检测肠炎沙门氏菌ELISA方法的建立与应用研究

从已建立的抗沙门氏茵单抗中筛选出适用于肠炎沙门氏菌ELISA检验的3—47—26单抗．采用筛选强阳性杂交瘤细胞株制备了高效价的3—47—26单抗．改进用辣根过氧化物酶标记高效价单抗的方法，进行了HRP标记单抗的免疫生物学特性的鉴定。确定了包被浓度和酶标抗体的工作浓度：HRP-3—47—26为1：100；LPS的包被浓度为400ng／mL。试验中采用LPS与多聚赖氨酸的结合，解决了包被过程中的解吸附作用，增强了包被的稳定性，同时也减少了假阳性反应，优化了包被过程。建立了检测肠炎沙门氏菌的抗原竞争ELISA方法，利用样品中的LPS抑制酶标抗体与包被的LPS结合，以降低底物反应的颜色从而达到检测的目的。通过对210份肠炎沙门氏菌感染鸡泄殖腔棉拭子、羽毛和组织样品先筛选后鉴定的方法进行检测具有明显的抑制作用，通过与国标法对大量的样品检测结果比较表明，竞争ELISA方法的检出率为18．09％；检出阳性样品36份，国标法检出率为17．14％；两者符合率为97．14％。竞争ELISA敏感性和特异性分别为94．4％和97．7‰从而为肠炎沙门氏菌的检测提供了一种敏感、特异的检测方法。     

肠炎沙门氏菌快速检测方法的建立

利用针对肠炎沙门氏菌主要O因子抗原O9的3－47－26单克隆抗体建立了快速检测肠炎沙门氏菌的竞争ELISA方法（C－ELISA）。将被检样品与McAb作用，竞争性抑制了McAb与包被肠炎沙门氏菌的结合，同时，也减弱了酶标二抗与McAb的结合，以降低底物反应的颜色，从而达到检测样品的目的。试验结果表明，本方法只选择性地与肠炎沙门氏菌反应，而与其它沙门氏菌均不反应。通过与国标法对210份样品检测结果比较表明，竞争ELISA方法的敏感性和特异性分别为94．4％和97．7％，从而为肠炎沙门氏菌的检测提供了一种快速、敏感、特异的检测方法。     

单抗竞争ELISA检测猪沙门氏菌感染方法的建立与应用

以猪霍乱沙门氏菌作为包被抗原 ,利用沙门氏菌O7单克隆抗体酶结合物建立了竞争ELISA方法检测猪霍乱沙门氏菌抗体。经过研究确定抗原包被浓度为 2×1 0 8CFU/ml,血清检测稀释度为 1∶1 6,酶标抗体浓度为 1∶70 0 ,包被液为碳酸盐缓冲液 ( 0 .0 5MpH9.6)。应用单抗竞争ELISA和平板凝集试验 (PAT)同时对 5 0 6份血清进行猪霍乱抗体检测 ,PAT的检出率为 3 .60 % ,ELISA检出率为 5 .75 % ,两者符合率达 96.4%。试验结果表明 ,ELISA方法敏感性高 ,特异性强 ,稳定性和重复性好 ,操作简便。本方法的建立在猪群沙门氏菌感染的检疫、实验室诊断的标准化及流行病学调查方面具有应用价值。     

抗鸡白痢沙门氏菌单抗及其在鸡白痢病检疫中的应用研究

本研究应用淋巴细胞杂交瘤技术制备了一组针对鸡白痢沙门氏菌O_9抗原的单抗。以单抗3-47-26酶结合物和鸡白痢沙门氏菌脂多糖抗原建立了一种抗体竞争ELISA试验以检疫鸡白痢病,该法通过样品抑制酶标单抗与LPS的结合,以检测鸡白痢抗体。试验结果表明,79份鸡白痢阳性血清具有显著抑制作用(95.0±4.3％),而130份鸡白痢阴性血清以及48份SPF鸡血清抑制不明显,它们的抑制率分别为12.3±10.6％和6.2±5.5％。对94份种鸡血清的检疫结果表明,抗体竞争ELISA比PAT、AGPT、PHA敏感和特异,并进一步讨论了该法在血清流行病学调查方面的应用前景。     

