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05 December 2024, Volume 51 Issue 12
Biotechnology
Whole Genome Sequencing of Bacteriocin-producing Bacillus velezensis CM7-4 and Its Bacteriostatic Activity
MA Haotian, LI Yang, YU Mengbo, PAN Ruixue, LONG Yuner, ZHAO Yining, PENG Jinju, MA Yi
2024, 51(12):  5117-5127.  doi:10.16431/j.cnki.1671-7236.2024.12.001
Abstract ( 127 )   PDF (14441KB) ( 93 )  
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【Objective】 Bacillus velezensis CM7-4 was isolated from seawater and had been demonstrated to possess good antibacterial activity.This study was to investigate the antimicrobial mechanism of Bacillus velezensis CM7-4 and analyze the whole genome sequence in order to explore its antibacterial and other functional genes.【Method】 The bacteriocin crude extract of Bacillus velezensis CM7-4 was obtained by acid precipitation,and the effect of proteinase K on its antibacterial effect was determined by the solid agar perforation method.Subsequently,the complete genome of Bacillus velezensis CM7-4 was sequenced on the MGI DNBSEQ-T7 platform.Their bacteriocin production potential was analyzed by sequence assembly,gene prediction and functional annotation.【Result】 The bacteriocin crude extract of Bacillus velezensis CM7-4 demonstrated a notable bacteriostatic effect on a diverse range of 8 kinds of Gram-negative and positive bacteria,including Escherichia coli,Salmonella,Enterococcus faecalis,Listeria monocytogenes and others. Compared with control group, the bacteriocin crude extract demonstrated a extremely significantly diminished inhibitory effect on the eight indicator bacteria following treatment with proteinase K (P<0.01).Whole genome sequencing revealed that the genome size of Bacillus velezensis CM7-4 was 3 953 124 bp with a 47.04% GC content.A total of 3 870 protein-coding genes,9 rRNA, 81 tRNA and 1 ncRNA were identified.And 11 bacteriocin synthesis genes,10 antioxidant activity genes,and 4 immune and inflammatory signalling pathway regulation genes were predicted.【Conclusion】 The results of this experiment indicated that Bacillus velezensis CM7-4 was a potential bacteriocin-producing strain.This study provided a theoretical basis for further study of its antibacterial properties.
Construction of Brucella Effector Protein BspE Gene Deletion Strain and Its Biological Characteristics
ZHANG Silan, ZHI Feijie, DING Jian, SONG Yinjuan, ZHENG Fuying, FU Qiang, CHU Yuefeng, SHI Huijun, XU Jian
2024, 51(12):  5128-5137.  doi:10.16431/j.cnki.1671-7236.2024.12.002
Abstract ( 83 )   PDF (6428KB) ( 54 )  
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【Objective】 The purpose of this study was to construct the BspE gene deletion strain of Brucella abortus (B.abortus) A19 strain,explore the growth characteristics of survival,adhesion and invasion in host cells,and also observe the subcellular localization of BspE protein of B.abortus A19 during infection.【Method】 B.abortus A19 was used as the research object.The Brucella effector protein BspE gene deletion strain A19ΔBspE was constructed by homologous recombination and SacB reverse screening technology,and its complement strain A19CΔBspE was constructed by plasmid complementation method.The growth of three strains was measured and the influence of BspE gene deletion on the survival,adhesion and invasion of Brucella in cells was analyzed.The localization of BspE protein in RAW264.7 cells was observed by indirect immunofluorescence assay.【Result】 PCR results of the bacterial solution showed that a specific band with a size of 2 060 bp was obtained,and the BspE gene deletion strain A19ΔBspE was successfully constructed.Western blotting results showed that a BspE-flag band with a size of approximately 14.59 ku was obtained,and the complementation strain A19CΔBspE was successfully constructed.The growth curve results showed that under the same culture conditions,there was no significant difference among B.abortus A19,A19ΔBspE and A19CΔBspE (P>0.05),all of them reached the logarithmic growth phase at 8 h and entered the platform phase at 32 h.The results of intracellular proliferation test showed that the viability was not significantly different from B.abortus A19,A19ΔBspE and A19CΔBspE during 48 h post infection in RAW264.7 cells (P>0.05).The results of bacterial adhesion and invasion test showed that the ability of A19ΔBspE to adhere and invade RAW264.7 cells was not significantly different from that of B.abortus A19 and A19CΔBspE (P>0.05).The result of indirect immunofluorescence assay showed that BspE protein was mainly distributed in the perinuclear of RAW264.7.【Conclusion】 This study proved that the deletion of BspE gene did not affect the growth of Brucella in vitro and the survival,adhesion and invasion in RAW264.7 cells,and BspE protein was mainly located in the perinuclear region,which laid a foundation for the biological function and pathogenic mechanism of Brucella effector protein BspE.
Screening of Candidate Proteins Regulating Egg Green-shell Traits Based on TMT Quantitative Proteomics Technology
ZHAO Depeng, XIAO Tao, LONG Xia, LUO Wei, YE Tao, YU Huan, CHEN Youbo, SHI Yushi, WANG Wenliang, LI Hui
2024, 51(12):  5138-5149.  doi:10.16431/j.cnki.1671-7236.2024.12.003
Abstract ( 76 )   PDF (11328KB) ( 104 )  
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【Objective】 As an important economic trait of green-shell eggs,eggshell color not only affects consumers’ purchase preference,but also directly affects the market pricing of green-shell eggs,and the color of green-shell eggs is regulated by multiple genes.The purpose of this study was to explore the candidate proteins and key signaling pathways that regulated the color of green-shell eggs,and provide materials for optimizing the selection and breeding of green-shell chickens and improving the economic value of green-shell eggs.【Method】 Chishui Black-bone chickens in the high-yield period of the pure line were used as the research objects,the differentially expressed proteins in the eggshell gland tissues of Chishui Black-bone chickens producing light green eggs (QL) and dark green eggs (SL) were analyzed by tandem mass tags (TMT) quantitative proteomics technology.GO function and KEGG pathway enrichment analysis were performed to screen candidate proteins that regulated the color depth of green shell eggs and verify them by Western blotting.【Result】 A total of 5 958 quantifiable proteins were identified,including 102 differentially expressed proteins,of which 46 were up-regulated and 56 were down-regulated.GO functional analysis showed that differentially expressed proteins were mainly enriched in the receptor signaling pathway,vitamin A response and response to estrogen in biological process.Molecular function was primarily enriched in the items of serine hydrolase activity,carbohydrate transmembrane transporter activity,retinol-binding,etc.Cellular component was mainly enriched in items such as egg yolk,collagen trimer and blood particles.Six pathways were significantly enriched in KEGG pathway analysis,among which steroid hormone biosynthesis pathway might change the formation of eggshell color by affecting the eggshell calcification process.Western blotting results showed that the expression of HSD17B7 and TXNRD2 proteins in eggshell glands of Chishui Black-bone chickens in SL group were extremely significantly higher than that in QL group (P<0.01),and the expression trend was consistent with the proteomics results.【Conclusion】 The significant up-regulation of SLC22A15b,SLC35A,SLC2A1,SLC4A4,HSD17B7 and TXNRD2 proteins,and the significant down-regulation of KNG1 protein in differentially expressed proteins might be relate to the formation of eggshell color.The results could provide reference for elucidating the molecular mechanism of pigmentation in green-shell eggs.
Phylogenetic and Bioinformatics Analysis of F Gene of Pigeon Paramyxovirus Type Ⅰ from Shanghai Area
LIU Jian, ZHOU Jinping, SHEN Liping, GUI Yaping, GE Feifei, WANG Xiaoxu, YANG Xianchao, LIU Peihong, WANG Jian
2024, 51(12):  5150-5161.  doi:10.16431/j.cnki.1671-7236.2024.12.004
Abstract ( 69 )   PDF (9288KB) ( 34 )  
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【Objective】 The purpose of this study was to understand the genetic variation of F gene of Pigeon paramyxovirus type Ⅰ (PPMV-1) epidemic strain in Shanghai area,analyze the bioinformatic characteristics of F protein,and provide technical support for the prevention and control of PPMV-1.【Method】 F gene of the isolate was amplified by RT-PCR,and sequencing and genetic evolution analysis were performed.The structure and function of F protein were predicted by online bioinformatics software.【Result】 The total length of the F gene coding region of the isolate was 1 662 bp,encoding 553 amino acids,and the F protein cleavage site sequence was 112R-R-Q-K-R-F117,which was consistent with the sequence structure of the virulent strain.Phylogenetic tree analysis showed that the isolate belonged to Class Ⅱ group Ⅵ.2.1.1.2.2 subtype of Newcastle disease virus (NDV).The isolate was in a different evolutionary branch from the vaccine strains La Sota and Mukteswar,and belonged to the same branch Ⅵ.2.1.1.2.2 with the domestic isolates Pi/SH/CH/0617/2013 and pigeon/Ningxia/2068/2016.The results of bioinformatics analysis showed that F protein was a hydrophilic protein with molecular mass of 59.03 ku,theoretical isoelectric point (pI) of 7.87,half-life period of 30 h,instability coefficient of 35.06 and fat coefficient of 109.73.F protein was mainly distributed in the plasma membrane and endoplasmic reticulum of the cell,and had a transmembrane structure and signaling peptides,including 6 potential N-glycosylation sites,13 cysteine residues,103 O-glycosylation sites,and 67 phosphorylation sites.In the secondary structure,alpha helix,random coil,extended chain and beta turn account for 46.11%,31.46%,18.08% and 4.34%,respectively.The predicted result of tertiary structure was consistent with the secondary structure.B-cell antigen site prediction showed that F protein contained 11 epitopes (amino acid number ≥7).【Conclusion】 The PPMV-1 isolate belonged to NDV Class Ⅱ group Ⅵ.2.1.1.2.2 subtype of NDV,and its F protein had a good epitope,which could be used as the target protein for PPMV-1 vaccine development and antiviral therapy.The results provided a certain reference for effective prevention and control of PPMV-1 infection,and also laid a foundation for further research and development of new vaccines.
Metagenomic Analysis of Calf Intestinal Viral Population in Daqing Region
CHEN Tianjie, LI Yuduo, ZHOU Hualiang, ZHAO Huijun, ZHAO Jianjun
2024, 51(12):  5162-5173.  doi:10.16431/j.cnki.1671-7236.2024.12.005
Abstract ( 66 )   PDF (26813KB) ( 25 )  
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【Objective】 The experiment was aimed to comprehend the composition and genetic variability of calf intestinal viruses within the dairy cattle population in the Daqing region,and furnish a theoretical framework for the detection of viral pathogens responsible for diarrhea in local calves.【Method】 A total of 155 fecal samples of calves were collected from dairy farms surrounding Daqing,which were divided into six diarrhea sample pools and three clinically asymptomatic sample pools.Total nucleic acid was extracted,high-throughput sequencing was performed based on Illumina sequencing platform,SPAdes and SOAPdenovo were used for sequence splicing and assembly,and viral population composition and genetic variation were analyzed according to the obtained sequences.【Result】 A total of 6 517 147 viral-related sequences from 21 viral families were successfully annotated.In the calf intestinal viral community,except for bacteriophaes,Coronaviridae exhibited the highest abundance,followed by Picornaviridae.Comparison of annotation results of the diarrhea samples with asymptomatic samples,the diversity of the virus population in the asymptomatic samples was significantly higher than that in the diarrhea samples,and it was found that the relative abundance of Parvoviridae, Picornaviridae and Reoviridae was higher in the diarrhea group samples compared to the non-diarrhea group.This study annotated multiple novel sequences of calf diarrhea viruses such as Bovine torovirus (BToV),Bovine kobuvirus (BKV) and Bovine norovirus (BNoV).In addition,for the first time,Bovine nebovirus was identified in calf feces in Heilongjiang region.Genetic evolution analysis on the assembled genomes of BRV,BCoV and BoAstV was conducted,and it showed that the predominant genotypes of BRV were identified as G6,G10,P[5] and P[11].The assembled full-length genome of BCoV showed the closest phylogenetic relationship with Coronavirus/Bo/BCoV6/2021/CHN,a prevalent strain in China.BoAstV sequences clustered consistently within the branch of enteric tissue tropism strains.【Conclusion】 The abundance of Coronaviraceae was the highest in the calf enterovirus population in Daqing area.The abundance of viral population in the diarrhea group samples was lower than that in the non-diarrhea group samples.Several calf diarrhea viruses,such as BToV,BKV and BNoV were annotated in the fecal samples.Additionally,BNeV was first annotated in calf feces from Heilongjiang province.Genetic evolution characteristics of local prevalent viruses such as BRV,BCoV and BoAstV were analyzed.These findings provided a theoretical basis for the detection of viral pathogens causing diarrhea in local calves.
Cloning and Bioinformatics Analysis of CERS4 Gene in Hetian Sheep and the Effect of Androgens on Its Expression Distribution in the Skin
FENG Zhiya, PENG Wanwan, YOU Xiaosong, WU Zhenhui, ZHANG Jianping, ZHANG Nan, SHI Ruijun, LI Shuwei
2024, 51(12):  5174-5184.  doi:10.16431/j.cnki.1671-7236.2024.12.006
Abstract ( 70 )   PDF (16363KB) ( 36 )  
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【Objective】 The aim of this study was to investigate the effect of androgens on the skin structure of Hetian sheep,and understand the role of ceramide synthases 4 (CERS4) gene and protein in the influence of androgens on the skin structure of Hetian sheep.【Method】 15 2-year-old Hetian ewes were selected and randomly divided into 5 groups.Every three days,0,1,2,3 and 4 mg/kg of testosterone were injected into the muscles of Hetian ewes in the control group and experimental groups 1,2,3 and 4.The treatment lasted for 42 days,and the back skin tissue was collected on the 42nd day.Firstly,the skin samples from control group were selected for cloning and sequencing of the CDS region of the CERS4 gene.A phylogenetic tree of the amino acid sequence of the CERS4 protein in Hetian sheep was constructed,and the physicochemical properties and protein structure of the CERS4 protein in Hetian sheep were analyzed.Afterwards,by making skin tissue slices and conducting Masson staining,the variations in skin structure after different doses of testosterone treatment were investigated in this study.Real-time quantitative PCR was used to detect the expression of CERS4 gene in Hetian sheep skin treated with different doses of testosterone,and immunohistochemistry was used to observe the distribution of CERS4 protein in Hetian sheep skin.【Result】 The CDS of CERS4 gene in Hetian sheep had been successfully cloned,with a length of 1 182 bp,encoding 393 amino acids.Similarity analysis showed that CERS4 gene of Hetian sheep had the highest similarity with Ovis aries.The results of the phylogenetic tree analysis showed that Hetian sheep belonged to the same branch of evolution as Ovis aries,and had the farthest genetic relationship with Homo sapiens and Mus musculus.Bioinformatic analysis showed that CERS4 protein of Hetian sheep was a hydrophilic protein,with no signal peptide and might have 5 transmembrane domains.The CERS4 protein of Hetian sheep was mostly located in the endoplasmic reticulum and cytoplasmic membrane,with the least content in the Golgi apparatus,nucleus,and mitochondria.This protein had a correlation of 95% or more with W5Q2R1_SHEEP,ACER3,and SGMS1.It was found that the number and area of sebaceous glands in Hetian sheep evidently expanded after treatment with testosterone (P<0.05).The CERS4 gene expression of the testosterone-treated skin samples in Hetian sheep were outstandingly higher compared with the control skin samples (P<0.05).CERS4 protein was expressed in the skin mainly in the sebaceous gland site.【Conclusion】 The CERS4 gene of Hetian sheep was been successfully cloned.Androgen treatment promoted an extend in sebaceous gland area and quantity in Hetian sheep,and prominently enhanced the expression of skin CERS4 gene.This study provided a theoretical basis for further understanding the impact and mechanism of androgens on the proliferation and differentiation of Ovis aries sebaceous gland,and had theoretical reference significance for solving skin health problems caused by androgens.
