猪肌生成抑制素C末端80氨基酸编码基因的合成及其原核表达

将猪肌生成抑制素编码序列下游883-l126bp按大肠杆菌嗜好密码子进行优化，将优化后序列双链分成l2个单链片段(F1、F2、F3、R6、R5、R4、F4、F5、F7、R3、R2、R1)，采用化学合成方法合成，每对相邻互补片段之间有20bp序列交叉重叠。F1长38bp，Rl长36bp，其他片段均40bp长，Fl和R1片段两端分别加上限制性内切酶NcoI和XhoI的识别位点序列。经PCR扩增出254bp片段，该片段与pMDl8—T载体连接，转化JMl09感受态细胞，所得阳性克隆进行测序分析，得到的克隆序列与设计的序列完全一致，表明成功地获得了猪肌生成抑制素下游编码序列克隆载体。从阳性细菌中提取质粒，经NcoI和XhoI酶切，回收254bp目的片段，定向克隆到pET28a中，转化DH5α受体菌，提取质粒，再转化到BL2l(DE3)中，成功筛选出阳性克隆。阳性菌经IPTG诱导，通过SDS—PAGE，检测出猪肌生成抑制素的表达。     

溆浦鹅卵泡抑制素α亚基编码区cDNA克隆及其原核表达

根据GenBank中鸡卵泡抑制素α亚基序列设计引物，对该序列按大肠杆菌嗜好密码子进行优化，在设计的引物两端分别加上限制性内切酶SacⅠ和XhoⅡ的识别位点序列。利用RT-PCR技术从溆浦鹅卵泡的颗粒细胞总RNA中扩增出抑制素α亚基成熟区序列，经PCR扩增出354bp片段，该片段与pMD18-T载体连接，转化JM109感受态细胞，所得阳性克隆进行双酶切鉴定和PCR鉴定，并进行了测序分析，得到的克隆序列与设计的序列基本一致。表明成功地获得了抑制素α亚基编码序列的克隆载体。从阳性细菌中提取质粒，经SacⅠ和XhoⅠ酶切，回收354bp目的片段，定向克隆到pET28a中，转化DH5α受体菌，提取质粒，再转化到BL21(DE3)中，成功筛选出阳性克隆。阳性菌经IPTG诱导，通过SDS-PAGE，检测出溆浦鹅卵泡抑制素α亚基编码区的表达。     

猪肌生成抑制素成熟蛋白编码序列真核表达载体的构建

从猪肌生成抑制素(MSTN)cDNA序列中设计引物。以pMD18-T-MSTN质粒为模板，PCR扩增猪MSTN成熟蛋白编码序列，该片段全长1095bp。将所得片段与pMD18-T载体连接，构建重组质粒pMT—mpMSTN，将其转化到大肠杆菌DH5a中，成功地筛选到阳性克隆。双酶切重组质粒pMT—mpMSTN和真核表达载体pcDNA3．1，连接目的片段与载体pcDNA3．1，转化大肠杆菌DH5a，经酶切、PCR及测序分析，表明成功地构建了真核表达质粒pcDNA3．1-mpMSTN。     

猪生成抑制素(MSTN) cDNA的克隆

从猪的肌生成抑制素(MSTN)编码序列中设计引物,以军牧一号猪肌细胞总RNA为模板,利用RT-PCR和嵌套PCR技术,扩增出MSTNcDNA片段.该片段全长1277bp,包含猪MSTN基因的全部编码序列.将所得片段与pMD18-T载体连接,转化到JM109大肠杆菌中,成功地筛选到阳性克隆,其质粒测序结果与文献报道的一致.经EcoRⅠ和PstⅠ酶解分析,cDNA片段与pMD18-T载体之间既有正向插入的克隆,也有反向插入的克隆,所得到的MSTNcDNA可用于原核和真核表达载体的构建.     

猪肌生成抑制素基因成熟蛋白编码序列表达载体的构建及其转录水平的检测

构建猪肌生成抑制素(MSTN)成熟蛋白编码序列的克隆与表达载体，并对mRNA进行转录水平的检测，为获得较好的猪肌生成抑制素抗原、制备抗体打下良好的基础。猪MSTN基因的cDNA去除信号肽后，用PCR扩增的1．2kb目的片段与pMDl8-T载体连接，将克隆载体的质粒DNA与同样用BamH Ⅰ和Sal Ⅰ限制性内切酶酶解的表达载体pET28a( )质粒定向连接，对筛选出的阳性克隆用酶切及测序法鉴定。对猪MSTN基因成熟蛋白编码序列进行反转录，对mRNA Northern杂交进行转录水平的检测。结果，所得到的序列与所设计的序列完全一致，表明成功地进行了猪MSTN基因编码序列的克隆及筛选；目的片段定向插入，阅读框架正确；RT-PCR成功地反转录得到约1．2kb的目的DNA片段，Northern杂交后对mRNA印迹放射自显影，见到清晰的1．2kb特异带。     

猪肌生成抑制素（MSTN）cDNA的克隆

从猪的肌生成抑制素（MSTN）编码序列中设计引物，以军牧一号猪肌细胞总PNA为模板，利用RT－PCR和嵌套PCR技术，扩增出MSTN cDNA片段。该片段全长1277bp，包含猪MSTN基因的全部编码序列。将所得片段与pMD18-T载体连接，转化到JM109大肠杆菌中，成功地筛选到阳性克隆，其质粒测序结果与文献报道的一致。经EcoR Ⅰ和Pst Ⅰ酶解分析，cDNA片段与pMD18-T载本之间既有正向插入的克隆，也有反向插入的克隆，所得到的MSTN cDNA可用于原核和真核表达载体的构建。     

猪Ghrelin基因的克隆及原核表达

从猪下丘脑、胃等组织中提取总RNA，根据已发表的猪的Ghrelin mRNA序列设计合成引物，通过RT-PCR进行cDNA扩增，获得了282bp的片段。将该片段克隆于pMD-18T载体后进行序列分析，确认PCR产物为Ghrelin cDNA。从阳性克隆中提取质粒，经NheⅠ和XhoⅠ双酶切，回收282bp的目的片段，定向克隆到pET-28a表达载体中，提取质粒并再次转化到BL21（DE3）中，成功地筛选出阳性克隆。经IPTG诱导阳性菌，通过SDS-PAGE检测出猪Ghrelin基因的表达．     

鹅副粘病毒F和HN基因的克隆及原核表达载体的构建

根据发表的鹅副粘病毒SF02株全基因组序列,分别设计合成一对引物和二对引物,分别采用RT-PCR方法和RT-套式PCR扩增出与预期设计大小相符的F基因和HN基因特异性条带.将扩增出的DNA片段克隆到pMD18-T载体中,转化TGI感受态细胞,经AMP/IPTG/X-Gal平板筛选,酶切鉴定,获得阳性重组质粒.对重组质粒进行序列测定,结果表明:鹅副粘病毒JS/1/97/Go株F基因全长1662bp,编码553个氨基酸残基;F蛋白裂解位点的氨基酸顺序为112R-R-Q-K-R-F117,与NDV的强毒株特征相符.HN基因全长1716bp,编码571个氨基酸残基.将F基因插入到原核表达质粒pGEX-6P-1的EcoR Ⅰ、Xho I多克隆位点之间,转化到TGI感受态细胞中,获得了表达F基因的阳性亚克隆重组质粒;将HN堪因插入原核表达质粒pProEX HTb的XBaⅠ、XhoⅠ多克隆位点之间,转化到TGI感受态细胞中,获得了表达HN基因的阳性亚克隆重组质粒.     

猪肌生成抑制素基因的克隆和序列测定

根据文献报道的猪肌生成抑制素基因cDNA序列，分别从第1、第2和第3外显子中设计引物，以大约克猪染色体DNA为模板，分段扩增出含内含子和外显子的序列。将所得各序列回收、克隆到pGEM-easyT载体中，然后进行序列测定。合并所有测得的猪肌生成抑制素因序列，共获得6kb连续序列。分析结果表明，猪肌生成抑制素基因第1内含子长1809bp，第2外显子长374bp，第2内含子长1978bp。所测得的外显子序列与已发表的cDNA序列同源分析比较，没有出现引起氨基酸改变的突变。表明瘦肉型猪（大约克猪）肌生成抑制素基因仍很完整，可通过改变基因结构等降低肌生成抑制素活性的方法提高瘦肉率。     