单抗酶联试剂盒与PCR方法快速检测沙门氏菌的比较

本文对单抗酶联试剂盒与PCR方法快速检测沙门氏菌进行了比较。将沙门氏菌与大肠杆菌以不同比例混合后 ,分别用ELISA和PCR法检测 ,在 1∶1 0 0的比例时 ,用这 2种方法均能检测出阳性样品 ;在 1∶1 0 0 0的比例时 ,用ELISA法检测为阴性 ,而用PCR法则能检测出。检测鲜牛奶、龙虾、虾仁、熟食制品等样品 ,2种方法的符合率达 97 6 %。对冻虾仁、人粪便样品的前增菌、选择性增菌、后增菌过程进行同步检测 ,在样品的前增菌液 ,直接ELISA法检测的OD值小于 0 5 ,而PCR法扩增能出现特异条带 ,经国标方法验证为阳性 ,证实PCR法的敏感性优于直接ELISA法 ,而在选择性增菌液和M 肉汤后增菌液中 ,2种方法均能检出阳性样品。     

食源性沙门氏菌快速检测技术的应用研究

通过样品试验,得到优化的沙门氏菌检验方法,了解上海地区沙门氏菌 污染情况.在免疫胶体金技术、单抗直接ELISA、PCR法检测沙门氏菌的基础上,将 PCR法和单抗直接ELISA三种快速检测方法进行比较研究.用免疫胶体金技术、直接E LISA、PCR法对鸡蛋、水样、牛奶、猪肉、牛肉、虾仁等样品401份进行沙门氏菌检测,并与 常规国标方法对比,直接ELISA法的敏感性和特异性达100%和97.3%,免疫胶体金技术敏感 性和特异性达100%和95.3%,PCR法的敏感性和特异性均为100%.通过对样品的检测, 最终确定较优化的沙门氏菌检测程序.PCR法的特异性优于免疫胶体金技术和直接ELISA法.对大量样品检测,可采用免疫胶体金技术或直接ELISA法筛检,除去大量阴性样品,阳性样品用PCR法作进一步鉴定;需要确定血清型时则用国标法.     

牛奶中孕酮直接竞争ELISA检测方法的建立

采用直接竞争ELISA方法检测牛奶中孕酮含量.采用碳化二亚胺法,应用辣根过氧化物酶(HRP)标记11-α羟基孕酮琥珀酸酯(11α-OH-P4-HS),制备孕酮酶标抗原;酶标抗原与牛奶样品中的孕酮共同竞争结合固相包被的孕酮单克隆抗体,建立直接竞争ELISA反应体系.试验结果表明,最佳抗体包被浓度为1∶2 000,最佳酶标抗原工作浓度为1∶16 000,建立的标准曲线为y=-2.4598x+0.999(R2=0.996),牛奶中孕酮检测范围0.12～40 ng/mL,最低检测量为0.12 ng/mL,批内和批间变异系数分别为1.63%和2.49%.成功建立了可用于牛乳中孕酮快速检测的直接竞争ELISA方法.     

用多粘菌素布酶免法快速、简便测定鸡粪中的沙门氏菌

作者用多粘菌素布酶免法（P－CEIA）测出鸡粪中的三株沙门氏菌。用吸附有多粘菌素B的大孔隙聚酯布结合热抽提脂多糖（LPS）抗原，并检测此抗原，全部过程仅需22小时。此法也可测定接种沙门氏菌鸡粪便中的内源性沙门氏菌及其含量。这种测定鸡粪中沙门氏菌的方法简单、快速、敏感，不仅用于普查鸡群中的沙门氏菌，而且也用于测定病人和食品加工者大便中的沙门氏菌。1材料与方法1．1细菌鼠伤寒沙门氏菌、肠炎沙门氏菌、哈达尔沙门氏菌分别保存于胰蛋白酶大豆琼脂（TSA）中，挑取少量于由汤蛋白陈中37℃培养16～18小时备用。1．2测定粪便沙…     

