PCR检测感染牛血液中边缘无浆体DNA

以边缘无浆体表面保护抗原的ｍｓｐｌβ基因设计和合成的１对２０ｍｅｒ寡核苷酸作为引物，用耐热的Ｔａｇ－ＤＮＡ聚合酶经５０个循环扩增边缘无浆体模板，扩增产物直接用凝胶电泳检测，并用标准分子量确定。结果表明，只有用边缘无浆体ＤＮＡ模板进行扩增时，扩增产物才能生成，而以血液原虫，如牛巴贝斯虫、双芽巴贝斯虫、锥虫和牛白细胞ＤＮＡ作模板扩增，无扩增产物生成。ＰＣＲ检测边缘无浆体的灵敏度可达约３００个无浆体的ＤＮＡ。边缘无浆体ＰＣＲ适用于牛边缘无浆体病的病因鉴定和检测     

PRV特异性糖蛋白gp50基因片段的克隆探针制备

在ＢＨＫ２１细胞中增殖伪狂犬病病毒（ＰＲＶ），提纯ＰＲＶＤＮＡ，选编码特异性糖蛋白ｇｐ５０基因中的２６２ｂｐ片段进行ＰＣＲ扩增。扩增产物经ＫｐｎⅠ和ＳａｌⅠ双酶切后，将２２２ｂｐ片段与质粒ｐＵＣ１９连接构成重组子ｐＵＰ。通过转化大肠杆菌ＪＭ８３、Ａｍｐｒ和α互补效应筛选后，扩增、提纯重组子ｐＵＰ，用ＫｐｎⅠ和ＳａｌⅠ酶切，电泳回收克隆的２２２ｂｐ片段。用光敏生物素标记２２２ｂｐ片段和重组子ｐＵＰ制备的探针，分别检出４６．８ｐｇ和９３．６ｐｇ的ＰＲＶＤＮＡ，且ｐＵＰ探针只与ＰＲＶ呈杂交阳性反应，与ＨＳＶ－１、ＨＳＶ－２及ＣＭＶ呈阴性反应。     

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     

传染性法氏囊病病毒VP2基因的克隆和鉴定

以鸡胚成纤维细胞培养ＩＢＤＶ弱毒疫苗株Ｄ７８，经蔗糖密度梯度离心纯化，电镜下见典型ＩＢＤＶ粒子。用ＳＤＳ－蛋白酶Ｋ法提取病毒基因组，低熔点胶回收，琼脂糖凝胶电泳，可见ＩＢＤＶ典型双节段基因组。根据已发表的ＩＢＤＶ基因组序列，设计并合成了１对特异扩增ＩＢＤＶＶＰ２基因的引物。以ＩＢＤＶ基因组为模板，利用ＲＴ－ＰＣＲ技术扩增出了１．５ｋｂ的ｃＤＮＡ产物，将ＰＣＲ产物适当处理后与经ＳｍａⅠ酶切的ｐＵＣ１９质粒平端连接，转化大肠杆菌ＤＨ５α，在含Ａｍｐ、Χ－ｇａｌ和ＩＰＴＧ的琼脂平板上培养，产生大量白色菌落。挑取白色菌落，经质粒大小比较分析初步筛选到一重组质粒。以重组质粒ＤＮＡ为模板作ＰＣＲ检测和部分酶切分析证实，该质粒为含Ｄ７８ＩＢＤＶＶＰ２基因的重组ｐＵＣ１９     

