耐氟喹诺酮类药物鸡毒支原体gyrA基因突变的研究

鸡毒支原体(MG)是引发鸡慢性呼吸道疾病的主要病原,临床常采用抗菌药物进行控制[1]。FQ s作为控制鸡毒支原体感染的主要药物,随着药物的广泛使用,尤其不合理用药,临床已反映该类药物疗效并不理想。但有关MG临床株对FQ s耐药性的研究却鲜有报道。仅有的几篇报道也限于诱导耐药株     

鸡毒支原体红霉素和替米考星耐药体外诱导及其23S rRNA基因V域突变特征分析

鸡毒支原体（Mycoplasma gallisepticum，MG）是禽类主要病原菌之一，其感染引起鸡慢性呼吸道病（CRD）和火鸡传染性窦炎。目前预防鸡毒支原体感染仍主要依靠抗菌药物，但长期广泛应用抗菌药物造成鸡毒支原体耐药性不断发展。有调查表明，鸡毒支原体临床株对四环素类、大环内酯类及氟喹诺酮类等抗菌药物已产生了耐药性，造成药物疗效下降甚至失效。由于支原体分离培养困难，国内外对动物源支原体耐药性研究十分有限，至今未见有鸡毒支原体大环内酯类抗生素耐药机制的研究报道。本文以鸡毒支原体S6株为模式菌，研究红霉素和替米考星在体外诱导鸡毒支原体的耐药情况，并分析耐药菌株23S rRNA基因V域的耐药突变特征。     

禽支原体病最新诊断标准

鸡毒支原体(MG)和滑液支原体(MS)属于柔膜体纲,支原体目,支原体科.虽然火鸡支原体和衣阿华支原体也可以引起家禽的疾病,但MG和MS是致病支原体中最重要的,并且在全球广泛传播.     

广西鸡毒支原体的分子流行病学研究的概述

研究分别建立了鸡毒支原体（MG）、滑液霉形体（MS）、禽衣阿华支原体（MI）、火鸡支原体（MM）及鉴别MG强毒株和弱毒疫苗株单一PCR和多重PCR检测鉴定技术,以及核酸探针检测MG的技术。应用多种分子生物学技术即SDS—PAGE、PCR—RFLP、RAPD和29KD多肽基因序列分析等方法对广西MG流行株的分子病原学进行研究,摸清了广西MG流行野毒株之问以及与现用疫苗株和国际参考株之间的差异和遗传相关性;在此基础上制定的综合防制措施经生产的实施与应用,取得了显著的效果。     

中国农业科学院哈尔滨兽医研究所——动物支原体病创新团队

<正>研究内容1.动物支原体天然免疫机制的研究2.动物支原体致病性和毒力致弱机制的研究3.动物支原体诊断技术,流行病学检测和耐药机制的研究4.动物支原体疫苗的研制与应用。5.主要研究对象包括:丝状支原体丝状亚种(Mmm),山羊支原体山羊肺炎亚种(Mccp),牛支原体(M.bovis),鸡毒支原体(MG),猪肺炎支原体(Mhp)     

中国农业科学院哈尔滨兽医研究所——动物支原体病创新团队

<正>研究内容1.动物支原体天然免疫机制的研究2.动物支原体致病性和毒力致弱机制的研究3.动物支原体诊断技术,流行病学检测和耐药机制的研究4.动物支原体疫苗的研制与应用。5.主要研究对象包括:丝状支原体丝状亚种(Mmm),山羊支原体山羊肺炎亚种(Mccp),牛支原体(M.bovis),鸡毒支原体(MG),猪肺炎支原体(Mhp)     

科技动态

防制新城疫新型疫苗——病毒颗粒疫苗当前预防侵入性新城疫(ExoticNewcastleDisease,END)主要采用弱毒疫苗或灭活疫苗,虽然效果良好但不良反应较大,常导致家禽的生产性能下降。最近,美国农业部的专家采用分子生物学技术获得了一种新型病毒颗粒疫苗(virosomevaccine)。该疫苗的特点是有关病毒复制的遗传物质被去除,因此病毒粒子在机体内不能复制,也不会在家禽中传播,能良好的诱发机体的保护性免疫应答,并能有效区分疫苗株和野毒感染。在研究中,采用1日龄雏鸡接种该疫苗,鸡群再暴露在END环境中,接种了弱毒疫苗和病毒颗粒疫苗的试验鸡全部…     

