犬瘟热病毒OP株囊膜糖蛋白F基因片段表达产物的免疫原性

根据已发表的犬瘟热病毒OP株核酸序列设计2对引物，以犬瘟热病毒OP株感染Vero细胞收获病毒提取的RNA为模板，应用RT—PCR扩增出F基因的769、832bp片段。将PCR产物按正确的阅读框架定向克隆进pGKX—6p—1载体中谷胱甘肽—S—转移酶(GST)基因的下游，再将重组质粒转化入宿主菌BL21中，在37℃ 1．0mmo1/L IPTG诱导下，F基因2个片段获得了良好的表达。表达产物经聚丙烯酰胺凝胶电泳鉴定，确定所表达的融合多肽大小分别为54000、56000。将表达产物回收后混合免疫小鼠，IFA显示，免疫小鼠血清能与病毒感染细胞呈现特异反应，并表现出1：16的中和抗体活性，表明体外表达的F多肽保留了天然蛋白的部分抗原性。     

犬瘟热病毒OP株囊膜糖蛋白F基因片段表达产物的免疫原性

根据已发表的犬瘟热病毒OP株核酸序列设计2对引物,以犬瘟热病毒OP株感染Vero细胞收获病毒提取的RNA为模板,应用RT-PCR扩增出F基因的769、832 bp片段.将PCR产物按正确的阅读框架定向克隆进pGEX-6p-1载体中谷胱甘肽-S-转移酶(GST)基因的下游,再将重组质粒转化入宿主菌BL21中,在37℃1.0 mmol/LPTG诱导下,F基因2个片段获得了良好的表达.表达产物经聚丙烯酰胺凝胶电泳鉴定,确定所表达的融合多肽大小分别为54 000、56 000.将表达产物回收后混合免疫小鼠,IFA显示,免疫小鼠血清能与病毒感染细胞呈现特异反应,并表现出116的中和抗体活性,表明体外表达的F多肽保留了天然蛋白的部分抗原性.     

犬瘟热病毒Yunnan株H基因的克隆与表达

根据已发表的犬瘟热病毒(CDV)Onderstepoort株的序列设计两对引物,以犬瘟热病毒云南株感染的Vero细胞收获病毒提取的总RNA为模板,RT-PCR扩增出H基因的939 bp(H基因前段,H1)和920 bp(H基因后段,H2)片段,分别将其定向克隆于PET-28a( )中,将重组质粒转化宿主菌BL21(DE3)plysS,在35℃1、.0 mmol/L IPTG诱导下获得良好表达,经SDS-PAGE鉴定,表达的融合蛋白为34 ku,与预期大小一致。免疫印迹试验显示,该重组蛋白可被CDV Onderstepoort株多克隆抗体识别,表明该重组蛋白具有抗原性。     

犬瘟热病毒H蛋白的原核表达及免疫原性的初步鉴定

本研究旨在对犬瘟热病毒(CDV)疫苗株H基因进行克隆及原核表达,并对产物的免疫原性做初步鉴定。根据犬瘟热病毒参考株Ondetstepoort的H基因序列,去除信号肽序列并选取其主要抗原表位设计引物,用反转录—聚合酶链式反应(RT-PCR)扩增目的片段;产物克隆至表达载体pET28b并转化宿主菌Rosetta^TM,优化诱导表达条件,纯化目的蛋白,并进行SDS-PAGE鉴定、Western blotting分析及间接酶联免疫吸附试验(ELISA);并将纯化的H蛋白免疫小鼠,进行中和试验检测抗体效价。结果显示,PCR扩增得到1113 bp DNA片段;在37 ℃、1.0 mmol/L IPTG诱导条件下可获得较高水平的表达;经SDS-PAGE鉴定,表达的H蛋白分子质量为42.38 ku,与预期值相符;Western blotting显示,在42.38 ku出现特异性目的条带;ELISA结果显示,表达的H蛋白能被抗CDV抗体识别,但与正常血清未发生非特异性反应;中和试验结果表明,血清中和抗体效价约为2^－3.3。结果提示,H蛋白获得了正确表达,对CDV抗血清具有特异反应性,可作为实时检测动物机体免疫状况检测的候选抗原,为进一步研制犬瘟热抗体检测ELISA试剂盒和新型疫苗奠定基础。     

犬瘟热病毒的静水压灭活

以250MPa的静水压力对犬瘟热病毒（CDV）处理30min之后，CDV被灭活，由其制成的灭活疫苗诱导犬产生中和抗体的能力明显优于传统的福尔马林灭活疫苗，免疫保护率达90％。电镜观察显示，经不同程度静水压力处理后的CDV粒子均发生变形，表面呈现凸起，但CDV H基因部分片段的RT－PCR扩增结果揭示，高达510MPa的静水压力作用30min也未能使其基因片段受到破坏。     

犬瘟热病毒H基因的序列分析与表达

以临床疑似犬瘟热病犬外周血总RNA为模板,采用RT-PCR方法扩增犬瘟热病毒(CDV)血凝素(H)基因。序列测定和分析表明该病料犬瘟热病毒H基因序列与国内其他地区分离到的毒株同源性较高(94.6%~99.2%),而与Onderstepoort株、Convac株等疫苗毒株的同源性较低(90.4%~91.2%)。分子遗传进化分析显示所有毒株可分为7个大的分支,且各分支间具有一定的地域性,其中待检病料中CDV与大多数中国分离株一样处于Ⅰ型。进一步以H全基因为模板,截短表达其3′端816 bp、459 bp两个片段,并克隆入原核表达载体pET30 a进行表达。结果显示它们分别能表达大小约为36.4 ku和22.2 ku的融合蛋白。免疫转印表明该纯化蛋白均可与犬瘟热病毒抗血清发生阳性反应,说明重组蛋白具备抗原性,可用于进一步的血清学研究。     

犬瘟热病毒小熊猫株H、F和N基因的克隆及表达

根据GenBank中发表的犬瘟热病毒（CDV）的核苷酸序列，设计并合成了扩增CDVH、F和N基因的3对引物，经RT—PCR分别扩增获得了CDV小熊猫株（LP株）H、F和N基因，并对H、F及N基因进行了克隆和序列测定。序列分析表明，CDV LP株属于强毒谱系，与CDV流行株的亲缘关系近．H基因含有较多潜在的糖基化位点．F和N基因相对比较保守。将CDV LP株H、F和N基因克隆入真核表达栽体pVAX1的CMV启动子下游，构建了CDV基因疫苗表达载体pVAXLPH、pVAXLPF、pVAXLPN，体外转染BHK-21细胞．用间接ELISA方法检测到目的蛋白的表达。用构建的3个表达质粒免疫小鼠，从小鼠血清中检测到了抗CDV抗体．初步证实用CDVH、F和N基因作为核酸疫苗免疫动物，可以激活机体的免疫应答。     

犬瘟热病毒中国分离株囊膜糖蛋白基因免疫小鼠诱发的抗体应答

将CDV中国分离株(YZ0101)的两囊膜糖蛋白基因F和H与pGEM—rreasy载体构建的重组质粒分别采用EcoRI和Kprd、BamHI和Kpnl双酶切后定向克隆进真核表达载体pcDNA3．1(-)中，DNA测序和限制性酶切分析筛选阳性克隆pcDNA-H和pcDNA-F，以重组质粒DNA和脂质体共转染COS-7细胞，用间接免疫荧光试验验证转染的COS-7细胞胞浆中分别表达了CDV的H和F蛋白。以聚乙烯胺(PEI)为佐剂，将犬瘟热病毒F和H基因重组质粒pcDNA-H和pcDNA-F的DNA分别或混合肌注6周龄BABI／c小鼠，同时设pcDNA原载体DNA对照。以2周为间隔共免疫4次。最后一次免疫后3周，采血分离血清，酶联免疫吸附试验(ELISA)和病毒中和试验(SN)分析基因免疫在小鼠体内诱导的免疫应答。结果显示：pcDNA-H和pcDNA-F试验组免疫4次后分别激发了10^2.99 0.134、10^2.93 0.164滴度的ELISA抗体；两种质粒DNA混合免疫后产生了1：32～1：128的抗CDV的中和抗体；而原载体DNA免疫后，用ELISA和SN未检测出特异的CDV抗体。表明犬瘟热病毒囊膜糖蛋白F和H基因免疫小鼠可诱发产生特异性的体液免疫。     

犬瘟热病毒GZ-Z株H基因原核表达载体的构建

利用实验室构建的含有犬瘟热病毒H基因的p MD18-H质粒,根据其序列设计带有Bam HⅠ和HindⅢ酶切位点的引物,对H基因ORF进行PCR扩增,得到约1 949 bp的片段。将该片段克隆到p MD18-T载体内,用Bam HⅠ和HindⅢ进行双酶切鉴定、质粒PCR鉴定,筛选出阳性克隆。将阳性克隆再用Bam HⅠ和HindⅢ进行双酶切,纯化回收CDV H基因ORF片段;将原核表达载体p ET32a(+)用同样的方法酶切,回收载体片段,并用T4连接酶将以上两回收片段连接,构建原核表达载体质粒p ET32-H,后经Bam HⅠ和HindⅢ双酶切、质粒PCR、测序进行系列鉴定。结果表明,p ET32-H原核表达质粒构建成功,其中插入片段大小为1 842 bp,可编码607个氨基酸残基。该实验为下一步H蛋白的原核表达及单克隆抗体的制备奠定了基础。     

犬瘟热病毒重组H蛋白间接ELISA检测方法的建立

为建立一种检测水貂犬瘟热病毒(CDV)血清抗体的间接ELISA方法,将CDV的血凝素蛋白(H)基因的主要抗原表位片段克隆至p ET-30a-c(+)载体中,转化至感受态细胞Rosetta(DE3)中进行诱导表达,并对表达产物进行SDS-PAGE分析和Western-blot鉴定。以纯化的重组H蛋白作为包被抗原,建立了检测CDV抗体的间接ELISA方法,并证实该方法具有良好的特异性、重复性和敏感性。采用建立的方法与国外商品化的犬瘟热抗体检测试剂盒对80份血清进行检测,两者符合率为92.5%。本研究建立的间接ELISA方法为水貂抗体水平检测和评价犬瘟热疫苗免疫效力提供了简便快速的方法。     

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.     

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.     

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.     

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.     

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.