截短侧耳素类药物侧链改进专利分析

截短侧耳素类药物作为一类经典抗生素,研究人员不断对其进行结构改造以期获得更好的抗菌活性和更低的耐药性。截短侧耳素的C14位侧链是有效的改造位点。本文针对近年来对于C14位侧链的专利申请进行介绍,并给出了相关领域的专利申请建议。     

截短侧耳素及其衍生物的研究进展

截短侧耳素是一种具有抗菌活性的三环二萜类化合物,它的两种衍生物泰妙菌素和沃尼妙林已在兽医临床得到广泛应用。由于该类化合物独特的抗菌机制,近年来截短侧耳素衍生物备受药物研究工作者的关注。文章简要综述了截短侧耳素衍生物的抗菌作用机制、构效关系及耐药性,以期为该类动物专用抗菌药的研发提供参考。     

截短侧耳素的萃取及结晶

研究截短侧耳素的萃取和结晶。截短侧耳素闪蒸粉分别用甲醇、乙醇和K酮在20℃、40℃和60℃条件下萃取,萃取时间60min,比较萃取率;60℃的K酮饱和溶液,分别降温至50℃、45RE和40℃,加入晶种保温3h,然后以5-8℃／h的速度降温至0℃,然后保温2h,离心获得晶体,50℃干燥1h,获得截短侧耳素粉,分析干燥失重、含量、杂质和收率。K酮在60℃时的萃取率最高,均值为92％;40℃加入晶种保温3h获得产品收率最高95．01％。K酮为更加理想的截短侧耳素萃取剂,萃取温度60℃,晶体成长的第一过程选择40℃。     

苯甲磺酰截短侧耳素国家对照品的研制

研制首批苯甲磺酰截短侧耳素甲磺酰截短侧耳素对照品为白色或类白色粉末,红外光吸收图谱与USP溯源对照品图谱一致,质谱法测定分子离子峰为555.19 Da([M+Na]~+),高效液相色谱法测定其平均纯度为99.7%,可作为系统适用性对照品,用于延胡索酸泰妙菌素及其制剂含量测定的系统使用性检查。     

高效液相色谱法测定二甲基半胱胺截短侧耳素的研究

建立了以高效液相色谱法测定盐酸沃尼妙林关键中间体二甲基半胱胺截短侧耳素含量的检测方法。色谱条件为SepaxC18色谱柱(150×4.6mm,3μm)流动相为乙腈-磷酸缓冲液(pH2.5)(V/V= 40∶60), 流速为1ml/min,检测波长为205nm。 并对该检测方法进行了系统适用性、线性、精密度、重现性稳定性、检测限、定量限以及回收率的考察,结果均良好,可以用于盐酸沃尼妙林关键中间体的含量检测。     

截短侧耳素类抗生素的研究进展

截短侧耳素(pleuromutilin,化合物1)是由担子菌产生的一种三环二萜类兽用抗生素,对革兰阳性菌和支原体有强抗菌活性[1]。自1950年发现以来,以截短侧耳素为先导化合物,已成功开发出泰妙菌素(tiamulin,化合物2)和沃尼妙林(valnemulin,化     

用PCR对鸡毒支原体感染的检测

应用已建立的检测 MG和 MS的 PCR方法 ,对人工接种鸡毒支原体后 2～ 2 0 d的 SPF鸡气管棉拭样品和气管、肺、肝、脾、胸肌、腿肌等器官和组织进行了检测 ;对现场采集的样品同样作 PCR检测。结果表明 ,上述人工样品均检测到病原 ,以气管棉拭样品检出最多 ;现场样品PCR的阳性检出率为 1 0 .2 8% ,分离培养的阳性率为 2 .8% ,敏感性前者高于后者。     