鸡毒支原体人工感染SPF鸡胚研究

试验使用低剂量的鸡毒支原体(MG)培养液(液稀释至10~4CCU/mL)经卵黄囊接种5~7日胚龄SPF鸡胚,结果:鸡胚死亡高峰时间发生在孵化后期(17d以后),疫苗6/85株导致SPF鸡胚死亡情况及死亡鸡胚病理变化与MG强毒S6株和临床分离株无明显差异。从孵化后期死胚中采集不同样品进行MG分离鉴定,最终确定死胚最佳分离部位为卵黄囊膜。     

鸡毒支原体(MG)疫苗株和野毒株的PCR鉴定

针对由于鸡毒支原体(Mycoplasma gallisepticum, MG)不同毒株(针对TS-11、6/85、F株疫苗株和S6、R株野毒株)的特定序列,设计出特异性检测引物,对临床免疫MG疫苗株TS-11的鸡群以及父母代(A～H场)鸡群和祖代(I场)鸡群采集毛蛋气囊拭子,A场父母代种鸡群和I场祖代鸡群同时采集咽喉拭子进行MG的PCR鉴别诊断,对临床送检免疫6/85的客户鸡群(J场)采集病料,F株疫苗株进行MG的鉴别诊断,同时采集SPF鸡群咽喉拭子作为阴性对照群,验证该鉴别引物的特异性。结果显示:鉴别引物具有非常好的特异性,SPF鸡群MG的检测均为阴性,不存在假阳性的弊端;临床免疫MG疫苗株6/85的鸡群(J场)存在MG野毒株S6的感染,在免疫后1个月左右采集病料中仍能检出MG 6/85疫苗株。对免疫TS-11的父母代种鸡群(A～H场)和祖代种鸡群(I场)死亡的毛蛋气囊拭子,用PCR检测MG阳性率,结果显示:祖代鸡群(I场)后代死亡的毛蛋气囊评估结果为优,毛蛋气囊拭子MG的PCR鉴别引物检测均为阴性;而父母代鸡群后代死亡的毛蛋气囊从50周龄开始出现浑浊,MG的PCR鉴别引物可同时检出TS-11疫苗毒和野毒株,部分鸡群同时存在R株和S6株两种野毒感染;采集A场父母代种鸡群的咽喉拭子和后代啄壳死亡毛蛋气囊拭子,PCR检测结果显示种鸡群和后代可同时检出野毒株S6和疫苗株TS-11。这表明种鸡群MG的感染,可通过垂直传播感染其后代。本研究设计的MG鉴别引物,可将不同MG疫苗株(TS-11、F株、6/85)与野毒株(S6株、R株)区分,为临床MG的鉴别诊断和种鸡群的MG感染状态评估提供积极的指导意义。     

禽支原体病

败血性支原体(MG)和滑液支原体 (MS)是一种没有细胞壁的细菌,可以感染鸡和其它家禽,在某些情况下能引发疾病。有些感染虽不表现临床症状但仍对生产造成影响。譬如减少总产蛋和产雏数,或降低肉鸡生产性能。败血性支原体(MG)通常更易致病, 因而比滑液支原体(MS)对生产造成的影响更大。然而,不同的种类和分离株之间的特性差异很大,包括毒力与动力学血清学反应。     

畜禽支原体耐药性及耐药机制研究进展

支原体感染给畜禽养殖造成的危害已受到国内外科研人员的广泛重视。目前控制畜禽支原体感染、降低畜禽养殖经济损失的切实有效方法是大量使用抗生素,其中四环素、大环内酯类及喹诺酮类药物是现阶段临床兽医首选的3类抗菌药。但短短几十年的临床实践表明,滥用抗生素已经严重威胁畜禽健康,许多畜禽支原体临床分离株不仅仅对单一抗生素产生耐药性,而且同时对多种抗生素耐药。作者从药物靶位点改变、外排泵系统形成、抗菌药物灭活酶产生等方面对上述抗畜禽支原体药物的耐药机制进行阐述,以期为建立支原体耐药性检测体系、指导临床合理用药、开发新的药物等提供理论依据和策略。     

