鹅大肠埃希菌的分离鉴定及系统进化树分析

为对发病鹅感染致病性大肠埃希菌的临床治疗提供指导并防控致病性大肠埃希菌感染,本研究无菌采集腹泻鹅及病死鹅脾脏、肝脏等组织器官进行病原菌分离培养、染色观察、生化鉴定、致病性试验,对菌株部分基因进行测序、构建遗传进化树并进行药敏试验。结果显示,普通琼脂培养基上生长出灰白色、圆形、凸起、边缘整齐菌落,麦康凯琼脂培养基上长出玫红色、光滑圆润菌落;分离菌株经革兰氏染色,镜下可见革兰氏阴性菌,两端钝圆、大小中等,多呈单个存在的杆状菌;致病性试验表明,该分离菌株对小鼠、雏鹅均有较高致死率;遗传进化树结果显示,分离菌株与患者粪便分离株（GenBank登录号：CP024992.1）同源性最高,达99.5%;体外抑菌试验发现菌株耐药情况较严重,对大多数抗菌药均有较强抗性,仅头孢噻呋对该分离株有较强的抑菌作用,合理用药后病情得到有效控制。本研究为有效防治鹅大肠埃希菌病提供了理论依据,对规模化鹅场防控大肠埃希菌等其他细菌性疾病具有重要价值。     

猪戊型肝炎病毒ORF3蛋白影响HepG2细胞核黄素代谢的lncRNA-mRNA调控网络的鉴定

为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪 HEV-ORF3在HepG2细胞中实现过表达。Western blotting检测ORF3蛋白过表达成功后,运用lncRNA高通量组学测序,筛选出差异表达的lncRNA并进行靶向差异基因预测,对lncRNA靶向差异基因进行GO功能和KEGG通路富集分析,初步鉴定出与核黄素代谢信号通路相关的lncRNA-mRNA调控网络。Western blotting结果显示,在约为12 ku处出现目的条带,说明成功实现腺病毒介导猪HEV-ORF3在HepG2细胞中过表达。lncRNA高通量组学测序结果显示,共发现102个显著差异表达lncRNAs表达量上调,80个显著差异表达lncRNAs表达量下调。GO功能和KEGG通路富集分析显示,初步挖掘出lncRNA(MSTRG.13995.2)和lncRNA(MSTRG.1960.1)可能是与核黄素代谢信号通路相关的显著差异表达lncRNA,分别通过顺式调控其靶向基因APC-5和FLAD1来影响核黄素代谢信号通路。本试验初步鉴定出lncRNA(MSTRG.13995.2)-APC5和lncRNA(MSTRG.1960.1)-FLAD1可能是影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络。     

Effects of periodical application of bioactive peptides derived from cottonseed on performance,immunity, total antioxidant activity of serum and intestinal development of broilers

This experiment aimed to examine the effect of periodical application of bioactive peptides derived from cottonseed (BPC) in comparison with using sub-therapeutic doses of lincomycin and the excessive inclusion of vitamin E on performance, immunity, total antioxidant capacity of serum and intestinal morphology of broiler chickens. A total of 240 one-d-old male broiler chicks with similar initial weight (Ross strain) were randomly assigned to 6 groups (8 chicks/pen): non-treated group (basal diet), basal diet supplemented with 2 mg/kg lincomycin, basal diet supplemented with 50 IU vitamin E, basal diet supplemented with 6 g BPC/kg in starter period, basal diet supplemented with 6 g BPC/kg in starter and grower periods and basal diet supplemented with 6 g BPC/kg throughout the whole experiment. The highest final body weight was obtained in the group supplemented with BPC in starter and grower periods. In the finisher phase, broilers fed the diet containing BPC in the starter period and in the whole trial had significantly (P < 0.05) better feed conversion ratios (FCR). Jejunal villus height was significantly elevated in broilers supplemented with antibiotic (P < 0.001), furthermore it tended to be greater in broilers fed BPC in the starter period. The jejunal villus height-to-crypt depth ratio was significantly (P < 0.01) higher in broilers fed the diet containing antibiotic in comparison to other groups. Humoral immune response against Newcastle disease vaccine tended to be elevated in broilers fed the diet containing BPC in the whole trial (P > 0.05). Broilers supplemented with BPC in starter and grower, and in the whole trial had significantly (P < 0.05) higher antibody titers against sheep red blood cells (SRBC). The highest total antioxidant capacity was obtained in broilers supplemented with the excessive level of vitamin E, furthermore it tended to improve in broilers fed the diet containing BPC in the whole trial. In summary, the results of the study indicated that addition of BPC in broiler diets in the whole trial could improve FCR, immune responses and total antioxidant activity of serum, and BPC could be used in broiler diets as an alternative to in-feed antibiotics.     

