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20 April 2014, Volume 41 Issue 4
Bioinformatics Analysis of MSTN Gene in Sansui Duck
YI Heng-jie,LI Hui,YANG Sheng-lin,ZHAO Zhong-hai,PENG Bang-xing
2014, 41(4):  1-6. 
Abstract ( 264 )  
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In this study, the coding region of MSTN gene was amplified by PCR and sequenced by direct sequencing. Some characters of the MSTN gene encoding protein, including the physico-chemical properties, singal peptide, ransmembrane structure, secondary structure, high structure and functional domains, were analyzed by bioinformatics tools. The results showed that MSTN protein of Sansui duck was a kind of labile protein with high hydrophobicity, which was probably being singnal peptide. The protein contained 22 α-helics,26 β-sheets,26 T-turns,20 irregular coils and 1 transmembrane domain, and it had the main feature of TGF-β superfamily. The result provided an important reference for its further research.
Establishment and Application of a Duplex PCR Method for Detecting Brucella and Chlamydia psittaci
PENG Wu-li, LUO Zhan-rong, SHI Qian, YUN Li-juan, YU Xue-hui, JI Xin-cheng
2014, 41(4):  7-11. 
Abstract ( 203 )  
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To establish a duplex PCR method for simultaneously detecting Brucella and Chlamydia psittaci, this study obtained two genus-specific gene sequences according to the GenBank, including bp26 of Brucella spp. and 23S rRNA of Chlamydia psittaci. Two pairs of specific primers were respectively designed. After optimizing the reaction condition, the duplex method was established for simultaneously detecting Brucella and Chlamydia psittaci. The method had better specificity and repeatability, the detection sensitivity of simplex method for each gene both could reach 3.1×102 copies per reaction, the detection sensitivity of duplex method for each gene could reach 3.1×103 copies per reaction. Using this duplex method to detect 172 samples including bloods, sera, placentas of abortion cattle and milk suspected infecting Brucella in clinic,53 Brucella positive samples and 2 Chlamydia psittaci positive samples were detected out, the positive rates were 30.8% and 1.2%, separately. 2 co-infection positive samples were detected out else and the positive rate was 1.2%. The above results indicated that this method could be used for simultaneously, rapidly and sensibly detecting the two pathogens of Brucella and Chlamydia psittaci.
Sequence Analysis of TK Gene of Porcine Pseudorabies Virus Isolated from Shanxi Area
FAN Zhen-hua, MENG Fan, WU Xin, LIU Wen-jun, YAO Jing-ming, WANG Juan-ping, HAN Yi-chao, MI Rui-juan, LEI Yu-ping
2014, 41(4):  12-17. 
Abstract ( 188 )  
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TK genes of two pseudorabies virus (PRV) strains that had been isolated from Shanxi province were amplified by RT-PCR among 2009 and 2011, cloned and sequenced. The obtained sequences and the deduced amino acid were analyzed and compared with the domestic and international major epidemic strains of PRV Bartha strain, Becker strain, Ea strain, Kaplan strain, LA strain, Min-A strain, NIA-3 strain, SH strain, SL strain and Yangsan strain by homologous analysis. Sequence analysis showed that the nucleotide homologies of TK genes were 99.7%, 99.6%, 99.8%, 99.7%, 99.7%, 94.4%, 99.1%, 57.2%, 99.5% and 99.7%, respectively, and the amino acid homologies were 99.1%, 99.1%, 99.7%, 99.4%, 99.4%, 90.3%, 98.1%, 49.7%, 98.8% and 99.1%. 3 sections of GC box like sequence upstream of the initiation codon and poly adenosine polyadenylation signal AATAAA downstream of the stop codon were found in nucleotide sequence, TK gene was composed of 320 amino acids,with a conserved sequence of TK gene of herpesvirus common amino acid sequence,namely -R*Y*DG**G*GK*T- and -FDRHP*A***C*P*AR-.
Stydy on Expression Regulation and Regulatory Functions of miRNA-205 in Pig Oocyte in vitro Maturation
CHEN Chao,LI Wan-hong, SUN Li-na,CHEN Shu-xiong,CHEN Lu,LI Chun-jin,ZHOU Xu
2014, 41(4):  18-22. 
Abstract ( 212 )  
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The research aimed to assess the expression profile and regulatory functions of miRNA-205 in pig oocyte in vitro maturation. The potential target of miRNA-205 was predicted by biology software including TargetScan and microRNA.org. The expression profile of miRNA-205 and its target gene were detected by Real-time PCR in pig cumulus-oocyte complexes(COCs) in vitro maturation 0, 24 and 48 h. The expression of miRNA-205 in pig COCs maturation gradually increased, 0 h lowest, 48 h maximum. The expression of miRNA-205 was significantly reduced in mature solution added EFG, FSH and LH compared with which in a basic mature solution, the expression of PTX3 and cumulus proliferation-related genes including TNFAIP6 and HAS2 were significantly increased. The expression of PTX3 was negative correlation with that of miRNA-205 in porcine oocyte maturation. From these results could be concluded that the miRNA-205 conserved sequence specifically bound to 3′-UTR region of PTX3 gene might be participated in the regulation of oocyte maturation.
Development and Application of a Duplex PCR Assay for the Detection of Mycoplasma mycoides Cluster and Pasteurella multocida
WANG Cheng-long, WU Yu-xi, FENG Xu-fei, YANG Fa-long
2014, 41(4):  22-26. 
Abstract ( 187 )  
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The aim of this study was to establish a duplex PCR method for simultaneous detection of Mycoplasma mycoides cluster and Pasteurella multocida, so as to provide a rapid, accurate and convenient detection tool for these pathogens. Two pairs of primers which were specific to Mycoplasma mycoides cluster and Pasteurella multocida,respectively, were used to develop the method. Following optimization of PCR components and reaction profile, specificity and sensitivity of the assay were evaluated. A total of 52 clinical samples were tested using the duplex PCR. The results showed that the assay could specifically amplify Mycoplasma mycoides cluster and Pasteurella multocida, while no-target pathogens were not detected. The detection limits of the assay were determined to be 24.8 and 28.9 pg for Mycoplasma mycoides cluster and Pasteurella multocida, respectively. The duplex PCR could detect Mycoplasma mycoides cluster and Pasteurella multocida from clinical samples. These results suggested that the duplex PCR assay was specific and sensitive, and would be useful for clinical detection, identification and epidemiological investigation of Mycoplasma mycoides cluster and Pasteurella multocida.
Study on Prokaryotic Expression and Immunogenicity of Toxoplasma gondii Embryogenesis-related Protein
TIAN Wei-peng, ZHANG Nian-zhang, GAO Qi, LU Li, ZHU Xing-quan, SONG Ming-xin
2014, 41(4):  27-31. 
Abstract ( 225 )  
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In order to analyze the immunogenicity of Toxoplasma gondii embryogenesis-related protein (TgERP), the TgERP gene was amplified from genomic DNA of T.gondii RH strain by PCR and then cloned into prokaryotic expression plasmid pET-30a (+). The recombinant plasmid pET-30-ERP was transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The expressed product was purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blotting. The polyclonal antibody against the recombinant protein was prepared by immunizing New Zealand White rabbits with the purified protein. PCR amplification and restriction analysis proved that the restriction prokaryotic expression plasmid pET-30a (+) was constructed correctly and sequencing results showed that the cloned TgERP gene was identified to be the corresponding sequence reported on the website (http: //toxodb.org). The TgERP protein was expressed under 1.0 mmol/L IPTG at 37 ℃, shaking for 6 h. SDS-PAGE analysis showed the protein product was successfully purified with a molecular weight of about 16.7 ku. Western blotting and ELISA analyses indicated that the TgERP protein reacted with sera from vaccinated rabbit. In conclusion, it could be a potential candidate antigen for developing new detection methods or new sub-unit vaccine against toxoplasmosis.
Segmented Expression of Aleutian Disease Virus (ADV) NS1 Gene Fragment in E.coli
LIU Hong-na, WANG Wen-yu, ZHANG Yun, YANG Yu-hang, HAO Jun-wei, SHI Kun, LI Jian-ming, DU Rui
2014, 41(4):  32-36. 
Abstract ( 284 )  
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We predicted the non-structural protein NS1 of aleutian disease virus (ADV) by DNAStar software Protean module. Then three fragments of NS1 gene named as L1N1, L2N2 and L3N3 were amplified using the laboratory saved NS1-pMD18-T plasmid as template. We constructed three cloning plasmids pMD18-T-L1N1, pGEM-T-L2N2 and pMD18-T-L3N3. After identification, they were linked to pGEX-4T-1 prokaryotic expression vector by two enzymes EcoRⅠ and SalⅠ, respectively, and transformed into E.coli BL21 (DE3) inducing with IPTG. The expressed recombinant proteins were analyzed by SDS-PAGE and Western blotting. The results showed that three recombination prokaryotic expression plasmids pGEX-4T-L1N1, pGEX-4T-L2N2 and pGEX-4T-L3N3 were successfully constructed and the SDS-PAGE results showed that three purpose proteins were expressed about 57, 55 and 44 ku as expected. Furthermore, the proteins were recognized by ADV positive sera and had the reactionogenicity. In the study, the NS1 gene of ADV was expressed segmentedly in E.coli for the first time and laid a foundation for the further study of ADV subunit vaccines.
