猪3种重要病毒寡核苷酸芯片诊断方法的建立

将猪细小病毒(PPV)、猪圆环病毒-Ⅱ(PCV-2)、猪瘟病毒(CSFV)分别应用生物信息学方法,针对病毒基因组保守序列设计特异性强的60-mer寡核苷酸探针,并将其按所设计阵列固定于表面经氨基化修饰的玻片上,制备出寡核苷酸芯片.分别设计出相应的引物,对待测样本进行不对称PCR扩增,从而产生大量可与寡核苷酸探针特异性互补的单链DNA片段,并通过间接荧光标记技术使扩增产物标记上荧光染料.将标有荧光染料的扩增产物与芯片上寡核苷酸探针杂交.扫描、分析芯片上荧光信号.试验结果表明,芯片上各样本对应探针位点呈现阳性荧光信号.而阴性对照和空白对照则基本不能检测到荧光信号.不对称PCR技术制备的单链DNA片段与寡核苷酸芯片进行杂交反应可同时、快速、特异性地检测多种猪疫病病毒.     

猪瘟病毒基因分型寡核苷酸芯片方法的建立

根据猪瘟病毒（CSFV）E2基因序列,设计41条针对CSFV 3个基因群共10个亚群各亚群寡核苷酸探针。利用欧盟猪瘟诊断手册推荐的CSFV E2基因套式RT-PCR方法,在内套PCR过程中进行Cy3-dCTP掺入荧光标记,制备芯片杂交样品。用标记的PCR产物与寡核苷酸探针阵列杂交,置于GenePix 4100A扫描仪中扫描,利用Ge-nePix Pro 6.0软件分析杂交图像。特异性和灵敏度试验显示,芯片方法与本室发表的CSFV real-time RT-PCR方法的灵敏度相近,芯片探针与猪繁殖与呼吸综合征病毒（PRRSV）、猪2型圆环病毒（PCV2）、猪伪狂犬病毒（PRV）样品无非特异性杂交。以包括1.1、2.1、2.2、2.3亚群的8份CSF阳性样品进行芯片的检测验证,结果表明,通过特异性的杂交图谱或杂交信号分析可准确判定样品所属的基因亚群,寡核苷酸芯片的检测结果与测序的分型结果全部符合。本研究为将寡核苷酸芯片技术用于猪瘟病毒的基因分型和分子流行病学研究奠定了基础。     

猪瘟、猪圆环病毒病和猪繁殖与呼吸综合征寡核苷酸芯片诊断方法的建立与初步应用

猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）和猪圆环病毒Ⅱ型（PCV-2）是引发猪繁殖障碍的主要病原,建立其基因芯片快速检测体系,对开发猪繁殖障碍快速检测试剂盒具有重要意义。根据GenBank中已发表的PRRSV、CSFV和PCV-2的病毒基因组序列,设计合成特异性引物和特异性较强的60mer的寡核苷酸探针,并将探针按所设计阵列固定于表面经氨基化修饰的玻片上,制备出寡核苷酸芯片。通过引物标记及特异性验证,建立了带标记引物的多重PCR检测体系,从而产生大量可与寡核苷酸探针特异性互补的带标记的DNA片段。将标有荧光染料的扩增产物与芯片上寡核苷酸探针杂交,扫描、分析芯片上荧光信号。结果表明,芯片上各样本对应探针位点呈现阳性荧光信号,而阴性对照和空白对照则基本不能检测到荧光信号。分别用基因芯片检测方法和PCR/RT-PCR检测60份临床病料,两者符合率高达92%,表明该检测技术能够用于临床病料的检测及快速诊断PRRSV、CSFV和PCV-2。     

猪病毒性腹泻检测基因芯片的两种样品标记技术比较

对猪病毒性腹泻检测芯片的两种样品标记方法进行比较。分别选取猪流行性腹泻病毒(PEDV)的S和M基因,猪传染性胃肠炎病毒(TGEV)的N和S基因,A型猪轮状病毒(GAR)的VP7和NSP4基因设计引物,用PCR扩增制备靶基因,纯化后制备猪病毒性腹泻联合检测基因芯片。提取样品核酸,采用Cy3直接标记法和间接标记法进行PCR扩增标记,标记的样品再与芯片杂交、扫描和数据分析,同时对两种标记方法的特异性、敏感性等进行了比较。结果表明,两种方法标记的样品均能与基因芯片特异性杂交,但直接法的中位信号值(median)均高于间接法的中位信号值,直接法的芯片杂交信噪比是用间接法的5倍以上。两种标记方法制备样品特异性好,猪蓝耳病毒、猪瘟病毒、猪乙型脑炎病毒、猪伪狂犬病毒验证检测为阴性;直接法和间接法的最低检测浓度分别为10~5和10~7 copies·μL~(-1),前者的灵敏性是后者的100倍。应用优化的直接标记法处理56份临床样品,进行芯片的检测应用,结果与RT-PCR一致。本研究结果表明直接法荧光标记样品可明显提高病毒性腹泻检测芯片的检测效果。     

