克伦特罗单克隆抗体的制备及其初步鉴定

目的：制备克伦特罗单克隆抗体(McAb)建立克伦特罗ELISA检测方法，为研制检测试剂盒奠定基础。方法：用偶氮化法将克伦特罗与钥孔血蓝蛋白(KLH)牛血清白蛋白(BSA)及卵清白蛋白(OVA)偶联．制备完全抗原，免疫Ballb／c小鼠，建立杂交瘤细胞株以备单抗，并对单抗特异性、交叉反应性及抗原识别位点等特性进行初步鉴定。用HRP标记CL，建立ELISA方法。结果：获得了1H5、1H7、1D6、2F10四株杂交瘤。其中1H5、1H7培养上清效价都在1：2000以上．腹水效价在1：100000以上。对沙丁胺醇交叉反应测定，最小的是1D6，只有2．32％的交叉反应性。最后建立了检测CL的固相抗体竞争ELISA方法。     

恩诺沙星免疫抗原与检测抗原的制备及鉴定

采用N-羟基琥珀酰亚胺法将恩诺沙星（Enro）半抗原与载体蛋白-牛血清白蛋白（BSA）相偶联，制备出了抗Enro单克隆抗体的免疫抗原Enro-BSA。同样的方法，将Enro与卵清白蛋白（OVA）相偶联制备了抗Enro单克隆抗体的检测抗原Enro-OVA。紫外扫描光谱及动物免疫试验结果表明，其偶联成功。     

鸡肝脏和肌肉组织中常山酮残留的ELISA检测

将常山酮进行人工改造,制备了半抗原常山酮琥珀酸衍生物（Hal-suc）.采用N-羟基琥珀酰亚胺活性酯法将半抗原与牛血清白蛋白（BSA）、卵清白蛋白（OVA）偶联,制备免疫原和包被原.动物免疫6次后采血制备抗血清,以间接ELISA法测定血清效价,测得抗血清的最佳工作浓度为1∶102 400.建立了常山酮在鸡肝脏和肌肉组织中检测的ELISA法,该方法在50、100、500 ng/g 的添加浓度水平下,在鸡肝脏和肌肉组织中测得的平均回收率范围分别为74.2%～96.8%和74.3%～90.0%,检测限分别为28和19 ng/g.     

洛克沙胂完全抗原的制备及其单克隆抗体的研制

本研究以洛克沙胂(ROX)的结构类似物3-氨基-4-羟基苯胂酸(HAPA)为小分子半抗原,采用碳二亚胺法,分别将其与载体蛋白——牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)偶联,制备免疫抗原HAPA-BSA和检测抗原HAPA-OVA。经紫外光谱扫描及聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定后,以20μg/只剂量的HAPA-BSA免疫BALB/c小鼠;选择血清效价高、敏感性强的小鼠,取脾脏进行细胞融合,制备杂交瘤细胞株。利用间接ELISA法筛选得到1株可以稳定分泌抗ROX单克隆抗体的细胞株,命名为G12;间接ELISA法测定其细胞上清效价为1:512,间接竞争ELISA测定其半数抑制浓度IC_(50)为3.147 ng/μL。经小鼠体内诱生腹水,辛酸-硫酸铵法纯化获得纯度较高的单克隆抗体,间接ELISA测定其效价为1:102 400,IC_(50)为1.433 ng/μL,且具有良好的特异性。本试验成功合成了ROX人工抗原,并制备了可以分泌高敏感性单克隆抗体的杂交瘤细胞株,为ROX免疫学快速检测方法的建立奠定了基础。     

氯霉素全抗原合成及多克隆抗体的制备

用重氮化方法将氯霉素与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联，制备免疫抗原和包被抗原，用ELISA方法进行鉴定。将偶联抗原用弗氏佐剂乳化后免疫德国大白兔。制备多克隆抗体，并用硫酸铵沉淀法初步纯化。用亲和层析法进一步纯化。用所得抗体建立竞争酶联免疫吸附分析法（ELISA）用于氯霉素检测，最低检查限为0.3ng/mL。     

达氟沙星间接竞争ELISA检测方法的建立

将达氟沙星（danofloxacin, DFLX）与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联分别作为免疫原与包被原, 达氟沙星为竞争的半抗原，建立间接竞争ELISA检测方法。试验结果表明,理想的包被抗原浓度为1.25 μg/L, 多抗(DFLX-PcAb)工作浓度为1∶10000,酶标二抗的工作浓度为1∶4000,最适检测范围为0.1～10 ng/L,最低检测限为0.2 ng/L, 批内和批间变异系数分别为3.81%和6.25%。得到回归方程y=0.7975-0.125x（R2=0.989）和标准曲线，从而建立了DFLX的快速检测残留的间接竞争酶联免疫吸附试验（ci-ELISA）。     

氨基脲单克隆抗体制备及其ELISA检测方法的建立

以氨基脲(SEM)和对醛基苯甲酸(CP)为原料合成半抗原(CP-SEM),将半抗原和载体蛋白偶联后免疫Balb/C小鼠,应用杂交瘤技术建立稳定分泌抗氨基脲的杂交瘤细胞株。常规制备腹水,用辛酸-硫酸铵法纯化,并对纯化的mAb进行特异性鉴定。通过对不同抗体组合的分析和条件的优化,建立检测氨基脲的间接竞争ELISA方法。利用对醛基苯甲酸衍生草鱼肌肉提取的氨基脲,用建立的ELISA方法进行检测,并计算回收率和最低检测限。经细胞融合、筛选及克隆化,共获得7株稳定分泌抗氨基脲的杂交瘤细胞株,其中4株亲和力较高。建立了间接竞争ELISA检测方法,该方法灵敏度达到0.1ng/mL,平均回收率为98.47%,在草鱼组织中的最低检测限为0.76ng/g,回收率为70.55-100.56%,可用于肌肉中氨基脲残留的检测。     

盐酸克伦特罗单克隆抗体的制备和鉴定

为进一步建立克伦特罗(CL)残留检测方法,给制备CL残留检测试剂盒打下基础,进行了CL单克隆抗体(McAb)的制备研究。以重氮反应,将CL与牛血清白蛋白(BSA)、鸡卵清白蛋白(OVA)偶联,分别制备免疫原和检测原,并通过杂交瘤技术,获得了5株能稳定分泌特异结合CL小分子的单克隆抗体杂交瘤细胞株(1E9A9、2A9C1、3F2E9、6G2E9、6E10D9)。这5株细胞株经过体外传代培养和冻存复苏后均能稳定分泌抗体。用间接酶联免疫吸附试验(ELISA)测定的这5株细胞上清液抗体效价分别为1:1.28×10~5、1:5.12×10~5、1:1.28×10~5、1:5.12×10~5、1:2.56×10~5;选用3F2E9、6E10D9细胞株,制备CL单克隆抗体腹水并纯化,其纯化后的腹水抗体效价分别为1:1.28×10~5、1:2.56×10~5,BCA(2,2-联喹啉-4,4-二甲酸二钠)方法检测的浓度分别为1.60 mg/mL、1.54 mg/mL;最终通过斑点ELISA(Dot-ELISA)方法测定CL抗原与这2株抗体有较强的结合力。在上述方法的制备及验证下,成功获得了特异性较高的CL单克隆抗体,为进一步研制CL残留检测试剂盒奠定了基础。     

抗氧氟沙星单克隆抗体的制备及ELISA快速试剂盒的初步研制

本试验通过碳二亚胺法,将半抗原氧氟沙星与载体牛血清白蛋白（BSA）和卵清白蛋白（OVA）偶联,制备得到氧氟沙星免疫原和包被原,并制备抗氧氟沙星的单克隆抗体,初步研制测定氧氟沙星的ELISA试剂盒,经过测试,对鱼肉样本中氧氟沙星的检测限为1 μg/kg,添加回收率在62.8%～97.7%之间,批内、批间试验的相对标准偏差均小于10%。     

己烯雌酚完全抗原及其单抗的制备与分析

为建立己烯雌酚免疫学检测技术,试验采用混合酸酐法制备完全抗原DES-HS-BSA,并对制备产物进行紫外光谱、红外光谱、SDS-PAGE、MS分析鉴定,以DES-HS-BSA免疫Balb/c小鼠,用ELISA试验测定抗体.结果表明:半抗原衍生物DES-HS已被成功地偶联到牛血清白蛋白上,计算得每分子牛血清白蛋白上结合26个DES-HS;ELISA试验结果证实免疫小鼠血清中含有抗己烯雌酚的抗体,将免疫好的小鼠脾脏细胞和SP2/0细胞融合,筛选出了1株能稳定分泌己烯雌酚单抗的杂交瘤细胞株18G5,抗体亚型为IgG3.     

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 -