REVIEW
Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations
Robert S.Dungan ÆApril B.Leytem
Received:12August 2008/Accepted:8April 2009/Published online:26April 2009ÓUS Government 2009
Abstract The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs)is a concern from a human and animal health perspective.To better understand the airborne microorganisms found in the environments,a number of collection and analytical techniques have been utilized and will be discusd in this review.The most commonly ud bioaerosol collection method is the liquid impingement format,which is suitable with a number of culture-bad and non-culture molecular-bad approaches,such as polymera chain reaction.However,the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-bad analys.Becau of the limitations often associated with culture-bad analys,we focud our discussion on the applica-tion of molecular-bad techniques to identify and/or quantify microorganisms,as they have promising applica-tion in bioaerosol rearch.The ability to rapidly charac-terize airborne microorganisms will help
to ensure protection of public and environmental health.
Keywords Airborne microorganisms ÁBioaerosol ÁConcentrated animal-feeding operations ÁImpaction ÁImpingement ÁNucleic acid ÁPolymera chain reaction ÁReal-time PCR
Introduction
Modern animal husbandry has changed from one that was low density pasture-bad to one that predominately employs confinement of animals at high stocking density.Confined or concentrated animal-feeding operations (CA-FOs)concentrate a large population of single species in one area to increa production and reduce costs.During recent decades,CAFOs have become common in many countries including The Netherlands,Denmark,France,USA,Can-ada,China,Germany,and Poland (Schulze et al.2006).A conquence of high stocking densities combined with enclod rearing facilities,in some cas,is that the air may contain bioaerosol levels that are sufficiently high to cau adver health effects in both animals and workers (Thorne et al.1992).Crook and Sherwood-Higham (1997)indicated that inhalation of airborne microorganisms and their constituents can be detrimental to health through infection,allergy,or toxicosis.As the environment within CAFOs can be potentially hazardous to both human and animal health at the facility as well as in surroundin
g areas,rearch is being pursued in order to quantify,characterize,and control the relea of bioaerosols from CAFOs.
毛笔的简笔画Bioaerosols is a term commonly ud to describe via-ble and non-viable airborne biological particles,such as fungal spores,bacteria,pollen,and virus and their fragments and byproducts (Grinshpun et al.2007).Fungal spores,bacteria,and pollen are typically 1–30,0.25–8,and 17–58l m in diameter,respectively,while virus generally have diameters \0.3l m (Jones and Harrison 2004).Matthais-Mar et al.(2000)suggested that up to 28%(by volume)of the particulate matter suspended over remote land surfaces is comprid of biological particles.Womiloju et al.(2003)concluded that fungal cells and pollen accounted for 4–11%of the total mass of airborne
The u or mention of any commercial products does not imply any endorment of that product by either the authors or the US Department of Agriculture.
R.S.Dungan (&)ÁA.B.Leytem
USDA-Agricultural Rearch Service,Northwest Irrigation and Soils Rearch Laboratory,3793North 3600East,Kimberly,ID 83341,USA
e-mail:robert.dungan@v
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World J Microbiol Biotechnol (2009)25:1505–1518DOI 10.1007/s11274-009-0043-1
particulate matter\2.5l m(PM2.5).Although microor-ganisms are ubiquitous in the ambient environment,pre-vious studies have shown higher airborne microorganism concentrations in animal hous than in industrial,resi-dential,or ambient ttings(Clark et al.1983;Thorne et al.1992;Griffiths et al.1997).
Bioaerosols are typically associated with particulate matter or surrounded by a thin layer of water,having an aerodynamic diameter range of0.5–100l m(Lighthart 1994;Cox1995).Bioaerosol particles1–5l m in diameter prent the most concern since they are readily transported into the lung,with the greatest retention of the1–2l m particles in the alveoli(Salem and Gardner1994).The microbial component of respirable bioaerosols contributes significantly to the pulmonary dias associated with inhalation of agricultural dusts(Merchant1987;Lacy and Crook1988).The allergenic,toxic,and inflammatory respons are caud by exposure to not only viable but also non-viable microorganisms prent in bioaerosols (Robbins et al.2000;Gorny et al.2002).An estimation of occupational and residential risks from bioaerosol exposure have been addresd by Brooks et al.(2
005a,b)and Tanner et al.(2008).As the generation of bioaerosols from CAFOs is a concern from a human and animal health perspective, the sampling and analysis of airborne microorganisms is of great interest.Protection of public and environmental health is dependent upon the ability to efficiently collect bioaerosol samples,then accurately identify and quantify the airborne microorganisms.
