinant proteins GgTrx1 and GgTrx2 were purified with a purity of more than 90% with concentration of 4.0 and 5.0 mg/mL, respectively.The purified recombinant proteins GgTrx1 and GgTrx2 both had higher ability to reduce insulin disulfide bonds in a concentration-dependent manner.In vitro cell assays showed that both GgTrx1 and GgTrx2 significantly reduced hydrogen peroxide-induced BRL-3A membrane lipid peroxidation and protected antioxidant enzymes SOD and CAT activities,and GgTrx2 was more effective.The results of this experiment indicated that both purified GgTrx1 and GgTrx2 had biological activity and good anti-oxidative stress characteristics,and mitochondrial Trx2 provided more possibilities for clinical treatment of oxidative stress diseases.

Effect of Transport Stress on Haemotological Indices of Donkey
GAO Weiping, JIANG Guimiao, LIANG Weichao, LI Junqiao, JI Chuanliang, ZHAO Fuwei, DENG Lixin
2019, 46(2):  373-379.  doi:10.16431/j.cnki.1671-7236.2019.02.006
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This study was designed to investigate the effects of long-distance transport stress on blood parameters of donkey.The study was conducted with 12 heads of Dezhou donkeys with weight of (140.8±5.2) kg,the transportation distance was about 950 km,the departure temperature was -20℃,and the destination temperature was -10℃,which lasted for 21 h,including 10 parking observations (≤ 20 min).15 mL of jugular venous blood was collected before loading,0 h after transportation and 3,5,7,9 and 15 d after transportation,and blood hormone indexes,blood routine indexes and blood biochemical indexes were detected,and the weights before transport,0 h after transport and 10,20 and 30 d after transport were recorded.The results showed that the concentrations of adrenocorticotropic hormone (ACTH),heat shock protein 90 (HSP90) and cortisol in the serum at 0 h after landing were significantly higher than those before transportation (P<0.05),and gradually decreased within 3,5,7,9 and 15 d after transportation,and ACTH and HSP90 recovered to pre-carriage levels.The number of neutrophils (Neu),the total number of red blood cells (RBC) and the hemoglobin concentration (HGB) increased significantly after 3,5,7,9 and 15 d (P<0.05),the total number of white blood cells (WBC) increased but not significantly different from that before transportation (P>0.05),the total number of lymphocyte (LYM) was significantly lower than before transport (P<0.05).There were no significant changes in serum total protein (TP),serum albumin (ALB),creatinine (CREA) and lactate dehydrogenase (LDH) before and after transport (P>0.05).Aspartate aminotransferase (AST),creatine kinase (CK) and urea (UREA) were significantly higher than before transport (P<0.05).The body weight decreased significantly (P<0.05),and recovered to the pre-transport level within 10 d after transportation.The above results indicated that long-distance transport induced stress response to sputum,liver damage and elevated levels of metabolism,but the immune system was not significantly damaged.Within 15 d after landing,the body gradually recovered from the stress response to the pre-shipment state.

Effects of Oleic Acid and Linoleic Acid on Triglyceride Content and Gene Expression Involved in Milk Fat Synthesis in Goat Mammary Epithelial Cells
LI Jun, YANG Wenzhuo, HOU Xiafei, WANG Xiaoxiao, JI Xiangbo, LI Xinfeng, QUAN Kai
2019, 46(2):  380-386.  doi:10.16431/j.cnki.1671-7236.2019.02.007
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The aim of this study was to investigate the effects of oleic acid and linoleic acid on triglyceride (TG) content and the expression of genes involved in milk fat synthesis in dairy goat mammary epithelial cells in vitro.The goat mammary epithelial cells were used as the research object,and different concentrations of oleic acid (0,50,100,200 μmol/L) and linoleic acid (0,20,40,80 μmol/L) were added in culture medium for a 24 h cultivation.The intracellular TG content was measured by using triglyceride determination kit,and gene expressions were detected by Real-time quantitative PCR.The results showed that 100,200 μmol/L oleic acid and 40,80 μmol/L linoleic acid significantly increased TG content (P<0.05).The expression of DGAT2,ACC and FASN genes were significantly up-regulated by addition of 100 and 200 μmol/L oleic acid (P<0.05),while the SCD1 gene expression was significantly inhibited by oleic acid (P<0.05).However,oleic acid had no significant effect on the expression of PPARγ gene (P>0.05).The results of linoleic acid addition showed that 20 μmol/L linoleic acid could significantly inhibit the expressions of ACC and FASN genes (P<0.05).40 and 80 μmol/L linoleic acid could significantly promote the expression of DGAT2 gene (P<0.05),inhibit the expression of SCD1 and SREBP1 genes (P<0.05),and had no significant effect on the expression of ACC,FASN and PPARγ genes (P>0.05).In conclusion,100 to 200 μmol/L oleic acid and 40 to 80 μmol/L linoleic acid were optimal levels considering its improvement effects on milk fat synthesis in dairy goat mammary epithelial cells.

Research Progress on the Development and Regulation of Mammalian Hair Follicles
GUO Xuefeng, BAO Pengjia, CHANG Yongfang, ZHANG Yongfeng, LI Zhongbang, LEI Lei, YAN Ping, PAN Heping
2019, 46(2):  387-394.  doi:10.16431/j.cnki.1671-7236.2019.02.008
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The hair follicle is a specific structure of mammalian skin and the organ which has a highly self-renewing ability and grows periodically for the lifetime.The development of hair follicles begins at the embryonic stage,and a series of interactions which are between epidermal and hypodermal cells induce the formation of hair follicles.The follicle will enter the periodic cycle,which contains three stages:Anagen,catagen and telogen,each of which is regulated by complex genetic networks.In recent years,great progresses have been made in the study of mammalian hair follicle development and regulation mechanisms.Some previous studies have shown that the development and circulation of hair follicles regulated by various factors,different signaling pathways and the miRNA,lncRNA and related genes,which constitute a large and complex network map.Mutual promotion and restriction between each control factor provide the necessary guarantee for the development of the hair follicle and cycle.This paper briefly describes the morphogenesis,periodic cycling and related regulatory factors of mammalian hair follicles in humans,sheep and mice in order to improve the comprehensive understanding of mammalian hair follicle development and regulation,and provide a reference and an idea for artificially controlling the cycle growth of the villus to improve the yield and quality of the villus.

Animal Nutrition and Feed Science
Study on Improved Method of Iodimetry for Determination of Cysteamine Hydrochloride in Diets,Intestinal Digesta and Excreta
LIU Baoliang, ZHAO Feng, LI Zhefeng, CHEN Kaixuan, ZHANG Hu, ZHAO Wei, YANG Ling, ZHANG Hongfu
2019, 46(2):  395-403.  doi:10.16431/j.cnki.1671-7236.2019.02.009
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This experiment was conducted to improve the method of iodimetry for the determination of cysteamine hydrochloride (CSH) in diet,digesta,and excreta,which will be used to assay the CSH for evaluating the releasing process of encapsulated cysteamine in gastro-intestine of animal.Experiment 1 was to examine the linear relationship between iodine consumption on the amount of CSH with the iodimetry.Six levels of 28.28,113.12,226.24,339.36,452.48,565.61 mg of CSH was adopted in a single-factor completely randomized design.Experiment 2 was to investigate the effect of pretreatment on eliminating interference from diet,digesta and excreta in the determination of CSH with iodimetry.Fifteen samples were composed that in each 2 g of diet,duodenal digesta,jejunaldigesta,ilealdigesta or excreta of chicken,the amounts of CSH (reagent grade) were 30,100 and 300 mg.The volume of 0.1,0.5 or 1 mL of 6 mol/L sulphuric acid solution was adopted in single-factor completely randomized design to pretreat each sample.Experiment 3 was to test the differences between conventional iodimetry and the improved method established in the experiment 2 to determine CSH in diet,digesta and excreta.The amounts of CSH (provided by the encapsulated product) of 28.73,84.84,141.47 and 200.56 mg were mixed in each 2 g of diets,duodenal digesta,jejunaldigesta,ilealdigesta,and excreta of chicken.A two-sample experimental design was adopted to compare 2 methods to determine the content of CSH.The results showed as follows:①There was a significant linear relationship between the iodine consumption or the determined CSH on actual amount of CSH (P<0.01,R2=0.9999);② In the diet mixed with CSH,sulfuric acid pretreatment for iodimetry made the regression of determined on actual amount of CSH consistent with Y=X (P>0.05).In the duodenal or ilealdigesta mixed with CSH,sulfuric acid pretreatment for iodimetry didn't made the regression of determined on actual amount of CSH consistent with Y=X (P<0.05),however,the pretreatment with 0.5 or 1 mL sulfuric acid made the determinred value closer to the actual value than the pretreatment with 0.1 mL.In the jejunaldigesta or excreta mixed with CSH,sulfuric acid pretreatment made the regression of determined on actual amount of CSH consistent with Y=X (P>0.05),whereas,the pretreatment with 0.1 mL sulfuric acid made the determined value significantly lower than actual value of CSH (P<0.01).③In diet,duodenal,ileal and jejunaldigesta or excreta mixed with CSH,conventional method made the regression of determined on actual amount of CSH deviate from Y=X (P<0.05),whereas,pretreatment with 1.0 mL sulfuric acid made the regression of determined on actual amount of CSH consistent with Y=X (P>0.05).In conclusion,pretreatment with an appropriate amount of sulphuric acid solution cculd improve the determination of CSH by eliminating the interference from diet,digesta or excreta.