Cloning,Bioinformatics Analysis and Expression Vector Construction of CEBPA Gene in Rugao Yellow Chicken
KONG Ruihong, XIE Ke, WANG Xu, WU Han, LIU Jiying, WANG Yingjie
2024, 51(12):  5185-5197.  doi:10.16431/j.cnki.1671-7236.2024.12.007
Abstract ( 70 )   PDF (12661KB) ( 51 )  
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【Objective】 The aim of this experiment was to investigate the biological characteristics and tissue expression level of CCAAT/enhancer binding protein alpha (CEBPA) gene in Rugao Yellow chicken,and obtain its expression vector.【Method】 The CDS region of CEBPA gene of Rugao Yellow chicken was cloned and sequenced,homology comparison between Rugao Yellow chicken and other species was performed,and phylogenetic tree was constructed.The physicochemical properties,phosphorylation sites,glycoylation sites,functional domains,secondary and tertiary structures,interacting proteins of CEBPA protein of Rugao Yellow chicken were predicted by bioinformatics software.The expression of CEBPA gene in the heart,liver,lung,kidney,duodenum,jejunum,glandular stomach,muscle and testis of Rugao Yellow chicken was detected by Real-time quantitative PCR.Finally,the overexpression vector (OE-CEBPA) and interference vectors(CEBPA-sh12,CEBPA-sh167 and CEBPA-sh727) of CEBPA gene were constructed,and the activities of each vector were detected.【Result】 The CDS region of CEBPA gene in Rugao Yellow chicken was 975 bp in length,encoding 324 amino acids.Homology comparison results showed that the amino acid sequence similarity of CEBPA in Rugao Yellow chicken with Coturnix japonica,Anser cygnoides,Columba livia and Anas platyrhynchos was more than 94%,with the similarity with Coturnix japonica was the highest (99.4%).The phylogenetic tree results showed that Rugao Yellow chicken was closely related to Coturnix japonica,but far related to mammals.The CEBPA protein of Rugao Yellow chicken was hydrophilic and unstable,with 25 phosphorylation sites,including 9 N-glycosylation sites and 109 O-glycosylation sites.CEBPA protein had a transcriptional activation region and a DNA-binding leucine zipper structure bZIP.The secondary structure of CEBPA protein of Rugao Yellow chicken was mainly composed of 193 random coil,93 alpha helix,25 extension chains and 13 beta turn,and the prediction results of tertiary structure were basically consistent with the secondary structure.CEBPA protein mainly interacted with TRIB1,PPARG,RXRA,MAPK9,MAPK10,JUN,ESR1 and other proteins.CEBPA gene was expressed in all tissues of Rugao Yellow chicken,and the expression was the highest in liver.The CEBPA overexpression vector and three interfering vectors were successfully constructed and transfected into chicken fibroblasts,respectively.Compared with the control group,the CEBPA gene expression was significantly increased in the OE-CEBPA group (P<0.05),and significantly decreased in the CEBPA-Sh167 group (P<0.05).【Conclusion】 The complete CDS region of CEBPA gene in Rugao Yellow chicken was obtained in this experiment,and the chicken CEBPA expression vector with the best overexpression/interference activity was successfully constructed and screened.The gene was widely expressed in various tissues of Rugao Yellow chicken,and the relative expression of CEBPA gene was higher in liver.The results provided a reference for further study on the function of CEBPA in chicken.
Physiological and Biochemical
Study on the Interaction Between Dihydrotestosterone and Melatonin of Hair Follicle Cells in Yak
ZHANG Xiaolan, ZHAO Zhidong, SHI Bingang, HU Jiang
2024, 51(12):  5198-5208.  doi:10.16431/j.cnki.1671-7236.2024.12.008
Abstract ( 70 )   PDF (6277KB) ( 37 )  
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【Objective】 This study was aimed to reveal the interaction effect between dihydrotestosterone (DHT) and melatonin (MLT) of hair follicle cells in yak,so as to provide a basis for elucidating the regulatory mechanism of the two hormones in the development of hair follicle cycle and their application in hair trait selection of yak.【Method】 Using the dermal papilla cells (DPCs) and hair matrix cells (HMCs) of yak as research subjects,the effects of different concentrations of DHT (0,0.01,0.1,1,10,20,40 and 80 μmol/L) and MLT (0,250,500,600,750,1 000 and 1 500 pg/mL) on the proliferation of DPCs and HMCs in yak were determined by MTT assay.The concentrations of DHT and MLT that had the most significant effect on hair follicle cells were selected for combined treatment of HMCs and their interaction effect of the two hormones in cell proliferation was detected using MTT method.The test result was further verified by EdU method.The expression of several genes related to cell proliferation and hair follicle development were detected by Real-time quantitative PCR after combined treatment of DHT and MLT,and analyze the underlying mechanisms.【Result】 Compared with control group (0 μmol/L),DHT concentrations ranging from 10 to 40 μmol/L could significantly or extremely significantly promote the proliferation of DPCs (P<0.05 or P<0.01),and the concentration of DHT at 40-80 μmol/L could extremely significantly promote the proliferation of HMCs (P<0.01).40 μmol/L DHT was the optimal concentration to promote the proliferation of DPCs and HMCs,while low concentration of DHT (1 μmol/L) could extremely significantly inhibit the proliferation of HMCs (P<0.01).Compared with control group (0 pg/mL),500 pg/mL MLT had the most significant proliferative effect on DPCs (P<0.01),while all concentrations of MLT had no significant effect on the proliferation of HMCs (P>0.05).When HMCs were combining treated with 40 μmol/L DHT and 500 pg/mL MLT for 72 h,compared with DHT group,DHT+MLT extremely significantly reduced the proliferative effect of HMCs (P<0.01).Real-time quantitative PCR results showed that compared with control group,the expression of LEF1,SFRP1,TGFB2 and BCL2 genes related to hair follicle development were extremely significantly or significantly changed after DHT treatment of HMCs (P<0.01 or P<0.05),indicating that DHT might be involve in the regulation of hair follicle development by affecting of these genes.【Conclusion】 DPCs and HMCs of yak had different sensitivities to DHT and MLT.A certain concentration of DHT could significantly promote the proliferation of both DPCs and HMCs,while a low concentration of DHT could inhibit the proliferation of HMCs.500 pg/mL MLT had the most significant effect on the proliferation of DPCs,and MLT had no significant effect on HMCs at different concentrations.Finally,MLT might have a function of anti-DHT in HMCs.
Advances on Paracellular Permeability Based on Intestinal Tight Junctions
LI Ming, SUN Juan, WANG Manxi, LI Qinghao, LI Yilei, MA Junxing, JIN Xin
2024, 51(12):  5209-5217.  doi:10.16431/j.cnki.1671-7236.2024.12.009
Abstract ( 65 )   PDF (1172KB) ( 36 )  
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As an important component of the intestinal barrier,the intestinal epithelial cell tight junction not only determines the paracellular permeability of the intestinal tract,but also plays an important role in maintaining the stability of the intestinal environment and preventing the invasion of harmful substances.When the structure or function of the tight junctions is damaged,the integrity of the intestinal barrier is disrupted,resulting in a significant increase in paracellular permeability,bacteria,endotoxins,viruses and macromolecules can enter tissues and organs through the paracellular pathway,leading to the occurrence of intestinal diseases.However,different molecules have different ways to pass through the intestinal tight junctions,and there are two primary paracellular pathways,the pore pathway and the leak pathway.These two pathways are different in terms of molecular size,permeability and regulatory mechanisms.The pore pathway mainly involves the passage of small molecules,and regulating the expression of tight junction protein Claudin-2 affects the permeability of the pore pathway,which in turn affects the function of the intestinal barrier. Whereas the leak pathway mainly involves the passage of large molecules,and tight junction proteins,such as Occludin,Tricellulin and ZO-1,play a key role in this pathway.Occludin,Tricellulin,ZO-1 and other tight junction proteins play a key role in this pathway to maintain the integrity of the intestinal barrier.Taking the relevant proteins affecting the permeability of the pore pathway and the leak pathway as the starting point,the author elucidated the regulatory mechanisms of these proteins affecting the permeability of the pore pathway and the leak pathway,thus preventing the entry of toxic and harmful substances into the body,with a view to provide theoretical basis for the prevention and treatment of intestinal diseases caused by the disruption of the intestinal epithelial tight junction barrier.
Research Progress on the Mechanisms of Autophagy in Mastitis,Ketosis and Fatty Liver in Dairy Cows
ZHANG Bo, GENG Yang, LI Tianzun, AO Yingnan, LU Yulai, WU Yinga, QI Zhili
2024, 51(12):  5218-5224.  doi:10.16431/j.cnki.1671-7236.2024.12.010
Abstract ( 75 )   PDF (1133KB) ( 69 )  
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Mastitis,ketosis and fatty liver are common diseases in dairy cows that not only severely harm their health but also cause significant economic losses to the dairy industry.Autophagy,a highly conserved process in eukaryotic life,helps clear harmful substances within cells,ensuring homeostasis and playing a crucial role in maintaining organismal health.There are various pathways for activating autophagy,including lysosomal autophagy mediated by AMPK and mTOR signaling pathways,as well as mitophagy mediated by the PINK1/PARK2 pathway.Additionally,autophagy activation is influenced by various nutrients,such as plant extracts,amino acids and vitamins.The authors discussed the mechanisms of autophagy in common diseases such as mastitis,ketosis and fatty liver in dairy cows,and reviewed recent research developments,aiming to provide a scientific basis for clinical interventions in common dairy cow diseases.
Nutrition and Feed
Effects of Different Light Sources on Growth Performance,Slaughter Performance and Sexual Development of Beijing-You Chicken
MA Zhong, LI Yunlei, LI Dongli, CHANG Zhuo, YANG Yuze, CHEN Chao, SUN Yanyan, GUO Yanli, CHEN Jilan
2024, 51(12):  5225-5233.  doi:10.16431/j.cnki.1671-7236.2024.12.011
Abstract ( 60 )   PDF (2944KB) ( 36 )  
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【Objective】 This experiment was aimed to explore the optimal light source for Beijing-You chickens by comparing the growth performance,slaughter performance,and sex development-related indicators under four different light sources.【Method】 A total of 720 1-day-old commercial Beijing-You chickens with similar body weights were randomly assigned to four light treatment groups:Incandescent lamp,fluorescent lamp,warm white LED lamp and cool white LED lamp.Each treatment group was divided into 5 replicates,with 36 chickens per replicate.At the end of the 4th,8th and 13th weeks of age,production performance was measured for each replicate.One chicken was randomly selected from each replicate,and a total of 5 chickens in each treatment group were measured for sex hormone-related indexes.At the end of 13 weeks of age,3 chickens with body weight close to the average of each replicate were selected from each replicate,and a total of 15 chickens were collected per treatment group,for the measurement of slaughter performance and sexual organ index.【Result】 The results showed that throughout the growth period,the feed intake of chickens in incandescent lamp and cool white LED lamp groups were significantly higher than that of fluorescent lamp group and warm white LED lamp group (P<0.05),the feed to gain ratio in warm white LED lamp group was significantly lower than that of cool white LED lamp and fluorescent lamp groups (P<0.05),and incandescent lamp group had the highest feed to gain ratio among the four groups (P<0.05).The four light sources had no significant effect on the slaughter performance of Beijing-You chicken (P>0.05).In terms of sexual development,the secretion of testosterone in the incandescent lamp and the cool white LED lamp groups were significantly higher than that of fluorescent lamp group (P<0.05),and it was at an intermediate level in warm white LED lamp group.The secretion of melatonin in incandescent lamp,cool white LED lamp and warm white LED lamp groups were significantly higher than that of fluorescent lamp group (P<0.05).There was no significant difference in testicular organ index and cockscomb thickness of roosters among the four light sources groups (P>0.05),the length of chicken comb in warm white LED and fluorescent lamp groups were significantly greater than that in incandescent lamp group (P<0.05),and it was at an intermediate level in cool white LED lamp group.Similarly,the cockscomb height was the highest in warm white LED lamp group,and it was significantly larger than that in incandescent lamp group (P<0.05),and it was at an intermediate level in fluorescent lamp and cool white LED lamp groups.【Conclusion】 Using LED lamps in the lighting system for Beijing-You chickens could significantly improve the growth performance.Moreover,LED lamps could also promote the secretion of sex development-related hormones,indirectly facilitating sexual development.
