辽宁地区副猪嗜血杆菌的分离鉴定及耐药性研究
发布时间:2018-06-22 08:33
本文选题:副猪嗜血杆菌 + 分离鉴定 ; 参考:《沈阳农业大学》2017年硕士论文
【摘要】:副猪嗜血杆菌(Haemiophilus parasuis,HPS)于 1992 年被 Schermer 和 Ehrlich 证实是HPS病的病原,患HPS病的猪在临床上主要表现为发热、呼吸困难、跛行、关节肿大和后肢瘫痪,剖检可见大量的纤维素性多发性浆膜炎、心包炎、脑膜炎、关节炎等病理变化。在辽宁地区,HPS病已经成为仔猪发病和死亡的主要原因之一,并且成年猪也有大面积感染的现象,给养猪行业造成了非常严重的经济损失。因此,有必要对辽宁地区HPS流行株进行分离鉴定和分析,以便于为地区性的防制奠定基础。本研究自2015年4月至2016年12月,从辽宁省部分市区县,采集疑似HPS病猪的样品137份,利用细菌分离培养、革兰氏染色镜检、生化实验、PCR鉴定和16SrRNA序列分析及药物敏感性分析等方法进行研究。并且由于HPS具有不易培养的特性,在一定程度上影响了分离率,因此本研究还进行了培养基的优化实验,添加不同浓度的血清及NAD含量,每6h测定分离株的OD600值并绘制生长曲线,由OD600值反映不同培养时间及浓度的活菌数、生长速度等。主要结果如下:1.从收集的样品中共分离出9株HPS,分别命名为HN1512、XM1601、HC1604A、HC1604B、DD1604、YK1603、HN1604、TL1607 和 LY1611。并运用多重 PCR 检测方法,鉴定血清型,分离得到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未测定出血清型。2.培养优化实验表明:在血清浓度为5%,NAD终浓度为10 μg/mL时,HPS生长良好。如果将NAD浓度提高到20 μg/mL时,对菌液浓度影响不大,将血清浓度增加到7%或9%时,可以使菌液浓度略有提高。3.为了解辽宁地区HPS分离株对药物敏感情况,本研究检测了 9株HPS辽宁分离株对25种药物的敏感性。结果显示:①辽宁分离株对氨苄西林、米诺环素和头孢唑啉等高度敏感,该结果能够指导临床合理用药,避免猪场损失。②同时发现辽宁分离株对一些药物存在较为严重的耐药现象,所有分离株都表现出了多重耐药,其中对药物产生最少耐受的菌株为HN1604菌株,对3种药物药物产生耐受性,而对药物产生最严重耐受性的菌株为YK1603达到对10种药物耐受。③通过PCR方法筛查HPS分离株的23种耐药基因,发现9株HPS分离株都具有耐药基因,且基因组DNA和质粒DNA分别检测出7种和9种耐药基因。耐药表型的测定结果与耐药基因检测结果基本保持一致。该研究为辽宁株HPS研究及耐药机制奠定基础。
[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.
【学位授予单位】:沈阳农业大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:S858.28
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本文编号:2052245
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