伪狂犬病病毒HS株tk基因的PCR扩增与克隆

参照伪狂犬病病毒（Ｐｓｅｕｄｏｒａｂｉｅｓｖｉｒｕｓ,ＰＲＶ）ＮＩＡ－３株ｔｋ基因序列,设计并合成１对长２２ｍｅｒ的引物,引物间距１．５ｋｂ,其内包含完整的ＰＲＶｔｋ基因。以ＢＨＫ２１细胞繁殖的ＰＲＶ湖北地方强毒株（ＨＳ－９３０４）基因组为模板进行ＰＣＲ扩增。琼脂糖凝胶电泳检测显示扩增主带清晰,长约１．５ｋｂ,符合设计要求。扩增片段克隆至由ｐＵＣ１８质粒改制而成的Ｔ载体中,限制性内切酶ＢａｍＨＩ、ＳｍａⅠ、ＸｈｏⅠ、ＨｉｎｄⅢ酶切分析证实,扩增片段的酶切位点与ｔｋ基因一致,说明扩增和克隆片段包含ＰＲＶｔｋ基因。     

用反转录—聚合酶链反应检测猪繁殖与呼吸综合征病毒的研究

根据猪繁殖与呼吸综合征病毒美洲型标准毒株的ＯＲＦ６及部分ＯＲＦ７的序列，设计合成了一对引物２６３７４ ＰＳ／２６３７５ＰＲ，用反转录－聚合酶链反应技术对６株ＰＲＲＳＶ北京分离株进行了检测。结果该引物对６株病毒均能扩增出与预期大小相符的ＲＴ－ＰＣＲ产物，并与ＡＴＣＣ ＶＲ－２３３２株扩增产物的大小相当，而欧洲型标准毒株未获扩增片段。     

应用多重反转录—聚合酶链反应检测鸡新城疫病毒和传染？…

本文建立了一种同时检测ＮＤＶ和ＩＢＶ二种病原体的多重ＰＣＲ。根据ＮＤＶ和ＩＢＶ的基因文库，设计了两对分别与ＮＤＶ与ＩＢＶ某段基因序列互补的引物，用这两对引物对同一样品ＮＤＶ和ＩＢＶＲＮＡ模板进行多重ＲＴ－ＣＰＲ扩增，结果均得到了两条特异性大步与实验设计相符的３１０ｂｐ和１７２０ｂｐ多重的ＲＴ－ＣＰＲ扩增带，而对其他６种禽病病原的ＰＣＲ扩增结果均为阴性，敏感性测定结果表明，该多重ＲＴ－ＰＣＲ技术能同     

鸡传染性喉气管炎病毒中国王岗株gX基因的克隆及鉴定

以ｐＵＣ１９质粒为载体克隆鸡传染性喉气管炎病毒（ＩＬＴＶ）中国王岗株的ＤＮＡ，构建了鸡传染性喉气管炎病毒ＤＮＡ的ＫｐｎⅠＤＮＡ文库。参考ＩＬＴＶ－ＳＡ２株ｇＸ基因的核酸序列，设计并合成了分别为１２ｂｐ和１３ｂｐ的１对引物。以ＩＬＴＶ中国王岗株ＤＮＡ为模板，用ＰＣＲ方法特异性地扩增出０．８４ｋｂ的ＩＬＴＶ－ｇＸ基因片段。以地高辛标记该０．８４ｋｂ的片段为探针，经Ｓｏｕｔｈｅｒｎ杂交从ＩＬＴＶ中国王岗株ＫｐｎⅠＤＮＡ文库中筛选出３个含５．２ｋｂ外源ＩＬＴＶＤＮＡ片段的ｇＸ基因阳性重组子。经酶切分析、Ｓｏｕｔｈｅｒｎ杂交、ＰＣＲ检测和该片段部分酶谱分析表明，ＩＬＴＶ中国王岗株ＤＮＡ５．２ｋｂ的ＫｐｎⅠ片段无论是片段大小还是酶切图谱均与ＩＬＴＶ－ＳＡ２株完全相同，而且Ｓｏｕｔｈｅｒｎ杂交和ＰＣＲ检测均为ｇＸ阳性，证明其中含有完整的ｇＸ基因     

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     

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。     

白术、微米白术和白术多糖对断奶仔猪生长性能和肠道形态及微生态区系的影响

试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。     

Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia

Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.     

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.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.     

Feline intracranial neoplasia: retrospective review of 160 cases (1985-2001)

The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.     

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.