Microbiological Aerosol Of Animal Houses And Their Transmission To The Surroundings

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.