Effects of IGF-Ⅰ, HGF, TGF-β1, IFN-γ on Proliferation of Bovine Mammary Epithelial Cells
LI Ying-ying, XIA Xiao-jing, WU Yun-di, HE Bo-ping, YAN Xin-wu, GAO Yuan-yuan, WANG Yu-ping, LEI Lian-cheng
2014, 41(4):  37-42. 
Abstract ( 206 )  
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This experiment was conducted to investigate the regulation of insulin-like growth factor-Ⅰ(IGF-Ⅰ), hepatocyte growth factor (HGF), transforming growth factor-β1 (TGF-β1) and interferon-γ (IFN-γ) in the growth of bovine mammary epithelial cells in vitro.The bovine mammary epithelial cells were isolated by explant culture technique and differential adhesion method and identified by immunochemistry.The effect of cytokines regulating the growth of bovine mammary epithelial cells in different concentrations was measured by MTT.The results showed that when the concentrations of IGF-Ⅰ and HGF were 10 to 200 and 0.1 to 100 ng/mL, respectively, the growth of bovine mammary epithelial cells increased. While when the concentrations of TGF-β1 and IFN-γ were 2.5 to 100 and 5 to 160 ng/mL, respectively, the growth of bovine mammary epithelial cells decreased in a dose-dependent manner.These results indicated that IGF-Ⅰ and HGF could upregulate the proliferation of bovine mammary epithelial cells in vitro in a dose-dependent manner.TGF-β1 and IFN-γ could inhibit the proliferation of bovine mammary epithelial cells in vitro.
Development of an Indirect ELISA Assay for Detecting Antibody to Porcine Reproductive and Respiratory Syndrome Virus with Recombinant GP5-encoding-epitopes Protein
CHEN Ru-jing, WU Xue-min, CHE Yong-liang, WANG Long-bai, LIU Yu-tao, YAN Shan, WEI Hong, ZHUANG Xiang-sheng,ZHOU Lun-jiang
2014, 41(4):  43-47. 
Abstract ( 213 )  
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An indirect ELISA assay was constructed to detect porcine reproductive and respiratory syndrome virus (PRRSV) antibodies with recombinant GP5-encoding-epitopes protein.The optimal concentration of antigen was 7.5 μg/mL; The blocking buffer was 5% defatted milk powder with the blocking time 2 h,at 37 ℃;The serum sample was diluted in 1∶100 with incubated for 2 h at 37 ℃;The dilution of rabbit-anti-porcine IgG labeled by HRP was diluted in 1∶3000 with incubated for 2 h at 37 ℃;The TMB substrate was added and incubated at 37 ℃ for 15 min and then terminated with stopping solution. The standard of judgment was that the serum sample D450 nm value with S/P≥0.254 was positive, and the sample D450 nm value with S/P≤0.212 was negative. The recombinant antigen had no cross-reaction with antibodies of other five porcine diseases. 70 serum samples collected from pigs which were vaccinated with Ingelvac PRRS MLV were detected by this assay, the D450 nm value of which were higher than 0.85. The results demonstrated that the establishment indirect ELISA method could be used for monitoring PRRSV antibody.
Construction and High Level Expression of Prokaryotic Expression Vector of Chicken Interferon-α Gene
LIU Cheng-qian, WANG Jing, LI Hong, YU Zong-xing, YI Jian-zhong, CHEN Lei
2014, 41(4):  47-50. 
Abstract ( 216 )  
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Interferon (IFN)-α had received much more attention for its comprehensive antiviral activity. According to the relevant sequence from GenBank, a pair of specific primers was designed for amplifying chicken IFN-α gene with PCR method. The IFN-α gene was cloned into prokaryotic expression vector pET-32a and pET-30a-DsbA and the protein’s expression was identified by SDS-PAGE. Immunity characteristic of the protein was analyzed by Western blotting. The cloned pET-30a-DsbA-IFN-α/E.coli was found to produce a 40.4 ku protein at the high level. The expression products existed mainly in the form of inclusion body. These expression products could specificly react with His-tag monoclonal antibody.
Cloning and Sequence Analysis of c-Myc Gene in Goat
AN Chen-rui,AN Tie-zhu,ZHANG Qiu-ting,LI Hao,PIAO Shan-hua,WANG Chun-sheng
2014, 41(4):  51-56. 
Abstract ( 352 )  
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In order to clone sequence of the coding region of the goat c-Myc gene, this study designed specific primers by refering to c-Myc genes coding sequence reported in GenBank including sheep,cattle,pigs,horses,etc.,and then amplified c-Myc coding sequence from embryonic skin tissue of goats by using RT-PCR technique and had sequence analysis. The result indicated that CDS of goat c-Myc gene had 1320 bp length including the stop codon. For only 10 differences comparing to sheep c-Myc gene nucleotide sequence (GenBank accession number:Z68501.1),homology reached 99.17% and homology of deduced amino acid sequence reached 99.09%. Analysis of homology with other species showed that homology of goat c-Myc gene coding region nucleotide sequence was 91.2%,92.0%,90.2%,86.9%,86.0% with pigs,wolves,humans,rats,mice,and homology of deduced amino acid sequence was 93.6%,96.1%,92.3%,91.1%,89.6%. Using bioinformatics software analysis showed that goat c-Myc protein was localized in the nucleus and contained a HLH domain. This study further researched the function of c-Myc gene and laid the foundation of investigating the effect in the process of goat somatic cell reprogramming.
Prokaryotic Expression of the Truncated 43K OMP Genes of Bovine Fusobacterium necrophorum
LV Si-wen, ZHANG Hong, SUN Dong-bo, HU Bai-dong, GUO Dong-hua
2014, 41(4):  56-60. 
Abstract ( 211 )  
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Four pairs of primers containing BamHⅠ and XhoⅠ sites were designed for amplification of 43 ku outer membrane protein (43K OMP) of bovine Fusobacterium necrophorum strain H05 according to the GenBank. The PCR products of the truncated 43K OMP genes were digested with BamHⅠ and XhoⅠrestriction endonuclease, and then the digested products were ligated to the pET-32a vector with His tag. The positive plasmids of four truncated 43K OMP genes were transformed into E.coli BL21(DE3). Protein expression of four truncated 43K OMP genes were induced using 1.0 mmol/L IPTG. The result indicated that the four truncated 43K OMP genes were successfully expressed in E.coli, and molecular weights of the expressed proteins were all about 30 ku. This study would provide some basis for further research of immunogenicity of the outer membrane protein of Fusobacterium necrophorum.
Cloning and Sequence Analysis of Nucleoprotein, Matrix Protein and Nonstructural Protein Gene of H1N1 Subtype Swine Influenza Virus Isolated from Tianjin
SUN Ying-feng, YAN Ming-hua, LU Chao, LI Xiu-li, ZHANG Li, HAN Wei, REN Wei-ke, TIAN Xiang-xue
2014, 41(4):  61-66. 
Abstract ( 267 )  
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Six strains of H1N1 subtype swine influenza viruses were isolated from pigs with influenza-like signs in Tianjin. Three pairs of primers were designed based on nucleoprotein (NP), matrix protein (M) and nonstructural protein (NS) genes of the influenza virus H1N1 subtype in GenBank. The NP, M and NS genes were amplified by RT-PCR, which were cloned into pMD18-T vector, sequenced and analyzed. As demonstrated by homology and phylogenic analysis, it was found that the NP, M and NS genes of A/swine/Tianjin/TJ2/2005 (H1N1) and A/swine/Tianjin/TJ4/2006 (H1N1) were higher homologues with A/swine/Guangdong/33/2006, belonging to the classical H1N1 swine lineage. While the A/swine/Tianjin/TJ3/2006 (H1N1) and A/swine/Tianjin/TJ8/2006 (H1N1) were higher homologues with A/Dunedin/2/2000 (H1N1), maybe belonging to human H1N1 lineage. A/swine/Tianjin/TJ6/2009 (H1N1) and A/swine/Tianjin/TJ7/2009 (H1N1) were higher homologues with A/swine/Jiangsu/s15/2011 (H1N1), belonging to the class of avian H1N1 swine lineage. This study showed the evolution and epidemic situation of swine influenza viruses in Tianjin area.
Polymorphisms and Bio-informatics Analysis of Ovis aries and Homo sapiens DRB1 Genes Exon2
CHEN Yue-e, LI Jian-hua, GOU Ya-feng, WANG Wen-wen, ZHU Jun-bao, GAO Jian-feng
2014, 41(4):  66-70. 
Abstract ( 213 )  
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This study was aimed to detect the single nucleotide polymorphisms (SNPs) and single amino acid polymorphisms (SAPs) of Ovis aries and Homo sapiens DRB1 genes exon2, and analyze the structure and characteristic change of OLA-DRB1 protein exon2 by bio-informatics. The sequence polymorphisms of Homo sapiens and Ovis aries MHC-DRB1 exon2 were analyzed with comparative genomics and biological databases. The protein structures and functions of Ovis aries MHC-DRB1 were predicted and analyzed with bio-informatics softwares. The result showed that there were rich polymorphisms and SAPs both in HLA-DRB1 and OLA-DRB1 exon2. The number of singleton polymorphic and parsimony informative polymorphic sites were different from each other. There were only 14 same SNPs loci compared with each other, the rest of the site were different. Sequence analysis indicated that they were highly homology on MHC-DRB1 exon2. The results of the bio-informatics analysis indicated that caused by sequence variation of single amino acid triggering protein structure’s change secondary structure and tertiary structure, could lead to obnormal gene function, and result in disease resistance and disease susceptibility mutation.
Effects of Cinobufagin on the Major Histocompatibility Complex ClassⅠ Molecules and Expression of their Related Genes in B16 and RMA-S Cells
ZHANG Jin, SHI Qi-yun, CHENG Sha, CAO Shu-fang, CHANG Hao, SONG Yu
2014, 41(4):  71-75. 
Abstract ( 240 )  
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The study was aimed to investigate the effects of cinobufagin on the major histocompatibility complex classⅠ(MHC-Ⅰ) molecules and expression of their related genes in B16 and RMA-S cells. The effect of CBG on cell proliferation was determined by MTT assay. The expression of MHC-Ⅰ molecules was assayed by flow cytometry (FCM). The mRNA expression of LMP2, LMP7, TAP1 and TAP2 genes were deteceted by Real-time PCR. The results showed that the expression of MHC-Ⅰ was not affected significantly by CBG both in B16 and RMA-S cells(P>0.05), while the mRNA expression of LMP2, LMP7, TAP1, TAP2 genes were increased remarkably by CBG of high concentrations. CBG may have the potential to enhance the immunogenicity of tumor by means of increasing the expression of LMP2, LMP7, TAP1 and TAP2 genes.
Extraction,Purification and Activity Analysis of Lipopolysaccharide of Salmonella paratyphi C
YANG Cheng-lan, LUO Wei, LIU Nei-sheng, YANG Yang
2014, 41(4):  75-78. 
Abstract ( 247 )  
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This study was to obtain lipopolysaccharide (LPS) from Salmonella paratyphi C and analyze its activity. In this study, LPS was extracted from Salmonella paratyphi C by the hot phenol-water method,and purified with DNaseⅠ, RNase A and proteinase K. The contents of the purified polysaccharose, protein and nucleic acid of the purified LPS were detected, its bioactivity was detected with tachypleus amebocyte lysate (TAL). The results showed that the productivity of the purified LPS was 1.48%, the proportions of the average polysaccharose, protein and nucleic acid were 3.84%, 1.49% and 5.45%. The sequence of nucleic acid was lower than 100 bp. SDS-PAGE and silver stain showed that strip size was approximately 10 to 15 ku, the agglutinate activity of TAL was 10.99 ng/mL. The chromogenic limulus amobocyte lysate assay showed that the activity of LPS was 9.82×105 EU/mg. In conclusion,the purified LPS of Salmonella paratyphi C in this study was with high purity and bioactivity.
Advances on Molecular Biology Technology of the Water Buffalo (Bubalusbubalus)
DENG Ting-xian, PANG Chun-ying, WANG Jian, YANG Bing-zhuang, ZHANG Xiu-fang, LIANG Xian-wei
2014, 41(4):  79-84. 
Abstract ( 279 )  
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The water buffalo (Bubalusbubalus) is an important economic specie in southern provinces that was acclaimed as the most potential and valuable development on the livestock by FAO. The new molecular biology technology will bring the new challenge and opportunity for buffalo researches in recent years. Many major breakthroughs have been made in the research area on the molecular biology technology of the water buffalo, which should reveal its great superiority and development potential on the buffalo genetics and breeding. Consequently, the molecular biology technology applied to the buffalo genetics and breeding and its prospect will be discussed, mainly including the molecular marker, gene chip, transgenic technology and high-throughput sequencing technology, etc. It will lay a foundation for further research on the molecular biology technology of the water buffalo and play an important role in promoting the rapidly development of the buffalo industry.
Construction and Expression of Prokaryotic Expression Vector of SLA-2-YTH Derived from Yantai Black Pig
JIANG Wei, DONG Song-peng, LI Zi-bin, JIANG Long3, FENG Lei, GAO Feng-shan
2014, 41(4):  85-89. 
Abstract ( 207 )  
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In order to construct the SLA-2-YTH gene prokaryotic expression vector of Yantai Black pig, a pair of primers for amplifying the extracellular domain of SLA-2-YTH by PCR was designed, followed by cloning the gene into pMD 19-T Simple vector, and then the positive clones could be analyzed directly. After cleavage with NdeⅠand XhoⅠ, the positive clone was successfully inserted into pET-28a(+) and then the recombinant plasmids were transformed into competent cell BL21 (Rosseta).After induction, the protein expression could be detected by SDS-PAGE. The results showed that sub-clone of the extracellular domain of SLA-2-YTH was about 834 bp, and it was successfully cloned into pMD 19-T Simple vector showed by the enzyme analysis. By SDS-PAGE, SLA-2-YTH gene was successfully expressed in Escherichia coli BL21 (Rosseta) and the target protein was about 31.0 ku, which was consistent with prediction. After optimization, the relative expressed content of recombinant SLA-2 protein reached more than 25%. Through the research, we successfully constructed the SLA-2-YTH gene prokaryotic expression vector of Yantai Black pig, and then gained the expressed protein, which would lay the foundation for the future study of structure and function of SLA-2.
Prokaryotic Expression Vector Construction and Immunogenicity Analysis of Glycosylated Envelope Protein 5 from Porcine Reproductive and Respiratory Syndrome Virus
LI Xin-xian, ZHANG Xiao-dan1, LIU Jing-jing, MIAO Zeng-min2, ZHANG Tao-tao, CHAI Tong-jie
2014, 41(4):  89-94. 
Abstract ( 248 )  
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In order to analyze the immunogenicity of glycosylated envelope protein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV), ORF5 gene fragment was amplified by RT-PCR from the PRRSV (GenBank: HQ701732.1). Based on the ORF5 gene sequence, two pairs of primers were used to amplify two gene fragments, excluding the signal peptide sequence and transmembrane regions. The two gene fragments were cloned into the prokaryotic expression vector pET-28a(+), and then the recombinant plasmid was transformed into Escherichia coli. The results of Western blotting and ELISA respectively showed that the expression of GP5 could be recognized by positive serum antibody of PRRSV, and corresponding antibodies of GP5 protein could produce in the immunized BALB/c mice. Therefore, the recombinant GP5 protein had good biological activity, and could provide fundamental data for the further study on the structure and function of GP5 protein of PRRSV.
Establishment of Indirect ELISA Method to Detect Q Fever Coxiella burnetii
WU Wen-jun,JIA Guang-le,LIAO Juan-hong2,MEI Lin,HAN Xue-qing,LIN Xiang-mei,ZHANG Ying
2014, 41(4):  95-101. 
Abstract ( 224 )  
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In this study, the whole bacteria protein of the Coxiella burnetii attenuated strain phase Ⅱ was used as the antigen and then the indirect ELISA to detect Q fever Coxiella burnetii was established. Through optimizing the concentration of the coated phase Ⅱ antigen, the concentration of the second antibody and the reaction condition, we confirmed that the critical value of this method was 0.44. 393 clinical bovine sera were detected by this method and IDEXX Q fever (Coxiella burnetii) Antibody Test Kit. The results showed that the sera positive detection rates were 11.45% and 6.10%, respectively. Compared with the results of the two detection methods, the coincidence was 94.66%. The results demonstrated that sensitivity and specificity of the established indirect ELISA detection method were better and it could be used for the clinical diagnosis of the antibody of Q fever Coxiella burnetii.
Establishment and Preliminary Application of Nested PCR Method for Detection of Porcine Proliferative Enteropathy
ZHANG Wen-bo, DAI Yi-min, JIANG Xin-hua, HU Yang, XU Chang-man, CHEN Song-chang, DENG Shun-zhou
2014, 41(4):  102-106. 
Abstract ( 172 )  
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Porcine proliferative enteropathy (PPE), caused by Lawsonia intraeellularis (LI), was a kind of swine contagious infectious disease. In order to establish a nested PCR method to detect LI, two pairs of primers were designed according to the genome of LI, and the nested PCR was established for detection of LI in ileum or feces of pigs. The sequencing result analysis showed that the homologies of the amplified nucleotide sequences to the LI references sequence were above 99%. The nested PCR method established in this study was rapid, easy and sensitive, could be used in clinical diagnosis of PPE.
Effects of Wheat and Fermented Sesame Meal on Growth Performance,Meat Quality and Serum Biochemical Indexes in Broilers
XU Xin,WU Dong,QIAN Kun,CHEN Li-yuan,ZHOU Fen
2014, 41(4):  107-112. 
Abstract ( 393 )  
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This study was to investigate the effects of different proportions of wheat and fermented sesame meal on growth performance,meat quality and serum biochemical indexes in broilers under the condition of adding exogenous enzymes in the diet.A total of 520 21-day-old broilers (fast)with an average body weight of (330±10)g were randomly assigned to 5 groups with 4 replicates in each group and each replicate contained 26 broilers. Broilers in the 5 groups were fed experimental diets containing 0 (control group),20% wheat +5% fermented sesame meal (group Ⅰ),40% wheat +10% fermented sesame meal (group Ⅱ),20% wheat + soybean meal (group Ⅲ) and 40% wheat + soybean meal (group Ⅳ),respectively.The pre-test period lasted for 3 days and the trial period lasted for 34 days.The results showed that compared with the control group,the ADG in groups Ⅰ,Ⅱ,Ⅲ and Ⅳ was significantly decreased by 9.40%,7.15%,8.87%,5.46% (P<0.05),respectively,while the ADFI and F/G showed no significant differences (P>0.05).There were no significant differences on meat quality among all experimental groups(P>0.05), respectively, but group Ⅱ had better meat quality than others.Compared with the control group,the content of alanine aminotransferase in serum in groups Ⅲ, Ⅳ was significantly decreased by 30.10%,25.36% (P<0.05),but there were no significant differences in the contents of total protein,urea nitrogen and aspartate aminotransferase in serum in all experimental groups(P>0.05).In conclusion,under the experimental conditions,adding appropriate proportions of wheat and sesame meal could replace part of the corn and soybean meal in the diet,and the better supplementation level of wheat and fermented sesame meal was 40% and 10%,respectively.
