Induction of tibial dyschondroplasia by carbamate and thiocarbamate pesticides

Tibial dyschondroplasia (TD) is a major poultry leg problem, the natural etiology of which is unknown. Certain dithiocarbamate pesticides such as tetramethyl thiuram disulfide (thiram) have been shown to induce the disease in chickens. Because many different carbamate and thiocarbamate chemicals are used in a number of agricultural, industrial, and household applications, the objective of this study was to determine whether all chemicals of these categories induce TD and whether there is a concentration-dependent relationship between the ingestion of these chemicals and the incidences and the severity of the disease. Week-old broiler chicks were fed diets containing thiram or other assorted carbamate and thiocarbamate pesticides mixed in feed for 24-48 hr between ages 8 and 10 days. The birds were killed on day 15 and the proximal tibial and tarsometatarsal growth plates were evaluated for the presence and severity of TD lesions. TD was distinguished by broadening of growth plates; upon histologic exam chondrocytes appeared to be shrunken and dead. When compared by including equimolar concentrations of these chemicals in the feed, the dithiocarbamates with more than two sulfide groups, such as disulfiram, ferbam, thiram, and ziram were potent inducers of TD, whereas those with two sulfides to no sulfide group appeared ineffective at inducing TD. Both thiram and ferbam also reduced the bird's body weights. Thiram increased the incidence and the severity of the disease, denoted by TD index, in a dose-dependent manner. These results suggest that inadvertent contamination of feed or litter with some of these or similar chemicals may cause leg problems in poultry.     

福美双诱发肉鸡胫骨软骨发育不良的组织病理学变化

120羽1日龄健康AA肉鸡预饲1周后随机分为2组,对照组饲以基础日粮,试验组饲以基础日粮添加100mg/kg福美双,进行了肉鸡胫骨长度、生长板厚度、肉鸡胫骨软骨发育不良(TD)指数及TD发病率等指标的检测,并进行了形态学和组织病理学观察。结果显示,患病鸡胫骨长度、生长板厚度和TD指数均有显著变化(P<0.01),TD发病率显著上升(P<0.05);病鸡胫骨近端的纵切面有玉白色楔状软骨团块深入干骺端甚至骨髓腔,呈现典型的胫骨软骨发育不良病理学变化。结果提示,100 mg/kg福美双可显著提高AA肉鸡TD发病率,并引起相应的组织病理学变化,为TD分子机理的研究提供了一个理想的实验动物模型。     

肉鸡胫骨软骨发育不良早期钙黏蛋白1(CDH1)差异表达研究

为研究福美双诱发肉鸡胫骨软骨发育不良(Tibial Dyschondroplasia,TD)早期钙黏蛋白1(CDH1)的差异表达,基础日粮中添加福美双,在试验第1、2、6天,对试验鸡进行剖杀,迅速采取胫骨生长板故入4％多聚甲醛溶液于4℃固定,做CDH1免疫组化分析;提取对照组和饲喂福美双组的生长板总RNA,采用Real-time PCR对CDH1 基因进行差异表达验证.结果钙黏蛋白1 (CDH1/E-cadherin)基因在TD生长板表达上调,在对照和TD软骨生长板,其蛋白合成主要在前肥大、肥大区软骨细胞质,增殖区软骨细胞无表达,钙化区软骨细胞表达少,在饲喂福美双第1、2、6天其表达增加与定量PCR变化结果相一致,且在饲喂福美双第2、6天呈明显高表达.结果表明CDH1 参与细胞黏附、血管入侵及调节Wnt/β-cat的信号传递,和其它分子共同调节软骨内骨化.     

新城疫病毒感染鸡肾脏组织蛋白质组学方法条件的优化

为研究新城疫病毒(NDV)与宿主组织细胞之间的相互作用关系,本研究以NDV疫苗(La Sota)接种的鸡肾脏组织为材料,对其差异蛋白的表达进行分析,通过条件的优化,建立了NDV感染鸡肾脏组织的双向凝胶电泳检测方法。采用ImageMaster2D Platinum version 6.0软件对电泳图谱分析后显示:24 cm IPG胶条(pH4～pH7)对应的凝胶中可检测到约1 400个蛋白点,蛋白点匹配率达90%,表明NDV感染鸡肾脏组织蛋白质组双向凝胶电泳模型稳定、分辨率高、重复性好,在对4个时间点对照和人工感染凝胶分析过程中发现差异蛋白点主要集中于接种后7 d,该样品的2-DE电泳图谱中共有29个差异表达明显的蛋白点,其中上调表达蛋白点有15个,下调表达蛋白点有14个,利用荧光定量PCR对其中4个差异蛋白在mRNA水平进行检测,结果与双向电泳结果一致,NDV接种鸡肾脏组织蛋白质组学方法的建立为NDV致病机理的研究提供有效的方法。     