瘦蛋白基因的克隆及在大肠杆菌中的表达

采用 RT- PCR方法从猪脂肪组织扩增出瘦蛋白基因 ,将其插入 p MD1 8- T克隆载体 ,经序列分析证明 ,所获得的目的基因为 4 4 1 bp,与预期大小一致。提取质粒后用 Eco R 和 Xho 双酶切 ,克隆入 p ET- 2 8a表达载体 ,将阳性重组质粒转化表达受体菌 E.coli BL 2 1 ( DE3) ,经 IPTG诱导 ,SDS- PAGE检测 ,证明在 E.coli BL 2 1中正确表达了重组猪瘦蛋白     

白术、微米白术和白术多糖对断奶仔猪生长性能和肠道形态及微生态区系的影响

试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。     

Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia

Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

A brief history of the prevention of infectious diseases by immunisations

Infectious diseases have always been a terrible scourge for humans. The appearance of these plagues, as they were called without distinction, was generally connected to various conditions: asters, climatic changes or religious reasons. The concept of contagious, and then infectious, diseases came slowly. Variolation, i.e. transmission of ‘virulent’ matter to induce a natural disease and the immunity against it, was brought from Constantinople to England by Lady Montague, in 1721. This ‘variolation’ technique was also often performed in veterinary medicine against diseases like sheep-pox or pleuropneumonia. As ‘vaccination’ is the term generally accepted for ‘immunisation’, variolation can be the word designating such a technique. The second period of the history of immunisation began, in 1880, with the studies of Pasteur and his collaborators. A great number of bacterial vaccines were developed: dead, live but attenuated or only parts of pathogens. The viruses were produced in animals, then in eggs and at last, in tissue cultures. Second generation vaccines appeared with genetic engineering: recombinant vaccines, vector vaccines, nucleic acids vaccines, and markers vaccines, among others. These novel technologies can permit the development of new ones and improve the quality of the vaccines already existing.     

Micronutrient Content of Certain Tropical Conventional and Unconventional Feed Resources of Southern India

The ash, silica and certain important micronutrients were estimated in conventional and unconventional feed and fodder resources available in Southern India. Commonly used dry roughages, such as paddy straw, ragi straw, maize kadbi/stalk, jowar kadbi/stalk, bajra stalk and wheat straw, were high in ash (9.9% +/- 0.77%) and silica (6.4% +/- 0.65%) and low in most of the other micronutrients, except iron, with paddy straw containing most silica (>9%). Cultivated non-leguminous (maize, jowar) and improved green crops (hybrid napier, guinea, green panic, NB-21, CO-1) were also high in ash (10.5% +/- 0.60% and 12.5% +/- 0.51%) but were moderate sources of P, Mg and Cu and good sources of Zn (98 +/- 13.8 ppm and 55 +/- 6.7 ppm). Leguminous green fodders (stylosanthus, lucerne, cow pea, soyabean) were excellent sources of Ca (1.9% +/- 0.16%), Mg (0.40% +/- 0.05%), Cu (30 +/- 5.2 ppm), Zn (121 +/- 14.7 ppm) and Fe (1234 +/- 166 ppm) and moderate sources of P. Mixed local grasses and weeds were high in silica (6.9% +/- 1.00%) but were good sources of Cu, Zn and Fe. Cereal grains (maize, wheat, rice, ragi) were low in ash (2.9% +/- 0.33%) and were relatively poor sources of Ca (0.22% +/- 0.03%), Mg (0.19% +/- 0.03%) and Cu (13 +/- 3.1 ppm). Pulses were low to medium sources of most minerals and good sources of Fe (1230 +/- 293 ppm). Oil seed cake/extractions (groundnut cake, cotton seed cake, soyabean meal, sunflower cake, safflower cake) and cereal by-products (rice polish, rice bran, wheat bran) were excellent sources of P (1.1% +/- 0.47% and 2.3% +/- 0.19%) and good sources of Zn (65 +/- 3.9 ppm and 66 +/- 10.7 ppm) and Fe (938 +/- 130 ppm and 662 +/- 126 ppm). Among the unconventional feeds screened, orange peel, sunflower heads, meat meal, rubber seed cake, spirulina algae and sea weeds contained plentiful Ca, Zn and Fe: tree leaves/top feeds (mulberry, erythrina, glyricidia, banana, subabul, groundnut haulms) were excellent sources of Ca (1.5% +/- 0.13%), Zn (120 +/- 22.9 ppm) and Fe (1033 +/- 133 ppm) but relatively poor sources of P. Soyabean husk, cocoa seed husk, rubber seed cake and meat meal were moderate to good sources of P (1.0% and 0.33%). The high Zn and Fe values of most feeds/fodders were probably due to soil contamination. This account of the micronutrient content of feed/fodder resources should help in strategic supplementation intended to alleviate local deficiencies.     

