本文選題：副豬嗜血桿菌 + 分離鑒定�。� 參考：《沈陽農業(yè)大學》2017年碩士論文

【摘要】：副豬嗜血桿菌(Haemiophilus parasuis,HPS)于 1992 年被 Schermer 和 Ehrlich 證實是HPS病的病原,患HPS病的豬在臨床上主要表現(xiàn)為發(fā)熱、呼吸困難、跛行、關節(jié)腫大和后肢癱瘓,剖檢可見大量的纖維素性多發(fā)性漿膜炎、心包炎、腦膜炎、關節(jié)炎等病理變化。在遼寧地區(qū),HPS病已經(jīng)成為仔豬發(fā)病和死亡的主要原因之一,并且成年豬也有大面積感染的現(xiàn)象,給養(yǎng)豬行業(yè)造成了非常嚴重的經(jīng)濟損失。因此,有必要對遼寧地區(qū)HPS流行株進行分離鑒定和分析,以便于為地區(qū)性的防制奠定基礎。本研究自2015年4月至2016年12月,從遼寧省部分市區(qū)縣,采集疑似HPS病豬的樣品137份,利用細菌分離培養(yǎng)、革蘭氏染色鏡檢、生化實驗、PCR鑒定和16SrRNA序列分析及藥物敏感性分析等方法進行研究。并且由于HPS具有不易培養(yǎng)的特性,在一定程度上影響了分離率,因此本研究還進行了培養(yǎng)基的優(yōu)化實驗,添加不同濃度的血清及NAD含量,每6h測定分離株的OD600值并繪制生長曲線,由OD600值反映不同培養(yǎng)時間及濃度的活菌數(shù)、生長速度等。主要結果如下:1.從收集的樣品中共分離出9株HPS,分別命名為HN1512、XM1601、HC1604A、HC1604B、DD1604、YK1603、HN1604、TL1607 和 LY1611。并運用多重 PCR 檢測方法,鑒定血清型,分離得到1株血清1型、3株血清7型、2株血清9型、1株血清11型和1株血清14型,且有一株HPS未測定出血清型。2.培養(yǎng)優(yōu)化實驗表明:在血清濃度為5%,NAD終濃度為10 μg/mL時,HPS生長良好。如果將NAD濃度提高到20 μg/mL時,對菌液濃度影響不大,將血清濃度增加到7%或9%時,可以使菌液濃度略有提高。3.為了解遼寧地區(qū)HPS分離株對藥物敏感情況,本研究檢測了 9株HPS遼寧分離株對25種藥物的敏感性。結果顯示:①遼寧分離株對氨芐西林、米諾環(huán)素和頭孢唑啉等高度敏感,該結果能夠指導臨床合理用藥,避免豬場損失。②同時發(fā)現(xiàn)遼寧分離株對一些藥物存在較為嚴重的耐藥現(xiàn)象,所有分離株都表現(xiàn)出了多重耐藥,其中對藥物產生最少耐受的菌株為HN1604菌株,對3種藥物藥物產生耐受性,而對藥物產生最嚴重耐受性的菌株為YK1603達到對10種藥物耐受。③通過PCR方法篩查HPS分離株的23種耐藥基因,發(fā)現(xiàn)9株HPS分離株都具有耐藥基因,且基因組DNA和質粒DNA分別檢測出7種和9種耐藥基因。耐藥表型的測定結果與耐藥基因檢測結果基本保持一致。該研究為遼寧株HPS研究及耐藥機制奠定基礎。
[Abstract]:Haemiophilus parasuis HPS was confirmed by Schermer and Ehrlich as the pathogen of HPS in 1992. The main clinical manifestations of HPS were fever, dyspnea, claudication, swelling of joints and paralysis of hind limbs. A large number of cellulosic polyserositis pericarditis meningitis arthritis and other pathological changes were observed. HPS disease has become one of the main causes of morbidity and mortality of piglets in Liaoning Province, and adult pigs also have a large area of infection, which has caused very serious economic losses to pig industry. Therefore, it is necessary to isolate, identify and analyze HPS epidemic strains in Liaoning in order to lay a foundation for regional prevention and control. From April 2015 to December 2016, 137 samples of suspected HPS pigs were collected from some cities and counties of Liaoning Province. The methods of PCR identification, 16s rRNA sequence analysis and drug sensitivity analysis were studied. Because HPS has the characteristic of not easy to culture, it affects the separation rate to a certain extent, so the experiment of medium optimization was carried out in this study, adding different concentrations of serum and NAD content. The OD600 value of the isolated strain was measured every 6 hours and the growth curve was drawn. The OD600 value reflected the number of live bacteria and the growth rate of different culture time and concentration. The main results are as follows: 1. Nine strains of HPSs were isolated from the collected samples and named HN1512 XM1601 HN1512 XM1601 HN1512 XM1601 HN1512 XM1601, HN1604TL1607 and LY161111, respectively. Multiplex PCR was used to identify the serotypes. A serotype of serotype 1, serotype 7, serotype 2, serotype 9, serotype 11, and serotype 14, were isolated, and one HPS strain was not determined. The optimum culture experiment showed that HPS grew well when the final concentration of NAD was 10 渭 g / mL. If the concentration of NAD was increased to 20 渭 g / mL, the concentration of bacterial liquid was not affected. When the concentration of serum was increased to 7% or 9%, the concentration of bacterial liquid increased slightly by 0.3%. In order to understand the drug sensitivity of HPS isolates in Liaoning area, the sensitivity of 9 HPS isolates to 25 drugs was studied. The results showed that the Liaoning strain was highly sensitive to ampicillin, minocycline and cefazolin. At the same time, it was found that Liaoning isolate had more serious drug resistance to some drugs, and all the isolates showed multidrug resistance, among which HN1604 strain was the least resistant to the drug. The strain YK1603 that had the most serious tolerance to the drug was YK1603, which was resistant to 10 drugs. 23 resistant genes of HPS isolates were screened by PCR, and 9 HPS isolates were found to have drug resistance genes. Seven and nine resistant genes were identified by genomic DNA and plasmid DNA, respectively. The results of drug resistance phenotypes were consistent with those of drug resistance genes. This study laid a foundation for the study and drug resistance mechanism of Liaoning strain HPS.
【學位授予單位】：沈陽農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.28
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本文編號：2052245