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Microbial exposure and respiratory dysfunction in poultry hatchery workers ¨ckel* Elena Martin, Solveig Ernst, Gabriele Lotz, Gunter Linsel and Udo Ja Today's modern animal confinement with high stocking density of a single species has resulted in new workplaces that are rarely characterised in regard to microbial exposure. In this study we determine the personal microbial exposure by long term monitoring in a duck hatchery. Four hatchery workers were accompanied for four weeks and on every working day personal bioaerosol sampling and lung function tests were performed. Quantitative and qualitative molecular methods were used for analysing
Received 12th September 2012 Accepted 1st December 2012 DOI: 10.1039/c2em30758h rsc.li/process-impacts
bioaerosol samples. Restriction Fragment Length Polymorphism (RFLP) analyses showed a unique microbial exposure on eclosion days. By 16S rRNA gene sequence cloning analysis we detected Staphylococcus, Acinetobacter and Enterococcus as predominant bacterial genera. Ducklings' down was identified as a medium for bacterial contamination. Furthermore on eclosion days the four workers showed a decline in lung function over their working shift causing an average FEV1 decrease.
Environmental impact This study presents health risks of occupational exposure for workers in a duck hatchery measured by a long term monitoring. Quantitative and qualitative analyses of the airborne microorganisms in the inhalable dust fraction indicate that the workers are on eclosion days permanently exposed to a high work related microbial exposure level (WoRMEL). The study design could be an example for a comprehensive analysis of the work related microbial exposure level in agricultural environments.
Introduction Exposure to agricultural organic dust containing e.g. bacteria and fungi and animal allergens may cause a variety of lung reactions and diseases, such as Organic Dust Toxic Syndrome (ODTS), chronic bronchitis, and Extrinsic Allergic Alveolitis (EAA).1 In several epidemiological studies it has been shown that severe chronic respiratory diseases and respiratory dysfunctions appear with high prevalence among workers in the agricultural environment.2–12 Since animal husbandry has changed from low density pasture-based to predominately modern animal connement at high stocking density of a single species, new workplaces have emerged that are rarely characterised. Due to the densely stocked and enclosed animal production buildings, bioaerosols can reach high exposure levels and therefore affect via inhalation the workers respiratory system.13 The term bioaerosol describes a mixture of viable and non-viable airborne particles of biological origin, such as fungal spores and hyphen, bacteria, pollen, and viruses and their fragments and byproducts. The bacterial community in the agricultural bioaerosol exposures is
Federal Institute for Occupational Safety and Health, N¨oldnerstr. 40-42, 10317 Berlin, Germany. E-mail: [email protected]; Fax: +49 30 51548 4171; Tel: +49 30 51548 4788
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not characterized well enough in order to understand the etiology of respiratory diseases. Even so, the knowledge of occupational sources of common respiratory diseases is important not only for the purpose of diagnosis but also for prevention. Hence the study of compositions and dynamics of airborne microbial communities in the agricultural environment is a matter of particular interest. Little is known about the composition and dynamics of agricultural airborne microbial communities in general. Difficulties in choosing an applicable collection and detection method are reasons for the poor characterisation. Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count non-cultivable or non-viable bacteria. Consequently, the culture-dependent quantitative assessment of microorganisms is oen underestimated.14 So far molecular biological methods such uorescence microscopy and PCR may offer an appropriate alternative to study across-shi microbial exposure in bioaerosol samples collected using a personal ltration system.15 The study was carried out in the context of an ongoing study in a German duck producing enterprise, regarding assessment of exposure to airborne microorganisms as well as workers health effects in different departments from hatchery, animal husbandry to slaughterhouse, rst cross-sectional analysis
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Paper suggests a workplace-dependent effect of bioaerosol exposure on lung function impairment, with regard to hatchery workers.16 The aim of the present study was to measure changes in workers lung function and their relationship to the exposure levels to bioaerosols by long term monitoring. In a preliminary study in the same duck hatchery there have been detected high microbial exposure levels on ducklings' eclosion days.15 This had been the occasion to start studying the microbial exposure level also on those days without duckling eclosion with the intention to test the predicted lower exposure level at those days. The intent was to measure changes in workers lung function depending on the exposure level. Additionally it was of special interest to investigate if these effects persist even at lower exposure levels.
