3种禽源支原体替米考星耐药株的体外诱导及23SrRNA基因V域碱基突变分析

作者拟探讨禽源支原体对替米考星耐药的分子机制.体外诱导得到鸡毒支原体(R株、PG31株和S6株)、鸡滑液支原体、衣阿华支原体的替米考星耐药株;PCR扩增原始敏感株和诱导耐药株的23S rRNA基因V域,测序分析耐药相关碱基突变情况.结果鸡毒支原体R株、PG31株、S6株、鸡滑液支原体、衣阿华支原体分别通过9代、8代、6代、14代、9代诱导获得替米考星耐药(≥128 μg·mL-1)株;3种禽源支原体替米考星诱导耐药株均对大环内酯类药物表现交叉耐药,23S rRNA基因发生A2503T突变的诱导耐药株对截短侧耳素类、氯霉素类药物的敏感性明显降低.诱导获得的替米考星耐药鸡毒支原体R株发生了A2058G和A2503T突变,PG31株发生了A2058G和A2059G突变,S6株发生了A2058G和A2503T突变;而诱导获得的替米考星耐药鸡滑液支原体发生了G2162A突变,衣阿华支原体发生了A2059C突变.本研究表明鸡毒支原体和衣阿华支原体在体外较易经替米考星诱导产生耐药性,而鸡滑液支原体相对较难.菌株23S rRNA基因V域2 058、2 059、2 503位点的碱基突变与替米考星耐药表型有密切关系.     

替米考星--新霉素对鸡毒支原体病的临床疗效试验

鸡毒支原体(Mycoplasnm gallisepticam，MG)感染常称为鸡慢性呼吸道病(CRD)，感染通常危害整个鸡群，导致鸡群食欲减退、体重减轻，产蛋鸡群产蛋量下降，是导致养禽业明显经济损失的疾病之一。替米考星(Tilmicosin)是英国Elanco公司20世纪80年代开发的一种动物专用的大环内酯类抗生素，用于多种动物由敏感菌引起的感染性疾病，特别是畜禽呼吸道感染。     

替米考星-新霉素对实验性鸡毒支原体病疗效研究

替米考星(tilmicosin)是英国Elanco公司20世纪80年代开发的一种动物专用的大环内酯类抗生素。目前该药在世界许多国家都有上市，被批准使用治疗牛、山羊、绵羊、奶牛、猪、鸡等动物由敏感菌引起的感染性疾病，特别是畜禽呼吸道感染。鸡毒支原体感染(Myeoplasma gallisepticam Infection，MG)常称为慢性呼吸道病和火鸡传染性窦炎，其所导致的经济     

替米考星用于鸡毒支原体和大肠杆菌混合感染的临床试验

试验以人工诱发鸡毒支原体和大肠杆菌混合感染为模型,以酒石酸泰乐菌素为对照药物,评价替米考星溶液的疗效。按每升水加入400m g、200m g、100m g替米考星及500m g酒石酸泰乐菌素的用量给病鸡饮水给药,连用5天。试验表明:替米考星大剂量和中剂量组料肉比与药物对照差异不显著(P>0.05),与其余各组差异极显著(P<0.01)。大剂量组与中剂量组气囊损伤评分与其他各组比较差异极显著(P<0.01),表明大、中剂量均能明显减轻支原体和大肠杆菌混合感染引起的气囊损伤。大剂量和中剂量组的死亡率与药物对照组相比差异均不显著(P>0.05),与其余各组相比差异极显著(P<0.01)。从治愈率来看,大剂量组中剂量组与其他各组相比差异极显著(P<0.01),而小剂量组与药物对照组的治愈率相当(P>0.05)。数据分析表明:替米考星溶液中剂量组能有效地降低气囊损伤度,提高饲料转化率。     

替米考星的体外抑菌试验和其磷酸盐预混剂对鸡慢性呼吸道病的疗效试验

替米考星的体外抑菌试验表明，替米考星对鸡毒霉形体MGS6株、金黄色葡萄球菌CMCC26112、禽巴氏杆菌临床分离株有较强的体外抗菌活性，MIC值分别为0．125、0．500和0．800pg／mL。体内试验结果表明，磷酸替米考星预混剂对鸡慢性呼吸道病有很好的疗效，饲料中磷酸替米考星预混剂含量为l～2g/kg时，能明显提高感染鸡的成活率和增加平均体重，显著降低气囊的病理损伤程度及抗体检出率，其综合疗效较同类磷酸泰乐菌素强。体内外试验结果说明，该药在防治鸡毒霉形体引起的鸡慢性呼吸道病方面有实际应用价值，值得进一步推广使用。     

