【作者】 李庆雷；

【导师】 柴同杰；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 微生物是动物舍环境污染的主要因素,动物舍的生物污染可以引起一系列传染病的流行。近几年的研究表明,一些气载病原微生物能够通过空气传播很远的距离,造成传染病的流行。过去对畜禽养殖环境微生物气溶胶的传播主要研究对象是细菌,是通过舍内外环境中的细菌浓度的变化以及细菌耐药性及某些致病菌含量等来确认的。然而,这些方法受到的影响因素很多,难以保证分类的准确性和科学性。未能证明舍内外环境分离的微生物气溶胶的起源及其同源性,没能获得充分的证据证明微生物气溶胶起源于养殖环境并向周边及社区环境传播。近些年,以ERIC-PCR和PFGE(脉冲场凝胶电泳)为代表的分子生物学细菌分型技术得以应用,以鉴定不同来源细菌的同源性,追溯其来源。并且,随着分子生物学技术的不断变革,对畜禽舍养殖环境病毒气溶胶的传播研究也越来越广泛。本研究分别对细菌和病毒的气溶胶发生和传播做了相关研究。对细菌,以大肠杆菌为指示菌,采用Andersen-6级空气微生物样品收集器,分别在5个鸡场舍内空气、舍外上风向10m, 50m和下风向10m, 50m, 100m, 200m, 400m的距离处收集空气样品,每个动物舍设三点采样,每次重复采集5个样本。同时采集鸡的粪便,分离、鉴定大肠杆菌,计算每一个采样点大肠杆菌的浓度(CFU·m?3空气)。然后,对每个鸡舍,先采用ERIC-PCR对不同来源(粪便、舍内、外不同距离空气样品)的大肠杆菌分离株进行基因分型,然后对ERIC-PCR相似系数在90%以上的菌株,再采用脉冲场凝胶电泳(PFGE)技术,进行同源性鉴定,准确确认细菌的来源。根据每个采样点大肠杆菌浓度的变化以及在不同采样点分离的大肠杆菌的遗传距离,确认动物舍内微生物气溶胶的起源及其向舍外环境传播。对病毒,分别在山东省德州、济宁、莱芜、临沂、青岛、潍坊、烟台、东营等地的养猪场的舍内和舍外下风向10m、20m,用AGI-30采集空气样品,每个采样点重复采集3个样品,并采集棉拭子。在泰安市岱岳区采集猪血样品,进行血凝及血凝抑制试验,以检测抗体效价。在山东农业大学门诊及富农门诊收集病死猪病料、棉拭子、血液进行处理。运用荧光定量RT-PCR技术检测新型H1N1病毒的含量,探讨猪舍中新型H1N1病毒的气溶胶的传播。本实验共分离到118株大肠杆菌。从浓度来看,5个鸡场舍内空气中大肠杆菌的浓度为11-56 CFU·m-3空气,远远高于舍外上风和舍外下风处的大肠杆菌浓度(P<0.05),显示了舍内微生物气溶胶不断产生和集聚;然而,舍外下风不同距离间的大肠杆菌浓度差异并不显著(P>0.05),显示来源于鸡舍内的气载大肠杆菌能够传播到一定了距离。通过ERIC-PCR筛选出来26组大肠杆菌,共82株大肠杆菌同源性高于90%。各组内菌株均需要运用脉冲场凝胶电泳技术进行进一步基因分型。ERIC-PCR和PFGE两种方法相结合进行同源性鉴定显示,从鸡的粪便中分离到的大肠杆菌与从舍内空气中分离到的部分大肠杆菌(17.1%)具有相同来源;从鸡场舍外下风方向(10m, 50m, 100m, 200m)分离到的多数大肠杆菌(59.1%)与舍内空气或粪便中分离的大肠杆菌来源相同。而从鸡舍上风分离到的大肠杆菌与舍内空气或粪便中分离的大肠杆菌相似指数较小(<80%),表明这些细菌并非来源于该鸡舍。舍外下风向各采样点分离的大肠杆菌与上风向10m,50m分离得的大肠杆菌同源性较低,排除下风向采得的大肠杆菌来自上风向的可能。而很多从舍内空气和舍外下风方向分离到的大肠杆菌与粪便中的相似指数较高(>85%),说明粪便中的细菌能够形成气溶胶,并且通过舍内外气体交换传播到舍外,依气象条件传播到舍外不同的距离。造成周边环境的生物污染以及病原微生物的扩散,给邻近的社区居民形成感染风险。本研究建立了在线检测新型H1N1病毒的荧光定量RT-PCR方法,本次试验共对山东省九个地区40个养猪场,及山东农业大学门诊进行了样品采集,共采集到空气样品和棉拭子样品162份,血液样品27份。其中,舍外20米的37个样品均为阴性,舍外十米37个样品有3个样品为阳性,潍坊1个,德州2个。本实验共收集40个猪场157个空气样品,其中41个样品为阳性,阳性率为26%。27份血清样品进行血凝血凝抑制实验,阳性11个,效价分别为3、3、5、3、8、2、8、8、7、4、4。初步说明新型H1N1病毒可以通过空气传播一定的距离(至少10m),为新甲流的防控提供了必要的依据。更多还原

【Abstract】 Microorganisms and their products in bioaerosol from animal houses can cause serious air pollution. They may also affect the health and the production capability of the animals and induce prevalence of aerosol infectious diseases. The polluted air in livestock farms is often associated with the outbreak of the epidemic diseases and the environmental problems. It is known that many airborne pathogen microorganisms, including viruses and bacteria, can spread over a large area through the air. Bioaerosol disseminated from animal houses to their environments has been studied with an emphasis on total bacterium amount, pathogenic bacteria and antibiotic resistances of the bacteria in animal houses and their ambient air. It is difficult to differentiate between two strains that have very close genetic relationship using traditional bacterial taxonomy, and can not get the enough evidence to prove the transmission of microorganism from animal houses to their sorroudings. In recent years, the bacteria classification technologies of molecular biology represented from ERIC-PCR and PFGE(Pulse field gel electrophoresis) were used to identify the bacteria homology.To get in-depth understanding of microbial aerosol development and transmission to the surroundings of a chicken house, using Escherichia coli as indicator bacteria in this study, air samples were collected by the Andersen-Grade 6 Air Microorganism Sampler from air in the 5 chicken houses, and at 10- and 50-m upwind sites and at 10-, 50-, 100-, 200-, and 400-m downwind sites outside of the chicken houses; meanwhile, chicken manure samples were collected to