正交设计优化山野豌豆ＳＲＡＰ－ＰＣＲ反应体系与引物筛选

 采用正交试验设计,以山野豌豆叶片ＤＮＡ 为模板,从Ｍｇ^２＋ 、ｄＮＴＰ、引物和ＴａｑＤＮＡ 聚合酶４种因素３个水平,对山野豌豆ＳＲＡＰ－ＰＣＲ 反应体系进行优化,并比较了不同浓度模板ＤＮＡ 对扩增效果的影响,建立了山野豌豆的ＳＲＡＰ－ＰＣＲ 最佳反应体系。结果表明,山野豌豆ＳＲＡＰ－ＰＣＲ 最佳反应体系为：２μＬ１０×ＰＣＲｂｕｆｆｅｒ（不含Ｍｇ^２＋ ）、３０ｎｇ的模板ＤＮＡ、引物２．０μｍｏｌ／Ｌ、Ｍｇ^２＋ ２．０ｍｍｏｌ／Ｌ、ＴａｑＤＮＡ 聚合酶１．５Ｕ、ｄＮＴＰ０．２ｍｍｏｌ／Ｌ,总体积为２５μＬ。各因素对扩增反应结果均有不同影响,其中以引物浓度影响最大,ｄＮＴＰ 浓度的影响最小。运用该体系对３份山野豌豆种源进行验证,证明该体系稳定可靠,并从９８对ＳＲＡＰ 引物组合中筛选出扩增条带清晰、多态性丰富的２０对引物组合。这一体系的建立及多态性引物组合的筛选为ＳＲＡＰ分子标记技术进行山野豌豆分子遗传学研究奠定了基础。     

家蚕RAPD的扩增条件、重复性及遗传模型研究

发现当ＰＣＲ体系中Ｍｇ￣（２＋）为２ｍｍｏｌ／Ｌ、ｄＮＴＰｓ为１５０μｍｏｌ／Ｌ、Ｔａｇ酶为１Ｕ、引物为０．２μｍｏｌ／Ｌ时，以４０～４２℃退火可得到稳定结果；８％的ＤＭＳＯ对提高扩增特异性有利；分别从蚕卵、蚁蚕、５龄蚕丝腺、体壁及生殖腺、蛹、蛾中提取模板ＤＮＡ，其ＲＡＰＤ结果一致；以Ｃ＿（１０８）×大造为材料，发现其Ｆ＿１ＲＡＰＤ为共显性。     

牛Y染色体特异DNA序列的体外扩增

本试验建立了用聚合酶链反应（Ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）体外扩增牛Ｙ染色体特异ＤＮＡ序列的方法。在牛、山羊和绵羊Ｙ染色体特异ＤＮＡ同源序列高度保守区设计一对引物，两条引物均为２０ＮＴ，对公牛Ｙ染色体特异的３０７ｂｐ序列进行体外扩增。从屠宰的成年公牛、母牛肝提取ＤＮＡ，用作ＰＣＲ扩增的模板。ＰＣＲ反应总体积为５０μＬ，各成分含量为：２５ｍｍｏ１／ＬＴｒｉｓ－ＨＣＩ（ｐＨ８．２），２５ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４、２ｍｍｏｌ／ＬＭｇＣｌ２、０．１ｍｇ／ｍＬｇｅｌａｔｉｎ、５％Ｆｏｒｍａｍｉｄｅ、ｌμｇ模板、引物各为２５ｐｍｏｌ、ｄＮＴＰ各为２００μｍｏｌ／Ｌ、ＤＮＡ聚合酶２ＩＵ。反应在微机控制ＰＣＲ仪上进行，采用９２．５℃变性，５５℃退火，７２℃延伸，共３５个循环。扩增产物在２％琼脂糖凝胶上电泳，用ｐＧＥＭ－７ｚｆ（＋）Ｈａｅ　Ⅲ作分子量标准。电泳结果，公牛个体样品显示清晰可见的特异ＤＮＡ扩增带，而母牛个体样品无扩增片段出现。扩增产物用ＰｓｔⅠ酶解后出现３条区带，与预期结果一致。     

应用聚合酶链反应技术检测沙门氏菌

利用聚合酶链反应（ＰＣＲ）检测沙门氏菌,使用了２对引物ＨＩ和ｐｈｏＰ。ＨＩ引物对各长２０ＮＴ,根据鞭毛Ｉ相抗原基因自行设计,扩增序列长２６９ｂｐ;ｐｈｏＰ引物对各长２１ＮＴ,根据ｐｈｏＰ／ｐｈｏＱ基因设计,扩增片段长２９９ｂｐ。ＰＣＲ采用５０μＬ反应体系。ｄＮＴＰＳ各１００μｍｏｌ／Ｌ,引物各１μｍｏｌ／Ｌ,Ｍｇ２＋２．５ｍｍｏｌ／Ｌ,酶２Ｕ。三温段ＰＣＲ循环条件为：９７℃预变性７ｍｉｎ;９４℃变性６０ｓ、５５℃复性４０ｓ、７２℃延伸６０ｓ,３５个循环;７２℃延伸７ｍｉｎ。检测８株标准阳性菌,结果都出现了ＨＩ引物和ｐｈｏＰ引物特异带,标准阴性菌３株只出现ｐｈｏＰ特异带,说明ＨＩ引物特异性很强。     

结缕草属植物ISSR-PCR反应体系研究

以SDS法提取的结缕草(Zoysia spp.)叶片DNA为模板,应用正交试验设计,从Mg²、dNTP、引物和DNA聚合酶并通过比较不同浓度的模板DNA对PCR扩增的影响,确立结缕草属植物ISSR-PCR反应的最佳体系。结果表明:各因素不同浓度对PCR反应结果有显著影响,正交设计可以用于ISSR反应体系建立,该方法建立的结缕草属植物ISSR-PCR优化反应体系为10X Buffer7、0 ng模板DNA、Mg²+2.0 mmol·L^-1、dNTP 150μmol·L^-1、Primer 0.3μmol·L^-1、Taq DNA聚合酶1.0 U,总体积20μL;经对11份结缕草属植物进行检验,证明该体系稳定可靠。该体系的建立可为今后利用ISSR标记技术进行结缕草属遗传多样性研究、种质资源鉴定和亲缘关系分析等提供依据。     

应用复合多聚酶链反应方法快速鉴别伪狂犬病弱毒疫苗与野毒

本研究根据伪狂犬病病毒（ＰｒＶ）共有ｇＢ和疫苗株缺失的ｇＥ基因序列分别设计合成１对通用（ＰＢ１／ＰＢ２）和１对鉴别引物（ＰＥ１／ＰＥ２）,以在我国广泛使用的疫苗株Ｂａｒｔｈａ－Ｋ６１及从国内外收集的野毒株Ｓ、ＳＵ、Ｆ、Ｌ、Ｙ、Ｍｉｎ－Ａ、Ｓｈｏｐｅ、Ｓ（川）、ＳＬ１、１０＃、ＥＡ（鄂Ａ）ＤＮＡ为模板在同一反应管中同时扩增ｇＢ和ｇＥ基因序列建立了复合多聚酶链反应（ＰＣＲ）方法。ＰＣＲ产物经２．０％琼     

４２份高粱与苏丹草及其２个杂交种犇犖犃指纹图谱的构建

选用１００个ＲＡＰＤ 引物和９５对ＳＳＲ 引物进行ＰＣＲ 扩增,旨在构建４２份高粱和苏丹草品种资源及２份国审品种高粱－苏丹草杂交种的ＤＮＡ 指纹图谱。结果表明,从１００个ＲＡＰＤ 引物中筛选到９个多态性高、重复性好的引物,多态性条带比率为６４．０６％,利用４个核心ＲＡＰＤ 引物可以为每份品种构建１张特定的数字指纹,并通过其中１个引物Ｆ ０１构建了１张能鉴别２个杂交种的ＲＡＰＤ 指纹图谱,不过该图谱不能区别皖草３ 号与其父本Ｓａ。从９５对ＳＳＲ 引物中筛选出多态性丰富的引物７３对,多态性条带比率为８６．０６％,通过３对核心ＳＳＲ 引物就可以构建４２份高粱和苏丹草的ＳＳＲ 数字指纹,同时利用其中１对ＳＳＲ 引物狋狓狆１８,寻找到２个杂交种的互补带,从而构建了２个高粱－苏丹草杂交种的ＳＳＲ 指纹图谱,这张ＳＳＲ 指纹图谱不仅能鉴别皖草２号和３号,还可以把杂交种与其亲本区别开来。     