Methods For a four consecutive week period four workers in an industrial duck hatchery were observed during their work. The duck hatchery comprises different rooms like the sorting room, cleaning room and breeding rooms. During the two eclosion days a week the working comprises handling and sorting of approximately 80 000 ducklings per day. Manual sorting takes place at an assembly line in the sorting room. Here, ducklings were separated from egg shells and transferred into transport boxes. The three non-eclosion days include works like cleaning and disinfection of production machines and surfaces, egg washing and egg candling for detecting dead or unfertilized eggs in the whole hatchery. Egg washing means that the eggs were exposed to hydrogen peroxide and then washed in an automated washing system with chlorine water to remove coarse contamination like faeces and straw. Sampling of bioaerosols The personal exposure to airborne microorganisms was measured via personal air sampling (developed by Institute for Occupational Safety and Health of the German Social Accident Insurance: http://www.dguv.de/ifa/de/fac/ring/probenahme/ pgp_ueberblick/index.jsp) in February 2009.17 Therefore four workers carried each working day a ltration system (Gilian Personal Air Sampling Pump, HFS-513A, Sensidyne) for collecting the inhalable dust fraction including the airborne microorganisms on polycarbonate lters (pore size: 0.8 mm, Ø 37 mm, Whatman, Schleicher & Schuell) according to the EN 481 regulation. The ow rate was 3.5 l min1 and time of sampling was between 300 and 500 min. In parallel, every working day an outside air sample was taken. Therefore one personal air sampling system was placed stationary in the luv side of the hatchery. Study subjects Four female workers were investigated during a four week period. They were 39.5 (8.9) years old and for 40 (20) month employed at the duck hatchery. The mean height was 1.65 (0.07) m and the mean was BMI 27.4 (3.2) kg m2. They were without severe acute or chronic diseases and without
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Environmental Science: Processes & Impacts chronic respiratory symptoms. They had normal spirometric values before the beginning of the follow up. Two workers were smokers. Three workers kept a few animals for there private use. Health examination Immediately before (5.00–7.00 h) and aer work on the same day (15.00–16.00 h), the workers were examined. The examination included once in the whole survey a standardized interview regarding occupational history, smoking habits, and health history concerning in particular about respiratory diseases. Additionally, a daily questionnaire before and aer work was performed to consider acute respiratory symptoms. Pulmonary function testing with regard to vital capacity (VC) and forced expiratory volume in one second (FEV1) using a portable asthma monitor AM1 (J¨ ager/VIASYS Healthcare) was carried out by the workers themselves both immediately before and aer the work-day. Thereby, the optimum value of a triple serially conducted expiration of breath was automatically saved. Healthy unexposed men showed a slight diurnal increase in both FVC and FEV1 (about 5%),18 which was conrmed in the comprehensive study.16 No external control group was therefore used in the present study. The study was approved by the Committee on Ethics in Human Research of the Berlin General Medical Council in Germany. All workers took part voluntarily in the study. Cultivation independent approach For the cultivation independent approach polycarbonate lters were transferred to sterile plastic bags. 10 ml of cell free isotonic NaCl solutions were added into the bags and the cells were detached from the lter surface by using a Stomacher (Stomacher, 80 Biomaster, Seward, UK) at the highest level for 60 s. The quantication of airborne microorganisms was performed via total cell counting aer DAPI staining using 5 ml of cell suspension.19 The other 5 ml of the cell suspension was used for qualitative analyses with molecular methods. Therefore the cell solutions were concentrated by centrifugation (21 380g, 15 minutes). The following steps were DNA extraction and subsequent amplication of 16S rRNA genes of “all” bacteria (primers 27F, 1492R)20 as described elsewhere.19 For qualitative characterisation we applied rst the restriction fragment length polymorphism (RFLP) in combination with the chip based automated capillary electrophoresis (Biorad Experion system) and secondly 16S rRNA gene sequence clone libraries. RFLP analyses Aer amplifying the 16S rRNA gene sequence we puried the PCR products with the QIAquick PCR purication Kit (Qiagen). To determine the concentrations of the PCR products we used a Qubit Fluorometer (Invitrogen). For the restriction digest we used two FastDigest restriction endonucleases (Fermentas; Hin6I and HpaII) and each digest was performed with 300 ng PCR products of each bioaerosol sample aer manufacture's
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Environmental Science: Processes & Impacts instruction with a protocol of 11 ml H2ODEPC, 15 ml PCR product (20 ng ml1), 3 ml 10 FastDigest buffer and 1 ml FastDigest enzyme (1 FDU per ml). For the chip based automated capillary electrophoresis (Biorad Experion system) we used the DNA 1K assay (Biorad) and followed the manufacture's instruction. With this system DNA bands between 15 bp and 1500 bp could be separated and identied with an internal bp-marker. The results were obtained in an electropherogram. Cloning approach For cloning approach we used bioaerosol samples which were taken in 2007 at the same workplaces under similar conditions and the same sampling procedure. Furthermore we collected in this study the ducklings' down during one examination day (29.01.09). Therefore we collected with sterile instruments the ducklings down directly from ducklings' incubator trolley, which was disinfected at the beginning of the production process. Two replicates of DNA extraction and 16S rRNA PCR were done from ducklings' down sample. Aer 16S rRNA gene sequence amplication we did cloning approach like described elsewhere.15 For each cloning approach of a bioaerosol sample 64 clone inserts were sequenced (appr. 800 bp). For the cloning approach of ducklings' down 30 clone inserts were sequenced (appr. 800 bp). The 16S rRNA gene sequences were compared with available sequences in GenBank using BLAST (Basic local alignment search tool) provided by the NCBI (National Center for Biotechnology Information Server). Phylogenetic calculations were performed as described by Martin et al. (2010).19 Note RFLP analyses and cloning approach were done only with bioaerosol samples from eclosion days. It was not possible to get
Paper any DNA extract from bioaerosol samples from non-eclosion days because there were too little bacteria in each sample. Statistical methods The (dependent) pairwise, bidirectional t-test was performed to verify the presence of the paired value groups. P # 0.05 was regarded as the level of signicance.
Results In total, measurements were taken on 8 eclosion days and 8 non-eclosion days. Numbers (n) of individual working days (Fig. 1 and Fig. 2) could differ because of the absence of hatchery workers. Quantitative assessment of microbial exposure The total cell counts show that there are regular differences in the exposure level depending on the working step. On ducklings' eclosion days the total cell count ranges from 2.6 106 cells per m3 air to a maximum of 9.8 107 cells per m3 air. In contrast, on days without ducklings' eclosion there have always been detected moderate exposure levels ranging from 3.6 103 cells per m3 air to 9.6 104 cells per m3 air and therefore are comparable to those values detected in outside air controls (3.6 104 cells per m3). The overall mean cell count value on ducklings' eclosion days amounts to 3.0 107 cells per m3 air gained by DAPI staining (see Fig. 1). In contrast, on days without ducklings' eclosion the mean cell count value amount was 2.6 104 cells per m3 air. Despite the different positions of the workers at sorting of ducklings and even totally different tasks on non-eclosion days the mean exposure of all examined workers was quite similar on eclosion days (3.0 107 cells per m3 air) and on non-eclosion days (2.6 104 cells per m3 air), respectively.