Effects of Phytase Supplementation in Low Phosphorus Diet on Growth Performance, Plasma Biochemical Parameters,and the Contents of Total Nitrogen and Total Phosphorus in the Feces of Geese
LIU Zuolan, CHEN Ying, XUE Jiajia, HUANG Xiaofeng, ZHONG Hang, LUO Yi, XIE Qun, WANG Qigui, WANG Chao
2024, 51(12):  5234-5243.  doi:10.16431/j.cnki.1671-7236.2024.12.012
Abstract ( 57 )   PDF (1167KB) ( 49 )  
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【Objective】 This experiment was conducted to investigate the effects of phytase supplementation in low phosphorus diet on growth performance,plasma biochemical parameters,carcass traits,the contents of total nitrogen and total phosphorus in the feces of geese from 28 to 70 days of age.【Method】 A total of 200 28-day-old geese were randomly allotted to 5 groups with 5 replicates per group and 8 goslings per replicate.Geese in the positive control group (PC group) were fed a routine diet containing 0.7% total phosphorus (available phosphorus level were 0.35%),geese in the negative control group (NC group) were fed a low phosphorus diet containing 0.5% total phosphorus (available phosphorus level were 0.15%),and others in experimental groups were fed low phosphorus diets supplemented with 1 000 (NC1000 group),2 000 (NC2000 group) and 4 000 IU/kg (NC4000 group) phytase,respectively.The experiment lasted for 42 days.The feed intake,initial body weight,and final body weight of each replicate were recorded to calculate the average daily gain,average daily feed intake,and feed/gain.At the age of 70 days,two geese were selected from each replicate for blood collection from the wing vein to determine plasma biochemical parameter.One goose was selected from each replicate according to the average body weight of corresponding replicate and were slaughtered for carcass trait measurement.Two geese were selected from each replicate according to the average body weight for droppings collection,and determination of total nitrogen and total phosphorus.【Result】 ① The final body weight,average daily gain and average daily feed intake of geese in PC,NC1000,NC2000 and NC4000 groups were significantly higher than those in NC group (P<0.05),there were no differences in feed/gain among the groups (P>0.05).② The plasma phosphorus content of geese in PC,NC1000,NC2000 and NC4000 groups were significantly higher than those in NC group (P<0.05),the plasma alkaline phosphatase activity of geese in NC2000 and NC4000 groups were significantly lower than those in NC and NC1000 groups (P<0.05),phytase supplementation in low phosphorus diet had no significant effect on plasma total protein,albumin,globulin,glucose,triglyceride,cholesterol and calcium concentrations and alanine amino transferase,aspartate amino transferase,lactate dehydrogenase activities of geese (P>0.05).③ The dressed percentage and half eviscerated ratio of geese in the PC,NC1000,NC2000 and NC4000 groups were significantly higher than those in NC group (P<0.05),phytase supplementation in low phosphorus diet had no significant effect on eviscerated ratio,breast muscle ratio,leg muscle ratio,lean meat ratio,abdominal fat ratio,skin fat ratio and intestinal weight and length of geese (P>0.05).④ Phytase supplementation in low phosphorus diet had no significant effect on the total nitrogen content in the feces of geese (P>0.05),the total phosphorus content in the feces in NC,NC1000,NC2000 and NC4000 groups were significantly lower than those in PC group (P<0.05).【Conclusion】 The phytase supplementation in low phosphorus diet could improve the growth performance and carcass trait of geese from 28 to 70 days of age,and decrease emission of phosphorus.Under this experiment condition,the suitable addition of phytase was 1 000 IU/kg in low phosphorus diet that total phosphorus was 0.5%.
Effects of Fermented Soybean Meal on the Production Performance,Antioxidant Capacity and Gut Microbiota Diversity of Laying Hens in the Late Laying Period
GUO Fangchao, JIA Ling, CHEN Wenfeng, CHEN Liang, MU Qingqing, XU Shuying, WANG Yongjuan
2024, 51(12):  5244-5253.  doi:10.16431/j.cnki.1671-7236.2024.12.013
Abstract ( 78 )   PDF (3728KB) ( 46 )  
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【Objective】 The effect of replacing soybean meal in the basic diet with 5% fermented soybean meal on the production performance,antioxidant capacity,intestinal tissue morphology and intestinal flora diversity of laying hens in the late laying period was studied.【Method】 330-day-old Nongda No.3 laying hens were selected,with 9 000 in control group and 6 800 in experimental group.The hens in control group were fed basal diet,and in experimental group were fed experimental diet in which 5% fermented soybean meal was substituted for the soybean meal in the basal diet.The experimental period was 42 days.During the period,the number of eggs laid and abnormal eggs,the number of dead and culled chickens,and the feed intake were recorded.On the 21st and 42nd days of the experiment,the egg quality of 100 eggs was randomly measured,and the serum biochemical,immune and antioxidant indicators of 12 chickens were tested in each group.On the 42nd day of the experiment,12 laying hens were randomly slaughtered in each group,and duodenum and jejunum segments were collected for analysis of intestinal tissue morphological changes,and cecal contents were collected for determining intestinal microbiota diversity.【Result】 Compared with control group,on the 21st day,the feed-to-egg ratio was significantly increased (P<0.05),the egg yolk color became significantly lighter (P<0.05),and the serum AST activity was significantly increased (P<0.05),while the other indicators for investigation had no significant difference (P>0.05) of hens in experimental group. Compared with control group,on the 42nd day,the feed-to-egg ratio,egg yolk color and serum AST activity were not significantly different (P>0.05),while the abnormal egg rate and egg production rate were significantly reduced (P<0.05),eggshell strength,egg weight and protein height were significantly increased (P<0.05),serum total antioxidant capacity (T-AOC) was significantly increased (P<0.05),the relative abundance of the genus Cryptobacteroides in the gut microbiota of laying hens in experimental group was significantly reduced (P<0.05).【Conclusion】 Replacing 5% of the soybean meal in the basal diet with fermented soybean meal could significantly improve the production performance and egg quality of laying hens in the late laying period,enhance the antioxidant capacity,and have a certain impact on the gut microbiota structure of the laying hens.
Toxic Effects of Ochratoxin A on Livestock and Poultry and Its Mitigation Measures
WU Chenhui, TU Yuang, LIU Haoyang, SHAN Tizhong
2024, 51(12):  5254-5264.  doi:10.16431/j.cnki.1671-7236.2024.12.014
Abstract ( 77 )   PDF (2910KB) ( 76 )  
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Ochratoxin a (OTA) is a type of mycotoxin that has a wide range of contamination and widely exists in wheat,corn and other feed ingredients.After absorbed from the gastrointestinal tract,OTA diffuses to the liver and kidney through the circulatory system of animals,causing nephrotoxicity,hepatotoxicity,enterotoxicity,immunotoxin,carcinogenicity and teratogenicity and other toxic effects to livestock and poultry,thus causing serious harm to the health and production of livestock and poultry.Therefore,it is urgent to study how to alleviate the toxic effect of OTA.Biological mitigation strategies mainly included microbial mitigation measures such as yeast,Bacillus and enzymatic degradation measures.In addition to biological mitigation,nutritional mitigation measures including plant extracts,vitamins,trace elements,amino acids,etc.,which can also have a good mitigation effect on OTA toxicity.The authors summarize the toxic effects of OTA on livestock and poultry,and focuse on the research progress on biological and nutritional mitigation strategies to alleviate the toxic effects of OTA,in order to provide a reference for alleviating the harm of mycotoxins to the healthy breeding of livestock and poultry and promoting the high-quality development of livestock and poultry.
Effects of Compound Enzyme Preparation Added to Distiller’s Grains Diet on Growth, Intestinal Development,Meat Quality and Nutrients Utilization in Meat Ducks
LU Zhentao, WU Zhanyue, GUO Yanhong, WANG Ning, REN Wenwen, WU Yongbao, CAO Junting, WU Xuezhuang, WEN Zhiguo
2024, 51(12):  5265-5276.  doi:10.16431/j.cnki.1671-7236.2024.12.015
Abstract ( 66 )   PDF (1223KB) ( 52 )  
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【Objective】 This experiment was conducted to investigate the effects of compound enzyme preparation in distillers’ grains diet on growth performance,meat quality,intestinal development,blood indexes and apparent nutrient availability of meat ducks.【Method】 A total of 144 1-day-old male Peking ducks with similar body weight were randomly divided into 3 groups with 6 replicates per group and 8 ducks per replicate.Ducks in the control group were fed a corn-soybean meal basal diet,ducks in the distiller’s grains group were fed a diet supplemented with 18% distiller’s grains,and ducks in the compound enzyme preparation group were fed a diet supplemented with 500 mg/kg compound enzyme preparation on the basis of distiller’s grains group.The experimental period was divided into two feeding stages of 1-14 days and 15-35 days.After the experiment,blood,pectoral muscle,excreta and small intestine were collected,and the quality of pectoral muscle,blood indexes and apparent availability of nutrients were measured.【Result】 ①Compared with the control group, the 14-day body weight and 1-14 day ADG of ducks in distiller’s grains group and compound enzyme preparation group were significantly decreased, and the 1-14 day F/G of ducks in compound enzyme preparation group was significantly increased (P<0.05), and the 1-14 day F/G of ducks in compound enzyme preparation group was significantly lower than that in distiller’s grains group (P<0.05). Compared with the control group, ADFI and F/G of ducks in distilling grains group and compound enzyme preparation group were significantly increased from 15 to 35 days and from 1 to 35 days (P<0.05), and ADFI and F/G of ducks in compound enzyme preparation group were significantly lower than those in distilling grains group from 1 to 35 days (P<0.05). ② Compared with the control group, the chest muscle percentage of ducks in compound enzyme preparation group was significantly decreased (P<0.05), the abdominal fat percentage of ducks in distiller’s grains group and compound enzyme preparation group was significantly increased (P<0.05), the pancreas index of ducks in distiller’s grains group and compound enzyme preparation group, and the muscle stomach index of ducks in compound enzyme preparation group were significantly increased (P<0.05). ③ Compared with the control group, the relative weights of duodenum and jejunum of ducks in distiller’s grains group and compound enzyme preparation group were significantly increased (P<0.05). ④ Compared with the control group, the energy utilization rate of ducks in distiller’s grains group and compound enzyme preparation group was significantly decreased (P<0.05). Compared with distiller’s grains group, crude protein utilization rate and energy utilization rate of ducks in compound enzyme preparation group were significantly increased (P<0.05). ⑤ Compared with the control group, pH24 h and drip loss of breast muscle of ducks in distiller’s grains group and pH24 h of breast muscle of ducks in compound enzyme preparation group were significantly increased (P<0.05), and pH24 h of breast muscle of ducks in compound enzyme preparation group was significantly higher than that in distiller’s grains group, while drip loss was significantly lower than that in distiller’s grains group (P<0.05). Compared with the control group, the pH45 min of the two experimental groups was significantly decreased (P<0.05). ⑥ There were no significant differences in blood routine indexes among groups (P>0.05). Compared with the control group, the activities of AST and LDH, AST/ALT, ALB and UA in plasma of ducks in distiller’s grains group were significantly decreased (P<0.05), and AST/ALT in compound enzyme preparation group was significantly decreased (P<0.05). Compared with distiller’s grains group, the contents of GLU, TP, GLB and UA in plasma of ducks in compound enzyme preparation group were significantly increased (P<0.05).【Conclusion】 In conclusion,the compound enzyme preparation could reduce the feed/gain and improve nutrient utilization rate when the basal diet of Beijing ducks was supplemented with 18% distiller’s grains.
Application of Near-infrared Reflectance Spectroscopy Technology in Animal Production
CHENG Liuyang, ZHONG Chongliang, LUO Yaping, LIU Yang, XIN Hangshu
2024, 51(12):  5277-5289.  doi:10.16431/j.cnki.1671-7236.2024.12.016
Abstract ( 62 )   PDF (1230KB) ( 89 )  
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With the swift advancement of modern information technology,near-infrared reflectance spectroscopy (NIRS) has gained widespread application in the field of agriculture,favored for its capabilities in preserving sample integrity and rapidly analyzing multiple components during testing.Within the wavelength range of 800 to 2 500 nm,NIRS captures spectral data through the harmonic and combination vibrational absorption of specific frequencies of light by hydrogen-containing functional groups,which yields overtone and combinational vibrations indicative of molecular composition.This spectral data is subsequently processed to obtain qualitative and quantitative attributes of the sample.The animal production process is intimately connected to human sustenance and apparel,in this domain,the assessment of nutritional value in animal feed and the analysis of the quality of animal products are of critical importance.The amalgamation of NIRS with animal husbandry encourages the expeditious evaluation of standard nutrients in feed,ensuring the equilibrium of nutrition in animals and the monitoring of product quality to meet market demands.The authors chronicle the developmental history and principles of application of NIRS,with a focused synthesis of its application and effectiveness in animal production,providing a reference for the future utilization of NIRS in this vital field.
Effects of Grape Seed Proanthocyanidins on Growth Performance,Serum Biochemical Indices,and Rumen Environment of Beef Cattle
MA Jing, DENG Jiahan, WANG Juze, YANG Zhimei, LI Xuefeng, ZAN Linsen
2024, 51(12):  5290-5301.  doi:10.16431/j.cnki.1671-7236.2024.12.017
Abstract ( 68 )   PDF (4372KB) ( 56 )  
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【Objective】 This experiment was conducted to investigate the effects of different dietary levels of grape seed procyanidins (GSPs) on growth performance,nutrient digestion,serum biochemical indices and rumen environment of beef cattle.【Method】 Twenty Simmental bulls aged 12 months with good health and body weight of about 400 kg were randomly divided into 4 treatment groups with 5 replicates per group.The cattle in control group was fed a basal diet,in experimental groups was fed the basal diets supplemented with 20 mg/kg (20 GSPs group),40 mg/kg(40 GSPs group) and 80 mg/kg GSPs(80 GSPs group),respectively.The pre-feeding period was 15 days,and the normal test period was 55 days.The initial body weight and final body weight of each cattle were measured,and the average daily feed intake and feed to gain ratio were recorded.Three days before the end of experiment, the nutrient digestibility was measured using the full fecal collection method. After the experiment,15 mL of blood and 50 mL of rumen fluid were collected from each cattle for the determination of serum biochemical indexes,rumen fermentation parameters and rumen 16S rRNA gene sequence.【Result】 Compared with control group,①the final weight of Simmental cattle in 20 GSPs and 40 GSPs groups were significantly increased (P<0.05).The apparent digestibility of dry matter and neutral detergent fiber in 40 GSPs group were significantly enhanced (P<0.05),and crude protein apparent digestibility in 20 GSPs and 40 GSPs groups were significantly improved (P<0.05).②The activities of serum aspartate aminotransferase and alanine aminotransferase in 40 GSPs group were remarkably decreased (P<0.05),serum urea nitrogen contents in all groups supplemented with GSPs were significantly decreased (P<0.05).③The total antioxidant capacity in 40 GSPs group was significantly increased (P<0.05),the serum glutathione peroxidase activity in 40 GSPs and 80 GSPs groups were significantly improved (P<0.05).④Serum immunoglobulin G and complement 3 contents in 80 GSPs group were significantly increased (P<0.05). The serum complement 3 and complement 4 contents were significantly increased (P<0.05),while the serum interleukin-6 was significantly reduced (P<0.05) in 40 GSPs group. The serum tumor necrosis factor was significantly decreased in 80 GSPs group (P<0.05).⑤The propionic acid content of 40 GSPs and 80 GSPs groups were significantly enhanced (P<0.05),the bacterial Chao1 index in 20 GSPs group were significantly increased (P<0.05).The relative abundance of Spirochaetes and Clostridium in 20 GSPs group were significantly increased (P<0.05).The relative abundance of Fibrobacteres,Actinobacteria and Spirochaetes in 80 GSPs group were significantly increased (P<0.05),the relative abundance of Succiniclasticum in 40 GSPs and 80 GSPs groups were significantly decreased (P<0.05).【Conclusion】 The supplementation of 40 mg/kg GSPs could increase the final body weight,serum immune and antioxidant levels and rumen bacterial diversity, and improve the composition of rumen microbial community of Simmental cattle.
