本文關(guān)鍵詞：雞滑液囊支原體JS1株的分離鑒定及禽支原體、大腸桿菌、沙門菌多重PCR檢測方法的建立　出處：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 滑液支原體 分離鑒定 攻毒模型 多重PCR

【摘要】：雞滑液囊支原體感染是由滑液支原體(Mycoplasma synoviae,MS)引起的,雞和火雞的一種急慢性傳染病,其臨床以病雞關(guān)節(jié)或足墊腫大、滑液囊和腱鞘發(fā)炎為主要特征,也可引起呼吸道疾病及氣囊炎,嚴(yán)重的引起全身性感染,也可導(dǎo)致新城疫病毒、傳染性支氣管炎病毒、大腸桿菌等病原微生物的混合或繼發(fā)感染。該病在世界范圍內(nèi)流行,多個(gè)國家的流行病學(xué)監(jiān)測表明MS在商品代雞群中有很高的流行率。近幾年滑液支原體在中國的發(fā)病率有增多趨勢,很多地區(qū)季節(jié)性發(fā)病相當(dāng)嚴(yán)重。MS感染雞群可導(dǎo)致跛行、產(chǎn)蛋量下降、生長發(fā)育遲緩及酮體降級等危害,嚴(yán)重影響雞的生產(chǎn)性能,給世界商品養(yǎng)雞產(chǎn)業(yè)造成巨大的經(jīng)濟(jì)損失。本研究從江蘇某雞場收集病雞,對其進(jìn)行支原體分離,獲得一株疑似支原體的分離物,通過菌落觀察、血清學(xué)實(shí)驗(yàn)、支原體16S r RNA通用引物測序鑒定及與實(shí)驗(yàn)室標(biāo)準(zhǔn)株MS WVU1853差異性鑒定,確定該分離物為滑液支原體,且不同于本實(shí)驗(yàn)室的MS標(biāo)準(zhǔn)株(MS WVU1853),因此將其命名為JS1株。本研究對滑液支原體SPF雞攻毒模型進(jìn)行了初步探索,將分離株MS JS1對21日齡SPF雞進(jìn)行攻毒,分別采用關(guān)節(jié)注射、肌肉注射、足墊注射和滴鼻四組不同攻毒方式,每組10只SPF雞,攻毒劑量為每只1×109 CCU。結(jié)果表明攻毒方式不同,其發(fā)病率也不一致,其中關(guān)節(jié)攻毒組發(fā)病率最高,達(dá)9/10,其后依次是足墊攻毒組發(fā)病率為8/10,滴鼻攻毒組發(fā)病率為4/10,肌肉攻毒組發(fā)病率為0。除肌肉攻毒組無發(fā)病癥狀外,其余三種攻毒方式均能引起不同程度的特征性病變,即雞關(guān)節(jié)囊腫、足墊腫大、胸部皮下囊腫、跛足以及雞冠蒼白等,對發(fā)病雞部分剖檢也能從關(guān)節(jié)、足墊或肺組織中分離到滑液支原體。針對滑液支原體(Mycoplasma synoviae,MS)的pdh A基因,雞毒支原體(Mycopl asma gallisepticum,MG)的gap A基因,大腸桿菌(Escherichia coli,EC)的pho A基因,沙門菌(Salmonella,SA)的inv A基因分別設(shè)計(jì)2-3對特異性引物,通過篩選引物組合、優(yōu)化體系條件,最后確定了一組最優(yōu)引物組合和反應(yīng)體系,建立了針對這四種禽常見致病菌的多重PCR檢測方法。特異性實(shí)驗(yàn)證明該方法能特異性擴(kuò)增出MS、MG、EC和SA的目的片段,其他非相關(guān)的禽致病菌則無擴(kuò)增產(chǎn)物。敏感性檢測發(fā)現(xiàn)MS和MG的檢測靈敏度均達(dá)到1×102 CCU/反應(yīng),EC的檢測靈敏度1×102 CFU/反應(yīng),SA的檢測靈敏度1×103 CFU/反應(yīng)。臨床感染組織樣本檢測結(jié)果顯示,多重PCR檢測結(jié)果與分離鑒定結(jié)果一致,檢測率可達(dá)83.8%。本實(shí)驗(yàn)對MS分離株JS1進(jìn)行了分離鑒定,初步建立了滑液支原體對SPF雞的攻毒模型,并成功建立能同時(shí)檢測禽常見致病菌MS、MG、EC和SA的多重PCR檢測方法,為滑液支原體感染流行病學(xué)調(diào)查研究以及進(jìn)一步開展滑液支原體疫苗的研制奠定了基礎(chǔ)。
[Abstract]:Mycoplasma synoviae infection by Mycoplasma synoviae (Mycoplasma synoviae, MS) caused by the chicken and Turkey for acute and chronic infectious diseases, the clinical disease in chicken joint or footpad swelling, synovial capsule and tendon sheath inflammation as the main feature, can also cause respiratory disease and airsacculitis, caused by severe systemic infection also, can lead to Newcastle disease virus, infectious bronchitis virus, Escherichia coli and other pathogenic microorganisms or mixed infection. The epidemic is prevalent throughout the world, and epidemiological surveillance in many countries shows that MS has a high prevalence rate in commercial chicken flocks. In recent years, the incidence of Mycoplasma synovium in China has been increasing, and the seasonal incidence of the disease is very serious in many areas. MS infected chickens can cause lameness, egg production, growth retardation and ketone body degradation, which seriously affect the production performance of chicken and cause huge economic losses to the world's chicken industry. This study collected the sick chicken from a chicken farm in Jiangsu, for the isolation of Mycoplasma isolates, obtained a strain of