【作者】 段会勇；

【导师】 柴同杰； 常维山；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2008， 博士

【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方面来确认的。然而,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畜禽舍微生物气溶胶向环境的传播。因此,本课题测量了19个动物舍(5个鸡舍、5个猪舍、6个牛舍和3个兔舍)舍内及舍外不同距离(上风10、50m和下风10、50、100、200、400m)的大肠杆菌和肠球菌含量,在此基础上,(1)统计舍内、舍外气载需氧菌的含量;(2)对大肠杆菌的耐药性及其肠毒素的检测;(3)采用分子生物学方法(ERIC-PCR和REP-PCR)对不同地点分离的大肠杆菌和肠球菌的遗传相似性进行比较;(4)对肠球菌的耐药基因进行了检测。根据以上结果确定动物舍微生物气溶胶的危害性及其向环境中的传播。1动物舍内微生物气溶胶的含量及其向舍外环境的传播本试验采用ANDERSEN-6级空气微生物样品收集器和RCS离心式采样器在16个动物舍舍内空气、舍外上风10、50m和下风10、50、100、200、400m不同距离收集微生物气溶胶,一方面,通过对动物舍环境中气载需氧菌含量、气载大肠杆菌含量、气载肠球菌含量的检测,以及它们在ANDERSEN六级采样器上的分布规律来推断其对饲养员及动物自身可能造成的危害,从而使人们对动物产生的微生物气溶胶及其健康威胁的高度重视;另一方面,通过对畜禽舍内、外需氧菌含量的比较分析,从而初步观察菌群随着距离增长变化的规律。研究结果表明,(1)在所有检测的养殖环境中微生物气溶胶粒子浓度要远远高于一般的原野环境,而且大部分粒子的空气动力学直径较小,更容易进入呼吸道深部。虽然我们没有对养殖环境的动物及饲养员的健康作系统的调查,但是长期处在这种高浓度的微生物气溶胶环境下,对动物体和饲养人员感染压增大,导致很大的感染风险,能够产生有明显临床症状的传染发生,或隐性感染,或发展为慢性呼吸道疾病以及继发感染等,该领域还有待于进一步研究;(2)通过比较舍内外的气载需氧菌的含量发现,动物舍舍内气载需氧菌含量远远高于动物舍上风和舍外下风方向(＞50m)不同距离空气中需氧菌的浓度(P＜0.05),该结果表明,舍内微生物气溶胶含量较高,随着舍内外气体的交换而不断传播到舍外一定距离,特别是下风50 m内。另外,下风不同距离细菌浓度与上风处(一般原野)比较,差异显著(P＜0.05),舍外下风方向在100、200和400 m(鸡舍A和牛舍C″除外)之间需氧菌的浓度差异不显著(P＞0.05),表明气载需氧菌在一定的气象条件下也可以传播到舍外下风较远的距离(≥200 m)。2 ERIC-PCR对鸡舍和牛舍环境中气载大肠杆菌向舍外环境传播的鉴定本课题测量了5个鸡场和6个牛场舍内及舍外不同距离的大肠杆菌含量,在细菌学鉴定的基础上,采用ERIC-PCR方法对不同地点分离的大肠杆菌鉴定其同源性,获得大肠杆菌ERIC片段指纹图谱,通过该片段在细菌基因组内的数量和分布之间的关系,比较其遗传相似性,确定动物舍微生物气溶胶的向环境中的传播。ERIC-PCR结果表明,从动物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(鸡舍为34.1%;牛舍为)相似性可达100%,从动物舍外下风方向(10、50、100和200m)分离到的多数大肠杆菌(鸡舍为54.5%;牛舍为)与舍内空气或粪便中分离的大肠杆菌相似性可达100%。而从动物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性较小(＜90%)。所以,得出结论,从上风分离到的多数大肠杆菌并非来源于动物的粪便或者舍内空气,而很多从舍内空气和舍外下风方向分离到的大肠杆菌来源于动物的粪便,说明源于动物舍的微生物气溶胶能够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。3鸡舍环境中大肠杆菌耐药性向舍外环境传播的鉴定通过对同一鸡舍内外环境中大肠杆菌耐药性的调查、分析,证明鸡舍内环境中的大肠杆菌可以通过舍内外气体的交换而传播到舍外环境中去,可能对附近养殖场及其周边居民的健康构成潜在的威胁。结果表明,鸡舍粪便中及舍内、舍外空气中分离到的大肠杆菌都对P-G和RIF完全耐药;对GEN和TOB都敏感,并且没有发现对这两种药物的耐药菌株。特别是从舍外下风方向上分离到的菌株对药物的敏感性与从舍内空气中或者是鸡的粪便中分离到的大肠杆菌的耐药性基本一致,说明这些耐药菌株来源于动物舍,它们能够从舍内向舍外环境传播。4 ERIC-PCR和REP-PCR对猪舍环境气载大肠杆菌向舍外环境传播的鉴定采用ERIC-PCR和REP-PCR两种方法进一步对同一猪舍不同地点分离到的大肠杆菌进行同源性比较鉴定,一方面,可以验证两种方法的可靠性;另一方面,为研究猪舍养殖环境产生的大肠杆菌气溶胶向其周围环境的传播提供可靠的方法。在本实验中,我们分析了从5个猪舍环境中分离到的120株大肠杆菌,ERIC-PCR和REP-PCR两种方法都显示出了很高的菌株间的区分性。REP-PCR表现出的指纹图谱与ERIC-PCR表现出的指纹图谱显示出极高的相似性,从而也说明了这两种方法的可靠性。结果表明,有35.1%(20/57)的从粪便中分离的大肠杆菌与舍内空气或舍外下风分离的大肠杆菌的相似性≥90%,然而从上风方向分离的大肠杆菌与舍内空气或粪便中分离的大肠杆菌的相似性较低(61%-69%)。可见,很多从舍内空气和舍外下风方向分离到的大肠杆菌来源于动物的粪便。5动物舍环境大肠杆菌主要毒力基因的检测及其向舍外环境传播的鉴定本研究采用多重PCR方法分别对5个鸡舍及其周围环境中分离到的117株大肠杆菌,5个猪舍及其周围环境中分离到的120株大肠杆菌和6个牛舍及其周围环境中分离到的143株大肠杆菌进行了5种不同的毒力基因(STa、STb、LTa、Stx1和Stx2/Stx2e)的多重PCR检测,不仅调查了不同动物舍舍内环境中大肠杆菌的5种主要毒力基因的携带情况,而且还通过对舍内、舍外环境中大肠杆菌的5种主要毒力基因的比较检测分析,研究舍内大肠杆菌5中主要毒力基因向舍外环境中的传播。结果表明,不同的动物舍环境中大肠杆菌其携带的5种毒力基因型虽然不同,但是都有一定数量的大肠杆菌携带毒力基因,而且很多是携带2种或2种以上的毒力基因,这些致病性大肠杆菌可以通过舍内外气体的交换而传播到舍外环境。6 REP-PCR对动物舍内气载肠球菌向舍外环境传播的鉴定为了给畜禽舍微生物气溶胶的产生与传播提供更充分的证据,以另一种微生物一肠球菌作为指示菌,以REP-PCR方法来研究三种动物舍(鸡舍、猪舍和牛舍)环境中气载肠球菌向舍外环境中的传播,从而与大肠杆菌作为指示菌来做对比,进一步证明动物舍舍内微生物气溶胶向舍外环境传播的必然性,同时也证明了以肠球菌作为指示菌研究微生物气溶胶在环境中传播的可行性。