In this conci review,we focus our discussion on bio-aerosol sampling and sample processing methods that are most suitable to quantitatively and qualitatively determine airborne microorganisms at CAFOs,although their appli-cation to other situations is not limited.The majorfindings of bioaerosol studies conducted at CAFOs are also dis-cusd.While this is not meant to be an exhaustive review of the literature,the reader willfind an excellent array of peer-reviewed articles on aerosol science and molecular biology and their application to studies of air quality.This review will be very uful to tho interested in conducting bioaerosol rearch using both traditional microbiological and molecular techniques.
Airborne microorganism sampling
The collection of airborne microorganisms is performed through active air sampling,which results in t
he efficient removal and collection of biological particles from the air in a manner that maximizes the ability to detect the organisms.Airborne microorganisms can be collected using a number of different techniques(Lundholm1982; Juozaitis et al.1994;Grinshpun et al.1996;Terzieva et al.1996;Duchaine et al.2001),but two inertial techniques,surface impaction and liquid impingement, are ud in the majority of outdoor aerosol studies.Fil-tration is a non-inertial technique that parates particles from the airstream when air is pasd through a porous medium,such asfibrousfilters,membranefilters,or etched membranes(Crook1995a).For airborne microor-ganisms,however,filtration pos two major disadvan-tages:(a)dehydration of cells and therefore loss of viability and/or culturability due to the large volume of air passing over the particle that is deposited on a dry medium,and(b)inconsistent and poor recovery of the deposited material from certainfilter types.Two addi-tional techniques,gravity sampling and electrostatic precipitation,have been employed for airborne microor-ganism collection but are not routinely ud due to cali-bration errors and unknown performance characteristics (Pillai and Ricke2002).
燕然未勒归无计
The most common bioaerosol sampling techniques uti-lized at cattle,poultry and swine CAFOs are prented in Table1.Direct impaction of airborne microorganisms on filters was ud in*40%of the studies,while a combi-nation of liquid impingement and multistage or single stage impaction was u
去泰国要办护照吗d in*33%of the studies.Other sam-pling techniques included u of a personal slide sampler to measure fungi in a cattle shed(Adhikari et al.2004)and drag swab for determination of Salmonella in a poultry hou(Endley et al.2001).The target organisms in the studies included Wallemia bi,total bacteria and fungi, Gram-negative bacteria,li,enteric bac-teria,Salmonella,yeast,and molds.
Impaction samplers
The surface impaction method parates particles from the airstream by utilizing the inertia of the particles to force their deposition onto a collection surface(Grinshpun et al. 2007).The collection surface is usually an agar medium for culture-bad analysis or an adhesive-coated surface that can be analyzed microscopically.A commonly ud impaction system is the multi-stage Andern viable sam-pler(Thermo Scientific,Waltham,MA,USA)that con-centrates bioaerosols bad on their size characteristics. Two-stage and six-stage Andern models are available. The six-stage Andern sampler is capable of concentrating particles in the size range of0.65–7.0l m in diameter (Grinshpun et al.2007).Air enters the sampler through an inlet nozzle and heavier particles are deposited on thefirst stage.Lighter particles not deposited on thefirst stage are carried by the airstream onto the successive stages.