猪细小病毒检测基因芯片的构建及初步应用

本研究以猪细小病毒VP2基因为目的基因设计引物和探针,通过不对称PCR扩增Cy3标记的DNA片段与固定于芯片上的探针进行杂交,对杂交芯片进行扫描分析,根据荧光信号的强度来确定是否存在猪细小病毒。结果表明,采用浓度为5μmol/L的探针与PCR产物于47℃杂交1 h即可得到清晰的荧光信号,检测灵敏度可达34.5 ng/μL,同时用制备的基因芯片对临床20份疑似猪细小病毒病感染的病料进行检测,检测结果与PCR检测结果符合率达100%,表明基因芯片检侧技术是一种灵敏度高、特异好的检侧方法。该方法的建立可以快速有效地对猪细小病毒做出诊断,具有较好的应用前景。     

猪瘟病毒和猪细小病毒检测基因芯片的构建

本试验克隆猪瘟病毒(Classical swine fever virus,CSFV)和猪细小病毒(Porcine parvovirus,PV)各自病毒基因保守序列,提取质粒中的模板、扩增纯化作为探针,应用微量点样技术将探针固定在硝酸纤维素膜上,制备诊断基因芯片;同时对制备的基因芯片进行有效性监控和特异性、重复性的质量控制;然后提取样品核酸,PCR扩增并用生物素标记,并将所获得的扩增产物与诊断基因芯片进行特异性的逆向点杂交,最后通过芯片扫描来实现对病原的高效检测和分析判断。试验结果表明病料中抽提的核酸与芯片杂交的信号为阳性且较明显,说明制备的猪瘟病毒和猪细小病毒检测基因芯片有效性监控正常且两种病原的特异性和重复性均良好。     

皮毛中5种病毒基因芯片检测方法的研究

根据GenBank上蓝舌病病毒(BTV)、O型口蹄疫病毒(FMDV)、山羊痘病毒(GPV)、绵羊痘病毒(SPPV)和牛病毒性腹泻病毒(BVDV)等5种病毒的特异性保守序列,分别设计多重荧光标记引物和相应寡核苷酸探针.使用芯片点样液稀释各探针至终浓度30μmol/L,点样制备11×11阵列芯片.核酸杂交后,建立并优化基因芯片检测方法.结果显示,使用470 mL/L甲酰胺杂交液,42℃摇转杂交4h为最佳基因芯片杂交条件.建立的基因芯片检测方法与伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、禽流感病毒(AIV)和新城疫病毒(NDV)等无交叉反应,检测敏感性可达20拷贝病毒核酸.制备的基因芯片稳定,保存6个月可用.对151份临床样品进行基因芯片和商业化PCR试剂盒平行检测,两者的符合率为100％.     

耐氟喹诺酮类病原菌gyrA基因突变的寡核苷酸芯片检测

根据大肠杆菌、沙门菌、无乳链球菌和鸡毒霉形体的gyrA基因序列，设计了通用引物和ll条寡核苷酸探针；利用点样仪将探针点在基片上，制成寡核苷酸芯片；采用PCR荧光标记靶基因，与芯片杂交，用荧光扫描仪检测信号；同时以PCR一测序法进行gyrA基因突变的检测。结果，PCR反应体系能特异性地扩增出靶基因；寡核苷酸芯片能同时检测不同病原菌GyrA第83、87位发生的突变，芯片检测结果与测序结果较为一致。结果表明，使用寡核苷酸芯片技术检测病原菌耐氟喹诺酮类基因突变是可行的；研究结果为基因芯片技术应用于兽医临床耐药性检测提供了基础。     

几种动物病毒的基因芯片检测技术

分别用水疱性口炎病毒、蓝舌病病毒、口蹄疫病毒、猪瘟病毒、牛病毒性腹泻病毒、鹿流行性出血热病毒和赤羽病病毒各一段高度保守的基因片段构建质粒，在此基础上制备了芯片探针。提取样品中的核酸，经反转录和荧光标记后滴加到芯片上进行特异性杂交，对杂交结果扫描检测，可同时对上述7种动物传染病进行快速、准确的诊断，此方法敏感性高，特异性强，适合于大批动物高通量检疫。     

