The Fluorescent Characterization and Analysis of Two Brucella Attenuated Vaccine Infecting Mouse Macrophagocyte

The experiment was conducted to discuss the difference of binding time of green fluorescent protein B.melitensis M5 (GFP-M5) and B.abortus S19 (GFP-S19) infecting the mouse macrophagocyte (RAW264.7),lysosome,endoplasmic reticulum and golgi body in the initial stage and compare the binding rate of GFP-M5,GFP-S19 with organelle in different timeline,respectively,by confocal laser scanning microscope (CLSM) and flow cytometry.The result showed that GFP-M5 and GFP-S19 were successfully constructed.The intracellular survival ability of Brucella M5,Brucella S19,GFP-M5 and GFP-S19 were not obvisouly affected after infecting RAW264.7.GFP-M5 and GFP-S19 could enter the macrophagocyte in 30 mins,and in 2 h the Brucella could reach lysosome,endoplasmic reticulum and golgi body.In addition,the binding time for two attenuated vaccine did not show differences in 1,2,3 and 4 h.The content of GFP⁺ cell produced by RAW264.7 infected by GFP-M5 and GFP-S19 did not show significant differences (P>0.05).Therefore,the two strains did not have significant differences in the invasion ability in the initial stage of infecting host cell.     

Effects of Donor Cells Treated with Egg Extracts on the Development Competence of Cloned Embryos

Japanese Balck cattle fetal fibroblasts (JBCFF) were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer (SCNT) was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin+6-DMAP and A23187+6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158 μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7 μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7 d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion (92.83% vs 96.04%),cleavage (89.64% vs 89.78%) and blastocyst formation (24.06% vs 23.12%) of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells (P>0.05).Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos (cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%).Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.     

Isolation and Identification of Cellulase-producing Bacillus in Yak Rumen

To investigate the species of cellulase-producing Bacillus in yak rumen,10 samples of rumen content were aseptically collected from 10 adult Maiwa yaks to isolate the heat-resistant Bacillus by water bath at 80 ℃ for 20 min.The cellulase-producing strains were screened using the CMC-Na medium and Congo red staining.The 16S rRNA gene sequence of those cellulose-producing strains were amplified and sequenced.The results showed that 64 strains were isolated from the 10 samples.Total 23 strains were identified as cellulase-producing bacillus,including 16 strains of Bacillus cereus,7 strains of Bacillus thuringiensis.Furthermore,phylogenetic analysis showed that the 16 Bacillus cereus strains were clustered into two branches:One isolate was clustered into a branch alone,the other 15 isolates were clustered into a branch which clustered into 5 small branches,showing that there was certain genetic diversity in the isolates of Bacillus cereus.And all 7 Bacillus thuringiensis strains were clustered into a branch.Hence,the results layed the foundation of investigating the species of cellulase-producing Bacillus in yak rumen and developing probiotics special for yak.     

Effects of Supplemental Complex Water Soluble Vitamin on Production Performance,Blood Biochemical Parameters and Oxidation Resistance of Dairy Cows

The objective of the study was to investigate the effects of supplemental complex water soluble vitamins on production performance, blood biochemical parameters and oxidative resistance of dairy cows in spring and summer.The experiment was designed by 2×2 factorial randomized blocks design.Eighty healthy dairy cows with similar parity and body weight were randomly divided to four groups, with 7 days of adaptation and 70 d experimental phase.The dairy cows in group Ⅲ and Ⅳ were fed a basal diet supplemented with complex water soluble vitamins in spring.The dairy cows in group Ⅱ and Ⅳ were fed a basal diet supplemented with complex water soluble vitamins in summer.The results showed that the ADFI and daily milk yield of dairy cows receiving water soluble vitamin in spring were extremely significantly higher (P<0.01);The ADFI of dairy cows receiving water soluble vitamin was significantly higher in summer (P<0.05) and daily milk yield were extremely significantly higher (P<0.01);The butterfat rate was extremely significantly increased and SCC was extremely significantly decreased in spring and summer (P<0.01);Supplementation of water soluble vitamin was also significantly increased LYM (P<0.05).In summer, T-AOC, T-SOD were extremely significantly increased (P<0.01), and TP, CK, LDH, TP, HDL and ALB were extremely significantly decreased (P<0.01) by water soluble vitamin supplementation than that of control group.In conclusion, complex water soluble vitamins could increase the production performance and non-specific immune function, improve the oxidative resistance and relieved the heat stress of the dairy cows.     

Isolation and Identification of BPIV3 Genotype C

To identify the infection agents from Ningxia Hui Autonomous region, where feedlot cattle indicated bovine respiratory disease complex (BRDC), the M gene of the bovine parainfluenza virus type 3 was amplified by RT-PCR.The PCR product was ligated to pMD18-T vector and cloned to E.coli DH5α.The positive clones were sequenced and compared with the reference strains in GenBank by the molecular biology software.Sequence alignment results showed that a BPIV3 strain was isolated from the samples and named NX49, the M gene of NX49 included 1 056 nucleotides.Evolutionary analysis showed that the NX49 belonged to BPIV3 C genotype and shared 99.4% nucleotide identity with that of the SD0835 isolated in Shandong province.The characterization of the NX49 demonstrated that it was sensitive to temperature, acid and organic matter.The presence of Mg²⁺ showed no protection against the treatment at high temperature.The HA test suggested that the NX49 enables to agglutinate the guinea pig RBC at 4 ℃ and the titer was 1∶4.The study isolated a BPIV3 genotype C strain successfully, which facilitate the study of molecular evolution and epidemiology of BPIV3 in China.     