甲磺酸培氟沙星对体外鸡毒支原体的抑菌试验和对鸡毒支原体病的治疗试验

利用甲磺酸培氟沙星水溶性粉剂对鸡毒支原体分别进行体内外的抑菌治疗试验。体外抑菌试验结果表明：该药物能有效地抑制试管中鸡毒支原体的生长繁殖。体内试验结果表明：当饮水中药物浓度达到１００ｍｇ／Ｌ时,能有效地防止由于鸡毒支原体引起的死亡;当饮水中药物浓度达到５０ｍｇ／Ｌ以上时,能明显地减轻以至部分消除鸡毒支原体强毒引起的气囊损伤;和感染不治疗对照组相比,该药物能提高鸡体增重;用甲磺酸培氟沙星治疗,能抑制感染鸡体内的鸡毒支原体,从而使部分鸡体内检测不到鸡毒支原体抗体。     

氧氟沙星对体外鸡毒支原体的抑菌试验和对鸡体内鸡毒支原体病的治疗试验

利用氧氟沙星水溶液对鸡毒支原体分别进行了体外抑菌试验和体内鸡毒支原体病的治疗试验。体外试验结果表明： 当饮水中药物浓度达到２５ｐｐｍ , 能较好地防止由于鸡毒支原体病引起的死亡;当浓度达到５０ｐｐ ｍ 时, 对提高鸡体增重、抑制体内鸡毒支原体产生方面也有一定的作用; 当浓度增至１００ｐｐｍ 时, 能有效地减轻感染鸡的气囊损伤。其作用优于强力米先。     

Pathogenicity and immunogenicity of three Mycoplasma gallisepticum isolates in house finches (Carpodacus mexicanus)

Mycoplasma gallisepticum (MG) has become a common cause of conjunctivitis in free-living house finches (Carpodacus mexicanus) since its emergence in the early 1990s. To date, temporal and spatial genotypic variation in MG has been documented, but phenotypic variation in pathogenicity and immunogenicity has not been examined. House finches were inoculated with MG isolates Virginia (VA)1994, California (CA)2006, or North Carolina (NC)2006, which were cultured from free-living house finches with conjunctivitis in 1994, 2006, and 2006, respectively. Infection with NC2006 resulted in the most severe eye lesions, highest pathogen loads, and highest levels of pathogen-specific lachrymal and serum antibodies. Infection with CA2006 caused the least severe eye lesions, lowest pathogen load, and lowest levels of antibodies. A small number of birds in each group developed protracted, severe disease in spite of robust antibody responses, suggesting that immunopathology may contribute to the lesions. Immunoblot analyses indicated that isolates are antigenically similar; thus, there may be partial cross-protection if a house finch encounters two or more strains of MG throughout the course of its lifetime. This study provides evidence that MG strains or strain variants circulating in house finch populations vary in their ability to cause disease, induce antibody responses, and persist in the host.     

抗菌药物联合使用对耐达氟沙星鸡毒支原体的体外抑制作用研究

为探究抗菌药物联合使用对耐达氟沙星鸡毒支原体的体外抑制效果,选择鸡毒支原体标准株S6和对达氟沙星MIC升高程度不同的突变株M1、M2和M4,分别测定泰乐菌素、大观霉素、林可霉素、泰妙菌素、替米考星和多西环素种对4株鸡毒支原体的最低抑菌浓度,并使用达氟沙星分别联合上述6种抗菌药物进行体外抑菌试验.结果显示:除达氟沙星外,...     

The effect of including anti-Ig sera in the haemagglutination inhibition test forMycoplasma gallisepticum

Addition of anti-immunoglobulin M (anti-IgM), G (anti-IgG) and A (anti-IgA) sera to the haemagglutination-inhibition (HI) test (anti-Ig HI test) forMycoplasma gallisepticum resulted in 2- to 8-fold increases in the HI titres. On investigating the anti-Ig HI reaction using IgM and IgG antibodies separated by affinity chromatography, it was confirmed that, in the enhanced HI titres, specificity existed between the chicken Ig classes having antibody activity and the antisera used in the test. Four days after inoculation ofM. gallisepticum, the anti-Ig HI reaction was markedly enhanced by anti-IgM antiserum in the intravenously inoculated chickens and by anti-IgA serum in the nasally inoculated chickens. Ten days after inoculation ofM. gallisepticum marked enhancement of the reaction was produced by anti-IgG serum in both intravenously and nasally inoculated chickens, but the enhancement of the anti-Ig HI reaction diminished from the second week after inoculation.     

复方硫氰酸红霉素可溶性粉对人工感染鸡慢性呼吸道病的疗效试验

用复方硫氰酸红霉素可溶性粉以每升水中加0.5、0.6、0.7、0.8、0.9 g 5个剂量对人工感染鸡慢性呼吸道病的鸡群混饮治疗,同时设立硫氰酸红霉素可溶性粉对照组.试验结果表明,用药5d后,复方硫氰酸红霉素可溶性粉每升水加0.6、0.7、0.8、0.9g剂量组的有效率和治愈率皆高于硫氰酸红霉素可溶性粉组.     

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

Background

A field trial was carried out with two Mycoplasma hyopneumoniae vaccines in order to investigate the benefit of vaccination under field conditions in modern Danish pig production facilities with pigs being positive for M. hyopneumoniae. The M. hyopneumoniae infection of the herd was confirmed through blood samples that were positive for antibodies against M. hyopneumoniae combined with gross lesions of the lungs related to M. hyopneumoniae at slaughter and detection of M. hyopneumoniae by polymerace chain reaction in these lesions.

Results

A total of 2,256 pigs from two herds were randomly divided into three groups. Group 1 received 2 mL ThoroVAX^®VET, Group 2 received 1 mL Ingelvac^®MycoFLEX, and Group 3 was a non-vaccinated control group. The vaccination was performed by a person who was not involved in the rest of the trial and vaccination status thereby blinded to the evaluators.The prevalence of lung lesions related to M. hyopneumoniae were significantly lower for pigs vaccinated with ThoroVAX^®VET but not for pigs vaccinated with Ingelvac^®MycoFLEX^®, when compared to non-vaccinated pigs. There was no significant effect of vaccination on growth rate, antibiotic consumption or mortality.

Conclusion

This trial demonstrated that vaccination with Thoro^®VAX VET was effective in reducing the prevalence of lung lesion in pig units infected with M. hyopneumoniae.     

解淀粉芽孢杆菌的分离鉴定及其对嗜水气单胞菌抑制作用研究

本试验从鲫鱼肠道中分离到1株芽孢杆菌,经生物学特性测定及16S rRNA测序分析,鉴定该分离菌株为高产蛋白酶的解淀粉芽孢杆菌(命名为JX001).对该分离菌进行嗜水气单胞菌体外抑菌试验,结果发现有明显的抑菌圈和抑菌带,说明该分离菌对嗜水气单胞菌具有较强的抑制作用,在养殖水中加入分离菌JX001可防止嗜水气单胞菌的暴发;同时分离菌JX001在生长代谢过程中产生大量的蛋白酶,可有效地促进蛋白质水解,用于蛋白质饲料的降解.     

鸡毒霉形体HS株pMGA多基因族的研究

鸡毒霉形体（Mycoplasma gallisekpticum,GM)基因组中存在着编码细胞表面血凝粘附素蛋白（protein of Mycoplasma gallisepticum adhesin,pMGA)相关基因组的多基因族。为筛选出含有完整的pMGA基因片段，并估算出鸡毒霉形体HS株基因组中pMGA多基因族的基因数，本试验以pUC18为载体，用EcoR I限制性内切酶构建了鸡毒霉形体HS株的基因组文库。根据pMGA基因引导序列的保守区和pMGA基因间隔区的序列，人工合成了2个寡核苷酸探针（探针I、探针Ⅱ），经标记后，双筛选所建的基因文库，从600个转化子中共得到的38个含有pMGA基因的阳性克隆子，选其中1个7．5kb的片段（17号阳性克隆子，W17），用4种限制性内切酶（EcoR I,Hind Ⅲ，Pst I,Bgl Ⅱ）酶切消化，绘制了该片段的物理图谱。该片段酶切产物经电泳、转膜后与探针I和探针Ⅱ杂交，发现2个探针杂交图谱基本相同，根据杂交带及放射信号的强度值，估测出该片段含有4个pMGA基因，以这个片段所含的pMGA基因数为标准，估算出鸡毒霉形体HS株含有39个pMGA基因。     

4种佐剂对绵羊肺炎支原体的免疫辅佐效力研究

不同种类的佐剂对同种抗原的免疫辅佐效力不同。为筛选绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)灭活疫苗的理想佐剂,本研究制备了4种不同佐剂的MO灭活疫苗,分别免疫成年家兔,用MO间接血凝试剂盒测定免疫后1～9周血清抗体效价。检测结果显示,4种疫苗在免疫后都能迅速产生抗体,其中用MO Buonavoglial's和ISA-760佐剂疫苗免疫家兔,产生抗体均于免疫后第3周达到高峰((5.67±0.70) log2,6.00 log2),到第9周时维持抗体高峰(6.00 log2,(6.00±1.00) log2);MO ISA-206和白油佐剂疫苗免疫抗体均于免疫后第4周达到高峰((4.33±0.57) log2,5.00 log2),至第9周下降约1个滴度。统计学分析结果显示,ISA-760、Buonavoglial's佐剂对MO的免疫增强效力显著优于ISA-206和白油佐剂(P<0.05),是MO灭活疫苗的理想候选佐剂,为高效MO灭活疫苗的研发提供了科学依据。     

以重组PvpA蛋白作为抗原的鸡毒支原体抗体间接ELISA检测方法的建立

为建立鸡毒支原体(MG)种特异性检测的间接ELISA检测方法,本研究以原核表达的PvpA蛋白作为包被抗原,初步建立了检测MG抗体的间接ELISA检测方法.特异性试验结果表明,重组PvpA蛋白与鸡新城疫病毒、鸡传染性支气管炎病毒、滑液支原体、大肠杆菌“O”抗原、禽沙门氏茵“O”抗原阳性血清均不发生交叉反应.该方法的批内变异系数小于8％,批间变异系数小于11％,表明具有较好的重复性.采用该检测方法与进口试剂盒对不同省份送检的鸡血清进行检测比较,两者阳性和阴性符合率均达到90％以上.本研究建立的间接ELISA检测方法为MG抗体检测试剂盒的研制奠定了基础.     

Control of Mycoplasma hyopneumoniae infections in pigs

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. The organism adheres to and damages the ciliated epithelium of the respiratory tract. Affected pigs show chronic coughing, are more susceptible to other respiratory infections and have a reduced performance. Control of the disease can be accomplished in a number of ways. First, management practices and housing conditions in the herd should be optimized. These include all-in/all-out production, limiting factors that may destabilize herd immunity, maintaining optimal stocking densities, prevention of other respiratory diseases, and optimal housing and climatic conditions. Strategic medication with antimicrobials active against M. hyopneumoniae and, preferably, also against major secondary bacteria may be useful during periods when the pigs are at risk for respiratory disease. Finally, commercial bacterins are widely used to control M. hyopneumoniae infections. The main effects of vaccination include less clinical symptoms, lung lesions and medication use, and improved performance. However, bacterins provide only partial protection and do not prevent colonization of the organism. Different vaccination strategies (timing of vaccination, vaccination of sows, vaccination combined with antimicrobial medication) can be used, depending on the type of herd, the production system and management practices, the infection pattern and the preferences of the pig producer. Research on new vaccines is actively occurring, including aerosol and feed-based vaccines as well as subunit and DNA vaccines. Eradication of the infection at herd level based on age-segregation and medication is possible, but there is a permanent risk for re-infections.