Bovine Mastitis in Selected Areas of Southern Ethiopia

A study on bovine mastitis, designed to determine the causal agents, prevalence of infection and impact of risk factors in three cattle breeds, was conducted in selected areas of southern Ethiopia. A total of 307 lactating and non-lactating cows, of which 162 were indigenous Zebu, 85 Jersey and 60 Holstein-Friesian, were examined by clinical examination and the California mastitis (CMT) test. Of these, 40.4% were positive by CMT and bacteriology for clinical or subclinical mastitis, with prevalence rates of 37.1% and 62.9%, respectively. Out of 1133 quarters examined, 212 (18.7%) were found to be infected, 83 (39.2%) clinically and 129 (60.8%) subclinically. The prevalence of mastitis was significantly higher in Holstein-Friesian than in indigenous Zebu, in non-lactating cows than in lactating cows, in the early lactation stage than in the mid-lactation stage, in cows with lesions and/or tick infestation on skin of udder and/or teats than in cows without this factor, and in the wet season than in the dry season. Mastitis increased with parity number (R = 0.9). Of 248 CMT and clinically positive udder quarter samples analysed microbiologically, 212 were culturally positive for known mastitis pathogens and 36 were negative. Of the 199 positive samples, Staphylococcus accounted for 39.2%, Streptococcus for 23.6%, coliforms for 14.1%, Micrococcus and Bacillus species for 8.0% each and Actinomyces or Arcanobacterium (Corynebacterium) for 7.0%. It was concluded that there was a high prevalence of clinical and subclinical mastitis, mainly caused by Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli, in this study area.     

不同日粮氮水平对山羊氮代谢和微生物蛋白质合成的影响

本试验旨在探讨不同日粮氮水平对内蒙古白绒山羊氮代谢和微生物蛋白质合成(MCP)的影响,以达到提高氮利用率,减少环境污染和饲料资源的浪费。试验选用9只体况良好、体重为(43.83±2.95)kg,装有瘤胃瘘管的内蒙古白绒山羊,按体重随机分为3组,每组3只。日粮分为低氮7.5%、中氮10.5%、高氮13.5%3个氮水平。试验使用常规试验方法分析测定粪尿血液等生化指标,通过全收尿法和尿嘌呤衍生物法估测MCP。结果表明:随着氮水平增加,氨氮(NH3-N)浓度、氮摄入量、尿氮排出、尿中尿素氮(UUN)和MCP显著增加(P0.05),干物质采食量(DMI)显著降低(P0.01);沉积氮占总摄入氮的比例在中氮组最高,MCP在高氮组最高。综上所述,适当降低日粮氮水平可减少粪尿氮排放,提高反刍动物氮利用率。     

Microsporidial keratopathy in two dogs

A microsporidial keratopathy is described in two dogs. Both dogs presented with a unilateral stromal keratopathy characterized by multifocal coalescing opacities, and the diagnosis was made on histopathologic examination of keratectomy specimens. Transmission electron microscopy (TEM) on formalin‐fixed, paraffin‐embedded corneal tissue was performed in one dog, and the morphologic features were consistent with Nosema species infection. Both dogs were initially diagnosed and treated by superficial keratectomy. One dog received additional antifungal medication and underwent a penetrating keratoplasty following local recurrence two years later. No other systemic lesions attributable to the microsporidial infection were identified clinically. The clinical and diagnostic pathology findings, treatment, and follow‐up are discussed.     

