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1.
以柔嫩艾美耳球虫（Eimeria tenella）敏感株、地克珠利抗药株和马杜拉霉素抗药株孢子化卵囊为材料，用银染mRNA差异显示方法筛选和克隆与球虫抗药性相关的基因。首先提取这3个虫株孢子化卵囊的总RNA为模板．用Oligo（dT）12GG为锚定引物和2个10碱基随机引物组合进行RTPCR，产物经变性聚丙烯酰胺凝胶电泳后银染。分别切取5务差异条带，进行2次PCR扩增，产物回收后与PGEM—T—easy Vector连接转化。经PCR鉴定正确后，再进行斑点杂交试验、序列分析和同源性比较。结果发现，地克珠利抗药株和马杜拉霉素抗药株分离的差异片段中都有2个cDNA片段（可能为新的基因片段），这为克隆全长cDNA和探索球虫抗药性产生的分子机理奠定了一定的基础。  相似文献   

2.
为建立猫杯状病毒(FCV)基于重组酶介导的等温核酸扩增(RAA)结合CRISPR-Cas12a系统及侧向流层析试纸条(LFS)(RAA-CRISPR/Cas12a-LFS)的快速检测的方法，本研究基于FCV衣壳蛋白(VP1)基因序列保守区设计RAA引物、4对针对VP1靶标的扩增引物、4对VP1-cr RNA扩增引物和1条报告探针。利用4对VP1-crRNA扩增引物经退火和T7 RNA聚合酶转录合成4条靶标crRNA:VP1-1/2/3/4-crRNA，利用4对VP1靶标扩增引物经退火合成4条靶标双链DNA:VP1-1/2/3/4，经EnGen?Lba Cas12a (Cpf1)核酸酶试剂的切割反应和Cas12a试纸条检测，根据出现条带的强弱筛选最佳VP1-crRNA。结果显示，VP1-2-crRNA对应的T线条带最强且C线不完全切割的条带最弱。因此选择VP1-2-crRNA进行后续试验。分别以FCV VR-782株及6株分离的FCV RNA为模板，利用RAA引物扩增其VP1基因，并通过琼脂糖凝胶电泳检测并评估RAA引物的扩增效果；利用Cas12a (Cpf1)核酸...  相似文献   

3.
为了研究PCR检测感染小鼠血液中旋毛虫DNA的敏感性,应用旋毛虫1.6 kb重复序列为扩增靶序列对旋毛虫(T1)、乡土旋毛虫(T2)、布氏旋毛虫(T3)、伪旋毛虫(T4)和南方旋毛虫(T7)肌幼虫DNA进行PCR扩增,并检测小鼠感染20、100、300条T1肌幼虫后不同时间的外周血.结果表明,T1、T4和T7肌幼虫可扩增出特异性目的条带(510 bp),而T2和T3无扩增产物;1、0.04和0.02条T1、T4和T7肌幼虫均能扩增到清晰的目的条带(510 bp).20条幼虫感染小鼠后5 d～6 d,PCR阳性率均为7.69%;100条幼虫感染小鼠后5 d～12 d可检出旋毛虫DNA,其中感染后5 d～7 d的阳性率分别为30.77%、38.46%及30.77%;300条幼虫感染小鼠后5 d～15 d可检出旋毛虫DNA,感染后7 d的阳性率为61.54%,感染后6 d与8 d～10 d的阳性率均为53.85%. 3组旋毛虫感染小鼠PCR阳性率间的差异有统计学意义(p<0.01),PCR阳性率随感染剂量的增加而升高(p<0.01),100条与300条感染小鼠感染后不同时间的PCR阳性率与检测时间有相关性(p<0.01).以上实验结果表明PCR检测感染小鼠血液中旋毛虫DNA的敏感性与感染程度和检测时间有关,对感染早期旋毛虫抗体阴性宿主有一定诊断价值.  相似文献   

4.
选用Avine肉用仔鸡作为试验对象 ,饲喂硫酸锌和蛋氨酸锌 2种锌源。采用银染差异显示技术进行差异基因的初步筛选。结果成功回收扩增差异条带 2 6条 ;其中加锌组 (硫酸锌组和蛋氨酸锌组 ) 2 3条 ,3条为缺锌组所特有。在加锌组的 2 3条中 ,6条为蛋氨酸锌组所特有 ,1 3条为硫酸锌组所特有 ,其余 4条为 2组所共有。这些初步筛选得到的差异基因尚需进一步进行克隆、测序和杂交进行验证。  相似文献   

5.
本文采用Trizol试剂提取毒害艾美耳球虫第二代裂殖子总RNA,用oliogo(d T)磁珠法纯化m RNA后,采用SMART技术构建了毒害艾美耳球虫第二代裂殖子cDNA噬菌体表达文库。经鉴定,原始文库容量为2.14×106pfu/m L,扩增后的文库容量为4.47×1010pfu/m L,扩增文库的重组率为90%。随机挑取100个单克隆,通过菌液PCR检测得知文库插入片段长度以400~1000 bp为主。本研究结果为毒害艾美耳球虫新基因的筛选和功能研究奠定了基础。  相似文献   

6.
有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库的构建   总被引：10，自引：2，他引：8  
利用 Stratagene公司 Hybri ZAP- 2 .1建库试剂盒构建了经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库。采取健康鲤鱼外周血 ,分离白细胞 ,经 L PS、PHA和 Con A分组作用不同时间后 ,分别提取总 RNA,将各组样品混合后 ,以 Oligo(d T)纤维素柱分离纯化 m RNA,经逆转录合成 c DNA的第 1链和第 2链 ,与 Eco R 接头连接 ,酶切后 ,用CHROMA SPIN- 4 0 0柱离心层析纯化 ,与 Hybri ZAP- 2 .1载体连接 ,体外包装后转染 E.coli XL I- Blue宿主菌 ,进行滴度测试和文库扩增。结果构建的经有丝分裂原刺激的鲤鱼外周血白细胞 c DNA文库原始库容量为 1.6 7× 10 6 pfu,插入片段长度在 0 .4× 10 3～ 3.0× 10 3bp,重组率为 98.9% ,扩增后的文库滴度为 6 .16× 10 9pfu/m L ,该文库符合 c DNA文库构建的标准 ,为克隆和研究鱼类免疫应答相关基因提供了有效的工具  相似文献   

