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1.
为建立以茨城病病毒(IBAV)VP7蛋白为诊断抗原的茨城病(IBAD)血清学检测方法,本研究克隆了VP7蛋白抗原性、亲水性较强部分的编码基因,并在原核表达系统中表达了可溶性截短VP7蛋白。用截短VP7蛋白建立了IBAV间接ELISA检测方法。确定的阳性血清的判定标准为不低于0.32。特异性试验表明建立的ELISA方法可以特异性检测IBAV阳性血清。建立的ELISA方法与中和试验结果比较,符合率为77%。用建立的ELISA方法检测70份来自云南的牛血清样品进行检测,IBAV血清阳性率为45.7%。该方法的建立为IBAD的诊断提供了一种简单快速的辅助手段。  相似文献   

2.
研究旨在表达流行性出血病病毒（EHDV） VP7蛋白并制备其多克隆抗体，为EHDV检测方法的建立提供材料。试验通过大肠杆菌进行VP7重组蛋白的诱导表达，通过镍离子亲和层析与透析进行重组蛋白的纯化与复性并免疫家兔制备多克隆抗体，通过间接ELISA、Western blotting与间接免疫荧光试验（IFA）分析多克隆抗体的效价与反应原性。结果显示，在37℃与IPTG （0.1 mmol/L）的诱导下，VP7重组蛋白（分子质量46 ku）以包涵体形式高效表达，纯化与复性处理后的重组蛋白纯度达94.52%，可与不同血清型的EHDV阳性血清特异性结合。制备的兔抗VP7蛋白多克隆抗体效价达1∶24 000；以制备的多克隆抗体为一抗，通过IFA与Western blotting检测到EHDV感染细胞中VP7蛋白的表达。本研究在大肠杆菌中实现了EHDV VP7蛋白的高效表达，制备的兔抗VP7蛋白多克隆抗体具有良好的反应原性与特异性，为EHDV诊断抗原的制备与血清学检测方法的建立提供了试验材料。  相似文献   

3.
根据GenBank上发表的犬细小病毒（CPV）VP2蛋白基因序列，设计并合成1对引物，通过PCR扩增VP2基因全长。将其克隆到pET一30a载体中，构建了原核表达载体pET—VP2，转化大肠杆菌Rosetta，表达并纯化了重组蛋白，Western—blotting证明该重组蛋白具有免疫原性。利用重组蛋白为抗原，通过方阵试验确定了包被抗原的最佳包被量为15．8ng／孔，血清的最佳稀释倍数为1：100，建立了检测CPV血清抗体的间接ELISA方法。  相似文献   

4.
利用反转录－聚合酶链反应（RT－PCR）扩增出A群猪轮状病毒（PRV）G5型OSU株长度约为1．0kb的基因片段，将其克隆到pMD18-T载体上进行测序鉴定，证明为该PRV的VP7蛋白基因，与已发表的该PRV，VP7基因序列的同源性为99．8％，将该基因黏端克隆至表达载体PblusBAChis C质粒中，酶切及PCR鉴定表明获得了PRV－VP7蛋白基因与PblueBAChisC质粒的阳性重组子，纯化该重组质粒并与线性杆状病毒DNA分子Bac-N-Blue共转染昆虫细胞sf9,5d后收获重组病毒，通过对重组杆状病毒DNA分子的酶切及PCR鉴定，确定PRV－VP7基因已正确投入，将经3次蚀斑筛选纯化后的该重组杆状病毒接种于sf9细胞大量增殖后，获得了重组杆状病毒PRV－VP7蛋白的真核表达。  相似文献   

5.
采用pBlueBacHis2A系统筛选鹅细小病毒（GPV）的检测抗原，利用PCR扩增和双酶切方法分别构建了含有NS2基因和VPl与VP3非重复序列基因的重组质粒pBlueBacHis2A-GPV-NS2、pBlueBa-cHis2A-GPV-VP（1-3），分别转染sf9细胞，得到重组病毒，分别表达出了54ku的NS2融合蛋白和24ku的VP（1-3）融合蛋白。Western-blotting和Dot-ELISA检测结果证实，表达产物具有特异性；间接免疫荧光试验证实，重组蛋白在细胞中获得了表达。  相似文献   

