Acta Veterinaria et Zootechnica Sinica

Cloning, Expression and Functional Analysis of Osteopontin Gene in Large White Pig

ZHANG Dongjie;LIU Di ;WANG Xiaohong;YANG Guowei

2009, 40(7): 965-970. doi:

Abstract ( 761 )

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The Osteopontin cDNA in size of 909 bp was cloned by RTPCR and analyzed by bioinformatics. The results showed that Asp, Glu and Ser occupied the highest proportion and the special sequence ArgGlyAsp (RGD) existed in OPN primary structure, α helix occupied the higher proportion in secondary structure and hydrophile was better, OPN signature existed in the first homologous region and the mutual conservative sequence was ［KQ］x［TA］x(2)［GA］SSEEK in OPN secondary structure. Two phylogentic trees constructed based on OPN protein sequence showed that the relationship between pig and cow was the closest, but distant from chicken. OPN mRNA was expressed in many tissues of pig: higher in stomach, kidney, lung, small intestine and ovary, lower in heart, spleen and large intestine. The protein size was different in different tissues: 70 ku, 70 and 45 ku, 70, 45 and 24 ku.

The Optimal Multiple Traits Selection for Multiple QTL with Discrete Generation and Control of Inbreeding

TANG Guoqing;LI Xuewei;ZHU Li;LI Mingzhou;SHUAI Surong

2009, 40(7): 971-977. doi:

Abstract ( 792 )

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In a discrete generation population,the potential extra genetic gain was studied while the genetic contribution of candidate and the relative weight given to QTL were optimized using the random computer simulation. In a simulated example of practical breeding population for pig, the number born alive (NBA) and days to 100 kg (D100) were selected, two QTLs of each trait were segregating together with polygenes across multiple generations. When compared with standard QTL or conventional BLUP selection, QTL optimized selection combined optimization for weights given to QTL and contributions of candidates substantially increased more selection response at a fixed rate of inbreeding, and avoided the conflict between short term and long term response.

Cloning and Expression of Chicken Mx cDNA in Escherichia coli

YIN Chunguang; DU Lixin ;LI Shangang;WEI Caihong;LIU Tao;LI Hongbin;ZHAO Guiping

2009, 40(7): 978-981. doi:

Abstract ( 761 )

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This experiment was conducted to study Mx gene expression in E.coli under the condition of cloning Mx gene complete sequence. Chicken embryo fibroblast (CEF) were treated with poly I:C to stimulate Mx gene expression. Full length cDNA of Mx was cloned. Recombinant expression vector pGEXMx including Mx ORF was constructed using pGEX4t2 expression system to transfer Escherichia coli Rosetta (DE3) strain. The result showed that the relative molecular weight of expressed protein was about 75 ku after induction with IPTG, suggesting that Mx gene has been efficiently expressed. The research laid a good foundation for further studying on bioactivity assay, exploring new way of antiviral medication and transgenic study.

Expression Study and Establishment of pEGFPADSL Cloning Cell Lines ofShouguang Chicken Adenylosuccinate Lyase Gene

LIU Changqing ;ZHANG Honghai ;WEN Jie;MA Yuehui ;GUAN Weijun ;TANG Xuexi

2009, 40(7): 982-991. doi:

Abstract ( 763 )

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The specific expression of ADSL gene in 8 different tissues of Shouguang chicken was investigated by RTPCR and RACE in this study. The fusion expression vector pGEXADSL was constructed and transformed into Escherichia coli BL21(DE3), positive cloning screened, induced and expressed by IPTG. Eukaryotic expression vector pEGFPADSL was transfected into Shouguang chicken fibroblast cells using lipofectin method. The results of sequences analysis showed that the open reading frame of 1 455 nucleotides encode a protein of 485 amino acids; The promoter of chicken ADSL cDNA showed typical features of house keeping genes，there was a C28T mutation which caused the site mutate to NRF2 binding site. The SDSPAGE electrophoresis results showed that there were specific bands, about 805 ku and an isoelectric point of 679, and Westernblot analysis showed that the fused protein was ADSL. After transfection 24, 48 and 72 h, transfection rate of pEGFPADSL were between 264%-412%, and green fluorescent could be observed in cytoplasm and nucleus welldistributed except cryptomere vesicle. Both of RTPCR and Westernblot confirmed that the pEGFPADSL had been integrated into the Shouguang chicken fibroblast cell genome, and accessed to the normal fusion protein expression. This research revealed the ADSL gene structure of excellent native chickens of China, established the foundation for further research with its biological function.