Fig. 1 Personal microbial exposure given as total cell counts per m3 inhalable air after DAPI staining of four hatchery workers. Grey columns show the microorganisms concentration on eclosion days and white columns show the microorganisms concentration on non-eclosion days. Cell counts are given as mean values SD for each worker, n represents the number of examined individual working days.
480 | Environ. Sci.: Processes Impacts, 2013, 15, 478–484
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Environmental Science: Processes & Impacts
Fig. 2 The across shift changes in the lung function parameter FEV1 of 4 hatchery workers on work shifts with and without ducklings' eclosion. Bars show the mean values of n examined days of each worker as well as the average SD.
Lung function tests We determined a clear decrease of FEV1 across the work shis during eclosion days of 2.75% on average for the 4 workers. In contrast, on working days without ducklings' eclosion FEV1 increased on average by 0.46%, a signicant difference to eclosion days (t-test, p ¼ 0.03). Qualitative analysis of bacterial exposure The amount of extracted DNA from airborne microorganisms on non-eclosion days was below the limit for further qualitative analysis. For this reason we present qualitative results only from
eclosion days. In total, 28 bioaerosol samples from eclosion days were analysed regarding the bacterial composition. The virtual gel picture from single runs (n ¼ 28) in Fig. 3 shows unique restriction patterns from bioaerosol samples of eclosion days. Here, one band corresponds to one segment of digested 16S rRNA gene sequence PCR product. There are two size standards as an internal reference in the system with a length of 15 bp and 1500 bp. The peak threshold is $0.1 ng per segment. This RFLP method was used to show the unique pattern of the bacterial composition on eclosion days in this hatchery. Fig. 4 illustrates that there is a low variation within the microbial community on eclosion days independent of the year
Fig. 3 Virtual gel picture of single runs showing RFLP-patterns received from airborne bacterial communities of serially taken hatchery bioaerosol samples on ducklings' eclosion days (2008). Restrictions were performed by Hin6I digestion (FastDigest enzymes, Fermentas) of 16S rRNA gene PCR products generated from DNAextracts. Ladder is between 15 and 1500 bp.
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Paper
Fig. 4 Percentage distribution of genera detected via the 16S rRNA gene sequence cloning approach. One column represents one clone library with n sequenced clone inserts. Three independent clone libraries each (a, b and c) from personal and stationary sampling of hatcheries' air were generated. The personal bioaerosol sampling was done in 2007 and stationary sampling in 2008.15 Ducklings' down were collected in 2009 on one eclosion day and were used to generate two clone libraries (I and II).
of sampling. The dominant bacterial genus is Staphylococcus. At the species level mainly risk group 2 bacteria (TRBA 466) were detected (Staphylococcus aureus, Acinetobacter baumannii, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Staphylococcus intermedius (Martin and J¨ ackel, 2011).15 With the Good's coverage as a non-parametric estimator the proportion of phylotypes in a library of innite size could be estimated.21 The coverage between the clone libraries generated from personal bioaerosol sampling was between 93 and 97% and between the clone libraries generated from stationary bioaerosol sampling was between 97 and 98%.
Discussion Previous cross-sectional epidemiologic studies indicate that work in poultry farms is associated with the development of acute and chronic respiratory symptoms and changes of lung function.2,3,5,6,10,12,22,23 For example it has been shown that chicken catchers had signicant decrements over a work shi in forced expiratory volume in 1 s (3.4%) compared to reference groups and therefore are at high risk of respiratory dysfunction.6 In our present study we investigated with the help of a medical– molecular biological-interdisciplinary approach if there is a relationship between changes in lung function and the personal microbial exposure level of duck hatchery workers. Therefore we measured daily the personal microbial exposure level and the diurnal FEV1 change as an indicator of lung function in a one month study. In this study it was shown that the across shi changes of lung function of duck hatchery workers is observed when the microbial exposure level is high like on ducklings' eclosion days. On these eclosion days there has always been detected a high microbial air contamination. The average cell count value on those days amounts to 3.0 107 cells per m3 air gained by DAPI staining. These exposure values are similar to those taken at different working places of a duck farm24 and other working environments like compost facilities.25 The detection of the total cell count on non-eclosion days was with an average of 2.6 104 cells per m3 air, which is above the 482 | Environ. Sci.: Processes Impacts, 2013, 15, 478–484
detection limit of the DAPI staining method (103 cells per m3 air). The quantitative exposure on non-eclosion days is comparable to the detected outside background levels (3.6 104 cells per m3). These values are comparable to those found by Klug and J¨ ackel (2012)24 in their investigation on background levels in the outside air. On eclosion days the four workers showed on average a decline in lung function over their working day causing an average FEV1 decrease of 2.75% independent of whether they are smokers or not. In contrast to non-eclosion days an increase in the mean of diurnal lung function by 0.46% was detected, which might be connected to the fact that the airborne cell content in the hatchery on those days without ducklings eclosion is nearly identical to normal cell count values found in outside air controls. This corresponds to previously described changes in diurnal lung function of healthy men showing circadian FEV1 increases of about 5%.18,26 Previous studies have shown that increased daily and annual loss in lung function can be related to exposure to organic dust and endotoxins in farming.23,27 We conclude that diurnal lung function changes of duck hatchery workers are related to high microbial exposure levels because a decrease in lung function specically occurs on those days with high microbial exposure. But it is difficult to determine whether the adverse effect on workers lung function is due to the microbial exposure directly or if the microbial exposure acts as an indicator of the complex burden of bioaerosols on workplaces in agriculture.28 As shown in a previous study in the hatchery there was found high dust and endotoxin concentrations on eclosion days, too.16 Smid et al. (1994)29 reported in their study that lung function changes could be related to endotoxin exposure which indicates that endotoxin may lead to short-term lung function (FEV1) decrease, too. In this study we analysed only the bacterial composition to get an insight into the source of endotoxins. As shown in Fig. 4 the sources for airborne endotoxins seem to be Acinetobacter, Pseudomonas, Klebsiella, Janthinobacterium, Enterobacter, and Achromobacter. Furthermore we exclude an effect on decline of lung function caused by airborne fungi because the previous study indicated low amounts of them in the air of the hatchery.16