Effects of Different levels of Enzyme Mixture on Quality of Rice Straw Silage
CHEN Guo-fu,ZHANG Wen-chang,ZOU Chang-lian,ZHANG Guo-yi,YE Hang,ZHUANG Yi-fen
2014, 41(4):  113-118. 
Abstract ( 217 )  
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This trial aimed to study the effects of different levels of enzyme mixture (containing cellulose and xylanase) on quality of rice straw silage.Conventional moisture ensilaging (MC1) and low moisture ensilaging (MC2) were added 0(control group),25,50,100 mg/kg enzyme mixture,respectively. Each treatment was repeated 3 times.The materials were ensiled at room temperature and opened 60 days later. The results showed that pH,dry matter recovery and water soluble carbohydrate content in MC2 were extremely significantly higher than that in MC1 (P<0.01).The contents of lactic acid and ammonia nitrogen in MC2 were extremely significantly lower than that in MC1 (P<0.01).Compared with the control group,the contents of ammonia nitrogen (P<0.01) and neutral detergent fiber (P<0.05) in all the treatments of MC1 had significantly decreased.The pH in all the treatments of MC2 had extremely significantly decreased (P<0.01),the dry matter recovery and ammonia nitrogen content in 100 mg/kg group of MC1 and MC2 had extremely significantly increased and decreased (P<0.01), respectively,the water soluble carbohydrate content in 100 mg/kg group of MC1 and MC2 had significantly increased (P<0.05).The results indicated that enzyme mixture could improve the quality of both MC1 and MC2,the best level was 100 mg/kg in MC2.
Effects of Excessive Fluoride on Animal Organism
WANG Liang,FAN Hong-jie
2014, 41(4):  119-122. 
Abstract ( 193 )  
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Fluoride is the necessary element for animal organism. Appropriate fluoride is beneficial to the growth of bones and teeth. Excessive fluoride could cause chronic systemic poisoning,which has main influence on the normal growth of bones and teeth,and also has influence on digestive system, genitourinary system,nervous system,endocrine system and immune system. According to the internal and outernal research of present situation and progresses about the mechanism of fluorosis,this paper reviews the effects of excessive fluoride on animal organism,the objective is to provide scientific reference for the related research of animal fluorosis.
Study on the Optimized Conditions and Digestion in vitro of Wheat Bran with Solid-state Fermentation by Multi-strains
CAO Xiang-lin, CHEN Jian-jun
2014, 41(4):  123-127. 
Abstract ( 213 )  
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In this study, the best solid-state fermentation condition of wheat bran was optioned by using different ratio of Lactobacillus B, Lactobacillus W, Bacillus subtilis and Saccharomycetes, the digestion of fermentation wheat bran was analyzed by extraction of intestinal digestive enzyme from the grass carp. Experiment was arranged six groups according to the different microbial combination, the unfermented wheat bran acted as control group, and each treatment was repeated 3 times. The best condition was selected through measuring the acidity, bacteria colony and crude protein content of fermentation bran. The digestion of unfermented wheat bran and best fermentation wheat bran was carried out by using the digestive enzyme from the grass crap in vitro, the dry matter and crude protein digestibility and amino acid content were determined. The results showed that the crude protein content of group 3 was the highest, the content was improved by 56.56% compared with the control group. Moreover, the bacteria colonies were reached 7.8×108 CFU/g in 96 h in group 3, which was apparently higher than those in other groups, and the acidity was firstly increased and then decreased. All these exhibited the group 3 were the best one. In addition, the former intestine showed the highest dry matter and crude protein digestibility and amino acid content between unfermented wheat bran group and fermentation wheat bran group, followed by middle intestine, and posterior intestine was minimum, moreover, the digestibility of best fermentation wheat bran was significantly greater than that of unfermented wheat bran in vitroP<0.05).The results indicated that the fermentation wheat bran have the higher nutritional value and digestibility, which can partially replace the protein feed.
Effect of Glycogen Synthase Kinase 3β on Cell Proliferation and Cell Cycle in Dairy Cow Mammary Epithelial Cells
ZHANG Xia,GAO Xue-jun,ZHAO Feng,SI Yu,HUANG Yu-ling,LI Qing-zhang
2014, 41(4):  128-132. 
Abstract ( 233 )  
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Adding different concentrations Licl (0 (control group), 10,20,40 mmol/L),a specificity protein inhibitor of glycogen synthase kinase 3β (GSK3β),to the purification of the dairy cow mammary epithelial cells (DCMECs),after 24 h,this study detected the effects of GSK3β on cell proliferation and cell cycle in DCMECs,and using the Real-time quantitative PCR to test the effects of different concentrations Licl on GSK3β and Cyclin D1 mRNA level and Western blotting to test the effects of GSK3β/phosphorylated GSK3β (p-GSK3β) and Cyclin D1 protein expression levels in DCMECs. The results showed that Licl could improve proliferation in DCMECs,Licl inhibiting GSK3β certainly promote DCMECs proliferation and the optimal concentration of Licl was 20 mmol/L. Compared with the control group,GSK3β protein expression was restrained after adding Licl,p-GSK3β protein expression level up-regulation,Cyclin D1 protein expression level increased at the same time.These dates demonstrated that GSK3β was a negative regulatory factor of DCMECs on proliferation ability,the deactivation GSK3β through Cyclin D1 pathway could promote the process of cell cycle.
Isolation, Culture and Differentiation Potential of Canine Adipose-derived Mesenchymal Stem Cells
GE Xiu-guo, LI Ji-xia, JIN Tian-ming, MA Ji-fei
2014, 41(4):  132-136. 
Abstract ( 223 )  
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In order to obtain canine adipose-derived mesenchymal stem cells (ASCs), the dog inguinal subcutaneous adipose tissue was used to isolate ASCs by the methods of tissue culture and enzyme digestion, respectively. Different cell morphologies and proliferation characteristics were observed and the differentiation potential was tested by inducing cells into osteoblasts and adipocytes. The results showed that the adipose tissue from young dogs could get a lot of ASCs by tissue culture methods, which grew strong, uniform morphology, and could differentiate into osteoblasts with alkaline phosphatase positive and adipocytes with oil red O positive. The isolation and culture method of canine ASCs was easy to be established and could provide sufficient cell sources for the study of cells transplantation and therapy.
Effect of Colostrums Powder and Mature Milk Powder of Buffalo on Small Intestine Tissue Morphological Structure in Newborn Piglets
CHEN Rui-fang,GUO Ao-min,GENG Dan,ZHANG Hai-yan,WANG Shi-chang
2014, 41(4):  137-143. 
Abstract ( 189 )  
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The experiment was conducted to study the effect of colostrum powder and mature milk powder of buffalo on small intestinal tissue morphology development of newborn piglet.Newborn piglets within 72 h in treatments group were fed with buffalo colostrum powder (SCR group),buffalo mature milk powder (SC group) and glucose saline (PY group), respectively. 0-day-old newborn piglets as control (XD group).The results showed that intestinal villi of newborn piglets were densely,stout,arranged neatly, the thickness of muscular layer and membranaepropria were increased after taking buffalo colostrum powder. Compared with XD group,villus height,villus width,as well as membranaepropria thickness of duodenum,villus width and muscular layer thickness of jejunum,also and villus height of ileum were extremely significantly increased in SCR group (P<0.01).The villus width of duodenum, villus width and muscular layer thickness of jejunum in SCR group were extremely significantly increased than that in PY group (P<0.01).Buffalo mature milk powder caused damage to newborn piglets’ intestinal mucosal,including epithelial sheded,muscle layer thinned,membranaepropria naked and intestinal villi collapsed. Compared with XD group,the muscular layer thickness of duodenum,villus height, villus width, muscular layer thickness and membranaepropria thickness of jejunum, villus width and membranaepropria thickness of ileum in SC group were reduced,but there were no significant differences among them (P>0.05).However,the muscular layer thickness of jejunum in SC group was significantly decreased than that in PY group (P<0.05). In summary,the development of newborn piglets’ intestinal was promoted by feeding with buffalo colostrum powder. The biggest influence on jejunum,followed by duodenum,ileum. There was no effect on protecting intestinal mucosa and promoting its development by buffalo mature milk powder.The value of buffalo mature milk powder was less than glucose saline for development of newborn piglets within 72 h.
Study on Expression of L-type Amino Acid Transporter 1 in Development and Lactation of Mouse Mammary Gland
LIN Ye, HU Hong-liu, HOU Xiao-ming
2014, 41(4):  143-147. 
