阿维菌素类药物单克隆抗体的制备和鉴定

将阿维菌素与牛血清白蛋白偶联,免疫Balb／c小鼠,用杂交瘤技术获得了1株分泌抗阿维菌素特异性抗体的杂交瘤细胞株。用间接ELISA测定腹水的效价为1：160000,抗体的50％抑制药物浓度（IC50）为2．5ng／ml,抗体与伊维菌素的交叉反应为12．5％,与多拉菌素、莫西菌素均无交叉反应。     

抗氯霉素单克隆抗体的制备及其特性

用重氮化法人工合成氯霉素-牛血清蛋白（CAP-BSA）抗原免疫BALB/c小鼠,应用杂交瘤技术将免疫鼠脾细胞与SP2/0细胞融合,建立了分泌抗CAP单克隆抗体（McAb）的杂交瘤细胞株.经检测、鉴定,筛选出5株高亲和力的杂交瘤细胞株,其腹水效价均高于1：10^6,且5株杂交瘤细胞株分泌的单抗与其他几种抗生素无交叉反应,可用于氯霉素的快速检测.     

小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用

针对小反刍兽疫病毒核蛋白制备特异性的单克隆抗体,并对其进行生物学特性鉴定和初步应用。以纯化的Bacmid-PPRV-N重组蛋白为抗原免疫BALB/c小鼠,取免疫小鼠的致敏脾细胞与SP2/0骨髓瘤细胞在PEG作用下融合,获得单克隆抗体,并通过染色体技术等方法研究其生物学特性,将其作为竞争单抗,Bacmid-PPRV-N重组蛋白作为检测抗原建立竞争ELISA检测方法。结果表明:经克隆和间接ELISA筛选,获得了2株能稳定分泌抗小反刍兽疫病毒N蛋白抗体的杂交瘤细胞株,分别命名为5B11和3H10-3B8。生物学特性鉴定试验表明:5B11和3H10-3B8抗体类型和亚类均为IgG2b;5B11单抗腹水的效价达1∶819 200,3H10-3B8达1∶12 800;血清学试验证明2株单抗均能与Bacmid-PPRV-N重组蛋白抗原结合,具有高度的特异性;相加ELISA试验结果显示,5B11和3H10-3B8 2株单克隆抗体分别识别N蛋白上不同的抗原位点;2株杂交瘤细胞的染色体均为99～104。应用建立的c-ELISA检测方法对222份血清样品进行PPRV抗体的检测,与参考试剂盒比较得到98.20%的符合率。本研究获得了2株能稳定分泌抗PPRV N蛋白单克隆抗体的杂交瘤细胞株,以单抗5B11作为竞争抗体建立了PPRV的c-ELISA检测方法。     

水泡性口炎病毒单克隆抗体的制备与鉴定

将水泡性口炎病毒（VSV）经差速离心和蔗糖密度梯度离心法进行纯化后,以纯化的VSV作为免疫原免疫8～10周龄雌性BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA方法筛选能稳定分泌抗VSV单克隆抗体的杂交瘤细胞株,并对制备出的抗VSV单抗的特异性、抗体亚类等生物学特性进行鉴定。结果显示,试验成功筛选出2株能稳定分泌抗VSV单克隆抗体的杂交瘤细胞株,分别命名为1A2、4C3。ELISA鉴定结果表明,2株单抗均能特异性地与VSV结合,而与口蹄疫病毒（FMDV）、猪水泡病病毒（SVDV）均不发生交叉反应;诱生小鼠腹水产生的抗体效价可达1∶25 600～1∶51 200。杂交瘤细胞染色体核型鉴定结果显示,杂交瘤细胞染色体数为95～105,均高于2个亲本细胞的染色体数目,说明这2株细胞是两者的杂交产物。抗体亚类鉴定结果显示,所获得2株单抗1A2、4C3均为IgG1。Western blot分析结果表明,1A2可识别VSV G蛋白。2株抗VSV单克隆抗体的成功制备将为VSV快速检测方法的建立以及检测试剂的研制等奠定基础。     

非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定

为制备非洲猪瘟病毒(ASFV)p30蛋白的单克隆抗体,以原核表达的重组p30蛋白为免疫原免疫6～8周龄雌性BALB/c小鼠,3次免疫后分离小鼠脾细胞并将其与SP2/0细胞进行融合,通过间接ELISA方法筛选,获得2株能稳定分泌抗ASFV p30蛋白单克隆抗体的杂交瘤细胞株。2株杂交瘤细胞从第10代开始均能稳定分泌抗体,细胞上清抗体效价均为1∶6 400。亚型鉴定结果显示,2株单克隆抗体的重链均属于IgG1,轻链均为κ型。Western blot和间接免疫荧光试验(IFA)结果显示,2株单克隆抗体均能与ASFV感染的猪肺泡巨噬细胞发生特异性反应。试验结果为p30蛋白生物学和诊断方法研究提供了技术支持。     