通过对5个不同的鸡舍(127株)、5个不同的猪舍(135株)以及6个不同的牛舍(164株)舍内及其周围环境中肠球菌同源性的REP-PCR分析,结果表明,动物舍内动物的粪便中的肠球菌可以不断形成气溶胶,不仅在能够在舍内空气中传播,而且还能够随着气体的交换而传播到舍外较远的距离,特别是下风方向(≥100m)。7动物舍环境气载肠球菌主要耐药基因的检测及其向舍外环境传播的鉴定为了了解当前养殖环境中细菌的耐药现状,仍以肠球菌为指示菌,通过对该菌的耐药性调查,了解不同场的药物使用状况,细菌耐药谱及其程度,为此后用药提供指导。通过对不同动物舍环境中分离的肠球菌对四环素类(TetM)、氨基糖甙类抗生素(庆大霉素)及糖肽类抗生素(万古霉素VanA和VanB)主要耐药基因的检测,目的:(1)了解当前养殖环境中肠球菌的耐药现状,并为以后的用药提供理论基础;(2)通过对舍内、舍外不同环境中采集到的肠球菌主要耐药基因的检测与比较,研究耐药肠球菌在动物舍舍内及其周围环境中的传播。结果表明:本研究通过对16个动物舍426株肠球菌耐药基因的检测,动物舍环境中存在一定比例的(62/426,14.55%)对β-内酰胺酶耐药肠球菌;三种动物舍舍内及其舍外环境分离株都存在不同程度的对四环素类抗生素的耐药性,而且,耐药率较高。其中,鸡舍粪便分离株肠球菌TetM的检出率最高(79.03%),其次为猪舍粪便分离株;有很少几株肠球菌携带vanA和vanB基因,但是,也存在少量的vanA和vanB阳性菌株,这是在动物舍环境中首次检测到,所以应当引起我们足够的重视;绝大多数肠球菌携带AMEs基因的一种或几种,只有7.7%(33/426)的肠球菌不携带AME基因,可见,鸡、猪、牛舍环境中的肠球菌对氨基糖苷类抗生素耐药的普遍性;同时,统计结果也可以看出,大多数肠球菌都携带2种或2种以上的AME基因,最多者可以携带6种AME基因[aac(6′)-aph(2″)+ant(4′)-Ia+ant(9)-Ia+aph(3′)-Ⅲa+aac(6″)-Ii+ant(6)-Ia],可见,肠球菌对庆大霉素类抗生素耐药的严重性。因此,在兽医临床诊断以及治疗的过程中不得不引起我们足够的重视。通过对鸡、猪、牛舍舍内(动物粪便和舍内空气)及其周边环境中(上风方向10、50m,下风方向10、50、100、200、400m)肠球菌耐药基因(TEM,tetM,VanA和VanB,AMEs)的检测结果比较后可以看出,舍外环境中的,特别是下风方向分离到的很多肠球菌其携带的耐药基因类型与舍内或动物粪便中的肠球菌其携带的耐药基因类型相同。因此,结合REP-PCR同源性分析结果,可以确定肠球菌耐药性也可以在舍内及其周围环境中传播。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 surroundings.So,air samples,including indoor and outdoor air(upwind 10 and 50 m as well as downwind 10,50,100,200 and 400 m away) of 5 chicken houses,5 swine houses,6 cow houses and 3 rabbit houses were collected using six-stage Andersen microbial samplers and RCS samplers.E.coli and enterococcus concentrations(CFU/m~3 air) collected from different sampling sites were calculated.E.coli and enterococcus strains from animal feces samples were also isolated.In order to study microbiological aerosol spreading from animal houses to their surrounding air,the following methods were used:firstly,airborne aerobic bacteria concentrations collected from different sampling sites were calculated;secondly,the antibiotic-resistant and enterotoxin of E.coli were detected;thirdly,the enterobacterial repetitive intergenic consensus polymerise chain reaction(ERIC-PCR) and the repetitive extragenic palindromic(REP-PCR) approaches were used to study the genetic variability and to determine the strain relationships among E.coli or enterococcus isolated from different sites in each animal house;finally,the antibiotic-resistant factor genes of enterococcus were detected too.1.The concentration of microbiological aerosol in animal houses and their transmittion to the environmentAir samples,including indoor and outdoor air(upwind 10 and 50 m as well as downwind 10,50,100,200 and 400 m away) of 5 chicken houses,5 swine houses,6 cow houses and 3 rabbit houses were collected using six-stage Andersen microbial sampler and RCS.Firstly, through the detection of the concentrations of airborne aerobic bacteria,airborne E.coli and airborne enterococcus and their distribution in each stage of Andersen sampler,the possibly damage of microbiological aerosol to animal or farm workers should be given an enough importance;secondly,through comparing the concentrations of airborne aerobic bacteria indoor air with outdoor air of each animal house,the changeable law