Abstract ( 218 )  
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To investigate the underlying relationship between amino acid transporter expression and mammary gland development, the expression of LAT1 and 4F2hc in virgin, pregnancy, lactation and involution of mouse mammary gland was detected by RT-PCR and laser confocal microscope. The results showed that LAT1 and 4F2hc located on the membrane of ductal epithelial cell, myoepithelial cell and adipocyte in virgin mammary gland. The expression of LAT1 and 4F2hc was lower because duct development was slow in this period. In pregnancy, LAT1 and 4F2hc located on the basolateral of ductal epithelial cell. The expression of LAT1 and 4F2hc went up as ductal epithelial cell proliferation increased. In lactation, LAT1 and 4F2hc located on the basolateral of acinar epithelial cells. The expression of LAT1 and 4F2hc was the highest because of milk protein synthesis. In involution, acinar structure collapsed. The expression of LAT1 and 4F2hc decreased. All these results revealed that LAT1/4F2hc was an important amino acid transporter in mouse mammary gland. The expression of LAT1 and 4F2hc was related to amino acid requirement in mammary gland development and lactation.
Effect of BCG-PSN on Antioxidation and Immune Activity of Chicken Immuned by Newcastle Disease Vaccine
YU Min, WANG Hai-bo, MENG Ting, GAO Yue-xiu, DING Xiao-li, DONG Ya-qing
2014, 41(4):  148-152. 
Abstract ( 151 )  
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Polysaccharide nucleic acid fraction of Bacillus Calmette Guerin (BCG-PSN) was applied on chicken immuned by Newcastle disease (ND) vaccine to observe the influence on antioxidation and immune activities. Two hundreds 1-day-old AA chicken were randomly divided into 5 groups,BCG-PSN high dose (0.12 mg per chick), medium dose (0.06 mg per chick), low dose (0.03 mg per chick) groups, Astragalus polysaccharide group (APS, positive group, 0.06 mg per chick) and control group with 40 chicken each group. All chicken were injected with drugs for 7 days according to the group and immuned by ND vaccine in 7 days old, while the control group was injected with physiological saline instead. Chicken were killed after 5,10,15,20 days injection, and thymus, spleen, bursal and jugular vein blood were collected, while immune organ index, NDV antibody titer, SOD, LPO, GSH-Px, IgG and IgM were tested. The results showed that BCG-PSN could not only significantly increase the activities of SOD and GSH-Px while significantly reduce the concentration of LPO (P<0.05) which indicated a good function of antioxidation, but also significantly increase the immune organ index, NDV antibody titer, concentrations of IgG and IgM in serum (P<0.05) which indicated an effect of immune adjuvant.
Effect of Recombinant ISG15 Protein from Argali,Bashibai Sheep and their Hybrid Sheep on Sheep Peripheral Blood Lymphocyte Transformation
LU Hai-fu,SHEN Wen,JIANG Fang-pei,CUI Ru-peng,SUN Yan-ming
2014, 41(4):  153-155. 
Abstract ( 143 )  
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In order to compare the different effects of recombinant ISG15 proteins in different varieties of sheep on lymphocyte transformation,we compared the effects of five different concentrations recombinant ISG15 proteins of Argali,Bashibai sheep and their hybrid sheep on the sheep peripheral blood lymphocyte transformation.The results showed that when the recombinant ISG15 protein concentrations of Argali sheep were from 10 to 80 μg/mL,D570 nm were significantly higher than PBS control group (P<0.05); the recombinant ISG15 protein concentrations of Bashibai sheep were from 40 to 80 μg/mL,D570 nm were significantly higher than PBS control group (P<0.05); while the recombinant ISG15 protein of hybrid sheep,compared with PBS control group,had no significant difference (P>0.05). In conclusion,certain concentrations of the recombinant ISG15 proteins of Argali and Bashibai sheep could stimulate lymphocyte transformation,with the increase of protein concentrations,the effects of lymphocyte transformation were enhanced,while the recombinant ISG15 protein of hybrid sheep had no significant effect on peripheral blood lymphocyte transformation.
EPO Protein Expression and Hypoxic Adaptation Analysis in Tibetan Pig
WU Hong,DEJI Bazhuo,ZHANG Jian,WANG Ya-fei,ZHANG Hao,CHAMBA Yangzom
2014, 41(4):  156-160. 
Abstract ( 222 )  
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In order to explore the relationship between the expression of erythropoietin (EPO) in kidney tissue of Tibetan pig and hypoxia adaptation, the Western blotting technique was used to detect EPO protein and the kidney tissue which was from Tibetan pig and Large Yorkshire in different altitudes was used in this study. The high altitude Tibetan pigs and Large Yorkshire pigs were from Linzhi of Tibet, and the low altitude were from Beijing and Hefei of Anhui. The results showed that the EPO protein of ZZ,ZB,YZ,YH in A,B and C three groups of experiments was in different expression. Through comparative and analysis, the EPO protein expression was significant difference between high altitude Large Yorkshire pigs and the high altitude Tibetan pigs (P<0.05), and it was extremely significant difference between high altitude Large Yorkshire pigs and the low altitude of Tibetan pigs and Large Yorkshire pigs in group A (P<0.01);The EPO protein expression was no significant difference between high altitude Tibetan pigs and the low altitude Tibetan pigs (P>0.05), and it was significant difference between high altitude Tibetan pigs and the high and low altitude Large Yorkshire pigs in group B (P<0.05);The EPO protein expression was significant difference between Tibetan pigs (P<0.05), and it was extremely significant difference between low altitude Tibetan pigs and the high and low altitude Large Yorkshire pigs in group C (P<0.01). When Large Yorkshire pigs were in the high altitude emergency state, the EPO protein expression of Large Yorkshire was higher than Tibetan pigs. When Yorkshire pigs had lived for a long time in the highlands, the EPO protein expression was lower than Tibetan pigs, but higher than the low altitude Yorkshire pigs. The stable expression of EPO was an essential factor of Tibetan pigs to adapt to high altitude hypoxia environment. And the high expression of EPO protein in high altitude Large Yorkshire’s kidney tissue was the physical adjustment process which could help Yorkshire pigs adapt to the plateau low oxygen environment.
Synergistic Effects of 18β-glycyrrhetinic Acid in Combination with Antimycobacterial Drugs against Mycobacterium bovis
XING Li-qun, JIA Fang
2014, 41(4):  160-164. 
Abstract ( 191 )  
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In this study,we found that 18β-glycyrrhetinic acid had potent antimycobacterial properties against the drug-susceptible and drug-resistant Mycobacterium bovis. Minimum inhibitory concentration (MIC) were 50 and 100 μg/mL after appling 18β-glycyrrhetinic acid alone aginst ATCC 27294 and ATCC 19210; MICs were 25~50 and 100~200 μg/mL after appling 18β-glycyrrhetinic acid alone aginst 2 drug-susceptible and drug-resistant Mycobacterium bovis. More importantly, they exhibited synergistic effects with the first-line drugs isoniazid (INH), rifampicin (RIF) andstreptomycin (SM) against clinical Mycobacterium bovis isolates, including drug-resistant strains. In combination with asubinhibitory concentration of 18β-glycyrrhetinic acid, the minimum inhibitory concentrations (MICs) of the antituberculosisagents decreased, ranging from 2 to 32, 4 to 8 and 4 to 16 fold for INH (fractional inhibitory concentrationindex (FICIs) 0.125~0.375), RIF (FICIs 0.240~0.490) and SM (FICIs 0.165~0.460), 18β-glycyrrhetinic acid had lesser poisonous to BHK-21 cell and could restraint SMMC fission,respectively. These findings indicated that 18β-glycyrrhetinic acid might serve as potential therapeutic compounds for futureantimycobacterial drug development.
Effects of Measurement Time of CIDR,Different Models of Feeding and Management,Repeat Superovulation and Oestrus Time on Superovulation in Sheep
WANG Feng,WANG Shen-yuan,ZHANG Xian-bao,ZHOU Li-jun,ZHANG Dong,LIU Cai-yun,WANG Hai-tao,LIU Yi-yi,PAN Jing,HAN Li-dong,ZHOU Huan-min,ZHANG Li
2014, 41(4):  165-169. 
Abstract ( 141 )  
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This experiment was conducted to study the effects of measurement time of CIDR,different models of feeding and management,repeat superovulation and oestrus time on superovulation in sheep.The results showed that the average number of available embryos of the 10 days was significantly higher than those of the 13 days (P<0.05).In addition,there were extremely significantly differences on the rate of available embryos between the 10 and 13 days of CIDR (P<0.01).The average number of available embryos and available embryos rate of drylot feeding group were extremely significantly higher than those of pasture feeding group (P<0.01).The optimal result of repeated superovulation was two times,and there were no significant differences between the second and the third times (P>0.05),but there were significantly differences compared to other forth times (P<0.05).The quality of embryos in the group of 24 h after superovulation was the best.In conclusion,the measurement time of CIDR,the pattern of different feeding and management and repeat superovulation had significantly effects on sheep superovulation,and the best quality embryos of sheep was in 24 h oestrus after superovulation.
Research on Genetic Effects of Myogenin Gene SNPs on the Reproductive Traits in Jinghai Yellow Chicken
TANG Ying, WANG Jin-yu, ZHANG Gen-xi, GE Ying, XUE Qian, WANG Ya-nan, WANG Yong-juan
2014, 41(4):  170-174. 