磷酸替米考星饮水剂治疗鸡败血支原体的疗效试验

鸡慢性呼吸道病是由鸡败血支原体引起的一种接触性、传染性呼吸道病，以呼吸罗音、咳嗽、流鼻涕和窦部肿胀为特征，病情发展缓慢且周期长。在气候多变和寒冷的秋冬季多发。环境卫生条件差、通风不良、鸡群过密、气雾免疫等因素均可促使本病的发生。慢性呼吸道病常常伴发大肠杆菌病，造成鸡只大批死亡。在药物冶疗上通常选用泰乐菌素、红霉素，但是症状缓解慢，治疗周期长。我们选用磷酸替米考星对该病进行冶疗，发现冶疗效果良好，现将试验结果报告如下。     

氟喹诺酮类药物压力下鸡毒支原体gyrA基因突变特征分析

按常规方法提取鸡毒支原体参考株(S6-10)、疫苗株(F14-6)及在氟喹诺酮类药物压力下体外筛选出来的耐药株的基因组DNA，扩增各菌株DNA旋转酶gyrA基因片段，并克隆、测序，利用DNASIS分析软件对测序结果进行分析。结果表明，S6-10和F14-6在恩诺沙星或环丙沙星压力下均筛选出不同程度的耐药菌，而且恩诺沙星致鸡毒支原体发生耐药性突变的机率高于环丙沙星，S6-10株耐药突变频率高于F14-6。S6-10筛选出来的耐药菌株发生突变的位置在DNA旋转酶gyrA亚基的第83位(相对于大肠杆菌gyrA亚基中的位置，以下同)和87位上，取代模式为Ser83→Ile、Glu87→Gln，高水平耐药株(Se32M，MIC≥128)除了在第83位发生突变外，在第82位还增加一突变位点(Asp→Gly)；F14-6筛选出来的高水平耐药菌株(Fe32M)在DNA旋转酶gyrA亚基的第83、87位发生了双位点突变(Ser83→Ile，Glu87→Gly)，而低水平耐药株(Fe8M、Fc8M)在gyrA亚基未发生任何位点突变。     

鸡毒支原体病及其防控

鸡毒支原体感染,又称鸡败血支原体感染或鸡慢性呼吸道病(CRD),是由鸡毒支原体引起的鸡和火鸡的慢性呼吸道传染病。病鸡主要表现为呼吸道症状,如气管炎、气囊炎等,以咳嗽、气喘、流鼻液和呼吸锣音为特征。该病病程缓慢、病程长,成年鸡多     

多杀性巴氏杆菌对替米考星耐药性的体外诱导

新药替米考星（Tilmieosin）对巴氏杆菌具有较强的抗菌活性及抗菌后效应，并且具有动物机体吸收快、血药浓度半衰期长、表观分布容积大、组织（尤其是肺组织）药物浓度高等优良的药动学特征，对临床畜禽多杀性巴氏杆菌感染表现出良好疗效‘孔“，已成为防治巴氏杆菌感染的一个重要药物。但随着替米考星的应用，发现多杀性巴氏杆菌菌株对替米考星的敏感性开始下降，     

鸡毒支原体感染的诊断和防控

鸡毒支原体感染又称鸡败血支原体感染或鸡慢性呼吸道病(CRD),是有鸡毒支原体引起的鸡和火鸡的一种慢性呼吸道传染病。鸡主要表现为呼吸道症状,如气管炎、气囊炎等,以咳嗽、气喘、流鼻液和呼吸啰音为特征。该病程缓慢、病程长,成年鸡多呈隐性感染,可在鸡群中长期存在和蔓延。鸡毒支原体感染是世界性分布的,国内也很普遍。     

In vitro development of resistance to enrofloxacin,erythromycin, tylosin,tiamulin and oxytetracycline in Mycoplasma gallisepticum,Mycoplasma iowae and Mycoplasma synoviae

The in vitro emergence of resistance to enrofloxacin, erythromycin, tylosin, tiamulin, and oxytetracycline in three avian Mycoplasma species, Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae was studied. Mutants were selected stepwise and their MICs were determined after 10 passages in subinhibitory concentrations of antibiotic. High-level resistance to erythromycin and tylosin developed within 2-6 passages in the three Mycoplasma species. Resistance to enrofloxacin developed more gradually. No resistance to tiamulin or oxytetracycline could be evidenced in M. gallisepticum or M. synoviae after 10 passages whereas, resistant mutants were obtained with M. iowae. Cross-sensitivity tests performed on mutants demonstrated that mycoplasmas made resistant to tylosin were also resistant to erythromycin, whereas mutants made resistant to erythromycin were not always resistant to tylosin. Some M. iowae tiamulin-resistant mutants were also resistant to both macrolide antibiotics. Enrofloxacin and oxytetracycline did not induce any cross-resistance to the other antibiotics tested. These results show that Mycoplasma resistance to macrolides can be quickly selected in vitro, and thus, providing that similar results could be obtained under field conditions, that development of resistance to these antibiotics in vivo might also be a relatively frequent event.     

