鸡脾脏组织抑制消减cDNA文库构建及其差异表达基因的分析

为分离与鸡抗病性密切相关的差异表达基因,并进行功能分析,利用抑制性消减杂交技术,以大骨鸡和海兰褐商品代蛋鸡20周龄时的脾脏组织为试验材料构建消减cDNA文库。文库的插入片段集中在600 bp左右,挑取760个克隆进行PCR筛选获得663个阳性克隆,经过点杂交筛选后选择了531个阳性克隆,从中随机挑取100个阳性克隆进行测序。经过同源性比对归并后得到37个差异表达基因或ESTs序列,其中,32个是已知基因,包括一般抗病性或免疫性能、特异抗病相关基因、细胞信号分子、膜蛋白质、转录因子等差异表达基因;5个为功能尚未确定的基因。并对4个可能影响鸡抗病性能的差异表达基因进行了RT-PCR半定量检测鉴定。该试验结果为进一步研究这些差异表达基因在抗病过程中的重要功能及其调控作用机理奠定了基础。     

羊草叶片cDNA文库的构建及部分表达序列标签的分析

采用SMART技术,以品质优良羊草“吉生一号”叶片为材料,构建了高质量cDNA文库。原始文库滴度达到10⁶cfu/mL,扩增文库滴度接近10¹¹cfu/mL。随机抽样检查结果表明,插入片断大小在0.5~3.0kb,主要集中在1kb左右,其中检测到插入片断大于1kb的占70%,最大达到2.5kb,其重组率达到98%。同时在扩增文库中检测到了羊草维生素E合成途径中的关键酶基因α-生育酚环化酶(TC)及γ-生育酚甲基转移酶(γ-TMT)的特异信号,挑选307个筛选出了285条EST序列,将得到的117条非重复序列与GenBank中已知序列比对,获得了如3-磷酸甘油醛脱氢酶、光系统Ⅱ蛋白D1、翻译起始因子蛋白、翻译延生因子蛋白、RNaseS-likeproteinprecursor蛋白等基因。羊草高质量的cDNA文库的构建为进一步从分子水平研究羊草及开发利用这一基因资源提供了条件。     

Gateway技术构建结缕草低温和干旱诱导cDNA文库及其质量鉴定

为研究结缕草（Zoysia japonica）胁迫响应信号转导途径中的关键基因,揭示结缕草抗逆分子机制,建立结缕草功能基因组学研究基础平台,以经低温、干旱处理的结缕草为材料,采用Gateway技术构建了首个结缕草低温和干旱诱导的标准cDNA文库。文库质量分析表明,未扩增的原始文库滴度1.76×106 pfu·mL-1,库容7.04×106 pfu,插入片段平均大小大于1 kb,重组率90%。文库质量优良,可能包含大量新基因,不仅能为结缕草功能基因组分析提供必要资源,也可为后期进行高通量EST测序、发掘新抗逆相关基因、制作基因芯片等研究奠定基础。     

Analysis of blood flow in a third ventricular ependymoma and an olfactory bulb meningioma by using perfusion computed tomography

Brain perfusion computed tomography (CT) scanning was performed in a mongrel dog and a golden retriever that were diagnosed with third ventricular tumor and olfactory bulb tumor, respectively, by contrast-enhanced CT. The tumors were pathologically diagnosed as ependymoma and meningioma, respectively. Perfusion CT results revealed that the ependymoma in this study had a lower blood flow, higher blood volume, and greater transit time of blood than the adjacent brain tissue. Further, the meningioma in this study had a higher blood flow, higher blood volume, and greater transit time of blood than the adjacent brain tissue. Perfusion CT can potentially be used for the grading of brain tumors and narrowing differential diagnosis, provided the perfusion CT data of animals are accumulated.     