mycoplasma, through observation, serological test, Mycoplasma colony 16S R RNA universal primers and sequencing with standard laboratory strains of MS WVU1853 were identified, confirmed the isolate for Mycoplasma synoviae and is different from the laboratory. The MS standard strain (MS WVU1853), it will be named JS1 strain. This study conducted a preliminary exploration on Mycoplasma synoviae SPF chicken infection model, will isolate MS JS1 on the 21 day old SPF chickens were challenged by intraarticular injection, intramuscular injection, foot pad injection and intranasal inoculation of four different groups, 10 rats in each group of SPF chickens challenged dose for each 1 * 109 CCU. The results showed that the incidence of attack was different. The incidence of joint attack group was the highest, reaching 9/10, followed by foot pad attack group, the incidence rate was 8/10, the incidence of intranasal attack group was 4/10, and the incidence of muscle attack group was 0. In addition to the muscle inoculation group without symptoms, the other three kinds of virus attack methods can induce the characteristic lesions in different degrees, namely chicken articular cyst, foot pad swelling, chest subcutaneous cyst, lame and comb pale, on the incidence of chickens can be separated from the autopsy joint, foot pad or lung tissue to Mycoplasma synoviae. For Mycoplasma synoviae (Mycoplasma synoviae MS) PDH A gene of Mycoplasma gallisepticum (Mycopl ASMA, gallisepticum, MG) gap A gene in Escherichia coli (Escherichia, coli, EC) Pho A gene, Salmonella (Salmonella, SA) inv A gene specific primers were designed for 2-3, through the screening of primer combinations and the optimization of system conditions, finally identified a set of optimal primers and reaction system was established for the four common multiple PCR avian pathogen detection method. Specific experiments showed that the method could specifically amplify the target fragments of MS, MG, EC and SA, and other unrelated avian pathogenic bacteria had no amplification products. Sensitivity test showed that the detection sensitivity of MS and MG reached 1 * 102 CCU/ reaction. The sensitivity of EC was 1 * 102 CFU/, and the sensitivity of SA was 1 * 103 CFU/ reaction. The results of detection of tissue samples from clinical infection showed that the results of multiple PCR detection were in accordance with the results of isolation and identification, and the detection rate could reach 83.8%. JS1 of MS isolates were isolated and identified in the experiment, established the model of poison Mycoplasma synoviae to SPF chicken, and multiple PCR detection method for simultaneous detection of avian pathogenic bacteria MS, MG, EC and SA is established successfully, which laid the foundation for the epidemiological investigation of Mycoplasma synoviae infection and the development of the further development of Mycoplasma synoviae vaccine.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.31

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