The Determination of Growth Performance,Wool Quality and Blood Physiological and Biochemical Indexes of SDH Hybrid Sheep
SHI Huibin, WANG Yuqin, HE Wengtan, WU Zhibo, TIAN Zhilong, YU Zuhua, DING Ke, ZHAO Zhanqin
2019, 46(2):  404-413.  doi:10.16431/j.cnki.1671-7236.2019.02.010
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In order to study the growth and development of Suffolk×Dorper×Hu sheep (SDH) hybrid sheep and its adaptability to the local ecological environment,the body weight and body size indexes of sheep at different ages were measured by conventional measurement of production performance,microscopic measurement and automatic analysis of blood physiology and biochemistry analyzer.Wool quality (including net wool percentage,wool fineness,ratio of medullated and non-medullated wool,natural length and straight length of different parts,etc.) and physiological and biochemical indexes of adult sheep blood were measured.The results showed that the early growth rate of SDH hybrid sheep was fast,and the body length of rams was significant higher than that of ewes in primary stage and one year old (P<0.05).At three months of age,there was a significant difference in body height,body length and chest circumference between rams and ewes (P<0.05),and rams were higher than ewes.At the age of six months,the body weight of ewes was extremely significant higher than that of rams (P<0.01).The body weight and body height of rams were significantly higher than that of ewes in adulthood (P<0.05).There was no significant difference in body weight and body size between rams and ewes during the other growth stages (P>0.05).The average net wool rate,average natural length,straight length,fineness of the hybrid wool was 45.21%,4.55 cm,6.22 cm and 17.60 μm,respectively.There were some differences between blood physiological and biochemical indicators of male and female sheep and the reference value of domestic sheep.The difference in PLT and PCT of ewes were extremely significant higher than rams (P<0.01).There was a significant difference in the contents of triglyceride and albumin (P<0.05),and the ram was higher than the ewes.There was no significant difference between the other indicators (P>0.05).In conclusion,the SDH sheep had improved in various aspects in terms of growth and development,and the results could provide a reference for the further development and utilization of the sheep.

Effect of Moringa oleifera Lam. Extract on Production Performance,Slaughter Performance,Meat Quality and Serum Biochemical Indexes in AA Broilers
LIU Jiao, CHANG Wenhuan, CHEN Zhimin, ZHENG Aijuan, LIU Guohua, CAI Huiyi, ZHENG Yi
2019, 46(2):  414-423.  doi:10.16431/j.cnki.1671-7236.2019.02.011
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The study was to investigate the effect of Moringa oleifera Lam. extract (MOLE) on the production performance,slaughter performance,meat quality,antioxidant and serum biochemical indexes of AA broilers,and evaluate its availability as a feed additive and its optimal dosage.A total of 480 one-day-old healthy AA male broilers were selected and randomly divided into 5 groups with 6 replicates per group and 16 broilers per replicate which were control group (basal diet),antibiotic group (basal diet+50 mg/kg chlortetracycline) and 3 groups supplemented with MOLE of 0.25,0.50 and 1.00 g/kg in basal diet,respectively.The trial lasted for 42 days composed of two stages (1 to 21 d and 22 to 42 d).The results showed as follows:①Compared with the control group,the final body weight and ADG in antibiotic group were significantly improved (P<0.05),and the final body weight and ADG of 1 to 42 d in 0.25 and 0.50 g/kg MOLE groups were significantly increased (P<0.05) and 0.50 g/kg MOLE had a better effect.There were no significant differences in ADFI or F/G among treatment groups(P>0.05).②There was no significant difference in the slaughter performance and meat quality among all groups (P>0.05).③Compared with the control group,the T-AOC of broilers were significantly increased in antibiotic group and 0.50,1.00 g/kg MOLE groups (P<0.05),and 0.50 g/kg MOLE had a better effect.The serum MDA in MOLE groups were significant decreased compared with antibiotic group (P<0.05).④There was no significant difference in biochemical indices in serum among all groups (P>0.05).⑤0.25,0.50 and 1.00 g/kg MOLE supplementation decreased the death rate of broilers to 1.04%,3.13% and 2.08%.In conclusion,the supplementation of MOLE could improve weight gain and antioxidant capability,and reduce the death rate of the broilers.The optimal supplemental level of MOLE was 0.50 g/kg under the conditions of this experiment.

Effects of Recombinant E.coli Expressing Heat-stable Enterotoxin on Small Intestinal Function in 7 Days Old Piglets
WU Mengjun, LI Siyuan, JI Changzheng, YU Kui, DONG Yi, ZHAO Guangyu, ZHAO Di, WU Tao, HOU Yongqing
2019, 46(2):  424-432.  doi:10.16431/j.cnki.1671-7236.2019.02.012
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This experiment was aimed to investigate the effects of recombinant E.coli expressing heat-stable enterotoxin(STa) on small intestinal function in 7 days old piglets.Twenty-four 7 days old piglets were randomly divided into four treatments of control,STa,LMG194 and K88 groups,which were fed with artificial milk throughout the experiment.Pre-feeding period was 4 d.STa,LMG194 and K88 groups were orally administered with 2×109 CFU E.coli LMG194-pBAD-STa,LMG194 and K88 twice in the morning and evening on the 5th day,respectively.The control group was given the same amount of normal saline.On the 7th morning of the experiment,each group was given D-xylose and blood was collected.Then they were slaughtered and the samples of jejunum and ileum tissue were collected,blood routine indicators,the content of plasma D-xylose,the activity of diamine oxidase (DAO),and the expression of absorption and transport related genes and protein were measured.The results showed that compared with control group,the neutrophils and neutrophil ratio were significantly decreased in blood,the lymphocyte ratio was significantly increased in STa group (P<0.05),the neutrophil ratio was significantly decreased in blood,the lymphocyte ratio was significantly increased in LMG194 and K88 groups (P<0.05);The content of D-xylose in plasma was significantly decreased in STa and K88 groups (P<0.05);The activity of DAO in plasma was significantly increased in STa,LMG194 and K88 groups (P<0.05).In jejunum,compared with control group,the expression levels of AQP8, AQP10,KCNJ13 and b0,+AT genes were significantly reduced in STa and LMG194 groups (P<0.05),the expression levels of AQP8 and AQP10 genes were significantly increased in K88 group (P<0.05).In ileum,the expression level of AQP8 gene was significantly reduced,the expression levels of AQP10 and SGLT-1 genes were significantly increased in STa group (P<0.05).The expression level of AQP8 gene was significantly increased in LMG194 and K88 groups (P<0.05),the expression levels of AQP10,KCNJ13,b0,+AT and SGLT-1 genes were significantly reduced in K88 group (P<0.05).The relative expression level of HSP70 was significantly increased in STa,LMG194 and K88 groups (P<0.05).These results suggested that recombinant E.coli expressing STa could lead to inflammation in small intestinal,absorption and transfer capacity were decreased in 7 days old piglets.

