从动物毛发中提取DNA进行RAPD扩增的研究

经实验改进了一种快速，简便提取动物毛囊细胞中ＤＮＡ的方法，电泳检测，ＤＮＡ带型整齐集中清晰，无拖尾现象；进行ＲＡＰＤ扩增，扩增带多而清晰，证明改进后的方法简便可行。     

BVD-MDV细胞毒的快速粗提

采用聚乙二醇（ＰＥＧ２００００）沉淀法提取了２批牛病毒性腹泻－粘膜病病毒（ＢＶＤ－ＭＤＶ）细胞毒。经毒价及ＯＤ值测定，浓缩病毒较之浓缩前毒力明显增强，病毒含量及纯度均有较大提高。将其作为检测抗原及免疫抗原，应用到ＢＶＤ－ＭＤＶ单克隆抗体的研制中，效果均较理想。试验证明，该方法具有简便、省时、费用低，实用等优点，适合基层推广应用     

BVD—MDV细胞毒的快速粗提

采用聚乙二醇（ＰＥＧ２００００）沉淀法提取了２批牛病毒性腹泻－粘膜病病毒（ＢＶＤ－ＭＤＶ）细胞毒，经毒价及ＯＤ值测定，浓缩病毒较之浓缩毒力明显增强，病毒含量及纯度均有较大提高，将其作为检测抗原及免疫抗原，应用以ＢＶＤ－ＭＤＶ单克隆抗体的研制中，效果均较理想，试验证明，该方法具有简便，省时，费用低，实用等优点，适合基层推广应用。     

玉米品种真实性和纯度鉴定的SSR标记多重PCR体系优化

 为了建立稳定可靠的玉米真实性和纯度鉴定ＳＳＲ 标记,对ＤＮＡ 提取方法、ＳＳＲ 引物和多重ＰＣＲ 反应程序进行了优化。结果表明,用预热到７５℃以上的研钵和９５℃的１．５×ＣＴＡＢ提取缓冲液进行材料研磨,可得到纯度高、完整性好的ＤＮＡ,并且提取成本较低。利用软件ＰｒｉｍｅｒＰｒｅｍｉｅｒ５．０和Ｏｌｉｇｏ６．７２对玉米指纹鉴定的ＳＳＲ 核心引物进行重新分析与设计,建立了２１对ＳＳＲ 通用引物构成的８组多重ＰＣＲ 复合扩增体系和３步法扩增程序,均能在统一的ＰＣＲ 扩增条件下进行,扩增片段之间不存在交叉现象,扩增条带清晰,扩增结果稳定,这一扩增体系检测效率比单对ＳＳＲ 引物提高２．６倍以上。     

成人肝cDNA文库的构建

用异硫氰酸胍／盐酸胍法从人肝组织中提取ＲＮＡ，通过Ｏｌｉｇｏ（ｄＴ）纤维柱分离出ｍＲＮＡ。以ｍＲＮＡ为模板，含ＮｏｔⅠ接头的Ｏｌｉｇｏ（ｄＴ）为引物，在逆转录酶ＳｕｐｅｒＳｃｒｉｐｔⅡＲＴ催化下合成单链ｃＤＮＡ（ｓｓｃＤＮＡ），再在Ｅ．ｃｏｌｉＤＮＡ聚合酶Ⅰ与ＲＮａｓｅＨ和ＤＮＡＬｉｇａｓｅ共同作用下，ｓｃＤＮＡ拷贝成双链ｃＤＮＡ（ｄｓｃＤＮＡ）。经放射自显影测定，合成有全长ｃＤＮＡ片段。平末端ｄｓｃＤＮＡ与ＳａｌⅠ接头连接，ＮｏｔⅠ酶切后，通过过柱取掉小于５００ｂｐ的ｃＤＮＡ片段，大片段ｃＤＮＡ与载体ｐＳＰＯＲＴ１连接，转化受体菌ＤＨ５α，经稀释测定出含有重组子的文库大小为３．３×１０６。该文库适合于筛选低丰度ｍＲＮＡ的ｃＤＮＡ克隆。     

产蛋下降综合征（EDS－76）病毒DNA探针的制备及应用研究

用纯化的产蛋下降综合征（ＥＤＳ－７６）病毒（Ｈ９１毒株）悬液提取的核酸，经琼脂糖凝胶电泳只出现１条分子量较大、背景清晰的核酸带。用ＢａｍＨＩ酶消化产生４个片段，大小分别为１７ｋｂ、１０ｋｂ、４．０ｋｂ和２．０ｋｂ。将其中的２．０ｋｂ片段克隆进ｐＵＣ１８ＤＮＡ载体中，重组质粒ＤＮＡ用ｄｉｇｏｘｉｇｅｎｉｎ标记作为ＤＮＡ探针，在ｄｏｔｂｌｏｔ中该探针不与鹅胚尿囊液核酸抽提物、马立克氏病病毒（ＭＤＶ）ＤＮＡ、鸡传染性贫血病毒（ＣＡＶ）ＤＮＡ、ＳＰＦ鸡基因组ＤＮＡ等核酸反应，只与３株ＥＤＳ病毒（ＥＤＳ标准毒株ＡＶ－１２７、ＥＤＳＨ９１毒株和从健康鹅体中分离的ＥＤＳＹ８１毒株）ＤＮＡ呈阳性反应。人工感染产蛋母鸡和雏鸡后２４ｈ即可用该探针从泄殖腔棉拭子样品中检测出ＥＤＳ病毒ＤＮＡ，在感染后３５ｈ仍能从部分感染鸡样品中检出。对部分探针检测阳性鸡的泄殖腔棉拭子样品用ＳＰＦ鸡胚分离病毒，并用ＨＡ和ＨＩ试验检测分离病毒的特异性；在感染过程中，同时检测了血清中ＨＩ抗体的消长情况。结果表明，人工感染鸡排毒至少可以维持２个月左右，而且血清中ＨＩ抗体即使很高，也不能阻止感染鸡的排毒。     

家蚕微孢子虫孢子DNA的提取、克隆及部分DNA序列分析

报道家蚕微孢子虫（Ｎｏｓｅｍａｂｏｍｂｙｃｉｓ简称Ｎ．ｂ）孢子ＤＮＡ的提取、克隆及部分ＤＮＡ序列测定的结果。采用ＳＤＳ－蛋白酶Ｋ将孢子破碎，用苯酚一氯仿法提取孢子的ＤＮＡ。将Ｎ．ｂ孢子ＤＮＡ与ｐＴＺ１８Ｒ质粒重组，转化于大肠杆菌ＤＨ５，获得４个阳性克隆株：ｐＴＺ１８ＲＮ．ｂ１，插入片段为１ｋｂ；ｐＴＺ１８ＲＮ．ｂ２，为１．２ｋｂ；ｐＴＺ１８ＲＮ．ｂ３为１．８ｋｂ及ｐＴＺ１８ＲＮ．ｂ４为１．９ｋｂ，经Ｓｏｕｔｈｅｒｎ印迹杂交，证实插入的ＤＮＡ片段为家蚕Ｎ．ｂ孢子所特有。与ＭＧ１（Ｎｏｓｅｍａｓｐ．），蓖麻蚕微孢子虫、蓝萤叶甲微孢子虫及蚕卵的ＤＮＡ均无同源性。用双脱氧链终止法测定４个克隆株插入片段的部分ＤＮＡ的序列，经检索尚未发现同源性的基因序列。讨论了ＰＣＲ技术诊断家蚕微孢子虫孢子与近缘种的问题。     

鸡病原性大肠杆菌（血清型O78）纤毛亚单位苗免疫原性的研究

本实验利用透射电镜观察了鸡源大肠杆菌（血清型Ｏ７８）表达的纤毛。提取并初步纯化了纤毛。通过ＳＤＳ－聚丙烯酰胺凝胶电泳检测了提取物的纯度及其亚单位分子量。表明提取物属Ｉ型纤毛。用提取纤毛制备油佐剂苗。评估了该苗的免疫原性，免疫组和阳性对照组病级数在极显左异（Ｐ＜０．０１），苗的保护指数分别为一次免疫６４．２８％，两次免疫７４．６０％。用ＥＬＩＳＡ检测了免疫后６８天鸡的抗体消长。通过统计分析二免后抗体     

家蚕微孢子虫孢子DNA的提取、克隆及部分DNA序列分析

报道家蚕微孢子虫（Ｎｏｓｅｍａｂｏｍｂｙｃｉｓ简称Ｎ．ｂ）孢子ＤＮＡ的提取、克隆及部分ＤＮＡ序列测定的结果。采用ＳＤＳ－蛋白酶Ｋ将孢子破碎，用苯酚一氯仿法提取孢子的ＤＮＡ。将Ｎ．ｂ孢子ＤＮＡ与ｐＴＺ１８Ｒ质粒重组，转化于大肠杆菌ＤＨ５，获得４个阳性克隆株：ｐＴＺ１８ＲＮ．ｂ１，插入片段为１ｋｂ；ｐＴＺ１８ＲＮ．ｂ２，为１．２ｋｂ；ｐＴＺ１８ＲＮ．ｂ３为１．８ｋｂ及ｐＴＺ１８ＲＮ．ｂ４为１．９ｋｂ，经Ｓｏｕｔｈｅｒｎ印迹杂交，证实插入的ＤＮＡ片段为家蚕Ｎ．ｂ孢子所特有。与ＭＧ１（Ｎｏｓｅｍａｓｐ．），蓖麻蚕微孢子虫、蓝萤叶甲微孢子虫及蚕卵的ＤＮＡ均无同源性。用双脱氧链终止法测定４个克隆株插入片段的部分ＤＮＡ的序列，经检索尚未发现同源性的基因序列。讨论了ＰＣＲ技术诊断家蚕微孢子虫孢子与近缘种的问题。     

应用PAPD方法对鸡败血霉形体DNA多态性的研究

用随机扩增多态性ＤＮＡ（ＲＡＰＤ）方法,通过筛选的５个随机引物（ＯＰＨ－０２,ＯＰＨ－０５,ＯＰＨ－０７,ＯＰＨ－１３,ＯＰＧ－１６）,对１４株鸡败血霉形体（ＭＧ）国际标准株和国内分离株进行了ＤＮＡ多态性研究。结果表明,５个引物共产生２１种条带,其中有３个条带分别为２个菌株所特有,扩增产物片段的长度在１５０～４５００ｂｐ之间,所有菌株均有１条共同条带,以ＯＰＨ－０５扩增产物的多态性最丰富。根据样品ＤＮＡ所获得的菌株间相似性指数表明,Ｄ９６０３与Ｄ９６０７相似指数最高,Ｓ６,Ｋ３９１３和Ｄ９６０４三者相互间相似性指数最低。提示ＲＡＰＤ方法可用于霉形体遗传标记的分析。     

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.     

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.     

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.     

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

Tropical Animal Health and Production -     

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.     

Chemotaxis of macrophage in inflammation

Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.     

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