of them with the distance was found.The results showed that:(1) the concentrations of microbiological aerosol in animal houses were higher than the normal environment,and the aerodynamic diameters of most particulate matter(PM) were very small(＜2.5μm).So,these PM can penetrate into the lower respiratory tract easily.Though the health station of animals and farm workers could not be investigated,the health of them would be damaged living in this environment so long and maybe cause many subclinical symptoms which need investigated farther;(2) the concentrations of airborne aerobic bacteria in indoor air were more higher than those in upwind air and downwind air(sampled at the distances of more than 50 m away from the houses) was significant(P＜0.05).All these results indicated that concentrations of microbiological aerosols in the houses were much high,and that it was spread from indoor to outdoor through air exchange,especially to the downwind sites(≤50 m).In addition,there was no significant difference(P＞0.05) in the microbiological aerosol concentrations among the different downwind sites(100,200 and 400 m) of the animal houses(except chicken house A and cow house C″),indicating microbiological aerosol could be spread far away (≥200 m) based on the meteorological conditions.2.Transmission identification of the Escherichia coli aerosol in chicken houses and cow houses to their environments using ERIC-PCRIn order to study E.coli aerosol spreading from animal houses to their surrounding air,air samples,including indoor and outdoor air(upwind 10 and 50 m as well as downwind 10,50, 100,200 and 400 m away) of 5 chicken houses and 6 cow houses were collected using sixstage Andersen microbial samplers and RCS.E.coli concentrations(CFU/m~3 air) collected from different sampling sites were calculated.Furthermore,the enterobacterial repetitive intergenic consensus(ERIC)-PCR method was applied to amplify the isolated E.coli strain DNA samples.Through the genetic similarity analyses of the E.coli obtained from different sampling sites,the spreading of bioaerosol from animal houses to the ambient air was characterized.The results showed that most of the E.coli isolated from indoor air had 100% similarity with those isolated from feces,and that most of E.coli isolated from downwind at 10,50,100 or even 200 m had 100%similarity with those isolated from indoor air or feces too.But those isolated from upwind air had a lower similarity(＜90%) with corresponding strains isolated from indoor air or feces.Our results suggested that some strains isolated from downwind air and indoor air originated in the chicken feces,but most of isolates obtained from upwind air samples did not come from the chicken feces or indoor air.This also could be concluded that indoor microbiological aerosol aerosols in indoor air of animal houses could transmit to their surroundings via air exchange and cause microbiological contamination.3.Transmission identification of the antimicrobial’ resistance of Escherichia coli in chicken houses to their environmentsAirborne