Analysis of antibiotic resistance phenotypes and genes of Escherichia coli from healthy swine in Guizhou,China

This study was carried out to investigate the resistance phenotypes and resistance genes of Escherichia coli from swine in Guizhou, China. A total of 47 E. coli strains isolated between 2013 and 2018 were tested using the Kirby–Bauer (K–B) method to verify their resistance to 19 common clinical antimicrobials. Five classes consisting of 29 resistance genes were detected using polymerase chain reaction. The status regarding extended-spectrum β-lactamase (ESBL) and the relationship between ESBL CTX-M-type β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) genes were analysed. A total of 46 strains (97.9%) were found to be multidrug resistant. Amongst them, 27 strains (57.4%) were resistant to more than eight antimicrobials, and the maximum number of resistant antimicrobial agents was 16. Twenty antibiotic resistance genes were detected, including six β-lactamase genes blaTEM (74.5%), blaCTX-M-9G (29.8%), blaDHA (17.0%), blaCTX-M-1G (10.6%), blaSHV (8.5%), blaOXA (2.1%), five aminoglycoside-modifying enzyme genes aac(3′)-IV (93.6%), aadA1 (78.7%), aadA2 (76.6%), aac(3′)-II c (55.3%), aac(6′)-Ib (2.1%) and five amphenicol resistance genes floR (70.2%), cmlA (53.2%), cat2 (10.6%), cat1 (6.4%), cmlB (2.1%), three PMQR genes qnrS (55.3%), oqxA (53.2%), qepA (27.7%) and polypeptide resistance gene mcr-1 (40.4%). The detection rate of ESBL-positive strains was 80.9% (38/47) and ESBL TEM-type was the most abundant ESBLs. The percentage of the PMQR gene in blaCTX-M-positive strains was high, and the detection rate of blaCTX-M-9G was the highest in CTX-M type. It is clear that multiple drug resistant E. coli is common in healthy swine in this study. Extended-spectrum β-lactamase is very abundant in the E. coli strains isolated from swine and most of them are multiple compound genotypes.     

Isolation from turkey breeder hens of a reassortant H1N2 influenza virus with swine, human, and avian lineage genes

Type A influenza viruses can infect a wide range of birds and mammals, but influenza in a particular species is usually considered to be species specific. However, infection of turkeys with swine H1N1 viruses has been documented on several occasions. This report documents the isolation of an H1N2 influenza virus from a turkey breeder flock with a sudden drop in egg production. Sequence analysis of the virus showed that it was a complex reassortant virus with a mix of swine-, human-, and avian-origin influenza genes. A swine influenza virus with a similar gene complement was recently reported from pigs in Indiana. Isolation and identification of the virus required the use of nonconventional diagnostic procedures. The virus was isolated in embryonated chicken eggs by the yolk sac route of inoculation rather than by the typical chorioallantoic sac route. Interpretation of hemagglutination-inhibition test results required the use of turkey rather than chicken red blood cells, and identification of the neuraminidase subtype required the use of alternative reference sera in the neuraminidase-inhibition test. This report provides additional evidence that influenza viruses can cross species and cause a disease outbreak, and diagnosticians must be aware that the variability of influenza viruses can complicate the isolation and characterization of new isolates.     

我国2株野生动物源细小病毒VP2和NS1基因序列及所编码蛋白的分析

为了解我国野生动物源细小病毒VP2、NS1基因序列和进化特点,用PCR方法获得貉源细小病毒CR86106和猴源细小病毒BJ-22的目的基因片段,对其核苷酸和氨基酸序列进行测定分析.结果显示CR86106和BJ-22细小病毒基因组长均为4 269 nt,其中NS1基因全长2 007 nt,共编码668个氨基酸;VP2基因全长1 755 nt,共编码584个氨基酸.CR86106 VP2蛋白除第300位氨基酸为脯氨酸(P)以外,其余关键氨基酸位点均与猫泛白细胞减少症病毒(feline panleukopenia virus,FPLV)一致;BJ-22 VP2蛋白除第323位为天冬酰胺(N)、第564位为丝氨酸(S)以外,其余关键氨基酸位点均与FPLV一致.CR86106 NS1蛋白氨基酸序列与细小病毒参考毒株的相似性是98.4％～ 99.3％,VP2是97.6％～99.7％;BJ-22 NS1蛋白氨基酸序列与细小病毒参考毒株的相似性是98.5％～99.4％,VP2是98.1％～99.3％.种系发生分析结果显示,CR86106 VP2和NS1与FPLV亲缘关系较近;BJ-22NS1归到CPV分支,VP2归到FPLV分支且单独分在一支.结果表明,CR86106具有FPLV样细小病毒序列特征,BJ-22具有FPLV和CPV重组病毒特征,为猴源细小病毒的重组现象,研究结果同时也证实了基因重组在FPLV进化过程中起重要作用的推论.     