Effects of Dietary Probiotics and Acid Preparations on Growth Performance and Intestinal Flora of Weaned Piglets
WEI Haibo, LIU Yingchun, SUN Shuguang, LIU Xiwu, ZHU Guihua, DAI Zhenghao, LIU Zongzheng, WEN Fengyun
2024, 51(12):  5302-5316.  doi:10.16431/j.cnki.1671-7236.2024.12.018
Abstract ( 65 )   PDF (17231KB) ( 108 )  
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【Objective】 The aim of this experiment was to study the effects of adding probiotics and acid preparations to diets on growth performance and intestinal flora of weaned piglets.【Method】 120 healthy weaned piglets from the same batch,with good health and of similar weight were selected and randomly divided into 3 groups with 5 replicates of 8 piglets each.The piglets in control group (group A) were fed with basic feed normally. The piglets in groups B and C were fed with basic diet supplemented with 1 g/kg of acid preparation and 2 g/kg of probiotics,respectively.The experimental period was 30 d.【Result】 ①Compared with group A,the final weight,average daily feed intake (ADFI) and average daily gain (ADG) of piglets in groups B and C were extremely significantly increased (P<0.01),while the feed to gain ratio (F/G) of piglets in group C was significantly reduced (P<0.05). ②The analysis of intestinal flora diversity based on 16S rDNA sequencing showed that there was no significant difference among the three groups in Alpha diversity,but in Beta diversity,there was significant difference in gut microbiota structure of piglets between group A and groups B and C. In faecal microbial composition,the dominant phyla in the three groups were mainly Firmicutes and Bacteroidetes,and the relative abundance of different phyla in each group showed different trends,with the relative abundance of Bacteroidetes being extremely significantly higher in group B compared to group A (P<0.01) and that of Spirochaetes was significantly lower in groups B and C compared to group A (P<0.05).Compared to group A,the relative abundance of Prevotella was found to be significantly or extremely significantly higher in groups B and C (P<0.05 or P<0.01),while the relative abundance of Treponema and SMB53 was significantly or extremely significantly lower (P<0.05 or P<0.01).The relative abundance of Faecalibacterium and Roseburia was found to be significantly or extremely significantly higher in group C compared to group A (P<0.05 or P<0.01),whereas the relative abundance of Oscillospira was significantly lower in group B compared to group A(P<0.05).③A functional analysis of metabolic pathways in KEGG intergroup colonies revealed that retinol metabolism (ko00830) and ubiquinone and other terpene-quinone biosynthesis (ko00130) pathways were significantly up-regulated in group B compared to group A (P<0.05),toluene degradation (ko00623) and sphingolipid metabolism (ko00600) were extremely significantly upregulated in group C (P<0.01).Compared with group B,the degradation of chlorinated alkanes and degradation of chloro-olefins (ko00625) was significantly up-regulated in group C (P<0.05).【Conclusion】 The addition of 2 g/kg probiotics or 1 g/kg acid preparations to the diets of weaned piglets could improve their growth performance,which might be closely related to the addition of probiotics and acidifiers to regulate the intestinal flora.Among them,adding probiotics had a better effect than adding acid preparations.
Effects of Supplementing Chinese Herbal Medicine and Active Dry Yeast Preparations on Diarrhea Rate and Rectal Microflora Diversity in Yanbian Cattle Lactating Calves
LI Jiongkui, JIN Xijiu, GENG Kai, REN Fenglong, WANG Xinhong, GENG Chunyin, JIN Yinghai
2024, 51(12):  5317-5324.  doi:10.16431/j.cnki.1671-7236.2024.12.019
Abstract ( 57 )   PDF (3081KB) ( 48 )  
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【Objective】 The purpose of this experiment was to study the effects of supplementary feeding of Chinese herbal medicine and active dry yeast preparation on diarrhea rate and intestinal flora diversity of lactating calves,and to provide theoretical basis for exploring the mechanism of action on intestinal health of lactating calves.【Method】 Twenty-four lactating calves of Yanbian Yellow cattle with similar body weight,month age and good body condition were randomly divided into three groups,with 8 calves in each group (5 males and 3 females).The calves in control group (group Ⅰ) were fed with basal diet,while in the Chinese herbal medicine group (group Ⅱ) were supplemented with 0.5% Chinese herbal medicine preparation in the basal diet,and the yeast group (group Ⅲ) was supplemented with 1 g/d Raman active dry yeast in the basal diet.The feeding experiment lasted for 31 days,the pre-test period was 10 days and the formal test period was 21 days.Before the morning feeding on the end of the feeding trial,the fresh fecal samples from the rectum of calves in the pasture were collected aseptically with cotton swabs and stored in a refrigerator at ―80 ℃ for testing.The feces collected from the rectum of calves were sequenced to compare the differences in bacterial diversity between groups Ⅱ,Ⅲ and group Ⅰ.【Result】 ①Compared with group Ⅰ,ADG of calves in groups Ⅱ and Ⅲ was significantly increased (P<0.05),F/G,diarrhea rate and fecal score of calves were significantly decreased (P<0.05).②In terms of rumen flora diversity,compared with group Ⅰ,there was no significant difference in Alpha diversity indexes (ACE,Chao1,Coverage,Shannon and Simpson)in the two treatment groups (P>0.05).③In terms of rumen flora distribution,at phylum level,the dominant phylum in both treatment groups were Firmicutes and Bacteroidetes.At genus level,the dominant genus in the two treatment groups were Ruminococcaceae_UCG-005,Rikenellaceae_RC9_gut_group and [Eubacterium]_coprostanoligenes_group.Compared with group Ⅰ,the relative abundance of Rikenellaceae_RC9_gut_ group in groups Ⅱ and Ⅲ was significantly increased (P<0.05),while there was no significant difference in the relative abundance of other microbiota at phylum and genus levels (P>0.05).【Conclusion】 Supplementation of 0.5% Chinese herbal medicine feed additive or 1 g/d active dry yeast preparation could reduce the diarrhea symptoms of lactating calves to varying degrees,improve the diversity of intestinal flora,and promote the healthy development of the intestine.
Progress on Autophagy Regulation of Porcine Adipogenesis
HONG Chun, ZHU Xiangxing, LI Xinming, HUANG Qiuyan, LIU Wenhua, DU Zongliang, TANG Dongsheng, WANG Sutian
2024, 51(12):  5325-5334.  doi:10.16431/j.cnki.1671-7236.2024.12.020
Abstract ( 75 )   PDF (3286KB) ( 64 )  
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Adipose tissue is an energy storage station in the animal organism and an important endocrine organ.Obesity caused by excessive fat deposition brings about a series of metabolic diseases,such as fatty liver,cardiovascular disease and insulin resistance.Similarly,porcine adipogenesis influences traits such as disease resistance,reproductive performance,meat quality and productivity,which affect the economic efficiency of hog production.Mature adipocytes are obtained by differentiation of mesenchymal stem cells,a process in which many signaling pathways and transcription factors are involved via preadipocytes.Autophagy is usually the most important means of catabolizing redundant components of the organism and is involved in the maintenance of homeostasis.There is a close link between autophagy and adipogenesis,active adipogenesis is accompanied by elevated levels of autophagy,and impaired autophagy is accompanied by suppressed adipogenesis.There are many related transcription factors and signaling pathways that regulate porcine adipogenesis during autophagic activities,and many transcription factors are involved in the process of porcine adipogenesis.By summarizing the newly reported transcription factors and signaling pathways related to autophagy regulation of adipogenesis,the author elaborates the research progress on autophagy involved in the regulation of porcine adipogenesis,which provides a new idea and target point for the regulation of porcine nutrition and meat quality breeding.
Genetics and Breeding
Comparative Study on Meat Quality Characteristics of Suffolk×Hu and Southdown×Hu and Hu Sheep
SHI Haina, ZHANG Jinxia, LU Zengkui, XU Zhenfei, GENG Zhiguang, YANG Bohui, YUE Yaojing, ZHOU Yanhong
2024, 51(12):  5335-5347.  doi:10.16431/j.cnki.1671-7236.2024.12.021
Abstract ( 120 )   PDF (1205KB) ( 69 )  
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【Objective】 The meat quality characteristics of two-way hybrid sheep Suffolk×Hu (SH) and Southdown×Hu (NH) and Hu sheep were studied to provide basic data and theoretical support for the breeding of new varieties of 'Zhonghuan Mutton sheep’ and the development of high-quality mutton products.【Method】 Selecting Hu sheep as the maternal parent,5 SH and 5 NH sheep were bred through laparoscopic artificial insemination using frozen semen from White Suffolk and Southdown sheep.5 purebred Hu sheep were used as the control group.The slaughtering performance and meat quality (longissimus dorsi muscle,biceps femoris and arm triceps) of F1 generation in NH,SH and HH sheep were compared and analyzed.【Result】 The live body weight and carcass weight of NH and SH sheep were increased by 19.92%,12.90% and 24.20% and 15.55% compared with Hu sheep,respectively,and there was a significant difference between NH and Hu sheep (P<0.05).The fat content and energy level of NH sheep were significantly lower than those of Hu sheep (P<0.05).The contents of ash and moisture in NH sheep were significantly higher than those in SH and Hu sheep (P<0.05).The carbohydrate content in SH sheep was extremely significantly higher than that in NH and Hu sheep (P<0.01).The contents of potassium,phosphorus,magnesium and zinc were the highest in NH sheep,the sodium content was the highest in SH sheep,and the niacin content was the highest in Hu sheep.The compositions of fatty acids and amino acids were the same,and the total fatty acid content in NH sheep was the highest, which was extremely significantly higher than that in SH and Hu sheep (P<0.01). The saturated fatty acid (SFA) content in NH sheep was significantly higher than that in Hu sheep (P<0.05).The monounsaturated fatty acid (MUFA) content in NH sheep was extremely significantly higher than that in Hu and SH sheep (P<0.01).The polyunsaturated fatty acid (PUFA) content in NH sheep was extremely significantly higher than that in SH sheep (P<0.01).The content of essential amino acid (EAA) was in the order of NH>SH>Hu sheep,and the content of EAA in Hu sheep was extremely significantly lower than that of NH and SH sheep (P<0.01).The value of EAA/total amino acid (TAA) was 38.95%-39.65%,and the value of EAA/non-essential amino acid (NEAA) was 63.79%-65.70% among three sheep.Except valine and methionine+cystine,the scores of other amino acids were higher than the recommended values of FAO/WHO.【Conclusion】 F1 generation of NH and SH sheep had fast growth rate,good slaughter performance,rich content of amino acids,fatty acids and several major minerals in muscle,which was more in line with the characteristics of high protein and low fat,and more in line with people’s demand for meat quality.They could be scaled up locally.
Research Progress of Machine Learning and Its Application in Animal Genetics and Breeding
ZHOU Bohan, MEI Bujun, LYU Qi, WANG Zhiying, SU Rui
2024, 51(12):  5348-5358.  doi:10.16431/j.cnki.1671-7236.2024.12.022
Abstract ( 83 )   PDF (1207KB) ( 87 )  
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With the progress of research on intelligent animal husbandry and the development of high-throughput omics platforms,animal genetic breeding has been gradually entered the “Breeding 3.0” in the era of big data.Machine learning is an indispensable and effective means of big data research.Machine learning,the automatic acquisition of knowledge by computers,is a multidisciplinary cross-discipline,involving many disciplines such as probability theory and statistics.As one of the popular algorithms in artificial intelligence and data mining,it has many advantages such as high learning rate,good generalization ability and high accuracy.It has been an important tool in the field of bioinformatics analysis for processing big data and making predictions.Nowadays,machine learning is widely used for the integration and analysis of genomic,transcriptomic,proteomic,metabolomic and other multi-omics data,especially in the computing of genomic estimated breeding value (GEBV),genotype imputation,the prediction of protein structure and function of livestock,and other outstanding achievements.Firstly,the author introduced the concepts and principles of several common machine learning algorithms.Besides,the outstanding research results achieved by machine learning algorithms in genetic breeding of important livestock (pigs,cattle,sheep,and goats) were reviewed and further discussed the advantages and disadvantages of certain machine learning algorithms as well as some of the problems in animal genetic breeding.Finally,the future development of machine learning was summarized and prospected,aiming to improve the accuracy and efficiency of estimation,accelerate the genetic progress of populations,and rapidly implement precision breeding.
Analysis of Reproductive Performance of Binary Crosses Between Dabao and Shiqi Pigeons and Production Performance of Crossbred Offspring
XI Jinquan, LIU Shihong, GU Lihong, XU Tieshan, QI Yanxia, ZHANG Xiaohui
2024, 51(12):  5359-5370.  doi:10.16431/j.cnki.1671-7236.2024.12.023
Abstract ( 87 )   PDF (3590KB) ( 21 )  
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【Objective】 This study was aimed to understand the reproductive performance of binary crosses between Dabao and Shiqi pigeons and the production performance of crossbred offspring,so as to provide data support for the subsequent establishment of high production performance matching lines.【Method】 The experiment established orthogonal (the offspring from cross between male Dabao pigeons and female Shiqi pigeons were DS pigeon) and anticross (the offspring from cross between male Shiqi pigeons and female Dabao pigeons were SD pigeon) cross groups to measure and analyze the reproductive performance of breeding pigeons,the growth performance of cross offspring from 0-28 days of age,the slaughter performance of squabs at 28 days of age,meat quality and conventional nutrients,amino acid and fatty acid contents in breast muscle,as well as the serum biochemical indicators of squabs.【Result】 The fertilization and hatching rates of DS breeding pigeons were significantly higher than that of SD breeding pigeons (P<0.05).The body weight of SD pigeons at 14 days of age was significantly higher than that of DS pigeons (P<0.05),the carcass rate,semi-eviscerated rate and eviscerated rate were significantly lower than that of DS pigeons (P<0.05).pH45 min of SD pigeons was significantly higher than that of DS pigeons (P<0.05). The a* value and drip loss of DS pigeons were significantly higher than that of SD pigeon (P<0.05).SD pigeons had a smaller diameter,a greater total number of pectoral muscle fibers,and a greater density of pectoral muscle fibers compared with DS pigeons (P<0.05).The total protein and triglyceride levels in serum of SD pigeons were significantly lower than that of DS pigeons (P<0.05).The blood glucose levels and total antioxidant capacity of SD pigeons were significantly higher than that of DS pigeons (P<0.05).【Conclusion】 DS and SD pigeons had excellent production performance and slaughtering traits,as well as good meat quality characteristics.The results provided a scientific theoretical basis and reliable data support for the breeding of high-yielding supporting lines for meat pigeons.
