猪带绦虫不同发育阶段的cDNA合成及含量测定

本文以猪带绦虫成熟虫卵、孵化激活的六钩蚴以及人工感染的囊尾蚴为原始材料，采用Promega试剂盒法分别分离提取总RNA和mRNA，反转录合成cDNA，并用a-^32P CTP掺入法放射测定其含量。实验表明：合成的猪带绦虫虫卵、六钩蚴和囊尾蚴的cDNA含量高，均可达2500ng，片段大小在300bp以上，可满足构建不同发育阶段cDNA基因文库的需要。     

从羊皮肤组织中提取总RNA方法的改进

在借鉴动物组织RNA提取方法的基础上,结合使用RNAlater保存液和Trizol试剂,改进了提取步骤和反应体系。利用改良的总RNA提取方法,从羊皮肤组织中快速、高效地提取了高质量总RNA,提取的总RNA可满足基于RNA分析技术的相关分子生物学试验需要。     

猪肌生成抑制素在COS-7细胞中的表达及检测

本研究室构建的猪肌生成抑制素(MSTN)成熟蛋白编码序列的真核表达质粒pef-dhfr1a-MSTN,转染到COS-7细胞中,使猪MSTN成熟蛋白编码序列全长358个氨基酸在COS-7细胞中进行表达,利用Trizol试剂提取转染细胞的总RNA,借助RT-PCR和SDS-PAGE电泳方法,分别从mRNA水平和蛋白质水平上检测到了猪MSTN在COS-7细胞中的表达,并应用Western-blotting方法证实了猪MSTN表达的特异性。     

五种核酸提取法对猪繁殖与呼吸综合征病毒提取效率的比较

目的：研究Trizol RNA提取法、杭州博日吸附柱RNA提取法、Promega核酸提取仪磁珠RNA提取法、西安天隆核酸提取仪磁珠RNA提取法、Thermo核酸提取仪磁珠RNA提取法等五种不同核酸提取方法对不同含量猪繁殖与呼吸综合征病毒（PRRSV）阳性样品的相对提取效率,为动物疫病监测诊断实验室核酸提取方式的选择提供技术参考。方法用五种不同核酸提取方法对4份10倍梯度稀释的PRRSV阳性血清、2份PRRSV阳性组织悬液及其离心后的上清液共8份样品同时进行核酸提取,用荧光RT-PCR方法进行检测,获取各样品PRRSV荧光RT-PCR结果的CP值,分析五种提取方式的相对提取效率。结果对合适的提取样品,磁珠提取法相对提取效率最高,吸附柱法其次,Trizol提取法相对效率较差;在三种磁珠提取法中,提取效率相差在2个CP值内,差异不明显,但以天隆磁珠提取法提取效率更高。结论预装试剂结合仪器自动化操作的磁珠法提取效果稳定,效率最高;国产仪器国产试剂提取效率不亚于进口仪器和试剂。     

猪精子RNA提取及cDNA质量检测

为了有效分离猪精子RNA,试验通过精子上游法获得纯净精子样本,采用改良TRIzol法提取精子总RNA,经核酸测定仪及琼脂糖凝胶电泳检测RNA的纯度和质量,RT-PCR检测相应体细胞标记基因E-Cadherin、CD4和c-kit。结果表明:研究建立的精子RNA提取法可获得高质量的RNA,浓度约为203 ng/μL,OD260/280值为1.99,并且无其他体细胞标记基因扩增产物,可用于猪精子mRNA的研究。     