多重PCR结合基因芯片技术检测5种猪繁殖障碍性病毒病方法的研究

为建立运用多重PCR和基因芯片技术同时检测5种猪繁殖障碍性病毒病的方法。本研究根据GenBank中登录的猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪日本乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的基因序列设计特异性引物与探针,制备相应的寡核苷酸芯片,检测了5种猪繁殖障碍性疾病病毒的标准毒株,并对16份临床样品进行检测。通过多重PCR扩增出带有荧光标记的5种病毒的特异性基因片段,并与固定有特异性探针的基因芯片杂交。结果显示,本研究建立的多重PCR结合基因芯片检测方法特异性强、稳定性好,灵敏度可达10~2拷贝/μL。16份临床样品检测结果显示阳性率达87.5%(14/16)。以上结果表明该方法特异性好、灵敏度高,可高效检测以上5种病毒,为其临床诊断及流行病学调查提供了有效的检测方法。     

Concentrations of strontium,barium, cadmium,copper, zinc,manganese, chromium,antimony, selenium,and lead in the liver and kidneys of dogs according to age,gender, and the occurrence of chronic kidney disease

This study was conducted to measure the concentrations of strontium (Sr), barium (Ba), cadmium (Cd), copper (Cu), zinc (Zn), manganese (Mn), chromium (Cr), antimony (Sb), selenium (Se), and lead (Pb) in canine liver, renal cortex, and renal medulla, and the association of these concentrations with age, gender, and occurrence of chronic kidney disease (CKD). Tissues from 50 dogs were analyzed using inductively coupled plasma mass spectrometry. Cu, Zn, and Mn levels were highest in the liver followed by the renal cortex and renal medulla. The highest Sr, Cd, and Se concentrations were measured in the renal cortex while lower levels were found in the renal medulla and liver. Female dogs had higher tissue concentrations of Sr (liver and renal medulla), Cd (liver), Zn (liver and renal cortex), Cr (liver, renal cortex, and renal medulla), and Pb (liver) than male animals. Except for Mn and Sb, age-dependent variations were observed for all element concentrations in the canine tissues. Hepatic Cd and Cr concentrations were higher in dogs with CKD. In conclusion, the present results provide new knowledge about the storage of specific elements in canine liver and kidneys, and can be considered important reference data for diagnostic methods and further investigations.     

Comparisons of Angus, Charolais, Galloway, Hereford, Longhorn, Nellore, Piedmontese, Salers, and Shorthorn breeds for weight, weight adjusted for condition score, height, and condition score of cows

Breed differences for weight (CW), height (CH), and condition score (CS) were estimated from records (n = 12,188) of 2- to 6-yr-old cows (n = 744) from Cycle IV of the U.S. Meat Animal Research Center's Germplasm Evaluation (GPE) Program. Cows were produced from mating Angus and Hereford dams to Angus, Hereford, Charolais, Shorthorn, Galloway, Longhorn, Nellore, Piedmontese, and Salers sires. Samples of Angus and Hereford sires were 1) reference sires born from 1962 through 1970 and 2) 1980s sires born in 1980 through 1987. The mixed model included cow age, season of measurement and their interactions, year of birth, pregnancy-lactation code (PL), and breedgroup as fixed effects for CW and CS. Analyses of weight adjusted for condition score included CS as a linear covariate. The model for CH excluded PL. Random effects were additive genetic and permanent environmental effects associated with the cow. Differences among breed groups were significant (P < 0.05) for all traits and were maintained through maturity with few interchanges in ranking. The order of F1 cows for weight was as follows: Charolais (506 to 635 kg for different ages), Shorthorn and Salers, reciprocal Hereford-Angus (HA) with 1980s sires, Nellore, HA with reference sires, Galloway, Piedmontese, and Longhorn (412 to 525 kg for different ages). Order for height was as follows: Nellore (136 to 140 cm), Charolais, Shorthorn, Salers, HA with 1980s sires, Piedmontese, Longhorn, Galloway and HA with reference sires (126 to 128 cm). Hereford and Angus cows with reference sires were generally lighter than those with 1980s sires. In general, breed differences for height followed those for weight except that F1 Nellore cows were tallest, which may in part be due to Bos taurus-Bos indicus heterosis for size.     

Supplementation of broodmares with copper, zinc, iron, manganese, cobalt, iodine, and selenium

In experiment 1, 6 pregnant mares received a concentrate that contained a trace mineral premix that provided 14.3 mg Cu, 40 mg Zn, 28 mg Fe, 28 mg Mn, 0.08 mg Co, 0.16 mg I, and 0.16 mg Se/kg concentrate (group A). Seven mares received the same concentrate plus 502 mg Zn and 127 mg Cu once daily (group B). No differences (P > .05) in foal growth data, or Cu, Zn, and Fe concentrations of mare milk, mare serum, or foal serum were observed. In experiment 2, 6 pregnant mares received the same concentrate as group A (group C), and 8 mares received the same concentrate fortified with 4× the trace mineral premix (group D). Group C mares had higher serum Zn concentration at 1 day (P < 0.01) and 56 days (P < 0.04). Group C mares had higher milk Fe concentration at 28 days (P < .01), and group D mares had higher milk Cu concentration at 56 days (P < .01). Group C foals had higher serum Cu concentration at 14 days (P < .03). The results from this study provide no evidence to indicate that supplementing late gestating and lactating mares with higher dietary trace mineral levels than those recommended currently by NRC has any influence on foal growth and development, or on the Cu, Zn, and Fe concentrations of the mare milk, mare serum, or foal serum.     