E.coli was spread from indoor to outdoor via air exchange by using the method of analyzing the antimicrobial resistance of E.coli isolated from the same chicken house which can cause the ambient air outside of the animal houses polluted and further threaten the neighboring inhabitants and the chickens themselves.The results showed all E.coli strains isolated from indoor and outdoor(downwind 10,50,100,200 m) air samples in the chicken houses against RIF and P-G,while they were sensitive to TOB and GEN.The sensitivity of E. coli isolated from downwind air was in relation to the E.coli isolated from indoor air or fecal samples,which indicated that the airborne E.coli can spread from indoor to outdoor.4.Source identification of airborne Escherichia Coli of swine house surroundings using ERIC-PCR and REP-PCRThe ERIC-PCR and REP-PCR approaches were used to study the genetic variability and to determine the strain relationships among E.coli isolated from different sites in each swine house.The aim of this experiment was as follows:firstly,the reliability of the two approaches could be tested;secondly,the two approaches could be used to study the transmission of E. coli aerosol in swine houses to their environments.In the present study,we analyzed 120 E. coli isolates originating from five swine houses.The grouping of REP-PCR fingerprints was highly similar with those obtained from ERIC-PCR.This showed that the two approaches yield an analogous taxonomic resolution,suggesting that the two approaches can be fully applied for the study of the transmission of bioaerosols from animal houses to their environments.Results showed that 35.1%of the bacterial DNA fingerprints from the fecal isolates matched with those isolated from indoor and downwind air samples(similarity≥90%).E.coli strains from the indoor and downwind air samples were closely related to the E. coli strains isolated from feces,while those isolated from upwind air samples(house C) had low similarity(61%-69%).Our results suggest that some strains isolated from downwind and indoor air originated in the swine feces. 5.The detecting of virulence factor genes in Escherichia coli in animal houses and the transmission identification to their environmentsThe 5 virulence factor genes of 117 E.coli isolates isolated from 5 chicken houses,120 E.coli isolates isolated from 5 swine houses and 143 E.coli isolates isolated from 6 cow houses were detected using multiplex PCR.Not only have the differences of the 5 virulence factored genes in E.coli in each animal houses been detected,but also through comparing the differences of the 5 virulence factored genes in E.coli isolated from indoor air to outdoor air in one houses,the transmission of virulence factor genes in E.coli could have been found. Results showed that the 5 virulence factor genes in E.coli in different animal houses were different,but there had a number of E.coli carried one or more than two virulence factor genes in each animal houses,and these pathogen E.coli could be spread outdoor air via air exchange.6.Transmission