三种抗原检测H1N1猪流感病毒抗体的比较

本研究采用昆虫杆状病毒表达系统制备了H1N1亚型猪流感病毒的HA蛋白、类病毒脂质体、病毒样颗粒,分别作为抗原进行包被,采用间接酶联免疫吸附实验（ELISA）检测H1N1猪流感病毒抗体.特异性实验结果表明,单独表达的HA蛋白和类病毒脂质体特异性良好,但是病毒样颗粒不能区分不同亚型的流感病毒;敏感性实验结果表明,类病毒脂质体作为抗原的敏感性最好;重复性实验结果表明,三种抗原的重复性好,ELISA实验的批间和批内变异系数均小于10％;稳定性实验结果表明,在4℃可至少稳定保存1年.综上所述,类病毒脂质体有较高的免疫反应性,与灭活全病毒相比有更好的安全性,在抗体水平测定方面有较好的应用前景.     

H5亚型禽流感病毒HA1基因在Sf9昆虫细胞中的表达及其抗原性检测

利用pBlueBacHis2杆状病毒载体在昆虫细胞Sf9中表达H5亚型禽流感病毒（AIV）的HA1基因。Western-blotting证明HA1在昆虫细胞中得到了表达，其产物具有抗原性；且不与H7、H9AIV的抗血清发生交叉反应，具有良好的特异性。Dot-ELISA结果显示，表达蛋白可以在非变性条件下与相应抗体作用，同样具有良好的抗原性。试验结果证明，表达的H5亚型AIV的HA1蛋白，为检测禽类体内是否感染H5亚型AIV和监测免疫禽类血清H5抗体水平提供了优良的检测抗原。     

H9N2亚型禽流感病毒荧光定量PCR方法的建立及初步应用

H9N2亚型禽流感病毒（Avian influenza virus, AIV）是我国主要流行的低致病性禽流感病毒亚型,因而对其进行病毒的监测与诊断显得极其重要。通过比对GenBank上H9亚型禽流感病毒HA基因序列,设计了一个特异性针对H9 AIV HA的探针。特异性和扩增曲线实验结果显示,该探针具有较好的扩增效率,且仅特异性识别H9亚型AIV。通过重组质粒pMD19-T-H9HA 10倍梯度稀释液为模板,进行荧光定量PCR。实验结果表明,本研究所建立的荧光定量PCR方法敏感性能达到100个DNA拷贝数,比传统PCR方法检测敏感性提高了1000倍。此外,本方法还能检测到10EID50个病毒,比传统PCR方法检测敏感性也提高了1000倍。通过批间和批内试验,结果表明,这两个试验的变异系数分别小于5%和1%,说明本研究的方法具有较好的重复性。此外,通过检测人工感染动物咽拭子,结果表明H9亚型AIV荧光定量PCR方法比传统PCR的方法检测敏感性提高了100倍。在临床样品的检测中,H9亚型AIV荧光定量PCR方法检测的阳性率比传统PCR法检测的阳性率提高了大约30%。综上所述,本研究所建立的H9N2亚型禽流感病毒荧光定量PCR方法具有较高的特异性、准确性和敏感性,对H9亚型禽流感疫情防控及其流行病学调查具有积极的科学意义。     