巴戟天多糖对胫骨软骨发育不良的肉鸡生产性能、胫骨指数、胫骨钙磷灰分和血清电解质的影响

目的旨在探究巴戟天多糖（Morinda officinalis How polysaccharides,MOP）对福美双诱导的胫骨软骨发育不良（tibial dyschondroplasia,TD）肉鸡生产性能、胫骨指数、胫骨钙磷灰分和血清电解质的影响。方法选取1日龄健康AA肉仔鸡120只,随机分为3组,每组4个重复,每个重复10只肉鸡。对照组（CON组）饲喂基础日粮;福美双诱导TD组（TD组）饲喂基础饲料,在第4天到第7天的饲料中添加100 mg/kg的福美双进行TD模型建立;巴戟天多糖组（MOP组）在接受TD造模处理后,在第7天到第15天的饲料中添加400 mg/kg的巴戟天多糖。结果与CON组相比,从试验开始到试验第7天,TD组肉鸡的平均采食量和平均日增重显著（P<0.05）降低,料肉比增加（P>0.05）;在试验第7天、第11天和第15天时,TD组肉鸡的胫骨生长板宽度和生长板宽度系数显著（P<0.05）增加,胫骨重量和胫骨长度显著（P<0.05）降低,胫骨重量指数降低（P>0.05）,胫骨灰分含量显著（P<0.05）降低,并且血清中电解质紊乱;与TD组相比,在试验第11天和第15天时,MOP组肉鸡的料肉比有降低的趋势（P>0.05）,MOP组肉鸡的生长板宽度和生长板宽度系数显著降低（P<0.05）,胫骨重量、胫骨长度和胫骨重量指数均显著（P<0.05）提高;在试验第11天时,MOP组肉鸡的胫骨灰分含量显著（P<0.05）提高;随着试验时间的延长,MOP组肉鸡的血清电解质含量趋于平衡。结论饲料中添加巴戟天多糖可以有效缓解和改善由福美双诱导的肉鸡TD症状。     

健康奶牛与临床型乳腺炎奶牛乳腺线粒体蛋白组差异表达分析

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构再进行蛋白质组学研究,可提高低丰度蛋白在双向凝胶电泳中的检出率。通过对比分析乳腺炎奶牛乳腺与正常奶牛乳腺线粒体蛋白质组的表达变化,为奶牛乳腺炎的生物学治疗及抗病育种工作筛选出目基因和蛋白。超速离心法分离线粒体,双向凝胶电泳分离蛋白,PDQuest7．4软件分析差异蛋白斑点,高效液相色谱串联离子阱质谱鉴定差异蛋白。从奶牛乳腺线粒体蛋白2-DE图谱中筛选出17个差异表达的蛋白质斑点,质谱鉴定出17个差异表达蛋白（6个蛋白在奶牛乳腺炎发生过程中下调,8个上调,1个只在正常情况下表达,2个只在乳腺炎乳腺组织中表达）。筛选出的差异蛋白质涉及到细胞的能量代谢、蛋白质合成、mRNA的加工成熟及调亡调控等许多方面,表明奶牛乳腺炎发生时乳腺线粒体组织结构和代谢状态都发生了明显的变化。     

Proteomic alteration of Marc-145 cells and PAMs after infection by porcine reproductive and respiratory syndrome virus

Viral infections usually result in alterations in the host cell proteome, which determine the fate of infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus (PRRSV), infected pulmonary alveolar macrophages (PAMs) and Marc-145 cells were subjected to proteomic analysis involving two-dimensional electrophoresis (2-DE) followed by MALDI-TOF-MS/MS identification. Altered expression of 44 protein spots in infected cells was identified in 2D gels, of which the 29 characterised by MALDI-TOF-MS/MS included 17 up-regulated and 12 down-regulated proteins. Some of these proteins were further confirmed at the mRNA level using real-time RT-PCR. Moreover, Western blot analysis confirmed the up-regulation of HSP27, vimentin and the down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected Marc-145 cells using proteomics to provide valuable information about the effects of PRRSV-induced alterations on Marc-145 cell function. Further study of the affected proteins may facilitate our understanding of the mechanisms of PRRSV infection and pathogenesis.     

福美双对肉仔鸡肝功能和胫骨软骨发育不良的影响

研究了福美双对肉鸡胫骨软骨发育不良、血清相关酶活性、肝脏SOD、MDA及增重的影响。120只1日龄AA肉仔鸡随机分为3组，对照组、低浓度福美双组（50mg／kg）、高浓度福美双组（100mg／kg）。每组40只鸡，每组4个重复，每个重复10只鸡，试验进行28d。结果表明，饲料中添加福美双可以显著提高肉鸡TD发病率（P〈0．05），升高血清中AST、ALT和ALP的水平和肝脏MDA的含量（P〈0．05），降低肝脏SOD水平和肉鸡的生产性能。福美双可能通过改变肝脏的抗氧化状态，损害肉仔鸡的肝脏功能，影响骨代谢的相关酶活性，导致肉仔鸡胫骨软骨发育不良的发生。     

Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate

Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.     

Evaluation of the efficacy of vitamin D3 or its metabolites on thiram-induced tibial dyschondroplasia in chickens

Two trials were conducted to determine if thiram-induced tibial dyschondroplasia (TD) in chickens was linked to a vitamin D deficiency and calcium homeostasis dysregulation, and whether feeding vitamin D fortified diets may prevent it. Day-old chickens were given grower diets containing different vitamin D products throughout the experiment until necropsy on day 16. Half of the birds in each feed group received thiram at levels of 100 ppm (trial 1) or 50 ppm (trial 2) between days 7-9 to induce TD. The birds were weighed, bled, and euthanized to determine TD incidences and severity by examining the growth plates. Tibial bones were used to measure biomechanical strength and ash content. Blood concentrations of 25-hydroxyvitamin D, Ca, P, alkaline phosphatase, and creatine kinase were measured in serum that showed no differences between different groups. Thiram reduced body weight and induced TD regardless of any vitamin D treatment to the same extent as untreated birds.     

Effect of diet with thiram on liver antioxidant capacity and tibial dyschondroplasia in broilers

1. An experiment was conducted to study the effect of thiram on liver antioxidant capacity and incidence of tibial dyschondroplasia in broilers. 2. One hundred and twenty Avian commercial broilers were allotted at random to three treatments: control group, low thiram group (50 mg/kg) and high thiram group (100 mg/kg). 3. Blood samples were collected to determine the activity of AST (aspartate aminotransferase). At the end of the trial, broilers were killed and liver samples were collected to determine the activity of SOD (superoxide dismutase), GSH-Px (glutathione peroxidase) and MDA (malondialdehyde) content, while the right proximal tibiotarsi were dissected in longitudinal section for assessment of tibial dyschondroplasia (TD) incidence and TD score. 4. The results showed that thiram increased the incidence of TD and TD scores, increased serum AST activity and MDA content of liver, and decreased the activity of SOD and GSH-Px in the liver. 5. They suggest that thiram causes TD in broilers by reducing liver antioxidation capability and damaging liver function; this may be one of the mechanisms by which thiram causes TD in broilers.     

福美双对肉鸡生产性能、血清相关酶活性及胫骨软骨发育不良的影响

将120只1日龄AA肉鸡随机分为对照组、低浓度福美双组（50mg／kg福美双）、高浓度福美双组（100mg／kg福美双），试验进行28d。结果显示，饲料中添加福美双显著升高了血清AST、ALT和ALP水平（P〈0．05），提高了肉鸡胫骨软骨发育不良（TD）的发病率（P〈O．05），降低了肉鸡生产性能。表明福美双能通过损害肉鸡肝脏功能，影响骨代谢相关酶活性，而致发肉鸡TD。     