Protection of Gynostemma pentaphyllum Polysaccharides on Mouse Oocytes and Mitochondria Damaged by H2O2
DU Qiansheng, LIU Keke, TANG Feitai, TANG Xiaochuan, HE Jiakang, WANG Xiaoli
2024, 51(12):  5371-5379.  doi:10.16431/j.cnki.1671-7236.2024.12.024
Abstract ( 66 )   PDF (6872KB) ( 22 )  
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【Objective】 This study was aimed to investigate the protection of Gynostemma penttaphyllum polysaccharides (GPP) on mouse oocytes and mitochondria damaged by hydrogen peroxide (H2O2).【Method】 Mouse oocyte maturation oxidative stress model was constructed with H2O2,and GPP concentration was selected.Then oocytes at MⅡ stage were randomly divided into blank control group,H2O2 group (H2O2 was added in mature medium) and H2O2+GPP group (GPP was added in mature medium containing H2O2).The cleavage rate,4-cell rate,8-cell rate,morula rate and blastocyst rate of oocytes were measured after in vitro fertilization (IVF).Mito Tracker Red CMXRos was used to detect mitochondrial distribution and content,JC-1 mitochondrial membrane potential detection kit was used to detect mitochondrial membrane potential,the ATP detection kit was used to detect mitochondrial ATP content,and the morphology and chromosome distribution of mouse oocytes were observed by immunofluorescence staining.【Result】 The cleavage rate,4-cell rate,8-cell rate,morula rate and blastocyst rate,the mitochondrial content,mitochondrial membrane potential and ATP contents of oocytes in H2O2+GPP group were significantly higher than those in H2O2 group (P<0.05),and the rates of spindle abnormality and chromosome abnormality were significantly lower than those in H2O2 group (P<0.05),but there was no significant difference between H2O2+GPP and control groups(P>0.05).【Conclusion】 GPP could improve the cleavage rate and blastocyst rate of H2O2-damaged mouse oocytes IVF,as well as the mitochondrial content,mitochondrial normal distribution,mitochondrial membrane potential and ATP content in oocytes,and reduce the spindle morphological abnormalities,chromosome morphological abnormalities and disordered arrangement,reducing mitochondrial dysfunction.
Sequence Characteristics,Tissue Expression of IGF1 Gene and Correlation with Production Trait in Huanghuai Sheep
LI Jun, SONG Mengke, GAO Fuxian, HAN Wanli, HAN Haoyuan, SHI Huibin, QUAN Kai
2024, 51(12):  5380-5391.  doi:10.16431/j.cnki.1671-7236.2024.12.025
Abstract ( 56 )   PDF (6243KB) ( 42 )  
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【Objective】 This study was aimed to research the sequence characteristics and tissue expression patterns of insulin like growth factor 1 (IGF1) gene in Huanghuai sheep,and conduct correlation analysis between the expression of IGF1 gene in longissimus dorsi muscle and meat production traits.【Method】 The 9 male Huanghuai sheep at the ages of 3,9,and 18 months each were selected.The meat production performance were determined and semitendinosus,quadriceps femoris,triceps brachii,longissimus dorsi muscle,subcutaneous fat,rumen,kidney,spleen,liver,testis and small intestine tissue samples were collected after slaughter.the IGF1 gene of Huanghuai sheep was cloned and sequenced,and analyzed by bioinformatics analysis.The mRNA levels of IGF1 gene in various tissues at 9-month-old and in the longissimus dorsi muscle at different age stages were detected by Real-time quantitative PCR,and the correlation between IGF1 expression and meat production traits were analyzed through Pearson correlation coefficient.【Result】 The CDS region size of IGF1 gene in Huanghuai sheep was 465 bp,encoding 154 amino acids,indicating the closest genetic relationship with Capra hircus.IGF1 protein of Huanghuai sheep was a hydrophilic and unstable protein with no transmembrane structure.It contained signal peptides and had 29 potential phosphorylation sites.The secondary structure of IGF1 protein in Huanghuai sheep mainly contained random coil (54.55%) and alpha helix (29.87%),and the predicted results of the tertiary structure were consistent with the secondary structure.There were interactions between IGF1 and IGF1R,IGFBP3,IGF2 and other proteins.The results of Real-time quantitative PCR showed that IGF1 gene was widely expressed in the tissues of Huanghuai sheep,with the highest expression in liver and the lowest expression in triceps brachii.The expression level of IGF1 gene was highest in the longissimus dorsi muscle of 3-month-old Huanghuai sheep,significantly higher than that of 9-month-old and 18-month-old sheep (P<0.05),while the expression of 9-month-old sheep was significantly lower than that of 18-month-old sheep (P<0.05).The correlation analysis results showed that the expression of IGF1 gene was not significantly correlated with growth traits and meat quality indicators (P>0.05).There was a significant or extremely significant positive correlation between body height,body length,chest girth,cannon circumference,body weight,and carcass weight (P<0.05 or P<0.01).There was also a significant or extremely significant positive correlation between dressing percentage,meat color,marble texture,and loin muscle area (P<0.05 or P<0.01).【Conclusion】 The CDS sequence of IGF1 gene in Huanghuai sheep was successfully cloned.The IGF1 protein was the hydrophilic and unstable alkaline protein,mainly expressed in the liver,and the general trend in the longissimus dorsi muscle muscle were first decreasing and then rising with age increasing,and was not significantly correlated with growth and development and meat quality indicators.These results provided a reference for further exploring the role of IGF1 gene in muscle development and meat quality regulation in Huanghuai sheep.
Screening of Genes Related to Lactation in Kazakh Horses Based on Transcriptomic
MENG Chen, ZENG Yaqi, WANG Jianwen, YAO Xinkui, LUO Penghui, XIE Xiaoyu, LI Pengcheng, LIU Xiaoxiao, WANG Chuankun, MENG Jun
2024, 51(12):  5392-5404.  doi:10.16431/j.cnki.1671-7236.2024.12.026
Abstract ( 61 )   PDF (19135KB) ( 76 )  
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【Objective】 The aim of this experiment was to screen the genes affecting lactation in Kazakh horses and provide data support for the selection of Kazakh horses for dairy type.【Method】 Transcriptome sequencing was performed on whole blood from the jugular vein of 12 Kazakh horses at peak lactation to screen differentially expressed gene (DEG) related to lactation in Kazakh horses.GO function and KEGG pathway enrichment analysis were performed on DEGs,and Sankey bubble maps of the enriched pathways were plotted to screen for genes related to lactation in Kazakh horses and construct a gene interaction network.6 DEGs were randomly selected for Real-time quantitative PCR to verify the accuracy of transcriptome data.【Result】 A total of 286 DEGs were identified in the high and low lactation group,including 198 up-regulated genes and 98 down-regulated genes.27 terms were enriched by GO enrichment analysis,which included cell structure,cellular enzyme activity,substance transport,etc.13 pathways were enriched by KEGG enrichment analysis,which included cellular processes related to cellular autophagy,proliferation and migration as well as neural signaling,hormone secretion,protein metabolism,environmental adaptation and immune diseases.A regulatory network of 95 DEGs was constructed by STRING program,and a Hub group of 10 DEGs was screened out.Real-time quantitative PCR results showed that the trends of the detected genes in the high and low lactation groups of Kazakh horses were basically consistent with RNA-Seq results.【Conclusion】 This study screened the genes KCNN4,CAMK2B, CACNA1D,CACNA1E and GRIA4 to be related to the amount of lactation of Kazakh horses during peak lactation,and these genes regulated lactation of Kazakh horses through reciprocal regulation of transmembrane transport,insulin and cortisol secretion and ErbB signaling pathway.
Haplotype Diversity,Genetic Differentiation and Population Expansion of Haemaphysalis longicornis Isolated from Different Districts
LI Zhongbo, CHENG Tianyin
2024, 51(12):  5405-5415.  doi:10.16431/j.cnki.1671-7236.2024.12.027
Abstract ( 54 )   PDF (4303KB) ( 49 )  
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【Objective】 This study was aimed to explore the haplotype diversity (Hd),genetic differentiation and population expansion of Haemaphysalis longicornis (H.longicornis) collected from Liuyang and Xinyang districts,so as to provide data support and theoretical basis for subsequent research on the population genetic structure of H.longicornis. 【Method】 A total of 30 H.longicornis were used as samples in this study.The sequences of cox1 and nad5 genes were amplified by PCR,and were then sequenced.The haplotype diversity of H.longicornis from two districts were analyzed using Clustal X,Dnasp 5.0 and Network 4.6 software.The genetic differentiation and population expansion of H.longicornis from two districts were analyzed by Dnasp 5.0 and Arlequin version 3.0 softwares.【Result】 There were 4 mutational sites and 5 haplotypes (A1-A5,Hd=0.3609) in the sequence of cox1 gene (776 bp),and the gene differentiation coefficient (Gst),fixation index (Fst) and gene flow (Nm) of cox1 gene were ―0.0154,―0.019 and ―26.82,respectively.There were 33 mutational sites and 17 haplotypes (B1-B17,Hd=0.9402) in the sequence of nad5 gene (519 bp),the Gst, Fst and Nm of nad5 gene were 0.0316,0.0503 and 9.44,respectively.There were 37 mutational sites and 20 haplotypes (C1-C20,Hd=0.9563) in the sequence of cox1+nad5 gene (1 295 bp),the Gst,Fst and Nm of the combined gene were 0.0184,0.0451 and 10.59,respectively.All results indicated that H.longicornis from Liuyang and Xinyang districts didn’t appear genetic differentiation.In the population expansion analysis of H.longicornis using cox1,nad5 and cox1+nad5 gene sequences,it was found that H.longicornis from Liuyang and Xinyang districts experienced multiple population expansions.【Conclusion】 H.longicornis from Liuyang and Xinyang districts had low level of genetic variation and haplotype diversity,but there was higher frequency of gene exchange among individuals,no obvious genetic differentiation was found among H.longicornis individuals,and there were population expansions for H.longicornis from Liuyang and Xinyang districts.
Comparative Study on Slaughter Performance and Meat Quality Characteristics of Longling Yellow Goat and Its Hybrid Posterity
JIANG Yanting, NI Xiaojun, KUANG Jicai, YANG Weilin, SUN Dawei, YANG Qingran, DONG Pengfei, TU Xingtiao, LI Wenjun, SHAO Qingyong
2024, 51(12):  5416-5424.  doi:10.16431/j.cnki.1671-7236.2024.12.028
Abstract ( 143 )   PDF (1153KB) ( 25 )  
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【Objective】 The purpose of this study was to explore the slaughter performance and meat quality of different cross combinations of Longling Yellow goat,and select best cross combinations,which provided scientific basis for the development and utilization of the variety resources of Longling Yellow goat.【Method】 6-month-old Longling Yellow goat,Boer×Longling Yellow goat F1 generation and Nubi goat×Longling Yellow goat F1 generation were selected,which were divided into three experimental groups of Longling Yellow goat,Bolong goat and Nulong goat with 12 goats in each group.The three experimental groups were raised under drylot feeding conditions,with a pre-trial period of 10 days and trial period of 180 days.After the experiment,6 goats were selected from each group for slaughter,their slaughter performance and meat quality and nutritional composition of longissimus dorsi muscle were analyzed.【Result】 The slaughter performance indexes of Bolong and Nulong goats were improved compared with Longling Yellow goat,and the back fat thickness of hybrid goats were extremely significantly higher than that of Longling Yellow goat (P<0.01).In terms of meat quality,the pH,drip loss and cooking meat rate of Bolong goats were extremely significantly higher than Longling Yellow goat (P<0.01),and the above indexes were not significantly different between Nulong goat and Longling Yellow goat (P>0.05).At the same time,there were no significant difference in shear force among the three groups (P>0.05).In the detection of major nutrient,the crude protein content of longissimus dorsi muscle of Longling Yellow goat was 23.33 g/100 g,there were no significant difference comparing with Bolong goat (P>0.05),and its crude fat was 0.88 g/100 g that was significantly lower than Bolong goat (P<0.05);The cholesterol content of longissimus dorsi muscle of Longling Yellow goat was 65.80 mg/100 g,which was not significantly different from Bolong goat (P>0.05),but significantly higher than Nulong goat (P<0.05).There were no significant differences in the contents of 7 trace elements and 10 fatty acids of longissimus dorsi muscle in the three groups (P>0.05).Sixteen kinds of amino acids were detected in the longissimus dorsi muscle of three groups,including 7 essential amino acids and 9 non-essential amino acids.The content of amino acids showed more in Longling Yellow goat,but there was no significant difference in content between Longling Yellow goat and Bolong goat(P>0.05).【Conclusion】 The slaughter performance of the hybrid goats had been improved to some extent,and the meat quality and nutrient composition of hybrid goats also could maintain the flavor of Longling Yellow goat.The results provided a theoretical basis for futher improvement and utilization of Longling Yellow goat.
