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Measurement of culturable airborne staphylococci downwind from a naturally ventilated broiler house

Article in Aerobiologia ? December 2011

DOI: 10.1007/s10453-011-9202-6

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Aerobiologia (2011) 27:311?318 DOI 10.1007/s10453-011-9202-6

ORIGINAL PAPER

Measurement of culturable airborne staphylococci downwind from a naturally ventilated broiler house

J. Schulz ? L. Formosa ? J. Seedorf ? J. Hartung

Received: 26 January 2011 / Accepted: 8 March 2011 / Published online: 22 March 2011 ? Springer Science+Business Media B.V. 2011

Abstract The objective of this study was to estimate the possible travel distance of airborne bacteria emitted from a naturally ventilated broiler house by using staphylococci as indicator organisms. Air samples were taken during the second half of three fattening periods with Impinger (AGI-30) in the barn and simultaneously upwind and downwind from the building. Staphylococci concentrations varied between 1 9 106 and 1 9 107 cfu m-3 in the barn. No Staphylococci were detected in air samples at the upwind side. A strong exponential decrease of these bacteria was observed at three sampling heights (1.5, 4.0 and 9.5 m) in the main wind direction downwind of the barn. Staphylococci concentrations up to 5.9 9 103 cfu m-3 were detected at the farthest sampling point (333 m) downwind. Identification to the species level by means of a 16S?23S ITS PCR confirmed that Staphylococcus spp. from downwind samples originated from the barn. Staphylococci served as an useful

J. Schulz (&) ? J. Hartung Institute for Animal Hygiene, Animal Welfare and Farm Animal Behaviour, University of Veterinary Medicine Hannover, Foundation, Bu?nteweg 17p, 30559 Hannover, Germany e-mail: Jochen.Schulz@tiho-hannover.de

L. Formosa Department of Environment, Climate Change and Water, Sydney, NSW, Australia

J. Seedorf University of Applied Sciences, Osnabru?ck, Germany

indicator to demonstrate the travel distance of bacterial emissions originating from a naturally ventilated broiler house. These findings indicate that airborne transmission of viable bacteria from this type of housing system to adjacent residential dwellings or animal houses several hundred metres away is possible.

Keywords Bioaerosols ? Emission ? Airborne ? Staphylococci ? Dispersion ? Broiler house

1 Introduction

The air of broiler houses can contain high amounts of air pollutants such as gases, dust, fungi and bacteria including zoonotic agents, endotoxins and allergens (Dungan and Leytem 2009; Seedorf and Hartung 2002) which are also addressed as bioaerosols (Hirst 1995). These bioaerosols are supposed to present a health risk for the animals in confined buildings and for humans working in this atmosphere (Bull et al. 2006; Hartung and Schulz 2008; Madelin and Wathes 1989; Whyte 1993). In recent years, concerns are rising that these bioaerosols when emitted from the animal houses by way of the exhaust air may also pose a health risk for nearby residents or animals in neighbouring farms (Millner 2009). However, there is a considerable lack of knowledge about the travel distance of bioaerosols and their compounds like bacteria from poultry farms and especially from naturally ventilated poultry houses. Some earlier

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experiments measured viable bacteria emitted from forced ventilated laying hen houses and found micrococci in concentrations of about 300 cfu m-3 250 m downwind from the barns in the prevailing wind direction. The strongest decrease took place within the first 100 m from the barns (Platz 1979; Mu?ller and Wieser 1987).

These results cannot be simply applied to naturally ventilated broiler barns because the average emission rates from broiler barns are distinctly higher than from laying hen houses and emission characteristics of naturally ventilated barns are fundamentally different from forced ventilated barns (Schneider et al. 2006; Seedorf 2004). Furthermore, the air exchange rates of naturally ventilated broiler barns can only be calculated by approximation (Formosa 2005). Consequently, usual dispersion models based on defined emission rates are less useful to estimate the spread of airborne microorganisms from naturally ventilated broiler barns. Therefore, direct measurements in the ambient air at different distances around animal houses seem to be the most promising method to characterise the bacterial dispersion qualitatively and quantitatively. In order to verify that the measured bacteria are really originating from the barn, the identification of indicator bacteria is useful. Staphylococcus spp. seem to be suitable for this purpose because they are the dominating airborne bacteria in broiler barns (Hartung and Saleh 2007; Oppliger et al. 2008) originating from the animals and the litter (Devrise et al. 1985; Lu et al. 2003; Shimizu et al. 1992). They attach to skin debris, broken feather barbules and particles from contaminated litter and become airborne due to the activity of the birds and the airflow within the barn (Whyte 1993). Wiegand et al. (1993) and Hartung and Saleh (2007) measured mean concentrations of staphylococci of 1.2 9 109 and 6 9 109 cfu in 1 g of airborne broiler house dust. Hence and due to their relatively high tenacity in the airborne state (Mu?ller and Wieser 1987), high emissions of culturable staphylococci can be expected from broiler houses. On the other hand, staphylococci apparently do not belong to the typical airborne microflora in the ambient air of rural areas (Depre?s et al. 2007; Harrison et al. 2005; Shaffer and Lighthart 1997). This study will investigate the spread of Staphylococcus spp. from a naturally ventilated broiler barn in order to estimate their possible travel distances in a viable state in the prevailing wind direction.