旋毛虫新生幼虫p46 000抗原基因的克隆及序列分析

应用旋毛虫感染猪血清，对旋毛虫新生幼虫cDNA文库进行了免疫筛选。对阳性克隆pBK-cMV-WN10的序列分析结果表明。cDNA全长为1352bp。含有1个1218bp的完整的开放阅读框架(ORF)，编码的多肽由406个氨基酸残基组成，其相对分子质量理论推导值为45900，等电点为5．43，N末端的信号肽及糖基化位点(NCS)表明其可能为分泌性糖蛋白，氨基酸序列19～156与158～295为重复区域，相似性为74％．C末端有1个半胱氨酸蛋白酶抑制剂结构域，但旋毛虫p46000抗原与其他线虫的半胱氨酸蛋白酶抑制蛋白结构有很大差异，可能已经失去半胱氨酸蛋白酶抑制蛋白的功能。PCR结果显示。从旋毛虫新生幼虫、肌幼虫、3日龄成虫和5日龄成虫cDNA中均扩增出此基因，表明此基因在旋毛虫各个时期均有表达。     

犬弓首蛔虫雄虫cDNA文库构建及EST分析

为构建T.canis雄虫cDNA文库,采用Trizol法提取T.canis雄虫的总RNA,合成cDNA,连接到λTripEx2载体上,通过包装蛋白对连接产物的包装,接种到大肠杆菌XL-1-Blue中进行原始文库和扩增文库的滴度测定.经质量鉴定表明:初始文库的滴度为5.25×106 pfu·mL-1,扩增后文库的滴度为6.90×109 pfu ·mL-1.文库的插入片段大小在500～2 000 bp,平均片段大小为1 000 bp,重组率为99.47％.所有指标均显示已成功构建了T.canis雄虫的cDNA文库.利用该文库获得了189条5'有效表达序列标签(EST).对ESTs拼接后代表了101个Unigenes,含有27个Contigs和74个Singletons.其Unigenes在GenBank中的序列号为HO348195～HO348295.同源性分析检索到有56个Unigenes与已知基因同源,其中具有已知或推测功能的基因有40个,未知功能基因有16个,未比对上的基因45个.未比对上的基因与NR数据库中的蛋白序列没有任何意义的匹配,为研究中发现的新基因.这些结果为进一步开展犬弓首蛔虫功能基因及分子机制研究奠定了基础.     

Characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation

A low-density cell population was isolated from skin explants of pigs and characterized as a highly enriched dendritic cell (DC) population based on phenotypical and functional properties. The skin-derived DCs were identified by their characteristic ultrastructural properties as well as by consistent co-expression of the CD1 and SWC3a antigens that clearly differentiate them from other porcine leukocytes. These cells exhibit higher expression of porcine MHC class II (SLAII) and CD80/86 antigens as compared to macrophage/monocyte cells. They consistently expressed the S100 beta antigen at high levels and did not express the lymphoid markers CD3, CD4 or CD8. Within this population of skin-derived DCs there was variable expression of CD11c, CD14 and CD16. Functional characterization of this DC population revealed that they are efficient in uptake and processing of soluble protein antigens and in endocytosis of small (0.02 microm) but not large (2 microm) polystyrene beads. Further, these cells were efficient inducers of primary allogeneic responses and in stimulating antigen-specific and mitogen-induced proliferation and IFN gamma responses in autologous lymphocytes. This study provides important information to further characterize the cutaneous DCs and develop models to analyze the role of these cells in immune responses in vivo.     

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.     

