Bioaerosol Health Effects and Exposure Assessment: Progress and Prospects

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• ls of Occupational Hygiene

• Volume 47, Number 3

• Pp. 187-200

Ann. occup. Hyg., Vol. 47, No. 3, pp. 187-200, 2003

© 2003 British Occupational Hygiene Society

Published by Oxford University Press

Review

Bioaerosol Health Effects and Exposure Assessment: Progress and

Prospects

J. DOUWES1,2,*, P. THORNE3, N. PEARCE2 and D. HEEDERIK1

1 Institute for Risk Assessment Sciences (IRAS), Division of

Environmental and Occupational Health, Utrecht University, The

Netherlands; 2 Centre for Public Health Research, Massey University

Wellington Campus, Wellington, New Zealand; 3 University of Iowa

College of Public Health, Department of Occupational and

Environmental Health, IA, USA

Received 19 July 2002; in final form 20 December 2002

ABSTRACT

TOP

ABSTRACT

INTRODUCTION

HEALTH EFFECTS

SPECIFIC EXPOSURES

PROTECTIVE EFFECTS OF MICROBIAL...

EXPOSURE ASSESSMENT

ASSESSMENT METHODS FOR...

ASSESSMENT METHODS FOR MICROBIAL...

ASSESSMENT METHODS FOR BIO...

BIOMARKERS OF EXPOSURE

STANDARD SETTING

RESEARCH NEEDS

CONCLUSIONS

REFERENCES

Exposures to bioaerosols in the occupational environment are

associated with a wide range of health effects with major public

health impact, including infectious diseases, acute toxic effects,

allergies and cancer. Respiratory symptoms and lung function

impairment are the most widely studied and probably among the most

important bioaerosol-associated health effects. In addition to these

adverse health effects some protective effects of microbial exposure

on atopy and atopic conditions has also been suggested. New

industrial activities have emerged in recent years in which

exposures to bioaerosols can be abundant, e.g. the waste recycling

and composting industry, biotechnology industries producing highly

purified enzymes and the detergent and food industries that make use

of these enzymes. Dose–response relationships have not been

established for most biological agents and knowledge about threshold

values is sparse. Exposure limits are available for some

contaminants, e.g. wood dust, subtilisins (bacterial enzymes) and

flour dust. Exposure limits for bacterial endotoxin have been

proposed. Risk assessment is seriously hampered by the lack of valid

quantitative exposure assessment methods. Traditional culture

methods to quantify microbial exposures have proven to be of limited

use. Non-culture methods and assessment methods for microbial

constituents [e.g. allergens, endotoxin, ß(1 3)-glucans, fungal

extracellular polysaccharides] appear more successful; however,

experience with these methods is generally limited. Therefore, more

research is needed to establish better exposure assessment tools and

validate newly developed methods. Other important areas that require

further research include: potential protective effects of microbial

exposures on atopy and atopic diseases, inter-individual

susceptibility for biological exposures, interactions of bioaerosols

with non-biological agents and other potential health effects such

as skin and neurological conditions and birth effects.

Keywords: asthma; ß(1,3)-glucans; bioaerosols; cancer; endotoxin;

exposure assessment; infections; microorganisms

INTRODUCTION

TOP

ABSTRACT

INTRODUCTION

HEALTH EFFECTS

SPECIFIC EXPOSURES

PROTECTIVE EFFECTS OF MICROBIAL...

EXPOSURE ASSESSMENT

ASSESSMENT METHODS FOR...

ASSESSMENT METHODS FOR MICROBIAL...

ASSESSMENT METHODS FOR BIO...

BIOMARKERS OF EXPOSURE

STANDARD SETTING

RESEARCH NEEDS

CONCLUSIONS

REFERENCES

Bioaerosols are usually defined as aerosols or particulate matter of

microbial, plant or animal origin that is often used synonymously

with organic dust. Bioaerosols or organic dust may consist of

pathogenic or non-pathogenic live or dead bacteria and fungi,

viruses, high molecular weight (HMW) allergens, bacterial

endotoxins, mycotoxins, peptidoglycans, ß(1 3)-glucans, pollen,

plant fibres, etc.

The interest in bioaerosol exposure has increased over the last few

decades. This is largely because it is now appropriately recognized

that exposures to biological agents in both the occupational and

residential indoor environment are associated with a wide range of

adverse health effects with major public health impact, including

contagious infectious diseases, acute toxic effects, allergies and

cancer. Several new industrial activities have emerged in recent

years in which exposures to biological agents can be abundant. One

example is the waste recycling industry. Workers in this industry

(e.g. waste sorting, organic waste collection and composting) are

often exposed to very high levels of microorganisms (van Tongeren et

al., 1997; Douwes et al., 2000a) and several studies have indicated

a high prevalence of respiratory symptoms and airway inflammation in

these industries (Sigsgaard et al., 1994; Poulsen et al., 1995;

Thorn and Rylander, 1998a; Douwes et al., 2000a; Wouters et al.,

2002). Another example is the production of highly purified

biological substances such as microbial enzymes that are used

particularly in the food processing industry (e.g. -amylase in the

bread making industry) and detergent industry (Sandiford et al.,

1994; Schweigert et al., 2000). Today, there is a clear trend to

increase production and use of these enzymes. Many of these enzymes

are potent allergens that may cause allergic asthma and rhinitis in

workers handling the enzymes and/or intermediate products that

contain enzymes (Cullinan et al., 2000, 2001). The increased

insulation of buildings combined with poor ventilation has also

created environments with elevated exposures to bioaerosols (mainly

moulds) and several studies have suggested that a significant

portion of building-related disease occurrence (e.g. `sick building

syndrome') is associated with these exposures (Walinder et al.,

2001). Finally, the widespread use of antibiotics in livestock has

accelerated the development of antibiotic-resistant pathogens which

may increase the risk of severe infectious diseases in workers

handling and processing livestock.

