Abstract
Coxiella burnetii is thought to infect humans primarily via airborne transmission. However, air measurements of C. burnetii are sparse. We detected C. burnetii DNA in inhalable and PM10 (particulate matter with an aerodynamic size of 10 μm or less) dust samples collected at three affected goat farms, demonstrating that low levels of C. burnetii DNA are present in inhalable size fractions.
TEXT
Q fever is a zoonotic disease caused by the Gram-negative intracellular bacterium Coxiella burnetii (1). Transmission of C. burnetii from animals to humans is considered to be primarily through air via inhalation of particles containing viable C. burnetii cells (1, 7, 8, 10). However, only a few published studies have actually demonstrated airborne C. burnetii (2, 3, 5, 12) and most did not take into account the size fraction of the dust particles collected (2, 3, 5). Given the respiratory route of infection, dust particles that can penetrate the respiratory organ should be considered. Examples of these are PM10 (particulate matter with an aerodynamic size of 10 μm or less) or inhalable dust particles (particulate matter with a 50% cutoff of 30 μm). One study detected C. burnetii in airborne inhalable dust samples in a sheep barn during shearing of an infected herd; however, the number of samples measured was very limited (12).
The motivation for this study was a human Q fever outbreak in the Netherlands. Infected dairy goats were identified as the primary source for the epidemic (8, 11), and to control the spread of C. burnetii, all pregnant goats on bulk tank milk (BTM)-positive farms were culled (6, 13, 14). The aim of the present study was to detect C. burnetii DNA in inhalable dust samples on a larger scale and to describe a method to detect C. burnetii DNA in inhalable dust and PM10 dust samples
Collection of samples.
The presence of C. burnetii DNA was investigated in air samples collected on three BTM-positive goat farms. Mandatory culling of pregnant goats had taken place 32 to 76 days prior to sampling. Some does had already kidded prior to the culling date; therefore, those does and their kids were still present on the farm during sampling. In addition, does that were not pregnant (both lactating and nonlactating) as well as bucks were present. Sampling was conducted between 12 March and 6 April 2010. PM10 samples were captured on Teflon filters (2.0 μm pore size; Air Diagnostics and Engineering Inc., United States) with Personal Environmental Monitor collection heads (SKC Inc., United States), which were connected to a BGI pump (BGI USA, Waltham, MA) (airflow, 4 liters per min). Inhalable dust samples were captured on 37-mm-diameter glass fiber filters (GF/A; Whatman, USA) with GSP collection heads (JS Holdings, United Kingdom) connected to a Gilliair pump (Gillian, United Kingdom) (airflow, 3.5 liters per min). Sampling heads were installed at 1.50 m aboveground using a tripod. Both sampling sets (inhalable dust and PM10) were installed side by side. Several sets were placed at different sites around the farm, namely, in the milking parlor, milking doe pen, young stock pen, and buck pen, as well as inside the farmer's house and upwind and downwind of the farm (distance to farm between 20 and 70 m).
Sampling was performed during 4 h in a period of minimal activity. To investigate if routine farm activities would affect the outcome, sampling was also performed at one farm during two periods of activity: (i) during milking of the goats and (ii) when deep straw bedding was being removed from the stables. Due to logistical constraints, sampling during activity was not performed on all three farms.
In addition to the airborne dust samples, settled dust samples were collected with a clean (ethanol) plastic scraper from horizontal surfaces in the stables.
After sampling, filters and dust samples were stored at −20°C until processing began.
Sample processing and DNA extraction procedures.
Filters were transferred to 50-ml tubes (Greiner Bio-one) containing 4 ml pyrogen-free water (Aqua B. Braun) with 0.05% Tween 20 (Calbiochem, United States). Tubes were agitated for 1 h on an end-over-end roller and centrifuged for 15 min at 1,000 × g. NucliSens lysis buffer (36 ml) (bioMérieux, France) was added to the tubes, followed by agitation on the end-over-end roller for 1 h. Samples were centrifuged for 15 min at 2,000 × g. The supernatant was transferred to a new tube. As an internal control for both DNA extraction and qPCR amplification, 7 × 104 spores of Bacillus thuringiensis (50 μl of a 1:10 diluted spore suspension; Raven Labs, Omaha, NE) was added to the supernatant, followed by DNA extraction with the NucliSens Magnetic Extraction Kit (bioMérieux, France) according to the manufacturer's protocol.
Multiplex qPCR.
C. burnetii DNA was determined by multiplex quantitative PCR (qPCR) as described previously by de Bruin et al. (4). Briefly, the qPCR detects one B. thuringiensis internal control target (cry1b) and two C. burnetii targets: a single-copy outer membrane coding gene, com1, and a multicopy insertion element, IS1111. The multiplex qPCR assays were carried out on a LightCycler 480 instrument (Roche Diagnostics Nederland B.V.), and analysis was performed with the instrument software (LightCycler 480 software release 1.5.0. SP3).
C. burnetii DNA-positive samples were categorized into two classes, with increasing C. burnetii DNA content: (i) IS1111 positive (+) and (ii) IS1111 + com1 positive (++), as amplification of multicopy target IS1111 is expected to occur before amplification of single-copy target com1 (9). Samples were scored as negative (−) when neither of the C. burnetii targets showed a positive signal in the presence of a positive internal control (cry1b). Samples were scored undetermined when the internal control did not amplify.
Detection of Coxiella burnetii DNA in various samples collected at three farms.
During a period of minimal activity, C. burnetii DNA was found in three out of a total of 23 PM10 samples (13.0%) and in five out of 23 inhalable dust samples (21.7%). In contrast, almost all settled dust samples were positive (Table 1).