Isolation, Sequence Analysis and Expression Profile of IDH1 in Goose Liver

ZHAO Ayong;;CHEN Guohong ;LU Lizhi ;ZHOU Qi

2009, 40(7): 992-998. doi:

Abstract ( 761 )

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To obtain an understanding of fattening mechanism of fatty liver, mRNA differential display reverse transcription PCR (DDRTPCR) was applied to study the differences of gene expression in French Landes Grey goose and Xupu White goose in different conditions of overfeeding and normal feeding. One gene was found to be significantly downregulated in fatty liver in both breeds（P<005）, which has a CDS length of 1 269 bp and 95% identity to chicken IDH1. The sequence analysis revealed that its 1 248 bpinlength open reading frame (ORF) encodes a 415aminoacids protein containning a putative conserved domain of Icd and has high homology with chicken(99%), rat(89%), human(90%), monkey(90%), cattle(88%),and mouse(88%)，respectively. The structure and function of this protein were analyzed using bioinformatics methods.The tissue expression analysis indicated that goose IDH1 mRNA was higher expressed in liver than that in other tested tissues. The present results suggested that overfeeding could decrease the mRNA expression level of goose IDH1.

Molecular Cloning and Sequence Analysis of the Yak Growth Differentiation Factor 9 Gene

YIN Ronghua;ZI Xiangdong ;MA Zhijie ;CHEN Shaowei ;ZHANG Dawei ;LIANG Guannan

2009, 40(7): 999-1006. doi:

Abstract ( 801 )

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In order to amplify the cDNA of yak growth differentiation factor 9, the primers were designed according to the GenBank sequence of cow GDF9 gene (AF 307092). Total RNA was extracted from the oocytes of the Maiwa yak and the cDNA encoding GDF9 was obtained by the reverse transcription PCR (RTPCR). The purified RTPCR product was cloned into T vector, and then the sequence was analyzed. The results demonstrated that the 1 399 bp product was the yak GDF9 cDNA, including the complete CDS and part of 3′ noncoding region. The size of the yak GDF9 gene coding region was 1 362 bp which encode 453 amino acids. Comparing GDF9 nucleotide sequences and deduced amino acid sequences of coding region of the yak to those of other species including cow, buffalo, sheep, goat, human and chimpanzee retrieved from GenBank, the size of the yak nucleotide and amino acid was the same with that of cow, buffalo, sheep and goat, and only one base was different, and this difference influenced polypeptide sequence after translation. The homologies of nucleotide sequences of the coding region of GDF9 gene between the yak and cow, buffalo, sheep, goat, human and chimpanzee were 999％, 984%, 970%, 968%, 856% and 851%, respectively, and the homologies of the deduced amino acid sequences of the coding region were 998％, 971％, 951％, 954％, 794％ and 795％, respectively. The molecular phylogenetic trees among species were constructed according to the nucleotide sequence of the coding region of GDF9 gene. The result indicated that yak, cow, and buffalo, sheep and goat assembled separately, and then assembled to a genus with human and chimpanzee. This result of phylogenetic clustering was identical to the genetic distance and the zoological classification, which indicated that the GDF9 gene was also fit to construct molecular phylogenetic tree among different species.

Effect of Trichostatin A on Reprogramming of Bovine Fetal Fibroblast Cells

ZHANG Dong;YANG Lu;WANG Yongsheng;LIU Gensheng;LIU Lijie;WAN Min;ZHANG Yong

2009, 40(7): 1007-1012. doi:

Abstract ( 1124 )

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It has been widely suggested that poor ability of oocyte cytoplasmic factors to globally erase the epigenetic modifications of a somatic cell is the major reason of the inefficiency of nuclear transfer. In this study, bovine fetal fibroblasts passaged 5 times were seeded into DMEM plus 10% FBS containing 75 nmol·L-1 of TSA for 6, 12, 24 h , respectively. These levels were selected to produce timedependent effects. Cells were subsequently subjected to essay for histone acetylation of H3K18 levels by confocal microscope. The effect of Trichostatin A treatment donor cells on in vitro development of bovine nuclear transfer (NT) embryos which were constructed with bovine fetal fibroblast cells and cell cycle was investigated. These results showed that TSA increased the levels of histone acetylation of H3K18 after bovine fetal fibroblast cells treated with 75 nmol·L-1 TSA for 12 or 24 h(P＜005);75 nmol·L-1 TSA treatment of donor cells for 12 h promoted blastocyst development compared to control (235% vs157%, P＜005) ; Marked difference existed in the percentage of cells at G0/G1 and S stages between treated and control groups(P＜005). It was concluded that TSA enhances the reprogramming in terms of high histone acetylation of doner cells.