Preventive Veterinary Medicine
Design of Multi-epitope Vaccines for Avian Pathogenic Escherichia coli O1 and O78 Serotypes Based on Subtractive Proteomics and Reverse Vaccinology
CHEN Hong, WU Shuang, CHE Yegui, QIU Shulei, ZHOU Zixiang, WANG Yi, WANG Yongjuan, YUAN Cheng
2024, 51(12):  5425-5438.  doi:10.16431/j.cnki.1671-7236.2024.12.029
Abstract ( 63 )   PDF (11456KB) ( 62 )  
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【Objective】 This study was aimed to design a multi-epitope vaccine (MEV) against avian pathogenic Escherichia coli (APEC) O1 and O78 serotypes,laying a foundation for the development of new vaccines for APEC.【Method】 This study combined subtraction proteomics and reverse vaccinology.Redundant and non-similar proteins in the protein sequences of APEC O1 and O78 were removed using CD-HIT and BLASTP tools.Similar proteins in APEC O1 and O78 were removed by BLASTP and compared with reference proteomes of chicken,retaining non-homologous proteins.Essential toxigenic proteins were screened using DEG,VFDB,etc.databases.Candidate proteins were selected using PSORTb and VaxiJen v 2.0.T-cell major histocompatibility complex (MHC) class Ⅰ and Ⅱ molecule-binding epitopes were predicted using NetCTL 1.2 and NetMHCⅡpan 4.0,and B-cell epitopes were predicted using IEDB.Multi-epitope vaccine were evaluated for antigenicity by VaxiJen v 2.0,and the qualified epitopes were linked together by flexible linkers to form a multi-epitope vaccine.The antigenicity,physical and chemical properties,N-glycosylation sites,secondary structure and tertiary structure of the constructed multi-epitope vaccine were predicted.The binding ability of the multi-epitope vaccine to immune receptors was evaluated by molecular docking,and the immune effect was evaluated by immune simulation.Finally,the codons were optimized for cloning and expression.【Result】 After screening,12 MHC Ⅰ,12 MHC Ⅱ and 12 B lymphocyte epitope-dominant epitopes were selected to construct the multi-epitope vaccine MEV-O1O78.The molecular mass of the multi-epitope vaccine MEV-O1O78 was 69.81 ku,a stable hydrophilic protein with good antigenicity,containing 7 potential N-glycosylation sites.In the secondary structure,alpha-helix,extended chain and random coil accounted for 7.93%,10.81% and 81.27%,respectively.The Ramachandran plot of the tertiary structure showed that the epitope-rich region contained 95.6% of the residues.The immune simulation results showed that multi-epitope MEV-O1O78 could induce good humoral immunity and enhance the expression of some cytokines.The codon optimization ensured that the designed multi-epitope MEV-O1O78 was efficiently and stably expressed in the Escherichia coli K12 expression system.【Conclusion】 In this study,the APEC O1 and O78 multi-epitope vaccine MEV-O1O78 containing 36 dominant epitopes was successfully designed,which provided theoretical basis and data support for the development of multi-epitope vaccine for avian pathogenic colibacillosis.
Preparation of Monoclonal Antibody Against gB Protein of Porcine Pseudorabies Virus and Development of a Competitive ELISA Detection Method
YANG Dawei, LIU Shuyi, LIN Yating, CHEN Hu, ZHANG Hongliang, LI Guimei
2024, 51(12):  5439-5448.  doi:10.16431/j.cnki.1671-7236.2024.12.030
Abstract ( 64 )   PDF (3442KB) ( 56 )  
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【Objective】 This study was aimed to prepare monoclonal antibodies against Porcine pseudorabies virus (PRV) gB protein and establish a competitive ELISA for detection of PRV antibody,provide reference for antibody detection of PRV and development of kits.【Method】 In this study,BALB/c mice were immunized with PRV gB protein,and PEG1500 was used to facilitate cell fusion between mice spleen cells and SP2/0 cells.Positive hybridoma cells were screened by indirect ELISA.Through two rounds of sub-cloning using the limiting dilution method,positive hybridoma cells were induced in vivo to produce ascites,and monoclonal antibodies from the ascites were purified by ammonium sulfate precipitation.Furthermore,the monoclonal antibodies were used to develop a competitive ELISA.The chessboard method was used to optimize the reaction conditions and the negative sera were tested to calculate the cutoff value.The assay was evaluated and compared with commercial kits.【Result】 Two strains of hybridoma cells,named as 2D6 and 2D8 were obtained.Both were IgG1 type with κ-chain light chain.The optimal dilution of monoclonal antibody of the established competitive ELISA method was 1∶400,the optimal coating concentration of protein was 1 μg/mL,the optimal dilution of serum to be examined was 1∶4.The optimal blocking solution was 1% BSA,and the optimal dilution of enzyme-labeled secondary antibody was 1∶4 000.It exhibited good specificity with no cross-reactivity with other porcine viruses. It remained positive when positive serum diluted to 1∶256,indicating that the method had high sensitivity.The coefficients of variation for both intra-assay and inter-assay were less than 10%,demonstrating that the established competitive ELISA method showed high repeatability.Compared with the commercialized kit,the consistency between the established ELISA and commercial test kit reached 93.3%.【Conclusion】 In this study,monoclonal antibodies against the PRV gB protein were successfully prepared,and a PRV gB antibody competitive ELISA detection method with high specificity and sensitivity was established.
Isolation and Identification of Actinobacillus pleuropneumoniae Serotype 1 from a Swine Farm in Fujian Province
YANG Yuan, YU Hui, HE Le, HUANG Wenlin, DAI Ailing, WEI Chunhua, LIU Jiankui
2024, 51(12):  5449-5458.  doi:10.16431/j.cnki.1671-7236.2024.12.031
Abstract ( 60 )   PDF (3980KB) ( 69 )  
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【Objective】 The aim of this experiment was to isolate and identify Actinobacillus pleuropneumoniae (APP) from the lungs of dead pigs suspected to have contracted contagious pleuropneumoniae from a pig farm in Fujian province,and to determine the serotype and biological characteristics of APP strain.In order to provide scientific basis for clinical rational drug use and epidemic prevention strategy in pig farms.【Method】 In this study,the lung tissue of a suspected to have died from APP infection was isolated,cultured,stained and biochemical identified.PCR amplification of 16S rRNA and Apx Ⅳ toxin genes and serotype analysis were performed.Drug susceptibility test and animal pathogenicity test were used to investigate the drug resistance and virulence.【Result】 A strain from diseased pig lung was successfully isolated.The isolate was round colonies with smooth,orderly,greyish-white translucent,Gram staining was negative with short bacilli with polymorphic,which had the culture characteristics of APP.Biochemical identification results showed that the isolate could use xylose,glucose and mannitol,urease,nitrate reduction and methyl red tests were positive,which was consistent with the biochemical characteristics of APP.The sequencing results of 16S rRNA and Apx Ⅳ genes showed that the similarity between the amplified sequence and the APP sequence in GenBank was more than 98%,which further confirmed that the isolate was APP and named it APP FJ2022.Serotype identification showed that the isolate was growth-dependent serotype 1 strain.The results of drug sensitivity test showed that the isolate was sensitive to cefotaxime,cefuroxime and cefmetazole,amoxicillin,and resistant to tetracycline,ampicillin,ciprofloxacin and flufenicol.The results of animal pathogenicity test showed that the isolate was pathogenic to mice,and its median lethal dose (LD50) was 7.71×108 CFU.【Conclusion】 In this study,a serotype 1 APP strain was isolated from dead pig suspected to be infected with pleuropneumonia in a large-scale pig farm in Fujian province.The isolate was resistant to many common antibiotics and showed strong pathogenicity.The experimental results were helpful to understand the serotype of APP in Fujian province,and provide reference for the prevention and control of APP infection in pig farms.
Development and Evaluation of an Indirect ELISA Based on Recombinant E2 Protein for Detection of Bovine Viral Diarrhea Virus Antibody
ZHANG Yichen, SUN Mingjun, DENG Changshun, DING Jiabo, XU Baoliang, CHEN Lei, QUAN Chunlan, ZHAO Kang, FAN Xuezheng, QIN Tong
2024, 51(12):  5459-5469.  doi:10.16431/j.cnki.1671-7236.2024.12.032
Abstract ( 55 )   PDF (2701KB) ( 51 )  
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【Objective】 To provide an effective technique for serological investigation,prevention and control of bovine viral diarrhea (BVD) in China,the E2 protein of Bovine viral diarrhea virus (BVDV) was expressed by eukaryotic system,and used as the coating material to establish a BVDV indirect ELISA antibody detection method for clinic detection.【Method】 The BVDV E2 gene was cloned into the eukaryotic expression vector pTT5,and 293F cells were transfected.After 50% of 293F cells died,the cells were collected,ultrasonicated and the recombinant E2 (rE2) protein was purified by using Ni ion affinity chromatography.rE2 protein was used as coating antigen,and the conditions of each reaction were optimized,an indirect ELISA method for detection of BVDV antibody was developed,and its specificity,sensitivity,repeatability and conformity with the classical neutralisation test were evaluated.The established method was used for detection of bovine serum samples from different provinces of China.【Result】 The rE2 protein was expressed by 293F cells and purified by affinity chromatography,and the purity was 99% with a concentration of 1.74 mg/mL.Through the optimization of each reaction condition,an indirect ELISA antibody detection method for BVDV was successfully established.The method was sensitive enough to detect the weak antibody potency serum (VN=1),which was consistent with that of the commercial kit.It had good specificity and no cross-reaction with the Infections bovine rhinotracheitis virus (IBRV),Bovine parainfluenza virus 3,(BPIV-3),Bovine respiratory syncytial virus (BRSV) and Bovine herpesvirus 1 (BHV-1) antibody-positive sera.The coefficients of variation of the intra- and inter-batch reproducibility tests were within 5%,which showed good reproducibility.And the conformity with the classical neutralisation test was 97.5%.Bovine serum samples from different provinces were detected and total 364 sera were positive and 64 sera were negative sera.【Conclusion】 The indirect ELISA detection method for antibody against BVDV based on eukaryotic expression of rE2 protein had good specificity and sensitivity,and provided an effective means for the prevention and control of BVD in China.
Subcellular Localization of Nonstructural Proteins of Duck Tembusu Virus and Their Role in IFN-β Signaling Pathway
ZHANG Rongrong, PAN Ailuan, WU Juan, FANG Bingbing, WANG Zui, LU Qin, ZHANG Tengfei, WEN Guoyuan, LUO Qingping
2024, 51(12):  5470-5478.  doi:10.16431/j.cnki.1671-7236.2024.12.033
Abstract ( 60 )   PDF (13591KB) ( 34 )  
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【Objective】 This study was aimed to explore subcellular localization of nonstructural proteins of Duck Tembusu virus (DTMUV) and their role in the β-interferon (IFN-β) signaling pathway.【Method】 7 nonstructural protein genes (NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5) of DTMUV were amplified by RT-PCR,cloned into eukaryotic expression vector pCAGGS-HA,constructed recombinant eukaryotic expression plasmid,and transfected into HEK-293T cells,respectively.The proteins expression was detected by Western blotting.The subcellular localization of nonstructural proteins was detected by indirect immunofluorescence assay,and the effect of DTMUV nonstructural proteins on the activity of IFN-β promoter from ducks was studied by dual luciferase reporting assay.【Result】 The experiment successfully constructed 7 eukaryotic expression plasmids of DTMUV with HA tags for nonstructural proteins.Western blotting results showed that the eukaryotic expression of nonstructural proteins was normal,and the molecular weights of NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5 proteins were 38,25,14.4,68,13.9,28 and 100 ku,respectively.The results of indirect immunofluorescence assay showed that the expression patterns of the 7 nonstructural proteins were different in the cell,mainly located in the cytoplasm.The results of dual luciferase reporting assay showed that compared with control group, the activity of IFN-β promoter from ducks was extremely significantly decreased after overexpression of NS2B and NS4B proteins (P<0.01).【Conclusion】 In this study,the eukaryotic expression plasmid of 7 nonstructural proteins of DTMUV was successfully constructed,and the nonstructural proteins were mainly expressed in cytoplasm,among which NS2B and NS4B proteins had the function of opposing IFN-β activity.The results provided a theoretical basis for the study of immune escape of DTMUV,and provided experimental materials for further exploring the mechanism of action of DTMUV in host natural immune signaling pathway.
Construction and Growth Characteristics of Recombinant Lumpy Skin Disease Virus Carrying eGFP Gene
XIN Ruolan, ZHOU Zhiyu, ZHANG Jiawen, DU Jige, YIN Chunsheng, WANG Fengxue, CHEN Xiaoyun, WEN Yongjun, ZHU Zhen
2024, 51(12):  5479-5488.  doi:10.16431/j.cnki.1671-7236.2024.12.034
Abstract ( 52 )   PDF (11067KB) ( 37 )  
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【Objective】 The aim of this experiment was to screen different promoter/cell combinations that could efficiently express recombinant Lumpy skin disease virus (LSDV),and thus construct recombinant LSDV carrying enhanced green fluorescent protein (eGFP) gene.The growth characteristics of the virus were studied,which laid the foundation for the development of antiviral drugs and the study of viral replication and mechanism of action.【Method】 The Orf virus (ORFV) promoter and LSDV promoter were linked to eGFP gene by fusion PCR to replace LSDV 005 gene and construct homologous recombinant fragment,and transfect it into African green monkey kidney cells (Vero),African green monkey kidney cell derived line (Vero-E6),Madin-Darby bovine kidney cell (MDBK),hamster ovary cells (CHO-K1),lamp testicular cells (LT),and primary lamp testicular cells (PLT) infected with LSDV,respectively.The transfection effect was observed by fluorescence microscopy,and the optimal promoter/cell combination was selected by using ImageJ software to calculate the percentage of fluorescence area.The recombinant virus was transfected with the optimal combination,and the purification effect of the virus was tested.The growth characteristics and stability of recombinant virus were further studied.【Result】 The transfection effect of LSDV promoter was superior to that of ORFV promoter in all 6 kinds of cells,and the fluorescence area percentage of LSDV promoter in PLT cells was the highest (8.38%),which was significantly different from that of other groups (P < 0.05).The recombinant virus LSDV-Δ005/eGFP was constructed using the combination of LSDV promoter/PLT cells.After purification,green fluorescence was observed in all the lesion sites under inverted fluorescence microscope,and the fluorescence area percentage was about 50.8%.The growth characteristics and stability analysis of the virus showed that the pathological effect of the recombinant virus LSDV-Δ005/eGFP in 6 kinds of cells and the trend of viral titer change over time was similar to that of the wild virus LSDV/Heilongjiang/2022,and the eGFP gene was statically expressed in PLT cells after 10 consecutive generations.【Conclusion】 The recombinant LSDV was constructed using the combination of LSDV promoter/PLT cells,and the growth characteristics of the recombinant LSDV-Δ005/eGFP were consistent with those of wild LSDV,and the genetic stability was good after 10 passages.
Isolation and Identification of Bovine Enterovirus Types E and F in Guangxi
LIU Huanghao, HUANG Yanhua, LUO Yuhang, REN Tongwei, QIN Yifeng, WEI Zuzhang, OUYANG Kang, CHEN Ying, XIE Jiang, LI Fengmei, CHEN Jicheng, WANG Xiaoling, PAN Yan, HUANG Weijian
2024, 51(12):  5489-5498.  doi:10.16431/j.cnki.1671-7236.2024.12.035
Abstract ( 51 )   PDF (9098KB) ( 49 )  
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【Objective】 By isolating Bovine enterovirus (BEV),this study was aimed to understand its transmission routes and pathogenic characteristics in Guangxi,provide data support for epidemic monitoring and vaccine development,and further promoting the prevention and control of the virus in Guangxi.【Method】 The BEV-positive samples were inoculated into fetal bovine kidney cells (MDBK).After continuous passage,the isolated strains were identified through cytopathic effects (CPE),RT-PCR and indirect immunofluorescence assay (IFA).The identified strains were subjected to plaque purification,virus titer determination,and multi-step growth curve experiments.Specific primers were designed to amplify the full-length genome sequence of the virus.The nucleotide and amino acid similarity between the isolated strains and reference strains were analyzed using MegAlign software.Phylogenetic trees of the viral whole genome and VP1 gene sequences were constructed.【Result】 The third passage of inoculated cells showed significant CPE.RT-PCR results indicated the presence of target bands (239 bp),and IFA results showed that specific green fluorescence could be observed in MDBK cells inoculated with the isolated strains,confirming the isolation of two BEV strains,named GXHC2317 and GXWZ2317, respectively.50% tissue culture infective dose (TCID50) results showed that the TCID50 of strains GXHC2317 and GXWZ2317 were 107.47 and 105.75/0.1 mL,respectively.Multi-step growth curve experiments demonstrated that strains GXHC2317 and GXWZ2317 replicated well in MDBK cells,reaching peak viral replication capacity within 24 h.Similarity analysis showed that the nucleotide and amino acid sequence similarities between strain GXHC2317 and the reference strain were 69.5% to 80.1% and 81.1% to 96.5%,respectively.The amino acid sequence similarity between strain GXHC2317 and strain NX-FY40 isolated from Ningxia region was the highest.The nucleotide and amino acid sequence similarities between strain GXWZ2317 and reference strain were 69.3% to 87.3% and 80.3% to 96.4%,respectively,with the highest amino acid sequence similarity between GXWZ2317 strain and SD-S67 strain isolated from Shandong region.Phylogenetic analysis of the viral whole genome and VP1 gene sequences indicated that the GXHC2317 strain belonged to the BEV E3 subtype,and the GXWZ2317 strain belonged to the BEV F1 subtype.【Conclusion】 This study isolated BEV E3 and F1 subtypes from Guangxi,enriching the investigation of BEV epidemiology and pathogenesis in China.It laid a virological foundation for basic and applied research related to BEV in Guangxi.
Isolation, Identification, Pathogenicity Analysis and Inactivated Vaccine Preparation of a Strain of Riemerella anatipestifer Serotype 2
DAI Tingting, LIU Rongchang, CHEN Haiyu, ZHUANG Liyun, WEI Yufan, TAN Huimin, LIU Yang, TANG Fuchuan, HUANG Yu, CHENG Yanqing, BAO Yinli, ZHENG Xintian
2024, 51(12):  5499-5506.  doi:10.16431/j.cnki.1671-7236.2024.12.036
Abstract ( 66 )   PDF (8287KB) ( 74 )  
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【Objective】 The objective of this study was to analyze the biological characteristics and vaccine effectiveness of Riemerella anatipestifer isolated from a dead duckling,in order to provide guidance for the prevention and treatment of infectious serositis of duck.【Method】 The brain,heart and liver tissues of the duckling were collected for bacterial isolation and culture.The strain was identified by colony morphology observation,Gram staining microscopy,16S rDNA PCR amplification and sequencing,and the genetic evolution relationship was analyzed.The serotype of the strain was identified by slide agglutination method,and the susceptibility to common antibiotics and pathogenicity of the strain were detected.The inactivated vaccine was prepared by the isolate and its immunoprotective effect was measured.【Result】 The isolate showed semi-transparent,dewdrop-like round colonies on the blood plate.Gram staining showed multiple filamentous arrangement of negative short bacilli,and Wright’s staining showed two stages of intensely stained short bacilli.16S rDNA sequencing showed that the isolate was in the same branch as the reference strain of Riemerella anatipestifer CZ-RA03,and the similarity was over 99.9%.Serotype identification showed that the isolate was Riemerella anatipestifer serotype 2.The results of drug sensitivity test showed that the isolate was resistant to ofloxacin,streptomycin and kanamycin,and sensitive to flufenicol,tetracycline and doxycycline.The results of pathogenicity test showed that the median lethal dose (LD50)of the isolate to 7-day-old duckling was 1.51×107 CFU,and the kidney bacterial load of infected duckling was the highest (2.15×106 CFU/g).The oil emulsion inactivated vaccine was used to immunize 7-day-old duckling,and the surviral rate was 86.67%.【Conclusion】 In this study,a high pathogenicity strain of Riemerella anatipestifer serotype 2 was isolated and identified from infected ducklings,and the oil emulsion inactivated vaccine prepared by the strain had good immune protection effect.The results provided a theoretical basis for the prevention and treatment of infectious serositis of duck.
Research Progress on Prevention and Control of Diarrhea of Piglets Infected with ETEC by Traditional Chinese Medicine
YANG Zhi, WANG Yuxuan, WANG Zhiwen, LU Hongde, WANG Luhao, HE Zhiyuan, DONG Hong
2024, 51(12):  5507-5517.  doi:10.16431/j.cnki.1671-7236.2024.12.037
Abstract ( 63 )   PDF (3583KB) ( 135 )  
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Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) seriously affects the health of piglets,which is an important cause of death of piglets,and brings huge economic losses to pig breeding industry.The main virulence factors of ETEC are enterotoxin and adhesin.Enterotoxin is mainly divided into heat-stable enterotoxin and heat-labile enterotoxin,which act on the surface of small intestinal epithelial cells in different ways,causing water and electrolyte accumulation and metabolic disorders in small intestine,resulting in diarrhea of piglets,while adhesin promotes the colonization of ETEC in small intestinal epithelial cells of piglets.The current vaccines only include part of fimbrial adhesin and enterotoxin,and the immune effect is not comprehensive and lacks broad spectrum.At present,the effect of traditional Chinese medicine in clinical application is good,which can greatly improve the survival rate of infected piglets,inhibit the growth of ETEC and the expression of virulence genes,alleviate the inflammatory response,reduce intestinal damage,improve the immunity of infected piglets,regulate the balance of intestinal microorganisms caused by ETEC infection,and regulate the intestinal chemical barrier of piglets,to prevent and control ETEC infection from various aspects.Under the background of total forage prohibition,consumers are increasingly favoring green and non-resistant pig products,and veterinary Chinese medicine provides new ideas and strategies for the clinical prevention and control of piglet diarrhea disease. Therefore,the author summarized the pathogenic mechanism of ETEC invasion into the intestinal tract of piglets and the application and therapeutic effect of Chinese medicine in the treatment of this disease.
Basic Veterinary Medicine
Alleviation of Airway Inflammation Induced by Chronic Obstructive Pulmonary Disease in Rats by Total Flavonoids of Gnaphalium affine Combination with Antibiotics
CHEN Wei, CHEN Xiaolan, ZHOU Siyang, XIE Xiaoyuting, WANG Hao, YANG Haifeng
2024, 51(12):  5518-5527.  doi:10.16431/j.cnki.1671-7236.2024.12.038
Abstract ( 58 )   PDF (12067KB) ( 47 )  
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【Objective】 This study was aimed to investigate the therapeutic effect of total flavonoids of Gnaphalium affine (G.affine) combined with antibiotics on chronic obstructive pulmonary disease induced by Klebsiella pneumoniae and lipopolysaccharides (LPS),and to explore the related anti-inflammatory mechanisms at the level of related proteins and genes to provide a new idea for the treatment of this disease.【Method】 54 Wistar rats were divided into 6 groups: Control group,model group,aminophylline(AM)group,total flavonoids of G.affine (TFG) group,antibiotic (AB) group,and total flavonoids of G.affine+antibiotic (TFG+AB) group,with 9 rats in each group.Among them,chronic obstructive pulmonary disease (COPD) was established in all groups except for control group by tracheal instillation of Klebsiella pneumoniae and lipopolysaccharide (LPS). Rats in control group was treated with equal volume of normal saline instead of Klebsiella pneumoniae and LPS.After successful modeling of COPD for 12 weeks,the rats in treatment groups were treated with aminophylline(50 mg/kg BW) and total flavonoids of G.affine(50 mg/kg BW)by gavage,and ceftazidime(200 mg/kg BW)by intramuscular injection every day.The rats in TFG+antibiotic group were treated with total flavonoids of G.affine (50 mg/kg BW) and ceftazidime (200 mg/kg BW) every day. Control and model groups were not treated. After 7 days of continuous treatment,peak expiratory flow (PEF),peak inspiratory flow (PIF),and the ratio of forced expiratory volume in 0.3 s to forced vital capacity (FEV0.3/FVC) were measured by pulmonary function tester.HE staining and TUNEL staining were used to detect the pathological morphology of rat lung tissue and the level of apoptosis in rat lung tissue, respectively.Western blotting was used to detect the protein expression of Bax,Bcl-2,Cleaved Caspase-3,MMP-2,MMP-9 and TIMP-1 in lung tissues of rats.Real-time quantitative PCR was used to detect the expression of MMP-2,MMP-9 and TIMP-1 genes mRNA in lung tissues of rats.ELISA was used to detect the levels of IL-1β,IL-6 and TNF-α in serum and BALF of rats.Flow cytometry was used to detect the proportion of Th17 and Treg cells in lung tissues of rats.【Result】 Compared with control group,PEF,PIF,FEV0.3/FVC,Bcl-2 and Treg cell ratio of rats in model group were significantly decreased (P<0.05).While the positive rate of TUNEL,the expression of Bax and Cleaved Caspase-3 proteins,Th17 cell ratio,Th17/Treg ratio,the expression of MMP-2,MMP-9,TIMP-1,IL-1β,IL-6 and TNF-α were significantly increased (P<0.05).Compared with model group,PEF,PIF,FEV0.3/FVC,Bcl-2 and Treg cell ratio in aminophylline,total flavonoids of G.affine,antibiotic and total flavonoids of G.affine+antibiotics groups were significantly increased (P<0.05).While the positive rate of TUNEL,the expression of Bax,Cleaved Caspase-3,Th17 cell ratio,Th17/Treg ratio,expression of MMP-2,MMP-9,TIMP-1,IL-1β,IL-6 and TNF-α were significantly decreased (P<0.05).Compared with total flavonoids of G.affine and antibiotic groups,all the proportion of PEF,PIF,FEV0.3/FVC,Bcl-2 and Treg cell ratio in total flavonoids of G.affine+antibiotics group were significantly increased (P<0.05).While the positive rate of TUNEL,the expression of Bax and Cleaved Caspase-3,Th17 cell ratio,Th17/Treg ratio, the expression of MMP-2,MMP-9,TIMP-1,IL-1β,IL-6 and TNF-α were significantly decreased (P<0.05).【Conclusion】 Both total flavonoids of G.affine and antibiotics could reduce airway inflammatory injury in rats with chronic obstructive pulmonary disease,and the combination of them showed better effect.The mechanism was related to the inhibition of apoptosis,the inhibition of matrix metalloproteinases (MMPs) and inflammatory response,and also the reduction of Th17/Treg ratio by total flavonoids of G.affine and antibiotics.
Synergistic Antibacterial Activity and Mechanism of Cinnamaldehyde and Thymol Against Staphylococcus aureus
CHEN Xiaohui, WANG Guiqin, WANG Jianchong, HE Xiaoqiang, CHEN Haorong
2024, 51(12):  5528-5538.  doi:10.16431/j.cnki.1671-7236.2024.12.039
Abstract ( 55 )   PDF (12535KB) ( 100 )  
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【Objective】 This study was aimed to explore the antibacterial activity and mechanism of the combined action of cinnamaldehyde (CA) and thymol (Thy) on Staphylococcus aureus (S.aureus),develop a novel plant-derived essential oil antibacterial agent,establish a theoretical foundation for the clinical application in preventing and treating mastitis in dairy cows.【Method】 In this study,the fractional inhibitory concentration index (FICI) of CA and Thy was determined using the micro-checkerboard dilution method.The time-sterilization curve was further used to verify the synergistic bactericidal ability.The effects of CA and Thy on the bacterial cell membrane structure were studied by observing the membrane integrity,membrane permeability,cell membrane potential,biofilm formation,and alkaline phosphatase (AKP) activity of S.aureus.The effect of CA and Thy on reactive oxygen species (ROS) production was also examined.【Result】 CA and Thy demonstrated significant synergistic antibacterial activity,with a FICI between 0.1875 and 0.375. The results of the time-killing test confirmed the synergistic effect of the CA and Thy combination on S.aureus.The cell membrane integrity significantly decreased,the cell membrane permeability significantly increased (P<0.05),the cell membrane potential depolarized,and the biofilm formation significantly decreased under the synergistic effect of CA and Thy (P<0.05).The synergistic effect of CA and Thy significantly increased the AKP extravasation of S.aureus (P<0.05) and significantly increased ROS level (P<0.05),leading to cell damage and death.【Conclusion】 In summary,the synergistic combination of CA and Thy exerted antibacterial activity by inducing cell membrane damage,leading to leakage of cell contents and disruption of intracellular homeostasis.This study provided a reference for the clinical application of the natural plant essential oil antibacterial agents CA and Thy in the treatment of mastitis in dairy cows.