Despite the recognition of the importance of bioaerosol exposure on

human health, the precise role of biological agents in the

development and aggravation of symptoms and diseases is only poorly

understood. It is not clear (with the exception of specific

pathogens and a few individual components such as bacterial

endotoxin and specific allergens; see below) which specific component

(s) primarily accounts for the presumed health effects. Dose–

response relationships have often not been described and knowledge

about threshold values is (with the exception of a few agents) not

available. This relative lack of knowledge is mainly due to the lack

of valid quantitative exposure assessment methods.

In this paper we will give an overview of the health effects

associated with bioaerosol exposure in the occupational environment.

In addition, we will review exposure assessment methods with a focus

on non-culturable methods. Finally, we will discuss the potential

for standard setting and identify relevant future research areas.

HEALTH EFFECTS

TOP

ABSTRACT

INTRODUCTION

HEALTH EFFECTS

SPECIFIC EXPOSURES

PROTECTIVE EFFECTS OF MICROBIAL...

EXPOSURE ASSESSMENT

ASSESSMENT METHODS FOR...

ASSESSMENT METHODS FOR MICROBIAL...

ASSESSMENT METHODS FOR BIO...

BIOMARKERS OF EXPOSURE

STANDARD SETTING

RESEARCH NEEDS

CONCLUSIONS

REFERENCES

In most situations exposure occurs to complex mixtures of toxins and

allergens (and chemicals) and a wide range of potential health

effects have to be considered. Three major groups of diseases

associated with bioaerosol exposure can be

distinguished: `infectious diseases', `respiratory diseases'

and `cancer'. Infectious and respiratory diseases are most common;

however, valid incidence or prevalence data for most diseases caused

by biological agents are lacking. In addition to these major disease

groups (discussed in more detail below) other adverse health effects

have been described [e.g dermatitis in latex exposed workers

(Turjanmaa, 1987; Charous et al., 1994) or pre-term births or late

abortions in farm women exposed to mycotoxins with immunotoxic and

hormone-like effects (sen et al., 2000)]. However, to date

these effects have not been studied extensively and therefore only

limited information is available on these issues. In this section we

will discuss the general types of health effects that may occur, and

these will then be considered in more detail when examining specific

exposures in the following sections. Viruses will be discussed as a

cause of infections and cancer but will not be described in more

detail since exposure and risk assessment for viruses have hardly

been developed for the occupational environment.

Infectious diseases

Infectious diseases arise from viruses, bacteria, fungi, protozoa

and helminths and involve the transmission of an infectious agent

from a reservoir to a susceptible host through direct contact, a

common vehicle, airborne transmission or vector-borne transmission.

Since the focus of our paper is on bioaerosols we will discuss

occupational infectious diseases caused by airborne exposures only.

These may be attributable to: (i) occupation-specific exposures such

as may occur in, for example, health workers (tuberculosis, winter

stomach flu, measles), farmers, abattoir workers, veterinarians (Q-

fever, swine influenza, anthrax) and forestry workers (tularaemia);

(ii) clustering of people in the workplace such as in the case of

office, military or aviation workers (influenza, winter stomach flu,

TB, etc.) (Driver et al., 1994; Van den Ende et al., 1998). In

addition, Legionnaires disease and Pontiac fever are high profile

bioaerosol transmitted infections that are caused by occupational

(as well as non-occupational) exposures to Legionellae (particularly

Legionella pneumophila). Legionellae are Gram-negative bacteria that

inhabit many water environments including man-made water systems

(often in bio-films in, for example, cooling towers, air

conditioning systems, etc.) that can cause pneumonia which may be

fatal, particularly in susceptible subjects (e.g. elderly or immuno-

compromised subjects). Legionnellae become airborne often as a

result of active aerosolizing processes (e.g. aeration of

contaminated water). Outbreaks have been reported in a variety of

workplaces including those associated with cooling towers

(Castellani Pastoris et al., 1997; Brown et al., 1999), hospital

bathrooms (Kool et al., 1999), meat packers (Osterholm et al.,

1983), workplaces where water mist systems are used (fruit and

vegetable stores; Mahoney et al., 1992) or whirlpool spas and

sprinklers (Den Boer et al., 2002). Finally, several diseases may

arise from inhalation of fungal spores in the course of handling

decaying matter, faeces, compost or soil. These diseases include

aspergillosis, histoplasmosis, blastomycosis, coccidioidomycosis and

adiaspiromycosis (MMWR, 1993, 1999; NIOSH, 1997).

Thus, high-risk occupations for occupational infectious diseases due

to bioaerosol exposure include farmers, veterinarians, health care

workers and biomedical workers studying infectious agents. For a

more complete overview of occupational infections the reader is

referred to textbooks (Garibaldi and Janis, 1992; Gerberding and

Holmes, 1994) and the NIOSH website (National Occupational Research

Agenda: infectious diseases at www.cdc.gov/niosh/ nrinfo.html).

Respiratory diseases

Respiratory symptoms and lung function impairment are probably the

most widely studied among organic dust-associated health effects.

They can range from acute mild conditions that (at least initially)

hardly affect daily life, to severe chronic respiratory diseases

that require specialist care. Generally, occupationally related

respiratory symptoms result from airway inflammation caused by

specific exposures to toxins, pro-inflammatory agents or allergens.

Based on the underlying inflammatory mechanisms and subsequent

symptoms, a distinction between allergic and non-allergic

respiratory diseases can be made. Non-allergic respiratory symptoms

reflect a non-immune-specific airway inflammation, whereas allergic

respiratory symptoms reflect an immune-specific inflammation in

which various antibodies (IgE, IgG) can play a major role in the

inflammatory response. In occupational medicine it has long since

been recognized that a substantial proportion of work-related asthma

symptoms are non-allergic. This type of asthma is often referred to

as `asthma-like disorder or syndrome' or `irritant-induced asthma'

(Bernstein et al., 1999) and is highly prevalent in farmers and farm-

related occupations and is in these occupations assumed to be caused

by bioaerosol exposures (particularly endotoxin) (Anonymous, 1998).