The Influence of SelW on GSH and GPx after PostTranscriptional Gene Silencing in Mouse Skeletal Muscle Cell

WANG Xiaolong;XU Kai;QIN Ouju;YANG Chuanping

2009, 40(7): 1013-1018. doi:

Abstract ( 1012 )

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The expression of Selenoprotein W (SelW) in C2C12 skeletal muscle cells was specifically decreased to examine its influence on the amount of glutathione (GSH) and the activity of glutathione peroxidase (GPx). SelW knockdown was performed by RNA interference (RNAi) in cultured muscle cells and verified by Realtime PCR and Western blotting. In addition, cell viability, GSH content and GPx activity were assayed. The results showed that the mRNA level and protein expression of SelW were decreased successfully by 719% and 688% relative to control values. WST assay showed that to compare with blank control, the value of positive group dropped 215%; In GSH and GPx assay, to compare with blank control the positive group increased 2976% and 4758% separately. In conclusion, SelW knockdown by RNAi caused significant cytotoxity in skeletal muscle cells and led to compensatory increases in GSH content and GPx activity. These findings are consistent with the suggestions from bioinformatics indicating an antioxidative role for SelW in skeletal muscle cells.

Effects of Dietary Energy Level on Intramuscular Fat Content, Fatty Acids Composition of Intramuscular Fat and Backfat in Finishing Pigs

XU Haijun;;DU Wen;LI Yajun;TANG Wenjie;LIU Zhiqiang;TAN Bie;KONG Xiangfeng;HUNAG Ruilin;LIU Yulan;YIN Yulong;

2009, 40(7): 1019-1027. doi:

Abstract ( 819 )

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Effects of increasing dietary energy by supplementation of soy oil on intramuscular fat (IMF) content, fatty acids composition of IMF and backfat in pigs were investigated. Thirty six Duroc×Landrace×Large White female pigs at a mean initial body weight of 40 kg were equally and randomly assigned into three treatment groups. The digestible energy of the diets was 1282, 1424 and 1566 MJ·kg-1 for three groups respectively and the energy level of diet was adjusted mainly by the supplementation of soy oil. Five pigs of each group were slaughtered at the body weight of about 90 kg. Longissimus dorsi muscle (LM) and backfat were sampled for determination of IMF content and fatty acids composition. There was no significant effect of dietary energy level on IMF content in LM (P>005). The percentages of most saturated (SFA) and monounsaturated fatty acids (MUFA) and of most polyunsaturated fatty acids (PUFA) decreased and increased significantly (P<005) respectively both in LM and backfat as the dietary energy level increased. The IMF content in LM was negatively correlated with the percentages of C20∶0, C18∶3n6, C20∶4n6, C22∶5n3 and C24∶1. Most fatty acids in LM were positively correlated with those in backfat while there were no significant relationships for C18:3n6,CLAc9t11,C20∶0,C20∶4n6,C22∶5n3 and C24∶1 between the two tissues. In general, SFAs were positively correlated with MUFAs and both of them were negatively correlated with PUFAs while PUFAs were positively correlated with each other in both tissues. In conclusion, increasing dietary energy level by supplementation of soy oil is an effective way to improve IMF fatty acids composition without decreasing IMF content, thus improving the nutritional value of pork without deleteriously affecting meat quality.

The Influence of Acetate and Propionate Molar Ration in Ruminally Infused Volatile Fatty Acid Mixtures on Milk Fat Synthesis in Lactating Goats

CHENG Guangmin;LIN Xueyan;LI Fuchang;CHEN Fengmei;WANG Zhonghua﹡;LIU Jiansheng;FU Guihua

2009, 40(7): 1028-1036. doi:

Abstract ( 845 )

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Four midlate lactation Wendeng goats equipped with permanent ruminal fistulae and temporary carotid and mammary vein catheters were assigned into a 4×4 Latin square experiment. The four volatile fatty acid mixtures (VFAs) were ruminally infused with different acetate/propionate/butyrate molar ratios (75∶15∶10,VFA1; 65∶25∶10,VFA2; 55∶35∶10,VFA3; 45∶45∶10,VFA4). The food each day was delivered equally in particle by automatic feeder per 2 h and the ratios of concentrate to forage was 45∶55. Among them, the diet should meet the demand of the sheep, the VFAs infused provides of an additional 30% of MEm. The present study results showed that milk yields and milk lactose content were not influenced , but milk fat percent and yields decreased significantly by treatments (P<005). Plasma insulin increased insignificantly and there was no effect on blood glucose(P>005). With the propionate molar ratios increasing in the VFAs, arterial acetate，NEFA and BHBA consistency decreased significantly(P<005), arterial propionate increased（P<005）, but TG or butyrate was not changed in treatments (P>005). In the meantime, mammary uptake ratios of BHBA，NEFA，TG and butyrate were decreased（P<001）, propionate was increased（P<005）, but acetate was no effect (P>005). With increasing propionate molar ratio in the VFAs, contents of short and medium chain fatty acids in milk fat were increased (P<005), and that of long chain fatty acids was decreased significantly (P<005). There was no effect on trans10，cis12 CLA. Results of the present study implied that increased propionate molar ratio in ruminally infused VFAs could decrease milk fat content, depress the mammary uptake of NEFA, increase the contents of short and medium chain fatty acids and decrease long chain fatty acids in milk fat.

Seroprevalence of Hepatitis E Virus Infections in Pigs from Zhejiang Province During 2005-2008

SHUAI Jiangbing;ZHANG Xiaofeng;XU Jingjing;CHEN Ning;FANG Weihuan

2009, 40(7): 1037-1042. doi:

Abstract ( 1052 )

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The study was aimed to establish an indirect ELISA for epidemiological investigation of hepatitis E virus（HEV） infections in pigs from the major pig-producing regions of Zhejiang Province. The gene fragment covering immunogenic epitopes of HEV ORF2 was synthesized and expressed in Escherichia coli. Western blot analysis revealed that the purified protein reacted to HEV positive sera, but not to positive sera of other common viruses infecting pigs. An indirect ELISA system was then developed using truncated HEV ORF2 protein as the coating antigen and had diagnostic accuracy of 915 % as compared with a diagnostic kit for HEV antibodies. A total of 1 330 serum samples were collected from 46 pig farms in Zhejiang province during 2005-2008 and tested for seroprevalence of HEV using the indirect ELISA. The average HEVpositive rate was 557% (741/1 330). The positive rate of 2005 and 2006 was 627% (175/279) and 614% (301/490) respectively, much higher than those of 2007 (431%, 59/137) and 2008 (486%, 206/424). Meanwhile, the pigs aged 66-100 days had a statistically higher positive rate (582%, 110/189）as compared with that of pigs of 30-65 (397%, 73/184） and 101-160 (441%, 83/188) days. Only three herds in the study were HEV antibodynegative, indicating high seroprevalence of HEV in the pig populations in Zhejiang Province.

The Quantitative Research on the mRNA Transcription Level of the Prion Receptor-37kDa/67kDa LRP/LR in Ovine Tissues

QIAO Junwen;SU Xiaoou;WANG Yiqin;ZHOU Xiangmei;YANG Jianmin;YIN Xiaomin;MA Liying;ZHAO Deming

2009, 40(7): 1043-1047. doi:

Abstract ( 1161 )

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The aim of the present study was to detect the mRNA transcription level of prion receptor-37kDa/67kDa LRP/LR in ovine tissues. The realtime RTPCR was employed for the quantitation of mRNA transcription level of 11 tissues from six sheep with the same background. The synthesis of cDNA for each sample was firstly performanced by RTPCR after the isolation of RNA from various tissues in sheep. The results showed that the highest LRP/LR mRNA transcription level was found in neocortex (P<005) , which exhibiting as much as tenfold higher than in lung, followed by heart and obex(P<005); intermedia transcription was occured in hippocampus, cerebellum, spleen, thalamus, mesenteric lymph node; the lower transcription was occured in liver and kidney, and the lung expressed extremely lowest but detectable level of LRP/LR mRNA (P<005). These results indicated that altered expression profiles of laminin receptor in various ovine normal tissues are basically corresponding to the degree of PrPSc accumulation, and tissues expressing high levels of LRP/LR may have higher potential susceptibility to PrPSc. Our results also suggest that in ovine tissues， the degree of PrPSc propagation was correlated with the expression level of prion receptor，37kDa/67kDa LRP/LR.

Study on DNA Delivery Methods: Increased in vivo Immunological Potency of DNA Vaccine by Tattooing

ZENG Weiwei;SHI Xingming;HUANG Tingting;WANG Mei;GAO Hongbo;SUN Yan;LI Jisong;WANG Yunfeng

2009, 40(7): 1048-1053. doi:

Abstract ( 1116 )

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Tattooing is one of the economical and effective DNA delivery methods，which is an invasive procedure involving a solid vibrating needle that repeatedly punctures the skin, wounding both the epidermis and the upper dermis in the process and causing cutaneous inflammation followed by healing, and thus nonspecifically stimulates the immune system. In this study, we focused on revealing the difference of two routes of administrations of DNA vaccine (intradermal tattoo versus intramuscular injection) on induction of immune response on BALB/c mice and SPF chickens, aiming at providing a new idea for DNA immunization. For comparison,we used eukaryotic expression vectors pCDNA3.1gJ (containing gJ gene of infectious laryngotracheitis virus) and pVAX1F (containing F gene of Newcastle disease virus) as the model antigen. BALB/c mice were inoculated with recombinant plasmid pCDNA3.1gJ and SPF chickens were inoculated with recombinant plasmid pVAX1F via intradermally by tattooing and intramuscularly by needle injection. Serum samples were collected regularly postvaccination for detection of the specific IgG antibody level. The chickens were challenged by NDV F48E9 strain with a dose of 105EID50 two weeks after the third immunization, then their morbidity and mortality were recorded. The results showed that the humoral immune responses elicited by tattooing are significantly stronger than elicited by intramuscular injection both on BALB/c mice and on chickens. In addition, low dose(50 μg )DNA via tattooing can induce higher specific antibody level than high dose(100 μg) via intramuscular injection in the same period,and tattooing immunizations can provide better immune protection than intramuscular injection. In summary, the tattoo delivery of DNA vaccine is a costeffective method and can induce more rapid and more robust immune responses that may meet the laboratory conditions need,which also provides a new idea for the future research directions on DNA immunization.

Development and Application of McAbbased Competitive ELISA Kit for Shigalike Toxin IIe

ZHANG Jianmin;WU Bin;ZHAO Zhanqin;LU Shun;HE Hua;XIANG Min; LI Liya;ZHANG Futao;CHEN Huanchun

2009, 40(7): 1054-1058. doi:

Abstract ( 1022 )

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The study aimed to establish a rapid and simple assay to detect Shigalike toxin IIe (SLTIIe). Monoclonal antibodies (McAbs) against SLTIIe A subunit were produced by using Escherichia coli (E. coli) expressed recombinant SLTIIe A subunit protein as antigen to immunize BALB/c mice. A competitive ELISA for detecting antibodies against SLTIIe was developed on the basis of recombinant protein expressed by E. coli and HRPlabelled McAbs against SLTIIe A subunit. The optimum conditions of the ELISA were developed as following: the concentration of recombinant for coating ELISA plates was 032 μg·mL-1; the best dilution of serum to be tested was 1∶2; the working titer for HRPlabelled McAbs was 1∶3 200; and the inhibition rate above 40% was selected as the positive judging standard. A total of 60 serum samples were detected in parallel by both competitive ELISA and vitro neutralization assay. 33.8% of them were detected as positive by competitive ELISA, while 309% of them were positive by vitro neutralization assay. The coincidence rate of the two assays was 882%. The results showed that the competitive ELISA had the advantages of higher sensitivity, specificity, reproducibility, stability and easy operation. It would be very useful in diagnosis, surveillance of SLTIIe antibodies and epidemiological survey.

Analysis of the Secreted Protein Encoding Genes in Genome of Brucella melitensis 16M

YANG Yu;WU QingMin

2009, 40(7): 1059-1062. doi:

Abstract ( 703 )

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The complete 3 197 open reading frames (ORFs) of the Brucella melitensis’s whole genome sequence has been analyzed. Among them, there are 191 proteins with Nterminal signal peptides based on the combination of prediction algorithms SignalP v30 and TatP, transmembrane domains prediction algorithms TMHMM v20 and Phobius. And there are also 391 nonsignal peptide secretion proteins based on the analyzed by SecretomeP. The functions of most of these proteins have not been identified clearly. For the important effect of secreted proteins have in bacteria pathology, the research of secreted proteins of brucella would be very useful to understand the pathogenic mechanism of brucella and to develope new methods for diagnosis.

Studies on Comparison of 18S rRNA Gene Sequences of Theileria Species Infective to Cattle

LIU Aihong;GUAN Guiquan;LIU Junlong;LI Youquan;MA Miling;NIU Qingli;REN Qiaoyun;YIN Hong;LUO Jianxun

2009, 40(7): 1063-1068. doi:

Abstract ( 827 )

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Ribosomal smallsubunit RNA gene sequence of ten Chinese Theileria strains infective to cattle, including four isolates of Theileria annulata, three isolates of T. sergenti and three isolates of T. sinensis, collected in the protozoon resource library at the centerconservation of strain in China veterinary microbion, were cloned and analyzed and then the phylogenetic trees were constructed based on 18S rRNA sequence of the Chinese isolates as well as other species of Theileria available in GenBank. The result indicated that the length of the 18S rRNA gene of all Theileria species involved in this study were varied between 1 740-1 890 bp and Chinese isolates of three Theileria isolates were located in the different phylogenetic branch, moreover, the percent identities of the different isolates in species were much higher than among species. The results were basically consistent with traditional classification as well as molecular classification based on 18S rRNA gene sequence for the bovine Theileria. This study will provide a foundation for PCR detected method for bovine theileriosis.