PRRSV抗体竞争ELISA检测方法的建立与标准化研究

以猪繁殖与呼吸综合征病毒(PRRSV)核衣壳蛋白(N蛋白)基因的原核表达产物重组N蛋白为包被抗原,利用兔抗重组N蛋白血清和PRRS猪血清竞争该抗原,建立间接竞争ELISA方法检测PRRS抗体。经研究确定重组N蛋白的包被浓度为0·3μg/mL,检测血清不用稀释,兔抗重组N蛋白血清的工作浓度为1∶3000,酶标抗体的工作浓度为1∶10000,包被液为0·05mol/L、pH9·6的碳酸盐缓冲液。该方法特异性强,稳定性和重复性好,整个检测过程可在3h内完成。以IDEXX试剂盒的检测结果为参照标准,该方法的敏感性为79·76%,特异性为90·9%,符合率为83·59%。将该方法按试剂盒要求标准化,4℃放置5个月,检测效果不变。     

酶标单抗阻断ELISA检测鸡白痢和鸡伤寒抗体

以辣根过氧化物酶标记的沙门氏菌O9单抗3-47-0与包被的鸡白痢沙门氏菌脂多糖抗原建立了一种抗体阻断EL ISA以检测鸡白痢和鸡伤寒沙门氏菌感染。该方法基于待检血清样品抑制酶标单抗与脂多糖的结合,以检测鸡白痢和鸡伤寒特异性抗体。对72份已知鸡白痢阳性血清(抑制率为(98.5±4.0)%)、54份已知鸡白痢阴性血清(抑制率为(15.3±8.0)%)、42份SPF鸡血清(抑制率为(0.8±7.2)%)检测的结果表明,该方法具有很强的区分能力。在人工感染试验中,从第2周开始,该方法能从全部鸡只中检测到特异性抗体,比全血玻板凝集试验(PAT)早1周时间。对344份种鸡血清的检测结果表明,单抗阻断EL ISA比PAT特异性好、敏感性高。另外,该方法也能检测出与鸡白痢和鸡伤寒沙门氏菌有共同抗原的沙门氏菌感染产生的抗体。     

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。     

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.     

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.     

The Prevalence and Intensity of Helminth and Coccidial Infections in Dairy Cattle in Central Kenya

A survey of gastrointestinal parasite infections of young (<6 months old), immature (6–12 months old) and adult (>12 months old) dairy cattle on 16 farms in Kiambu District, Kenya was conducted during a dry season (September 1991 to January 1992) and during a wet season (March to July 1992). The survey was based on monthly coproparasitological examination of cohorts and worm counts in tracer calves. The effects of age, sex, farm and season on the prevalence and intensity of helminth and coccidial infections were determined. Faecal egg and oocyst counts revealed that the overall prevalences were: strongyles (including trichostrongyles) (85.5%), liver flukes (Fasciola gigantica) (34.0%), coccidia (30.9%) and tapeworms (9.6%). Eight species of the protozoan Eimeria were identified, the most prevalent species being E. bovis and E. zuernii. The most prevalent nematode genera were Haemonchus, Cooperia, Oesophagostomum and Trichostrongylus. Season, farm and age of the animals had a significant (p<0.05) influence on the intensity of infection with strongyles, liver flukes and coccidia, whereas the sex of the animals had no significant (p>0.05) effect on the prevalence or intensity of infections. A higher intensity of infection with strongyles and coccidia was found in the wet season than in the dry season (p<0.05). The age-specific intensity was in the following order: for strongyles, immature animals of 6–12 months of age had the highest egg counts, followed by young calves and adults. Calves had significantly (p<0.05) higher oocyst counts than immatures or adults. Liver fluke egg counts did not differ significant (p>0.05) between immatures and adult cattle.     

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.     

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。