H5亚型AIV HA抗原表位重组蛋白McAbs的制备及应用

目的以H5亚型禽流感病毒(avian influenza virus,AIV)的血凝素(HA)抗原表位重组表达蛋白为抗原,探索H5亚型AIV单克隆抗体制备的简便有效途径。方法利用H5亚型AIV的HA抗原表位原核表达重组蛋白为抗原,4次免疫8～10周龄雌性BALB/c小鼠后,取小鼠脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA方法筛选能分泌单克隆抗体的杂交瘤细胞株,并对单克隆抗体的特性及初步应用价值进行测试。结果得到一株能稳定分泌针对H5亚型AIVHA抗原表位的特异性单克隆抗体杂交瘤细胞株,单克隆抗体ELISA效价达1:5×104,为具有IgK轻链的IgM亚类。该单克隆抗体能特异性地与H5亚型AIV产生肉眼可见的WesternBlot免疫印迹反应,与其它供试的禽病抗原无反应。H5N1亚型AIV阳性血清能有效阻断辣根过氧化酶标记的单克隆抗体与HA抗原表位重组蛋白的结合。结论本研究探索出一条利用H5亚型AIV的HA抗原表位重组表达蛋白为抗原的单克隆抗体制备的简便、高效途径,所制备的单克隆抗体稳定性好、效价高、特异性强,在H5亚型禽流感病毒及其血清学检测中有较高的应用价值。     

H9N2亚型禽流感荧光定量RT-PCR检测方法的建立及应用

为了研究H9N2亚型禽流感病毒(AIV)在家禽体内各个器官的分布,试验针对H9N2亚型AIV的HA基因设计1对特异性引物,同时设计1对扩增内参基因β-actin的引物,以阳性重组质粒作为标准品做标准曲线,建立荧光定量RT-PCR检测方法,并对人工感染H9N2的SPF雏鸡气管、胸腺、肺脏、肝脏、脾脏、肾脏、肠等器官进行3次重复检测。结果表明:该法线性关系R值均在0.99以上,检测极限均为10拷贝质粒DNA,比RT-PCR灵敏100倍以上;特异性好,批内和批间重复性试验变异系数均小于1%。通过比较各器官HA相对表达量,得出肠和胰腺中HA相对表达量最高,腺胃次之,脾脏、肝脏、胸腺、法氏囊中含量也比较高,气管、肺脏、肾脏中含量较低,其中肾脏含量最低。说明研究建立的H9N2亚型禽流感病毒荧光定量RT-PCR方法灵敏度高,特异性强。     

禽流感病毒HA1蛋白的原核表达及其免疫原性的初步研究

从H9N2亚型禽流感病毒感染的鸡胚尿囊液中提取病毒RNA,RT-PCR克隆全长血凝素(Hemagglutinin,HA)基因,并进一步克隆HA的主要抗原区HA1基因.将HA1克隆到原核表达载体pGEXKG,与谷胱苷肽(GST)融合表达,表达的融合蛋白GST-HA1以包涵体形式存在.包涵体经变性、复性处理,Western blot分析表明,表达蛋白具有良好的免疫学活性.ELISA检测发现,GST-HA1只能与H9亚型禽流感病毒抗体发生反应,而与H5和H7亚型禽流感病毒抗体无交叉反应.进一步将纯化的融合蛋白与佐剂混合后免疫Balb/c小鼠,免疫小鼠体内产生了较高滴度的特异性抗体.制备的HA1蛋白特异性强,具有良好的免疫原性,为禽流感病毒的鉴别诊断和禽流感疫苗开发奠定了基础.     

表达分泌型与膜结合型HH7N9 AIV血凝素蛋白重组NDV载体疫苗的免疫原性比较

研究以新城疫病毒(NDV)弱毒株LX为载体,构建了一株表达H7N9亚型禽流感病毒(H7N9 AIV)血凝素(HA)胞外区的重组病毒(LXs HA),并与一株前期构建的表达膜结合型HA的重组病毒(LXHAF)进行比较。生物学特性评价显示,两株病毒均为弱毒且在鸡胚中的繁殖性能较强。此外,LXs HA主要以分泌形式表达HA蛋白,而LXHAF则以膜结合形式表达HA。鸡的免疫试验显示,两株重组病毒在一次免疫后均能诱导针对NDV载体的血凝抑制抗体,但LXHAF能够诱导较高水平的H7N9 AIV特异性Ig Y抗体,而LXs HA不能诱导H7N9 AIV抗体应答。结果表明,表达膜结合型HA的重组病毒具有针对H7N9 AIV较好的免疫原性,而表达分泌型HA的重组病毒免疫原性较差。这为研发H7N9亚型禽流感载体疫苗提供新的信息与参考。     