牦牛卵泡液差异蛋白质组双向电泳图谱的构建与质谱分析

从蛋白质水平了解牦牛季节性繁殖规律,利用双向电泳与质谱鉴定技术分析牦牛卵泡液与血浆蛋白质组分变化。以青海高原牦牛卵泡液与血浆为研究对象,采用双向电泳技术构建牦牛卵泡液与血浆蛋白质双向电泳图谱,银染后利用Image Master 2D Platinum软件分析并采用MALDI-TOF-MS进行质谱鉴定。用试剂盒ProteoExtract Albumin/IgG Removal Kit去除高丰度蛋白质后,利用2-DE技术获得了分辨率较高的卵泡液与血浆蛋白质电泳图谱,卵泡液与血浆蛋白质图谱对比分析共发现了24个差异表达蛋白质点,其中2个蛋白质点表达上调,22个蛋白质点表达下调。经质谱分析,最终成功鉴定出8个蛋白质点、5个未知蛋白质点。本研究成功构建了蛋白质图谱及分离鉴定的差异蛋白质,为从蛋白质水平揭示牦牛卵泡发育规律及了解卵母细胞发育的微环境提供了试验依据。     

Study on Skin Differential Expression Proteins of Tan Sheep Within One Year Old

In order to study different expressed proteins of one-year-old Tan sheep skin, two-dimensional gel electrophoresis (2-DE) was used to separate the Tan sheep skin proteins of different growth stages by Coomassie blue staining, with PDQuest 8.0 software to detect and analyze differences in protein spots,and were identified by MALDITOF/TOF-MS/MS.Protein maps of four growth stages were performed between pairwise, after testing found a total of 19 differentially expressed proteins, mass spectrometry successfully identified 15 protein spots and a total of 13 proteins, of which 14-3-3 σ protein isoforms, annexin A2, keratin 25 and tubulin α chain might be related to growth regulation sheep skin. Those different expressed proteins might be associated with different stages of growth of Tan sheep skin follicles cyclical changes. The different proteins of one-year-old Tan sheep skin provided theoretical basis for annual variation of Tan sheep skin.     

一周岁内滩羊皮肤差异表达蛋白研究

为研究一周岁内滩羊皮肤中的差异表达蛋白,本试验利用双向凝胶电泳（2-DE）技术分离了滩羊不同生长阶段皮肤蛋白,经考马斯亮蓝染色,用PDQuest 8.0软件检测并分析差异蛋白点,并经MALDITOF/TOF-MS/MS鉴定。对4个生长时期的蛋白图谱进行两两比对,共发现了19个差异蛋白点,质谱成功鉴定出15个蛋白点共13种蛋白,可能与滩羊皮肤生长调控有关的蛋白有14-3-3蛋白σ亚型（14-3-3 protein sigma）、膜联蛋白A2（annexin A2）、角蛋白25（keratin 25）、微管蛋白α链（tubulin alpha chain）。对差异表达蛋白进行分析,发现这些差异表达蛋白可能与滩羊皮肤不同生长阶段的毛囊周期性变化有关。一周岁内滩羊皮肤差异蛋白的发现为滩羊皮肤的年变化规律研究奠定理论基础。     

牛冻、鲜精子差异蛋白的双向电泳和质谱鉴定的初步研究

采用适合于精子裂解的热Trizol法制备牛精子全蛋白质,应用线性固相IPG胶条（pH 3～7、24 cm）进行精子全蛋白2-DE电泳,在冻精和鲜精蛋白图谱中分别检测到839±34个蛋白点和564±16个蛋白点,经差异比较和显著性检验,在冻、鲜精子中共得到19个具有显著差异的蛋白点,在冻精中表达上调的蛋白点有9个,表达下调的蛋白点有1个,只在冻精中表达的蛋白点有9个。经过酶解和质谱鉴定,获得了3个差异蛋白点的质谱信息,生物信息学分析结果发现,这3个差异点分别为中性鞘磷脂酶激活结合因子（FAN）、谷胱甘肽S转移酶（GST-Mu5）及锌离子结合细胞色素氧化酶,分别与细胞应激和凋亡有关。推测认为,这3种与细胞应激反应及凋亡相关的蛋白在冻精中显著增高可能与精子冷冻损伤有关。     

A gel-based reference map of the porcine hepatocyte proteome

The overall goal of our research is to characterize and identify gene expression profiles of porcine hepatic cells. In this study, we have prepared two-dimensional electrophoresis maps of cytosol and membrane fractions from freshly prepared hepatocytes which were pooled from three crossbred pigs (35-69kg). Following isoelectric focusing with three pH range immobilized pH gradient strips (pH 3-6, 5-8 and 7-10) and staining the second dimension gels with colloidal Coomassie blue, 728 protein spots were picked and digested with trypsin. Extracted tryptic peptides were initially subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis for identification of proteins by peptide mass fingerprinting (PMF). Proteins which were not identified by PMF were analyzed by liquid chromatography-tandem MS. Utilizing publicly available databases [NCBInr, Swiss Prot and expressed sequence tags (EST)], 648 proteins were identified. Of those, 282 were unique proteins and greater than 90% of proteins spots contained single proteins. These data represent the first comprehensive proteomic analysis of porcine hepatocytes and will provide a database for future investigations of endocrine regulation of gene expression and metabolic processes in vitro.     

Identification of porcine serum proteins modified in response to HP-PRRSV HuN4 infection by two-dimensional differential gel electrophoresis

Since 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has become the major pathogen attributed to the prevalent porcine reproductive and respiratory syndrome (PRRS) in China. The present study aims to identify serum proteins modified in response to infection of HuN4, a HP-PRRSV strain isolated from a farm in 2006. 2-D DIGE analysis allowed for the detection of 19 differentially expressed protein spots, of which 18 were identified by MALDI-TOF/TOF MS. These 18 spots represented for a total of 9 proteins (6 up-regulated and 3 down-regulated), most of which belonged to the acute phase proteins in swine and showed a trend of regression in the late phase of the experiment. One of a series of AGP spots was identified for the first time to be decreased in acute phase of PRRSV infection in swine. But the whole level of the protein in the serum did not show significant changes by Western blot. The rising tendency of Hp was confirmed by Western blot and ELISA. These altered proteins were probably involved in the inflammatory process triggered by HuN4 and in alleviating the oxidative damage occurring in the process. In summary, these results may provide new insights into understanding the mechanisms of HP-PRRSV infection.     

柞蚕胚胎发育期蛋白质的2D-PAGE图谱分析

采用SDS-PAGE和2D-PAGE技术分析柞蚕胚胎期不同发育阶段的蛋白质组成和含量变化,为从蛋白质水平探讨柞蚕胚胎发育过程中的基因顺序表达模式积累基础信息。SDS-PAGE电泳结果显示:在柞蚕胚胎的发育过程中共分离到相对明显的32条蛋白带,包括分子质量约70、60、30 kD的高丰度蛋白条带,随着胚胎的发育,这些蛋白质含量逐渐减少,而40、15 kD左右的蛋白质含量逐渐增加,到孵化成蚁蚕(324 h)时,分子质量约70、60 kD的蛋白条带基本消失。进一步分析柞蚕胚胎发育不同时期蛋白质的2D-PAGE图谱:蛋白点数量随胚胎发育逐渐增多,胚胎发育12 h检测到370个蛋白点,胚胎发育300 h的蛋白点达到422个;胚胎发育132 h及276 h的蛋白点与胚胎发育早期36 h蛋白点的匹配率分别为52.4%和28.4%。此外,孵化蚁蚕总蛋白2D-PAGE图谱与胚胎期相比存在较大差异。研究结果提示,柞蚕胚胎发育前期,从胚胎形成期到附肢发生期(12～60 h),蛋白质种类相对较少,且变化不大;胚胎发育的气管形成期(228～276 h)开始,蛋白质种类变化剧烈,偏酸性蛋白质增加,蛋白点的匹配率下降。     

Comparative proteomic analysis of Salmonella enterica serovars Enteritidis, Typhimurium and Gallinarum

Salmonella enterica includes several related serovars which have different host ranges and cause diseases of different severities. However, their pathogenic potential is unknown, and it is not clear what mechanisms are activated or inhibited during adaptation to a specific host environment. Some proteins are involved in the mechanism of pathogenicity at a molecular level and provide the functional aspects that create the diverse phenotypes. To compare proteomic analyses of the total proteins of Salmonella Enteriditis (SE), Typhimurium (ST), and Gallinarum (SG), two-dimensional gel electrophoresis (2-DGE) was performed using a pH 4-10 immobilized pH gradient (IPG) strip, and some proteins were identified by mass spectrometry (MS). After staining the gels, the proteins that were expressed at 10-fold or higher levels compared to other spots on the gel were characterized. Some of the identified proteins were related to virulence, such as β-lactamase, RfbH protein, and shikimate kinase. Additionally, there was a high level of variation between serovars despite the similarities in the expression patterns. Furthermore, this study shows that 2-DGE combined with MS is a useful tool for identifying proteins differentially expressed between serovars with different host ranges and pathogenic potential.