Effects of Aflatoxin B1 and M1 on Intestinal Bacterial Diversity in Mice
ZHANG Muchen, ZHENG Nan, ZHAO Shengguo, ZHANG Yangdong, LI Songli, WANG Jiaqi
2019, 46(2):  433-441.  doi:10.16431/j.cnki.1671-7236.2019.02.013
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To study the effect of aflatoxin B1 and M1 on intestinal bacterial diversity in mice,40 ICR male mice (4 weeks) were randomized into four groups of 10 each and received an oral administration of 0.3 mg/(kg·BW) AFB1,3.0 mg/(kg·BW) AFM1,mixture of AFB1 and AFM1 (0.3 mg/(kg·BW) AFB1,3.0 mg/(kg·BW) AFM1) and vehicle (1.0% DMSO) for 28 days (once a day at 9:00 am).Mice were executed after gavage,and intestinal contents were collected.The bacterial diversity of intestinal contents was analyzed by 16S rRNA sequencing.The results showed that compared with control group,there was no significant change in the dominant bacteria microflora in three experimental groups,regardless of the phylum level,family level or genus level(P>0.05).However,different toxin treatments still resulted in significant changes in the abundance of different bacteria microflora:Pathogenic bacteria or opportunistic pathogens abundance in the AFB1 treatment and its combined treatment group with AFM1,such as Facklamia,Staphylococcus and Corynebacterium were significantly increased compared with the control group (P<0.05),but not for AFM1 group.Comprehensive analysis showed that individual AFB1 or combined with AFM1 could increase the abundance of pathogenic bacteria or opportunistic pathogens,and could change the healthy intestinal bacterial diversity,damage the function of intestinal microbial barrier.

Analysis of Wool Traits and Its Relationship in Tan Sheep During the Er-mao Period
LI Xianglong, TAO Jinzhong, DING Wei, GUO Yansheng, LIU Guolin, CHEN Yonghong, LIU Huanhuan
2019, 46(2):  442-448.  doi:10.16431/j.cnki.1671-7236.2019.02.014
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The aim of this study was to analyze the wool traits and their correlation in Tan sheep during the er-mao period.The samples were collected from 511 individuals (260 rams and 251 ewes) in the primary stage at 30 to 40 days of age (er-mao period).The lateral hair length,the number of crimps of wool collected in the primary and second wool periods were measured,and wool traits were determined including mean fiber diameter (MFD),mean fiber curvature (MFC),coefficient of variation of fiber diameter (CVFD),fiber diameter standard deviation (FDSD) of the heterotypic wool and fine wool.The characteristics of wool about descriptive statistics and correlation were analyzed.The results showed that there was an extremely significant moderate positive correlation between the number of crimps and the lateral hair length in the primary stage (r=0.410,P<0.001).There was an extremely significant moderate positive correlation between the number of crimps in the primary and the number of lateral flexion in the er-mao period (r=0.618,P<0.001).There was a strong positive correlation between FDSD and CVFD of both heterotypic wool and fine wool (r=0.796,P<0.001;r=0.942,P<0.001),respectively;The MFD and FDSD of both heterotypic wool and fine wool were significantly positively correlated (r=0.511,P<0.001;r=0.660,P<0.001),respectively;There was an extremely significant medium positive correlation between MFD and CVFD of fine wool (r=0.410,P<0.001),there was a weak negative correlation between MFD and CVFD of heterotypic wool (r=-0.106,P<0.05);There was a strong significant medium negative correlation between MFD and MFC of fine wool (r=-0.497,P<0.001),and there was a weak positive correlation between MFD and MFC of heterotypic wool,and the correlation was not significant (r=0.011,P>0.05).This study firstly examined the indicators of wool in Tan sheep during the er-mao period,and the correlation between the indicators were determined,which could provide data for the breeding of fur traits in Tan sheep.

Effects of Chicory Acid on Growth Performance,Serum Biochemical Indices and Antioxidant Ability of Grazing Yak in Perinatal Period
A Shunxian, LUO Zenghai, ZHANG Wenying, WANG Aichao, ZHANG Yuanxin, WU Hua
2019, 46(2):  449-457.  doi:10.16431/j.cnki.1671-7236.2019.02.015
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The aim of this experiment was to study the effects of chicory acid on the growth performance,serum biochemical indices and antioxidant capacity of grazing yak in perinatal period.Sixteen perinatal grazing yak with good body condition and similar weight (195 to 205 kg) were chosen and randomly divided into two groups,each group had eight yak,the control group was fed with concentrate supplement and oat green hay,and the experimental group was fed with concentrate supplement,0.15 kg/d chicory acid and oat green hay.The trial period was 60 days (from 30 days before delivery to 30 days after delivery),and the blood samples were collected on the 15th day before delivery,7th day before delivery,day of childbirth,7th day after delivery,and 15th day after delivery,and the corresponding indicators were determined.The results showed that,compared with the control group, on the 15th day after delivery, the contents of serum urea nitrogen (BUN) was significantly lower (P<0.05);On the day of childbirth and the 7th day after delivery,the activities of aspartate transaminase (AST) was significantly lower (P<0.05),while the contents of serum glucose (GLU), the activity of serum alkaline phosphatase (ALP) and total antioxidant capacity (T-AOC) were significantly higher (P<0.05); And on the day of childbirth, the activities of serum glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) were significantly higher, and malondialdehyde (MDA) content was significantly lower (P<0.05) in experimental group. The results of this study indicated that adding chicory acid to the perinatal gr azing yak supplement diet could effectively improve the blood biochemical indexes, alleviate the oxidative stress state and improve the antioxidant capacity of grazing yak during perinatal period.

Genetics and Breeding
Polymorphism of DRD2 Gene and Its Associations with Egg Traits in Xinhua Chicken
QU Ke, HUANG Tao, GONG Yanzhang
2019, 46(2):  458-469.  doi:10.16431/j.cnki.1671-7236.2019.02.016
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This study was aimed to investigate the polymorphism of dopamine receptor D2 (DRD2) gene and its correlation with egg traits in Xinhua chicken,and find the molecular genetic markers for the breeding of egg traits in Xinhua chicken.4 pairs of primers were designed using Primer Premier 5.0,the genotype of 473 individuals in Xinhua E-line chicken were identified by PCR-RFLP method,the correlation were analyzed between egg traits of Xinhua chicken and polymorphism of DRD2 gene using SPSS 19.0 software.Population polymorphism analysis results showed that there were 4 SNPs of A-16105G (SNP1),G-12510T (SNP2),G+3360A(SNP3) and T+5042C(SNP4),SNP1 and SNP2 were in Hardy-Weinberg equilibrium (P>0.05),and the heterozygosity were low,SNP3 and SNP4 significantly deviated from the Hardy-Weinberg equilibrium (P<0.05).Correlation analysis results showed that 4 SNPs of DRD2 gene were associated with egg traits in Xinhua E-line chicken,for egg production traits,SNP1 was significantly associated with age at the first egg (P<0.05);SNP2 was extremely significantly associated with egg production at 33 weeks old and the total number of eggs at 300 days of age (P<0.01);SNP3 was associated with body weight at sexual maturity (P<0.05),the total number of eggs at 300 days of age (P<0.01),average clucth size (P<0.01) and the largest clucth size (P<0.05);SNP4 was extremely significantly associated with age at the first egg (P<0.01).For egg quality traits,SNP1 was associated with egg-shaped index (P<0.01),yolk color (P<0.01) and Haugh units (P<0.05);SNP2 was associated with yolk weight (P<0.05);SNP3 was extremely significantly associated with eggshell strength (P<0.01);SNP4 was significantly associated with yolk weight,albumen weight and Haugh units (P<0.05).The haplotype analysis results showed that the different haplotype combinations of 4 SNPs of DRD2 gene were significantly correlated with the longest clutch size in chicken (P<0.01) and haugh units of eggs (P<0.05).The tissue expression profiling results indicated that DRD2 gene was mainly expressed of pituitary gland in Xinhua chicken,and was also highly expressed in chest muscle at 58 weeks old,with a low expression in brain at 36 weeks old.The results suggested that DRD2 gene in Xinhua E-line chicken could be used to assist the genetic improvement of egg traits.

