本文關(guān)鍵詞：豬支原體肺炎活疫苗RM48株氣霧免疫技術(shù)的研究　出處：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 豬支原體肺炎 豬肺炎支原體活疫苗RM48株 疫苗 佐劑 氣霧免疫 研究

【摘要】：豬支原體肺炎(Mycoplasmal pneumonia of swine,MPS)即豬的喘氣病,由豬肺炎支原體(Mycoplasma hyopneumoniae,MHP)引起的一種常見(jiàn)的慢性呼吸道疾病,一旦感染MHP后常常會(huì)繼發(fā)感染別的呼吸道疾病很難被治愈,給養(yǎng)豬業(yè)造成很大的經(jīng)濟(jì)損失。對(duì)于該病的根治理論上是完全可行的,但是在實(shí)際的操作過(guò)程中基本無(wú)法實(shí)現(xiàn)。目前對(duì)該病的控制措施最有效的方法是接種疫苗。目前我國(guó)市面上的疫苗主要是滅活苗和弱毒苗,滅活苗多為進(jìn)口疫苗價(jià)格高昂成為了其在各大豬場(chǎng)部署的障礙,而我國(guó)國(guó)產(chǎn)的弱毒疫苗多為肺內(nèi)注射,操作復(fù)雜且不被大多數(shù)養(yǎng)殖戶(hù)所接受同樣影響了其在個(gè)大養(yǎng)殖場(chǎng)的部署。鑒于此,本課題以豬肺炎支原體RM48疫苗株為研究對(duì)象,并對(duì)弱毒苗的佐劑進(jìn)行篩選,旨在驗(yàn)證MPS的氣霧免疫的可行性以及篩選出合適的疫苗佐劑。具體的實(shí)驗(yàn)分為以下的三個(gè)步驟實(shí)施:1.霧化對(duì)RM48疫苗株的影響及霧化器的篩選分別使用購(gòu)買(mǎi)的超聲霧化器和壓縮空氣霧化器在無(wú)菌實(shí)驗(yàn)室中對(duì)RM48疫苗株進(jìn)行霧化,無(wú)菌保存?zhèn)溆�。使用CCU法對(duì)收集到的疫苗進(jìn)行支原體計(jì)數(shù),并設(shè)立未霧化對(duì)照組。在生化培養(yǎng)箱中培養(yǎng)12d后最后一瓶不再變色。結(jié)果顯示超聲和壓縮空氣霧化組的活菌數(shù)均和對(duì)照組無(wú)差別,說(shuō)明霧化對(duì)RM48疫苗株并無(wú)太大損害。通過(guò)比對(duì)兩種霧化器的霧化速度、霧化后氣霧的粒徑以及噴射距離后選擇了壓縮空氣霧化器作為噴霧免疫的霧化器。2.利用家兔模型評(píng)價(jià)各種佐劑對(duì)豬支原體肺炎活疫苗的免疫增強(qiáng)作用以及活疫苗噴霧免疫的可行性佐劑的初步篩選及用量確定:將事先準(zhǔn)備并配制好的黃芪多糖、天蠶素、板藍(lán)根+烏梅黃酮、CPG、天冬提取物佐劑按一定的濃度與RM48活疫苗混合稀釋,分別室溫下放置30min、1h和2h進(jìn)行CCU活性計(jì)數(shù)看佐劑對(duì)活疫苗的毒性。結(jié)果顯示板藍(lán)根+烏梅黃酮對(duì)疫苗的活性影響較大棄用。其余佐劑對(duì)活疫苗基本無(wú)毒性作用。天蠶素和CPG的用量由實(shí)驗(yàn)室提供分別為20μg/頭和10μg/頭。通過(guò)活性計(jì)數(shù)實(shí)驗(yàn)確定黃芪多糖和天冬提取物的用量為1mg/頭和5mg/頭。家兔免疫后體內(nèi)疫苗株的PCR檢測(cè):在免疫后第7d和14d每組分別剖殺一只兔子取肺臟、脾臟和肝。研磨處理后提取DNA然后進(jìn)行PCR實(shí)驗(yàn)檢測(cè)體內(nèi)疫苗株分布。結(jié)果顯示在第7d肺內(nèi)的PCR結(jié)果為陽(yáng)性,其他組織均為陰性,第14d只有一組的肺呈弱陽(yáng)性。免疫后體內(nèi)細(xì)胞因子及抗體的檢測(cè):于首次免疫后第7d、14d、21、28d分別取兔鼻拭子和血液,處理后用事先買(mǎi)好的試劑盒分別測(cè)定IL-4、IFN-γ和SIgA,結(jié)果顯示各佐劑組均能誘導(dǎo)兔子產(chǎn)生高水平的細(xì)胞免疫、體液免疫以及黏膜免疫水平,其中尤其以CPG佐劑組最高。3.用噴霧的方式對(duì)仔豬進(jìn)行免疫在本動(dòng)物試驗(yàn)研究中,實(shí)驗(yàn)分為天冬提取物噴霧免疫組、CPG噴霧免疫組、黃芪多糖噴霧免疫組、天冬提取物噴霧免疫組、肺內(nèi)注射無(wú)佐劑組、無(wú)佐劑噴霧免疫組和對(duì)照組。分別在免疫前測(cè)定每組四只初生仔豬的體重,并在免疫后7d、14d、21d和28d采鼻拭子并測(cè)體重。用試劑盒測(cè)定鼻拭子中的SIgA和IFN-γ。結(jié)果顯示除對(duì)照組外,各組SIgA和IFN-γ水平均明顯升高,其中以CPG組最高與兔子實(shí)驗(yàn)相符。各免疫組間豬的體重均無(wú)太大差異。綜上所述,本試驗(yàn)結(jié)果顯示了豬肺炎支原體氣霧免疫的可行性,為以后豬肺炎支原體氣霧免疫的大規(guī)模推廣提供了基礎(chǔ)使人們更加關(guān)注這一疫苗預(yù)防接種的新方式。
[Abstract]:Mycoplasma hyopneumoniae (Mycoplasmal pneumonia of swine, MPS) swine enzootic pneumonia, mycoplasmal pneumonia of swine (Mycoplasma hyopneumoniae MHP) is a common chronic respiratory disease caused by MHP infection, once after secondary infection of other respiratory diseases are often difficult to be cured, which caused great economic losses to the pig industry for this. The radical theory of disease is feasible, but in the actual operation of the process can not be achieved. The basic method of control measures for this disease is currently the most effective vaccination. At present on the market the vaccine is mainly inactivated vaccine and attenuated vaccine, inactivated vaccine for the high import price has become a vaccine the obstacle is deployed in all large farms, and China's domestic attenuated vaccine for pulmonary injection, complex operation and will not be accepted by most farmers also affected the large farms in this deployment, This subject to Mycoplasma hyopneumoniae vaccine strain RM48 as the research object, and adjuvant on the attenuated vaccine were selected to verify the feasibility of MPS aerosol immunization and screened vaccine adjuvant appropriate. The implementation of specific experiments can be divided into three steps: ultrasonic atomizer screening 1. atomization on the effect of RM48 vaccine strain and atomizer are used to buy and compressed air atomizer atomization of RM48 vaccine strains