维生素C废水厌氧处理生产运行的新技术研究

对生产规模上流式厌氧污泥过滤器处理维生素Ｃ废水的工艺流程和设备进行了合理的改革，以厌氧出水回流与高浓度废水混合稀释降低进水ＣＯＤ浓度，提高进水的总碱度，同时控制反应器水力负荷，从而提高了反应器的处理能力。     

Effect of Vitamin E on Bovine Granulosa Cells Apoptosis and Proliferation through Cx43

The aim of this study was to determine the effect of vitamin E on Cx43,the mechanism and function of vitamin E on bovine granulosa cells apoptosis and proliferation.In this study,granulosa cells were isolated from bovine ovary and cultivated in vitro by adding different concentration of vitamin E (0,25,50,100,200 and 500 μmol/L) for 24 h.After cultured,apoptotic cells were detected by FCM,mRNA expression levels of BCL2/BAX、P53 and Cx43 genes were determined by Real-time PCR and cell proliferation was detected by CCK8.The results showed that compared to control group,100 μmol/L vitamin E could significantly inhibit the apoptosis of granulosa cells (P<0.05).Real-time PCR detection results showed that vitamin E significantly changed the mRNA expression levels of BCL2/BAX,P53,Cx43 genes (P<0.05).Vitamin E could significantly improve granulosa cells proliferation when granulosa cells were treated for 24 and 36 h (P<0.05).The results provided a theoretical basis on further analysis for studing the influence mechanism of vitamin E on oocytes development and maturity,and improvement of female animal reproduction by influencing granulosa cells proliferation and apoptosis.     

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.     

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.     

Research Lactobacillus rhamnosusFermented Herbal and Bacillus subtilis Complex on Immune Performance,E.coli Infection of Broiler

This experiment was designed to study the complex of Lactobacillus rhamnosus fermented herbal and Bacillus subtilis on White Feather broiler immunity performance and impact of Escherichia coli infection.360 one-day-old broiler chickens were randomly divided into 3 groups with 4 replicates in each group and 30 chickens per replicate.The pretrial period lasted for 7 d,and the experiment lasted for 35 d.The chickens in the group Ⅰ(positive control group) and group Ⅱ(negative control group)were all only fed a basal diet,group Ⅲ was test group,by additive 1% fermented herbal preparations,groups Ⅱ and Ⅲ were intraperitoneal injection of 1 mL E.coli at 35 d,broiler mortality,immune organ index,cecalmicroflora content,immunoglobulin levels,IL-2 and IL-6 content were tested.The results showed that injection of E.coli caused massive death of chickens,group Ⅱtook up to 75.00%,it was significantly higher than groups Ⅰ and Ⅲ (P < 0.05),the mortality in group Ⅲ was significantly lower than group Ⅱ(P < 0.05),was only 23.33%.Injection of E.coli maked spleen index and thymus index of group Ⅱincreased significantly (P < 0.05),the spleen index and thymus index of groups Ⅰ and Ⅲ were no significant difference (P > 0.05).E.coli counts was significantly decreased after injectionin group Ⅲ (P < 0.05),but the number of intestinal Lactobacilli of group Ⅲ was significantly increased (P < 0.05),and inhibited the propagation of E.coli,the counts of E.coli in groups Ⅰ and Ⅲ were no significant difference (P > 0.05).At 42 d,the sIgA of the intestinal fluid in group Ⅲ were higher than that of groups Ⅰ and Ⅱ with 11.99% and 36.56%,respectively(P < 0.05).The serum IgG concentrations of group Ⅲ was higher than that of groupsⅠand Ⅱwith 14.68% and 28.15%,respectively(P < 0.05).At 42 d,the IL-2 content of group Ⅱ was the lowest,it was significantly lower than group Ⅲ(P < 0.05),the IL-6 of group Ⅲ was significantly lower than group Ⅱ(P < 0.05).     

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.     

维生素B_2对中华蜜蜂工蜂寿命及学习记忆能力的影响

本试验旨在研究维生素B_2对中华蜜蜂工蜂寿命及学习记忆能力的影响。试验分为工蜂饲养试验和学习记忆试验2部分。工蜂饲养试验:将1日龄中华蜜蜂工蜂分成3组,每组5个重复,每个重复约200只。对照组(Ⅰ组)饲喂1∶1的糖水,2个试验组在1∶1糖水的基础上分别添加100(Ⅱ组)、400 mg/kg(Ⅲ组)的维生素B_2,每天记录各组工蜂的死亡情况,直到全部死亡。工蜂学习记忆试验:分组方法同工蜂饲养试验,并按照工蜂饲养方法对1日龄工蜂喂养7 d后,利用吻伸反应方法测定工蜂短时学习记忆及长时学习记忆能力,并通过荧光定量PCR检测学习成功后工蜂学习记忆相关基因的相对表达量。结果表明:工蜂的平均寿命随维生素B_2添加量的升高而延长,而且试验组(Ⅱ组、Ⅲ组)的平均寿命显著高于对照组(P0.05),但Ⅱ组与Ⅲ组间差异不显著(P0.05)。Ⅲ组工蜂的长时学习记忆及短时学习记忆能力均显著高于对照组和Ⅱ组(P0.05),而且Ⅱ组工蜂的长时学习记忆能力也显著高于对照组(P0.05)。Ⅲ组工蜂的多巴胺受体基因2(Acdop3)、c AMP反应元件结合蛋白(Ac CREB)相对表达量均显著高于对照组和Ⅱ组(P0.05),而且Ⅱ组工蜂的Acdop3相对表达量也显著高于对照组(P0.05)。由此得出,维生素B_2影响中华蜜蜂工蜂的寿命及学习记忆能力,在人工饲喂蜂群时需要提供适量的维生素B_2。