Study on Inner Environment of Early Embryos Development in Racoon Dog
CAO Junguo, DUAN Baoning, XU Baozeng, FENG Huailiang
2019, 46(2):  470-479.  doi:10.16431/j.cnki.1671-7236.2019.02.017
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To investigate the micro-environment in vivo of preimplantation embryos in the raccoon dog,23 adult healthy females with the same age and similar body weight were selected.After 1-2 times of copulation during breeding season,starting with the first mating(0 h),the females were killed randomly at 29-99 h (n=7),100-126 h(n=7),169-268 h (n=9),respectively.The morphological and ultrastructural changes of the oviduct and uterus during early embryonic development were analyzed using immunohistochemistry,transmission and scanning electron microscopy.9 elements such as potassium,calcium,iron,zinc of sputum oviduct fluid and uterus fluid during early embryo development were determined by X-ray energy spectroscopy.The results showed that:① With the development of early embryos of raccoon dog,at the later stage,the length,mucosal thickness,duplicatures' height and epithelial height of oviduct were significantly reduced (P<0.05);Although the diameter of oviduct was also decreased,but there was no significant difference (P>0.05).On the other hand,the mucosal thickness,duplicatures' height and density of uterine gland were significantly increased (P<0.05);Although the diameter and length of uterus were enhanced,but the difference was not significant (P>0.05).②With the development of early embryos of raccoon dog,the numbers of microvilli,lipid droplets and lysosomes in the uterine mucosa epithelium were magically increased.③With the development of early embryos of raccoon dog,at the later stage,the contents of S,Ca,Fe,Cu and Zn in the oviduct fluids were significantly improved (P<0.05),while the content of P in the oviduct fluids was significantly decreased (P<0.05),the contents of K,Cl and Na in the oviduct fluids showed the fluctuation change with the development of embryos.The contents of S,Cl,K in the uterus fluids were decreased (P>0.05),the content of Zn in the uterus fluids was significantly decreased (P<0.05),the contents of P,Ca,Fe and Cu presented the fluctuation change with the development of embryos.This study preliminarily investigated the physical and chemical micro-environment in vivo of preimplantation embryos in raccoon dogs,which would provide reference for the establishment of culture system of preimplantation embryos in vitro in raccoon dogs.

Analysis of Genetic Structure of Yunshang Black Goat Based on SNP Chip
LAN Rong, ZHU Lan, YANG Hongyuan, WANG Peng, SHAO Qingyong, JIANG Yanting, HONG Qionghua
2019, 46(2):  480-488.  doi:10.16431/j.cnki.1671-7236.2019.02.018
Abstract ( 246 )   PDF (1902KB) ( 90 )  
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In order to evaluate the genetic structure in Yunshang Black goat at the molecular level,108 Yunshang Black goats from 10 families,11 Yunling Black goats,14 Nubian goats and 10 Boer goats (control group) were genotyped by Illumina Iselect Goat60k array (SNP chip).Genetic diversity parameters statistics,principal component analysis and phylogenetic tree construction were performed by GenAlEx 6.5,Cervus 3.0,SNPRelate,Plink 1.07 and Mega 4.0.The typing results at 48 116 SNPs loci in 143 samples had been gotten.There was a great genetic distance between Boer goat and Yunshang Black goat,and a large genetic distance also existed between Boer goat and Nubian goat.Bore goat was displayed as an independent branch on the cluster tree.The results proofed the method of this study was practical and reliable.108 samples of Yunshang Black goats were high consistency,and these samples were clustered into a large cluster in the phylogenetic tree.The samples of Nubian goats (14) and Yunling Black goats (11) were clustered into a cluster.UPGMA evolution tree displayed that Yunshang Black goats were closely related to Nubian goats,and had a little relationship with Yunling Black goats,which was consistent with the results of the breeding blood guide in Yunshang Black goat.Yunshang Black goat could be distinguished from the breeding material at the genomic level,which had genomic characteristics of an independent variety.

Analysis of Genetic Diversity and Paternal Type of Sika Deer Based on SRY Gene
DONG Yimeng, LIU Huamiao, Hidetoshi B. Tamate, LIU Huitao, JU Yan, XING Xiumei, HE Jinming, JU Guichun
2019, 46(2):  489-496.  doi:10.16431/j.cnki.1671-7236.2019.02.019
Abstract ( 179 )   PDF (1316KB) ( 69 )  
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This study was aimed to analyze the genetic diversity and paternal typec of sika deer based on SRY gene.144 individuals from five subspecies,such as Cervus nippon hortulorum,Cervus nippon yesoensis,Cervus nippon centralis,Cervus nippon nippon and Cervus nippon yakushimae,were selected for DNA extraction using kit and traditional phenol/imitation (1:1) methods.PCR amplification and PCR products sequence were used to analyze the nucleotide diversity (Pi),haplotype diversity (Hd),genetic distance and phylogenetic relationship of sika deer.The results showed that 6 SNPs polymorphisms were detected at positions 47,62,602,1 148,1 569 and 1 604 bp in the 1 613 bp sequences,which accounted for 0.3% of the total number of nucleotides.The base substitution was dominated by base conversion.6 haplotypes of Hap-1,Hap-2,Hap-3,Hap-4,Hap-5 and Hap-6 were identified by 6 SNPs polymorphisms,Hap-4,Hap-5 and Hap-6 were the new haplotypes.The sequence of nucleotide diversity from high to low were:Cervus nippon nippon, Cervus nippon yakushimae,Cervus nippon hortulorum,Cervus nippon centralis,Cervus nippon yesoensis.The smallest genetic distance among subspecies was 0.000056 between Cervus nippon yesoensis and Cervus nippon centralis,and the largest distance was 0.001733 between Cervus nippon hortulorum and Cervus nippon nippon.Phylogenetic trees were constructed based on haplotypes and all individuals using the adjacency method.The phylogenetic tree showed that compared with the Japanese sika deer,Cervus nipppon hortulorum was closer to Cervus elaphus,Cervus nippon hortulorum had two branches,and Japanese sika deer had no obvious branches,and the other haplotypes were evolved by Hap-3.The haplotype minimum span network map was consistent with the evolutionary tree.This results indicated that there were two patrilineal types in Cervus nippon hortulorum,there was a paternal type in Japanese sika deer,Hap-3 was the original haplotype of Japanese sika deer.

Effect of Psammaplin A on in vitro Development of Bovine Aging Oocytes
WANG Huaqing, GAO Qingshan, JIANG Wenjie, ZHAO Yuhan, JIN Qingguo, LI Yinghua, XU Yongnan
2019, 46(2):  497-503.  doi:10.16431/j.cnki.1671-7236.2019.02.020
Abstract ( 199 )   PDF (2148KB) ( 164 )  
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To investigate the effect of histone deacetylation inhibitor Psammaplin A (PsA) on the development of bovine aging oocytes in vitro,oocytes were randomly divided into control group,aging group and 50 mmol/L PsA treated aging group (PsA group).Immunofluorescence staining and JC-1 were used to detect the blastocyst rate of bovine oocytes after parthenogenetic activation,the number of cells in blastocysts,apoptosis,reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential intensity of embryos.The results showed that the blastocyst rate of the aging group was significantly lower than that of PsA and control groups (P<0.05).The blastocyst rate of PsA group was not significantly different from that of control group (P>0.05).The number of cells in the blastocysts of control group and PsA group were significantly higher than that of aging group (P<0.05).The number of cells in the blastocysts of PsA group was not significantly different from that of control group (P>0.05).The apoptosis rate in aging group was significantly higher than that of control and PsA groups (P<0.05),the apoptosis rate of PsA group was significantly higher than that of control group (P<0.05).The GSH level of MⅡ oocytes in aging group was significantly lower than that of control and PsA groups (P<0.05).There was no significant difference in GSH level between control and PsA groups (P>0.05).The ROS level of the embryos in aging group was significantly higher than that of control and PsA groups (P<0.05).The ROS level in PsA group was significantly higher than that of control group (P<0.05).The mitochondrial membrane potential of early embryos of aging group 4-8 cells was significantly lower than that of control and PsA groups (P<0.05).The mitochondrial membrane potential intensity of control group was significantly higher than that of PsA group (P<0.05).In summary,PsA could effectively delay the aging of bovine oocytes and improve the quality of oocytes.