Table 1.
Presence of C. burnetii DNA in dust samples by sample typea
| Sample location | Resultb for sample type |
||
|---|---|---|---|
| PM10 | Inhalable dust | Settled dust | |
| Kitchen | |||
| Farm 1 | − | − | n/a |
| Farm 2 | − | − | n/a |
| Farm 3 | + | + | n/a |
| Young stock | |||
| Farm 1 | + | − | ++ |
| Farm 2 | − | − | ++ |
| Farm 3 | − | ++ | ++ |
| Milking does | |||
| Farm 1 | − | − | ++ |
| Farm 1 | − | − | − |
| Farm 2 | ++ | − | ++ |
| Farm 3 | − | + | ++ |
| Farm 3 | − | − | ++ |
| Milking parlor | |||
| Farm 1 | − | − | ++ |
| Farm 2 | − | − | ++ |
| Farm 3 | − | − | ++ |
| Bucks | |||
| Farm 1 | − | + | ++ |
| Farm 2 | − | + | ++ |
| Farm 3 | − | Undeterminedc | ++ |
| Downwind | |||
| Farm 1 | − | − | n/a |
| Farm 2 | − | − | n/a |
| Farm 3 | − | − | n/a |
| Upwind | |||
| Farm 1 | − | − | n/a |
| Farm 2 | − | − | n/a |
| Farm 3 | − | − | n/a |
PM10, inhalable dust, and settled dust samples were collected during a period of minimal activity at various locations on three bulk tank milk-positive farms, 32 to 62 days after culling (35 days at farm 1, 62 days at farm 2, and 32 days at farm 3).
++, C. burnetii IS1111 + com1 positive; +, C. burnetiiIS1111 positive; −, C. burnetii DNA negative; n/a, not available.
Undetermined sample showed PCR inhibition and no signal for internal control (cry1b).
Coxiella burnetii DNA in air samples during periods of activity.
The number of C. burnetii DNA-positive air samples increased when there was more activity on the farm (milking and cleaning), compared to a period of minimal activity. Additionally, even downwind air samples were positive during activity (Table 2).
Table 2.
Presence of C. burnetii DNA in dust samples by activitya
| Sample location | Resultb for activity and sample type |
|||||
|---|---|---|---|---|---|---|
| Minimal activity, 62 days postculling |
Milking activity, 75 days postculling |
Bedding removal, 76 days postculling |
||||
| PM10 | Inhalable | PM10 | Inhalable | PM10 | Inhalable | |
| Young stock | − | − | − | + | − | + |
| Milking does | ++ | − | − | − | + | + |
| Milking parlor | − | − | − | + | − | − |
| Downwind of farm | − | − | − | + | + | + |
| Upwind of farm | − | − | − | − | − | − |
| Downwind of manure heap | n/a | n/a | − | + | + | − |
| Upwind of manure heap | n/a | n/a | − | − | − | − |
Presence of C. burnetii DNA in PM10 and inhalable dust samples collected during periods of minimal activity, during milking activity, and during deep straw bedding removal at bulk tank milk-positive farm 2 (62, 75, and 76 days after culling, respectively).
++, C. burnetiiIS1111 + com1 positive; +, C. burnetii IS1111 positive; −, C. burnetii DNA negative; n/a, not available.
In general, C. burnetii DNA was found more frequently in inhalable dust samples (12/37 [32.4%]) than in PM10 samples (6/37 [16.2%]). This suggests that C. burnetii binds or aggregates more frequently to larger dust particles. Moreover, the previous air sample studies in goat and sheep farms (2, 3, 5) detected C. burnetii DNA more often, which could be due to the larger particle size fractions collected in those studies. On the other hand, on farm 2 a single-copy gene of C. burnetii DNA was detected in a PM10 sample in the milking does area, but no C. burnetii DNA was found in the accompanying inhalable dust sample (Table 1). Future research is needed to look into the particle size distribution in relation to the presence of C. burnetii DNA.
Although this study is the largest study thus far investigating the presence of C. burnetii DNA in air samples that can enter the respiratory tract, the results should be interpreted with caution. The observed C. burnetii DNA levels were generally low, and most airborne samples were positive only for the multicopy gene. A previous study in the Netherlands also reported low C. burnetii DNA levels (3). It is not possible to determine whether low levels of airborne C. burnetii DNA are the result of minimal active shedding (high-risk goats had been culled) or minimal farm activity.
Interestingly, we collected more C. burnetii DNA-positive samples during periods with relatively higher activity, which suggests an increased formation of contaminated aerosols. These positive samples likely in part reflect resuspended dust. Whether this is of importance with regard to infectivity is unknown. In general, detection of C. burnetii DNA in air samples provides no information on the viability of the C. burnetii organism detected in the samples. Future research should look into the level of viable C. burnetii DNA in environmental samples to allow a proper human and animal infectiveness risk assessment from airborne exposures.
In conclusion, the results show that C. burnetii DNA can be detected in airborne dust samples of size fractions that can be inhaled by humans. This supports the general assertion that airborne transmission might indeed be a likely route of exposure, and it should be explored in more detail to understand the spread and transmission route of C. burnetii and the risk posed by C. burnetii for humans.
ACKNOWLEDGMENTS
We thank the farmers for their cooperation. Myrna de Rooij, Lianne de Heer, and Rozemarijn van der Plaats are acknowledged for their help with DNA extractions and qPCR analyses.
Footnotes
Published ahead of print 11 May 2012
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