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Aerobiologia (2011) 27:311?318

2 Materials and methods

The field experiments were carried out inside and in the vicinity of a naturally ventilated Louisiana type broiler barn, initially housing nearly 40,000 broilers (Ross hybrid broilers) on straw litter during three subsequent fattening periods (growing cycle) in a summer season. The barn is situated in a typical rural area of the north of Germany surrounded by arable land and meadows. Some high oak trees and older farm buildings are located north of the barn. Each fattening period was 5 to 6 weeks. Prior to housing of new one-day-old chicks, the animal house was cleaned, disinfected and dried within 2 weeks.

Air sampling was conducted simultaneously in the animal house as well as upwind and downwind from the barn by means of impingement.

2.1 Impingement

AGI-30 impingers (Ace Glass Inc., Vineland, N J) were used to sample bioaerosols including airborne staphylococci in 50 ml 1:1 glycerol?phosphate buffer solutions. The impingers were operated for 30 min inside the barn and for 90 min outside the barn with an air flow of 12.5 l min-1. The air flow was controlled before and after the end of the sampling time with the flow meter 044-14G from Analyt-MTC (Mu?llheim, Germany). Impingers used for ambient air sampling were protected by a white insulating envelop against direct sunshine and UV radiation.

2.2 Air sampling outside the animal house

Air samples were taken downwind and upwind the barn only on rainless days during summer season, when the three sampled flocks were in the second half of the growing cycle (period of a growing cycle = 35?39 days) and the prevailing wind came from western directions. Three pairs of impingers were fixed on horizontally mounted metal bars at three different sampling heights (1.5, 4.0 and 9.5 m above ground) on commercially available weather masts (Clark Masts Teksam NV, Belgium). Overall, favourite meteorological conditions were met at five different sampling days during the summer allowing thirteen complete sets of measurements having the weather masts positioned both in the prevailing wind direction downwind and upwind the barn. While the

Aerobiologia (2011) 27:311?318

downwind measurements took place at different distances from the barn, the masts had to be moved between the measurements to the next sampling point. One mast was erected approximately 40 m upwind the barn and was not moved (control). Wind direction and wind speed were recorded automatically every minute by a stationary weather station (UNIKLIMA 7, TOSS, Potsdam, Germany) which was positioned 200 m southeast of the barn. An optical range finder (TC 110 Tachymeter, Leica Geosystems, Switzerland) was used to measure the distance and the relative position of the sampling points in the surrounding field to the centre of the barn. Table 1 gives the distances between the sampling points downwind from the barn and the barn centre, the deviations of sampling points from the main wind direction, the average wind speed, the age and number of birds present during the samplings in the three fattening periods.

2.3 Air sampling inside the animal house

In order to identify and to quantify airborne Staphylococcus spp. in the animal house air, two consecutive impinger samples were taken in parallel to the samplings outside. Because of the shorter sampling time in the animal house, the samplings started 30 min later than the outside samplings. Always the same sampling place was chosen in the centre of the building at a height of 1.5 m above floor level.

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2.4 Cultivation and quantification of Staphylococcus spp.

Impingers were shaken for 30 s at full speed with a Vortex-Genie2 (Scientific Industries Inc., USA), and 1 ml aliquots were taken to prepare serial dilutions (10-1, 10-2 and 10-3) of the indoor samples. Aliquots (0.1 ml) of these dilutions and aliquots (0.1 ml) taken directly from the outdoor samples were plated on mannitol salt agar (OXOID LTD, Basingstoke, Hampshire, England). The plates were incubated for 24?48 h at 36?C. Typical staphylococci colonies were counted, and the number of airborne colony forming units per m3 (cfu m-3) was calculated by the equation from Lin et al. (1999). The detection limit was around 300 cfu m-3.

From each sampling (Table 1), all typical staphylococci colonies grown from the highest dilution step (10-2 or 10-3) of one indoor air sample (10?25 colonies) and from one outdoor air sample (15?40 colonies) were streaked on blood agar basis plates (OXOID LTD, Basingstoke, Hampshire, England) and incubated for 24?36 h at 36?C to generate pure cultures. These isolates were confirmed to the genus Staphylococcus spp. by gram reaction, morphology, motility, catalase test, oxidase test and lysostaphin susceptibility as described by Freney et al. (1999). In addition, the presence of the clumping factor was tested with mannitol-fermenting staphylococci by

Table 1 Characterisation of conditions during outdoor sampling

Sampling no.