电感耦合等离子-质谱法测定饲料中钠、镁、铬、锰、铁、铜、锌、砷、硒、镉和铅

应用电感耦合等离子-质谱技术(ICP-MS),建立饲料中钠、镁、铬、锰、铁、铜、锌、砷、硒、镉和铅等元素的测定方法。对饲料样品的前处理方法、仪器工作参数和11种元素标准曲线进行优化;并以加标回收、分析方法比对和重复测试说明方法的准确性和精密性。方法在0～1 000 ng/ml范围内线性良好,仪器检出限为0.557 7～5.072 ng/ml,具有良好的精密度,其回收率在88.1%～104.4%之间,相对标准偏差小于5.0%。同时与原子吸收和原子荧光方法进行比对,测定结果相近。所建立的方法简单、快速,可替代原子吸收和原子荧光方法测定饲料中的11种金属元素,为饲料的质量控制提供理想的元素分析方法。     

The effects of antibiotic,probiotic, organic acid,vitamin C,and Echinacea purpurea extract on performance,carcass characteristics,blood chemistry,microbiota, and immunity of broiler chickens

The present context investigated the comparative study on the supplementation of antibiotic, probiotic, organic acid, vitamin C, and herbal extract after vaccination into drinking water and their effects on performance, carcass quality, blood biochemical parameters, immune system, and intestinal flora in broiler chicks for 42 days. A total of 420 one-day-old male broiler chicks (Ross 308) were randomly assigned into 7 treatments with 3 replicates (pens) per treatment and 20 male chicks for each replicate (pen). The experimental treatments consisted of drinking water (control, without additive); drinking water + antibiotic sulfamet; drinking water + C-Vet-50; drinking water + antibiotic sulfamet + C-Vet-50; drinking water + probiotic Primalac; drinking water + butyric acid; and drinking water + extract of Echinacea purpurea Moench (coneflower). There were no differences observed among the treatments for feed intake, but during the whole experimental period, the highest body weight gain was found in the chicks fed with drinking water + antibiotic sulfamet + 50 cc vitamin C (P < 0.05). There were no differences (P > 0.05) observed among the treatments for feed conversion ratio (P > 0.05). Moreover, there were no differences reported among treatments for carcass characteristics at the end of the experiment. Among the treatments, drinking water + 50 cc vitamin C, and drinking water + extract of E. purpurea reduced (P < 0.05) the levels of cholesterol, triglycerides, and low-density lipoproteins. Drinking water + 50 cc vitamin C, drinking water + Primalac, and drinking water + extract of E. purpurea increased (P < 0.05) the lymphocytes count and decreased the heterophils count and heterophil:lymphocyte ratio. The highest Escherichia coli count and lowest Lactobacillus count in ileal content of the broilers were observed in the control group (P < 0.05). The additives used in this study may be incorporated in the drinking water of broiler chickens as growth promoters and for improved performance. A further, wider supplementation study is required to understand the performance, immune system, variation in the intestinal microbial counts, and any other possible alteration in the intestinal biota of the broilers.     

Granulocyte isolation from whole blood of goat, sheep, cattle, horse, dog, pig, and man

A simple two step procedure for the isolation of caprine, ovine, bovine, equine, canine, porcine and human peripheral blood granulocytes is described. After enrichment of granulocytes by centrifugation, contaminating erythrocytes are lysed hypotonically. Recovery, purity, and viability of the granulocyte suspensions are determined. FACScan analysis of the cell suspensions measuring cellular size by forward and sideward light scatter is compared with the corresponding analysis of whole blood leukocytes. Constituencies of the isolated cell suspensions and loss of granulocyte subpopulations through isolation procedure is discussed with regard to granulocyte function assays.     

Department of Clinical Veterinary Practices, School of Veterinary Medicine, University of Dublin, Trinity College, Ballsbridge, Dublin 4

Circular excised skin wounds in the thoracic and metatarsal regions of the dog were studied. A similar sequence of events took place in the two regions although differences did occur due to the different reactions of the tissues which surrounded the wounds. None of the wound cavities became filled with exudate during the early stages of healing. In the thoracic wounds the cavities were largely filled by the swelling and inward movement of adipose tissue. Epithelium first grew on the wound surface in the sector of the wound that was situated in the direction of hair flow. The average time to complete epithelization was similar in both sets of wounds. A zone of alopecia developed around the wounds.