Although it meets the clinical criteria of asthma (i.e. reversible

airways obstruction) it has been shown in some populations (e.g.

swine farmers) that these symptoms are not only associated with a

cross-shift reversible decrease in lung function (asthma) but also

with an accelerated chronic decline in lung function (COPD, chronic

obstructive pulmonary diseases) (Vogelzang et al., 1998). This is

clearly different from allergic asthma (caused by allergen exposure)

where a chronic lung function decline is generally only moderate. In

addition, cross-shift decline in lung function is usually smaller

than observed in typical type I allergic asthma. Table 1 gives an

overview of allergic and non-allergic respiratory diseases with

potential causal agents of biological origin. Pre-existing

respiratory conditions or other host factors (e.g. atopy, smoking)

may modify the risk of developing work-related respiratory symptoms

(Cullinan et al., 1999). For instance, an asthmatic worker with pre-

existing asthma may experience work-related exacerbation of asthma

symptoms due to organic dust exposure at levels that do not normally

induce any symptoms in other `healthy' workers.

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Table 1. Non-infectious respiratory diseases, potential

causal agents and work environments with known or suspected

increased risks

In addition to asthma and COPD (see above), organic dust exposed

workers may develop hypersensitivity pneumonitis (HP) and organic

dust toxic syndrome (ODTS) (Table 1). ODTS is an acute febrile non-

allergic illness, characterized by an increase in body temperature

(fever), shivering, dry cough, chest tightness, dyspnea, headache,

muscle and joint pains, fatique, nausea and general malaise (Donham

and Rylander, 1986; Von Essen et al., 1990; Rylander, 1997c). The

symptoms resemble those of influenza, but symptoms usually disappear

the following day. The disease is common among workers heavily

exposed to organic dust. HP is a generic term used to describe a

serious pulmonary condition with delayed febrile systemic symptoms,

manifested by an influx of inflammatory cells to the lung parenchyma

and the formation of granulomas there (Bourke et al., 2001). HP is

also known as extrinsic allergic alveolitis (EAA) and, depending on

the specific work environment where the disease has been observed,

various other names have been introduced to describe the disease,

e.g. farmer's lung, pigeon breeder's lung, mushroom grower's lung,

maple bark strippers disease, etc. Symptoms characteristic for HP

are very similar to those described for ODTS but are more serious

and in its chronic stage may lead to permanent lung damage and work

disability (Pickering and Newman , 1994). The underlying

immunological mechanisms of HP are complex and only partly

understood but both allergic and non-allergic immunological

responses are believed to play a role (Salvaggio, 1997; Bourke et

al., 2001).

Cancer

Cancer can be caused by a variety of factors including oncogenic

viruses and other biological agents. To date the only clearly

established non-viral biological occupational carcinogens are the

mycotoxins. These occur in industries in which mould-contaminated

materials are handled (Anonymous, 1998). Perhaps the best-known

carcinogenic mycotoxin is aflatoxin from Aspergillus flavus, which

is an established human carcinogen particularly with regard to liver

cancer ( et al., 1984; Sorenson et al., 1984; Bray and ,

1991). Ochratoxin A is also considered a possible human carcinogen

(National Toxicology Program, 1991). The most relevant route of

exposure to aflatoxin and ochratoxin is by ingestion, but exposure

can also occur by inhalation in industries such as peanut processing

or livestock feed processing and in industries in which grain dust

exposure occurs (Sorenson et al., 1984; Autrup et al., 1993).

Workers in livestock feed processing have an increased risk of liver

cancer as well as cancers of the biliary tract, salivary gland and

multiple myeloma (Olsen et al., 1988). Farmers are at increased risk

for certain specific cancers, including haematological cancers, lip,

stomach, prostate, connective tissue and brain cancer (Blair et al.,

1992; Khuder et al., 1998). Hypothesized explanations involve

exposure to pesticides or exposure to oncogenic viruses or other

biological agents carried by farm animals. Studies have shown a

consistent excess of lung cancer associated with abattoir workers

and butchers (Reif et al., 1989). Others also indicated increased

leukaemia risks with employment in the meat industries (Bethwaite et

al., 2001). Direct contact with animals was determined to be an

important factor suggesting that biological exposures (probably

zoonotic viral exposures such as herpes, avian leucosis and

papilloma viruses) are likely to be responsible. In addition, a

number of studies have found associations between exposure to wood

dust and various specific cancers, in particular sinonasal cancer in

furniture making, cabinet making, carpentry and joinery and in other

wood-related jobs including sawmills (Demers and Boffetta, 1998).

Finally, workers in several other industries that process biological

materials are at risk of developing various cancers, such as workers

in the rubber, textile, leather and boot and shoe industries.

However, it is currently unknown whether these excess risks occur

from exposures to biological agents or are due to various chemicals

used in these industries.

SPECIFIC EXPOSURES

TOP

ABSTRACT

INTRODUCTION

HEALTH EFFECTS

SPECIFIC EXPOSURES

PROTECTIVE EFFECTS OF MICROBIAL...

EXPOSURE ASSESSMENT

ASSESSMENT METHODS FOR...

ASSESSMENT METHODS FOR MICROBIAL...

ASSESSMENT METHODS FOR BIO...