Induction of Apoptosis by a Highly Pathogenic Avian Influenza Virus，A/duck/Guangdong/220/2004（H5N1）in Experimentally Infected Ducks

LI Yugu;YE Yuanlan;CUI Congying;ZHOU Quanhe;ZHANG Yuan;MA Yongjiang;LI Chuxuan

2009, 40(7): 1069-1073. doi:

Abstract ( 750 )

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Using terminal deoxyuridine transferase nickend labeling（TUNEL）and electron microscopy technique, the apoptosis were investigated in ducks inoculated with a highly pathogenic avian influenza virus, A/duck/Guangdong/220/2004(H5N1) by intravenous and ocularnasaloralcloacal routes. The results demonstrated that the virusinduced apoptosis occurred in the pancreas, kidney, liver, heart, lung, intestine, cerebrum, cerebellum, thymus, bursa of Fabricius and spleen, etc. The predominent apoptotic cell types were pancreatic acinar epithelial cells, renal tubular epithelial cells, hepatocytes and intestinal epithelial cells. The minority of neurons, glial cells, cardiac muscle cells, epithelial cells of lung air capillary, lymphocytes in the thymus, bursa of Fabricius and spleen also experienced apoptotic changes.

Anatomy and Histology of Pig Tonils

LIU Zhixue;YANG Qian

2009, 40(7): 1074-1081. doi:

Abstract ( 1278 )

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The tonsils of the pig were lymphoepithelial organs situated on the opening of both digestive and respiratory tracts. The morphology and ultramicrostructure of the various tonsils were studied on 15 pigs aged 5 months. The results showed that there were four tonsils in pig. The soft palatine tonsil and Paraepiglottic tonsil were both located on oropharynx tract. They consist mainly of secondary lymph nodules and were encapsulated in dense connective tissues. The stratified squamous epithelium covering the tonsils and their crypts was frequently infiltrated heavily by lymphocytes. The pharyngeal tonsil and tubal tonsil were situated on nasopharyngeal tract. The epithelium covering the tonsils was squamous epithelium that often distributes sparsely in some surface regions. The intercellular junction of the epithelium was looser, and the intercellular space was larger compared to other epithelium. These tonsils consist of scattered lymph nodules, aggregations of lymphocytes and diffuse lymphoid tissue. The feature of pharyngeal tonsil and tubal tonsil was benefit for the antigen absorption and presentation,and provide the sufficient evidence to intranasal immunization.

Effects of Verapamil on the Expression of cfos and cjun in Cardiac Myocytes of the Ascites Syndrome in Broilers

LI Kai;MA Liqin;XU Tong;WANG Jianlin;WANG Huiyu;QIAO Jian

2009, 40(7): 1082-1087. doi:

Abstract ( 736 )

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Right ventricular hypertrophy and failure is an important factor in the development of ascites syndrome. In this study, the technology of immunohistochemistry and in situ hybridization were adopted to observe the changes of cfos and cjun in cardiac myocytes of broilers under low temperature and treated with verapamil. At the same time, the mechanism of cfos and cjun in right ventricular hypertrophy and failure was discussed. Compared with the controls, the broilers under low temperature showed that the average optical density of cfos/cjun protein and mRNA of right ventricular myocytes significantly increased on 22, 29, 36, 43 and 50 days of age, and the percentage of positive area about cfos/cjun mRNA expression also significantly increased. Compared with broilers under low temperature, the broilers given verapamil showed that the average optical density of cfos/cjun protein and mRNA of right ventricular myocytes significantly decreased on 22, 29, 36, 43 and 50 days of age, and the percentage of positive area about cfos/cjun mRNA expression also significantly decreased. These date demonstrated that cfos and cjun are important factors in right ventricular hypertrophy and failure. The verapamil can inhibit the expression of cfos and cjun, and prevent right ventricular hypertrophy and failure.

Resistance to Aminoglycosides in Enterobacteriaceae Isolated from Pig Farms

CHEN Lin;LIU Jianhua;ZHANG Junfeng;CHEN Zhangliu ;ZENG Zhenling

2009, 40(7): 1088-1096. doi:

Abstract ( 778 )

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To investigate the resistance to aminoglycosides in Enterobacteriaceae isolated from two pig farms， Enterobacteriaceae isolated from two pig farms were screened for the presence of the rmtA, rmtB, armA，rmtC genes and 10 aminoglycosides modifying enzyme genes by PCR and sequencing. Conjugation experiments were carried out to study the horizontal transfer mechanism of rmtB and 10 AME genes. All isolates and their transconjugants were tested for susceptibility to 20 antimicrobial agents by the broth microdilution method. Of 152 Enterobacteriaceae isolates recovered from two pigs, 49 strains(323%) were positive for the rmtB gene, including 48 strains of Escherichia coli and a single isolate of Enterobacter cloacae. No other kind of the 16S rRNA methylase genes was detected. 7 AMES genes including aac(3)Ⅱ，aph(3′)Ⅶ，aph(3′)Ⅱ，aadA1，aac(6′)Ib，aac(3)Ⅳ and aph(4)Ⅰ were presented in 49 rmtBpositive isolates. The present rate of the 7 AMES genes were ordered as 935%（aac(3)Ⅱ）,796%（aph(3′)Ⅱ）,776%（aph(3′)Ⅶ）,345%（aadA1）,143%（aac(6′)Ib）,102%（aac(3)Ⅳ） and 82%（aph(4)  I）. Total of 46 rmtBpositive isolates transferred their rmtB, aac(3)Ⅱ，aph(3′)Ⅶ，aph(3′)Ⅱ and aadA1 easily to recipients of E.coli C600 and E.coli 488 Rifr. Conjugative transfer frequencies varied from 30×10-6 to 46×10-13 transconjugants per recipient. All 49 rmtBpositive isolates and 46 transconjugants showed extraordinarily highlevel resistance to 4,6substituted deoxystreptamine (MICs, >512 μg·mL-1) and other aminoglycosides .The results indicated that 16S rRNA methylases RmtB and 10 AMEs were widely spread in veterinary clinical isolates from two pig farms.They mediated the highlevel resistance to 4,6substituted deoxystreptamine and other aminoglycosides.They located on the conjugated plasmid and transfer resistance of aminoglycosides together.

Study on Targeting of Tilmicosin Gelatine Microsphere in Rabbits

YUAN Liguo;LI Chengming;BAO Jie;ZHU Lixiang;ZHANG Xiuying

2009, 40(7): 1097-1100. doi:

Abstract ( 785 )

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The purpose of this study was to evaluate tilmicosin gelatine microsphere targeting. The timed tilmicosin concentrations of organs from tilmicosin gelatine microsphere and injection administered rabbits were detected with high performance liquid chromatography, and the area under curve was calculated with no compartment model by 3p97, a software of pharmacokinetics. Targeting parameters of tilmicosin gelatine microsphere were analysed. Compared with tilmicosin injection, lung targeting efficiency to heart′s, muscle′s, liver′s and kidney′s was elevated by 518±013, 311±006, 394±006, 374±002 times, respectively. Relative intake rate of lung, heart, muscle, liver and kidney was 823±004, 159±003, 265±004, 209±002 and 220±001. The ratio of peak concentration of lung, heart, muscle, liver and kidney was 222±004, 042±002, 046±001, 061±002 and 071±002, respectively. It was concluded that tilmicosin gelatine microsphere had better lung targeting and alleviated the tilmicosininduced cardiotoxicity.

Quantitative Expression of Vascular Endothelial Growth Factor Gene and Hypoxic Adaptation in Tibetan Chicken

CHAMBA Yangzom;XIE Zhuang;SHANG Peng ;LI Qifa;ZHAI Mingxia;ZHANG Hao

2009, 40(7): 1101-1105. doi:

Abstract ( 749 )

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In this study, the expression of VEGF gene was measured in chorioallantoic membrane (CAM) tissue of Tibetan chickens and Dwarf Recessive White chickens which were incubated at normoxia (O2，21%) and hypoxia (O2，13%), respectively. The results showed that hypoxiainduced VEGF upregulation occurred in CAM tissue for both breeds. However, the extent of upregulation was significantly higher in Dwarf Recessive White chickens than that in Tibetan chickens. It concluded that hypoxia induced VEGF mRNA expression to a certain degree in Tibetan, which made for angiogenesis and adaptation to hypoxic environment for Tibetan chickens. However, the VEGF expression was excess in CAM tissue in Dwarf Recessive White chickens at hypoxic condition, which made the embryo of Dwarf Recessive White chickens against to development and survival.

Polymorphism of PRKAG3 Gene and Its Association with Carcass and Meat Quality Traits in Beef Cattle

LI Wufeng;YANG Runjun;GAN Qianfu;ZHANG Lupei;XU Shangzhong;LI Junya;GAO Xue

2009, 40(7): 1106-1111. doi:

Abstract ( 1165 )

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Polymorphisms of PRKAG3 gene were detected in 267 beef cattle in 7 beef cattle breeds (Simmental, Angus, Hereford, Charolais, Luxi cattle, Limousin and Jinnan cattle) by PCRRFLP. Two polymorphic sites T2643C and T2885C were found(DQ082736), both of them in intron 4. Association analysis of the two SNPs with 9 carcass and meat quality traits was carried out. The results showed that T2885C substitution of PRKAG3 gene is significantly associated(P＜005) with meat tenderness, and the mean value of individuals with genotype CC is lower than that of individuals with genotype CD and DD. The information found in the present research is very important for meat quality breeding in beef cattle by markerassisted selection.

