猪生殖与呼吸综合征病毒ORF6片段的cDNA克隆及鉴定

根据已发表的ＰＲＲＳＶ基因组序列，设计并合成了１对特异扩增ＡＴＣＣ ＶＲ－２３３２株的ＯＲＦ６的基因的引物，以ＡＴＣＣ ＶＲ－２３３２基因组为模板，通过ＲＴ－ＰＣＲ扩增出５８６ｂｐ的ｃＤＮＡ产物。将该ｃＤＮＡ片段经Ｔ－Ａ连接插入ｐＧＥＭ－Ｔｅａｓｙ载体中，用重组质粒转化大肠埃希氏菌ＪＭ１０９，获得重组质粒转化菌。对重南粒ＤＮＡ进行ＰＣＲ及酶切鉴定，证明插入的ＤＮＡ片段与ＰＣＲ扩增的ＤＮＡ片段大小相     

牛β—酪蛋白基因上游调控序列PCR扩增及克隆

采用蛋白酶Ｋ法从母牛肝中提取染色体ＤＮＡ,将其纯化后作为ＰＣＲ扩增模板。以引物设计软件从牛β－酪蛋白基因上游６００ｂｐ至第１个外显子设计引物,引物长度１９ｎｔ,跨度６３７ｂｐ。扩增产物电泳结果显示,在分子量Ｍａｒｋｅｒ６５７ｂｐ带附近,出现一明显亮带,这一结果与设计产物大小基本一致。用低温冷冻法纯化ＰＣＲ产物,煮沸裂解法提取载体ｐＢｌｕｅｓｃｒｉｐｔⅡＫｓ＋质粒,ＥｃｏＲＶ酶切质粒ＤＮＡ,Ｔ４ＤＮＡ连接酶连接ＰＣＲ扩增片段和质粒ＤＮＡ。将重组质粒ＤＮＡ转化到ＪＭ１０１大肠杆菌中,经筛选、酶切、测序鉴定,结果表明所克隆的片段为牛β－酪蛋白基因上游调控序列。     

猪生殖和呼吸综合征中国分离株ORF7基因的克隆及鉴定

本研究利用对应于ＡＴＣＣＶＲ－２３３２及ＬＶＯＲＦ７基因保守序列的１对均为２８个碱基的引物１００１１ＰＣＳ、１０１０ＰＣＲ对ＰＲＲＳＶ中国分离株Ｂ１３进行ＲＴ－ＰＣＲ，结果扩增出了１条包括完整ＯＲＦ７基因的５１０ｂｐ的ＤＮＡ片段。纯化此扩增产物，并对其进行ＥｃｏＲＩ／ＰｓｔＩ双酶切，与用ＥｃｏＲＩ、ＰｓｔＩ双酶切及碱性磷酸酶处理的ＰＵＣ１８载体连接。转化大肠杆菌。结果得到了１个Ｂ１３ＯＲＦ７基因与ＰＵＣ１８载体的重组质粒ＰＵＣ１８Ｂ１３ＯＲＦ７１０。通过ＥｃｏＲＩ／ＰｓｔＩ及ＰＣＲ证明此重组质粒即为Ｂ１３ＯＲＦ７基因与ＰＵＣ１８载体的重组质粒。从而为ＯＲＦ７基因及所表达的核衣壳蛋白进一步研究奠定了基础。     

猪瘟病毒兔化弱毒株5’端调控序列及P23基因的扩增与克隆

从猪瘟病毒兔化弱毒株（ＨＣＬＶ）的细胞培养液中直接提取ＲＮＡ，同时根据猪瘟病毒Ａｌｆｏｒｔ株的基因组序列化学合成了６条引物，应用“半巢式”ＰＣＲ方法扩增了ＨＣＬＶ两个ｃＤ－ＮＡ片段，其大小与推算的长度相符，依次为６９３ｂｎ和２７６ｂｐ。这２个片段包括了ＨＣＬＶ基因组５’端非编码区调控序列，编码具有蛋白酶活性的Ｐ２３蛋白的基因和编码大部分核衣壳蛋白Ｐ１４的基因。最后，按标准方法将这２个ｃＤＮＡ片段克隆到ｐＵＣ１８的ＳｍａＩ切点。     

番鸭细小病毒M91G27株病毒结构蛋白VP3编码基因的克隆与鉴定

纯化番鸭细小病毒Ｍ９１Ｇ２７毒株鹅胚尿囊液,通过ＰＣＲ技术,从病毒ＤＮＡ中扩增出病毒结构多肽ＶＰ３完整编码基因。将该ＰＣＲ扩增片在ＨｉｎｃⅡ和ＳａｃⅡ位点克隆进ｐＵＣ１８质料载体,酶切分析筛选到含１．６ｋｂ基因片段的重组质粒ＭＰ１３,进一步对该片段进行序列及用ＣＬＯＮＥ软件分析该序列。结果结盟,其与国外已报道毒株核苷酸序列有９８．８％的同源性,氨基酸序列有９８．１％的同源性,证明该重组质粒是ＶＰ３编码基因的克隆。     

