用PCR技术从一兔出血症病料扩增和检出2种病毒核酸

应用ＰＣＲ和ＲＴ－ＰＣＲ技术,对兔出血症病毒核酸作了鉴定,分别设计合成２对ＰＣＲ扩增特异性引物,即按兔出血症病毒中国分离的ＮＪ核酸酸序列合成１对引物（ＮＪ引物）,按国分离的ＦＲＧ株核酸序列民另１对引物（ＦＲＧ引物）,进行ＰＣＲ和ＲＴ－ＰＣＲ。     

鹅Ⅰ型禽副粘病毒融合蛋白基因3‘端cDNA片段的分子克隆

参考禽副粘病毒的融合蛋白基因（Ｆ基因）ｃＤＮＡ序列，设计并合成了１对引物，以他离到对鹅有致病力的Ⅰ型禽副粘病毒ＧＰＭＶ／ＱＹ９７－１株的核酸（ＲＮＡ）为模板，用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）对其融合蛋白基因３＇端进行扩增，获得了预计的８８４ｂｐ左右的片段。将该片段克隆进ｐＧＥＭ－Ｔ Ｅａｓｙ质粒载体，获得重组质料Ｔ－ＧＰＭＶＦ３＇，采用限制性内切酶和ＰＣＲ方法对该重组质粒进行鉴定，证明所克隆的     

分泌抗禽呼肠孤病毒单克隆抗体杂交瘤细胞株的建立与鉴定

采用腹腔的接种１次脾内注射禽呼肠孤病毒（ＡＲＶ）蛋白免疫小鼠，经３次融合后，共筛选８株分泌抗禽呼肠孤病毒（单克隆抗体杂交瘤细胞株，这８株ＭｃＡｂ均可与ＡＲＶＳ１１３３株，ＦＤＯ株发生反应，而与ＩＢＤＶ，ＭＤＶ，ＥＤＳ－７６病毒不发生反应，经亚类鉴定，ＡＥ７，ＡＦ８，ＢＤ１，ＤＨ１０，ＥＥ５为ＩｇＧ１；ＡＤ６，ＣＧ４为ＩｇＣ２ａ，ＡＧ７为ＩｇＧ２ｂ。腹水效价在１０＾３～１０＾５之间。     

应用聚合酶链反应检测鸡毒霉形体和滑液霉形形体的研究

利用鸡毒霉形体与滑液霉形体基因一定区域互补的序列,合成能分别对ＭＧ和ＭＳ目的基因的２对引物。和这２对引物对１０个国际标准的ＭＧ与ＭＳ菌株ＤＮＡ模板进行ＰＣＲ扩增,结果均得到与预期大小相一 的约７３２ｂｐ（ＭＧ）和２０７ｂｐ（ＭＳ）的ＰＣＲ产物。     

杜大长猪IGF-I基因的克隆及其序列分析

采用ＴＲＩzol法从杜大长杂交猪的肝脏中提出总ＲＮＡ,将其纯化后作为ＰＣＲ扩增模板 ,以设计的Ｐ１（５′－ＣＴＡＣＡＴＴＣＴＧＴＡＧＴＴＣＴＴＧＴＴＴＣＣ－３′）为引物合成ＩＧＦ－Ｉ基因cＤＮＡ的第１链,再以Ｐ１和Ｐ２（５′－ＡＴＧＧＣＣＣＴＧＴＧＣＴＴＧＣＴＣＴＣＣＴＴ－３′）为引物扩增到大小约为３６０bp的产和,并将其达隆至pＧＥＭ－Ｔ载体上。经筛选、酶切、我分析,表明该片段为ＩＧＦ－Ｉ基因的cＤＮＡ     

新城疫病毒（昌黎株）F蛋白基因的克隆和序列分析

以鸡胚尿囊液繁殖的新城疫病毒（昌黎株）,经差速离心浓缩后,提取ＲＮＡ,参考国外发表新城疫病毒Ｆ基因序列,设计并合成了一对长度皆为２６ｂｐ特异性引物,经ＲＴ－ＰＣＲ扩增出ＮＤＶ昌黎株部分Ｆ基因序列,将其插入本室构建的ＦｐＫＳ（－）,进行序列测定。序列分析表明该部分Ｆ基因长度为８１０ｂｐ,编码２６７个氨基酸,有４个半胱氨酸残基和２个糖基化位点,裂解位点区（１１２－１１７）氨基酸序列为Ｒ－Ｒ－Ｑ－Ｋ－Ｒ－Ｆ,与所有强毒株在这一区域的序列（Ｒ／Ｋ－Ｒ－Ｑ－Ｒ／Ｋ－Ｒ－Ｆ）相符,证明ＮＤＶ昌黎株为强毒株。     

