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。     

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。     

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。     

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.     

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.     

A brief history of the prevention of infectious diseases by immunisations

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.     

Micronutrient Content of Certain Tropical Conventional and Unconventional Feed Resources of Southern India

The ash, silica and certain important micronutrients were estimated in conventional and unconventional feed and fodder resources available in Southern India. Commonly used dry roughages, such as paddy straw, ragi straw, maize kadbi/stalk, jowar kadbi/stalk, bajra stalk and wheat straw, were high in ash (9.9% +/- 0.77%) and silica (6.4% +/- 0.65%) and low in most of the other micronutrients, except iron, with paddy straw containing most silica (>9%). Cultivated non-leguminous (maize, jowar) and improved green crops (hybrid napier, guinea, green panic, NB-21, CO-1) were also high in ash (10.5% +/- 0.60% and 12.5% +/- 0.51%) but were moderate sources of P, Mg and Cu and good sources of Zn (98 +/- 13.8 ppm and 55 +/- 6.7 ppm). Leguminous green fodders (stylosanthus, lucerne, cow pea, soyabean) were excellent sources of Ca (1.9% +/- 0.16%), Mg (0.40% +/- 0.05%), Cu (30 +/- 5.2 ppm), Zn (121 +/- 14.7 ppm) and Fe (1234 +/- 166 ppm) and moderate sources of P. Mixed local grasses and weeds were high in silica (6.9% +/- 1.00%) but were good sources of Cu, Zn and Fe. Cereal grains (maize, wheat, rice, ragi) were low in ash (2.9% +/- 0.33%) and were relatively poor sources of Ca (0.22% +/- 0.03%), Mg (0.19% +/- 0.03%) and Cu (13 +/- 3.1 ppm). Pulses were low to medium sources of most minerals and good sources of Fe (1230 +/- 293 ppm). Oil seed cake/extractions (groundnut cake, cotton seed cake, soyabean meal, sunflower cake, safflower cake) and cereal by-products (rice polish, rice bran, wheat bran) were excellent sources of P (1.1% +/- 0.47% and 2.3% +/- 0.19%) and good sources of Zn (65 +/- 3.9 ppm and 66 +/- 10.7 ppm) and Fe (938 +/- 130 ppm and 662 +/- 126 ppm). Among the unconventional feeds screened, orange peel, sunflower heads, meat meal, rubber seed cake, spirulina algae and sea weeds contained plentiful Ca, Zn and Fe: tree leaves/top feeds (mulberry, erythrina, glyricidia, banana, subabul, groundnut haulms) were excellent sources of Ca (1.5% +/- 0.13%), Zn (120 +/- 22.9 ppm) and Fe (1033 +/- 133 ppm) but relatively poor sources of P. Soyabean husk, cocoa seed husk, rubber seed cake and meat meal were moderate to good sources of P (1.0% and 0.33%). The high Zn and Fe values of most feeds/fodders were probably due to soil contamination. This account of the micronutrient content of feed/fodder resources should help in strategic supplementation intended to alleviate local deficiencies.     

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.     

Bovine Mastitis in Selected Areas of Southern Ethiopia

A study on bovine mastitis, designed to determine the causal agents, prevalence of infection and impact of risk factors in three cattle breeds, was conducted in selected areas of southern Ethiopia. A total of 307 lactating and non-lactating cows, of which 162 were indigenous Zebu, 85 Jersey and 60 Holstein-Friesian, were examined by clinical examination and the California mastitis (CMT) test. Of these, 40.4% were positive by CMT and bacteriology for clinical or subclinical mastitis, with prevalence rates of 37.1% and 62.9%, respectively. Out of 1133 quarters examined, 212 (18.7%) were found to be infected, 83 (39.2%) clinically and 129 (60.8%) subclinically. The prevalence of mastitis was significantly higher in Holstein-Friesian than in indigenous Zebu, in non-lactating cows than in lactating cows, in the early lactation stage than in the mid-lactation stage, in cows with lesions and/or tick infestation on skin of udder and/or teats than in cows without this factor, and in the wet season than in the dry season. Mastitis increased with parity number (R = 0.9). Of 248 CMT and clinically positive udder quarter samples analysed microbiologically, 212 were culturally positive for known mastitis pathogens and 36 were negative. Of the 199 positive samples, Staphylococcus accounted for 39.2%, Streptococcus for 23.6%, coliforms for 14.1%, Micrococcus and Bacillus species for 8.0% each and Actinomyces or Arcanobacterium (Corynebacterium) for 7.0%. It was concluded that there was a high prevalence of clinical and subclinical mastitis, mainly caused by Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli, in this study area.     

不同日粮氮水平对山羊氮代谢和微生物蛋白质合成的影响

本试验旨在探讨不同日粮氮水平对内蒙古白绒山羊氮代谢和微生物蛋白质合成(MCP)的影响,以达到提高氮利用率,减少环境污染和饲料资源的浪费。试验选用9只体况良好、体重为(43.83±2.95)kg,装有瘤胃瘘管的内蒙古白绒山羊,按体重随机分为3组,每组3只。日粮分为低氮7.5%、中氮10.5%、高氮13.5%3个氮水平。试验使用常规试验方法分析测定粪尿血液等生化指标,通过全收尿法和尿嘌呤衍生物法估测MCP。结果表明:随着氮水平增加,氨氮(NH3-N)浓度、氮摄入量、尿氮排出、尿中尿素氮(UUN)和MCP显著增加(P0.05),干物质采食量(DMI)显著降低(P0.01);沉积氮占总摄入氮的比例在中氮组最高,MCP在高氮组最高。综上所述,适当降低日粮氮水平可减少粪尿氮排放,提高反刍动物氮利用率。     

Microsporidial keratopathy in two dogs

A microsporidial keratopathy is described in two dogs. Both dogs presented with a unilateral stromal keratopathy characterized by multifocal coalescing opacities, and the diagnosis was made on histopathologic examination of keratectomy specimens. Transmission electron microscopy (TEM) on formalin‐fixed, paraffin‐embedded corneal tissue was performed in one dog, and the morphologic features were consistent with Nosema species infection. Both dogs were initially diagnosed and treated by superficial keratectomy. One dog received additional antifungal medication and underwent a penetrating keratoplasty following local recurrence two years later. No other systemic lesions attributable to the microsporidial infection were identified clinically. The clinical and diagnostic pathology findings, treatment, and follow‐up are discussed.