isolate and identify Escherichia coli, and calculate the Escherichia coli concentration (expressed in CFU/m3 air) at each sampling point. Then, for each chicken house, genotyping of Escherichia coli isolates of different origins were carried out by ERIC-PCR, and then homology identification of the strains with ERIC-PCR similarity coefficient >90% was carried out by pulsed field gel electrophoresis (PFGE ), to accurately determine the origin of the strains. The mode of microbial aerosols in a chicken house to transmit to its surroundings was determined, according to changes of Escherichia coli concentrations at each sampling point, as well as genetic distances of Escherichia coli strains isolated at different samplings. With respect to the concentrations, the Escherichia coli concentrations of the 5 chicken houses were 11-56 CFU/m3 air, far higher than those at the upwind and downwind sites outside of the chicken houses, suggesting that microbial aerosols were unceasingly produced and accumulated in the chicken houses. However, there was no significant difference of Escherichia coli concentrations (P>0.05) at the downwind sites with different distances, suggesting that airborne Escherichia coli derived from the chicken houses were possibly transmitted to a certain distance. Homology identification, carried out by the combination of the two methods, i.e. ERIC-PCR and PFGE, showed that, Escherichia coli isolated from chicken manure and some Escherichia coli (17.1%) isolated from air in the chicken houses were of the same origin. The majority of Escherichia coli (59.1%) isolated at the downwind sites (10-, 50-, 100-, and 200-m) outside of a chicken house, and Escherichia coli isolated from chicken manure, were of the same origin. However, Escherichia coli isolated at the upwind sites were of small similarity coefficient (<80%) with Escherichia coli isolated from chicken manure or from air in the chicken houses, suggesting that they were not derived from the chicken houses. Escherichia coli isolated at each downwind sampling point were of low homology with Escherichia coli isolated at 10- and 50-m upwind sites, excluding the possibility that Escherichia coli isolated at downward sites were from the upwind sites. However, most of Escherichia coli isolated from air in the chicken houses and at the downwind sites outside of the chicken houses were of high similarity coefficient (>85%), suggesting that bacteria in chicken manure was able to form into aerosols, and transmit to the surroundings of the chicken houses through air exchange to a different distance based on different meteorological conditions, leading to biological contaminations of surroundings, and transmission of microbial pathogens. Using the combination of ERIC and PFGE in this study, homology analysis of strains with ERIC-PCR similarity coefficients >90% was carried out by PFGE, to obtain more accurate results.The project established a method to detect the novel H1N1 influenza virus by Real-Time RT-PCR with a novel probe provides. We collected 135 samples in 20 pig farms in 5 areas in Shan Dong province. The experiment detected positive samples at the downwind 10m, which indicated the novel H1N1 influenza virus may transmit to the surroundings of the pig farms through air exchange to a different distance. The detection system also provided a powerful technical support for the study of the source, spread and infective dose of H1N1, and was significant for evaluation of public environmental sanitation and early warning of epidemic situation.更多还原