BIOMARKERS OF EXPOSURE

STANDARD SETTING

RESEARCH NEEDS

CONCLUSIONS

REFERENCES

Fungi and bacteria

Fungi and thermophilic bacteria are well-known sources of allergens

that play a role in the development of HP. The species involved

include many common genera such as Penicillium and Aspergillus,

which occur in some work environments usually at very high levels

(e.g composting facilities, farms, etc.). Hay contaminated with

thermophilic bacteria such as Saccharopolyspora rectivirgula or

Thermoactinomycetes vulgaris is the source of allergens causing

farmer's lung or HP (Pepys et al., 1990; Reboux et al., 2001), and

similar disorders have been observed among mushroom growers (Van den

Bogart et al., 1993) and, incidentally, among compost workers (Weber

et al., 1993; Allmers et al., 2000). A specific exposure with high

risk of occupational disease is that to Aspergillus fumigatus, a

fungus that not only induces allergic sensitization and symptomatic

allergic lung disease, but can also cause an infectious mycosis

(broncho-pulmonary aspergillosis), especially in immuno-compromised

subjects. Many fungal species have also been described as producers

of type I allergens (IgE binding allergens), and IgE sensitization

to common outdoor and indoor fungal genera like Alternaria,

Penicillium, Aspergillus and Cladosporium spp. is strongly

associated with allergic respiratory disease, especially asthma

(Halonen et al., 1997; Ostro et al., 2001). However, there is very

little evidence that supports an important role for type I allergy

to fungi in occupational respiratory disease. Fungi are also a

source of ß(1 3)-glucans which are suspected to cause non-allergic

respiratory symptoms (see below).

Most bacteria or bacterial agents are not very potent allergens,

with the exception of the spore-forming actinomycetes described

above. Bacterial cell wall components, such as endotoxin (present

only in Gram-negative bacteria; see below) and peptidoglycans (most

prevalent in Gram-positive bacteria), are agents with important pro-

inflammatory properties that may induce respiratory symptoms. The

effects of peptidoglycans are assumed to be very similar to those

observed with endotoxin exposure; however, this has not been

systematically studied.

Finally, bacteria and fungi include a number of well-known

pathogenic infectious microorganisms that may after inhalation cause

specific diseases as described above.

Endotoxin

Endotoxin is composed of lipopolysaccharides (LPS) and is a non-

allergenic cell wall component of Gram-negative bacteria with strong

pro-inflammatory properties. It is commonly present in many

occupational environments (Table 1) but also in the general

environment, and particularly in house dust ( et al., 1964;

Douwes et al., 2000b)

Endotoxin has been recognized as an important factor in the

aetiology of occupational lung diseases including (non-allergic)

asthma (Douwes and Heederik, 1997) and ODTS (see above). Subjects

exposed to endotoxin in inhalation experiments experience clinical

effects such as fever, shivering, arthralgia, influenza-like

symptoms (malaise), blood leukocytosis, neutrophilic airway

inflammation, asthma symptoms such as dry cough, dyspnea and chest

tightness, bronchial obstruction, as well as dose-dependent lung

function impairment (FVC, FEV1 and flow-volume variables) and

decreased lung diffusion capacity (Pernis et al., 1961; Castellan et

al., 1987; Michel et al., 1992, 1996, 1997; Clapp et al., 1994;

Jagielo et al., 1996; Michel, 1997; Thorn and Rylander et al.,

1998b). Many occupational studies have shown positive associations

between endotoxin exposure and health effects including both

reversible (asthma) and chronic airway obstruction, respiratory

symptoms (symptoms of asthma, bronchitis and byssinosis) and

increased airway responsiveness (Table 2). This was consistently

observed in a large variety of occupational environments (listed in

Table 2) characterized by different exposure levels and different

compositions of the bioaerosol exposures (Kennedy et al., 1987;

Milton et al., 1996; Douwes and Heederik, 1997). Several of these

studies reported clear exposure–response relationships (Smid et al.,

1992; Vogelzang et al., 1998). One study in the potato processing

industry showed that acute airway obstruction was already apparent

at levels of 50 endotoxin units (EU)/m3 ( 5 ng/m3) (Zock et al.,

1998). Subjects with increased bronchial hyper-responsiveness and/or

asthma are more sensitive to develop symptoms (Michel et al., 1989)

but interestingly large differences in airway responsiveness to

inhaled endotoxin also exist in healthy (non-allergic) subjects

suggesting that potentially only susceptible individuals are at risk

(Kline et al., 1999). Several studies in the indoor environment have

suggested a causal association between endotoxin and asthma

exacerbation in children and adults (Michel et al., 1996; Park et

al., 2001).

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Table 2. Work-related health effects of bacterial endotoxin

and various occupational environments in which those effects have

been demonstrated

ß(1 3)-glucans

ß(1 3)-glucans are glucose polymers with variable molecular weight

and degree of branching (, 1997) that originate from most

fungi, some bacteria, most higher plants and many lower plants

(Stone and e, 1992).

Results of several studies in which subjects were exposed to

airborne ß(1 3)-glucans suggest that these agents play a role in

bioaerosol-induced inflammatory responses and resulting respiratory

symptoms (Rylander et al., 1992; Fogelmark et al., 1994; Rylander,

1996). In a small group of garbage workers only minor inflammatory

responses in the nasal mucosa were demonstrated after nasal

instillation with ß(1 3)-glucan (Sigsgaard et al., 2000). However,

in a whole blood assay measuring cytokine release after in vitro

exposure to high concentrations of ß(1 3)-glucans, a significant

increase in all measured cytokines (TNF , IL-1ß, IL-6 and IL-8) was

found (Sigsgaard et al., 2000), thus demonstrating the pro-

inflammatory potential of ß(1 3)-glucans. Several, mostly small,

field studies have been performed in the home environment, day care

centres, an office building, schools and among household waste

collectors and paper mill workers, suggesting a relation with

respiratory symptoms, airway inflammation, lung function and atopy

in exposed individuals (Rylander et al., 1994, 1998, 1999; Rylander,

1997a,b; Thorn and Rylander, 1998a,b; Wan and Li, 1999). A recent

study in The Netherlands showed an association between peak flow

variability and ß(1 3)-glucan levels in house dust among children (n

= 159) with respiratory symptoms (Douwes et al., 2000b). Animal

studies showed that ß(1 3)-glucan may act synergistically with

endotoxin in causing airway inflammation (Fogelmark et al., 1992,

1994), and it was further suggested that ß(1 3)-glucan may enhance

the production of specific IgE. However, the results of those

studies were mixed (Rylander and Holt, 1998; Wan and Li et al.,

1999; Fogelmark et al., 2001). Health effects of ß(1 3)-glucan

exposure in the occupational environment thus seem plausible but the

evidence is currently still weak since most studies were small and

not always appropriately controlled for other potential causal

exposures.