Isolation and Identification of Pathogenic Bacteria Causing Dairy Cow Mastitis in Liaoning and Drug Sensibility Test of Pathogenic E.coli

In order to understand the main bacterial pathogen species causing dairy cow mastitis in Liaoning, as well as the characteristics of drug sensitivity of the pathogenic E.coli,the milk samples from 75 dairy cows with clinical manifestations for mastitis in certain large-scale dairy farm in Liaoning were collected.The bacteria in milk were cultured and isolated with biochemical methods and in vitro drug sensitivity tests were processed with the isolated E.coli strains.The results showed that the main bacterial pathogen for dairy cow mastitis were E.coli（separation rate 58.7%),S.aureus（64.0%）and S.agalactiae（54.7%),and multiple infection including double and triple infection were identified.The drug sensitivity tests on the isolated E.coli indicated that the E.coli isolates were highly resistant to sulfonamides（resistance rate>85%）and chloramphenicol（resistance rate>30%),and they were relatively low resistant to ampicillin（9.5%),ciprofloxacin（9.5%),ceftiofur（7.1%）and ofloxacin（4.8%).The results was able to provide reliable theoretical basis for prevention and control of dairy cow mastitis in Liaoning area.     

东北地区奶牛乳房炎表皮葡萄球菌和腐生葡萄球菌的分子流行病学及耐药性研究

为调查表皮葡萄球菌和腐生葡萄球菌在东北地区的流行病学情况和耐药性,本研究对来自东北地区3个大型奶牛场采集的330份奶样进行葡萄球菌的分离、鉴定及其耐药表型的检测,并采用脉冲场凝胶电泳(PFGE)进行分离株的亲缘性分析,对表皮葡萄球菌进行多位点序列分型(MLST),同时应用PCR扩增分离株中携带的相关耐药基因。研究结果表明,在330份奶样中共分离到表皮葡萄球菌32株(9.7%),腐生葡萄球菌34株(10.3%);PFGE分析共获得9种不同谱型的表皮葡萄球菌和11种不同谱型的腐生葡萄球菌。药敏试验结果显示,两种菌对青霉素(70%)、苯唑西林(60%)和林克霉素(55%)的耐药率较高,主要耐药基因为lnu(B)(40%)、erm(B)(30%)和mec A(25%)。本研究结果揭示了东北地区奶牛乳房炎病原菌表皮葡萄球菌和腐生葡萄球菌的耐药谱和流行情况,为临床合理用药及奶牛乳房炎的防控提供了实验依据。     

9种牧草对青海同德牧区土壤特性的影响

通过对青海省高寒牧区常见的9种多年生牧草单播2年后耕层0~15cm土壤理化(pH、容重(BD)、有机碳(SOC)、全氮(TN)、无机碳(C)及微生物学性质(微生物生物量碳(Cmic)、氮(Nmic)和群落代谢功能)等指标的测定分析,结果表明,研究区域只有在种植披碱草2年后土壤有机碳含量有所增加,说明与其他草种相比,种植披碱草利于有机质的积累;试验在每年施肥1次的情况下,土壤氮含量仍然偏低,说明此区氮素被过度利用,处于缺乏水平,因此每年增施氮肥数量、频率以及时间上应加强管理。通过对不同牧草种植区土壤各因子的聚类分析,发现贫花鹅观草、无芒雀麦、紫野麦草和扁穗冰草之间相似度较高,表明其对土壤养分及微生物群落功能的影响较为接近,故在大面积种植的时候可根据牧草地上生物量/质量的高低进行选择性播种。从土壤质量方向考虑,种植杂花苜蓿、红豆草和西北羊茅不利于土地的改良。     

鸭IFIT5基因(544G>A)多态性与部分免疫指标的关联分析

探索IFIT5基因作为抗性性状的候选标记的可能性,为家禽抗病育种提供一些参考依据。以樱桃谷北京鸭、苏牧麻鸭(樱桃谷鸭×金定鸭)、金定鸭、白羽番鸭为试验材料,利用DNA直接测序技术扫描IFIT5基因单核苷酸多态性(SNP),并分析其与部分免疫指标的关联性。结果表明:仅在IFIT5基因外显子2上检测到一个错义突变(G544A),产生2种等位基因,2种基因型(GG、GA),未发现有AA基因型。经χ2检验,4个鸭群体只有金定鸭和苏牧麻鸭处于Hardy-Weinberg平衡状态。关联分析结果显示:樱桃谷鸭体内H5含量GG型显著高于GA型(P0.05),而Ig M含量GG型显著低于GA型(P0.05),其余免疫指标均无显著差异(P0.05);苏牧麻鸭体内H5含量GG型极显著高于GA型(P0.01),其余免疫指标均无显著差异(P0.05);金定鸭不同基因型各免疫指标均无显著差异(P0.05)。白羽番鸭在+544位点均为GG纯合基因型。由此可以看出,IFIT5(544GA)可作为樱桃谷鸭体内H5含量的候选分子标记。     

Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse

Brucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial growth curves and resistance to environmental stress factors showed that Pyk plays an important role in B. abortus growth, especially under the conditions of nutrition deprivation, and resistance to oxidative stress. Additionally, cell infection assay showed that Pyk is necessary for B. abortus survival and evading fusion with lysosomes within RAW264.7 cells. Moreover, animal experiments exhibited that the Pyk deletion significantly reduced B. abortus virulence in a mouse infection model. Our results elucidated the role of the Pyk in B. abortus virulence and provided information for further investigation of Brucella virulence associated carbon metabolism.