鸭疫里默氏杆菌基因组文库的构建及免疫原性基因初步筛选

以血清1型鸭疫里默氏杆菌(RA)CH-1株为材料提取基因组DNA,采用限制性内切酶Sau3A进行部分酶切消化,纯化回收1.0~6.5 kb片段,用T4DNA连接酶将回收片段与经BamH酶切并去磷酸化的ZAP Express载体进行连接,用噬菌体包装蛋白包装。经测定包装滴度为5.23×106pfu/mL,蓝白斑筛选重组率为96.8%,表明已成功构建血清1型RA CH-1株部分基因组文库。在此基础上以RA抗血清为抗体探针进行免疫筛选,获得3个阳性克隆,经多次重复筛选后再体内删除,得到含有插入片段的质粒并测序。生物信息学分析结果显示,该序列(GenBank登录号:DQ151838)含有1个编码369个氨基酸的完整开放阅读框架,是尚未见报道的RA基因序列,并且存在多个预测的抗原表位。     

猪肺炎支原体基因组DNA文库的构建

将猪肺炎支原体Z菌株接种于A26液体培养基中培养，在收获菌体细胞后，提取和纯化其基因组DNA。并经EcoRI大量酶切获得4．0－8．0kb的DNA片段，与λgtll载体臂连接为重组λDNA，然后进行包装，感染于Y1090宿主菌，构建了猪肺炎支原体基因组DNA文库，并得以表达。     

瘤胃中小韦荣球菌H2基因文库的构建

为了构建小韦荣球菌H2基因的文库,研究提取小韦荣球菌H2总基因组,采用机械剪切方法获得DNA随机片段,将获得的DNA片段与Sma Ⅰ酶切质粒pUC18体外连接并电转化至大肠杆菌DH5α,在含有IPTG、X-gal和氨苄西林的LB琼脂平板上用蓝白斑法筛选含重组质粒的大肠杆菌,并进行扩增分析.结果表明:重组质粒经鉴定分析证实包含小韦荣球菌H2的3～4 kb插入片段,文库滴度为1×105 pfu/mL;根据公式N=ln(1-p)/(1-f)证明99%基因组包含在文库中.     

细菌人工染色体文库基因组DNA制备技术

试验旨在对基因组DNA的制备进行研究,为细菌人工染色体(bacterial artificial chromosome,BAC)文库的构建奠定基础。以豁眼鹅全血为试验材料,提取高质量的基因组DNA,分别采用HindⅢ、EcoRⅠ和BamHⅠ3种限制性内切酶对所提基因组DNA进行部分酶切,并利用控制酶切时间、设置不同的HindⅢ酶切浓度梯度对基因组DNA进行部分酶切。结果表明,HindⅢ为最佳限制性内切酶,并得到了最佳酶切用量(40U/μL)。该方法所制备的基因组DNA质量较好,可用于BAC文库的构建。     

吉氏巴贝斯虫cDNA文库的构建

用离心柱层析法除去以反转录合成的吉氏巴贝斯虫双链ｃＤＮＡ的短片段，加ＥｃｏＲＩ粘性末瑞，再以ＮｏｔＩ酶切，定向克隆到λｇｔ１１Ｓｆｉ－ＮｏｔＩ载体ＤＮＡ的ＬａｃＺ基因内。体外包装后，感染Ｙ１０９０菌，构建成含３．２８×１０５个重组子的表达型吉氏巴贝斯虫ｃＤＮＡ文库。酶切鉴定表明，白斑噬菌体ＤＮＡ均含有大小不等的ｃＤＮＡ片段，免疫学筛选显示文库中表达阳性的重组子数占总重组子数的８．９％。     

有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库的构建

利用 Stratagene公司 Hybri ZAP- 2 .1建库试剂盒构建了经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库。采取健康鲤鱼外周血 ,分离白细胞 ,经 L PS、PHA和 Con A分组作用不同时间后 ,分别提取总 RNA,将各组样品混合后 ,以 Oligo(d T)纤维素柱分离纯化 m RNA,经逆转录合成 c DNA的第 1链和第 2链 ,与 Eco R 接头连接 ,酶切后 ,用CHROMA SPIN- 4 0 0柱离心层析纯化 ,与 Hybri ZAP- 2 .1载体连接 ,体外包装后转染 E.coli XL I- Blue宿主菌 ,进行滴度测试和文库扩增。结果构建的经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库原始库容量为 1.6 7× 10 6 pfu,插入片段长度在 0 .4× 10 3～ 3.0× 10 3bp,重组率为 98.9% ,扩增后的文库滴度为 6 .16× 10 9pfu/m L ,该文库符合 c DNA文库构建的标准 ,为克隆和研究鱼类免疫应答相关基因提供了有效的工具     