Isolation and Identification of Staphylococcus chromogenes from Yak Milk and Analysis of Virulence and Drug Resistance
MENG Fanxing, ZENG Jiangyong, QIN Haofeng, ZHAO Congming, YUAN Zhenjie, WANG Dongjing, GESANG Zhuoga, ZHUO Ma, WU Qingxia, MA Hongcai
2024, 51(12):  5539-5548.  doi:10.16431/j.cnki.1671-7236.2024.12.040
Abstract ( 61 )   PDF (2746KB) ( 49 )  
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【Objective】 The aim of this study was to investigate the drug resistance of Staphylococcus chromogenes derived from yak milk in Lhasa and Nagqu,Tibet,and explore the carriage of virulence and drug resistance genes.【Method】 Staphylococcus chromogenes was isolated and cultured from yak milk,and its biochemical characteristics were analyzed.Molecular biological identification was performed by PCR and virulence genes and drug resistance genes carried by the isolates were detected.The drug sensitivity test was carried out by Kirby-Baue (K-B) method.【Result】 11 strains of Staphylococcus chromogenes were isolated and identified from 66 yak milk,and the isolation rate was 16.67%.On 5% defibrated sheep blood agar medium,the isolates formed milky white colonies with neat edges,round,smooth and opaque,without hemolysis. Gram staining microscopy showed positive cocci in a moniliform arrangement.Biochemical identification showed that the isolates could ferment fructose,lactose,mannose and sucrose,and the nitrate reduction reaction was positive.The results of 16S rRNA amplification sequencing showed that the similarity between the isolates and Staphylococcus chromogenes in GenBank was more than 98%.2 biofilm-related regulatory genes,icaA and bap,were detected among 13 virulence genes,and the detection rates were 81.82%.Among the 14 drug resistance genes,the detection rate of aminoglycoside resistance gene aph(3')-Ⅰa,macrolide resistance gene ermB and sulfonamine resistance gene sul1 was 63.64%,27.27% and 9.09%,respectively. The resistance rates to penicillin,ampicillin and tetracycline was 100.00%,63.64% and 36.36%,respectively.【Conclusion】 In this study,11 strains of Staphylococcus chromogenes were isolated from 66 yak milk samples collected from Lhasa and Nagqu.The isolates had different degrees of resistance to penicillin,ampicillin,tetracycline and other common antimicrobials,and carried fewer types of virulence and resistance genes.The results provided a theoretical basis for the prevention and treatment of Staphylococcus chromogenes in yaks.
Isolation,Identification and Biological Characteristics of Staphylococcus aureus, Escherichia coli and Proteus mirabilis from Chickens
XIE Changhong, LIU Guilan, WANG Ying, REN Jianle, NIU Sheng, ZHAO Yujun, FAN Ruiwen, TIAN Wenxia
2024, 51(12):  5549-5559.  doi:10.16431/j.cnki.1671-7236.2024.12.041
Abstract ( 59 )   PDF (6378KB) ( 74 )  
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【Objective】 In April 2023,chicks in a broiler farm in Taiyuan,Shanxi province showed depression,loss of appetite,rapid onset,short course of disease,and high mortality.This experiment was aimed at determining the pathogenic bacteria infection of dead chickens and studying its biological characteristics,in order to provide reference for the prevention and control of bacterial infection diseases in this area.【Method】 Pathological examination and observation were carried out on dead chickens.The heart,liver and spleen tissues of dead chickens were inoculated with BHI agar medium in sterile environment by plate streak method.The morphology of isolates were observed by Gram staining.The bacterial species were identified by biochemical characterization,16S rRNA gene and bacteria-specific primers PCR,and the phylogenetic tree was constructed by Mega 11.0 software.The sensitivity of the isolates to common antibiotics was detected by drug sensitivity test,and the pathogenicity of the isolates was analyzed.【Result】 Subcutaneous edema and exudate were found in the necropsy of the dead chicken.There were bleeding points in the heart,liver,spleen and pectoral muscle.12 isolates were obtained by isolation and purification,8 of them showed obvious hemolysis on blood agar medium,but did not grow on MacConkey agar medium and the Gram staining was purple.3 isolates showed purplish red colonies in MacConkey agar medium and Gram staining was red.1 strain was transparent white colony,Gram staining was red. Biochemical identification results showed that the biochemical characteristics of the isolates were consistent with Staphylococcus aureus,Escherichia coli and Proteus mirabilis,respectively.The plasma coagulase test of 8 isolates was positive and 4 isolates were negative.The results of 16S rRNA gene PCR amplification and sequencing showed that the isolates were more than 98% similar to Staphylococcus aureus,Escherichia coli and Proteus mirabilis in GenBank,and were named SA-1 to SA-8,E1 to E3,and P1,respectively. PCR identification of bacterial specific genes showed single bright bands at 280, 414 and 509 bp, which was consistent with expectations. The results of drug sensitivity test showed that isolates SA-1 to SA-8 were sensitive to imipenem, minocycline and ceftazidime, E1 to E3 were sensitive to flufenicol, imipenem and minocycline, and P1 was sensitive to imipenem, gentamicin, amikacin, ceftazidime and ampicillin. The results of pathogenicity test showed that all the chickens died within 20 h after single infection and mixed infection,and all the isolates had strong pathogenicity.【Conclusion】 In this study,8 strains of Staphylococcus aureus,3 strains of Escherichia coli and 1 strain of Proteus mirabilis were successfully isolated.All of the isolates showed multiple drug resistance and were highly pathogenic to chicks.The results provided important data support for the pathogenesis and prevention of mixed infection of Staphylococcus aureus,Escherichia coli and Proteus mirabilis.
Effects of MLKL on Secretion of Inflammatory Mediators and Activation of Inflammatory Signaling Pathway Induced by Staphylococcus aureus
LIU Bo, GONG Zhiguo, HASI Surong
2024, 51(12):  5560-5569.  doi:10.16431/j.cnki.1671-7236.2024.12.042
Abstract ( 58 )   PDF (6111KB) ( 42 )  
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【Objective】 Staphylococcus aureus (S.aureus),a Gram-positive facultative anaerobic bacterium,was an intracellular pathogen capable of causing various diseases in both humans and animal hosts through the utilization of necrotic apoptosis.This study was aimed to investigate whether the mixed lineage kinase domain-like protein (MLKL) was involved in regulating the inflammatory response induced by S.aureus infection,and provide new ideas and theoretical basis for the prevention and control of diseases caused by S.aureus infection.【Method】 In this study,genetic change in mouse macrophages before and after stimulation with Pam2CSK4 and S.aureus SA113 were analyzed by transcriptome sequencing.The docking modes and sites of Pam2CSK4 and MLKL were analyzed by molecular docking technique.The differentially expressed genes were verified by Real-time quantitative PCR.The phosphorylation levels of MLKL,p65,p38 and ERK were detected by Western blotting.The secretion levels of pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)),chemokine (RANTES) and anti-inflammatory factor (IL-10) were detected by ELISA.【Result】 The results of transcriptional analysis showed that stimulation with Pam2CSK4 and S.aureus SA113 induced the expression of necroptosis-related genes in macrophages,with a high correlation in MLKL gene(P<0.05).Molecular docking prediction results showed that Pam2CSK4 was well bound to MLKL protein and formed hydrogen bonds with ARG-304,ARG-301,GLN-331 and LYS-333 amino acids.Real-time quantitative PCR and Western blotting results showed that compared to unstimulated group, the stimulation with Pam2CSK4 and S.aureus SA113 led to an extremely significant upregulation in the phosphorylation and gene expression levels of MLKL in mouse macrophages (P<0.01).These findings suggested the involvement of MLKL in the S.aureus infection process.The impact of MLKL on the activation of the NF-κB and MAPK signaling pathways was examined using Western blotting.The results indicated that compared with C57BL/6J mice,MLKL-/- mouse macrophages exhibited a significant or extremely significant increase in p65 phosphorylation after S.aureus stimulation for 30 and 60 min (P<0.05 or P<0.01),while an extremely significant decrease was observed after Pam2CSK4 stimulation for 30 and 60 min (P<0.01).After 15,30 and 60 min of Pam2CSK4 stimulation,the phosphorylation level of ERK in MLKL-/- mouse macrophages was extremely significantly downregulated (P<0.01).After 60 min of stimulation with Pam2CSK4 and S.aureus SA113,the phosphorylation level of p38 in MLKL-/- mouse macrophages was significantly or extremely significantly downregulated (P<0.05 or P<0.01). Compared with C57BL/6J mice,after Pam2CSK4 stimulation,the secretion levels of TNF-α and RANTES in the supernatant of MLKL-/- mice macrophages were extremely significantly increased (P<0.01).After SA113 stimulation,the secretion levels of TNF-α,IL-1β and RANTES in the supernatant of MLKL-/- mouse macrophages were extremely significantly increased (P<0.01),while the secretion level of IL-10 was extremely significantly decreased (P<0.01).【Conclusion】 These results suggested that MLKL played a regulatory role in the secretion of inflammatory mediators and the activation of inflammatory signaling pathways induced by S.aureus.
Isolation,Identification and Pathogenicity Analysis of a Strain of Pasteurella multocida A∶L3 from Yaks
ZHUO Ma
2024, 51(12):  5570-5576.  doi:10.16431/j.cnki.1671-7236.2024.12.043
Abstract ( 57 )   PDF (4899KB) ( 44 )  
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【Objective】 The objective of this study was to explore the etiology of disease and death of yaks in a breeding farm in Ali area,Tibet,and analyze the biological characteristics of the pathogenic bacteria,so as to provide reference for effectively preventing and controlling the disease.【Method】 Tissues of dead yaks were collected aseptically,and laboratory diagnosis was carried out according to clinical symptoms,and pathogenic bacteria were isolated and cultured.The isolate was identified by morphological observation,biochemical identification,16S rRNA PCR amplification and sequencing,kmt gene identification,capsular serotyping and lipopolysaccharide typing.The drug sensitivity and pathogenicity of the isolate was investigated.【Result】 The isolate grew grayish white,transparent and dewdroplet colonies in 7% serum TSA medium.Gram staining showed strong Gram-negative brevibacterium at both ends.The results of biochemical tests showed that the isolate was positive in glucose,ornithine decarboxylase,oxidase,sorbitol and mannitol biochemical reaction tubes.The results of capsular serotyping and lipopolysaccharide typing showed that the isolate was Pasteurella multocida capsular type A,and its lipopolysaccharide type was L3.Drug susceptibility test showed that the isolate was sensitive to carbenicillin,ampicillin,cefazolin,gentamicin,nitrofurantoin,cefuroxime,amicana,minocycline and norfloxacin.The results of pathogenicity test showed that the mortality rate of mice was 100% (5/5) after injecting 0.2 mL 1×108 CFU/mL of the isolate bacterial solution,and the isolate had strong pathogenicity.【Conclusion】 In this study,a strain of serotype A lipopolysaccharide L3 Pasteurella multocida was successfully isolated and identified,which was sensitive to carbenicillin,ampicillin,cefazolin and other drugs,and was highly pathogenic.The results provided data reference for exploring the biological information and etiological characteristics of Pasteurella multocida from yaks.
Clinical Veterinary Medicine
Investigation and Analysis of Epidemiological Characteristics of Canine Respiratory Tract Infection
JU Houbin, YANG Dequan, LI Xin, JIN Jizexiao, WANG Jian, SHEN Sufang, YANG Xianchao, GE Jie, ZHU Jiuchao, SHEN Haixiao, LU Jun, TANG Wenhong, WU Xiujuan, TANG Yanting, SHENG Wenwei
2024, 51(12):  5577-5585.  doi:10.16431/j.cnki.1671-7236.2024.12.044
Abstract ( 61 )   PDF (1295KB) ( 26 )  
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【Objective】 The aim of this study was to understand the composition and infection characteristics of the pathogen spectrum of respiratory tract infections in dogs.【Method】 This study selected 400 cases of canine nasal and oropharyngeal swab samples with clinical respiratory symptoms from animal diagnosis and treatment institutions in 18 provinces of China in 2022.The nucleic acid of 6 kinds of dog respiratory pathogens were detected by constant temperature nucleic acid amplification chip,and the characteristics of their different prevalence among different genders,different seasons,different ages,different regions and different breeds were analyzed.【Result】 The total detection rate of canine respiratory tract pathogen nucleic acid in 400 samples was 97.25%,and the detection rate of Canine parainfluenza virus (CPIV) was the highest 35.00%,followed by Mycoplasma canis (M.canis, 31.00%),Canine respiratory coronavirus (CRCoV,28.50%),Canine distemper virus (CDV,18.50%),Brodetella bronchiseptica (B.b,10.50%),and Canine influenza virus (CIV,1.50%).There were 92 cases of mixed infection,and the mixed infection rate was 23.00%,among which 77 cases were double infection,accounting for 83.70% of the mixed infection cases.CPIV+M.canis,CRCoV+CPIV and CRCoV+M.canis were more common,the positive rates were 4.75%,4.25% and 3.75%,respectively.There were 12 cases of triple infection,accounting for 13.04% of mixed infection cases,2 cases of quadruple infection,accounting for 2.17% of mixed infection cases,and 1 case of quintuple infection,accounting for 1.08% of mixed infection cases.According to Pearson chi-square test,there were significant differences in CDV among different ages (P<0.05).There were no significant differences among the other five respiratory pathogens in different sexes,different ages,different regions and different varieties (P>0.05).【Conclusion】 The results showed that the composition of canine respiratory tract pathogen spectrum was complex,mainly composed of CPIV,M.canis,CRCoV,CDV and B.b,and it was easy to mixed infection,which could be infected all year around,especially in autumn and winter.Systematic and continuous research should be carried out to provide scientific basis for the accurate prevention and control of canine respiratory tract diseases.
Research Progress of Urinary Incontinence in Spayed Bitches
LI Yujue, ZHONG Yougang
2024, 51(12):  5586-5596.  doi:10.16431/j.cnki.1671-7236.2024.12.045
Abstract ( 118 )   PDF (1886KB) ( 70 )  
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Urinary incontinence (UI) is the involuntary loss of an animal during the filling phase of the bladder.Among the clinical diseases of small animals,UI is more common in bitches,especially in spayed bitches,with the incidence of 1.74%-20.10%.The risk factors of UI in bitches include neuter status,breed,weight,etc.Urethral sphincter mechanism incompetence (USMI) is the most common cause of UI in bitches.However,the pathogenesis is still unclear and influenced by multiple factors,including changes in sex hormones,structure and function.At present,the diagnosis of USMI is difficult in small animal clinical practice,usually confirmed by the use of α-adrenergic agents and estrogen products that alleviate the symptoms of UI after the exclusion of other differential diagnoses.Drug treatment is preferred for USMI,and surgical treatment can be considered for refractory cases.In addition,Chinese medicine therapy may be a potential effective treatment.The authors reviewed the research progress of UI in spayed bitches at home and abroad,in order to provide theoretical reference for the diagnosis and treatment of this disease,to deepen veterinarians’ understanding and make more efficient diagnosis of this disease,and provide new ideas for the research related to UI in spayed bitches.