Abstract ( 165 )  
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The objective of this study was to analyze the association of single nucleotide polymorphisms (SNPs) in MyoG gene with chicken reproductive traits. In this study, PCR-SSCP approach was applied to assess SNPs of MyoG. Genetic effects were analyzed between different genotypes and reproductive traits of 378 Jinghai Yellow chicken by using the least square analysis. The results showed that sequencing revealed one mutation (G102A) on exon 1 of MyoG gene in the genotype AA in comparison to the genotype BB while other mutation (T36C) on exon 3 of MyoG gene in the genotype CC in comparison to the genotype DD. The least square analysis showed that there was no significant difference among genotype AA,AB and BB (P>0.05). While individuals with DD genotype had advantages on weight at first egg than those with CC genotype (P<0.05). In other reproductive traits, there was no significant difference among genotype CC,CD and DD (P>0.05). It suggested that SNPs of MyoG gene had no significant effects on reproductive traits of Jinghai Yellow chicken and which could be used in marker-assisted selection to accelerate the chicken breeding progress.
Analysis of Genetic Diversity on Microsatellite in Six Sheep Breeds
WANG Ya-lei,LI Jing-xin,MAO Da-gan,WANG Hui-li,LI Yin-xia,QIAN Yong,MENG Chun-hua,CAO Shao-xian
2014, 41(4):  174-179. 
Abstract ( 191 )  
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To detect the genetic diversity of Black bone sheep in Lanping and its genetic relationship with the other five sheep breeds (Hu sheep, Small-tail Han sheep, Dorper sheep, Texel sheep and hornless Dorset sheep), nine microsatellite loci were selected to study a total of 117 individuals, PCR amplification products of the microsatellite loci were resolved by native-polyacrylamide gel electrophoresis, and POPGEN32 software was used to calculate genetic parameters. The results showed that nine microsatellite loci were high polymorphism. The mean polymorphism information content (PIC) in each sheep population was 0.7211 (Hu sheep), 0.6780(Dorset sheep), 0.6492 (Small-tail Han sheep), 0.6479 (Black bone sheep), 0.5930 (Texel sheep) and 0.5728 (Dorper sheep), respectively. Among the six breeds, the mean heterozygosity (H) of Black bone sheep was highest (0.9543), while Dorper sheep was the lowest (0.2556). UPGMA cluster analysis showed that Hu sheep and Small-tail Han sheep clustered into a first class; the later was Black bone sheep. The further genetic distance was between Black bone sheep and the three introduced species. These results suggested that Black bone sheep might be crossed with Hu sheep or Small-tail Han sheep to improve fertility, and crossed with the introduced species to improve meat performance.
Association Analysis of Polymorphism in the CYP2C45 Gene with Fatty Liver Production in Geese
ZHANG Rui, ZHANG Yi-hui, SUN Xiao-xian, GONG Dao-qing
2014, 41(4):  180-184. 
Abstract ( 159 )  
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In order to study the influence of genetic variation in CYP2C45 gene on fatty liver production performance, PCR and sequencing methods were used to identify polymorphism of CYP2C45 gene exons in 113 Landes goose, and the association of polymorphism with production performance of goose fatty liver were investigated. The results indicated that there were 4 SNPs in exon 5 at 111 bp of C>T transition and at 147 bp of C>T transition, and in exon 7 at 71 bp of A>G transition and at 135 bp of G>A transition, respectively. These SNPs were in the stage of complete linkage by linkage disequilibrium analysis. Combining 4 SNPs as one genotype, there were AA (CCAG/CCAG) and AB (CCAG/TTGA) genotypes, but BB genotype (TTGA/TTGA) was not found, the genotype frequencies were 0.9027 and 0.0973, respectively, and the gene frequencies of alleles A and B were 0.9513 and 0.0487, respectively. Analysis of variance showed that the body weight of AB genotype was higher significantly than AA genotype (P<0.05), although the liver weight and ratio of liver weight versus body weight of AA genotype were higher than AB genotype, there were no significant difference (P>0.05). It suggested that, in the case of the same yield of liver production, less body weight less feeding, AA genotype maybe contribute to the improvement of production efficiency.
Study on Genetic Diversity of Domestic Chicken in Guizhou
WANG Wen-tao,LIN Jia-dong,CHEN Juan-hua,ZHANG Fu-ping
2014, 41(4):  185-188. 
Abstract ( 180 )  
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PCR and direct sequencing methods were used to analyze the genetic diversity and systematic evolution in Guizhou domestic fowls by determining a 780 bp DNA fragment of control region of mtDNA from 90 individuals of 6 Guizhou domestic chicken breeds. The percentage of nucleotide A, G, C and T were 26.14%,13.38%,25.98% and 34.50%, respectively. There were 40 polymorphic sites accounting for 5.13% of total analyzed sites. The diversity of haplotypes in 6 Guizhou domestic chicken breeds was ranged from 0.506 to 0.976, which showed significant genetic differentiation and higher levels of population genetic polymorphism.
Screening the GHSR Gene for SNPs and Analyzing the Mutations in Guizhou Semi-fine Wool Sheep
ZHANG Qiong-di, SONG De-rong,ZHOU Da-rong,PENG Hua,LIU Ruo-yu,WU Rui-ru,JIANG Xing-mei,WU Ping
2014, 41(4):  189-193. 
Abstract ( 196 )  
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By studying the GHSR gene polymorphism, A basis of selection and breeding was provide on molecular biology of Guizhou Semi-fine Wool sheep. Guizhou Semi-fine Wool sheep were used to construct DNA pools in this study, the complete sequences of exon 2 and part of intron 1 and intron 2 region in GHSR gene were obtained by designing one pair of specific primers. The amplified PCR products would be directly purified and conducted bidirectional sequences subsequently. DNAStar and BLAST software were used to identify polymorphisms of GHSR gene. Moreover, the impact of SNPs on secondary structure of RNA, secondary structure of GHSR protein would be analyzed by the bioinformatics software. The results showed that two SNPs (T70G and G229A) were found in GHSR gene which were two synonymous mutations. The SNPs led to the changes of RNA secondary structure. GHSR gene polymorphism might affect the growth of Guizhou Semi-fine Wool sheep.
Comparative Study of Meat Quality between the Hybrid F1 and F2 Generations from Gallus gallus and Wen-chang Chicken
FU Chuan-song,WANG Yu,SONG Wen-qing,WANG Li-li,ZHU Yu-jie,WANG Xue-mei
2014, 41(4):  193-197. 
Abstract ( 243 )  
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This study was conducted to determine meat quality between the hybrid F1 and F2 generations from Gallus gallus (♂) and Wen-chang chicken (♀).60 1-day-old the hybrid F1 and F2 generations from Gallus gallus (♂)and Wen-chang chicken (♀) were randomly allotted to 2 groups with 3 replicates per group and 20 broilers per replicate,and were supplemented with the same diets. Ten hybrid F1 and F2 generations in each replicate were randomly selected to slaughter and sampled breast and leg meat to determine meat characteristics of meat on day 90 and 120.The results showed that meat traits of muscle between the hybrid F1 and F2 generations were significant differences at 90 and 120 days (P<0.05),but there were no significant differences in pH and warner-bratzler shear force (WBS) of breast and pH of leg meat at 90 old days and 24 h drip loss and WBS of breast and leg meat and pH24 h of leg meat at 120 old days (P>0.05). The diameter and density of muscle fibre were significant difference between the hybrid F1 and F2 generations (P<0.05), but there were no significant differences in that of leg meat at 120 old days (P>0.05),and hybrid F2 generation muscle fiber density was higher than that in hybrid F1 generation,especially the hen.There were no significant differences in the dry matter,crude protein and crude fat content and cholesterol level in muscle (P>0.05),however crude fat level of leg muscle was significant difference from hybrid F2 generation (P<0.05).In conclusion,meat quality of the hybrid F2 generation from Gallus gallus and Wen-chang chicken was excellent and utilizatied for meat,and slaughter at 120 days was more appropriate.
Studies on MⅡ Stage Porcine Oocytes Vitrification
QI Gui-long, REN Liang, ZHU Meng, FU Bo, MA Hong, LIU Di
2014, 41(4):  198-202. 
Abstract ( 115 )  
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The study was designed to select right cryoprotectants and carrier for porcine matured oocytes to improve oocyte viability after vitrification and lay a good foundation for porcine oocytes continue development. Using 40% EG, 15% EG+15% DMSO+20% FBS, 15% EG+15% DMSO+20% SPS as cryoprotectants and taking GMP and cryotop as carrier to do porcine oocytes vitrification. The results showed that VS3 including SPS made better active of FDA (59.40%±4.93%) than VS1 (42.12%±4.08%) and VS2 (37.57%±1.21%)(P<0.05);VS3 as cryoprotectants and cryotop as carrier made better active of FDA (83.33%±3.33%) than GMP (59.40%±4.93%)(P<0.05). The data demonstrated that use SPS to instead of FBS and cryotop as carrier could significantly improve active of oocyte after vitrification.
Effects of Nerve Growth Factor on Proliferation of Mouse Ovarian Granulosa Cells
HOU Xiao-feng,LI Wan-hong,SUN Li-na,CHEN Shu-xiong,CHEN Lu,LI Chun-jin,ZHOU Xu
2014, 41(4):  202-206. 