替米考星、红霉素体外诱导猪胸膜肺炎放线杆菌耐药性的初步研究

采用微量稀释法测定了替米考星和红霉素对5株临床分离猪胸膜肺炎放线杆菌(App)的最小抑菌浓度,并用药物浓度递增法体外诱导App对两种药物的耐药性。结果表明替米考星和红霉素对App都具有很高的体外抑菌活性;经15代诱导,App对替米考星的最高耐受浓度没发生明显变化,而对红霉素的最高耐受浓度有了较大程度的提高,表明App对替米考星不易产生耐药,而对红霉素可缓慢产生耐药。试验结果提示替米考星是治疗App感染的理想药物。     

Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA

A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.     

Examination of the 16S-23S rRNA intergenic spacer sequences of 'Candidatus Mycoplasma haemobos' and Mycoplasma haemofelis

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.     

Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes

Gallibacterium was recently established as a new genus including organisms previously reported as Pasteurella anatis, [Actinobacillus] salpingitidis and avian Pasteurella haemolytica-like organisms. The aim of the present study was to develop a PCR method allowing unambiguous identification of Gallibacterium. PCR primers positioned in the 16S rRNA (1133fgal) and 23S rRNA (114r) genes were defined and their specificity was subsequently tested on 122 strains. Twenty-five of the strains represented all of the presently available 15 phenotypic variants of Gallibacterium from different geographical locations, 22 other strains represented other poultry associated bacterial species or bacteria which could pose a differential diagnostic problem including members of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae, and finally 75 Gallibacterium field strains isolated from Mexican chicken egg-layers. Specific amplicons were generated in all 100 Gallibacterium strains tested, whereas none of the non-Gallibacterium strains tested positive. Correct identification was confirmed by hybridization with the Gallibacterium specific probe GAN850. Two internal amplification control strategies were successfully incorporated into the PCR assay, one based on amplification of the house-keeping gene rpoB (sharing target DNA) and another based on addition of trout DNA (foreign target DNA) and amplification with beta-actin specific primers. In conclusion, the described PCR assay enables specific identification of Gallibacterium and will thus stand as a strong alternative to the present diagnostic methods.     

Comparison of the pharmacokinetics of tilmicosin in plasma and lung tissue in healthy chickens and chickens experimentally infected with Mycoplasma gallisepticum

The objectives of this study were to compare the plasma and lung tissue pharmacokinetics of tilmicosin in healthy and Mycoplasma gallisepticum-infected chickens. Tilmicosin was orally administered at 4, 7.5 and 10 mg/kg body weight (b.w) for the infected and 7.5 mg/kg b.w for the uninfected control group. We found no significant differences in plasma tilmicosin pharmacokinetics between diseased and healthy control chickens. In contrast, the lung tissues in M. gallisepticum-infected chickens displayed a t_1/2 (elimination half-life) 1.76 times longer than for healthy chickens. The C_max (the maximum concentration of drug in samples) of tilmicosin in M. gallisepticum-infected chickens was lower than for controls at 7.5 mg/kg b.w (p < .05), and the AUC_inf (the area under the concentration–time curve from time 0 extrapolated to infinity) in infected chickens was higher than for the healthy chickens (p < .05). The mean residence time of tilmicosin in infected chickens was also higher than the healthy chickens. These results indicated that the lungs of healthy chickens had greater absorption of tilmicosin than the infected chickens, and the rate of elimination of tilmicosin from infected lungs was slower.     

恩诺沙星与其他抗菌药对鸡毒支原体的体外联合抑菌试验

采用2倍稀释法测定了恩诺沙星及其他8种抗菌药对鸡毒支原体BG44T株的最小抑菌浓度（MIC）,再以棋盘法测定恩诺沙星分别与其他8种抗菌药不同联合对鸡毒支原体BG44T株的敏感性。结果显示：恩诺沙星、替米考星、泰乐菌素、吉他霉素、林可霉素、沃尼妙林、泰妙菌素、氯霉素及氟苯尼考对鸡毒支原体BG44T株的MIC分别为0．063、0．004、0．016、0．063、16、〈0．004、0．008、8、8μg／mL。在8种不同联合用药对鸡毒支原体BG44T株的药敏试验中,恩诺沙星＋替米考星、恩诺沙星＋泰乐菌素、恩诺沙星＋吉他霉素、恩诺沙星＋林可霉素、恩诺沙星＋沃尼妙林、恩诺沙星＋泰妙菌素联合表现出相加作用,恩诺沙星＋氯霉素、恩诺沙星＋氟苯尼考表现出拮抗作用。