抑制性消减杂交技术分析猪链球菌2型毒力相关基因

以猪链球菌2型四川分离强毒株458#为检测子,国际参考无毒菌株1330#为驱动子,用抑制性消减杂交方法寻找其基因组水平的差异.采用3种不同的限制性内切酶分别酶切458#与1330#基因组获得3套酶切片段,经消减杂交后构建猪链球菌2型强毒株458#特异DNA的差异文库,并对差异序列进行比较分析.结果表明,试验获得了3套差异文库共计42个特异性差异片段,所有差异片段均与已发表的猪链球菌2型05ZYH33株全基因组序列高度同源.构建了具有代表性的猪链球菌2型强毒株与国际无毒参考菌株基因组差异DNA文库,差异片段通过Blast比对,部分差异片段与已知功能基因如转座子,耐药基因、1型限制酶修饰系统、表面蛋白和毒力相关蛋白等有同源性,尚有许多差异片段属于未知功能蛋白或假定蛋白,其中可能包括与猪链球菌2型毒力相关的基因,为进一步分析猪链球菌2型可能的毒力相关基因奠定了基础.     

家猪Fosmid基因组文库的构建

本研究采用蛋白酶K和苯酚抽提法提取基因组DNA，通过物理方法随机剪切DNA，经T₄DNA聚合酶和T₄多聚核苷酸激酶末端修饰，琼脂糖凝胶电泳回收大小为25~40 kb的片段，与pCC1FOS载体连接，噬菌体包装蛋白体外包装，转染大肠杆菌EPI300，构建了家猪Fosmid基因组文库。文库容量为8.50×10⁹CFU/ml，平均插入片段大小约25 kb，用家猪MC1卫星DNA做探针对文库进行初步筛选，获得含该卫星DNA的阳性克隆，该文库的构建为进一步筛选并研究猪卫星DNA序列奠定了基础。     

氨肽酶N鼠源单链抗体T7噬菌体展示文库的构建

氨肽酶N(pAPN)是一种常见的猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)的细胞受体。为研究抗pAPN的单链抗体(scFv),试验设计一系列简并引物,通过RT-PCR方法从免疫了天然pAPN的BALB/c小鼠脾中克隆了免疫球蛋白轻链可变区(VL)和重链可变区(VH)的编码基因。通过重叠延伸PCR(SOE-PCR)用12个氨基酸的连接肽将VL和VH的扩增产物随机连接,从而获得单链抗体基因。将整个单链抗体基因与T7Select10-3b载体连接,通过体外包装获得了针对pAPN的鼠源ScFv噬菌体文库。pAPN单链抗体初级文库包含2.0×107个重组噬菌体克隆,扩增后的文库滴度达到3.6×109 PFU/mL。Bst NⅠ酶切分析和DNA测序结果显示,28个pAPN抗体初级文库的噬菌体克隆多样性良好。以pAPN C亚基重组蛋白为包被抗原,用噬菌体ELISA进一步验证抗pAPN单链抗体库的活性。本研究主要介绍了采用T7噬菌体展示系统构建猪冠状病毒TGEV和PEDV通用受体pAPN的鼠源单链抗体库及其鉴定。     

猪传染性胸膜肺炎放线杆菌基因组表达文库的构建及对小鼠的免疫保护作用研究

本研究应用限制性内切酶MboⅠ消化胸膜肺炎放线杆菌血清7型的基因组DNA，回收500bp～3000bp的片段，插入到真核表达载体pCDNA3．1（＋）的BamHⅠ酶切位点构建重组质粒，电转化大肠杆菌TG1，获得胸膜肺炎放线杆菌的基因组表达文库（大约含有10^6个克隆）。将该文库随机分为10个亚文库（10个Clone pool），分别提取每个亚文库的重组质粒免疫小鼠（n=130只），免疫剂量为100μg，免疫3次后以1型胸膜肺炎放线杆菌进行攻毒，通过相对保护率、肺组织荷菌数、病理组织学3个指标测定其对小鼠的免疫保护作用。试验结果表明，Clone pool3、Clone pool2、Clone pool6和Clone pool7的免疫效果明显优于其他免疫组，尤其是Clone pool3的免疫保护效果最佳。本研究成功构建了7型胸膜肺炎放线杆菌的基因组表达文库，从而为筛选和获得新的保护性抗原基因奠定基础。     

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 -