Detection of Single Nucleotide Polymorphism and Methylation of CpG Island in Promoter Region of Chicken HSP90AA1 Gene
CHAO Zhe, WU Wangjun, XING Manping, HUANG Lili, SUN Ruiping, LIU Hailong, ZHENG Xinli
2019, 46(2):  504-511.  doi:10.16431/j.cnki.1671-7236.2019.02.021
Abstract ( 168 )   PDF (1273KB) ( 84 )  
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This study was aimed to analyze the structure of HSP90AA1 gene,reveal the distribution of SNP in Wenchang chicken and Beijing-You chicken,investigate the promoter CpG islands methylation status of HSP90AA1 gene,and explore the role of HSP90AA1 gene in muscle tissue growth and development.HSP90AA1 gene was amplified by PCR.Then the gene mutation was identified by sequencing.CpG island was predicted using online software MethPrimer.With MassArray system,the DNA methylation levels and patterns of CpG islands in HSP90AA1 gene promoter were analyzed between the breast muscles of Wenchang chicken and Beijing-You chicken.Seven mutations were detected in the HSP90AA1 gene genomic fragment,two SNPs in the promoter region (A-189G,C-109T),one SNP in the first exon (A+6G),one SNP in the second exon (C+343T),two SNPs in the second intron (A+634G,A+836G) and one SNP in the seventh intron (A+3449G).The chicken HSP90AA1 gene sequence was determined including ten exons and nine introns.We found a CpG island,extending from -1 802 to -469 bp,in the promoter of HSP90AA1 gene.Forty-two CpG dinucleotides in HSP90AA1 promoter were tested.A total of 9 CpG loci (CpG_16.17.18,CpG_21.22.23,CpG_32.33 and CpG_57) were significantly different in different developmental stages in Wenchang chicken.A total of 4 CpG loci (CpG_1,CpG_5.6 and CpG_57) were significantly different in different developmental stages in Beijing-You chicken.This study proved that sequence information and methylation levels of HSP90AA1 gene in Wenchang chicken and Beijing-You chicken were different.The results provided some useful epigenetic basis principles for growth and development,system selection of Wenchang chicken and Beijing-You chicken.

Research Progress on Application of Gonadotropin-releasing Hormone and Its Analogues in Porcine Reproduction
LIU Chuang, LI Nan, WEI Qiaoli, FANG Xiaohuan, TIAN Jianhui, WENG Shiqiao, LIU Yan, LI Junjie
2019, 46(2):  512-518.  doi:10.16431/j.cnki.1671-7236.2019.02.022
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Gonadotropin-releasing hormone (GnRH) is a reproductive hormone secreted by the hypothalamus.It mainly participates in regulating the reproductive activities of animals through the hypothalamic-pituitary-gonadal axis,and can also directly play an important role in animal gonads or other organs.GnRH in mammals has the same ten peptide structure.Different GnRH analogues can be synthesized by changing the sixth,ninth and tenth amino acids in the ten peptide structure.GnRH and its analogues can affect animal reproductive performance by stimulating luteinizing hormone (LH) secretion,inhibiting estrogen receptor dimerization and regulating the synthesis of proteins associated with embryo implantation.GnRH and its analogues in production have been shown to increase the fertility of pigs.The application of GnRH analogues still has some problems,such as the significant effect of the second pregnancy,the promotion of ovulation but not the increase of litter size.This paper introduced the application of GnRH and its analogues in sow breeding from such aspects:The source and function of GnRH,the structure of GnRH and its analogues,the structure and function of gonadotropin-releasing hormone receptor,the effect of GnRH and its analogues on reproductive performance of swine,existing problems and prospects.

Research Advances on DNA methylation and Demethylation to Regulate Adipose Deposition
WANG Hejie, QIN Benyuan, WANG Yuanyuan, GUO Yulong, SONG Pengkang, LIU Yadan, LE Baoyu, ZHANG Ningfang, CHENG Zhimin, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing
2019, 46(2):  519-525.  doi:10.16431/j.cnki.1671-7236.2019.02.023
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Adipose deposition is a complex biological process,and is regulated by genetic and epigenetic effects.DNA methylation and demethylation are important ways of epigenetic modification,can participate in important life processes such as growth and development of body and cell differentiation by interacting with transcription factors or altering the structure of chromatin.Animal adipose deposition is the result of adipocyte proliferation and differentiation and hypertrophy.Adipocyte differentiation is a process in which pluripotent stem cells are transformed into precursor adipocyte and then differentiated into mature adipocytes.Related studies have shown that the transcription factor peroxisome proliferator activiated receptor γ (PPARγ) and CCAAT enhancer binding protein family (CEBPs) play key regulatory roles in adipose deposition.Recent studies found that DNA methylation can participate in the differentiation of adipocytes and the growth and development of adipose tissue by regulating the expression of related genes in the process of adipogenesis.Demethylation can also affect the adipose deposition process in animals,but the specific mechanism is still unclear.This article introduced the definition,site of occurrence biological function,related enzyme and mechanism of DNA methylation and demethylation,then introduced the adipose deposition process and the role of transcriptional regulators such as PPARγ and C/EBPα in adipose deposition.The effects of DNA methylation and demethylation on the expression of genes involved in adipogenesis and adipocytes differentiation were highlighted.This paper provides a reference for clarifying the mechanism of adipose deposition and improving meat quality in animal production.

Preventive Veterinary Medicine
Epidemiological Survey of Six Major Viral Reproductive Disorders of Pigs in Guangxi During 2014 to 2018
DUAN Qunpeng, CHEN Zhongwei, HE Ying, ZHOU Yingning, LI Xiaoyu, LIANG Jiaxing, BI Bingfen, YANG Siyi, JIANG Dongfu, LU Jingzhuan, LI Bin, QIN Yibin, LU Bingxia, SU Qianlian, ZHAO Wu
2019, 46(2):  526-535.  doi:10.16431/j.cnki.1671-7236.2019.02.024
Abstract ( 178 )   PDF (1498KB) ( 93 )  
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The aim of the study was to understand the occurrence of major viral reproductive disorders of pigs in Guangxi and provide a scientific basis for the prevention and control of such diseases.6 major viral reproductive disease pathogens of swine fever virus (CSFV),porcine reproductive respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine Japanese encephalitis virus (JEV) from 349 aborted fetus samples from March 2014 to February 2018 in Guangxi were collected by RT-PCR/PCR,and the positive detection rate in different years and seasons and the mixed infection of pathogens were analyzed.The results showed that from 2014 to 2017,the positive rate of pig farms of six pathogens from high to low were JEV (36.00%),PRRSV (16.00%),PCV2 (9.00%),PRV (7.00%),PPV (6.00%) and CFSV (5.00%);The positive detection rate of samples from high to low were JEV (27.06%),PRRSV (6.18%),PRV (5.88%),PCV2 (3.82%),CSFV (2.35%) and PPV (2.35%).Seasonal factors had an impact on the occurrence of six viral reproductive disorders:The detection rate of JEV was higher in summer and autumn,and was lower in spring and winter;The detection rate of PRRSV was higher in all seasons,and there was no significant difference;PCV2 had higher detection rate in spring,followed by summer and winter, and the detection rate was lower in autumn;The detection rate of PRV was higher in spring and winter,and the detection rate was 0 in summer and autumn;The detection rate of PPV was higher in summer,followed by spring and autumn,and the detection rate was 0 in winter;CSFV had highest detection rate in winter,followed by summer,and the detection rate was 0 in spring and autumn.The abortion fetus was dominated by a single infection.The six viruses had different degrees of single infection.The single infection rate was the highest in JEV (23.82%) and the lowest in PCV2 (0.59%).The mixed infection was mainly caused by double infection.Among them,the double infection was more in 2016,and the infection combination was diverse.The triple infection was less,only in 2016.One case was found in the samples submitted,and no quadruple or more infections were found.The results showed that from 2014 to 2017,JEV had a high positive detection rate,which had become the most important viral pathogen in sow abortion in some pig farms in Guangxi.PRRSV and PRV still were the main viral pathogens causing abortion in sows in Guangxi.

Effects of Artificially Infected Duck Newcastle Disease Virus on the Expression of β-defensins and Cytokines in Chinese Shaoxing Ducks
XU Liwen, WANG Qiuling, WANG Fangfang, LIU Chenggang, MA Deying
2019, 46(2):  536-547.  doi:10.16431/j.cnki.1671-7236.2019.02.025
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To investigate the effects of artificial infected duck Newcastle disease virus (NDV) on the expression of avian β-defensins(AvBDs) and cytokines in Chinese Shaoxing duck,forty five 20-day-old SPF Chinese Shaoxing ducks were randomly divided into attack group (27) and control group (18).The ducks in attack group were intraocularly inoculated with 108.38 EID50 of virulent NDV strain (Md/CH/LGD/1/2005),others were mock-inoculated with phosphate-buffered saline (PBS).6 ducks in two groups were killed at 24 and 48 h,respectively.Nine tissues including spleen,lung,kidney,glandular stomach,bursa of Fabricius,cecal tonsil,trachea,harderian glands and marrow,were collected from ducks for Real-time quantitative PCR analysis to quantify the mRNA levels of AvBDs and cytokines.In addition,the remaining ducks were constantly observed every day,serum was collected,and specific antibody titres was evaluated at 4,8,12,16,20 and 24 d,respectively.Oropharyngeal and cloacal swabs were collected and specific antibody titres was evaluated at 24,48,72 and 120 h,respectively.The results showed that no obvious clinical signs were observed in the NDV infected ducks.The ducks began to detoxify at the 1st day after attack,and stopped at the 5th day,NDV was detected in the oropharyngeal and cloacal swabs of attack group.The antibody level test results showed that NDV could induce anti-NDV specific antibodies in ducks.Real-time quantitative PCR results showed that compared with control group,the expression levels of AvBD1,AvBD2,AvBD5,AvBD6,AvBD9 and AvBD16 were significantly increased in some tissues of NDV infected ducks at 24 h (P<0.05);The expression levels of AvBD1 and AvBD6 were significantly up-regulated in some tissues of NDV infected ducks at 48 h (P<0.05).The expression levels of IL-6 in bursa of Fabricius and IFN-γ in spleen at 48 h were lower than that at 24 h after infection.In summary,the duck-derived NDV strain infection could induce early natural immune responses in Chinese Shaoxing duck,and these reactions might be related to the replication of the virus in the host.

Isolation,Identification and Partial Biological Characteristics of Mannheimia haemolytica in Shipping Fever of Beef Cattle
HAN Xiaoli, REN Jingjing, YANG Mingwei, ZHU Ling, ZHANG Rui, YAN Genqiang
2019, 46(2):  548-556.  doi:10.16431/j.cnki.1671-7236.2019.02.026
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In order to identify the pathogens and biological characteristics of shipping fever of beef cattle.The bacteria of blood (heart),lung,liver and spleen were collected and isolated,biochemical tested,and PCR identified,the virulence gene detection and pathogenicity of the isolates were studied.The results showed that 7 isolates were Gram-negative brevibacterium with weak β-hemolysis,Wright's staining result showed that there was two-pole dense staining and obvious capsule.The biochemical test results showed that the isolated bacteria could ferment carbohydrates such as glucose,maltose,arabinose,mannitol,mannose,xylose etc.and do not ferment urease, MR-VP and indole.It could utilize carbohydrates such as glucose to produce a small amount of acid without gas production,and the results were consistent with the biochemical characteristics of Mannheimia haemolytica.The isolates were capsular serum A2 type by PCR,which contained three virulence genes of the type Ⅳ fimbriae-related gene ptfA,the copy-related gene dnaN,and the interleukin related gene LktC.The LD50 values of the bacteria in mice ranged from 107.83 to 108.50CFU/mL,there were some difference of LD50 values in different strains.The results showed that the pathogen which caused the shipping fever in the batch of beef cattle was Mannheimia haemolytica with capsular serum A2 type,which provided a reference for further study of the pathogenic mechanism of Mannheimia haemolytica.

Isolation,Identification and Pathogenicity Analysis of Bovine Viral Diarrhea Virus JLS-01 Strain from Porcine
ZHANG Shuqin, TAN Bin, LIU Zeyu, WANG Chao, GUO Li, SUN Na, CHENG Shipeng
2019, 46(2):  557-564.  doi:10.16431/j.cnki.1671-7236.2019.02.027
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In order to understand the molecular characteristics and pathogenicity of bovine viral diarrhea virus (BVDV),in this study,RT-PCR was used to detect BVDV nucleic acid positive in piglets with severe diarrhea symptoms in a pig farm in Jilin province.The treated BVDV positive samples were inoculated into MDBK cells,and one strain of virus was isolated and designated as BVDV JLS-01.The molecular evolution characteristics were analyzed by immunofluorescence detection and 5'UTR and Npro RT-PCR amplification.The results showed that the isolated strain was blindly transmitted to MDBK cells for 8 generations without cytopathic effect,and showed a positive fluorescent signal in the immunofluorescence assay.5'UTR and Npro fragments of 280 and 735 bp in size were obtained by RT-PCR amplification.The genetic evolution analysis of the 5'UTR and Npro sequences of BVDV JLS-01 strain showed that it was closely related to LN-1 and ZM-95,and the homology with the LN-1 of bovine source strain was 99.3%,suggesting that the strain might be derived from a bovine strain.BVDV and classical swine fever virus (CSFV) antibody negative pigs were artificially infected with BVDV JLS-01 strain F8 cell culture medium,the infected pigs did not show significant elevation of body temperature,but the number of white blood cells decreased,and the virus strain was isolated from the leukocyte extract of infected pigs,indicating that the virus strain had certain pathogenicity.The successful isolation of this strain was of great significance for further research on the epidemiological investigation and pathogenesis of BVDV.

Establishment and Application of Visual LAMP Detection Method for Infectious Laryngotracheitis Virus
XIE Jing, YU Jifeng, ZHOU Long, CAO Ye, LIN Yi, LI Xingyu, XIAO Lu, YE Yonggang, PAN Meng, KANG Runmin
2019, 46(2):  565-572.  doi:10.16431/j.cnki.1671-7236.2019.02.028
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This study was aimed to establish a simple,rapid and efficient method for detection and diagnosis of infectious laryngotracheitis virus (ILTV).The specific perimers were designed for the loop mediated isothermal amplification (LAMP) reaction based on the conservative region of ILTV TK gene in GenBank.The LAMP detection method for ILTV was established through the optimization of the LAMP reaction system and reaction conditions,as well as the specificity,sensitivity and detection of clinical samples.The results showed that the established LAMP reaction exhibited the highest amplification efficiency under the parameters of internal primers (ILT9 FIP and ILT9 BIP),outer primers (ILT9-F3 and ILT9-B3),ring primers (ILT9-LB and ILT9-LF) and reaction temperature (66℃).The established LAMP detection method could specifically detect ILTV (Hungarian and Wanggang strains),there was no cross-react with Newcastle disease virus (NDV,B strain),avian infectious bronchitis virus (IBV,H52 and H120 strains),Escherichia coli,Haemophilus parasuis, Pasteurella,etc.Meanwhile,the sensitivity of the established LAMP detection method was 0.06 pg/L,and its sensitivity was 100 times of the conventional PCR method.50 clinical samples were tested by establishing LAMP method,the positive rate was 14%,and the coincidence rate was 96% with PCR method.This study established a LAMP detection method with high specificity,high sensitivity and simple operation,which was suitable for rapid detection and diagnosis of ILTV in clinical application.