伪狂犬病弱毒疫苗与野毒株PCR鉴别诊断方法的建立

目前，在国内用于预防猪伪狂犬病的疫苗主要是基因缺失的弱毒（Ｂａｒｔｈａ－Ｋ６１）为了有效地地区弱毒免疫猪和野毒感染猪，本实验分别建立了一种通用和一种鉴别ＰＣＲ诊断方法，根据已发表的伪狂犬病病毒（ＰｒＶ）ｇＢ，ｇＥ基因的序列，设计并合成了两对引物：ＰＢ１／ＰＢ２和ＰＥ１／ＰＥ２，以疫苗株Ｂａｒｔｈａｋ－６１及野毒株Ｓ，Ｓｕ，Ｆ，Ｌ，Ｙ及闽Ａ（Ｍｉｎ－Ａ）ＤＮＡ为模板进行了ＰＣＲ扩增，结果显示用引物Ｐ     

结缕草属植物种间关系和遗传多样性的SRAP标记分析

 通过ＳＲＡＰ标记技术对结缕草属植物５种１变种共９６份种源进行遗传多样性分析。结果表明,１）结缕草属种质间存在丰富的遗传多样性,５４对ＳＲＡＰ引物共扩增出３６２条清晰的用于多样性分析的谱带,其中多态性条带３３７条,多态性比率（ＰＰＢ）为９３．０９％,Ｎｅｉ’ｓ基因多样性指数（犎）为０．２４０９,Ｓｈａｎｎｏｎ信息指数（犐）为０．３７４６。２）应用Ｎｅｉ－Ｌｉ相似系数法估算了９６份材料间的遗传相似系数（ＧＳ）,其ＧＳ为０．５９３９～０．９８３４,平均为０．７５８８。不同种之间,结缕草与中华结缕草、沟叶结缕草与细叶结缕草中的部分材料遗传相似系数较高,遗传距离较近。大穗结缕草Ｚ０１０和长花结缕草Ｚ１２２与其他结缕草的遗传相似系数都相对较低。同一个种内,结缕草、中华结缕草和沟叶结缕草种内遗传变异均较大,ＧＳ分别为０．５９９４～０．９８３４,０．６２４３～０．９８０７和０．６６３０～０．９４７５。３）通过ＵＰＧＭＡ 分子系统聚类法,将９６份种源分为７个大的类群,其中第１大类和第２大类均包含结缕草、中华结缕草和少量的沟叶结缕草种源;第３大类群主要包括４份美国引进的材料;第４大类主要包括沟叶结缕草和细叶结缕草;大穗结缕草Ｚ０１０、长花结缕草Ｚ１２２和结缕草Ｚ１１５都单独聚为一类。从聚类结果可以看出,结缕草、中华结缕草和沟叶结缕草均有交叉聚类现象,并不是同一个种的材料完全相聚,个别种源自行一类,遗传基础与其他材料差异较大,从遗传聚类图可以很明确地看出９６份种源间的遗传距离及亲缘关系。     

鸭不同品种的随机扩增多态性DNA分析

随机扩增多态性ＤＮＡ（ＲａｎｄｏｍＡｍｐｌｉｆｉｅｄＰｏｌｙｍｏｒｐｈｉｃＤＮＡＳ,ＲＡＰＤ）是利用含有１０个（或９个）碱基的随机引物（单引物）通过聚合酶链式反应（ＰＣＲ）对模板ＤＮＡ进行随机扩增,根据扩增的某一ＤＮＡ片段的有无来比较不同基因组ＤＮ...     

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