Mycotoxins

Mycotoxins or fungal toxins are low molecular weight biomolecules

produced by fungi that are toxic to both animals and humans. Some

(e.g. aflatoxin from Aspergillus) can be potent carcinogens (see

above). Numerous other mycotoxins have been classified ( et

al., 1983; Krough, 1984) possessing distinct chemical structures and

reactive functional groups, including primary and secondary amines,

hydroxyl or phenolic groups, lactams, carboxylic acids and amides.

Very little is known about occupational airborne exposures to

mycotoxins and respiratory health effects. Mycotoxins of Fusarium,

Aspergillus and Penicillium genera are known to be present in the

inhalable fraction of airborne corn dust (Sorenson, 1990), cotton

dust (Salvaggio et al., 1986) and grain dust (Lacey et al., 1994).

It is not clear, however, whether these components contribute to the

frequently reported respiratory symptoms in the cotton and grain

industries.

Allergens

Allergens can comprise a large variety of macromolecular structures

ranging from low (mainly chemicals such as di-isocyanates) to high

molecular weight sensitizers, which are most often proteins of

biological origin. Most potent occupational IgE binding allergens

include enzymes derived from fungi and bacteria produced by

biotechnological companies for use in, for example, washing powders

and both the human and animal food industries (Sandiford et al.,

1994; Schweigert et al., 2000; Cullinan et al., 2000, 2001).

Populations at risk are therefore not only workers in the enzyme

producing industries, but also those workers in for instance food

processing industries where enzyme preparations are used. Other well-

known IgE binding allergens are plant pollens, which may cause

allergies in greenhouse workers (van der Zee et al., 1999). Latex

allergens have received extensive attention during the last decade

with high numbers of health and hospital workers being sensitized

due to the use of latex gloves produced from sap from the rubber

tree Hevea brasiliensis (Turjanmaa et al., 1996). Finally, several

animal proteins (dust mite, cat, mouse and rat allergens) are known

to have strong allergenic properties. In particular, it is well

established that laboratory animal workers are at risk of developing

occupational type I (IgE-mediated) allergy to mice and rat allergens

(Cullinan et al., 1999). In addition to IgE binding allergens

workers may be exposed to IgG binding allergens. These allergens are

assumed to be involved in the pathogenesis of HP or farmer's lung

and are produced by moulds and actinomycetes (see above). However,

although an allergic immune response is suspected in HP the exact

pathology is not known and other mechanisms are assumed to play a

role as well.

PROTECTIVE EFFECTS OF MICROBIAL EXPOSURE?

TOP

ABSTRACT

INTRODUCTION

HEALTH EFFECTS

SPECIFIC EXPOSURES

PROTECTIVE EFFECTS OF MICROBIAL...

EXPOSURE ASSESSMENT

ASSESSMENT METHODS FOR...

ASSESSMENT METHODS FOR MICROBIAL...

ASSESSMENT METHODS FOR BIO...

BIOMARKERS OF EXPOSURE

STANDARD SETTING

RESEARCH NEEDS

CONCLUSIONS

REFERENCES

In recent years the `hygiene hypothesis' has shifted attention from

the adverse health effects to the potential beneficial effects of

microbial agents (ez, 1999). This postulates that growing up

in a more hygienic environment may enhance atopic (Th2) immune

responses (Holt et al., 1997; ez, 1999). It is believed that

microbial exposures, and particularly exposure to endotoxin early in

life, may protect from developing atopy and allergic asthma;

however, mechanisms are not well understood (Douwes et al., 2002).

This potential protective effect is remarkable since it is well

known from occupational studies that these agents may cause non-

allergic respiratory symptoms (see above). Some animal and in vitro

work (Tulic et al., 2000) and some population studies (Gereda et

al., 2000; Braun-Fahrlander et al., 2002) appear to support this

theory. In addition, several studies in farmers' children have shown

that growing up on a farm protects against atopy and asthma (Braun-

Fahrländer et al., 1999; Riedler et al., 2001). The reasons for this

are not established but one possible explanation is that high

endotoxin exposures in this environment may play a role (Von Mutius

et al., 2000). (Most of the studies in farmers' children were

conducted on small family farms that may not be representative of

large industrialized farms.) It is not clear whether these same

protective effects operate in the working population. Although

respiratory symptoms including (non-allergic) asthma are generally

more prevalent in working populations exposed to high levels of

microorganisms and endotoxins such as, for example, farmers

(Anonymous, 1998), some studies indicate that atopy is less

prevalent (Van Hage-Hamsten et al., 1987; Iversen and Pedersen,

1990) thus suggesting that these agents may protect from atopy and

atopic symptoms also in the working population. However, this

lowered prevalence might be the result of exposures in childhood

that are sustained up to adult age, although studies among farmers'

apprentices with an urban background suggest otherwise (Portengen et

al., 2002). Few studies have addressed this important area and the

results are inconclusive, studies in working populations without an

exposure history to endotoxins in childhood are therefore urgently

needed.

EXPOSURE ASSESSMENT

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ABSTRACT

INTRODUCTION

HEALTH EFFECTS

SPECIFIC EXPOSURES

PROTECTIVE EFFECTS OF MICROBIAL...

EXPOSURE ASSESSMENT

ASSESSMENT METHODS FOR...

ASSESSMENT METHODS FOR MICROBIAL...

ASSESSMENT METHODS FOR BIO...

BIOMARKERS OF EXPOSURE

STANDARD SETTING

RESEARCH NEEDS

CONCLUSIONS

REFERENCES

The assessment of exposures to bioaerosols offers distinct

challenges from those for inorganic aerosols and chemical agents.

Pathogenic microorganisms may be hazardous at extremely low levels

while other organisms may only become important health hazards at

orders of magnitude higher concentrations. Some organisms and spores

are extremely resilient while others may be easily degraded in the

sampling process. Certain fungal spores are easily identified and

counted while many bacteria are difficult to distinguish. Sensitive

and specific methods are available for the quantification of some

biological agents while there are no good methods for others. Many

of the newly developed methods [e.g. measurement of microbial agents

such as ß(1 3)-glucans or fungal extracellular polysaccharides; see

below] have not been well validated and are often not commercially

available. Even for some well-established methods (e.g. the LAL

assay to measure bacterial endotoxin; see below) significant

variations in exposure assessment between laboratories have been

demonstrated (Thorne et al., 1997; Chun et al., 2000; Reynolds et

al., 2002). Also, issues of storage and transport of bioaerosol

samples have often not been addressed whereas it is known that these

conditions may affect the activity of some biological agents, e.g.

endotoxin (Thorne et al., 1994; Douwes et al., 1995; Duchaine et

al., 2001). Finally, many biological agents that may cause health

effects are currently not identified. For instance, sewage treatment

workers have an increased risk of developing a wide range of

symptoms including respiratory, gastrointestinal and neurological

symptoms, whereas causal agents have not conclusively been

identified (Douwes et al., 2001). Thus, in order to establish a

complete picture of the bioaerosol exposure (and to appropriately

assess the risks associated with it) various exposure assessment

methods have to be explored.

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Measurement of microorganisms relies upon the collection of a sample

into or onto solid, liquid or agar media with subsequent

microscopic, microbiological, biochemical, immunochemical or

molecular biological analysis (Eduard and Heederik, 1998). Two

distinctly different approaches are being distinguished for the

evaluation of microbial exposure: `culture-based methods' and `non-

culture methods'.

Culture-based methods

Airborne exposure to microorganisms in the environment can be

studied by counting culturable propagules in air samples (or in

settled dust samples). A variety of devices for microbial bioaerosol

sampling have been developed and described previously (Eduard and

Heederik, 1998). Sampling of culturable bioaerosols is based on

impactor (microorganism are collected directly on a culture medium),

liquid impinger (microorganisms are collected in liquid collection

fluid) or air filtration methods (microorganisms are collected on a

filter). After sample collection colonies of bacteria and fungi are

grown on culture media at a defined temperature over a 3–7 day

period. Colonies are counted manually or with the aid of image

analysis techniques.

Counting of culturable microorganisms has some serious drawbacks

including poor reproducibility, selection for certain species due to

chosen culture media, temperature etc. and the fact that dead

microorganisms, cell debris and microbial components are not

detected, while they too may have toxic and/or allergenic

properties. In addition, good methods for personal air sampling of

culturable microorganisms are not available, and air sampling for a

period of more than 15 min is often not possible, whereas air

concentrations usually vary largely in time. On the other hand,

counting of culturable microorganisms is potentially a very

sensitive technique and many different species can be identified.

Traditionally used culture methods have proven to be of limited use

for quantitative exposure assessment. Culture-based techniques thus

usually provide qualitative rather than quantitative data that can,

however, be important in risk assessment, since not all fungal and

bacterial species pose the same hazard. An extensive review on

techniques for sampling and culturing microorganisms has recently

been published (Eduard and Heederik, 1998).

Non-culture methods

Non-culture-based methods enumerate organisms without regard to

viability. Sampling of non-culturable bioaerosols is generally based

on air filtration or liquid impinger methods. Microorganisms can be

stained with a fluorochrome, e.g. acridine orange, and counted with

an epifluorescence microscope (Thorne et al., 1994). Possibilities

of classifying microorganisms taxanomically are limited because

little structure can be observed. Electron microscopy (EM) or

scanning EM can also be used and allow better determination (Eduard

et al., 1988; Karlsson and Malmberg, 1989). Simple light microscopy

may be used to count microorganisms, but counting is based only on

morphological recognition, which may result in severe measurement

errors. Bacteria collected with impingers or filters can be counted

by flow cytometry after staining with 4',6-diamino-2-phenylindole

(DAPI) or by applying fluorescent in situ hybridization (FISH)

(Lange et al., 1997). FISH involves the use of fluorochrome-labelled

nucleic acid probes to target rRNA within morphologically intact

cells, allowing taxonomic determination from kingdom to species

(Lange et al., 1997).

The main advantage of microscopy or flow cytometry is that both dead

and living microorganisms are quantified, selection effects are

limited, personal air sampling is possible and sampling time can be

varied over a large range. Disadvantages include laborious and

complicated procedures, high costs per sample, unknown validity, no

detection of possibly relevant toxic or allergenic components or

cell debris, while possibilities for the determination of

microorganisms for most of these techniques are limited. A more

extensive review on microscopy and flow cytometry methods for

counting non-culturable microorganisms has recently been published

(Eduard and Heederik, 1998).

