抗A型口蹄疫病毒单克隆抗体的制备及抗原捕获ELISA检测方法的建立

为建立A型口蹄疫病毒(FMDV)病原学诊断方法,本研究应用纯化的A型FMDV免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,筛选获得一株稳定分泌单克隆抗体(MAb)的杂交瘤细胞株5F7.经IFA特异性鉴定,5F7为一株血清型特异性MAb.亚类为IgG1/k.间接ELISA检测杂交瘤细胞上清和腹水抗体效价分别为1:12 800和1:105.硫氰酸盐洗脱法测定其相对亲和力常数为1.5 mol/L.应用该A型特异性MAb及实验室已制备的MAb 3D9初步建立了检测A型FMDV的抗原捕获ELISA方法.该方法可以检出2.0 ng纯化FMDV和1.0×104 TCID50病毒,与O型、Asial型FMDV及牛肠道病毒、牛呼肠孤病毒、牛传染性鼻气管炎病毒等均无交叉反应,组内及组间重复性试验显示变异系数小于8％.本研究建立的抗原捕获ELISA方法为A型FMDV抗原检测提供了一种实用有效的技术手段.     

抗Asia1型口蹄疫病毒单克隆抗体的制备及生物学特性鉴定

用纯化的Asia1型口蹄疫病毒免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA和间接免疫荧光(IFA)筛选,有限稀释法克隆,获得了2株稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为3H6、5G3,其细胞培养上清效价分别为1:64和1:128,小鼠腹水效价分别为1×10~(-4)和8×10~(-3);ELISA和IFA结果显示,2株单抗仅与Asial型口蹄疫病毒反应,不与O型口蹄疫病毒反应,表明它们均为抗Asial型口蹄疫病毒的型特异性单克隆抗体。westem blot结果显示,2株单克隆抗体均不与全病毒抗原反应,表明它们所针对的抗原表位均为构象表位。相加ELISA试验表明,两株单抗识别不同的抗原表位。经硫氰酸盐洗脱法测定,3H6和5G3的相对亲和力指数分别为1.0 mol/L和1.5 mol/L。这2株单抗的获得为建立口蹄疫病毒检测方法提供了强有力的工具。     

Asia1型口蹄疫病毒单克隆抗体的制备及双抗体夹心ELISA方法的建立

为建立Asia1型口蹄疫(FMD)快速病原学诊断方法,本研究采用纯化的Asia1型口蹄疫病毒(FMDV)免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合并筛选到一株能够稳定分泌抗Asia l型FMDV单克隆抗体(MAb)2E10。经间接免疫荧光试验特异性鉴定,2E10为FMDV型特异型MAb。其抗体亚类为Ig G1/资,杂交瘤细胞上清和腹水抗体效价分别为1颐256和1颐104,相对亲和力常数为2.5 mol/L。在此基础上,以本实验室前期制备的MAb 2D8为包被抗体,以HRP标记的MAb 2E10为检测抗体建立了检测Asia1型FMDV抗原的双抗体夹心ELISA(DAS-ELISA)方法。该方法针对FMDV细胞毒的最低检出量为103TCID50,对O型FMDV、A型FMDV、牛肠道病毒、牛呼肠孤病毒及牛传染性鼻气管炎病毒检测结果均为阴性。本研究建立的Asia1型FMDV DAS-ELISA方法为Asia1型FMD病原学诊断试剂盒的研发奠定了基础。     

牛口蹄疫病毒VP2结构蛋白抗体间接ELISA方法的建立

为建立牛口蹄疫(FMD)抗体的检测方法,本研究将口蹄疫病毒(FMDV)的VP2基因,通过pPROEXTM HTb表达载体在大肠杆菌DH5α中表达,获得大小为35ku的重组VP2蛋白(rVP2),western blot证实rVP2可与FMDV5种血清型的牛阳性血清发生特异性反应。以纯化复性的rVP2为抗原建立了FMDVrVP2间接ELISA方法。重复性试验证实批内、批间变异系数均小于10%。特异性交叉试验表明,该抗原不与常见的其他7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫国家牛阴性血清的特异性为100%;检测感染血清敏感性为97.3%;检测O-AsiaⅠ的二价苗免疫牛血清,与4种商品化试剂盒比较,其符合率分别为69.0%、95.0%、90.4%和86.8%。实验结果表明建立的ELISA方法可以用于口蹄疫感染和免疫抗体检测。     

O型口蹄疫病毒单克隆抗体的制备和鉴定

利用O型口蹄疫病毒(FMDV)免疫BALB/c小鼠,取其脾细胞与Sp2/0细胞进行融合,经间接ELISA方法筛选和7次亚克隆后获得2株能稳定分泌单克隆抗体(MeAb)的杂交瘤细胞株.经间接ELISA检测,2株杂交瘤细胞株(3E1、7E6)的腹水效价分别为1:25 600、1:102 400.间接免疫荧光试验和特异性试验表明2株McAb可与不同株的O型FMDV反应,而不与其他血清型的FMDV反应.Western blot和叠加试验表明2株单抗可识别0型FMDV结构蛋白VP1上的同一个抗原表位.此特异性McAb的获得将为O型FMDV疫苗的研制和诊断方法的建立奠定基础.     

猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

为建立一种检测口蹄疫病毒（foot-and-mouth disease virus,FMDV）特异性IgA抗体的检测方法,本研究以原核表达系统表达纯化的FMDV结构蛋白VP1作为包被抗原,以鼠抗猪IgA单克隆抗体为二抗,辣根过氧化酶标记的羊抗鼠IgG抗体为三抗,建立猪A型FMDV特异性IgA抗体间接ELISA检测方法。确定抗原包被浓度为3.50 μg/mL,二抗与三抗的最佳稀释度为1∶10 000,二抗和三抗作用时间均为30 min。所建立的方法与抗猪瘟病毒、猪繁殖与呼吸道综合征病毒等病原的特异性IgA抗体间无交叉反应,A型口蹄疫感染样品的阳性检出率在90%以上,批内和批间重复性试验的变异系数介于3.16%~9.76%。该方法为监测FMDV特异性IgA抗体水平变化规律及猪的黏膜免疫效果评价及口蹄疫的早期诊断提供了一种新方法。     

基于单克隆抗体的O型口蹄疫病毒抗体固相竞争ELISA检测方法的建立

为建立评价O型口蹄疫病毒(FMDV)疫苗免疫水平的方法,本研究以单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 8E8作为检测MAb,经过条件优化建立了基于MAb的检测O型FMDV抗体的固相竞争ELISA(SPCE)方法。对该方法进行了特异性、敏感性、重复性试验。结果显示,MAb 3D9的最佳稀释度为1:25 000,灭活O型FMDV抗原的最佳稀释浓度为1:3,HRP标记的MAb 8E8的最佳稀释度为15 000,当血清1:32稀释时,检测的临界值确定为45%。该方法分别检测A型FMDV抗体阳性参考血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清,检测结果均为阴性,未出现交叉反应。经检测,当阳性标准血清的抗体稀释度在1:512时,该方法仍具有较好的敏感性;批内和批间重复性试验的变异系数均小于10%,表明其重复性较好。并将该方法与液相阻断ELISA(LPBE)方法和病毒中和试验(VNT)的相关性进行了比较,结果显示该方法与LPBE和VNT的相关性分别为0.896和0.923。本研究为国内评价O型FMDV疫苗免疫水平建立了一种新的方法。     

Asial型和O型口蹄疫病毒中和性单克隆抗体的免疫保护性

本研究用微量中和试验鉴定具有中和活性的口蹄疫病毒(FMDV)单克隆抗体,进而用乳鼠保护试验在体内验证单克隆抗体对FMDV感染的免疫保护作用.结果表明,针对Asial型和O型FMDV的单克隆抗体3E11和8E8腹水的半数保护量(PD50)分别为1995和2371;仅用8PD50单抗3E11和11PD50单抗8E8,就可完全保护乳鼠耐受致死剂量Asial型和O型FMDV的攻击.被动免疫的乳鼠在攻毒后体重增长的百分率随单抗腹水浓度的升高而增加,而且随着单抗腹水浓度的升高,乳鼠的起始死亡时刻后延、终点死亡时刻前移.     

A型口蹄疫抗体固相竞争ELISA检测方法的建立

为评价A型口蹄疫疫苗免疫水平,本研究以本实验室前期制备的口蹄疫病毒(FMDV)的单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 9A9作为检测抗体,建立了基于MAb的检测A型FMDV抗体的固相竞争ELISA(SPCE)方法,并对其条件进行优化。结果显示,MAb 3D9的包被浓度为1.16μg/mL,A型FMDV抗原的最佳稀释度为1:5,HRP标记的MAb 9A9的最佳稀释度为1:5 000,当血清132稀释时,检测的临界值确定为35%。利用该方法分别检测A型、O型口蹄疫抗体阳性标准血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清。结果显示,除A型口蹄疫阳性标准血清为阳性结果外其余均为阴性结果,未出现交叉反应,表明本研究建立的方法特异性强。该方法对经病毒中和试验(VNT)检测为阳性的3份血清进行敏感性试验,敏感性分别为1:1 024、1:256、1:128,均高于VNT,表明其敏感性较高;重复性试验结果显示,该方法批内和批间重复试验的变异系数均小于10%,表明其重复性较好。利用该方法与液相阻断ELISA方法(LPBE)和VNT对112份血清样品同时检测,分析三者相关性。结果显示,本实验建立的SPCE方法与二者的相关系数分别为0.901和0.916,表明该方法可靠性较好且与VNT的相关性更高。进一步利用本研究建立的SPCE方法和韩国Jeno A型FMDV ELISA抗体检测试剂盒同时检测470份临床血清样品(90份羊血清、170份牛血清和210份猪血清),结果显示该方法检测羊血清、牛血清和猪血清与Jeno试剂盒总体符合率分别为90.0%、91.8%和89.0%。表明该方法的检测结果较为准确。本研究为建立A型口蹄疫检测方法奠定了基础。     

牛病毒性腹泻病毒抗原捕获ELISA方法的建立

用兔抗牛病毒性腹泻病毒(BVDV)多抗作为包被抗体,BVDV NS3单克隆抗体作为捕获抗体,建立了检测BVDV抗原的捕获ELISA方法,对各项反应条件进行优化,最终获得最佳工作条件为兔多抗1∶1 600稀释包被,NS3单抗1∶2 000稀释,酶标抗体工作浓度为1∶4 000稀释。特异性和敏感性试验结果表明,该方法对牛轮状病毒、牛传染性鼻气管炎病毒、牛分支杆菌无特异性交叉反应,其最低可检测7.9×103个TCID50的病毒量,与RT-PCR方法的相比较,符合率为100%。所建立的BVDV抗原捕获ELISA方法快速、特异、敏感,可用于BVDV抗原的检测。     

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 -