H5亚型禽流感病毒反应原性差异及ELISA检测方法的研究

在获得禽流感病毒多克隆抗体及H5亚型特异性单克隆抗体的基础上,建立了H5亚型特异性抗原捕捉ELISA检测方法。通过分析不同单克隆抗体与不同禽流感毒株的反应性差异,筛选高特异性和高敏感性的H5亚型单克隆抗体。优化反应条件,确定包被抗体、检测抗体及酶标抗体的最佳工作浓度,进行敏感性、特异性、重复性及稳定性分析,并与斑点ELISA、H5N1多重RT-PCR或病毒分离鉴定的结果相比较,同时使用该方法对野外样品进行了检测。结果表明,该方法敏感、特异,具有良好的重复性和稳定性,可用于检测临床样品、鸡胚培养物及细胞培养物中的H5亚型禽流感病毒。     

H5亚型禽流感病毒荧光检测试纸条的研制

为了对引起家禽重大经济损失和具有公共卫生意义的H5亚型禽流感病毒(AIV)进行快速超敏检测,本研究研制了针对HA蛋白的4株单克隆抗体(MAb)杂交瘤细胞株,通过MAb的两两配对试验,以荧光量子点作为抗体标记生物材料,基于夹心法原理建立了H5亚型AIV荧光免疫层析检测试纸条。结果显示,该方法能够特异性检测出H5N1、H5N2等H5亚型AIV,而与H1N1、H3N2、H7N9、H9N2、NDV等其他病原无交叉反应;对标准检测抗原H5N1(Re-6)的最低检测量为1 ng/mL,比市售H5亚型AIV胶体金检测试纸条灵敏度高100倍;该方法的变异系数(CV)值在10%以内,与商品化的试剂盒检测结果符合率达99.8%。本文建立的H5亚型AIV荧光检测试纸条快速、敏感,检测结果准确可靠,为H5亚型禽流感的流行病学调查和快速诊断提供了基础。     

H3、H9亚型禽流感病毒双重荧光定量PCR方法的建立及初步应用

虽然H3、H9亚型禽流感病毒（Avian influenza virus, AIV）是低致病性禽流感病毒,但其具有跨宿主感染哺乳动物的能力,对社会公共卫生安全具有重大的潜在危害,因而对这些亚型病毒的监测极其重要。通过比对GenBank上H3、H9亚型禽流感病毒HA基因序列,设计了分别针对H3 AIV和H9 AIV的特异性探针。特异性试验和标准曲线试验的结果显示,这两个探针仅分别特异性识别H3、H9亚型AIV,且具有较好的扩增效率。通过重组质粒pMD19-T-H3HA和pMD19-T-H9HA 10倍梯度稀释液为模板,进行敏感性实验。结果表明,本研究所建立的荧光定量PCR方法能检测到100个DNA拷贝数H3 AIV和10个DNA拷贝数的H9 AIV,比传统PCR方法敏感性分别提高了100倍和1000倍。此外,本方法还能检测到10 EID50的H3亚型AIV或H9亚型AIV,比传统PCR方法检测敏感性均提高了1000倍。批间和批内试验的变异系数均小于3%,表明本研究建立的方法具有较好的重复性。此外,与传统PCR方法相比,用H3、H9亚型AIV双重荧光定量PCR方法检测人工感染动物的肺组织时,针对H3 AIV的敏感性提高了10~100倍,针对H9 AIV的敏感性提高了100倍。综上所述,本研究所建立的H3、H9亚型禽流感病毒双重荧光定量PCR方法具有较高的特异性和敏感性,对H3、H9亚型禽流感病毒监测具有重要的应用价值。     

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.     

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.     

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.     

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.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -