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1.
鹅源副黏病毒NA-1株全长cDNA克隆的构建及鉴定   总被引:1,自引:1,他引:0  
应用cRACE法扩增鹅源副黏病毒NA-1株cDNA 5'末端.参照Peter等的方法设计引物,扩增鹅源副黏病毒NA-1株cDNA的3'末端.根据GenBank上发表的鹅源副黏病毒序列设计6对引物,应用RT-PCR方法分6段扩增此病毒结构基因序列,并将其连接完成后与连接有鹅源副黏病毒NA-1株末端序列的转录载体连接,构建得到全长cDNA克隆.鹅源副黏病毒NA-1株全长cDNA克隆成功构建,为下一步病毒的拯救奠定坚实的基础.  相似文献   

2.
鹅源副黏病毒NA-1株反向遗传操作体系的建立   总被引:1,自引:1,他引:0  
应用cRACE等方法扩增鹅源副黏病毒NA-1株cDNA5′末端和3′末端,分6段扩增得到病毒的结构基因序列,而后连接本室构建的TLH-T转录载体,构建其cDNA全长克隆。在引入分子标签和验证辅助质粒的功能后,4质粒系统共转染VT7细胞系,成功拯救出了具有感染性的鹅副黏病毒。鹅源副黏病毒NA-1株反向遗传操作体系的建立为进一步深入研究该病毒基因组的功能以及新型疫苗的研发奠定了基础。  相似文献   

3.
为了从分子水平上探讨企鹅源禽1型副黏病毒BP01的演化,根据GenBank登录的鹅源禽1型副黏病毒基因序列设计引物,运用RT-PCR方法于国内外首次测定了企鹅源禽1型副黏病毒全基因组序列,其全长为15 192nt.经分析表明BP01病毒基因组与SF02株和ZJ1株等鹅源禽1型副黏病毒同源性最高,分别为99.1%和99.0%;与IT-227/82和dove/Italy/2736/00等鸽源禽1型副黏病毒同源性分别为90.2%和88.1%;而与Hert/33和LaSota等鸡源禽1型副黏病毒的同源性最低,仅分别为87.9%/和83.2%.对F基因第47~435nt进行系统进化树分析和F基因第334~1 682 nt进行HinfⅠ、BstO Ⅰ、Rsa Ⅰ酶切位点分析,确定BP01株病毒为基因Ⅶ型.F蛋白裂解位点氨基酸序列为112R-R-Q-K-R-F117,符合禽1型副黏病毒强毒株的特性,经预测发现BP01株病毒F蛋白的七肽重复序列分剐在第143~189位氨基酸残基和第461~503位氨基酸.通过对主要毒力基因推导的氨基酸序列进行比较发现,水禽源与其它源F蛋白有14个氨基酸不同,HN蛋白上有2个氨基酸的不同,而P蛋白有20个氨基酸的不同,且在第151~166位氨基酸存在一个明显的突变域.  相似文献   

4.
以与报道的鹅源Ⅰ型副黏病毒和鸭源Ⅰ型副黏病毒基因序列同源性高、而与鸡新城疫病毒基因序列同源性低的鸭源Ⅰ型副黏病毒强毒株,通过紫外线照射和番鸭胚、SPF鸡胚及其成纤维细胞传接数十代后,经IVPI、ICPI测定和F基因序列分析为弱毒.  相似文献   

5.
为研究EIAV弱毒疫苗株(EIAV_(FCCV15))S2基因发生的稳定性突变对疫苗株特性的作用以及S2基因的功能,以EIAVFDDVl5感染性克隆质粒pFDDV3-8为模板,根据疫苗研制过程中S2基因发生的4个主要稳定性变异位点,构建及拯救出S2基因不同差异位点逆向突变为强毒株相应氨基酸的6株感染性克隆衍生毒株.经实时定量PCR、逆转录酶活性和western blot等检测表明,疫苗株S2基因4个稳定性变异位点全部逆向突变的感染性克隆衍生毒、,pFDDVS2rl-3-4-5在体外培养靶细胞中的复制水平低于亲本疫苗株感染性克隆衍生病毒和其他组合的逆向突变的感染性克隆衍生病毒,提示ELAV疫苗株S2蛋白4个稳定突变位点的综合作用可能是决定疫苗株和强毒株特性差异的因素之一.以上结果为体内感染S2基因逆向突变感染性克隆衍生病毒,进一步揭示S2基因在ELAV弱毒疫苗致弱过程中的作用提供了依据.  相似文献   

6.
李想  高超  李芹 《中国兽医学报》2014,(5):751-755,760
以新城疫病毒(NDV)LaSota株感染性克隆PCI-La为平台进行反向遗传操作,并利用PCR技术将其3′端和5′端非编码区分别替换为鹅源NDV NA-1株3′端和5′端非编码区,以及3′端和5′端非编码区同时替换,通过生物学软件DNAStar对替换后的序列进行比对分析,所替换的序列未出现基因突变现象,证明非编码区替换成功,从而分别获得3株突变全长感染性克隆pCI-rL-NL、pCI-rL-NT、pCI-rL-NL-NT;将所获得的这3株突变感染性克隆分别与辅助质粒pCI-NP、pCI-P、pCI-L共转染BSR细胞,进行重组病毒的拯救。结果显示,pCI-rL-NL、pCI-rL-NT两种质粒表达系统均可拯救到有感染活性的病毒粒子,其HA效价为24,23,HI试验及间接免疫荧光试验结果均为阳性,并且通过电镜观察到具有典型副黏病毒科特征的、有囊膜的NDV颗粒。本试验成功构建了2株NDV LaSota突变株的反向遗传操作系统,为后期进行重组NDV致病性的研究奠定基础,也为进一步研究筛选新型分子标记疫苗、病毒活载体疫苗和抗病毒药物提供了可能。  相似文献   

7.
为构建猫杯状病毒(FCV)弱毒株F9的感染性克隆,本研究利经PCR分段扩增了FCV各基因片段,并拼接出FCV F9全基因组序列。经测序显示,其1823位多出一个碱基造成该序列存在移码突变,经替换为pOK-2280 (已构建出的FCV强毒株的感染性克隆)相应序列并对差异氨基酸逐个突变,最终获得嵌合FCV F9全基因组的重组质粒pOK-F9H。将线性化的pOK-F9H体外转录,获得的加帽RNA转染F81细胞,得到了拯救病毒rFCV F9。经过对拯救病毒与亲本病毒的蚀斑形成能力、CPE形成能力和多步生长曲线分析,结果显示rFCV F9与亲本株在复制能力和体外生长能力方面基本一致,表明构建了嵌合FCV F9株的感染性克隆并拯救出重组病毒。本研究构建的嵌合FCV F9株感染性克隆可以为FCV的基因功能研究以及新型FCV疫苗的研发奠定基础。  相似文献   

8.
为构建禽网状内皮组织增生症病毒(REV)MD-2株的感染性克隆,将MD-2株的前病毒基因组全长克隆至pMD18-T Simple载体中构建MD-2全长质粒,经转染CEF细胞后进行病毒拯救及鉴定,并对拯救病毒进行了生物学特性研究。结果表明,成功构建了MD-2全长质粒,拯救病毒感染CEF细胞后用间接免疫荧光试验检测到病毒抗原;拯救病毒的生长特性与亲本病毒相比无明显差异。随后对env基因进行了多个位点的定点突变,并拯救出相应的突变株,结果显示env基因第1433位碱基突变后不能拯救出病毒,推断1433位的突变为致死性突变。MD-2株感染性克隆的构建和病毒拯救的研究为继续研究REV基因和蛋白功能提供了技术支持。  相似文献   

9.
为构建高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)与致弱PRRSV的重组病毒,本研究在PRRSV致弱株APRRS的全长感染性克隆pAPRRS与HP-PRRSV感染性克隆pJX143的基础上,利用SOE-PCR技术将强弱毒的nsp2区域进行互换,构建了nsp2基因替换的强弱毒PRRSV嵌合感染性克隆.将构建的嵌合克隆转染MA104细胞,4d后观察到典型的细胞病变.通过RT-PCR和间接免疫荧光证明获得了强弱病毒株之间nsp2基因互换的嵌合病毒.这些嵌合病毒的构建和相应反向遗传操作平台的建立,为进一步研究HP-PRRSV的致病机制及相关疫苗的研发奠定了基础.  相似文献   

10.
一株经鸭胚传递的鸭副黏病毒的分离鉴定及生物学特性   总被引:1,自引:0,他引:1  
从表现疑似副黏病毒症状的1日龄雏鸭和死亡鸭胚中分离到1株病毒,经HA、HI和病毒中和反应鉴定为副黏病毒,命名为SDFCH株。按常规方法测得SDFCH株的生物学毒力指标MDT、ICPI和IVPI分别为56.6 h、1.78和2.59,鸭胚半数致死量LD50为10-8.5,表明SDFCH株为副黏病毒强毒株。用分离病毒对11日龄鸭胚和7日龄雏鸭攻毒,孵化出的雏鸭和攻毒的雏鸭均出现明显的剖检变化。根据GenBank中NDV F基因序列设计合成了3对引物,通过RT-PCR扩增出了鸭源Ⅰ型禽副黏病毒(APMV-1)SDFCH株的F基因。与已发表副黏病毒F基因相关序列对比结果表明:SDFCH株与鸭、鹅源副黏病毒的同源性较高,核苷酸和氨基酸的同源性均在96.4%~98.0%之间,而与Lasota株同源性较低,核苷酸和氨基酸同源性仅为84.4%和87.9%。表明该毒株相对于传统的Lasota株已经发生了较大的变异。  相似文献   

11.
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。  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

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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.  相似文献   

20.
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.  相似文献   

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