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Instead of counting culturable or non-culturable microbial

propagules, constituents or metabolites of microorganisms can be

measured as an estimate of microbial exposure. Toxic (e.g.

mycotoxins) or pro-inflammatory (e.g. endotoxin) components can be

measured but also non-toxic molecules may serve as markers of either

large groups of microorganisms or of specific microbial genera or

species. The use of advanced methods, such as polymerase chain

reaction (PCR)-based technologies and immunoassays (see below), have

opened new avenues for detection and speciation regardless of

whether the organisms are culturable. Some markers for the

assessment of fungal biomass include ergosterol measured by gas

chromatography–mass spectrometry (GC-MS) ( and Young, 1997) or

fungal extracellular polysaccharides measured with specific enzyme

immunoassays (Douwes et al., 1999), allowing partial identification

of the mould genera present. Volatile organic compounds produced by

fungi may be suitable markers of fungal growth (Dillon et al.,

1996). Other agents such as ß(1 3)-glucans (Aketagawa et al., 1993;

Douwes et al., 1996) and bacterial endotoxin are being measured

because of their toxic potency. Endotoxin is measured by using a

Limulus amoebocyte lysate (LAL) test prepared from blood cells of

the horseshoe crab, Limulus polyphemus (Bang, 1956). Analytical

chemistry methods for quantification of LPS have also been developed

employing GC-MS (Sonesson et al., 1988, 1990). However, these

methods require special LPS extraction procedures and have not been

widely used. Two methods to measure ß(1 3)-glucans have been

described, one of which is based on the LAL assay (Aketagawa et al.,

1993) and the other on an enzyme immunoassay (Douwes et al., 1996).

Finally, PCR techniques have been developed for the identification

and quantitation of specific species of bacteria and fungi in the

air (Alvarez et al., 1994; Khan and Cerniglia, 1994). PCR allows

amplification of small quantities of target DNA, typically by 106–

1010 times, to determine in a qualitative manner the presence of

specific microorganisms. Application of quantitative PCR for

analysis of air samples containing microorganisms is still under

development but is expected to find applications in situations where

specific infectious microorganisms may be present. Table 3 gives an

overview of assessment methods for constituents of microorganisms.

View this table:

[in this window]

[in a new window]

Table 3. Assessment methods for constituents of

microorganisms in bioaerosol samples

Most of the methods to measure microbial constituents (with the

exception of the method to measure bacterial endotoxin) are in an

experimental phase and have as yet not been routinely applied and/or

are not commercially available. Important advantages of these

methods include: (i) the stability of most of the measured

components, allowing longer sampling times for airborne

measurements, and frozen storage of samples prior to analysis; (ii)

the use of standards in most of these methods; (iii) the enhanced

possibility to test for reproducibility.

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Antibody-based immunoassays, particularly enzyme-linked

immunosorbent assays (ELISA) are widely used for the measurement of

aeroallergens and allergens in settled dust in buildings. To date,

the house dust mite allergens, Der p I, Der f I and Der p/f II, have

been most widely investigated and the methods have been well

described (Luczynska et al., 1989; Price et al., 1990; Leaderer et

al., 2002). Methods for assessment of exposure to allergens from

animals (Swanson et al., 1985; Virtanen et al., 1986; Schou et al.,

1991; Hollander et al., 1997), cockroaches (Pollart et al., 1991),

storage mites (Iversen and Pedersen, 1990) and latex rubber (

et al., 1996) have also been published. Assays are also available

for the measurement of bio-technologically produced allergens such

as fungal -amylase (Houba et al., 1997).

For bioaerosols very few biomarkers of exposure or dose have been

identified and the validity for exposure assessment is often not

established. To our knowledge no direct methods to measure

biological agents or metabolites thereof in body fluids (blood or

urine) have been described. IgG antibodies in serum have been

suggested as an indirect marker of recent exposure to fungi (Burell

and Rylander, 1981; Eduard et al., 1992). However, little is known

about the quantitative relation between serum IgG levels and

airborne exposures. Therefore, IgG levels as a proxy of exposure or

dose should be interpreted with caution.

IgE and inflammatory markers in blood, sputum, nasal lavage and

exhaled breath condensate have been suggested as biomarkers of

exposure, but these are more appropriately addressed as markers (or

intermediates) of effect since they play a major role in the

pathophysiological events leading to symptoms and disabling disease.

Therefore these should not be considered markers of exposure.

Although health risks associated with bioaerosols have been

identified, exposure–response relationships have been described only

for a few agents, often with dust exposure as the proxy of exposure

to the aetiological agent. `No effect levels' (NEL) are therefore

available only for some well-identified exposures. For some agents

such as wood dust, standards have been adopted in several countries

[e.g. 5 mg/m3 in the USA and 2 mg/m3 in The Netherlands, based on 8

h time weighted averages (8-TWA) of inhalable dust]. In a recent

literature review by Demers et al. (1997) a standard of 1 mg/m3 for

softwoods was suggested to protect workers from non-malignant

effects. The American Conference of Governmental Industrial

Hygienists (ACGIH) has established a `threshold limit value' (TLV)

for 8-TWA of 4 mg/m3 total grain dust (wheat, oats, barley) since

1980 (ACGIH, 1980). The Ad Hoc Committee on Grain Dust of the

Canadian Thoracic Society Standards Committee considered

a `permissible exposure level' (PEL) of 5 mg/m3 advisable to control

short-term effects, even if these effects are transient (Becklake et

al., 1996).

For endotoxin `no observed effect levels' (NOEL) for various health

endpoints have been reported in the literature ranging from 50 to

several hundred EU/m3 (Douwes and Heederik, 1997; Rylander, 1997c).