Isolatiog,Identification and Study of a Bacillus sp.B13 with High Antibiotic Character
LIU Shaona, ZHANG Bin, XIANG Decai, ZHAO Zhiyong, ZHAO Yanguang
2019, 46(2):  573-581.  doi:10.16431/j.cnki.1671-7236.2019.02.029
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The aim of the experiment was to find a new antimicrobial bacterium,then analyze its main metabolic antimicrobial characterization.A strain was isolated from pig intestinal contents with resistance to E.coli K88,Staphylococcus aureus and Salmonella Typhimurium by gel punching.Its taxonomic status determined by colonial morphology,Gram staining and 16S rDNA sequence analysis.Then the fermentation medium and conditions for its growth and antimicrobial activity were optimized.The results showed that the isolated strain was identified as Bacillus sp.and showed an excellent antimicrobial activity against E.coli K88,Staphylococcus aureus and Salmonella Typhimurium,and then was named as Bacillus sp.B13.It was Gram-positive,single-birth or in pairs of growth.Auxanographic analysis showed that the optimal growth condition of Bacillus sp.B13 was that the pH was 6.0,temperature was 34℃,sucrose as carbon source and peptone as nitrogen source.Results of antimicrobial activity against E.coli K88,Staphylococcus aureus and Salmonella Typhimurium showed the optimal condition was that pH was 7.0,temperature was 35℃,maltose as carbon source and peptone as nitrogen source.In conclusion,Bacillus sp.B13 had good bacteriostatic activities and had great development potential applied prospects in the production.

Isolation and Identification of Porcine Epidemic Diarrhea Virus Strain JS2017 and Its S Gene Sequence Analysis
ZHU Haixia
2019, 46(2):  582-589.  doi:10.16431/j.cnki.1671-7236.2019.02.030
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In order to understand the genetic variation of porcine epidemic diarrhea virus (PEDV) in a large-scale pig farm in Jiangsu,the intestinal content of piglets with watery diarrhea were collected and detected for PEDV by RT-PCR.The diseased tissue detected as positive was treated with 20 μg/mL trypsin,and then inoculated into Vero cells for culture.After the cell culture was repeatedly frozen and thawed,the virus solution was harvested and identified by RT-PCR.The S gene was amplified by a segmentation overlap strategy,and the nucleotide homology and genetic evolution of S gene was analyzed.The results showed that the pathological tissue was positive for PEDV by RT-PCR.After 4 generations of inoculation with Vero cells,the positive pathogens showed obvious cytopathic changes,which showed cell syncytosis,vacuolization and finally lysis.After the harvested virus solution was identified by RT-PCR,it was confirmed that one PEDV was isolated and designated as JS2017 strain.Sequence analysis showed that the S gene sequence of JS2017 strain was in the same evolutionary branch as the American variant strain OH1414,Canadian variant strain ON-007,Chinese variant strain PEDV-LYG and CH/PDS/2015,and its nucleotide homology was as high as 99.0%.Among them,the Chinese mutant strain YC2014 had the closest relationship,and its nucleotide homology was 100.0%;JS2017 strain had low homology with the classical strains CV777 and DR13,and had a distant relationship.The results showed that the JS2017 strain isolated in this study was a PEDV local epidemic strain,which was closely related to the 2014 Jiangsu isolate (YC2014 strain), and had a far-reaching relationship with the current swine epidemic diarrhea vaccine strain.

Clinical Veterinary Medicine
Immunopotentiation Effect of Recombinant Chicken Interleukin-6/2 Fusion Protein on Newcastle Disease Virus (LaSota) Live Vaccine
LIU Meifen, LI Qinnan, MA Zhenyuan, WU Zhiming, BAN Fuguo, YAN Ruoqian, WANG Huajun
2019, 46(2):  590-599.  doi:10.16431/j.cnki.1671-7236.2019.02.031
Abstract ( 150 )   PDF (1601KB) ( 83 )  
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In order to study the immunopotentiation effect of recombinant chicken interleukin-6/2 fusion protein (rChIL-6-Linker-ChIL-2) on Newcastle disease vinus (NDV) live vaccine (LaSota vaccine),90 SPF chickens were chosen and randomly distributed into six groups which were the PBS control group,NDV live vaccine control group,rChIL-6-Linker-ChIL-2 immunopotentiation group,rChIL-6 protein immunopotentiation group,rChIL-2 protein immunopotentiation group and the rChIL-6+rChIL-2 fusion protein immunopotentiation group.The interleukin fusion protein fluid and NDV live vaccine (LaSota vaccine) were inoculated into SPF chickens.The indexes of proliferation activities of peripheral blood and splenic lymphocytes,percentages of the CD3+CD4+/CD3+CD8+ T-lymphocytes in peripheral blood,the contents of Th1/Th2-type cytokines and the specific immune antibody level were detected by assays of MTS,FCM,ELISA and HA/HI.The results showed that compared with the indexes of NDV vaccine inoculation control group,the lymphocytes proliferation activity,the percentage of CD3+CD4+/CD3+CD8+ T-lymphocytes,and the contents of Th1/Th2-type cytokines such as ChIL-4,ChIL-6,ChIL-2,ChIFN-α and ChIFN-γ of chickens in rChIL-6-Linker-ChIL-2 inoculation group were promoted obviously at 7 to 21 dpi,and the HI immune antibodies increased about 1.0 to 1.9 titer,which was 0.2 to 0.7 titer higher than that of rChIL-6 group and 0.2 to 1.1 titer higher than rChIL-2 that of group.In conclusion,the rChIL-6-Linker-ChIL-2 protein had an obvious immunopotentiation effect on NDV live vaccine (LaSota) in chickens.

Study on the Therapeutic Effect of Modified Yujin Powder on Transimissible Gastroenteritis in Landrace Pigs
SONG Chunjie, JI Mei, XU Jia, HUO Bin, CHI Yongkuan, SHEN Xiaoyun
2019, 46(2):  600-607.  doi:10.16431/j.cnki.1671-7236.2019.02.032
Abstract ( 207 )   PDF (934KB) ( 77 )  
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This experiment was to study the therapy of modified Yujin powder on transimissible gastroenteritis in Landrace pigs.20 adult Landrace pigs with gastroenteritis were selected from the Shunde farm in Zhangjiakou and randomly divided into low dose group,high dose group,western medicine group and control group with 5 pigs per group.The pigs in modified Yujin powder group were given Yujing powder once every morning and evening,6,7 mL/(kg·BW) for the low dose group and high dose group,respectively.The pigs in western medicine group were fed rimantadine and injected with florfenicol subcutaneously,once every morning and evening.The pigs in control group were fed normally and no medicine was given.After 7 d,the clinical symptoms and growth performance of the pigs were observed,and the blood routine,serum biochemical indexes,serum immune indexes and antioxidant indexes were measured.The results showed that Landrace pigs were sensitively reaction with normal body temperature,and the food intake was increased and feces returned to normal in the modified Yujin powder group.The healing rates were 80%,90% and 60% in low dose group,high dose group and western medicine group,respectively.The effects of modified Yujin powder was better than that in western medicine.The ADG and ADFI of high dose group were extremely significantly increased and the F/G was significantly reduced compared to control group (P<0.01).The contents of BUN,MDA and WBC in 3 drug delivery groups were extremely significantly lower than the control group (P<0.01),and the contents of K,TP,ALB,GLB and T-AOC in the 3 drug delivery groups were extremely significantly higher than that of control group (P<0.01),but there were no remarkable differences between the 3 groups (P>0.05).The serum CAT activity in the high dose group was extremely significantly higher than that in the low dose group,high dose group and control group (P<0.01).In conclusion,modified Yujin powder had a good therapeutic effect on the transimissible gastroenteritis in Landrace pigs,and the effect of the high dose group was better.

Basic Veterinary Medicine
Pharmacodynamics and Molecular Mechanism Analysis of Zixiekang Oral Liquid
LUO Lirong, CUI Dongan, WANG Hui, HAO Zhihui, WANG Shengyi
2019, 46(2):  608-620.  doi:10.16431/j.cnki.1671-7236.2019.02.033
Abstract ( 204 )   PDF (12372KB) ( 85 )  
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The purpose of the experiment was to explore the pharmacodynamics and molecular mechanism of Zixiekang oral liquid in treating piglet diarrhea,and provide experimental basis for the further development of the drug.The pharmacological effects of Zixiekang oral liquid were verified by small intestinal propulsion test,diarrhea control test,ear swelling inhibition test,analgesic test and in vitro bacteriostasis test.Meanwhile,the active components of Zixiekang oral liquid were screened by network pharmacology.Target prediction and bioinformatics were used to determine the specific target of Zixiekang oral liquid for treating diarrhea.The molecular mechanism of the treatment of diarrhea was discussed by signal pathway enrichment analysis.The results showed that the anti-inflammatory and analgesic effects of Zixiekang oral liquid increased with the increase of dosage,and low dose of Zixiekang oral liquid had significant difference compared with blank control group (P<0.05);Compared with blank control group,the low,middle and high dose groups could all inhibit the propelling distance and pushing rate of small intestine carbon ink in mice,and the low dose group had significant difference (P<0.05).Zixiekang oral liquid had bacteriostasis effect on both Escherichia coli and Staphylococcus aureus,and had the more obvious effect on Escherichia coli.In this experiment,the antidiarrhea mechanism of Zixiekang oral liquid was predicted by network pharmacology,and it participated in Wnt signaling pathway to regulate the bacterial invasive epithelial cells,and also affected pathogenic Escherichia coli infection signal pathway;MAPK signaling pathway regulated the production of other inflammatory mediators by promoting or inhibiting the transcription of genes.At the same time,inflammatory mediators could increase the expression of various adhesion molecules on the cell surface by activating the MAPK pathway,and promote the progress of intercellular adhesion and inflammation.NRG/ErbB signal was involved in many aspects of neuropathic pain.In the central nervous system,microglia were activated by MEK/ERK pathway and a series of cytokines and inflammatory mediators were released to promote neuropathic pain.Neuropathic pain was also involved in neuropathic pain by regulating synaptic plasticity and central disinhibition.Peripheral nervous system,NRG/ErbB signal played an important role in analgesia by regulating the function of non-myelin forming cells and myelin forming Schwann cells and participating in ErbB signal of peripheral neuralgia.In summary,Zixiekang oral liquid might inhibit diarrhea from four aspects:Intestinal peristalsis,anti-inflammation,analgesia and bacteriostasis.

Establishment and Application of Fluorescence Quantitive Immunochromatographic Assay for Canine C-reactive Protein
KANG Ye, WANG Yu, HU Shukun, WU Peidian, LI Wenmei, HE Xiaowei
2019, 46(2):  621-627.  doi:10.16431/j.cnki.1671-7236.2019.02.034
Abstract ( 353 )   PDF (2433KB) ( 214 )  
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The study focused on the establishment of fluorescent microsphere immunochromatography quantitative method of canine C-reactive protein (C-CRP) to a develop test strips of simple operation and high sensitivity for rapid quantitative detection of C-CRP.A fluorescent sandwich immunoassay via double antibody was set up,anti-C-CRP monoclonal antibody and goat anti-chicken IgY labeled with carboxy fluorescent microspheres were used as labeled antibody,the other anti-C-CRP monoclonal antibody and chicken IgY were used as capture antibody for detection lines and control line.The results showed that the detection range of the lateral flow assay was 0.5 to 250 mg/L,the detection limit was 0.5 mg/L,and the CV in intra-assay and inter-assay were 0.68% to 6.94% and 0.89% to 8.79%,respectively.The average recovery was 98.15% to 101.19%,and there was no cross-react with 9 interfering substances.The stressed condition stability test showed that the test strip could be stored for 24 months at room temperature.For clinical sample test,the test strips had a good correlation with the control reagent (R2=0.995).In conclusion,the prepared fluorescent immunochromatographic strips could be applied to clinical test of C-CRP concisely and conveniently.

Research Progress on the Relationship Between Gut Microbiota of Animals and Pathogenic Microorganism Infection
CHEN Xiuqin, HUANG Meiqing, ZHENG Min, CHEN Shaoying
2019, 46(2):  628-634.  doi:10.16431/j.cnki.1671-7236.2019.02.035
Abstract ( 252 )   PDF (759KB) ( 482 )  
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Animals harbor a densely populated resident microbial community,generally referred as the commensal microbioata,which are pivotal in determining the developmental,metabolic and immunological status of the host.They are active participants in the progression of many diseases.The invasion of viruses,bacteria and parasites result in a disturbance to the gut microbiota,mainly is shown by a decrease of probiotics,while outgrowth of pathogens.The mechanisms include eliciting inflammatory host response and inhibiting immune cell.Additionally,gut microbiota also accommodate pathogenic organisms.For example,it plays a dual role in viral replication,inhibits bacterial replication,but promotes parasite infection.The mechanisms that regulate the ability of the gut microbiaota to restrain pathogen growth including competive metabolic interactions and induction of host immune responses.And the mechanisms of viral promoting involved are listed as follow:Improving the stability of virus and enhancing their attachment to target cells,inhibiting the immune system,promoting the proliferation of target cells.The mechanisms of parasite promoting maybe decrease Th2 cytokines (such as IL-4 and IL-13),as well as with heightening the frequency expression of regulatory T cell.The interactions among gut microbiota,pathogen and host affect are ongoing and continuously evolving throughout the infection process.This review focused on the impact of viruses,bacteria and parasites on the abundance of animals' gut microbiota,the process of pathogen infection impacted by animals' gut microbiota and the relevant mechanism.A goal of this work will facilitate our understanding of the mechanism of infection.What's more,it can also provide novel insights and theory for developing vaccines adjuvants and designing more effective and focused prophylactic and therapeutic strategies.

Isolation,Identification and Drug Sensitivity Analysis of Salmonella from Minks
SUN Na, CHEN Qiang, CHENG Yuening, YANG Yong, WANG Gaili, LIU Zeyu, ZHANG Shuqin, CHENG Shipeng
2019, 46(2):  635-641.  doi:10.16431/j.cnki.1671-7236.2019.02.036
Abstract ( 170 )   PDF (2813KB) ( 94 )  
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In order to guide rational drug use and provide the basis for treatment of diseased mink,the bacteria of seven sick minks suffering from salmonellosis provided by mink farms in Shandong province were isolated and identified,and the drug sensitivity was tested.The strains were isolated from the samples by separation and purification methods,and the isolated strains were identified by Gram staining,biochemical identification and PCR.K-B susceptibility test was used to detect the sensitivity of strains to commonly used drugs,and the drug resistance gene and class Ⅰ integrants were detected by PCR.The results showed that 7 strains were isolated,all of which were Gram-negative and short bacilli.Biochemical reaction tests showed that the isolates were positive for glucose,maltose,mannitol,MR test,citrate and hydrogen sulfide test,and the isolates were preliminarily identified as Salmonella.The sequencing results of the PCR products further indicated that 7 isolated strains were Salmonella.The drug sensitivity test showed that 7 strains of Salmonella were sensitive to ceftriaxone,levofloxacin,florfenicol and polymyxin,and resistant to all of the aminoglycosides,tetracycline and ampicillin.The results of drug resistance gene test showed that 8 kinds of resistance genes were detected in 7 strains of Salmonella,blaTEM-1,blaCTX-M-1G,aadA1, aac(3')-Ⅳ,aac(3')-Ⅱc,aph(4')-Ⅰa,aph(3')-Ⅶ and oqxAB,and the class Ⅰ integrin of the gene cassettes aaA1,arr-3-aacA4 and blaPSE-1 were detected.According to the above results,7 strains of Salmonella were isolated from the liver and spleen samples of 7 diseased minks,and the isolates were all resistant strains,and mainly showed multi-drug resistance;The drug resistance genes were diverse,and the presence of drug resistance genes on the plasmid expanded the spread of drug resistance genes and increased the drug resistance of bacteria,which brought difficulties for clinical drug treatment.