无色孔雀石绿免疫原的合成与鉴定

用化学方法合成了硝基无色孔雀石绿（NO2-LMG），还原为氨基无色孔雀石绿（NH2-LMG），经红外光谱扫描鉴定NH2-LMG为所需化合物。氨基无色孔雀石绿经重氮法处理后与兔血清白蛋白（RSA）载体蛋白相连，通过紫外分光光谱鉴定，偶联产物与其前体物相比吸收峰发生了飘移，说明偶联成功。通过动物免疫试验和ELISA抗体特异性试验对该人工合成抗原免疫原性和与无色孔雀石绿反应原性的鉴定，表明成功合成了无色孔雀石绿免疫原。     

磺胺类药物广谱性多克隆抗体的制备

基于磺胺类药物(Sulfonamides,SAs)公共母环(对氨基苯磺酰胺)结构,合成了两种具有该结构的半抗原∶2-(对氨基苯磺酸氨基)-4-噻唑乙酸(TS)和对氨基苯磺酰胺基苯甲酸(SS).采用碳二亚胺法合成了免疫原和包被原,分别免疫10只新西兰大白兔,制备广谱性磺胺类药物抗体.应用间接竞争酶联免疫吸附分析(ELISA)检测血清的灵敏度和特异性,发现所有兔子对TS-BSA和SS-BSA均产生较高效价和特异性.其中SS-1号和TS-6号兔的血清灵敏度最好,效价分别为1∶80 000和1∶160 000,对10种磺胺类药物均有不同程度的识别能力.SS-BSA组IC50为257.9～6 590.9 ng/mL,而TS-BSA组IC50为12.9～586.1 ng/mL.结果表明,具有磺胺母环结构的半抗原TS和SS,可以应用于广谱性磺胺类抗体的制备.通过10只兔子免疫试验证明,应用SS获得的血清具有较高的簇特异性、相似的亲和性,优于TS.     

三聚氰胺半抗原及完全抗原的合成与鉴定

本研究合成了三聚氰胺半抗原及完全抗原并鉴定合成是否成功,为制备三聚氰胺抗体奠定基础。将2-氯-4,6-二氨基-1,3,5-三嗪和3-巯基丙酸的反应合成半抗原3-(4,6-二氨基-1,3,5-三嗪-2-基硫代)丙酸,采用质谱鉴定和红外光谱鉴定;通过活性酯法将半抗原分别与牛血清白蛋白、卵清蛋白进行偶联制备完全抗原,采用紫外光谱法初步鉴定,后经动物免疫进一步验证。结果表明半抗原与完全抗原合成成功,为后续的抗体制备奠定了基础。     

磺胺母核人工半抗原的合成与鉴定

合成磺胺母核人工半抗原,并对其产物进行鉴定。采用4-氨基苯甲酸甲酯（PBPA）和对乙酰氨基苯磺酰氯（ASC）为原料,经过亲和取代,酯的水解反应合成磺胺药物共有的母核结构苯甲酸对氨基苯磺酰胺（SH）,应用质谱法（ESI—MS）与核磁共振氢谱法（1H—NMR）鉴定SH。质谱结果显示[M-]=291.03766,与理论值[M]-292相符,表明SH合成成功;核磁共振氢谱（1H—NMR）的数据与SH化合物的结构相符。磺胺母核人工半抗原合成成功,为研制磺胺药多残留检测试剂盒和胶体金免疫层析试纸条奠定基础。     

三聚氰胺抗原合成及其多克隆抗体的制备

以2-氯-4,6-二氨基-1,3,5-三嗪为原料,强碱条件下与3-巯基丙酸反应,制备三聚氰胺半抗原并经质谱和红外鉴定;应用碳二亚胺法将半抗原与BSA和OVA偶联制备三聚氰胺免疫原和包被原,完全抗原经紫外扫描鉴定偶联成功,偶联比分别为16:1和10:1,通过免疫新西兰大白兔成功制备三聚氰胺多克隆抗体,抗体效价可达1:128000以上。本研究为三聚氰胺人工抗原的制备提供了有效途径,为三聚氰胺免疫检测方法的研发奠定了基础。     

一种磺胺类母环半抗原TS的合成

通过人工合成具有磺胺类药物对氨基苯磺酰胺公共母环的半抗原TS,以期建立动物源性食品中磺胺药物的簇特异性抗体。采用NHS、MA法合成人工免疫原、包被原,通过紫外扫描法对其偶联结果进行初步鉴定,结合免疫小鼠的动物试验,通过ELISA方法,检测鼠多抗产生针对磺胺类药物的抗体,确证偶联成功。     

环丙沙星人工抗原的合成及鉴定

为了合成环丙沙星抗原,试验应用3-溴丙氨氢溴酸盐制备半抗原环丙沙星-溴丙氨衍生物,采用戊二醛方法与碳二亚胺法合成环丙沙星免疫抗原(CIP-NH2-BSA)与环丙沙星包被抗原(CIP-NH2-OVA),其中环丙沙星-溴丙氨衍生物采用红外光谱(IR)、电喷雾电离质谱(ESI-MS)和核磁共振(1H-NMR、13C-NMR)进行结构确证,CIP-NH2-BSA与CIP-NH2-OVA采用紫外扫描法进行鉴定。结果表明:环丙沙星-溴丙氨衍生物合成成功,环丙沙星-溴丙氨衍生物与牛血清白蛋白(BSA)和卵清白蛋白(OVA)耦联成功。说明通过制备环丙沙星衍生物可以合成高效的抗原。     

达氟沙星单克隆抗体的制备与鉴定

采用碳二亚胺法将达氟沙星与BSA和OVA偶联制成免疫抗原DFLX-BSA、检测抗原DFLX-OVA。采用FeCl3显色反应、紫外扫描分析法对合成的抗原进行了鉴定。用免疫抗原DFLX-BSA腹腔免疫BALB/c小鼠,有限稀释法筛选获得分泌特异性抗体的杂交瘤细胞株1株4F9,单抗亚型鉴定为IgG1,并将此细胞株注射小鼠腹腔获得腹水,用辛酸-硫酸铵法对腹水进行了纯化。纯化后的抗体效价为2.48×10-6,紫外分析方法计算蛋白含量为1.348 mg/mL。     

青霉素钠完全抗原制备及其抗体水平检测

利用戊二醛法偶联青霉素钠（Penicillin,PEN）与BSA和OVA,制备完全抗原PEN-BSA和PEN-OVA。经紫外扫描及SDS-PAGE电泳鉴定完全抗原PEN-BSA和PEN-OVA偶联成功,并进行动物免疫制备抗血清,建立间接ELISA法测定免疫血清效价,经测定抗原PEN-BSA和PEN-OVA均达到理想的免疫效价,为进一步制备青霉素钠单克隆抗体及其青霉素钠药物残留快速检测试剂盒奠定基础。     

动物组织中四环素类抗生素残留的ELISA检测研究——土霉素抗体的制备

四环素类抗生素属小分子半抗原物质 ,必须与大分子物质结合才能产生免疫原性。本试验采用重氮化法合成土霉素 牛血清白蛋白 (BSA OTC)及土霉素 鼠血清白蛋白 (RSA OTC)人工抗原 ,用紫外扫描对其进行鉴定。以BSA OTC免疫家兔制备抗血清 ,以RSA OTC为包被抗原 ,采用双向免疫扩散法和ELISA方法对抗血清进行鉴定。结果表明用重氮化法合成的人工抗原对四环素类药物有特异的抗原抗体反应 ,抗血清效价达 1 2 8× 1 0 5,完全能够满足四环素类药物残留的ELISA检测要求     

Tail-biting in outdoor pig production

A study was performed in five identical outdoor production units in the same geographic area using growing-finishing pigs of similar genetic makeup, age, diet and feed management. The severity of tail-biting (TS) was scored 1-4. The average group prevalence of bitten tails at slaughter on different farms was between 14.1+/-2.1% and 20.1+/-3.0% (P<0.05). The odds of a barrow being bitten were 2.9 times higher than those for a gilt. The most frequently recorded score of bitten tails was TS3, indicating moderate wounds with low grade infection. The prevalence of bitten barrows was positively correlated with the percentage of gilts in a group (r = 0.54, P<0.001). Pigs with zero TS score had no significantly higher weights at slaughter compared to pigs with a score of TS1. As the TS increased from 1 to 4, weights decreased (TS 1 to TS 2 to 4, P<0.05). TS 3 and 4 were positively (P<0.001) associated with subsequent carcass condemnation. We concluded that outdoor rearing does not prevent tail-biting.     

Click and low-, middle-, and high-frequency toneburst stimulation of the canine cochlea

A method was developed to deliver tonebursts ranging in frequency from 1 to 32 kHz for frequency-specific assessment of the canine cochlea. Brainstem auditory-evoked responses (early latency responses, 0-10 ms) to a click (CS) and to 1-, 2-, 4-, 8-, 12-, 16-, 24-, and 32-kHz toneburst stimulations (TS) were compared at 80-dB sound pressure level stimulus (SPL) intensity in 10 adult dogs. All stimulations yielded a 5-7 positive wave pattern, with the exception of the 1-kHz TS, which evoked a frequency-following response (FFR). Thresholds were lowest for the CS and the 12- and 16-kHz TS. All individual peak latencies for TS were significantly (P < or = .05) longer than for CS. Peak I latencies were significantly (P < or = .05) shorter for the 12- and 16-kHz TS than for the other TS. Interpeak latencies I-V were significantly (P < or = .05) longer for the 4- to 32-kHz TS than for CS. Differences in interpeak latencies I-III were not significant. Amplitudes of waves I and V were significantly (P < or = .05) lower for TS than for CS, except for higher wave V amplitude (P < or = .05) at 2- and 32-kHz TS. Peak I-V amplitude ratios were significantly (P < or = .05) higher for the 2-, 4-, 16-, 24-, and 32-kHz TS and lower for the 8- and 12-kHz TS, compared to CS. We conclude that reproducible information on frequency specificity of the canine cochlea can be obtained by TS. This report provides a normative database for parameters needed to evaluate frequency-specific hearing loss in dogs.     

Immunocontraception in Eastern gray squirrels (Sciurus carolinensis): morphologic changes in reproductive organs

Eastern gray squirrels (EGS) (Sciurus carolinensis) damage trees through bark stripping or gnawing due to territorial marking or agonistic gnawing behavior in concert with higher densities. This study was conducted to determine the effects of a contraceptive vaccine on EGS and its reproductive organ histology. Free-ranging urban EGS were vaccinated with the immunocontraceptive GonaCon. All EGS were > or = 6 mo of age as determined by a combination of pelage characteristics and body weights. The vaccine was administered by injection at a dosage rate of 0.4 ml containing 400 microg of GnRH-mollusk protein conjugate i.m. in the thigh to 33 EGS (17 male [m], 16 female [f]) in trapping session 1 (TS1), 23 (14 m, 9 f) in trapping session 2 (TS2), and 11 (8 m, 3 f) in trapping session 3 (TS3). A sham injection containing 0.4 ml saline-AdjuVac was given as control to 22 EGS (16 m, 6 f) in TS1, 20 (12 m, 8 f) in TS2, and 8 (4 m, 4 f) in TS3. In the last trapping session (TS4), 35 EGS (16 treated, 19 control) were killed for necropsy to evaluate histologic changes in testes and ovaries. Treated EGS males had testicular, prostatic, and epididymal atrophy compared with control EGS males. The tubuli seminiferi and prostatic glandular lumen of treated EGS males were atrophic, and the epididymal lumen contained no sperm cells. No histologic changes were observed in treated EGS females; however, females likely were not collected when changes due to GonaCon would have been observed. There were no observable histologic differences in the pituitary gland of treated and control EGS. There were no statistically significant differences in either testosterone or progesterone concentrations between control and treated EGS. Although there were no serious side effects to the vaccine, six EGS developed injection site abscesses. GonaCon may be a potential tool for EGS population control.     

Ceftiofur sodium, a broad-spectrum cephalosporin: evaluation in vitro and in vivo in mice

Ceftiofur sodium, a broad-spectrum beta-lactamase-resistant cephalosporin, was evaluated in vitro and in vivo in mice. Ceftiofur is the sodium salt of (6R, 7R)-7[( 2-amino-4-thiazolyl)-Z- (methoxyimino)acetyl]amino)-3-[( (2-furanylcarbonyl)thio]methyl)-8-oxo-5- thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylate. Minimal inhibitory concentration values were obtained with 264 strains representing 9 genera and 17 species of bacterial pathogens from cattle, swine, sheep, horses, poultry, dogs, cats, and human beings. Ceftiofur was more active than was ampicillin against all strains tested including beta-lactamase-producing organisms. In mice with systemic infections, ceftiofur was more active than or equivalent to ampicillin, cephalothin, cefamandole, cloxacillin, cefoperazone, or pirlimycin. These protection tests included infections with Escherichia coli, Haemophilus pleuropneumoniae, H somnus, Pasteurella haemolytica, P multocida, Salmonella typhimurium, or Staphylococcus aureus. In infant mice with E coli-induced lethal diarrhea and in mice with S aureus and E coli-induced mastitis, ceftiofur was comparable or more active than was ampicillin.     

Active immunization of beef heifers against luteinizing hormone: II. Evaluation of conjugation technique on antigenicity of LH

This study evaluated the antigenicity of LH-ovalbumin complexes produced using different conjugation techniques. Two homobifunctional cross-linkers, glutaraldehyde (Glut) and carbodiimide (ECDI), were evaluated together with one heterobifunctional reagent, m-maleimido-benzoyl N-hydroxysuccinimide ester (MBS). Polyacrylamide gel electrophoresis and Western transfer techniques were used to confirm conjugation of LH. Forty-four beef heifers were assigned randomly to seven treatment groups. Two groups of heifers were immunized against glutaraldehyde conjugates (Glut-I and Glut-II), two against MBS conjugates (MBS-I and MBS-II) and one against a carbodiimide conjugate (ECDI). Control animals were immunized against nonconjugated LH (LH-only) or ovalbumin alone (Oval). Heifers received one primary injection of antigen followed by two boosters at a 3-wk interval. The Glut conjugates induced the highest (P less than .05) LH antibody activity (Glut-II, 18 +/- 4%; Glut-I, 14 +/- 4%). The ECDI (11 +/- 4%), and MBS-I (11 +/- 2%) conjugates induced greater LH binding than MBS-II (4 +/- 1%), LH-only (4 +/- 1%) or Oval (2 +/- 1%). Glutaraldehyde produced an LH-ovalbumin conjugate of greater LH immunogenicity than either ECDI or the heterobifunctional reagent, MBS.     

Attenuation of avian infectious bronchitis virus by cold-adaptation.

Avian infectious bronchitis virus (IBV) Arkansas-type DPI strain was passaged 10 times in specific-pathogen-free (SPF) chicken embryos incubated at 28 C and 37 C. Virus grown at 28 C acquired cold-adapted (CA) and temperature-sensitive (TS) characteristics based on more-rapid growth at 28 C and a reduced ability to grown at 41 C, respectively, compared with non-cold-adapted (non-CA) virus grown at 37 C. The pathogenicity and immunogenicity were determined for CA and non-CA IBV in 1-day-old SPF chickens following intratracheal inoculation. The percentage of CA IBV-vaccinated chicks exhibiting respiratory disease exceeded 30% on only 1 day postinoculation (PI) (day 5 PI), compared with 8 days (days 2-9 PI) for birds given non-CA IBV. Mortality was 0% for CA IBV-vaccinated chickens and 6% for non-CA virus-vaccinated chickens. Microscopically, both CA and non-CA IBV caused diffuse tracheal deciliation, although mucosal hyperplasia, necrosis, and heterophil infiltration were more severe with non-CA IBV. Virus was reisolated from kidneys of chickens given CA IBV, suggesting the loss of the TS property. The instability of the TS property was confirmed by growth of the reisolated virus at 41 C. Both CA and non-CA viruses induced complete protection against homologous challenge virus infection of the upper respiratory tract.     

Identification of a soluble UDP-Gal: Gal (beta 1-4)Glc (or GlcNAc) (alpha 1-3) galactosyltransferase of bovine colostrum

A soluble UDP-Gal: Gal (alpha 1-3) galactosyltransferase was first detected in bovine colostrum and this enzyme activity was simply assayed by using rho-nitrophenyl-beta-lactoside (Gal(beta 1-4)Glc-C6H5NO2, rho NP-lactoside) as an acceptor. Treating the radioactive product with alpha- or beta-galactosidase, the radioactivity (greater than 95%) was released by only alpha-galactosidase and was identified as [3H]galactose. This shows that galactosyl residue was alpha-linked to rho-nitrophenyl-beta-lactoside. Methylation, hydrolysis, thin layer chromatography and fluorography of the reaction product (Gal(alpha 1-)-[3H]Gal(beta 1-4)Glc-rho NP) yielded 2,4,6-tri-O-methyl[3H]galactose, indicating that galactosyl residue had been transferred to the carbon-3 position of the terminal nonreducing beta-galactosyl residue in rho-nitrophenyl-beta-lactoside. These results confirmed that the structure of the reaction product was Gal(alpha 1-3)Gal(beta 1-4)Glc-rho NP. The enzyme requires Mn2+ for its activity, and shows pH optimum from 6.5 to 7.5. rho-Nitrophenyl-beta-lactoside and asialo alpha 1-acid glycoprotein were more effective as an acceptor than N-acetyllactosamine. The bovine colostrum (alpha 1-3) galactosyltransferase could not convert human O red cells into B active cells, indicating that this enzyme preparation did not contain the activity to synthesize human blood group B erythrocytes.     

保护性耕作下蚕豆田土壤呼吸及碳平衡特性

土壤呼吸是碳循环的重要环节,为探讨垄作和覆盖对旱三熟蚕豆田土壤呼吸的影响,测定了平作(T)、垄作(R)、平作+半量覆盖(TS₁)、垄作+半量覆盖(RS₁)、平作+全量覆盖(TS₂)、垄作+全量覆盖(RS₂)6种处理下的西南紫色土丘陵区蚕豆/玉米/甘薯旱三熟体系中蚕豆生长季节的土壤呼吸变化,分析了蚕豆田碳平衡特性。结果表明,蚕豆生长季节农田土壤呼吸随作物生长一致,呈先增加后减弱的变化趋势,变化范围为0.885~10.213 μmol/(m²·s)。全生育期平均土壤呼吸速率差异显著,表现为TS₂>RS₂>RS₁ >TS₁>T>R;分别为4.096,3.780,3.441,3.104,2.850,2.439 μmol/(m²·s),较平作不覆盖处理增加了43.7%,32.6%,20.7%,8.9%,-14.4%。垄作显著降低了蚕豆农田土壤呼吸速率,而秸秆覆盖显著提高土壤呼吸速率,且随着覆盖量的增加而增加。不同生育阶段土壤呼吸总量存在差异,表现为成熟期<苗期<鼓粒期<分枝现蕾期<开花结荚期,其中开花结荚期约占50%;利用根排除法测得蚕豆田根系呼吸占土壤呼吸比例变幅为19.49%~54.23%,利用回归分析法测得结果为37.02%~60.64%,二者均值分别为38.62%和49.12%。不同耕作和覆盖处理下蚕豆田整个生长季均表现为碳汇,净碳汇为857.26~2236.25 kg/hm²。与平作不覆盖相比, RS₂、TS₂、RS₁、TS₁、R分别较平作不覆盖处理T高出160.86%,101.44%,30.78%,47.63%,110.41%,差异达显著水平。试验结果表明垄作和秸秆覆盖有利于蚕豆田生态系统的碳汇,以垄作+全量覆盖的效果最好。     

Identification of metoclopramide metabolites in the urine of cattle by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection

The urinary metabolites of metoclopramide (4-amino-5-chloro-N-[2-diethylaminoethyl]-2-methoxybenzamide) were identified in cows. The drug was administered intravenously, voided urine was collected, and individual urine extracts were analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection. The parent compound and one major metabolite (4-amino-5-chloro-N-[2-(ethylamino)ethyl]-2-methoxybenzamide) were common to all individuals. In addition to the parent and major metabolite, a second, minor metabolite was identified in two cows as 4-amino-5-chloro-N-[2-(diethylamino)ethyl]-2-hydroxybenzamide. The identity of the minor metabolite was confirmed by comparison with a standard synthesized by a new method. Metabolite identification and characterization in food animal species allows the design of safety and environmental impact studies and relative metabolite ratios between dose treatment groups.Abbreviations ¹H-NMR proton nuclear magnetic resonance - R _T retention time - D₁ dopamine-1 - D₂ dopamine-2 - 5-HT₃ 5-hydroxytryptamine - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - UV-vis ultraviolet-visible - ID internal diameter - m/z mass/charge ratio