Cloning and Molecular Characterization of VP26 Gene of Duck Plauge Virus

CAI Mingsheng;CHENG Anchun;WANG Mingshu;ZHU Dekang;LUO Qihui;ZHAO Lichan;CHEN Xiaoyue

2009, 40(7): 1112-1119. doi:

Abstract ( 799 )

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A complete open reading frame (ORF) of UL35 gene encoding the protein VP26 of duck plauge virus (DPV) was obtained according to the DNA sequencing information of a recombinant plasmid of the DPV CHv strain gene library constructed in our laboratory combining together with the analysis of ORF Finder and BLAST tool of NCBI. Then the DPV VP26 gene was cloned into pMD18T，and was strongly confirmed by PCR amplification, restriction digestion and oligonucleotide probe hybridization. Molecular characterization analysis indicated that the DPV VP26 gene was composed of 354 nucleotides,and encoding a polypeptide of 117 amino acid residues, which had a Herpes_UL35 conserved domain related to small nucleocapsid protein family and highly conserved among the Herpes_UL35 proteins. Moreover, the nucleic acid and amino acid sequence of DPV VP26 had higher homology with its homologous protein of Alphaherpesvirus than others. Phylogenetic tree analysis showed that on taxonomic status the DPV could be grouped in Alphaherpesvirus subfamily, and might be a new genus of Alphaherpesvirus. Subcellular location analysis demonstrated that the VP26 mainly located in nucleus. Besides, condon preference analysis demonstrated that the alternative codons for the same amino acid in VP26 had distinctly different frequency and the DPV VP26 had 18, 19 and 25 codons’ frequency evidently disagreeing with E. coli, yeast and human, respectively. These results provide a molecular biology evidence for the further study on the function and mechanism of DPV VP26.

Development of a Rea1Time Fluorescent Quantitative RTPCR Based on M Gene of Newcastle Disease Virus (NDV) to Detect NDV in Clinical Samples

CAO Junping;HU Shunlin;WU Shuang;LIU Huimou;LIU Xiaowen;WANG Xiaoquan;WU Yantao;LIU Xiufan

2009, 40(7): 1120-1125. doi:

Abstract ( 759 )

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Pair of primers and a TaqMan probe were synthesized according to M gene conservative sequence of Newcastle disease virus. The positive recombinant plasmid containing M gene of NDV ZJ1 strain isolated from goose was used as a positive quantitative template to establish a standard curve. And then a rea1time fluorescent quantitative RTPCR assay was established. The method has a good linear relationship within the 106 to 101 copies, with which 3 copies·μL-1 of the virus nucleic acid can be detected in the initial template, and has similar sensitivity with traditional virus isolation methods. The conforming rate of positive sum and negative sum with traditional virus isolation method was 900% and 998% respectively in detecting 500 clinic cloacal swab samples. The result showed that the constructed method paved the way for the early and rapid detection of NDV as well as quantitative analysis for the infection degree of NDV.

Rapid Detection and Virulence Identification of Airborne Chicken Newcastle Disease Virus by Degenerate Primers RT-PCR

LI Xiaoxia;QIU Yuyu;YU Ailian;CHAI Tongjie;WANG Hairong;WANG Zhiliang;LIU Jingbo

2009, 40(7): 1126-1130. doi:

Abstract ( 746 )

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In order to rapidly detect and identify airborne Newcastle disease virus (NDV) in the indoor air and outdoor air of largescale commercial broiler house, international standard air collectorAGI30 was used to collect the air, Oropharyngeal and cloacal swabs were also collected. NDVs in the air samples and in the swaps were detected and virulence identified by degenerate primers based RTPCR; at the same time NDVs in the samples were isolated and purified and then were virulence identified by conventional biological pathogenicity methods. The results showed that both virulent virus and avirulent virus could be detected simultaneously in 2/ 15 indoor air samples and 4/15 swaps, avirulent virus could be singly detected in 3/15 indoor air samples and 13/15 swaps, no virus could be detected in the air of 5 m upwind samples, avirulent virus could be singly detected in 1/3 samples of 5 m downwind air, no virus could be detected in all the other samples by degenerate primers based RTPCR. Three virulent NDV and four avirulent NDV were isolated and virulence identified in the RTPCR positive samples and no virus was isolated from the RTPCR negative samples. The results suggest that degenerate primers based RTPCR can detect and virulence identify NDV directly and rapidly.

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