6.
兔出血症病毒VP60蛋白的原核表达及检测方法初步应用   总被引：1，自引：2，他引：1  
VP60是免出血症病毒（RHDV）的主要结构蛋白，也是免疫原性蛋白，与诱导抗病毒免疫反应直接相关，因而也是检测用首选抗原。本实验在原核系统pPRoEX^TMHTa中表达了VP60基因，经SDS—PAGE电泳中可见表达带。Western blot鉴定结果表明，重组蛋白可被RHD阳性血清特异性识别，具有相应的抗原性。将表达蛋白经SDS—PAGE电泳切胶纯化后免疫BALB／c小鼠，免疫血清经全病毒ELISA检测，效价可达1：3200-1：6400，经琼脂扩散试验检测效价为1：16。重组蛋白纯化、复性后，作为包被抗原初步建立了间接ELISA方法，并检测了48份免血清样品，与全病毒间接ELSIA试剂盒检测结果相同。实验结果表明．用原核系统表达的VP60蛋白．可以作为RHD新型诊断试剂的候选抗原。  相似文献   

7.
将扩增得到的PPV SC-1株VP2基因产物插入转移载体质粒pPI-2．EGFP中，构建了含PPV VP2基因及其EGFP报告基因的PRV基因缺失表达载体质粒pPI-2．EGFP．VP2。采用磷酸钙转染系统，将pPI-2．EGFP．VP2 DNA和PRV DNA共转染Vero细胞后通过直接荧光观察、Dot-blot核酸杂交筛选得到几株重组病毒，将其中一株（命名为SA215-B）DNA用BamHI酶切和Southern转印杂交进一步验证重组病毒构建成功，并通过SDS-PAGE电泳和Western免疫印迹检测表明PPV VP2基因在重组病毒内获得表达，产生大小约93ku的融合蛋白，同时融合蛋白中的PPV VP2结构蛋白仍然保持了原有的反应原性，表明成功构建了含绿色荧光蛋白基因的猪细小病毒病-伪狂犬病重组病毒。  相似文献   

8.
参照GenBank中犬细小病毒VP2基因序列设计了一对添加EcoRⅠ和XhoⅠ酶切位点的引物,对CPVVP2基因进行PCR扩增,克隆至pGM—T载体,获得重组质粒pGM—T—VP2,将重组质粒亚克隆到表达载体pET-32a上,获得表达重组子pET-32a—VP2,经EcoRⅠ和XhoⅠ双酶切鉴定后,序列分析确定该重组子YM株VP2基因ORF正确,转化至E．coli Rosetta（DE3）中,获得了基因工程菌株E．coli Rosetta（CVP2）,并对重组菌进行了诱导表达,鉴定及纯化。结果表明纯化样品中含有87kDa目的蛋白,Western blot检测发现该蛋白与犬细小病毒多克隆抗体发生反应,出现特异条带。E．coli Rosetta（CVP2）以包涵体形式表达出了CPV—YM株VP2,从而为进一步制备检测抗体效价的胶体金检测试剂盒的开发研究打下了良好的基础。  相似文献   

9.
根据已克隆的O型口蹄疫病毒VP1基因序列，设计了1对带有Sac Ⅰ和HindⅢ酶切位点的引物，用其将pMD18-T-VP1质粒中的VP1基因亚克隆到真核表达栽体pBlueBacHis2A中，成功地构建了重组表达质粒pBlueBacHis2A—VP1（633bp）。将pBIueBacHis2A—VP1（633bp）质粒与Bac-N—Blue^TM DNA共转染sf9昆虫细胞，蚀班筛选重组病毒后，将纯化的重组病毒感染sf9细胞，获得了32．6ku的目的蛋白条带。Dot—ELISA分析结果表明，该表达产物具有反应活性，可用于建立间接ELISA方法，进行口蹄疫病毒抗体检测。  相似文献   

10.
O型口蹄疫病毒VP1嵌合基因的构建及原核表达   总被引：9，自引：0，他引：9  
首先合成我国O型口蹄疫病毒2个疫苗毒株VP1基因的3个抗原袁位，与另外扩增的流行毒株VP1基因末端273bp片段相连,构建出O型VP1嵌合基因片段（VP1O）。然后，将VPIO基因连接到原核表达载体pET28a上，构建了重组表达质粒pET28a-VP1O，转化大肠杆菌BL21（DE3）进行诱导表达。SDS-PAGE电泳表明。VP1O基因在大肠杆菌中获得表达。Western blotting检测证实表达的VP1O蛋白具有良好的生物学活性。对表达蛋白通过包涵体洗涤的方法进行初步纯化，获得了较高纯度的VP1O蛋白。  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.
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.
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   

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   

17.
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   

18.
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   

19.
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   

20.
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   

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