發(fā)布時間：2019-06-26 20:38

【摘要】：大腸桿菌可引發(fā)多種動物疾病,對畜牧業(yè)及人類健康構(gòu)成極大威脅。大腸桿菌耐藥現(xiàn)象已十分普遍,給大腸桿菌感染性疾病的有效防治帶來較大困難。細(xì)菌耐藥可以使其致病性發(fā)生變化,有關(guān)大腸桿菌耐藥對自身毒力影響的研究已引起學(xué)者們的廣泛關(guān)注。本研究系統(tǒng)分析了分離自遼寧地區(qū)患臨床型乳房炎奶牛的大腸桿菌耐藥性對其毒力的影響,以期為臨床科學(xué)防治奶牛乳房炎提供指導(dǎo)。本研究應(yīng)用PCR技術(shù),對79株分離自遼寧地區(qū)的患臨床型乳房炎奶牛的大腸桿菌進(jìn)行毒力基因的檢測,分析了遼寧地區(qū)奶牛乳房炎源大腸桿菌種系發(fā)育分型情況及毒力基因流行特點(diǎn)。結(jié)果顯示,79株大腸桿菌中,種系發(fā)育A型占59.5%,B1型占27.9%,B2型和D型各占6.3%。對分離自患臨床型乳房炎奶牛的大腸桿菌進(jìn)行了 22種毒力基因的篩查,共檢出10種毒力基因,分別為fimH(89.9%)、ECs3703(88.6%)、ompC(100%)、ompF(73.4%)、colV(5.1%)、irp2(2.5%)、fyuA(2.5%)、eaeA(1.3%)、ler(1.3%)和iucD(1.3%)。大腸桿菌分離株中A型大腸桿菌數(shù)量最多,所含毒力基因種類亦最多。采用實(shí)時熒光定量PCR技術(shù)測定4種不同耐藥表型的大腸桿菌毒力基因ompF和ECs3703mRNA表達(dá)量。結(jié)果表明,ompF基因mRNA表達(dá)量隨大腸桿菌耐抗菌藥物種類的增多而下降,耐2類抗菌藥組與敏感組相比,ompF基因mRNA表達(dá)量的差異顯著,P0.05;耐4類抗菌藥組、耐6類抗菌藥組與敏感組相比,ompF基因mRNA表達(dá)量的差異極顯著,P0.01。ECs3703基因mRNA表達(dá)量隨大腸桿菌耐抗菌藥物種類的增多變化不明顯;但是,與敏感組相比,耐6類抗菌藥物的大腸桿菌ECs3703基因mRNA表達(dá)量下降顯著,P0.05。采用小鼠致病性試驗(yàn)對4種不同耐藥表型的大腸桿菌的致病性進(jìn)行比較,分析不同耐藥表型對毒力的影響。攻毒后,敏感組、耐2類抗菌藥物組、耐4類抗菌藥物組、耐6類抗菌藥物組小鼠死亡率的均值分別為72.22%、61.11%、33.33%、16.67%。隨著奶牛乳房炎源大腸桿菌耐藥種類的增加,被接種的小鼠死亡率逐漸下降;且接種耐4類抗菌藥物大腸桿菌和耐6類抗菌藥物大腸桿菌小鼠的致死率,明顯低于接種對6類抗菌藥物均敏感大腸桿菌小鼠的致死率。研究表明,對于四環(huán)素類、β-內(nèi)酰胺類、氟喹諾酮類、氨基糖苷類、酰胺醇類、大環(huán)內(nèi)酯類6類抗生素,大腸桿菌隨著自身耐藥種類的增多,毒力減弱。本研究分析了遼寧地區(qū)奶牛臨床型乳房炎源大腸桿菌毒力因子的流行情況,及大腸桿菌耐藥性對其致病性的影響,將為該地區(qū)奶牛乳房炎的有效防控提供理論依據(jù)。
[Abstract]:Escherichia coli can cause a variety of animal diseases, which poses a great threat to animal husbandry and human health. The drug resistance of Escherichia coli has been very common, which brings great difficulties to the effective prevention and treatment of Escherichia coli infectious diseases. Bacterial drug resistance can change its pathogenicity, and the study on the effect of Escherichia coli resistance on its toxicity has attracted extensive attention of scholars. In this study, the effect of Escherichia coli resistance on the virulence of dairy cows with clinical mastitis isolated from Liaoning was systematically analyzed in order to provide guidance for clinical scientific prevention and treatment of mastitis in dairy cows. In this study, PCR technique was used to detect the virulence genes of 79 strains of Escherichia coli isolated from Liaoning dairy cows with clinical mastitis, and the developmental typing and epidemic characteristics of virulence genes of Escherichia coli from dairy cows from Liaoning area were analyzed. The results showed that among 79 strains of Escherichia coli, type A was 59.5%, type B1 was 27.9%, type B2 and type D were 6.3% respectively. 22 virulence genes were screened from Escherichia coli isolated from clinical mastitis cows. 10 virulence genes were detected as fimH (89.9%), ECs3703 (88.6%), ompC (100%), ompF (73.4%), colV (5.1%), irp2 (2.5%), fyuA (2.5%), eaeA (1.3%), ler (1.3%) and iucD (1.3%). The number of type A Escherichia coli isolated from Escherichia coli was the largest, and the kinds of virulence genes were the most. The expression of virulence genes ompF and ECs3703mRNA in E. coli with different drug resistance phenotypes was detected by real-time fluorescence quantitative PCR. The results showed that the expression of ompF gene mRNA decreased with the increase of antibiotic resistance in E. coli. The expression of ompF gene mRNA in resistant group was significantly different from that in sensitive group (P0.05), and the expression of ompF gene mRNA in resistant group was significantly different from that in sensitive group. The expression of P0.01.ECs3703 gene mRNA did not change significantly with the increase of antibiotic resistance in E. coli. However, compared with the sensitive group, the expression of ECs3703 gene mRNA in Escherichia coli resistant to 6 kinds of antibiotics decreased significantly (P 0.05). The pathogenicity of E. coli with different drug resistance phenotypes was compared by mouse pathogenicity test, and the effects of different drug resistance phenotypes on virulence were analyzed. After challenge, the average mortality of mice in sensitive group, resistant to two kinds of antibiotics group, resistant to 4 types of antibiotics group and resistant to 6 types of antibiotics group was 72.22%, 61.11%, 33.33% and 16.67%, respectively. the average mortality of mice was 72.22%, 61.11%, 33.33% and 16.67%, respectively. With the increase of resistant types of Escherichia coli from dairy cows, the mortality of inoculated mice decreased gradually, and the fatality rate of mice inoculated with Escherichia coli resistant to four kinds of antibiotics and resistant to 6 kinds of antibiotics was significantly lower than that of mice inoculated with Escherichia coli sensitive to 6 kinds of antibiotics. The results showed that for tetracycline, 尾-lactam, fluoroquinolone, aminoglycosides, amides and macrolides, Escherichia coli became less virulent with the increase of drug resistance. In this study, the prevalence of virulence factors of Escherichia coli from clinical mastitis in dairy cows in Liaoning Province and the effect of Escherichia coli drug resistance on its pathogenicity were analyzed, which will provide a theoretical basis for the effective prevention and control of mastitis in dairy cows in Liaoning Province.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23

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