本文選題：藏豬 + 鏈球菌�。� 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：豬鏈球菌病(Streptococcus suis S.Suis)是因為豬鏈球菌感染而導致的分布極其廣泛的傳染病,對養(yǎng)殖業(yè)和人類影響極大。該病含有35個血清型,其中2型(S.Suis 2)發(fā)病率最高,主要以淋巴結炎、關節(jié)炎、腦膜炎及敗血癥為主要臨床特征,已被國家規(guī)定為二類動物疫病。然而在近期的臨床實踐中發(fā)現(xiàn)部分西藏林芝地區(qū)的藏豬出現(xiàn)類似于S.Suis的臨床癥狀,故此擬通過血清學調查進一步掌握該病在林芝地區(qū)藏豬的流行情況,以期為后續(xù)的防疫工作和綜合性措施提供依據(jù)。腸球菌(Enterococci)存在廣泛,為條件性致病菌。然而近年不科學的抗生素應用使腸球菌耐藥越發(fā)嚴重,不僅對高水平氨基糖苷類藥物耐藥,對大環(huán)內酯類、β-內酰胺類、四環(huán)素類及糖肽類抗生素等的耐藥率也較高,腸球菌引起的各種傳染病也隨之增多。然而,要想研究其耐藥性,分離腸球菌是必不可少的一步,而腸球菌又與其他球菌表型相似,故很難區(qū)分,加之其分布隨地區(qū)的不同而不同,西藏林芝地區(qū)對此研究甚少,故本研究就腸球菌的分離鑒定及其耐藥問題展開研究。本研究分別對2014年12月份到2015年1月份間在西藏林芝地區(qū)所分離的438份血清進行ELISA試驗,完成對豬鏈球菌2型在該地區(qū)陽性率的初步調查,這對增強當?shù)鼐用駥ωi鏈球菌病的防控意識,具有重要的公共衛(wèi)生學意義。同時,本研究對2015年7月到9月份及2016年6月到8月期間所采取的藏豬內臟及糞便進行腸球菌分離,并對分離菌株進行耐藥及MIC試驗。以此為基礎,篩選出耐藥性腸球菌菌株并進行基因檢測。試驗結果與結論如下:1.豬鏈球菌的檢測結果本試驗采用雙抗原夾心酶聯(lián)免疫法(ELISA)對林芝縣及米林縣共438份血清進行檢測。檢測結果顯示:所有血清的抗體陽性率為60.05%(263/438)。其中,雌性、雄性分別為61.50%和58.96%,林芝縣與米林縣的陽性率分別為61.79%和57.81%。應用SPSS 13.0軟件統(tǒng)計分析得出,在本研究中,性別及兩地區(qū)間無顯著性差異(P≥0.05)。本研究說明林芝地區(qū)藏豬S.Suis 2隱性感染率較高,當?shù)鼐用駪訌娫摬〉姆揽匾庾R。2.藏豬源腸球菌的分離情況本研究采用胰酶大豆瓊脂(Tryptic Soy Ager,TSA)固體培養(yǎng)基(加入2%兔血清,50μg/ml氨曲南及10μg/ml多粘菌素B)用于選擇性分離培養(yǎng)腸球菌,進而通過觸酶試驗并結合生化特征初步鑒定為腸球菌,最后進一步結合16SrDNA以及腸球菌特保守的tuf基因通過pcr進一步驗證得出如下結果:2015年從105個樣品中共分離出51株、4種藏豬源腸球菌(48.6%):E.faecalis 15株(29.4%),E.faecium 4株(7.8%),E.hirae 27株(52.9%),E.durans 2株(3.9%)及其他腸球菌3株(5.9%),E.durans為首次在藏源豬獲得;2016年從120個樣品中共分離出45株、2種藏豬源腸球菌(37.5%):E.faecalis 12株(26.7%),E.faecium 6株(13.3%)及其他腸球菌27株(60%)。3.腸球菌耐藥檢測情況本研究通過采用K-B藥敏片法及MIC法對2015-2016年兩年分離菌株進行6類10種抗菌藥物的敏感性測定。結果顯示:所有腸球菌對卡那霉素、慶大霉素及鏈霉素高水平耐藥率高達60%到80%以上,而對萬古霉素的敏感率均為100%,紅霉素及四環(huán)素的耐藥率均接近50%。2016年與2015年相比,氨芐西林的耐藥率明顯增加。頭孢呋辛、青霉素及左氧氟沙星的耐藥率均為10%左右,敏感率較高,且兩年增減幅度不大。此外,兩年份的菌株均表現(xiàn)為嚴重的多重耐藥性,尤其為糞腸球菌,高達5耐,未發(fā)現(xiàn)6耐菌株。說明當?shù)鼐用褚院髴嗉幼⒁獠⒖茖W用藥。4.腸球菌耐藥基因的檢測與分析結合K-B藥敏片法及MIC的結果,本研究分別對兩年份5類藥物10種耐藥基因(aac(6’)-Ie-aph(2’)-Ia,aph(3’)-IIIa,aph(2’)-Ic,ant(4’)-Ia,ant(6’)-Ia,ant(3’)-(9),TEM,ermB,mef A,tetM)進行檢測,結果發(fā)現(xiàn):菌株的耐藥表型及耐藥基因之間并不呈正相關。比如2015年耐高水平鏈霉素菌株為31株,但并無攜帶ant(6’)-Ia和ant(3’)-(9)的腸球菌。2015年部分菌株攜帶aac(6’)-Ie-aph(2’)-Ia,ant(4’)-Ia,ermB及tetM的陽性率高達100%,沒有發(fā)現(xiàn)aph(3’)-IIIa,aph(2’)-Ic,ant(6’)-Ia,ant(3’)-(9)及mef A耐藥菌株,但2016年分離的腸球菌中發(fā)現(xiàn)有6株攜帶aph(3’)-IIIa,3株攜帶ant(6’)-Ia,但沒有檢測到ant(4’)-Ia,其余分離率與2015年相比有微增長現(xiàn)象。雖然分離率不高,但E.faecalis與E.faecium均比其他腸球菌的分離率高。該結果說明E.faecalis與E.faecium的耐藥性比較嚴重,且2016年與2015年相比并沒有下降趨勢。
[Abstract]:Streptococcus suis (Streptococcus suis S.Suis) is extremely widespread due to Streptococcus suis infection of infectious diseases, greatly on aquaculture and human impacts. The disease contains 35 serotypes, including type 2 (S.Suis 2) the highest incidence, mainly in lymphadenitis, arthritis, meningitis and sepsis were the main clinical features, has been the provisions of the state for the two categories of animal diseases in clinical practice. However, recently found in the Linzhi area part of Tibet Tibet pig had clinical symptoms similar to S.Suis, therefore intends to further understand the serological investigation of the epidemic situation in Linzhi area of Tibet pig, in order to provide the basis for further prevention work and comprehensive measures of Enterococcus. (Enterococci) exists widely, for opportunistic bacteria. However antibiotics not scientific that enterococci become more and more serious, not only for the high level aminoglycoside drugs Resistance to macrolides, beta lactams, tetracyclines resistant and glycopeptide antibiotics such as to have a higher rate of various infectious diseases caused by enterococci are increasing. However, in order to study the drug resistance of enterococci is a necessary step, but also with other similar phenotypes aureus