猪精子RNA提取的初步研究

实验采用Trizol一次法或试剂盒提取猪精子的总RNA,然后通过RT-PCR扩增的方法,检测精子和睾丸组织中鱼精蛋白(PRM-1)的mRNA;使用不同数量的精子,检测在本实验条件下能扩增出PRM-1带所需要的最小精子数量;通过OD260/280的测算,判断不同提取方法所得RNA的纯度;通过琼脂糖电泳得到精子RNA与睾丸、卵巢组织RNA的电泳图。实验结果表明:在使用RNA沉淀剂时,1.51×107精子可检测到PRM-1的mRNA,不使用沉淀剂则需要3.02×107精子才检测到;使用Trizol一次法提取的精子总RNA纯度较常规Mini试剂盒(W6221)高;本实验首次得到猪精子总RNA的普通琼脂糖凝胶电泳图。初步研究的结果表明,Trizol一次法可用来提取精子总RNA供进一步研究;猪精子RNA的28S和18S片段与睾丸、卵巢组织无明显差异。     

猪各组织器官内肌生成抑制素基因mRNA表达谱的研究

用Trizol试剂法提取军牧一号猪的骨骼肌、心肌、肝、肾、肺、脾、胰、脂肪的总RNA做RT-PCR和嵌套PCR,结果在军牧一号猪骨骼肌、心肌和脂肪组织内检测到了肌生成抑制素mRNA的表达,而在其他组织中未检测到.     

利用SMART技术构建猪血管内皮细胞酵母双杂交cDNA文库

本研究旨在建立猪血管内皮细胞酵母双杂交cDNA文库,进而为研究猪瘟病毒的基因功能及致病机理提供良好的试验体系。体外培养猪血管内皮细胞,提取细胞总RNA,并用Oligotex mRNA Mini Kit纯化PolyA+mRNA,利用SMART技术合成双链cDNA(ds cDNA)。经CHROMA SPINTM TE-400Column纯化后的dsDNA与线性化的pGADT7-Rec共转化酵母感受态细胞Y187中,以同源重组的方式在酵母细胞内构建猪血管内皮细胞cDNA文库。结果获得的文库容量在6×106 CFU/mL,随机挑选24个克隆进行PCR检测,插入的片段大小集中在400² 000bp之间,平均插入长度为1 000bp左右,文库重组率为100%。本研究为从文库中高通量地筛选出与猪瘟病毒互作的重组子,即寻找与该病毒相互作用的受体蛋白奠定了坚实的基础。     

抗猪囊尾蚴单克隆抗体轻重链可变区基因的扩增及序列分析

从分泌抗猪囊尾蚴单克隆抗体杂交瘤细胞提取总RNA,以此为摸板,RT-PCR扩增出抗猪囊尾蚴单克隆抗体轻重链可变区基因,琼脂糖凝胶电泳检测显示约350 bp,符合鼠抗体特征.PCR产物与pMD18-T连接后转化JM109,蓝白斑法筛选出阳性重组子.以载体通用引物对阳性重组子进行鉴定,对证明为阳性的重组子测序.结果显示,轻链可变区和重链可变区基因符合鼠抗体轻链可变区和重链可变区基因特征.VH和VL有3个比较明显的CDR区和FR区.     

猪囊尾蚴西昌分离株线粒体Cytb和nad4基因的序列测定与种系发育分析

利用PCR技术对2个猪囊尾蚴西昌分离株的线粒体细胞色素b(Cytb)基因全序列和烟酰胺腺嘌呤二核苷酸脱氢酶亚单位4(nad4)基因部分序列(pnad4)进行扩增,分析其遗传变异,并用MEGA 5.0程序NJ法绘制种系发育树,探讨不同地区来源的猪囊尾蚴种系发育关系。测序结果显示,2个猪囊尾蚴分离株的Cytb基因全序列长度均为1 068bp,nad4基因部分序列长度均为815bp,核苷酸序列同源性均为99.8%。种系发育分析结果显示,所有猪囊尾蚴分离株形成一个分支,2个西昌分离株均属于猪带绦虫亚洲基因型。结果表明,Cytb和nad4基因均可用于猪带绦虫的分子分类,为猪带绦虫病/猪囊尾蚴病的分子诊断奠定基础。     

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.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

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.     

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.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -