气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因,用纯化的重组蛋白建立其间接ELISA检测方法,a。利用大肠杆菌密码子的偏爱性优化CctA基因序列,克隆至原核表达载体pET-28a （+）,双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌,经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化,SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗,用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法,以复性后的重组CctA蛋白作为检测抗原,摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清,同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示,得到大小约853 bp的条带,与预期相符,且测序结果正确。SDS-PAGE结果表明,成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示,豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL,于4 ℃包被过夜;封闭液选择10%胎牛血清,37 ℃孵育2 h;二抗的稀释度为1：8 000;室温避光显色10 min后终止反应,测定D_{450 nm}值。当P/N>4.6时,间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白,建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法,有望成为气肿疽灭活疫苗免疫效果验证的替代方法。     

贮藏时间与蒙药黄芩对紫花苜蓿草颗粒维生素和化学成分的影响

为有效保全苜蓿草颗粒贮藏过程中维生素及营养品质,本研究以紫花苜蓿草颗粒为研究材料,研究贮藏时间(0,60,120,180,240 d,冷冻240 d)与蒙药黄芩(0%,0.5%,1.0%,1.5%)对其维生素含量和营养品质的影响。结果表明：在贮藏过程中,与其他添加量相比,添加1.5%黄芩有利于保存苜蓿草颗粒维生素B5、维生素C和α-维生素E含量。贮藏60 d及以上显著降低了苜蓿草颗粒维生素B1、维生素B3、维生素C和维生素E含量;贮藏120 d时苜蓿草颗粒DM含量趋于稳定;苜蓿草颗粒贮藏180 d后其CP含量显著降低;贮藏0～180 d期间,随着贮藏时间的延长,苜蓿草颗粒NDF和ADF含量显著升高。与常温贮藏240 d相比,冷冻贮藏相同时间有利于保存苜蓿草颗粒中维生素B1、维生素B5、α-维生素E和OM含量,显著降低DM、NDF和ADF含量。因此,1.5%黄芩添加剂有利于保存苜蓿草颗粒维生素含量,且常温贮藏时间不宜超过60天。     

Treatment of contaminated radial fracture in Sprague-Dawley rats by application of a degradable polymer releasing gentamicin

Fracture-related infections remain a leading cause of morbidity and mortality. We aimed to establish a simple contaminated radial osteotomy model to assess the efficacy of a biodegradable polymer poly(sebacic-co-ricinoleic acid) [p(SA-RA)] containing 20% w/w gentamicin. A unilateral transverse osteotomy was induced in Sprague-Dawley (SD) rats, followed by application of Staphylococcus aureus suspension over the fracture. After successfully establishing the contaminated open fracture model, we treated the rats either systemically (intraperitoneal cefuroxime), locally with p(SA-RA) containing gentamicin, or both. Control groups included non-contaminated group and contaminated groups that were either untreated or treated with the polymer alone. After 4 weeks, the bones were subjected to micro-CT scanning and microbiological and histopathology evaluations. Micro-CT analysis revealed similar changes in the group subjected to both local and systemic treatment as in the non-contaminated control group. Lack of detectable bacterial growth was noted in most animals of the group subjected to both local and systemic treatment, and all samples were negative for S. aureus. Histopathological evaluation revealed that all treatment modalities containing antibiotics were highly effective in reducing infection and promoting callus repair, resulting in early bone healing. While p(SA-RA) containing gentamicin treatment showed better results than cefuroxime, the combination of local and systemic treatment displayed the highest therapeutic potential in this model.     

Amoxicillin—current use in swine medicine

Amoxicillin has become a major antimicrobial substance in pig medicine for the treatment and control of severe, systemic infections such as Streptococcus suis. The minimum inhibitory concentration 90% (MIC 90) is 0.06 μg amoxicillin/ml, and the proposed epidemiological cut‐off value (ECOFF) is 0.5 μg/ml, giving only 0.7% of isolates above the ECOFF or of reduced susceptibility. Clinical breakpoints have not been set for amoxicillin against porcine pathogens yet, hence the use of ECOFFs. It has also been successfully used for bacterial respiratory infections caused by Actinobacillus pleuropneumoniae and Pasteurella multocida. The ECOFF for amoxicillin against A. pleuropneumoniae is also 0.5 μg/ml demonstrating only a reduced susceptibility in 11.3% of isolates. Similarly, P. multocida had an ECOFF of 1.0 μg/ml and a reduced susceptibility in only 2.6% of isolates. This reduced susceptibility disappears when combined with the beta‐lactamase inhibitor, clavulanic acid, demonstrating that it is primarily associated with beta‐lactamase production. In contrast, amoxicillin is active against Escherichia coli and Salmonella species but using ECOFFs of 8.0 and 4.0 μg/ml, respectively, reduced susceptibility can be seen in 70.9% and 67.7% of isolates. These high levels of reduced susceptibility are primarily due to beta‐lactamase production also, and most of this resistance can be overcome by the combination of amoxicillin with clavulanic acid. Currently, amoxicillin alone is considered an extremely valuable antimicrobial in both human and animal medicine and remains in the critically important category of antibiotics alongside the fluoroquinolones and macrolides by the World Health Organization as well as the third‐ and fourth‐generation cephalosporins, but these cephalosporins show marked resistance to basic beta‐lactamase production and are only destroyed by the extended‐spectrum beta‐lactamases. Amoxicillin alone and in combination with clavulanic acid are currently classed together in Category 2 in the European Union. By reviewing the pharmacodynamic data and comparing this with pharmacokinetic data from healthy and infected animals and clinical trial data, it can be seen that the product has a good efficacy against S. suis and A. pleuropneumoniae, in spite of usage over many years. However, it may be much less efficacious on its own against E. coli, due to reduced susceptibility and resistance associated with beta‐lactamase production, which is largely overcome by the use of clavulanic acid. It is felt that this differentiation may be useful in future classification of amoxicillin alone, in comparison with its combined use with clavulanic acid and thereby preserve the use of the more critically important antibiotics in veterinary medicine and reducing the risk of their resistance being transmitted to human.     

Acute oral toxicity of zinc phosphide: an assessment for wild house mice (Mus musculus)

Irregular plagues of house mice, Mus musculus, incur major economic impacts on agricultural production in Australia. The efficacy of zinc phosphide (ZnP), the only registered broadacre control agent for mice, is reported as increasingly variable. Have mice become less sensitive over time or are they taking a sub-lethal dose and developing aversion? In this laboratory study, the sensitivity of mice (wild caught; outbred laboratory strain) was assessed using oral gavage of a range of ZnP concentrations. The estimated LD₅₀ values (72–79 mg ZnP/kg body weight) were similar for each mouse group but are significantly higher than previously reported. The willingness of mice to consume ZnP-coated grains was determined. ZnP-coated grains (50 g ZnP/kg grain) presented in the absence of alternative food were consumed and 94% of wild mice died. Mice provided with alternative food and ZnP-coated wheat grains (either 25 or 50 g ZnP/kg grain) consumed toxic and non-toxic grains, and mortality was lower (33–55%). If a sublethal amount of ZnP-coated grain was consumed, aversion occurred, mostly when alternative food was present. The sensitivity of wild house mice to ZnP in Australia is significantly lower than previously assumed. Under laboratory conditions, ZnP-coated grains coated with a new higher dose (50 g ZnP/kg grain) were readily consumed. Consumption of toxic grain occurred when alternative food was available but was decreased. Our unambiguous findings for house mice indicate a re-assessment of the ZnP loading for baits used for control of many rodents around the world may be warranted.     

柏芩提取液的药效学及对奶牛子宫内膜炎的疗效评价

柏芩提取液是由黄柏、黄芩、红花、党参等组成,为了探讨其对奶牛子宫内膜炎的治疗作用,采用实验性家兔子宫内膜炎模型进行柏芩提取液的主要药效学研究。确定药效作用后,按照农业农村部颁布的《防治奶牛临床子宫内膜炎的抗微生物兽药的靶动物安全性和有效性试验指导原则》对其进行奶牛子宫内膜炎的临床疗效评价。药效学试验结果表明,与模型组比较,柏芩提取液能明显改善实验性子宫内膜炎家兔的临床症状,血液白细胞计数分类、子宫分泌物涂片镜检、尿液及子宫分泌物化学检查结果趋于正常,并且能够减轻子宫充血、炎性细胞浸润、上皮细胞脱落等组织病理学改变。临床疗效试验结果表明,给药后第10天,柏芩提取液高、中剂量组的治愈率极显著高于低剂量组（P<0.01）,且与阳性药物对照组无显著差异（P>0.05）;各试验组的总有效率差异均不显著（P>0.05）。给药后第21天,柏芩提取液高、中剂量组的治愈率显著高于低剂量组（P<0.05）,且与阳性药物对照组差异不显著（P>0.05）;各试验组的总有效率未见明显差异（P>0.05）。以上结果证明柏芩提取液对实验性家兔子宫内膜炎和临床奶牛子宫内膜炎具有良好的治疗作用,其中1.5和1.0 g/mL浓度（30 mL/次）对临床病例的治疗效果明显,与土霉素效果相似,0.5 g/mL浓度效果不明显。本研究为其进一步研究和开发应用奠定了基础。     

黄连解毒散固体分散制剂的药效学研究

为了对黄连解毒散固体分散制剂进行药效学研究,采用致病性大肠杆菌和金黄色葡萄球菌为试验菌株考察黄连解毒散固体分散制剂的体外抑菌效果,以仔猪为实验动物考察其临床药效。结果显示：黄连解毒散固体分散制剂的体外抑菌效果是普通散剂的2倍,临床治疗效果比普通散剂提高了30％。黄连解毒散固体分散制剂显著提高了黄连解毒散对猪大肠杆菌病的疗效。     

Glomerular filtration rate after tramadol, parecoxib and pindolol following anaesthesia and analgesia in comparison with morphine in dogs

ObjectiveTo compare the effects of morphine, parecoxib, tramadol and a combination of parecoxib, tramadol and pindolol on nociceptive thresholds in awake animals and their effect on glomerular filtration rate (GFR) in dogs subjected to 30 minutes of anesthesia.AnimalsEight adult mixed breed experimental dogs.Study designRandomized, controlled trial.MethodsDogs received 0.05 mg kg^?1 acepromazine subcutaneously (SC) as anaesthetic pre-medication. Thirty to sixty minutes later, they received either tramadol 3 mg kg^?1 intravenously, (IV), parecoxib (1 mg kg^?1 IV), a combination of tramadol 3 mg kg^?1 (IV), parecoxib 1 mg kg^?1 (IV) and pindolol 5 μg kg^?1 (SC), morphine (0.1 mg kg^?1 (IV) or 0.9% saline (2 mL). Anaesthesia was then induced with IV propofol to effect (2.9 ± 0.8 mg kg^?1) and maintained with halothane in oxygen for 30 minutes. Systolic arterial blood pressure was maintained above 90 mmHg with IV fluids and by adjusting the inspired halothane concentration. Post-treatment nociceptive thresholds to mechanical stimuli, expressed as percent of pre-treatment values, were compared between the treatments to assess the analgesic efficacy of the drugs. Plasma iohexol clearance (ICL), a measure of GFR, was estimated both before and 24 hours after induction of anaesthesia to study the drugs’ effects on renal perfusion. Nociceptive threshold and GFR data were compared using mixed model analysis in sas^®9.1.ResultsBoth tramadol and parecoxib produced similar analgesia, which was less than that of morphine. Their combination with pindolol produced analgesia comparable with morphine. None of the test drugs, either alone or in combination, reduced GFR.ConclusionTramadol and parecoxib (either alone or in combination) can increase nociceptive thresholds in awake dogs and have minimal effects on renal perfusion in normotensive dogs subjected to anaesthesia.     

中药复方壮腰散对小鼠睾丸发育及抗缺氧能力效果观察

以牦牛鞭为主,辅以其他中药制成中药复方壮腰散,分别饲喂小白鼠,观察其对小鼠睾丸发育和抗缺氧、抗疲劳、腹腔巨噬细胞吞噬能力的影响。结果表明,单用牦牛鞭能促进小白鼠睾丸发育和增强其免疫力;牦牛鞭配以其他中药组成复方,不仅能促进睾丸发育,而且对提高小鼠的抗缺氧能力、抗疲劳能力均有显著作用。     

烟草成分对其品质影响程度的粗糙集判别方法

运用粗糙集理论研究了烟草主要化学成分 (总糖、还原糖、总氮、烟碱、氧化钾和氯离子 )对烟草品质影响程度的判别方法。对比已有的研究结果 ,表明基于粗糙集理论的计算方法简单 ,结果明了 ,而且还能量化烟草成分对品质的影响程度。