泰妙菌素完全抗原制备及鉴定

为建立泰妙菌素(Tiamulin,TML)完全抗原制备方法,分别用混合酸酐法(Mixed anhydride,MA)和N,N-羰基二咪唑法(1,1′-Carbonyldiimidazole,CDI)将半抗原泰妙菌素偶联至载体牛血清白蛋白(Bovine serum albumen,BSA),以制备泰妙菌素完全抗原。对2种方法制备的抗原进行紫外扫描鉴定和SDS-PAGE凝胶电泳鉴定,根据SDS-PAGE凝胶电泳图估算出完全抗原的偶联比。结果均显示抗原偶联成功,估算出的偶联比分别约为1∶23.6和1∶8.1,说明,混合酸酐法制备泰妙菌素完全抗原时偶联率更高。     

Cessation of clinical disease and spirochete shedding after tiamulin treatment in pigs experimentally infected with “Brachyspira hampsonii”

With the emergence of “Brachyspira hampsonii” associated with swine dysentery in North America, identification of effective treatments and interventions is a pressing need. Denagard® (tiamulin hydrogen fumarate) Liquid Concentrate 12.5% is approved in the United States for treatment of dysentery caused by Brachyspira hyodysenteriae at 0.006% in the water. In this study, the effectiveness of tiamulin in resolving clinical disease, eliminating viable spirochete shedding, and reducing neutrophilic colitis following infection with either “B. hampsonii” or B. hyodysenteriae was evaluated. Seventy-eight 7-week-old crossbred pigs were divided into three groups [sham-inoculated (n = 18), “B. hampsonii”-inoculated (n = 30), and B. hyodysenteriae-inoculated (n = 30)]. Each inoculum group was divided into three subgroups which received either 0.006% tiamulin, 0.018% tiamulin, or no medication. Both levels of tiamulin resolved clinical disease within 24 h of treatment initiation, eliminated spirochete shedding within 72 h of treatment initiation, and resolved and/or prevented histologic lesions in pigs infected with either Brachyspira spp.     

泰妙菌素与马杜霉素联用对金葡菌体外PAE的研究

分别测定了泰妙菌素及其与马社霉素联用对金黄色葡萄球菌的体外PAE.当药物以2MIC、4MIC和8MIC浓度作用于金黄色葡萄球菌时,泰妙菌素的体外PAE分别为1.56h、2.15h、3.02h;马杜霉素的体外PAE分别为1.59h、2.44h、3.15h;泰妙菌素与马杜霉素联用的体外PAE分别为3.46h、4.81h、6.48h.结果表明：泰妙菌素和马杜霉素在体外对金黄色葡萄球菌均有较长的PAE,且随药物浓度的升高其PAE也相应地延长呈明显的剂量依赖性.联用对金黄色葡萄球菌也有较长的PAE,且呈现相加或协同作用.     

泰妙菌素生产菌种原生质体制备条件优化

[目的]探讨泰妙菌素生产菌种原生质体的最优制备方法。[方法]通过对原生质体制备条件进行优化,建立泰妙生产菌种原生质体制备体系。[结果]优化后制备条件:以培养3 d的菌丝体为材料,将其置于含质量浓度0.10%溶壁酶、0.60 mol/L MgSO_4的酶液中,25℃酶解3.0~3.5 h,将酶解液用G2砂芯漏斗过滤,滤液即为原生质体悬液。采用该方法,泰妙生产菌种原生质体制备率达1.5×10~6个/m L。[结论]试验结果为提高泰妙菌素生产菌种的产量提供了理论依据。     

Tiamulin resistance in porcine Brachyspira pilosicoli isolates

There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified.     

泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的影响

本研究通过建立人肝微粒体体外孵育试验，考察泰妙菌素对4种CYP450亚酶的探针底物睾酮、非那西丁、氯唑沙宗、氢溴酸右美沙芬代谢的影响，以反映泰妙菌素对人CYP3A4、CYP1A2、CYP2E1、CYP2D6酶活性的作用。试验分为3组：药物试验组、阳性对照组（不含NADPH）、阴性对照组（不含CYP450抑制剂），孵育体系为100 μL，孵育试验在96孔板中进行。终止反应后使用高效液相色谱串联质谱仪（LC-MS/MS），以内标法检测96孔板中孵育液的剩余探针底物浓度。根据药物试验组与阴性对照组的探针药物代谢浓度之比，计算药物试验组的探针药物代谢率。使用Graphpad Prism 6.0软件，以药物试验组相对代谢率为纵坐标，药物浓度对数值为横坐标作图，计算试验组药物IC₅₀值。针对泰妙菌素与CYP3A4的孵育试验设置多个孵育时间点观察孵育时间对IC₅₀值的影响。试验结果显示，酮康唑对CYP3A4的IC₅₀值为0.044 μmol/L，α-萘黄酮对CYP1A2的IC₅₀值为0.030 μmol/L、4-甲基吡唑对CYP2E1的IC₅₀值为0.022 μmol/L、奎尼丁对CYP2D6的IC₅₀值为0.096 μmol/L。泰妙菌素对CYP1A2及CYP2D6的IC₅₀值均大于50 μmol/L，对CYP2E1的IC₅₀值为0.045 μmol/L，对CYP3A4的IC₅₀值为1.609 μmol/L。延长泰妙菌素与CYP3A4的孵育时间（10~50 min）后，泰妙菌素对CYP3A4的IC₅₀值由1.609 μmol/L增加至 8.657 μmol/L。本研究中4种亚酶常用抑制剂的IC₅₀值与参照值相近，表明所建立人肝微粒体体外孵育试验方法可靠。以IC₅₀值为指标显示泰妙菌素对CYP1A2和CYP2D6无抑制作用，对CYP2E1和CYP3A4存在强抑制作用，泰妙菌素可能是CYP3A4的可逆性抑制剂。     

泰妙菌素中溶剂残留量的测定

采用顶空气相色谱法,检测延胡索酸泰妙菌素中甲醇和乙酸乙酯有机溶剂的残留量。结果表明,甲醇和乙酸乙酯在设定范围内有较好的线性,其相关系数均为1.0000,平均回收率分别为105.7%和109.95%,相对标准偏差分别为1.30%和1.50%。该方法准确灵敏,重现性好,满足药典要求,适用于延胡索酸泰妙菌素原料药中有机溶剂残留量的测定。     

延胡索酸泰妙菌素可溶性粉对猪支原体肺炎的疗效研究

[目的]评价延胡索酸泰妙菌素可溶性粉对猪支原体肺炎的治疗效果。[方法]在水中添加30、45、60mg／L延胡索酸泰妙菌素可溶性粉,对猪支原体肺炎进行混饮治疗,研究延胡索酸泰妙菌素可溶性粉对猪支原体肺炎的治疗效果。[结果]与支原净对照组相比,延胡索酸泰妙菌素可溶性粉Ⅱ、Ⅲ有效率、治愈率、肺损伤改善率、相对日增重率、料肉比与支原净基本相同。延胡索酸泰妙菌素可溶性粉剂量为60mg／L试验组中有效率为100％,治愈率为95％。延胡索酸泰妙茵素可溶性粉剂量为45瑚唔／L试验组中有效率95％,治愈率为90％。[结论]延胡索酸泰妙菌素可溶性粉在饮水中的推荐混合荆量为45—60mg／L。     

马杜霉素和泰妙菌素联合应用对雏鸡血液中微量元素的影响

[目的]研究药物对雏鸡血液中微量元素含量的影响。[方法]将不同剂量的马杜霉素和泰妙菌素添加到雏鸡的饲料和饮水中,比较雏鸡血液中K、Zn、Cu元素含量的变化。[结果]正常推荐剂量的马杜霉素和泰妙菌素联合使用时,雏鸡血液中K、Zn、Cu元素含量变化不大。加倍剂量的马杜霉素会产生中毒反应,与正常或加倍剂量的泰妙菌素联合应用时,雏鸡血液中K、Zn、Cu元素含量升高,使中毒反应显著加强。[结论]高剂量马杜霉素和泰妙菌素联合应用,会使雏鸡产生毒性反应。     

Recurrence of Chlamydia suis infection in pigs after short-term antimicrobial treatment

The effect of short-term antimicrobial treatment on natural excretion of Chlamydia suis in rectal swabs and C. suis and Chlamydophila psittaci in nasal swabs was investigated in 47 clinically normal piglets by quantitative real-time PCR. Pigs were treated IM with 4 mg/kg enrofloxacin for 5 days (n = 22) or 2.5 mg/kg enrofloxacin for 3 days followed by 100 mg/mL tiamulin (n = 25). Antimicrobial treatment reduced the number of pigs positive for chlamydiae and the quantity of chlamydial DNA in positive swabs for a few days, but chlamydial excretion recurred in both groups. Short-term antimicrobial treatment at dosages recommended for treatment of other bacterial infections in pig herds was not effective in eliminating naturally occurring subclinical chlamydial infection in pigs.