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．     

猪瘟病毒Erns基因的克隆及原核表达

从猪瘟病毒石门株血液样品中提取总RNA,通过R-PCR对Erns基因进行cDNA扩增,获得了696 bp的片段.将该片段克隆于T-easy载体后进行序列分析,确认PCR产物为猪瘟病毒Erns基因,从阳性克隆中提取质粒,经Bam H Ⅰ和Hin d Ⅲ双酶切,回收产物亚克隆到pET-32a表达载体中,提取质粒后转化BL21(DE3)感受态细胞,并筛选出阳性克隆,经IPTG诱导后通过SDS-PAGE检测出Erns基因的表达.     

大肠杆菌多重耐药调控基因soxS的克隆及其原核表达

根据GenBank中大肠杆菌的soxS基因序列设计引物,以大肠杆菌ATCC25922的基因组为模板,PCR扩增出约324 bp的soxS基因片段,将所得片段与pMD18-Tsi mple vector连接,转化至J M109大肠杆菌中,成功地筛选到阳性克隆,其质粒序列测序结果与GenBank中报道一致。从阳性克隆中提取质粒,经BamHⅠ和XhoⅠ酶解,回收324 bp的目的片段,定向克隆到pET-28a表达载体中,提取质粒,再次转化到大肠杆菌BL21(DE3)中,成功地筛选出阳性克隆。经IPTG诱导阳性菌,获得表达产物,通过SDS-PAGE检测出soxS基因的表达。     

大肠杆菌多药耐药基因AcrA的克隆及其原核表达

从大肠杆菌AcrA的编码序列中设计引物,以大肠杆菌基因组为模板,扩增出AcrA基因中约691 bp的cDNA片段,将所得片段与pMD-18T载体连接,转化到DH5a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一致.从阳性克隆中提取质粒,经HindⅢ和BamH I酶解,回收691 bp的目的片段,定向克隆到pET-28a表达载体中,提取质粒,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AcrA部分基因的表达.     

大肠杆菌多药耐药基因AcrA的克隆及其原核表达

从大肠杆菌AcrA的编码序列中设计引物，以大肠杆菌基因组为模板，扩增出AcrA基因中约691bp的cDNA片段，将所得片段与pMD—18T载体连接，转化到DH5α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一致。从阳性克隆中提取质粒，经HindⅢ和BamHI酶解，回收691bp的目的片段，定向克隆到pET—28a表达载体中，提取质粒，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AcrA部分基因的表达。     

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。     

牛干扰素-γ基因的RT-PCR扩增、序列分析及其在大肠杆菌中的表达

根据GenBank上发表的牛干扰素γ基因序列设计引物,从牛外周血淋巴细胞中提取基因组RNA,采用RTPCR技术,扩增出干扰素γ基因并进行序列分析。琼脂糖凝胶电泳显示牛干扰素γ扩增片段约为500bp。序列分析结果表明,牛干扰素γ基因序列全长517bp,开放阅读框架内501个核苷酸,共编码166个氨基酸,分子质量19.4ku,与参考序列完全一致。克隆序列经BamHⅠ/EcoRⅠ双酶切后连接到经同样双酶切的PBV220质粒中,转化入大肠杆菌DH5α中,筛选阳性克隆进行温度诱导表达,经SDSPAGE分析,在约19.4ku处有表达的蛋白带,表达产物经免疫荧光染色鉴定为阳性。     

大肠杆菌多重耐药调控基因marR的克隆及其原核表达

为获得MarR多克隆抗体,试验以大肠杆菌ATCC25922基因组为模板,根据GenBank中大肠杆菌的marR基因序列设计引物,PCR扩增出长约435bp的marR基因片段,将所得片段与pMD18-T载体连接,转化至JM109大肠杆菌中,筛选阳性克隆,其质粒中插入序列的测序结果与GenBank中报道一致,从阳性克隆中提取质粒,经BamHⅠ和XhoⅠ酶切回收435bp的目的片段,定向克隆到pET-28a(+)表达载体中,提取阳性质粒转化到大肠杆菌BL21(DE3)中获得阳性克隆。结果表明:经IPTG诱导阳性菌收集表达产物,通过SDS-PAGE分析证实marR基因得到表达;经Western-blot检测该蛋白具有良好的反应原性。     

鸡BCL10基因的克隆与表达

通过电子克隆手段,克隆到大小为959bp的cDNA序列。然后从鸡的脾脏提取RNA,用RT-PCR方法扩增出目的片段。将该片段连接到原核表达载体pET-32a和真核表达载体pEGFP-C1中,采用SDS-PAGE检测原核表达载体pET-BCL10在大肠杆菌中的表达,脂质体转染法将真核表达载体pEGFP-BCL10转入vero细胞,Western blotting检测其在细胞中的表达。结果显示,PCR扩增到735bp的DNA片段,原核表达载体和真核表达载体经双酶切和核酸测序表明载体构建成功。SDS-PAGE电泳检测到pET-BCL10重组质粒在大肠杆菌中表达。Western blotting检测到pEGFP-BCL10重组质粒在vero细胞中表达。结论表明,BCL10基因在鸡体内存在,该基因的重组质粒能在大肠杆菌和vero细胞中表达。     

鸡α-2,3-唾液酸转移酶Ⅰ基因的克隆和真核表达载体的构建

试验利用Trizol法从鸡肠道组织提取总RNA,采用特异性引物通过RT-PCR扩增出α-2,3-唾液酸转移酶Ⅰ(α-2,3-sialyltransferaseⅠ,ST3GALⅠ)基因的cDNA片段,并将其克隆至pGEM-T easy载体,获得阳性重组质粒,再以阳性重组质粒为模板亚克隆ST3GALⅠ基因的完整ORF区,定向插入到真核表达载体pcDNA3.1(+)上,进行PCR、限制性酶切和DNA序列分析鉴定。结果表明,ST3GALⅠ基因全长1029 bp,测序结果同GenBank数据库收录的序列一致,无任何密码子缺失与突变,插入到真核表达载体pcDNA3.1(+)上的目的基因大小方向均正确。本研究成功构建了pcDNA3.1(+)-ST3GALⅠ真核表达载体,为下一步的真核表达及对ST3GALⅠ基因的功能研究奠定了基础。     

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.     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

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.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Feline intracranial neoplasia: retrospective review of 160 cases (1985-2001)

The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.