Enterococcus, so it is difficult to distinguish, and its distribution varied by region, Linzhi area, Tibet has little study about it, so the research on isolation and identification of resistance of Enterococcus is studied. In this study were ELISA trial in 438 serum samples in 2014 December to 2015 January, isolated in Linzhi area of Tibet, the completion of the preliminary investigation of Streptococcus suis 2 type the positive rate in the region, which is to enhance the awareness of prevention and control of local residents of Streptococcus suis, has important significance in public health. At the same time, the study on July 2015 to September and 201 6 years from June to August during the Tibetan pig offal and feces were adopted in enterococci separation, and drug resistance and MIC test of isolates. On this basis, screening of drug resistance of Enterococcus strains and gene detection. The experimental results and conclusions are as follows: 1. the results of the test of Streptococcus suis by double antigen sandwich ELISA (ELISA) in Linzhi county and Milin County, a total of 438 serum samples were detected. The test results show that: the positive rate of antibody in all serum was 60.05% (263/438). Among them, female and male were 61.50% and 58.96%, the positive rate of Linzhi county and Milin county were 61.79% and 57.81%. SPSS 13 software was used in statistical analysis. In this study, there was no significant difference between gender and interval (P = 0.05). This study shows that the Linzhi area of Tibet pig S.Suis 2 recessive infection rate is high, the local residents should strengthen the awareness of disease prevention and control of Tibet pig intestinal.2. The separation aureus used in this study (Tryptic Soy Ager tryptic soy agar, TSA solid medium (2%) with rabbit serum, 50 g/ml aztreonam and 10 g/ml polymyxin B) was used to isolate enterococci selectivity, then through catalase test and combined with the identification of biochemical characteristics of enterococci, furthermore tuf gene 16SrDNA and Enterococcus special conservative through the PCR verify the results obtained are as follows: 105 in 2015 from a sample of the isolated 51 strains of Enterococcus, 4 Tibet pig (48.6%): E.faecalis 15 (29.4%) strains, 4 strains of E.faecium (7.8%), 27 strains of E.hirae (52.9%), 2 strains of E.durans (3.9%) he and 3 strains of Enterococcus (5.9%), E.durans for the first time in 2016 from the source of Tibetan Pig; 120 samples isolated from 45 strains of Enterococcus, 2 Tibet pig (37.5%): E.faecalis 12 (26.7%) strains, 6 strains of E.faecium (13.3%) and 27 strains of Enterococcus Enterococcus (60%).3. The study of drug resistance detection by using K-B method and MIC method of drug sensitivity of 2015-2016 strains were 6 years of 10 kinds of antibacterial drug sensitivity test. The results showed that all the isolates to kanamycin, gentamicin and high-level streptomycin resistance rate is as high as 60% to more than 80%, while the sensitive rate to vancomycin were 100%, resistance to erythromycin and tetracycline were close to 50%.2016 compared with 2015, ampicillin resistant rate increased significantly. Cefuroxime, penicillin and levofloxacin resistance rate was about 10%, higher sensitivity, and two changes little. In addition, two copies of the strains showed multiple drug resistance is serious, especially for Enterococcus faecalis, as high as 5 resistant, not found in the 6 resistant strains. After the detection and analysis of local residents should pay more attention and scientific use of.4. Enterococcus resistance gene with K-B susceptibility method and MIC 鐨勭粨鏋，

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