雌二醇单克隆抗体制备及间接竞争ELISA检测方法的建立

为建立敏感、特异、快速的雌二醇（estradiol,E₂）残留免疫检测方法,本研究利用雌二醇人工抗原（E₂-BSA）免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术制备特异性雌二醇单克隆抗体,利用高效价、高特异性单克隆抗体建立间接竞争ELISA（icELISA）方法。结果显示,试验成功筛选获得一株稳定分泌抗雌二醇抗体的杂交瘤细胞株（3H3）,抗体效价可达1∶320 000,利用3H3腹水抗体优化间接竞争icELISA反应条件,检测抗体的敏感度,IC₅₀为1.636 ng/mL,IC₂₀~IC₈₀线性范围为0.202~13.281 ng/mL。雌二醇腹水抗体与雌三醇、乙炔雌二醇的交叉反应率分别达0.31%和0.25%,而与雌酮、戊酸雌二醇、苯甲酸雌二醇、炔雌醚、己烯雌酚、壬基酚的交叉反应率均<0.1%。结果表明,利用本研究制备的单克隆抗体建立雌二醇间接竞争ELISA检测方法,可满足食品中雌二醇残留高灵敏度的检测要求。     

抗猪传染性胃肠炎病毒单克隆抗体杂交瘤细胞株的建立

用猪传染性胃肠炎（TGEV）华毒株弱毒H－155代感染iBRS2传代细胞，通过冻融、PEG6000沉淀、蔗糖密度梯度离心提纯制备的TGEV抗原，免疫BALB／C小鼠，取血清抗体阳性的免疫鼠的脾细胞，在50％PEG4000作用下，与SP2／0小鼠骨髓瘤细胞进行融合，产生杂交瘤，用ELISA间接法对杂交瘤细胞生长孔上清液进行检测，筛选阳性杂交瘤细胞株。通过有限稀释法进行3次克隆，获得了2株分泌抗TGEV的单克隆抗体杂交瘤细胞株，并对其所分泌的单克隆抗体免疫球蛋白亚类、特异性、中和反应性等特性作了初步鉴定。     

赤羽病ELISA检测方法的建立及试剂盒的研制

为了提高赤羽病的检测速度与效率,建立双抗夹心ELISA检测方法并进行试剂盒的研制。试验用纯化的AKV免疫BALB／c小鼠,细胞融合后用ELISA方法筛选出3株分泌抗AKV特异性单克隆抗体的杂交瘤细胞（1D1、1812和2G1）,各杂交瘤细胞株均可以稳定地分泌特异性单克隆抗体,且其单克隆抗体亚型测定的结果均为IgG1、轻链均为k链。建立DAS--ELISA检测方法,确定抗AKV单克隆抗体1812株的最佳包被浓度为4μg／mL,多克隆抗体最佳稀释比为1：500,二抗最佳工作浓度为1：5000,阴性／阳性结果判定临界值为0．238,具有较好的特异性,检测极限为10^-4。表明该方法及试剂盒具有较强的实用价值。     

猪繁殖与呼吸综合征病毒NVDC-JXA1株膜基质蛋白单克隆抗体的研制

为制备抗猪繁殖与呼吸综合征病毒NVDC-JXA1株单克隆抗体,用纯化的猪繁殖与呼吸综合征病毒NVDC-JXA1株免疫BALB/c小鼠,最后一次免疫后第3天取其脾细胞与SP2/0细胞在聚乙二醇作用下融合,通过酶联免疫吸附试验（ELISA）和间接免疫荧光试验（IFA）筛选,以有限稀释法克隆3次,制备单克隆抗体,并对制备的单克隆抗体进行鉴定。结果表明获得了2株分泌PRRSV膜基质蛋白特异性抗体的杂交瘤细胞,命名为1D12、5H5,经鉴定其抗体亚型分别为IgG2b、IgM,2株均为κ链,腹水ELISA效价分别为1∶107和1∶105。该单克隆抗体与PRV、PPV、PCV、CSFV、JEV无交叉反应。作者成功制备了抗猪繁殖与呼吸综合征病毒NVDC-JXA1株膜基质蛋白单克隆抗体,为进一步建立相关诊断方法奠定了基础。     

H5N1禽流感病毒PB1-F2特异性单克隆抗体的制备与鉴定

以重组的H5N1亚型禽流感病毒(AIV)PB1-F2的C端蛋白(cPB1-F2)为免疫原制备单克隆抗体,并鉴定其特异性。构建cPB1-F2的pGEX-6p-1原核表达载体,进行蛋白表达与纯化;采用杂交瘤技术制备筛选抗重组cPB1-F2蛋白的杂交瘤细胞株,利用间接免疫荧光和酶联免疫吸附试验筛选鉴定阳性细胞株,并对单克隆抗体特异性进行鉴定。结果显示,纯化的重组cPB1-F2蛋白具有良好的免疫原性,筛选获得了3株稳定分泌抗cPB1-F2单克隆抗体的杂交瘤细胞株,分别命名为6F4、2C5和4D3,经鉴定抗体均为IgG1亚类,且3株单克隆抗体均能特异性识别重组PB1-F2蛋白。结果表明成功制备了cPB1-F2特异性单克隆抗体,为研制针对流感病毒的抗体药物奠定了基础。     

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.     

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

Tropical Animal Health and Production -     

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.     

Chemotaxis of macrophage in inflammation

Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.     

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