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Paper To our knowledge this study shows for the rst time a decline in lung function in hatchery workers using a daily lung function protocol. Therefore our results demonstrate that the used study design allows for an examination of only a small group of probands. The results of this study invite us to repeat these analyses at further hatchery working places to verify our observations. Qualitative analyses of bacterial community were done by RFLP and cloning approaches. On days without ducklings' eclosion there could never be generated a DNA extract and therefore no 16S rRNA gene amplicons out of the bioaerosol samples although bacteria universal primers were used. For future analysis of the bacterial diversity on non-eclosion days by culture independent methods the sampling volume should be increased or another sampling system for these days should be proven. As RFLP-analysis of 16S gene sequence amplicons revealed, the bacterial exposition pattern is uniform over the whole survey on ducklings' eclosion days. Low bacterial diversity and a missing dynamics are main characteristics of an extreme microhabitat marked by a low water content in the air (data not shown) and additionally poor growth conditions due to regular cleaning and disinfection of all production surfaces. Further detailed bacteriological investigation of six independent clone libraries based on 16S rRNA genes from bioaerosol samples revealed Staphylococcus, Enterococcus and Acinetobacter as the predominant genera in the hatchery airborne bacterial community. The abundance of Enterococcus and Acinetobacter in the air of hatcheries was found in earlier studies.30,31 Furthermore the three abundant genera Staphylococcus, Enterococcus and Acinetobacter were also detected as abundant genera by cultivation dependent and independent methods in the preliminary study.15 The main part of the three independent clone libraries from stationary bioaerosol sampling show on a 97% sequence similarity level on species section where 56% of all successfully sequenced clones show high sequence similarity to the 16S rRNA gene sequence of Staphylococcus aureus. This is the only abundant species which was not detected by personal sampling in the year 2007. We cannot point out whether this is due the different year of sampling or different sampling measurements. Following 9% of gained 16S rRNA sequences are closely related to the 16S rRNA gene sequence of Enterococcus spp., whereas half of the sequences were next similar to the type strain sequence of Enterococcus gallinarum and the other half was next similar to the type strain sequence of Enterococcus faecalis. Additional 10% of the sequenced clones show a high identity with the sequence of Acinetobacter baumannii. All together, the main part of the achieved clone sequences belong to bacteria species which are characterised as risk 2 bacteria according to the Directive 2000/54 EC32 and the German safety classication TRBA 466 (ref. 33) which means those organisms “that can cause human disease and might be a hazard to workers; they are unlikely to spread to the community; there is usually effective prophylaxis or treatment available” (Directive 2000/54 EC).32 In particular Acinetobacter baumannii seems to be a high risk for occupational health as it has been already
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Environmental Science: Processes & Impacts described as a causative organism for nosocomial bacteremia.34 The coverage calculation showed that 93–98% of the abundant bacteria were detected. We can assume from the RFLP and cloning results that on eclosion days the workers are permanently exposed to these detected bacteria. To gure out from where the bacterial air contamination in the hatchery has derived, ducklings' down samples were taken directly from the ducklings' incubator trolley, which was disinfected at the beginning of the production process. As a matter of fact we received identical patterns from DNA-extracts of ducklings' down, which uncovers them as the bacteria carrier. Furthermore the cloning approach from ducklings' down shows a nearly identical bacterial genera distribution like the cloning results from bioaerosol sampling. Anyway it remains unclear from where the bacterial contamination hit into the egg or into the down of the freshly hatched ducklings. Potentially, the unsterile water which is used in the brood incubator for humidity regulation could be the vehicle for contamination (and therefore possibly contaminate the ducklings' via an established bacterial biolm in the water system). The optimal temperature conditions combined with high humidity in the ducklings' incubator could be due to the excessive bacterial growth in their down. Nevertheless, as rst origin of bacterial contamination it seems to be that the microora of the egg shell surface itself is the most presumably source for bacterial invasion. This has likewise been shown that e.g. hens eggs are covered with bacteria strains such as Staphylococcus warneri, Acinetobacter baumannii, Alcaligenes sp., Serratia marcescens, Carnobacterium sp., Pseudomonas sp. and Salmonella enteritidis.35 Hence it can be assumed that the egg surface disinfection process with chlorine water and H2O2 gassing is not sufficient to eliminate all living bacteria or that the identied predominant bacteria could be exceptionally notably resistant towards the washing and gassing process.
Conclusion These quantitative and qualitative results conrm the results from the preliminary study in the same duck hatchery.15 Workers are permanently exposed to a high load of airborne biological agents during handling of ducklings. We recommend optimising the technical and personal protection measures for this kind of work, e.g. improved ventilation and breathing protection, respectively, in order to prevent the workers from continuous respiratory dysfunction or acute respiratory chronic diseases in future.