Research Progress on Drug Resistance and Its Mechanism of Mycoplasma in Livestock and Poultry

The harm for livestock and poultry production caused by Mycoplasma infection has got widespread attention of researchers in the world. The practical ways to control the disease in order to reduce economic loss through Mycoplasma infection is the extensive use of antibiotics. Many groups of antibiotics including tetracyclines, macrolides and quinolones have been shown to be effective to Mycoplasma. Clinical practice shows many Mycoplasma isolated from livestock and poultry are characterized not only by single drug resistance but also by multiple antibiotic resistance due to the legacy of past decades of antimicrobial misuse, which threat the livestock and poultry production health. This paper reviews the mechanism of anti-Mycoplasma medicines through the alterations in the targets, the formation of efflux pump system, and the generation of inactivated enzymes of antibiotics, which provide theoretical basis and strategies for establishing resistance testing system, rational drug use and development of new drugs.     

Genotyping of Mycoplasma gallisepticum and M. synoviae by Amplified Fragment Length Polymorphism (AFLP) analysis and digitalized Random Amplified Polymorphic DNA (RAPD) analysis

Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the cause of considerable economic losses in the poultry industry. Molecular differentiation of avian Mycoplasma strains may be helpful in tracing infections and in the evaluation of implemented intervention strategies. Amplified Fragment Length Polymorphism (AFLP) has shown to be a powerful typing technique but the application for poultry Mycoplasma strains is very limited. The aim of this study was to evaluate the reproducibility and discriminatory power of AFLP HindIII/HhaI and AFLP BglII/Mfel for the inter- and intraspecies differentiation of avian mycoplasmas and to compare these test characteristics with digitalized Random Amplified Polymorphic DNA (RAPD) analysis. The reproducibility of RAPD, AFLP HindIII/HhaI and AFLP BglII/Mfel was 50-100, 97-98 and 86-99%, respectively. RAPD and both AFLP enzyme combinations were able to differentiate between five avian Mycoplasma species. For AFLP, five MG and four MS clusters could be identified. The phylogenetic tree for both enzyme combinations was comparable. For RAPD, four MG clusters could be identified. For MS, however, due to the poor reproducibility of the RAPD technique, no clear genogroups could be identified. On basis of the results of this study it can be concluded that AFLP is a powerful technique for the genotyping of avian mycoplasmas and that, although AFLP HindIII/HhaI generated patterns with less fragments, the final results showed homologous results.     

Effects of an S6 strain of Mycoplasma gallisepticum challenge before beginning of lay on various egg characteristics in commercial layers

Mycoplasma gallisepticum (MG) is a reproductive/respiratory disease in poultry implicated in suboptimum egg production and decreased hatchability. Commercial layer hens raised in a controlled environment were inoculated with the S6 strain of MG at 10 wk of age. Egg production and selected egg and egg quality parameters were quantitated over the entire lay cycle for inoculated and control birds. The S6 inoculation had no effect on bird weight, egg production, associated egg quality parameters, or histopathologic lesion scores. This study shows that in the absence of environmental stressors a prelay S6 MG inoculation does not produce detrimental effects on layer hen performance.     

禽蛋和禽肉中兽药残留检测技术研究进展

随着我国家禽产业的规模不断扩大,兽药残留问题也更加严峻。兽药残留对人体与环境危害重大。现对目前禽蛋和禽肉中常见的兽药残留种类及最大残留限量进行综述,探讨了(超)高效液相色谱法、(超)高效液相色谱-质谱法、气相色谱-质谱法和免疫学检测法、分子印迹技术、生物芯片技术、生物传感器检测法在禽蛋和禽肉中的应用价值,以期为兽药残留检测和监管提供依据。     

Molecular characterization and determination of antimicrobial resistance of Mycoplasma gallisepticum isolated from chickens