Single-stage impactors,which u an agar or adhesive-coated impacting surface,are available from a variety of
爆款图片
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股东证明T a b l e 1B i o a e r o s o l s t u d i e s c o n d u c t e d a t c o n c e n t r a t e d a n i m a l -f e e d i n g o p e r a t i o n s i n c l u d i n g t h e t y p e o f o p e r a t i o n ,t h e t a r g e t o r g a n i s m ,s a m p l i n g t e c h n i q u e s u t i l i z e d a n d t h e a n a l y t i c a l m e t h o d s u s e d f o r d e t e r m i n a t i o n o f m i c r o o r g a n i s m s
O p e r a t i o n T a r g e t o r g a n i s m s
S a m p l i n g t e c h n i q u e s
A n a l y t i c a l m e t h o d s R e f e r e n c e s
C o w h o u s e W a l l e m i a s e b i
D i r e c t i m p a c t i o n o n fil t e r s职业调查问卷
C u l t u r e t e c h n i q u e s ,c o n v e n t i o n a l a n d r e a l t i m e P C R Z e n g e t a l .2004
D u c k -f a t t e n i n g u n i t
T o t a l a n d a e r o b i c G r a m -n e g a t i v e b a c t e r i a ,f u n g i ,e n d o t o x i n s
L i q u i d i m p i n g e m e n t ,m u l t i -s t a g e i m p a c t i o n ,a n d d u s t s a m p l i n g
C u l t u r e t e c h n i q u e s ,w h o l e b l o o d a s s a y ,E L I S A ,l i m u l u s a m e b o c y t e l y s a t e a s s a y Z u c k e r e t a l .2006
C a t t l e f e e d l o t B a c t e r i a a n d f u n g i
M u l t i -s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
W i l s o n e t a l .2002b
C a t t l e s h e d F u n g i
M u l t i -s t a g e i m p a c t i o n a n d P e r s o n a l s l i d e s a m p l e r
C u l t u r e t e c h n i q u e s a n d m i c r o s c o p y
A d h i k a r i e t a l .2004
P i g g e r y s h e d s H e t e r o t r o p h s a n d E .c o l i
L i q u i d i m p i n g e m e n t a n d m u l t i -s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
C h i n i v a s a g a m a n d B l a c k a l l 2005
S w i n e b a r n s
T o t a l a n d G r a m -n e g a t i v e e n t e r i c b a c t e r i a ,t o t a l f u n g i
M u l t i -s t a g e i m p a c t i o n ,l i q u i d i m p i n g e m e n t ,d i r e c t i m p a c t i o n o n fil t e r s
C u l t u r e t e c h n i q u e s a n d flu o r e s c e n c e m i c r o s c o p y
T h o r n e e t a l .1992
S w i n e b a r n s
C u l t u r a l b a c t e r i a ,G r a m -n e g a t i v e b a c t e r i a ,f u n g i
L i q u i d i m p i n g e m e n t ,m u l t i -s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
C h a n g e t a l .2001
P o u l t r y H o u s e S a l m o n e l l a
D r a g s w a b ,d i r e c t i m p a c t i o n o n fil t e r s
C u l t u r e t e c h n i q u e s a n d P C R
E n d l e y e t a l .2001
S w i n e b a r n s H e t e r o t r o p h i c b a c t e r i a
D i r e c t i m p a c t i o n o n fil t e r s
C u l t u r e t e c h n i q u e s
P r e d i c a l a e t a l .2001
S w i n e b a r n s
T o t a l a n d r e s p i r a b l e m i c r o o r g a n i s m s
D i r e c t i m p a c t i o n o n fil t e r s ,m u l t i -s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
P r e d i c a l a e t a l .2002
S w i n e b a r n s T o t a l b a c t e r i a a n d f u n g i
S i n g l e s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
K i m e t a l .2006,2007
P o u l t r y H o u s e T o t a l b a c t e r i a
D i r e c t i m p a c t i o n o n fil t e r s ,l i q u i d i m p i n g e m e n t
C u l t u r e t e c h n i q u e s
W o o d w a r d e t a l .2004
S w i n e C A F O B a c t e r i a
M u l t i -s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
G r e e n e t a l .2006
P o u l t r y h o u s e T o t a l a e r o b i c b a c t e r i a
S i n g l e s t a g e i m p a c t i o n
C u l t u r e t e c h n i q u e s
V e n t e r e t a l .2004
P o u l t r y ,c o w ,a n d s w i n e h o u s e
A i r b o r n e m i c r o o r g a n i s m s
D i r e c t i m p a c t i o n o n fil t e r s
E p i flu o r e s c e n c e m i c r o s c o p y
H e l d a l e t a l .1996
S w i n e b a r n s T o t a l a n d c u l t u r a l b a c t e r i a
L i q u i d i m p i n g e m e n t ,i m p a c t i o n o n g e l a t i n m e m b r a n e s
C u l t u r e t e c h n i q u e s ,r e a l t i m e P C R ,d e n a t u r i n g g r a d i e n t g e l e l e c t r o p h o r e s i s ,p h y l o g e n e t i c a n a l y s i s
N e h m e e t a l .2008
D a i r y b a r n s
Y e a s t s ,m o l d s ,m e s o p h i l i c b a c t e r i a ,t h e r m o p h i l i c b a c t e r i a
L i q u i d i m p i n g e m e n t
C u l t u r e t e c h n i q u e s
L a n g e e t a l .1997
S w i n e C A F O V i a b l e b a c t e r i a
L i q u i d i m p i n g e m e n t
C u l t u r e t e c h n i q u e s
R u l e e t a l .2005
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manufacturers.Adhesive-coated impacting surfaces are ud for the detection of total fungal spores
and pollen.In addition to the Andern impactors,there are other impaction-bad devices,such as the rotating impactor,slit sampler,and sieve-type sampler(Crook1995b).Disad-vantages associated with culture-bad impactors are:(a) detection of microorganisms relies on their ability to grow after sampling and loss of culturability may occur due to sampling stress,(b)multiple particles each containing one or more organisms passing through a single impaction hole may be inaccurately counted as a single colony,and(c) culturable counts account for only0.0001–10%of the total population within environmental samples,which can verely underestimate the total population of microor-ganisms in the sample(Parkes and Taylor1985).This is also a problem when using culture-bad techniques with impingement samplers.
Impingement samplers
Impingement samplers remove bioaerosols over a wide range of airborne particle concentrations(Grinshpun et al. 2007).The primary difference between impingement and impaction is that the bioaerosols are trapped in a liquid (e.g.,water,mineral oil,buffered solution,or dilute pep-tone solution).In theory,buffered or dilute peptone solu-tions are ud to maintain the viability of the microbial cells.Most impingers are constructed from glass with a single collection chamber;though multi-stage glass liquid impinges are available(Crook1995b).The All-Glass Impinger(AGI)-30(Ace Gl
ass,Inc,Vineland,NJ,USA)is a single chamber design that has been widely ud to measure bioaerosols under various conditions(Pillai et al. 1996;Chang et al.2001;Rule et al.2005;Tanner et al. 2005;Taha et al.2006).The SKC BioSamplerÒ(SKC Inc, Eighty-Four,PA,USA)is an improved design over the AGI-30and can be operated for up to8h when mineral oil is ud as the collectionfluid(Lin et al.1999).Both the SKC BioSamplerÒand AGI-30operate under an airflow rate of12.5l min-1through the u of a vacuum pump. During operation of the impinger,the microorganisms are suspended in the collectionfluid,but the high airflow velocity required for efficient particle collection also cau-s re-aerosolization of the biological particles(Grinsphun et al.1997;Lin et al.1997)and stress that can lead to viability loss(Lin et al.1999,2000).One of the advantages of impingement samplers is the ability to utilize a variety of analytical methods.In addition to culture techniques, samples can also be analyzed via microscopy,flow cytometry,biochemical assays,immunoassays,and molecular techniques such as polymera chain reaction (PCR)providing better detection of airborne microorganisms which may be non-culturable due to sampling stress.
High-volume samplers
Another class of bioaerosol samplers that has recently evolved due to bioterrorism and biological warfare con-cerns is high-volume samplers.Some examples of the units are the SASSÒ2300(Res
earch International,Mon-roe,WA),BioCaptureÒ560(MesoSystems Technology, Inc,Albuquerque,NM),and the SpinconÒ(Sceptor Industries,Inc,Kansas City,MO).The samplers operate atflow rates of200–450l min-1and the bioaerosols are captured in a concentrated liquid sample.While the high-volume samplers are very costly when compared to units such as the AGI-30and SKC BioSamplerÒ,they are generally more amenable to PCR-bad analys.The ASAPÒmodel2800(Thermo Electron Corporation, Greenbush,NY,USA)sampler has an operationalflow rate of200l min-1,but collects aerosol particles by impaction on polyurethane foam.While the ASAP unit does not u a liquid impingement format like the other high-volume samples,it is currently being marketed as PCR-compatible. At this time,however,a arch of the literature reveals a scarcity of peer-reviewed studies with respect to the or comparable units and their operating efficiencies(Bergman et al.2005).For a comprehensive list of commercially available bioaerosol samplers e Grinshpun et al.(2007). Sample processing
Once samples have been collected,choosing the appro-priate analytical technique is important in order to best answer the question of interest.One of the most popular methods to asss microbial populations in aerosol samples has been the u of culture-bad techniques.Culture-bad techniques were employed in89%of the studies reported here(Table1).As mentioned above,culture-b
ad tech-niques can drastically underestimate the microbial popu-lations in environmental samples as less than10%of the populations may be culturable.In order to improve microorganism detection,some studies have combined the u of culture techniques with other methods such as PCR (16%),microscopy(16%),denaturing gradient gel elec-trophoresis(DGGE,5%),and immunoassays(5%).Sample preparation is important for all of the techniques,as microorganism populations in bioaerosol samples tend to be small and,therefore,concentration of samples is esntial.The most commonly ud sample preparation methods compatible with the molecular characterization of bioaerosols can be found below.
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Concentration andfilter elution
After bioaerosols are collected in a liquid impingement solution,it is necessary to concentrate the microorganisms before molecular methods,such as PCR,can be performed. This is necessary becau the impingement solution usually contains a relatively low microbial concentration,which must be maximized to ensure nsitivity and quantification for PCR are achieved.A variety offilter materials have been tested for their compatibility with PCR(Table2)such as polytetrafluoroethylene(P
TFE),polycarbonate,polyvi-nylidene difluoride,nylon,mixed cellulo ester,and nitrocellulo(Bej et al.1991a).Bej et al.(1991a)reported that PCR was not inhibited by the prence of PTFE and polyvinylidene difluoridefilters,with PTFE giving the greatest nsitivity,but was inhibited by polycarbonate, nitrocellulo,and cellulo acetatefilters.Both Nytran (Alvarez et al.1994)and nitrocellulo(Toranzos and Alvarez1992)filters have been successfully ud in solid-pha PCR,where cell lysis and PCR amplification are performed on the membrane.
Since DNA does bind to somefilters,it is recommended that allfilters be removed before cell lysis and PCR amplification.Filter materials that have been successfully ud in PCR-bad bioaerosol studies using liquid samples from glass impingers are Nytran(Alvarez et al.1994), polycarbonate(Paez-Rubio et al.2005),nylon(Alvarez et al.1995),and Teflon(Alvarez et al.1995).Aerosol samples can also be directly impinged ontofilters for subquent PCR analysis;filters ud for this purpo are tracked-etched polyester(Wilson et al.2002a),polycar-bonate(Zeng et al.2004),and polyethersulfone(Sta¨rk et al.1998).Thefilters are added to sterile distilled water (Alvarez et al.1995)or buffer solution(Wilson et al. 2002a;Zeng et al.2004;Paez-Rubio et al.2005)and then the microorganisms are eluted via agitation such as vor-texing,shaking,or sonication.
Cell lysis and nucleic acid purification
After elution,thefilter is removed and the cells are then prepared for lysis,which can be performed either through physical,chemical,or enzymatic methods.Physical meth-ods include bead beating,sonication,microwave heating, and thermal shock(Roo-Amsaleg et al.2001),but bead beating and sonication can cau significant DNA shearing (Picard et al.1992;Miller et al.1999;Bu¨rgmann et al. 2001).Freeze-thaw lysis has been shown to relea70–75% of DNA in bacterial cells after one cycle with complete lysis within six cycles(Bej et al.1991b).Chemical lysis,either alone or in combination with enzymatic methods,has been ud extensively.The most widely ud detergent is sodium dodecyl sulfate(SDS),who function is to break up and dissolve cell wall lipids.Detergents are ud in combination with heat treatments and chelating ,EDTA)and various buffers(Tris and phosphate).In addition to a detergent,many protocols include enzymatic lysis.Lyso-zyme is a commonly ud lytic enzyme that breaks the b-1,4-glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan,thereby weakening the cell wall.Some proteas,like proteina K,are also ud to remove contaminating ,nucleas)that might otherwi degrade nucleic acids during purification. The protea,achromopeptida,has been ud with
Table2Filters utilized for preparation of bioaerosol samples for molecular methods including thefilter type,type of sample,and the methods ud for sample preparation and analysis
Filter Sample type Methods References
Polytetrafluoroethylene, Polyvinylidene
difluoride Bacterial cells in water
collected onfilters
Freeze thaw lysis of cells fromfiltered samples,
PCR DNA amplification withfilters prent
Bej et al.1991a,b
Polycarbonate Direct impingement of
bioaerosols onfilter Filters washed in buffer to remove bacteria,DNA
extraction(chemical/enzymatic),RT-PCR
Zeng et al.2004
Polycarbonate Bioaerosols collected in liquid
impingers andfiltered Impinger solutionfiltered,DNA extraction,PCR,
cloning,quencing
Paez-Rubio et al.
2005
Track etched polyester Direct impingement of
bioaerosols onfilter Filters washed in buffer to remove bacteria,DNA
extraction(physical/chemical/enzymatic),
microarray analysis
防溺水手抄报简单好画Wilson et al.2002a
Mixed cellulo nylon Bioaerosols collected in liquid
impingers andfiltered Cell lysis and DNA extraction(chemical/
enzymatic)performed onfilters,solid-pha
PCR ud for amplification
Alvarez et al.1994
Nitrocellulo Filtration of bacterial cells in
water Cell lysis and DNA extraction(chemical/
enzymatic)performed onfilters,solid-pha
PCR ud for amplification
Toranzos and
南斯拉夫内战Alvarez1992
Polyethersulfone Direct impingement of
bioaerosols onfilter Filters were dried and dissolved in chloroform,
DNA extraction(chemical),nested PCR assay
Sta¨rk et al.1998
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