7.
禽流感病毒M2蛋白跨膜区基因的缺失   总被引：5，自引：1，他引：5  
根据禽流感病毒 (AIV ) A/ Chicken/ Korea/ MS96 / 96 (H9N2 )株的核苷酸序列 ,设计并合成引物 ,通过 RT- PCR,从AIV H9N1株感染的 MDCK细胞总 RNA中扩增出 2 94 bp的 AIV全长的 M2基因。通过软件分析其序列中的跨膜区后 ,将其与p GEM- T easy载体连接产物 p GEM- T/ M2为模板 ,通过 PCR扩增出约 90 bp左右 M2的膜外区编码序列和约 16 0 bp左右 M2的胞内区编码序列。将两个扩增产物同时作为模板 ,以 OE- PCR(overlap extension- PCR)扩增出约 2 5 0 bp的缺失跨膜区的 M2基因M2 d。测序结果表明 ,M2 d的序列除在跨膜区以 4个甘氨酸序列替代外 ,其余部分与 M2完全一致 ,由此说明 OE- PCR扩增法成功地将禽流感病毒 M2基因跨膜区缺失  相似文献   

8.
猪垂体特异性转录因子1基因cDNA的克隆及序列分析   总被引：2，自引：2，他引：0  
本试验根据公开发表的猪POU1F1基因序列设计1对引物,提取猪垂体总RNA,通过RT-PCR方法扩增出POU1F1基因cDNA全序列,扩增产物用琼脂糖凝胶检测为预期的876 bp特异性条带。将扩增产物克隆入PTZ57R/T载体进行序列测定。经DNAStar软件分析序列与已发表序列同源性为99.7%,克隆获得的序列为进一步对猪POU1F1基因不同拼接形式功能性研究奠定了基础。  相似文献   

9.
竞争性RT-PCR检测铜对Ctr1 mRNA表达的影响   总被引：3，自引：0，他引：3  
为了应用竞争 RT- PCR法检测铜对猪传代肾细胞 (PK15 )中特异性铜转运蛋白 Ctr1基因 m RNA表达水平的影响 ,经 2次克隆 ,将筛选出一段 4 0 0 bp的核苷酸序列 ,插入 RT- PCR扩增出的 5 30 bp Ctr1目的片段中 ,构建了插入突变型 Ctr1- c DNA竞争模板。再将竞争模板与各试验组 c DNA同时进行 PCR扩增。结果 ,猪肾 PK15细胞 Ctr1基因m RNA表达量在 7.8～ 31.2 μmol/ L Cu范围内随铜添加浓度的升高减少 ,当铜添加浓度达到 6 2 .5 μmol/ L 时 ,其表达量又有所回升 ,呈双相反应。  相似文献   

10.
为了获得意大利蜜蜂重要农艺性状的分子遗传标记 ,本研究以 30种RAPD随机引物对平湖浆蜂、美意、澳意、苏意等 4个在产浆、采蜜和抗病性上存在较大差异的意蜂品系的基因组DNA进行RAPD_PCR扩增筛选。试验结果显示 ,随机引物W 1 8(5′_CGGACGGCGG_3′)和W2 3(5′_GTACCGCCCG_3′)的扩增图谱呈多态性。其中 ,在引物W 1 8所扩增出的 9条片断中 ,2 35 2bp和 36 4bp条带为美意所特有 ,表明可将之用于鉴别美意 ;2 0 92bp条带仅出现于蜂蜜高产的美意和澳意中 ,意味着该条带为一个蜂蜜高产性状的遗传标记 ;而 6 32bp条带在王浆高产品系平湖浆蜂中出现的频率 (0 91 )显著高于 (P <0 0 1 )美意(0 0 4 )、澳意 (0 0 2 )和苏意 (0 0 0 ) ,说明 6 32bp条带可能为王浆高产性状的一个遗传标记。在W2 3引物扩增条带中 ,6 5 1bp条带为澳意所特有 ,可用于澳意鉴别  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.
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   

13.
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   

14.
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.  相似文献   

15.
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   

16.
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   

17.
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   

18.
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   

19.
Infectious diseases have always been a terrible scourge for humans. The appearance of these plagues, as they were called without distinction, was generally connected to various conditions: asters, climatic changes or religious reasons. The concept of contagious, and then infectious, diseases came slowly. Variolation, i.e. transmission of ‘virulent’ matter to induce a natural disease and the immunity against it, was brought from Constantinople to England by Lady Montague, in 1721. This ‘variolation’ technique was also often performed in veterinary medicine against diseases like sheep-pox or pleuropneumonia. As ‘vaccination’ is the term generally accepted for ‘immunisation’, variolation can be the word designating such a technique. The second period of the history of immunisation began, in 1880, with the studies of Pasteur and his collaborators. A great number of bacterial vaccines were developed: dead, live but attenuated or only parts of pathogens. The viruses were produced in animals, then in eggs and at last, in tissue cultures. Second generation vaccines appeared with genetic engineering: recombinant vaccines, vector vaccines, nucleic acids vaccines, and markers vaccines, among others. These novel technologies can permit the development of new ones and improve the quality of the vaccines already existing.  相似文献   

20.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
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