Abstract ( 133 )  
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To study the effect of nerve growth factor (NGF) on proliferation of mouse ovarian granulosa cells. Primary mouse ovarian granulosa cells were cultured successfully, immunofluorescence was used to identify granulosa cells and observe the distributions of NGF and TrkA in granulosa cells, the MTS was used to analysis the different concentrations of NGF on proliferation of mouse granulosa cells. The cells were treated with NGF of different concentrations (10, 50, 100, 500 ng/mL) for 24 h. The D490 nm value of the cell that was identified was determined by micro-plate reader.Compared with the control group, each treatment group promote the proliferation of granulosa cells with different degrees (P<0.05), the effect of NGF group (50 ng/mL) on the granulosa cells proliferation was the most significant (P<0.01). These results indicated that NGF could promote proliferation of mouse granulosa cells. Also,mouse ovarian granulosa cells could express NGF and TrkA.
Exploration on Electrical Fusion Parameters of Enucleated Zona-free Oocytes
ZHANG Bin, HUA Zai-dong, ZHENG Xin-min, XU Sheng-cheng
2014, 41(4):  206-210. 
Abstract ( 132 )  
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In the present study, we investigated the effect of electrical fusion parameters on pig oocytes of enucleated zona-free by using different electrical field strength and the pulse duration and then observed cell fusion. The results showed that the comparison study of different electrical fusion parametes indicated that the rate of oocytes using one DC-pulse of 0.8 kV/cm for 30 μs (94.7%) groups were significantly higher than the other treatment groups(P<0.05). The rate of fusion of the enucleated zona-free oocytes in one DC-pulse of 0.8 kV/cm for 30, 60 and 90 μs group were 94.7%, 86.5% and 83.8%, respectively, showing that 30 μs treatment group were significant higher than those of 60 and 90 μs treatment groups(P<0.05). The rate of fusion in one, double and three DC-pulse of 0.8 kV/cm for 30 μs were 94.7%, 95.6% and 81.7%, respectively, showing that double DC-pulse treatment groups were no significant higher than those of one DC-pulse treatment groups (P>0.05), but both were significant higher than three DC-pulse treatment groups, the fusion rate of them was significant higher than three DC-pulse treatment groups(P<0.05).
Fitting the Weight Growth Curve of Xinjiang Brown Cattle
Zainaguli?Junjulieke, TAN Rui, HUANG Xi-xia, WANG Ya-chun4, Rexiti?Abudoureyimu,Nuerbiye?Wubuli, CHENG Li-ming, FU Xue-feng, JIA Xu-sheng, ZENG Li
2014, 41(4):  211-215. 
Abstract ( 220 )  
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The aim of this study was to explore the principles for growth and development of Xinjiang Brown cattle, using this four common growth curve model named Logistic, Gompertz, Brody and Bertallanffy in the SAS 8.1 software to fitting the weight growth curve for 344 heads Xinjiang Brown cattles. The results showed that four kinds of model all could fit the weight growth of Xinjiang Brown cattle very well, the degree of fitting R2 was 0.9217, 0.9263, 0.9176 and 0.9261. The Gompertz model was the best to fitting the weight growth of Xinjiang Brown cattle. The inflection point of the growth curve of in model Logistic, Gompertz and Bertallanffy were (0.4937 year,502.10 kg),(1.3168 year,379.54 kg)and(1.0477 year,3115.25 kg),respectively. This study had the certain reference value to the actual production.
Research Progress on Promoting Effect and Mechanism of Melatonin on Cashmere Growth in Cashmere Goat
ZHAO Yan-hong, WANG Lin, ZHANG Tie-jia, LI Jin-quan
2014, 41(4):  215-219. 
Abstract ( 250 )  
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Melatonin can shorten the growth cycle of cashmere in cashmere goat, promote the growth of cashmere, increase the production of cashmere. The cashmere presents cyclical variation that includes anagen, catagen, telogen, which is related to cyclical variation of melatonin’s secretion. The paper reviewed the secretion and function of melatonin, cyclical variation of cashmere, discussed the mechanism that melatonin promotes cashmere growth. The paper aims to not only provide foundation for elaborating regulatory mechanisms of melatonin promoting cashmere growth at the molecular level, but also provide reference for effectively application of melatonin in cashmere’s production.
Drug Resistance of Escherichia coli Strains Isolated from the Production Chains of Main Animal Food
PENG Zhen,LIU Shu-liang,FAN Qin,ZHU Dong-mei,CHEN Xun,HOU Xiao-gang,HAN Xin-feng, ZHOU Kang
2014, 41(4):  220-225. 
Abstract ( 221 )  
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To understand drug resistance of Escherichia coliE.coli) isolated from the production chains of main animal food,using trace dilution method recommended by clinical and laboratory standards institute CLSI broth,350 strains E.coli isolated from the production chains of pork,eggs and chickens were examined for sensitiveness against 10 kinds of antimicrobial agents (combination). The results showed that 350 strains E.coli were mostly resistant to tetracycline (82.0%),followed by resistance to trimethoprim/sulfamethoxazole (75.4%),and the gentamycin drug resistant rate (26.9%)was the lowest. Despite the different origins of E.coli for the vast majority of these drugs were basically consistent, the resistance rate of the strains from different segments of animal food production chain was different.76.2% (267 strains) of the isolates were multi-drug resistant strains,the most of which were the eight kinds of drug-resistant strains (17.7%). The whole test strains had 108 kinds of drug spectra, the spectra of E.coli isolated from the production chains of pork,eggs and chickens were 40,46 and 55,respectively,whereas the advantage drug resistance spectra were not obvious. The main drug resistance spectra included NAL-CIP-AMP-AMC-CEF-SPT-GEN-SXT-TET (21/350), TET (20/350), NAL-CIP-AMP-AMC-CEF-SPT-SXT-TET(16/350), NAL-CIP-AMP-CEF-FLO-SPT-SXT-TET(16/350) and SPT-SXT-TET(15/350). The results suggested the drug resistance of E.coli from the production chain of main animal food was serious,so it was necessary to strengthen the continuous monitoring and control.
Isolation and Identification of Mink Canine Distemper Virus LD-1 Strain
ZHUANG Jin-qiu, MEI Jian-guo, WANG Jin-liang, ZHANG Qian, SHEN Zhi-qiang, DING Zhuang
2014, 41(4):  226-232. 
Abstract ( 174 )  
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Canine distemper virus (CDV) was detected positive by RT-PCR from a suspected CDV infected mink liver tissue. No mink parvovirus was detected from it. The virus was inoculated to 3 kinds of cells including primary CEF cells, Vero cells and DF1 cells for virus isolation. It was successfully isolated from Vero cells through the passage culture by optimizing the cells culture condition, and showed the dew shape typical CPE. The isolated virus was identified by RT-PCR, sequencing of the PCR products, serum neutralization test, indirect immunofluorescence test, virus inclusion body examination and other methods. The results showed that CDV was positive. It was proved that the isolated virus was a mink CDV, named CDV LD-1 strain.
Isolation, Idetification and Drug Resistance Analysis of Streptococcus from Lungs of Tibetan Sheep
FENG Xu-fei, WANG Zhi-min, WU Yu-xi, PAN Yu-kun, DANG Bin, YANG Fa-long
2014, 41(4):  232-235. 
Abstract ( 178 )  
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The lung samples with pneumoniae were collected from Tibetan sheep. Bacterial isolation and identification were performed by culture and PCR methods. Antibiotic sensitivity of the isolates was determined, so as to provide useful information for treatment of the disease. The results showed that the isolated bacteria were gram-positive and chain-forming coccus-shaped proven to be Streptococcus by specific PCR, clone and sequence. It was also demonstrated that the isolates were sensitivity to most of antibiotics, especially for trimethoprim-sulfamethoxazole, enrofloxacin, gentamicin and tetracycline. The results obtained in this study provided useful basis for control and treatment of Streptococcus infection of sheep in Tibetan.
Research Progress on Detection Method of Jaagsiekte Sheep Retrovirus
ZHU Fu-yu, YU Li-xin, YAO Hong-qiang, MA Xue-en
2014, 41(4):  236-241. 
Abstract ( 146 )  
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The ovine pulmonary adenomatosis (OPA) is composed of β-reverse transcription virus jaagsiekte sheep retrovirus (JSRV) caused by a tumor infectious disease, the disease has a long incubation, spreads through the respiratory tract, the winter of captive breeding disease incidence rate is high, at present there are no treatment measures, the mortality rate is 100%. The continuing presence of OPA is a potential threat for sheep production and has caused great economic losses, serious harm to the healthy development of sheep industry. Therefore, early precise diagnosis on OPA is the premise of the disease prevention and control, particularly in the entry exit inspection and quarantine process of import and export of sheep JSRV detecting is very important. With the development of molecular biology techniques, JSRV molecular biology detection method has constant innovation and improvement. This review will focus on the detection method of JSRV, polymerase chain reaction (PCR), nucleic acid hybridization, enzyme linked immunosorbent assay (ELISA), loop mediated isothermal amplification (LAMP) survey research methods in recent years, to provide reference methods for the detection of JSRV and further development of the research on new JSRV detection methods for rapid, accurate and suitable in the entry and exit inspection and quarantine process.