in sterile laboratory, aseptic preservation reserve. CCU method is used to collect the vaccine for Mycoplasma counts, and the establishment of non atomized control group. After 12D culture, the last bottle of no discoloration in biochemical incubator. The results showed that ultrasonic and compressed air atomization the number of live bacteria group and control group were no difference, indicating atomization of the RM48 vaccine strain is not much damage. Two kinds of velocity ratio by atomizing atomizer, atomization spray particle size and After the selection of jet distance immune compressed air atomizer as a spray atomizer using.2. immune rabbit model to evaluate various adjuvant of swine mycoplasma pneumonia vaccine to determine the enhancement effect and preliminary selection of vaccine immune adjuvant feasibility and spray dosage: advance preparation and preparation of Astragalus polysaccharide, cecropin, Radix Isatidis + ebony flavonoids, CPG, aspartate extract concentration and RM48 adjuvant according to certain vaccine dilution, 30min were placed at room temperature, 1H and 2H activity of CCU vaccine adjuvant on counter toxicity. The result shows that the influence of ebony flavonoids on activity of Radix Isatidis + vaccine adjuvant on large abandoned. The remaining live vaccine has no toxicity effect of cecropin. And the amount of CPG provided by the laboratory were 20 g/ and 10 g/ head head. Through the activity counting experiment to determine the astragalus polysaccharide and aspartate extract dosage of 1mg/ and 5m head The head of the g/. In vivo detection of vaccine strain PCR in rabbits after immunization: after immunization of 7D and 14d in each group were killed a rabbit from the lung, spleen and liver. Mechanicalattrition DNA extraction followed by PCR assay in vivo vaccine strain distribution. The results showed that in the 7d lung PCR were positive, which he organized the 14d was negative, only one set of lungs was weakly positive in vivo detection of cytokines and antibodies after immunization in the first immunization: after 7d, 14d, 21,28d were obtained from rabbit nasal swabs and blood after treatment with kit bought in advance were measured by IL-4, IFN- and SIgA. The results showed that the gamma. The adjuvant group can produce high levels of immune cells induced by rabbit, humoral and mucosal immune level, especially with CPG adjuvant group had the highest.3. spray way of piglets were immunized in the animal experimental study, the experiment was divided into aspartate extract spray immune group, CPG injection The fog immune group, astragalus polysaccharide extract spray spray immune group, ASP immune group, pulmonary injection without adjuvant group, no spray adjuvant immune group and control group respectively. Four rats in each group were measured in newborn piglets before immunization and post immunization in weight, 7d, 14d, 21d and 28d in nasal swabs and weight. Determination of nasal swabs of SIgA and IFN- gamma kit. The results showed that except the control group, SIgA group and IFN- gamma were significantly increased, especially in CPG group and the rabbit experimental results. There was no much difference in all immunized groups between the weight of the pigs. In conclusion, the experimental results show the feasibility of pneumonia of swine Mycoplasma aerosol immunization, for future large-scale promotion of Mycoplasma hyopneumoniae aerosol immunization provides the basis to make people pay more attention to this new way of vaccination.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S858.28

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