Growing cycle

Age of birds (days)

Number of birds

Distance of sampling points to the barn (m)

1

A

28

2

A

33

3

A

33

4

B

35

5

B

35

6

C

20

7

C

20

8

C

20

9

C

20

10

C

28

11

C

28

12

C

28

13

C

28

39,113

60

38,632

221

38,632

221

38,324

264

38,324

264

39,340

60

39,340

333

39,340

60

39,340

333

39,164

130

39,164

333

39,164

130

39,164

333

Deviation (?) of sampling points from the main wind direction

14 5 7 7 2 8 5

10 3

11 11

2 2

Average wind speed (m s-1]

3.6 2.3 2.6 2.2 2.5 2.1 2.1 2.3 2.3 2.2 2.2 1.6 1.6

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using the DR595 test kit (OXOID LTD, Basingstoke, Hampshire, England).

2.5 Identification of Staphylococcus species by 16S?23S rDNA intergenic spacer PCR

A 16S?23S rDNA intergenic spacer PCR was used to identify staphylococci species simultaneously found in the barn air and downwind from the barn. Staphylococcus isolates prior confirmed to the genus level as described above were used from samplings no. 1, 3, 5, 7, 10 and 13 (Table 1). A small part of a colony from a pure culture grown for 24?36 h on blood agar basis was transferred with an aseptic toothpick into a 200-ll PCR reaction tube and incubated with 5 ll of a 100 lg ml-1 lysostaphin solution for 15 min at 37?C. Then, 5 ll of a GeneReleaser (BioVentures Inc., USA) was added and a thermocycle programme following the manufacture's procedure was started to release DNA from cells. Thereupon, 5 ll Taq buffer incl. Mg2?, 10 ll Taq Master, 1 ll nucleotide mix (200 lmol of each dNTP), 1 ll primer L1 (100 pmol/ll), 1 ll primer G1 (100 pmol/ll), 0.5 ll Taq Polymerase (2.5l) and 21.5 ll water (PCR grade) were added to a final volume of 50 ll per reaction tube. All reagents were purchased from Eppendorf (Hamburg, Germany) with the exception of primer G1 (50-GAAGTC GTAACAAGG-30) and L1 (50-CAAGGCATCCACC GT-30) which were synthesised by Biometra (Go?ttingen, Germany). Amplification reaction was performed on a Mastercycler gradient apparatus (Eppendorf, Hamburg, Germany). The program used for amplification consisted of an initial denaturation step at 95?C for 2 min and 26 cycles, each with 30 s at 95?C, 2 min at 47?C and 2 min at 72?C, followed by a final elongation at 72?C for 10 min. The analysis of the amplified products and the identification of Staphylococcus spp. were carried out as described by Mendoza et al. (1998).

3 Results

3.1 Number of Staphylococcus spp. in the animal house air

Numbers of typical staphylococci colonies grown on mannitol salt agar were used to calculate the concentrations of airborne staphylococci. All isolates

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Aerobiologia (2011) 27:311?318

of these colonies could be confirmed as Staphylococcus spp. by biochemical methods. Table 2 shows the staphylococci concentrations measured in the animal house when the birds were 20?35 days old. The average bacterial count of two consecutive samplings varies between 1.1 9 106 (growing cycle C, 20 days) and 1.5 9 107 cfu m-3 (growing cycle, 35 days). The deviation between the first and the second sampling ranges from 10 to 30% which reflects typical measurement errors of the used air sampling technique (Terzieva et al. 1996).

3.2 Concentrations of airborne Staphylococcus spp. sampled downwind from the barn in the prevailing wind direction

Staphylococci were found in all 39 samples on the downwind side of the barn. During the measurement periods, the deviation of the wind direction from the main wind direction in relation to the sampling points was less than ?15? and the average wind speed was 2.3 m s-1 (1.6?3.6 m s-1) ensuring an almost isokinetic air sampling (VDI-Guideline 2008).

Figure 1 shows the concentrations of Staphylococcus spp. recovered from the air at five different locations downwind from the barn in three different heights above ground level. The regression grades for all three sampling heights demonstrate a strong exponential (N = N0 9 e-0.01x) decrease of the bacteria with increasing distance from the barn. At distances around 200 m and more downwind from the barn,\1% of the number of staphylococci which were emitted from the animal house was recovered and 333 m from the barn bacterial concentrations dropped down to a range between 1.2 9 103 and 5.9 9 103 cfu m-3. The regression lines in Fig. 1 suggest that staphylococci may even be distributed farer than 330 m from the source. Staphylococcus spp. were not detected in any of the samples taken at the reference position on the upwind side of the barn. Figure 1 also displays some differences between the three sampling heights at the same location. From the Wilcoxon? Wilcox test (Wilcoxon and Wilcox 1964), it turned out that there were no significant differences between the levels 1.5 and 4.0 m. A significant difference (P \ 0.05) was observed between the lowest (1.5 m) and the highest (9.5 m) level, indicating a slightly higher bacteria concentration near the ground level. These differences are probably caused by slight

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