一次试验中出现的问题及对特异性PCR反应条件的探讨(简报)

本试验根据Ｌａｍｂ等［１］报道的鸡生长激素基因序列，设计合成２对特异性引物ＰＭ３和ＰＭＰＳ１，它们的碱基序列分别是ＰＭ３ＦＯＲ５′－ａｔｃｃｃｃａｇｇｃａａａｃａｔｃｃｔｃ－３′ＲＥＶ５′－ｃｃｔｃｇａｃａｔｃｃａｇｃｔｃａｃａｔ－３′ＰＭＰＳ１ＦＯＲ５′－ｃｔａａａｇｇａｃｃｔｇｇａａｇａａｇｇｇ－３′ＲＥＶ５′－ａａｃｔｔｇｔｃｇｔａｇｇｔｇｇｇｔｃｔｇ－３′。通过ＰＣＲ反应从鸡基因组中扩增出含有ＧＨ基因内含子１和４的片段，再用ＭＳＰ１内切酶对产物做ＲＦＬＰｓ分析。由于特异性ＰＣＲ的条件要…     

犬γ干扰素基因的克隆与序列分析

根据已发表的犬γ干扰素（Ｃａ－ＩＦＮ－ｒ）ｃＤＮＡ序列设计引物,用植物血凝素（ＰＨＡ）有机锗（Ｇｅ－１３２）刺激体外培养的犬脾细胞产生ＩＦＮ,提取ＩＦＮｍＲＮＡ,并以其为模板进行返转录聚合酶链反应（ＲＴ－ＰＣＲ）扩增出４８０ｂｐ基因片段。经酶切鉴定后将其克隆到ｐＵＣ１９的ＳｍａⅠ,构建重组质粒ｐＵＣ．ＩＦＮ。经酶切、ＰＣＲ及序列分析鉴定,可证明克隆的基因为犬γ干扰素,与所发表的核苷酸序列同源率为９     

生长激素释放因子（GRF）的基因改造及化学合成

为了提高生长激素释放因子（ＧＲＦ）的体内活性,根据猪ＧＲＦ的天然序列,设计了改造后ＧＲＦ（１～３２）的基因序列（含信号肽）,两端加设ＥｃｏＲＩ、ＨｉｎｄⅢ的粘性末端,分４段由ＤＮＡ合成仪合成,每段长度分别为９５、９７、８６、１０６ＮＴ,２条链间有１５个碱基的粘端。用尿素变性ＰＡＧＥ纯化合成片段,用Ｔ４多核苷酸激酶对合成ＤＮＡ磷酸化,混合４个片段复性,然后用Ｔ４连接酶连接。提取ｐＢｌｕｓｃｒｉｐｔ质粒,用ＥｃｏＲＩ和ＨｉｎｄⅢ在Ｍｕｌｔｉｃｏｒｅｂｕｆｅｒ中酶切完全后,琼脂糖电泳,由ＧｅｎｅＥｌｕｔｅＡｇｒｏｓｅＳｐｉｎＣｏｌｕｍｓ回收酶切大片段。将该酶切片段与上述合成的ＧＲＦ基因由Ｔ４连接酶连接,并转化至氯化钙致敏的ＤＨ５α。选取重组菌落,提取质粒,用ＰＣＲ及双酶切鉴定阳性克隆。用Ｍｉｎｉｐｒｅｐ提取重组质粒,ＡＢＩ３７３自动测序仪测序。测序结果表明已克隆到合成的ＧＲＦ基因。     

分泌抗赭曲霉素A单克隆抗体杂交瘤细胞株的建立

用赭曲霉素Ａ（ＯＡ）－ＢＳＡ合成抗原免疫Ｂａｌｂ／ｃ小鼠，取免疫鼠脾细胞与ｓｐ２／０细胞融合，通过克隆和ＥＬＩＳＡ法筛选，建立三株泌抗ＯＡ单克隆抗体杂交瘤细胞株（３Ｃ１２，１Ｄ１２和２Ｇ７）。用间接ＥＬＩＳＡ法测定，细胞上清液抗体效价为１２８＾×（３Ｃ１２）和６４＾×（１Ｄ１２和２Ｇ７）腹水抗体效价为１０＾－７（３Ｃ１２，１Ｄ１２）和１０＾－６（２Ｇ７）。三株单抗（ＭｃＡｂ）均属ＩｇＧ类，分泌抗体     

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 -