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Microbial exposure and respiratory dysfunction in poultry hatchery workers ¨ckel* Elena Martin, Solveig Ernst, Gabriele Lotz, Gunter Linsel and Udo Ja Today's modern animal confinement with high stocking density of a single species has resulted in new workplaces that are rarely characterised in regard to microbial exposure. In this study we determine the personal microbial exposure by long term monitoring in a duck hatchery. Four hatchery workers were accompanied for four weeks and on every working day personal bioaerosol sampling and lung function tests were performed. Quantitative and qualitative molecular methods were used for analysing
Received 12th September 2012 Accepted 1st December 2012 DOI: 10.1039/c2em30758h rsc.li/process-impacts
bioaerosol samples. Restriction Fragment Length Polymorphism (RFLP) analyses showed a unique microbial exposure on eclosion days. By 16S rRNA gene sequence cloning analysis we detected Staphylococcus, Acinetobacter and Enterococcus as predominant bacterial genera. Ducklings' down was identified as a medium for bacterial contamination. Furthermore on eclosion days the four workers showed a decline in lung function over their working shift causing an average FEV1 decrease.
Environmental impact This study presents health risks of occupational exposure for workers in a duck hatchery measured by a long term monitoring. Quantitative and qualitative analyses of the airborne microorganisms in the inhalable dust fraction indicate that the workers are on eclosion days permanently exposed to a high work related microbial exposure level (WoRMEL). The study design could be an example for a comprehensive analysis of the work related microbial exposure level in agricultural environments.
Introduction Exposure to agricultural organic dust containing e.g. bacteria and fungi and animal allergens may cause a variety of lung reactions and diseases, such as Organic Dust Toxic Syndrome (ODTS), chronic bronchitis, and Extrinsic Allergic Alveolitis (EAA).1 In several epidemiological studies it has been shown that severe chronic respiratory diseases and respiratory dysfunctions appear with high prevalence among workers in the agricultural environment.2–12 Since animal husbandry has changed from low density pasture-based to predominately modern animal connement at high stocking density of a single species, new workplaces have emerged that are rarely characterised. Due to the densely stocked and enclosed animal production buildings, bioaerosols can reach high exposure levels and therefore affect via inhalation the workers respiratory system.13 The term bioaerosol describes a mixture of viable and non-viable airborne particles of biological origin, such as fungal spores and hyphen, bacteria, pollen, and viruses and their fragments and byproducts. The bacterial community in the agricultural bioaerosol exposures is
Federal Institute for Occupational Safety and Health, N¨oldnerstr. 40-42, 10317 Berlin, Germany. E-mail: [email protected]; Fax: +49 30 51548 4171; Tel: +49 30 51548 4788
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not characterized well enough in order to understand the etiology of respiratory diseases. Even so, the knowledge of occupational sources of common respiratory diseases is important not only for the purpose of diagnosis but also for prevention. Hence the study of compositions and dynamics of airborne microbial communities in the agricultural environment is a matter of particular interest. Little is known about the composition and dynamics of agricultural airborne microbial communities in general. Difficulties in choosing an applicable collection and detection method are reasons for the poor characterisation. Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count non-cultivable or non-viable bacteria. Consequently, the culture-dependent quantitative assessment of microorganisms is oen underestimated.14 So far molecular biological methods such uorescence microscopy and PCR may offer an appropriate alternative to study across-shi microbial exposure in bioaerosol samples collected using a personal ltration system.15 The study was carried out in the context of an ongoing study in a German duck producing enterprise, regarding assessment of exposure to airborne microorganisms as well as workers health effects in different departments from hatchery, animal husbandry to slaughterhouse, rst cross-sectional analysis
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Paper suggests a workplace-dependent effect of bioaerosol exposure on lung function impairment, with regard to hatchery workers.16 The aim of the present study was to measure changes in workers lung function and their relationship to the exposure levels to bioaerosols by long term monitoring. In a preliminary study in the same duck hatchery there have been detected high microbial exposure levels on ducklings' eclosion days.15 This had been the occasion to start studying the microbial exposure level also on those days without duckling eclosion with the intention to test the predicted lower exposure level at those days. The intent was to measure changes in workers lung function depending on the exposure level. Additionally it was of special interest to investigate if these effects persist even at lower exposure levels.
Methods For a four consecutive week period four workers in an industrial duck hatchery were observed during their work. The duck hatchery comprises different rooms like the sorting room, cleaning room and breeding rooms. During the two eclosion days a week the working comprises handling and sorting of approximately 80 000 ducklings per day. Manual sorting takes place at an assembly line in the sorting room. Here, ducklings were separated from egg shells and transferred into transport boxes. The three non-eclosion days include works like cleaning and disinfection of production machines and surfaces, egg washing and egg candling for detecting dead or unfertilized eggs in the whole hatchery. Egg washing means that the eggs were exposed to hydrogen peroxide and then washed in an automated washing system with chlorine water to remove coarse contamination like faeces and straw. Sampling of bioaerosols The personal exposure to airborne microorganisms was measured via personal air sampling (developed by Institute for Occupational Safety and Health of the German Social Accident Insurance: http://www.dguv.de/ifa/de/fac/ring/probenahme/ pgp_ueberblick/index.jsp) in February 2009.17 Therefore four workers carried each working day a ltration system (Gilian Personal Air Sampling Pump, HFS-513A, Sensidyne) for collecting the inhalable dust fraction including the airborne microorganisms on polycarbonate lters (pore size: 0.8 mm, Ø 37 mm, Whatman, Schleicher & Schuell) according to the EN 481 regulation. The ow rate was 3.5 l min1 and time of sampling was between 300 and 500 min. In parallel, every working day an outside air sample was taken. Therefore one personal air sampling system was placed stationary in the luv side of the hatchery. Study subjects Four female workers were investigated during a four week period. They were 39.5 (8.9) years old and for 40 (20) month employed at the duck hatchery. The mean height was 1.65 (0.07) m and the mean was BMI 27.4 (3.2) kg m2. They were without severe acute or chronic diseases and without
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Environmental Science: Processes & Impacts chronic respiratory symptoms. They had normal spirometric values before the beginning of the follow up. Two workers were smokers. Three workers kept a few animals for there private use. Health examination Immediately before (5.00–7.00 h) and aer work on the same day (15.00–16.00 h), the workers were examined. The examination included once in the whole survey a standardized interview regarding occupational history, smoking habits, and health history concerning in particular about respiratory diseases. Additionally, a daily questionnaire before and aer work was performed to consider acute respiratory symptoms. Pulmonary function testing with regard to vital capacity (VC) and forced expiratory volume in one second (FEV1) using a portable asthma monitor AM1 (J¨ ager/VIASYS Healthcare) was carried out by the workers themselves both immediately before and aer the work-day. Thereby, the optimum value of a triple serially conducted expiration of breath was automatically saved. Healthy unexposed men showed a slight diurnal increase in both FVC and FEV1 (about 5%),18 which was conrmed in the comprehensive study.16 No external control group was therefore used in the present study. The study was approved by the Committee on Ethics in Human Research of the Berlin General Medical Council in Germany. All workers took part voluntarily in the study. Cultivation independent approach For the cultivation independent approach polycarbonate lters were transferred to sterile plastic bags. 10 ml of cell free isotonic NaCl solutions were added into the bags and the cells were detached from the lter surface by using a Stomacher (Stomacher, 80 Biomaster, Seward, UK) at the highest level for 60 s. The quantication of airborne microorganisms was performed via total cell counting aer DAPI staining using 5 ml of cell suspension.19 The other 5 ml of the cell suspension was used for qualitative analyses with molecular methods. Therefore the cell solutions were concentrated by centrifugation (21 380g, 15 minutes). The following steps were DNA extraction and subsequent amplication of 16S rRNA genes of “all” bacteria (primers 27F, 1492R)20 as described elsewhere.19 For qualitative characterisation we applied rst the restriction fragment length polymorphism (RFLP) in combination with the chip based automated capillary electrophoresis (Biorad Experion system) and secondly 16S rRNA gene sequence clone libraries. RFLP analyses Aer amplifying the 16S rRNA gene sequence we puried the PCR products with the QIAquick PCR purication Kit (Qiagen). To determine the concentrations of the PCR products we used a Qubit Fluorometer (Invitrogen). For the restriction digest we used two FastDigest restriction endonucleases (Fermentas; Hin6I and HpaII) and each digest was performed with 300 ng PCR products of each bioaerosol sample aer manufacture's
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Environmental Science: Processes & Impacts instruction with a protocol of 11 ml H2ODEPC, 15 ml PCR product (20 ng ml1), 3 ml 10 FastDigest buffer and 1 ml FastDigest enzyme (1 FDU per ml). For the chip based automated capillary electrophoresis (Biorad Experion system) we used the DNA 1K assay (Biorad) and followed the manufacture's instruction. With this system DNA bands between 15 bp and 1500 bp could be separated and identied with an internal bp-marker. The results were obtained in an electropherogram. Cloning approach For cloning approach we used bioaerosol samples which were taken in 2007 at the same workplaces under similar conditions and the same sampling procedure. Furthermore we collected in this study the ducklings' down during one examination day (29.01.09). Therefore we collected with sterile instruments the ducklings down directly from ducklings' incubator trolley, which was disinfected at the beginning of the production process. Two replicates of DNA extraction and 16S rRNA PCR were done from ducklings' down sample. Aer 16S rRNA gene sequence amplication we did cloning approach like described elsewhere.15 For each cloning approach of a bioaerosol sample 64 clone inserts were sequenced (appr. 800 bp). For the cloning approach of ducklings' down 30 clone inserts were sequenced (appr. 800 bp). The 16S rRNA gene sequences were compared with available sequences in GenBank using BLAST (Basic local alignment search tool) provided by the NCBI (National Center for Biotechnology Information Server). Phylogenetic calculations were performed as described by Martin et al. (2010).19 Note RFLP analyses and cloning approach were done only with bioaerosol samples from eclosion days. It was not possible to get
Paper any DNA extract from bioaerosol samples from non-eclosion days because there were too little bacteria in each sample. Statistical methods The (dependent) pairwise, bidirectional t-test was performed to verify the presence of the paired value groups. P # 0.05 was regarded as the level of signicance.
Results In total, measurements were taken on 8 eclosion days and 8 non-eclosion days. Numbers (n) of individual working days (Fig. 1 and Fig. 2) could differ because of the absence of hatchery workers. Quantitative assessment of microbial exposure The total cell counts show that there are regular differences in the exposure level depending on the working step. On ducklings' eclosion days the total cell count ranges from 2.6 106 cells per m3 air to a maximum of 9.8 107 cells per m3 air. In contrast, on days without ducklings' eclosion there have always been detected moderate exposure levels ranging from 3.6 103 cells per m3 air to 9.6 104 cells per m3 air and therefore are comparable to those values detected in outside air controls (3.6 104 cells per m3). The overall mean cell count value on ducklings' eclosion days amounts to 3.0 107 cells per m3 air gained by DAPI staining (see Fig. 1). In contrast, on days without ducklings' eclosion the mean cell count value amount was 2.6 104 cells per m3 air. Despite the different positions of the workers at sorting of ducklings and even totally different tasks on non-eclosion days the mean exposure of all examined workers was quite similar on eclosion days (3.0 107 cells per m3 air) and on non-eclosion days (2.6 104 cells per m3 air), respectively.
Fig. 1 Personal microbial exposure given as total cell counts per m3 inhalable air after DAPI staining of four hatchery workers. Grey columns show the microorganisms concentration on eclosion days and white columns show the microorganisms concentration on non-eclosion days. Cell counts are given as mean values SD for each worker, n represents the number of examined individual working days.
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Paper
Environmental Science: Processes & Impacts
Fig. 2 The across shift changes in the lung function parameter FEV1 of 4 hatchery workers on work shifts with and without ducklings' eclosion. Bars show the mean values of n examined days of each worker as well as the average SD.
Lung function tests We determined a clear decrease of FEV1 across the work shis during eclosion days of 2.75% on average for the 4 workers. In contrast, on working days without ducklings' eclosion FEV1 increased on average by 0.46%, a signicant difference to eclosion days (t-test, p ¼ 0.03). Qualitative analysis of bacterial exposure The amount of extracted DNA from airborne microorganisms on non-eclosion days was below the limit for further qualitative analysis. For this reason we present qualitative results only from
eclosion days. In total, 28 bioaerosol samples from eclosion days were analysed regarding the bacterial composition. The virtual gel picture from single runs (n ¼ 28) in Fig. 3 shows unique restriction patterns from bioaerosol samples of eclosion days. Here, one band corresponds to one segment of digested 16S rRNA gene sequence PCR product. There are two size standards as an internal reference in the system with a length of 15 bp and 1500 bp. The peak threshold is $0.1 ng per segment. This RFLP method was used to show the unique pattern of the bacterial composition on eclosion days in this hatchery. Fig. 4 illustrates that there is a low variation within the microbial community on eclosion days independent of the year
Fig. 3 Virtual gel picture of single runs showing RFLP-patterns received from airborne bacterial communities of serially taken hatchery bioaerosol samples on ducklings' eclosion days (2008). Restrictions were performed by Hin6I digestion (FastDigest enzymes, Fermentas) of 16S rRNA gene PCR products generated from DNAextracts. Ladder is between 15 and 1500 bp.
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Paper
Fig. 4 Percentage distribution of genera detected via the 16S rRNA gene sequence cloning approach. One column represents one clone library with n sequenced clone inserts. Three independent clone libraries each (a, b and c) from personal and stationary sampling of hatcheries' air were generated. The personal bioaerosol sampling was done in 2007 and stationary sampling in 2008.15 Ducklings' down were collected in 2009 on one eclosion day and were used to generate two clone libraries (I and II).
of sampling. The dominant bacterial genus is Staphylococcus. At the species level mainly risk group 2 bacteria (TRBA 466) were detected (Staphylococcus aureus, Acinetobacter baumannii, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Staphylococcus intermedius (Martin and J¨ ackel, 2011).15 With the Good's coverage as a non-parametric estimator the proportion of phylotypes in a library of innite size could be estimated.21 The coverage between the clone libraries generated from personal bioaerosol sampling was between 93 and 97% and between the clone libraries generated from stationary bioaerosol sampling was between 97 and 98%.
Discussion Previous cross-sectional epidemiologic studies indicate that work in poultry farms is associated with the development of acute and chronic respiratory symptoms and changes of lung function.2,3,5,6,10,12,22,23 For example it has been shown that chicken catchers had signicant decrements over a work shi in forced expiratory volume in 1 s (3.4%) compared to reference groups and therefore are at high risk of respiratory dysfunction.6 In our present study we investigated with the help of a medical– molecular biological-interdisciplinary approach if there is a relationship between changes in lung function and the personal microbial exposure level of duck hatchery workers. Therefore we measured daily the personal microbial exposure level and the diurnal FEV1 change as an indicator of lung function in a one month study. In this study it was shown that the across shi changes of lung function of duck hatchery workers is observed when the microbial exposure level is high like on ducklings' eclosion days. On these eclosion days there has always been detected a high microbial air contamination. The average cell count value on those days amounts to 3.0 107 cells per m3 air gained by DAPI staining. These exposure values are similar to those taken at different working places of a duck farm24 and other working environments like compost facilities.25 The detection of the total cell count on non-eclosion days was with an average of 2.6 104 cells per m3 air, which is above the 482 | Environ. Sci.: Processes Impacts, 2013, 15, 478–484
detection limit of the DAPI staining method (103 cells per m3 air). The quantitative exposure on non-eclosion days is comparable to the detected outside background levels (3.6 104 cells per m3). These values are comparable to those found by Klug and J¨ ackel (2012)24 in their investigation on background levels in the outside air. On eclosion days the four workers showed on average a decline in lung function over their working day causing an average FEV1 decrease of 2.75% independent of whether they are smokers or not. In contrast to non-eclosion days an increase in the mean of diurnal lung function by 0.46% was detected, which might be connected to the fact that the airborne cell content in the hatchery on those days without ducklings eclosion is nearly identical to normal cell count values found in outside air controls. This corresponds to previously described changes in diurnal lung function of healthy men showing circadian FEV1 increases of about 5%.18,26 Previous studies have shown that increased daily and annual loss in lung function can be related to exposure to organic dust and endotoxins in farming.23,27 We conclude that diurnal lung function changes of duck hatchery workers are related to high microbial exposure levels because a decrease in lung function specically occurs on those days with high microbial exposure. But it is difficult to determine whether the adverse effect on workers lung function is due to the microbial exposure directly or if the microbial exposure acts as an indicator of the complex burden of bioaerosols on workplaces in agriculture.28 As shown in a previous study in the hatchery there was found high dust and endotoxin concentrations on eclosion days, too.16 Smid et al. (1994)29 reported in their study that lung function changes could be related to endotoxin exposure which indicates that endotoxin may lead to short-term lung function (FEV1) decrease, too. In this study we analysed only the bacterial composition to get an insight into the source of endotoxins. As shown in Fig. 4 the sources for airborne endotoxins seem to be Acinetobacter, Pseudomonas, Klebsiella, Janthinobacterium, Enterobacter, and Achromobacter. Furthermore we exclude an effect on decline of lung function caused by airborne fungi because the previous study indicated low amounts of them in the air of the hatchery.16