Descriptive analysis and spatial epidemiology of porcine reproductive and respiratory syndrome (PRRS) for swine sites participating in area regional control and elimination programs from 3 regions of Ontario

The objectives of this study were to describe demographics, basic biosecurity practices, ownership structure, and prevalence of porcine reproductive and respiratory syndrome (PRRS) in swine sites located in 3 regions in Ontario, and investigate the presence of spatial clustering and clusters of PRRS positive sites in the 3 regions. A total of 370 swine sites were enrolled in Area Regional Control and Elimination projects in Niagara, Watford, and Perth from 2010 to 2013. Demographics, biosecurity, and site ownership data were collected using a standardized questionnaire and site locations were obtained from an industry organization. Status was assigned on the basis of available diagnostic tests and/or assessment by site veterinarians. Spatial dependence was investigated using the D-function, the spatial scan statistic test and the spatial relative risk method. Results showed that the use of strict all-in all-out (AIAO) pig flow and shower before entry are uncommon biosecurity practices in swine sites, but a larger proportion of sites reported having a Danish entry. The prevalence of PRRS in the 3 regions ranged from 17% to 48% and localized high and low risk clusters were detected. Sites enrolled in the PRRS control projects were characterized by membership in multiple and overlapping ownership structures and networks, which complicates the way the results of monitoring and disease management measures are communicated to the target population.     

彭泽鲫葡萄糖激酶基因全长cDNA克隆及表达分析

本试验采用RT-PCR和cDNA末端快速扩增(RACE)方法克隆获得了彭泽鲫(Carassius auratus var.Pengze)葡萄糖激酶(GK)基因全长cDNA序列.结果表明,该cDNA全长2 050 bp,含1个1 43l bp的开放阅读框(ORF),编码476个氨基酸,GK蛋白计算分子量为53.78 ku...     

Evaluation of a bone biopsy technique for determining the calcium and phosphorus status of swine from weaning to market weight

Tibia biopsies were taken from 75 live pigs at 4-wk intervals and from 251 slaughtered pigs to evaluate bone biopsy as a procedure for determining Ca/P status in pigs fed 70, 85, 100, 115 and 130% of the NRC (1979) estimated dietary Ca and P percentage requirements from weaning to market. Least squares means and SE of live and slaughter biopsy wet weight, ash weight and dry, fat-free ash percentage (DFF%) were compared at each time in each trial and found not to differ. Diet and time effects on ash weight, ash percentage of wet weight and DFF% of the biopsy core also did not differ greatly between slaughter and live biopsies and generally responded linearly and quadratically (P less than .01) to increasing Ca/P level and time. Biopsy measures were correlated (P less than .05) with third and fourth metacarpal and metatarsal length, bending and shear stress and DFF%. Means for slaughter biopsy DFF% did not differ greatly from the average of third and fourth metacarpal and metatarsal DFF% from slaughter pigs. Means for live and slaughter biopsy DFF% were lower than those for whole bones for the 70 and 85% NRC estimated Ca/P levels, but not for the 100, 115 and 130% NRC levels. Bone biopsy offers potential as a reliable noninvasive procedure for monitoring Ca/P status of swine from weaning to market, but it needs further study for use in Ca/P research in swine.     

Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.     

家蚕中肠cDNA T7噬菌体展示文库的构建和免疫相关基因的淘选

中肠是昆虫重要的免疫防御器官之一。为探究家蚕中肠的分子免疫机制,尤其是针对不同病原体的模式识别分子或结合蛋白,以家蚕5龄第3天幼虫中肠为材料,构建家蚕中肠cDNA T7噬菌体展示文库,并分别以几丁质和脂多糖为配体,通过不同方法进行文库的生物淘选。结果共获得10个基因片段,其中在以脂多糖为配体的淘选中获得了β-1,3葡聚糖识别蛋白4,该分子是已知的模式识别受体。另外还获得了多个免疫相关候选基因,如分别编码几丁质结合蛋白、翻译控制的肿瘤蛋白、氨肽酶N、脂肪酰辅酶A结合蛋白等的几个基因。研究结果为深入开展家蚕中肠免疫功能的研究提供了重要线索。