A health-based exposure limit has been proposed in The Netherlands

by the Dutch Health Council of 50 EU/m3 (8-TWA) (Dutch Expert

Committee on Occupational Standards, 1998). The Minister of Social

Affairs is now considering adopting a legally binding limit of 200

EU/m3 since a limit of 50 EU/m3 was found not to be feasible because

of economic effects for some sectors of the industry. Since

differences in storage, extraction and analysis of endotoxin samples

may result in large differences in exposure estimates (Hollander et

al., 1993; Douwes et al., 1995; Thorne et al., 1997, 2003; Duchaine

et al., 2001) it was decided to adopt the European Standardization

Organisation (CEN) draft protocol for measurement of endotoxin (CEN,

2001). However, the CEN protocol does not describe extraction and

measurement procedures very specifically, thus potentially resulting

in significant variations in exposure assessment between

laboratories. Several international round robin tests have been

conducted showing good correlations between laboratories but

significant differences in absolute levels (Chun et al., 2000;

Reynolds et al., 2002). Therefore for standard setting purposes

further validation and standardization of sampling, extraction and

analytical procedures are urgently needed.

The possibility of establishing exposure limits for allergen

concentrations in the air has only been explored in some isolated

cases. Subtilisins are bacterial enzymes usually produced from

Bacillus subtilis and used in detergents. They are well-recognized

respiratory sensitizers, and a TLV of 60 ng/m3 (ceiling

concentration) for workplace airborne exposure has been adopted by

the ACGIH (2002). However, there is considerable doubt about the

underpinning of this TLV, and the proposed value rationale for the

TLV seems determined mainly by analytical limitations, i.e. by the

detection limits of some of the earlier methods for exposure

measurements. ACGIH (2002) now lists subtilisins as a substance

under study. The Health and Safety Executive (HSE) in the UK is

proposing to withdraw the British limit (OES, 60 ng/m3, TWA), which

is based on the ACGIH TLV, because no safe exposure limit for

subtilisins could be identified. The ACGIH has also adopted an

exposure standard of 0.5 mg/m3 inhalable flour dust (8-TWA) based on

the published exposure–response relationship for wheat allergens

(ACGIH, 1990). For several other allergens, exposure–response

relationships have now been established (Heederik et al., 1999).

This has been made possible due to use of newly developed

immunoassays to measure the allergens directly instead of crude

exposure proxies such as dust levels. It is to be expected that

these relationships will be used for the development of exposure

standards. However, before this is possible, standardization of

immunoassays for measurement of allergens is urgently required.

RESEARCH NEEDS

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REFERENCES

As described above, bioaerosol exposure is associated with a large

variety of symptoms and diseases. However, it is often not clear

which agents are primarily involved and even for known pathogenic

agents clear dose–response relationships have not been established.

This is mainly due to a lack of valid methods to assess exposure

quantitatively. Thus, there is a clear need for method development,

particularly based on non-culture techniques since culture methods

have been proven to be of limited use in population-based studies.

Secondly, existing methods need rigorous validation and subsequent

further development to make them more suitable for large-scale

epidemiological studies or for industrial hygiene purposes.

Validation of methods is particularly needed for those agents where

occupational exposure limits have been established (e.g. endotoxin,

allergens) resulting in internationally accepted protocols that

should include concise and uniform guidelines on sampling, storage,

extraction and analytical procedures. Recurrence of infectious

diseases and the recent threat of bio-terrorism have accelerated the

development of measurement methods for specific microorganisms using

DNA-based technology. Moreover, a clear need is developing for rapid

and direct reading assays for bioaerosols for immediate evaluation

of the presence of health risks. This is a new development that will

certainly have an important spin-off for the occupational and

environmental health fields. Application of new and better methods

will allow a more valid risk assessment for bioaerosols and

individual components thereof.

Other important areas that require further research include: (i) the

potential protective effect of endotoxin and other microbial agents

on atopy and atopic diseases, for which studies should address the

issue of timing and dose of endotoxin exposure with respect to both

these potentially protective effects and adverse non-atopic health

effects; (ii) the shape of exposure–response relationships for

allergens and development of tolerance; (iii) the issue of

individual susceptibility for allergens, endotoxin and other

biological exposures; (iv) the interaction effects between various

allergenic and non-allergenic agents in causing health effects; (v)

the identification of other biological agents that may cause adverse

(or protective) health effects; (vi) more research into other health

effects (e.g. skin conditions, neurological symptoms, pre-term

births or late abortions) and exposure routes (e.g. skin, gastro-

intestinal system).

CONCLUSIONS

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CONCLUSIONS

REFERENCES

Potential health effects of bioaerosol exposures are diverse

including infectious diseases, acute toxic effects, allergies and

cancer. Methods to assess bioaerosol exposures are available;

however, selection of the most appropriate method(s) is highly

dependent on the specific goals of the study. Most culture methods

provide important qualitative information but are at best only semi-

quantitative and they have proven to be of limited use in population-

based studies. Several non-culture methods have been developed with

promising results in epidemiological studies, however, the

experience with those new assays is still limited and they are

generally not widely available. Even some of the more established

methods to measure specific biological agents (e.g. endotoxin with a

LAL assay or allergens with an enzyme immunoassay) are only poorly

validated. Therefore, interpretation of exposure results is

impossible without detailed information about the sampling and

analytical procedures. Thus, due to large uncertainties in exposure

assessment (because of poorly developed quantitative exposure

assessment tools) risk assessment is complicated, hampering legal

exposure limits being developed (with the exception of a few

specific components such as specific allergens and endotoxin).

Therefore, more research is needed to establish better exposure

assessment tools and to validate newly developed methods.

Acknowledgements—J.D. is supported by a research fellowship from the

Netherlands Organization for Scientific Research (NWO). P.T. is

supported by NIEHS P30 ES05605. N.P. is supported by the New Zealand

Health Research Council.

FOOTNOTES

* Author to whom correspondence should be addressed. IRAS, PO Box

80176, 3508 TD, Utrecht, The Netherlands. Tel: +31-30-2535400; fax:

+31-30-2535077; e-mail: j.douwes@...

REFERENCES

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REFERENCES

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