优质肉鸡S3系体重与体尺性状指标的主成分分析

为研究地方优质肉鸡S3系体重和体尺性状之间的内在相关性,12周龄时选取同一饲养条件下的150只鸡(公母各半)进行体重和体尺性状等7个指标的测定。结果表明:S3系公鸡的体重、体斜长、龙骨长、胸角均显著高于母鸡(P0.05);公母鸡体重、体斜长、胸宽、胫围这4项指标变异系数较大,有较大选育空间;体重与体斜长、龙骨长、胸宽、胸角、胫围呈极显著正相关(P0.01),相关系数分别为0.57、0.52、0.51、0.49和0.47,与胸深、胫长呈显著相关(P0.05),相关系数分别为0.44和0.39;其他各体尺指标间也存在不同程度的正负相关性(P0.05或P0.01);体重和7个体尺性能指标综合成5个复合指标,累计贡献率达92.6355%,其中第l、2、3、4、5主成分分别解释总变异的36.5353%、22.8642%、13.3115%、10.4535%和8.4710%;各主成分的特征根分布较广,经统计计算入选的5个主成分所包含信息的侧重点各有相同,分别都在一定程度上反映了S3鸡的体型特征和生长发育规律,为今后选育出更加符合市场需求的屠宰加工优质型肉鸡指明了的方向。     

Efficacy of tropicamide,homatropine, cyclopentolate,atropine and hyoscine as mydriatics in Angora goats

Abstract

AIM: To document the efficacy of five commercially available mydriatics for their potential for diagnostic and therapeutic use in Angora goats.

METHODS: Over 8 weeks, the mydriatic effects of 1% tropicamide, 2% homatropine, 1% cyclopentolate, 1% atropine and 0.25% hyoscine were evaluated. Given as block treatments, drugs were applied randomly to one eye of 10 Angora goats, and the contralateral eye served as a control. Vertical and horizontal pupil diameters were measured to document onset ofeffect, time to reach a difference of 5 mm in the vertical/horizontal pupil diameter between eyes, time to maximum pupillary dilation, and duration of mydriatic action.

RESULTS: Onset of mydriasis for all drugs occurred within 15 minutes. Time to reach a difference of 5 mm in the vertical pupil diameter between eyes was shortest for 1% tropicamide and 0.25% hyoscine (0.5 h), then 2% homatropine and 1% atropine (0.75 h), and longest for 1% cyclopentolate (1.5 h). The maximum vertical pupillary dilation occurred earliest with 1% tropicamide and 1% atropine (2 h), followed by 0.25% hyoscine (3 h), 2% homatropine (4 h), and latest with 1% cyclopentolate (8 h). The duration of vertical dilation of the pupil was shortest with 1% tropicamide (6 h), then 2% homatropine (12 h), 1% cyclopentolate (12 h), 1% atropine (24 h), and longest for 0.25% hyoscine (96 h).

The time to reach maximum horizontal dilation of the pupil in treated eyes was shortest with 1% cyclopentolate (1 h), followed by 1% tropicamide (1.5 h), 0.25% hyoscine (3 h), 2% homatropine (3.5 h), and 1% atropine (4 h). The duration of horizontal pupil dilation was shortest with 1% tropicamide (4.5 h), and longest with 0.25% hyoscine (48 h).

CONCLUSION: All five mydriatics induced clinical dilation. Tropicamide (1%) had the shortest duration of effect, but gave incomplete dilation. Good dilation was achieved with 1% cyclopentolate and 2% homatropine, but took too long to reach maximum dilation for routine mydriasis. The largest vertical dilation of the pupil was achieved with 1% atropine and 0.25% hyoscine, but pupils remained dilated for more than 24 h.

CLINICAL RELEVANCE: For routine mydriasis in goats, it is recommended that 1% tropicamide be used, though there may be incomplete dilation. For a longer duration of mydriasis, such as in the treatment of anterior uveitis, 1% atropine or 0.25% hyoscine would be the drugs of choice.