In this study, three consecutive approaches of molecular characterization, determination of minimum inhibitory concentration (MIC) and antimicrobial tested on Mycoplasma gallisepticum (MG) isolated from chicken farms were investigated. These approaches were conducted between 2004 and 2005 to 134 MG samples collected from five different regions of the intensive farming area of Thailand. Twenty MG isolates and four reference strains including S6, F, ts-11, and 6/85 were classified according to Random Amplification of Polymorphic DNA (RAPD) patterns prior to the antimicrobial tests. These isolates exhibited 5 different genotypes (A-E). Consequently, MG isolates representing each genotype were tested on 11 registered antibiotics. The levels of MIC were determined. Three antibiotics, doxycycline (0.20 microg/ml), tiamulin (0.10 microg/ml), and tylosin (0.33 microg/ml), gave the least MICs among all effective drugs. Break point comparisons of each antimicrobial suggested that the MG isolates were most sensitive to lincomycin, oxytetracycline, tiamulin, and tylosin. Some MG isolates had an intermediate effect on josamycin and were resistant to enrofloxacin and erythromycin. Our results also indicated that MG isolated and collected from the region and nearby districts had similar RAPD patterns showing properties of antimicrobial resistance. The RAPD patterns may imply the frequent use of antibiotics and a resistant strain of MG. This is the first report of genetic characterization using RAPD reflected by the levels of MIC against MG. The information is useful to plan for prophylactic and therapeutic impacts on the poultry industry especially in the area of intensive use of antibiotics.     

Pathogenic effects on domestic poultry of a mycoplasma gallisepticum strain isolated from a wild house finch

Mycoplasma gallisepticum (MG) has been isolated from wild house finches. The pathogenic effects of MG finch strain (K4058) and MG R-strain were compared after exposure of chickens and turkeys. Gross and histologic lesions, reisolation of the organism, serology, and clinical disease were evaluated. Milder histologic and gross lesions, in addition to lower serologic titers, occurred in birds inoculated with the finch strain. Mortality, concurrent with clinical and gross respiratory signs and lesions, was observed only in chickens challenged with R-strain. Both the MG finch strain and MG R-strain were recovered from the respective challenge groups at 14 and 28 days postexposure. The results show that MG isolated from wild house finches may infect domestic poultry species but causes only mild disease and is less virulent than MG R-strain. Commercial enzyme-linked immunosorbent assay kits best detected the serologic response of chickens and turkeys to the MG finch strain.     

Evaluation and comparison of various PCR methods for detection of Mycoplasma gallisepticum infection in chickens

Four genetic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16s rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.     

多重聚合酶链反应快速检测鉴别鸡毒支原体强毒株和弱毒疫苗株的研究

根据鸡毒支原体强毒株和弱毒疫苗株基因组的结构特点，设计合成了二对引物XZ1，XZ2和XZ45、XZ46，建立了一种同时检测鉴别MG野毒株和弱毒疫苗株的多重PCR技术。试验结果表明，用这两对引物对MG强毒株和弱毒疫苗侏进行多重PCR，强毒株只扩增出732bp一条带，而弱毒疫苗株则可同时扩增出732bp、524bp二条带，而对其他种类鸡支原体和其它禽病病原的扩增不出现任何条带，结果均为阴性；敏感性测定结果表明，该多重PCR最低能检出1Pg的MG强毒株和弱毒疫苗株的DNA模板。     

Breeder turkey hens seropositive and culture-negative for Mycoplasma synoviae.

Four flocks of clinically normal turkey breeder hens were shown to have suspect and positive Mycoplasma synoviae (MS) hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay, and, in some cases, serum plate agglutination serology in the absence of MS isolation. In all cases, HI serology for Mycoplasma gallisepticum (MG) and M. meleagridis was negative. Acholeplasma laidlawii was isolated from some hens in each of these MS-seropositive culture-negative flocks. Immunoblotting was used to help determine if this positive MS serology was a result of cross-reactive antibodies to A. laidlawii or to some other Mycoplasma species. When sera from two of the flocks were reacted with MS antigen in immunoblotting, a strong and characteristic MS immunoblot profile was seen. Immunoblotting gave no evidence of a strong antibody response to A. laidlawii, M. iowae, or MG. This suggests the presence (or earlier presence) of MS in these flocks that is difficult to isolate by routine methods. Furthermore, this work shows that immunoblotting can be an important tool in the diagnosis of poultry diseases.