identification of the Enterococcus aerosol in animal houses to their environments using REP-PCRTransmission identification of the microbiological aerosol in animal houses to their environments using REP-PCR for fingerprinting and Enterococcus as a target organism compared E.coli as a target organism.At the same time,the fidelity of Enterococcus as a target organism was tested too.Through the genetic similarity analyses of the E.coli obtained from different sampling sites in 5 chicken houses,5 swine houses and 6 cow houses using REP-PCR approach,the spreading of bioaerosol from ahimal houses to the ambient air was characterized.The results showed that E.coli in feces was found aerosolized and spread to outdoor air,especially to downwind air of the chicken houses via air exchange(≥100 m).7.The detecting of antimicrobial resistance genes in Enterococcus in animal houses and the transmission identification to their environmentsThe aim of this experiment,by detecting antimicrobial resistance genes of TEM,TetM, AME,VanA and VanB,were as follows:firstly,the situation of antimicrobial resistance in Enterococcus in animal rear environment were found and could be used as basis for farm workers;secondly,through comparing the differences of the antimicrobial resistance genes in Enterococcus isolated from indoor air with outdoor air in one houses,the transmission of antimicrobial resistance genes in Enterococcus could be found.The antimicrobial resistance genes of 426 Enterococcus isolated from 16 animal houses has been detected.The results showed that:(1) there was 14.55%(62/426) Enterococcus carring antimicrobial resistance gene of TEM;(2) the TetM gene was detected at high frequency in Enterococcus,and the ratio of carring TetM gene was highest(79.03%) in fecal samples of chicken,in fecal samples of swine was in the next;(3) there was a few of Enterococcus carring antimicrobial resistance gene of vanA and vanB,but it was the first report about them in animal environment.So,we must pay much attention to this;(4) most of Enterococcus carring one or more than one AME,and there was 7.7%(33/426) not carring antimicrobial resistance gene of AME.It is obvious that it was very frequency of AME in Enterococcus.Furthermore,it was of note that 6 AME genes[aac(6′)-aph(2″)+ant(4′)-Ia+ ant(9)-Ia+aph(3′)-Ⅲa+aac(6″)-Ii+ant(6)-Ia]were detected in Enterococcus.So,it was very serious of antimicrobial resistance gene of AME in Enterococcus isolated from animal houses,and we must pay much attention to this station in our future.By comparing antimicrobial resistance genes of TEM,TetM,AME,VanA and VanB in Enterococcus isolated from indoor(air and fecal samples) with the isolates isolated from their environments(upwind 10 and 50 m as well as downwind 10,50,100,200 and 400 m away), Enterococcus isolated from downwind air with them isolated from indoor air or feces had the same antimicrobial resistance genes.So,Enterococcus carring antimicrobial resistance genes could spread to not only indoor air but also outdoor air based on the REP-PCR results.更多还原