Isolation and Identification of Sorbitol-positive Aeromonas sobria from Fish
LI Xu, DING Chu-chu, LI Xue, SHI Hui, CHEN Ye-xiang, HU Xiu-cai, ZHU Ai-hua, LV Ai-jun
2014, 41(4):  241-244. 
Abstract ( 280 )  
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A bacteria was isolated from the intestine of Carassius auratus and temporarily named as SR-1 strain with sorbitol-positive character. It was identified as Aeromonas sobria by morphological, physiological, biochemical characteristics, drug sennsitivity test, 16S rDNA sequence and phylogenetic tree analysis. Further the 16S rDNA sequence of SR-1 was amplified by PCR method and the results showed that the size of fragment was 1509 bp. Phylogenetic tree analysis showed that SR-1 strain had the highest similarity to Aeromonas sobria of 99.87% to 100%. Drug sensitivity test results revealed that SR-1 was sensitive to azithromycin, chloroamphenicol, tetracycline, norfloxacin, cefotaxime, cefixime and so on.
Analysis of Resistant Phenotypes and Resistant Genes of Pathogenic Salmonella from Avian
DAI Jian-hua, WU Zhi, YUAN Wei-feng
2014, 41(4):  245-248. 
Abstract ( 178 )  
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To investigate the presence of antibiotic resistance and resistant genes about avian Salmonella in Suzhong area, 22 isolates were evaluated for antimicrobial sensitivity by K-B disc diffusion method against 12 antimicrobial drug,and then 12 resistant genes of the isolates were detected by PCR. The resistant rates of all isolates were found from 72.73% to 100% to amikacin, norfloxacin and so on; 5 resistant phenotypes were detected, and each isolates were resistant to more than 4 of the 12 antimicrobial drugs,even 10 drugs; 9 different resistant genes were amplified, the homologies were very high compared with the corresponding genes in GenBank (≥98%); the resistant phenotypes and their resistance related genes were essential consistent, but no absolute correlation. The above results showed that the antibiotic resistance of Salmonella was very serious, and the resistant genes existed widely.
Study on the Effect of Different Holly bark Extracts Combined with Western Medicine on ESBLs-producing Bacterial
SONG Jian-wu,CHEN Qiu-ying,LU Yun,ZHENG Yan-qing,SI Hong-bin
2014, 41(4):  248-251. 
Abstract ( 170 )  
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This research was aimed to investigate four kinds of Holly bark extracts combined with antimicrobial drugs to induce bacteria in vitro antibacterial activity. Four kinds of Holly bark extracts were prepared, the bacterial passage of drug-resistant Escherichia coli induced by Holly bark extracts combined with antimicrobial drugs were tested by 96-well reaction plate twice micro-dilution method. Four kinds of Holly bark extracts combined with antimicrobial drugs to induce bacteria passage for drug-resistant Escherichia coli had different antibacterial activities, in which extractsⅠ,Ⅲ combination with antimicrobial drugs had the best effect. Holly bark extracts Ⅰ,Ⅲ combined with antimicrobial drugs to induce bacterial passage had anti-drug-resistant bacteria effect in vitro.
Research Progress on Porcine Circovirus Type 2 Vaccine
WANG Xian-wen
2014, 41(4):  252-255. 
Abstract ( 175 )  
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There are many epitopes that are important in immunology and virulence on the surface of porcine circovirus type 2 (PCV2), which offer insights for the direction of future research. Great development has been produced in the study on PCV2 vaccines, several efficacious vaccines have already been produced. Nutritive substance such as carboxymethylpachymaran, L-glutamine, arginine supplementation and Selenium can improve the preventive or curative effect in animals infected with PCV2.Research progress on PCV2 vaccine was introduced for better control of PCV2-associated diseases.
Effects of Astragalus Polysacchrides on Peripheral Blood in Blood Deficiency Syndrome Model Chicken
HAO Li-juan, LI Rui-juan, XI Nan, KANG Jia, SHI Wan-yu
2014, 41(4):  255-257. 
Abstract ( 147 )  
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The assay was aimed to investigate the effects of Astragalus polysaccharides on peripheral blood in blood deficiency syndrome model chicken. The blood deficiency syndrome model was made by celiac injection of cyclophosphamide for 6 d (80 mg/(kg·d)). Blood deficiency syndrome group were respectively drunk three different concentrations of Astragalus polysaccharides. Erythrocyte (RBC), hemoglobin (Hb), platelet (PLT) were measured and compared. Astragalus polysaccharides could extremely significantly increase contents of RBC, Hb, PLT of blood deficiency syndrome chicken (P<0.01). Astragalus polysaccharides reversed RBC, Hb, PLT levels of blood deficiency syndrome chicken, so that they recovered their health.
Isolation and Identification of Porcine Pseudorabies Virus ZJ Strain and Research of its Immunogenicity
YIN Xiu-feng,DING Mei-juan,ZHANG Hai-tao,QIN Jin-jin,SUN Yan,XU Xiu-mei,WANG Ai-fen,ZHANG Xiao-fei,XUE Jia-bin
2014, 41(4):  258-264. 
Abstract ( 228 )  
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The material from the clinical samples of cerebrum and spleen in the pigs characterized by pseudorabies, proved to be porcine pseudorabies virus (PRV) positive samples through the amplification of gE gene of PRV, was inoculated into the BHK-21 cells and serially passaged 4 times. The isolate could induce typical cytopathogenic effect in cells and the titer of the fourth passage was 107.0 TCID50/mL. Identification was done by PCR method and four rabbits were injected with the fourth generation cell cultures of the isolate, and the specific band could be amplified and the typical clinical syndrome could be observed. It was indicated that the isolated virus named ZJ strain was PRV. The virulent PRV ZJ strain was killed with formalin and used as vaccine antigen with oil adjuvant. The rabbits were injected with 1 mL vaccine, 28 days after inoculation, serum antibodies were detected and healthy rabbits were challenged by PRV ZJ virulent strain, and the protection rate and serum neutralization index of antibodies were tested. The results showed that protection rate and serum neutralization index in rabbits were 100% and 13490,respectively.This study showed that PRV ZJ strain had good immunogenicity, and provided a basis for further research on vaccination of PRV.
Seasonal Dynamic Research on Developmental Stages of Marshallagta in Sheep and on Grassland
Qiaogelasen,Cairenjiapu,SONG Ying-chun,Jiergeli,XI Nai
2014, 41(4):  262-265. 
Abstract ( 108 )  
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Through a monthly observation of the parasitic load of the imagoes, the output of parasite eggs from sheep’s feces and the survival rate of ectogenetic parasites of Marshallagta, which was a typical dominant species among the parasitic nematodes parasitic on the sheep living on Bayanbulak grassland,which was a subalpinealpine meadow grassland, with the addition of the observing records of air temperature,relative humidity,evaporation capacity,the paper obtained the seasonal dynamics of Marshallagta in sheep and on grassland in different developmental stages as well as its relationship with the changes of air temperature and humidity. Both the parasitic load in sheep and the incubation amount in vitro on grassland were the lowest from July to August of 2006.But they reached their peak period in November of 2006 for the first time, and then an obvious decrease in February of 2007 before another peak period again in April of 2007. In addition, the amount of the parasitic larva was also the lowest from July to August of 2006, but its peak period appeared in September of 2006 for the first time, and afterwards an obvious rise came again in February of 2007. The peak period of the parasitic larva of Marshallagta corresponded to the peak period of the adult. It turned out that the peak period of the adult came with the peak period of larva, while the peak value of the parasite eggs in feces would often come along with the peak period of the adult.The in vitro survival rate was obviously affected by air temperature and humidity. When the average humidity was lower than 50%, no peak would come.When the temperature was lower than 5.6 ℃, the survival rate was zero. When the humidity was lower than 79%, the survival rate was very low. The test made a study on the seasonal dynamic regularity of Marshallagta in sheep and on grassland, which supplied theoretical foundations for the formulation of proper prevention measures.
Study on the Application of Sewage Treatment Techniques of Livestock Farms in Northwest China
ZHOU Lei,DENG Xian-de,LIAO He-rong,ZHANG Qing-dong,XUE Xiao-yan
2014, 41(4):  266-270. 
Abstract ( 191 )  
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Based on the sewage treatment features of livestock farms in the extremely cold northwest region at high attitudes of China, a method called “solid-liquid separation-UASB-coagulation and sedimentation-SBR-absorption and filtration” was adopted to treat piggery wastewater. Moreover, the heating insulation measures were used to ensure that the operation of the project being normal. This project was started successfully after four months’ start-up process, and it works very well. The results showed that after the sewage treatment, the indices of CODcr, BOD5, SS, NH4-N and T-P meet the requirements of discharge standard of pollutants for livestock and poultry breeding (GB18596-2001).
Research Progress on Enrichment and Detection of Environmental Virus
WEI Xiao-bing,HE Hui-li,LIU Ming-cheng,GOU Xiao-jing,YAN Yi-ting,LIU Xing-you
2014, 41(4):  270-274. 
Abstract ( 193 )  
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With the development of intensive farming and increasing breeding density, environmental viruses can cause prevalence and spread of infectious diseases, which bring seriously harm to human and animal and huge economic losses to farming enterprises. To learn the situation of environmental viruses, it is urgent to strengthen monitor of pathogenic viruses and establish early warning systems. In this paper, we demonstrate research progress on enrichment and detection of environmental virus in order to lay the foundation for environmental monitoring systems.