新城疫病毒F48E9株HN基因的克隆与酶切分析

为深入探讨新城疫病毒（ＮＤＶ）的结构与功能的关系，扩增和克隆了Ｆ４８Ｅ９株ＮＤＶ的ＨＮ基因。首先以提取的Ｆ４８Ｅ９株ＮＤＶ基因组ＲＮＡ为模板逆转录合成第一链ｃＤＮＡ，然后用ＨＮ基因特异性引物作ＰＣＲ扩增ＨＮｃＤＮＡ，结果得到１条分子量约１．８ｋｂ的ＤＮＡ带，与ＮＤＶＨＮ基因的大小一致。Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交进一步证实其为ＨＮ特异性ｃＤＮＡ。随后采用平端连接法将其克隆到ｐＳＶ·Ｓｐｏｒｔｌ质粒中，应用限制性内切酶ＥｃｏＲＩ、ＳｃａⅠ、ＭｌｕⅠ、ＡｐａⅠ、ＰｓｔⅠ、和ＳｍａⅠ对ＮＤＶＦ４８Ｅ９株ＨＮｃＤＮＡ重组质粒作单酶或双酶切，结果表明，ＮＤＶＦ４８Ｅ９株ＨＮ基因较ＨｉｔｃｈｎｅｒＢ１株的ＨＮ基因缺少１个ＭｌｕⅠ位点（７０５ｂｐ左右），多１个ＰｓｔⅠ位点（８０２ｂｐ左右）。     

马立克氏病病毒（MDV）糖蛋白D基因的分离克隆,定序及其在大肠杆…

用聚合酶链式反应（ＰＣＲ）技术，从马立克氏病病毒（ＭＤＶ）ＧＡ株感染的鸡胚成纤维细胞（ＣＥＦ）基因组ＤＮＡ中扩增出ＭＤＶ糖蛋白（ｇＤ）抗原基因１２０９ｂｐ编码序列。将该ＰＣＲ扩增产物于ＥｃｏＲＩ和ＫｐｎⅠ位点克隆进行ｐＵＣ１８质粒载体中。将ｇＤ重组ｐＵＣ１８质粒ＤＮＡ用Ｄｉｇｏｘｉｇｅｎｉｎ（Ｄｉｇ）标记后，在Ｓｏｕｔｈｅｒｎ ｂｌｏｔ中，该探针能识别ＭＤＶ基因组ＤＮＡ的Ｂａｍ ＨＩ－Ａ克隆中的Ａ     

传染性支气管炎病毒Holte株S1基因的克隆及鉴定

为给鸡传染性支气管炎（ＩＢ）基因工程疫苗的研制提供基因储备,通过ＲＴ－ＰＣＲ扩增出ＩＢＶＨｏｌｔｅ株Ｓ１基因ｃＤＮＡ,其长度与理论值１７６２ｂｐ相符;Ｓ１基因内部位点经ＨａｅⅢ酶切所产生的片段,亦与理论值５４９、３４３、３２４、１９１、１８０、１７１ｂｐ６个片段大小一致。将Ｓ１基因ｃＤＮＡ克隆入ｐＵＣ１８,并对Ｓ１基因两翼进行了序列分析。结果表明,所克隆的Ｓ１基因与ＧｅｎＢａｎｋ中收录的ＩＢＶＨｏｌｔｅ株Ｓ１基因完全相符。     

产气荚膜梭菌β-毒素基因的克隆及CPB-ST融合基因的构建

用聚合酶链式反应（ＰＣＲ）技术,从Ｂ型产气荚膜梭菌染色体基因组中扩增了９３０ｂｐ的β－毒素基因。用限制性核酸内切ａｍＨ Ⅰ和ＥｃｏＲ Ⅰ对ＰＣＲ产物进行双酶切处理,然后,通过Ｔ４ ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的载体质粒ｐＥＴ－２８Ｃ（＋）的多克隆位点,转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨ Ⅰ和ＣｃｏＲ Ⅰ双酶切分析和ＰＣＲ扩增检测。证明重组质粒ｐＥＣＢ２中含有产气荚膜梭     

应用复合引物扩增大肠杆菌肠毒素基因的研究

以两对合成的不同寡核苷酸引物通过聚合酶链反应（ＰＣＲ）、扩增肠毒素大肠杆菌（ＥＴＥＣ）不耐热肠毒素（ＬＴ）和耐热肠毒素（ＳＴ）基因；两对引物从ＥＴＥＣＬＴ和ＳＴ基因中分别扩增出３１４ｂｐ，２３７ｂｐ的ＤＮＡ片段，均能与相应的ＬＴ和ＳＴ基因探针杂交。ＬＴ扩增产物（３１４ｂｐ）和ＳＴ扩增产物（２３７ｂｐ）分别和ＳｍａⅠ和ＨｉｎｃⅡ酶切后，产生１９０ｂｐ和１２４ｂｐ，１４７ｂｐ和９０ｂｐ的ＤＮＡ片段。同一扩增反应中应用ＬＴ和ＳＴ复合引物进行扩增，３种基因型ＬＴ、ＳＴ和ＬＴＳＴＥＴＥＣ从样品中鉴定出。１０９份动物腹泻粪样分别用ＰＣＲ，核酸杂交和ＥＬＩＳＡ进行测定，结果表明，ＰＣＲ是最为灵敏和快速的测定ＥＴＥＣ的方法。     

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.     

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 -