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Paper To our knowledge this study shows for the rst time a decline in lung function in hatchery workers using a daily lung function protocol. Therefore our results demonstrate that the used study design allows for an examination of only a small group of probands. The results of this study invite us to repeat these analyses at further hatchery working places to verify our observations. Qualitative analyses of bacterial community were done by RFLP and cloning approaches. On days without ducklings' eclosion there could never be generated a DNA extract and therefore no 16S rRNA gene amplicons out of the bioaerosol samples although bacteria universal primers were used. For future analysis of the bacterial diversity on non-eclosion days by culture independent methods the sampling volume should be increased or another sampling system for these days should be proven. As RFLP-analysis of 16S gene sequence amplicons revealed, the bacterial exposition pattern is uniform over the whole survey on ducklings' eclosion days. Low bacterial diversity and a missing dynamics are main characteristics of an extreme microhabitat marked by a low water content in the air (data not shown) and additionally poor growth conditions due to regular cleaning and disinfection of all production surfaces. Further detailed bacteriological investigation of six independent clone libraries based on 16S rRNA genes from bioaerosol samples revealed Staphylococcus, Enterococcus and Acinetobacter as the predominant genera in the hatchery airborne bacterial community. The abundance of Enterococcus and Acinetobacter in the air of hatcheries was found in earlier studies.30,31 Furthermore the three abundant genera Staphylococcus, Enterococcus and Acinetobacter were also detected as abundant genera by cultivation dependent and independent methods in the preliminary study.15 The main part of the three independent clone libraries from stationary bioaerosol sampling show on a 97% sequence similarity level on species section where 56% of all successfully sequenced clones show high sequence similarity to the 16S rRNA gene sequence of Staphylococcus aureus. This is the only abundant species which was not detected by personal sampling in the year 2007. We cannot point out whether this is due the different year of sampling or different sampling measurements. Following 9% of gained 16S rRNA sequences are closely related to the 16S rRNA gene sequence of Enterococcus spp., whereas half of the sequences were next similar to the type strain sequence of Enterococcus gallinarum and the other half was next similar to the type strain sequence of Enterococcus faecalis. Additional 10% of the sequenced clones show a high identity with the sequence of Acinetobacter baumannii. All together, the main part of the achieved clone sequences belong to bacteria species which are characterised as risk 2 bacteria according to the Directive 2000/54 EC32 and the German safety classication TRBA 466 (ref. 33) which means those organisms “that can cause human disease and might be a hazard to workers; they are unlikely to spread to the community; there is usually effective prophylaxis or treatment available” (Directive 2000/54 EC).32 In particular Acinetobacter baumannii seems to be a high risk for occupational health as it has been already
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Environmental Science: Processes & Impacts described as a causative organism for nosocomial bacteremia.34 The coverage calculation showed that 93–98% of the abundant bacteria were detected. We can assume from the RFLP and cloning results that on eclosion days the workers are permanently exposed to these detected bacteria. To gure out from where the bacterial air contamination in the hatchery has derived, ducklings' down samples were taken directly from the ducklings' incubator trolley, which was disinfected at the beginning of the production process. As a matter of fact we received identical patterns from DNA-extracts of ducklings' down, which uncovers them as the bacteria carrier. Furthermore the cloning approach from ducklings' down shows a nearly identical bacterial genera distribution like the cloning results from bioaerosol sampling. Anyway it remains unclear from where the bacterial contamination hit into the egg or into the down of the freshly hatched ducklings. Potentially, the unsterile water which is used in the brood incubator for humidity regulation could be the vehicle for contamination (and therefore possibly contaminate the ducklings' via an established bacterial biolm in the water system). The optimal temperature conditions combined with high humidity in the ducklings' incubator could be due to the excessive bacterial growth in their down. Nevertheless, as rst origin of bacterial contamination it seems to be that the microora of the egg shell surface itself is the most presumably source for bacterial invasion. This has likewise been shown that e.g. hens eggs are covered with bacteria strains such as Staphylococcus warneri, Acinetobacter baumannii, Alcaligenes sp., Serratia marcescens, Carnobacterium sp., Pseudomonas sp. and Salmonella enteritidis.35 Hence it can be assumed that the egg surface disinfection process with chlorine water and H2O2 gassing is not sufficient to eliminate all living bacteria or that the identied predominant bacteria could be exceptionally notably resistant towards the washing and gassing process.
Conclusion These quantitative and qualitative results conrm the results from the preliminary study in the same duck hatchery.15 Workers are permanently exposed to a high load of airborne biological agents during handling of ducklings. We recommend optimising the technical and personal protection measures for this kind of work, e.g. improved ventilation and breathing protection, respectively, in order to prevent the workers from continuous respiratory dysfunction or acute respiratory chronic diseases in future.
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