The Q fever outbreak of 2007 was the first documented outbreak in the Netherlands. A case control study in 2007 revealed several risk factors for acquiring Q fever, however, a direct link with a particular source could not be established
. In this study we show that the most common MLVA genotype in both animal and environmental samples obtained from 45 farm locations is MLVA genotype A. The environmental sampling categories, surface swabs and aerosols, are of particular interest for establishing a source hypothesis of C. burnetii infection, since they may provide the link between C. burnetii in animals and humans. Coxiella burnetii laden dust can originate from the decomposition of C. burnetii contaminated aerosols and vice versa, re-aerosolisation of contaminated dust might occur. Contaminated aerosols are regarded as one of the most important transmission routes for C. burnetii to humans, especially when environmental conditions for aerosol dispersion are favourable
[3, 16, 18, 19]. Surface swabs were collected on 20 out of the 45 sampling locations and showed the occurrence of MLVA genotype A at 16 of these locations. Two aerosol samples collected at two of these locations were suitable for typing and MLVA genotype A was observed in these samples as well. As MLVA genotype A was also the dominant genotype in animal samples obtained from goats, sheep, and rats (Table
4), these data show that the most common genotype can be detected in both the animal hosts and the environmental matrices that are considered to play a dominant role in the direct or indirect transmission from animals to humans.
The finding of a single most common C. burnetii MLVA genotype in both environmental and animal samples in this study, supports the findings of two other MLVA genotyping studies in the Netherlands
[14, 15]. The 33 clinical samples that were successfully typed by Tilburg et al. using a panel of six MLVA markers, revealed a most common MLVA type referred to as MLVA genotype G (in 18 out of 33 samples). The four MLVA markers (Ms24, Ms27, Ms28, and Ms34) that can be compared to our study showed tandem repeats numbers that were identical to our most common MLVA genotype A. The most common MLVA genotype in the veterinary study by Roest et al. was referred to as MLVA genotype CbNL01, and was detected in 112 out of 126 animal samples obtained from 14 dairy goat farms, one dairy cattle farm and two sheep farms. Roest et al.
 used 11 MLVA markers for genotyping C. burnetii, as described by
. Five of these markers, Ms24, Ms27, Ms28, Ms31, and Ms34, can be compared to our study, and MLVA genotype CbNL01 showed identical numbers of tandem repeats within these loci when compared to our most common MLVA genotype A
The MLVA loci that can be compared between the three molecular typing studies of C. burnetii during the Q fever epidemic in the Netherlands show identical results for the numbers of tandem repeats within these loci for the most common genotypes. However, even when the different MLVA assays investigate the same markers, differences in primers used for amplification and other laboratory-specific conditions may affect the outcome of the analyses. In addition, the scoring of the numbers of tandem repeats may differ between individual researchers. This was illustrated by the outcome of an interlaboratory comparison with 7 European participants
. Finally, the sequence information regarding the repeat motif for a number of MLVA loci was not published
. This lead to the development of new primers for marker Ms20 in our study, which target a slightly different region than published by
[9, 21]. Therefore, comparisons of C. burnetii MLVA typing between laboratories requires a consensus approach, and confirmation of the number of tandem repeats by sequencing will play an essential role in providing the basis for confident calculations. This is of particular importance when the differences between MLVA genotypes, both within and between studies, are rather small.
Establishing a direct relationship between (clusters of) human Q fever cases and a single (or a cluster) of C. burnetii affected farms remains a challenge. Two epidemiological studies performed during the Q fever outbreaks in 2008 and 2009 in the Netherlands showed a strong association between clusters of human Q fever cases and a commercial dairy goat farm
 and a non-dairy sheep farm
. Although these studies focused on the best described clusters of human Q fever cases observed during the outbreaks, information about the number of C. burnetii genotypes circulating in patients, the environment, and potential veterinary sources in the Netherlands was not available at that time. Molecular typing of all samples obtained from animals and surface swabs from the dairy goat farm that was identified as the most probable source of a cluster of human Q fever cases
, revealed the presence of MLVA genotype A only. Unfortunately, molecular typing of the samples obtained from the non-dairy sheep farm that had been identified as the primary source for the cluster of human Q fever cases in its vicinity in 2009
, was not successful due to low C. burnetii DNA content (Cq values for target IS1111 >31). Investigations of C. burnetii MLVA genotypes in patients
, dairy live-stock
 mentioned earlier, and environmental and animal matrices in this study, showed that a single C. burnetii MLVA genotype is dominant in the Netherlands. Based on these data we conclude that the resolution attained by using MLVA is insufficient for pinpointing individual sources in most cases, as it is likely that the dominant C. burnetii type will be detected on several farms and in different patients in a particular area.
Another factor that complicates source-finding investigations, is that these studies are often biased by the selection of farms. For instance, in 2009 about 350,000 goats were registered on 3916 farms in the Netherlands. About 274,000 goats were distributed over 370 large commercial dairy goat farms.
The number of sheep was even larger in 2009, with over one million sheep present in the Netherlands, distributed over 12,833 registered farms (Statistics Netherlands: CBS). Only a small subset of these farms were selected for source-finding investigations in 2008 (29 farms) and 2009 (56 farms). Therefore, the number of MLVA genotypes observed in this study is an underestimation, because only 9.2% of commercial dairy goat farms and less than 1% of sheep farms present in the Netherlands in 2009 were included. Since dairy goat farms were quickly identified as the most probable sources for human Q fever in the Netherlands
[1, 5, 6, 23], the numbers of investigated dairy goat farms were much higher when compared to other potential sources for human Q fever (e.g. sheep or cattle). Primarily, those farms located in close proximity to human Q fever cases were selected. Within these clusters, farms were encountered with large numbers of samples positive for C. burnetii, as well as farms without any positive samples
. Furthermore, it is an interesting observation that rats, a potential reservoir for C. burnetii, collected on a cattle farm showed a different MLVA genotype (type E) in comparison to rats collected on a dairy goat farm (MLVA genotype A). Genotype E was also encountered in environmental samples obtained from a dairy goat farm, but only when genotype A was also detected. MLVA genotype F was observed on a single dairy sheep farm only and not on dairy goat farms, or non-dairy sheep farms. Although these data may suggest a preferential association of particular genotypes with specific hosts, further studies are needed to substantiate such a link. Finally, adequate source-finding studies for accurate source-attribution in the Dutch situation require a more detailed analysis of the strains present in animals, the environment and humans. Such studies would benefit from a method that enables strain differentiation at a higher resolution, both in time and in space. This requires improved genotyping methods specifically designed for the genotypes encountered in the Dutch outbreaks.