Samples were collected in six different states, which are identified by their geographic regions in Table . Approximately 270 samples were taken in each state, with a total of 1,622 samples included in the analysis. Out of these 1,622 samples, 386 were positive by a PCR assay that specifically detects the IS1111
insertion sequence of C. burnetii
). Thus, 23.8% of the samples were positive for C. burnetii
DNA. The percentage of positive samples in each state varied widely. The highest percentage was found at the sites in the Rocky Mountains region, where 44.6% of the samples analyzed indicated the presence of C. burnetii
. On the low end, the East Coast state had only 6% of samples positive. Overall, these data indicate that background levels of C. burnetii
in the environment are quite high and that C. burnetii
DNA is found over a broad geographic distribution.
Determination of the presence of C. burnetii DNA in environmental samples from six states
To confirm that these samples were positive for C. burnetii
, 23 of the IS1111
-positive samples were analyzed using a real-time PCR assay that targets the C. burnetii com1
). Because com1
is a single-copy gene, this assay is less sensitive than the IS1111
PCR assay, so it was expected that many of the IS1111
-positive samples would be negative by the com1
assay due to limits of detection. In this analysis, 15 out of 23 samples were positive for C. burnetii com1
. This suggests that the IS1111
assay is reliably detecting C. burnetii
genomic DNA in these environmental samples. The samples that were positive for IS1111
but negative for com1
all had IS1111 CT
values that were greater than 30.7 and most likely did not have a sufficient number of genomes to be detected by the less sensitive com1
assay. A 438-bp section of the com1
gene was also amplified from three of the environmental DNA samples by PCR, and these com1
gene fragments were sequenced. In all three cases, the environmental com1
gene fragment had 100% identity to published C. burnetii com1
Within each state, samples were taken at three different sites. The sites were roughly equivalent to a county and were the source of 90 samples. Each site consisted of nine locations (small farm, office building, school, etc.) from which 10 samples each were collected. We considered the possibility that the percentage of positive samples in some states could be strongly influenced by only one of the sites having a very high or very low percentage of positive samples. However, the percentage of positive samples taken in the different sites was fairly consistent within a state (Fig. ), the largest exception being a relatively low percentage of positive samples in one of the Rocky Mountain sites. These results suggest that factors affecting the presence of C. burnetii at a given site are not site specific and influence a fairly large geographic area.
FIG. 1. Percentage of Coxiella-positive samples at each of the 18 sampling sites. Environmental samples were evaluated for the presence of C. burnetii DNA using an IS1111 PCR assay. Approximately 90 samples were acquired at each of the 18 sites, and the percentages (more ...)
The presence of C. burnetii
in herds of livestock and the known association of animal contact with human exposure (2
) raise the possibility that the numbers of livestock at a given site could influence the ability to find Coxiella
-positive samples. To investigate this possibility, the percentage of positive samples at each site was compared to the density of cows and sheep at each site. As shown in Fig. , there was a very poor correlation between livestock density and the percentage of positive samples. This indicates that the overall numbers of livestock at a given site are not a major factor in the ability of Coxiella
to infiltrate the local environment. This suggests that factors influencing much larger geographic areas are more important, as indicated by the differences between states. Inclusion of goat populations in this analysis did not influence the results, as goat populations are small compared to populations of sheep and cows. Analysis of goat densities alone also did not correlate with the percentage of Coxiella
-positive samples (data not shown).
FIG. 2. Comparison of the percentages of Coxiella-positive samples to environmental factors. The percentages of samples with C. burnetii DNA at each of the 18 sampling sites were plotted against livestock density (A), human population density (B), annual mean (more ...)
Other site-specific factors were compared to the percentage of positive samples at the sites. The human population density (Fig. ), mean annual temperature (Fig. ), and annual precipitation (Fig. ) did not correlate with the ability to detect positive samples at the sites. Other factors such as median household income, percentage of Hispanic persons, and percentage of residents living in poverty also had a very weak correlation with the presence of C. burnetii DNA (correlation coefficients less than 0.52 [data not shown]). This further supports the idea that the characteristics of the regional environment are not a large factor in determining the prevalence of C. burnetii at these sites.
Although the general environment of the sites analyzed did not make a substantial difference, the known association of C. burnetii
with wild and domesticated animals (2
) led us to consider the effect of livestock directly at the sampling locations on the ability to detect C. burnetii
DNA at those locations. For this analysis, all of the samples were categorized as coming from a location where livestock were likely to be encountered (dairy farm, veterinary hospital, etc.) or from a location where livestock were unlikely to be found (post office, grocery store, etc.). When the percentages of positive samples coming from these two types of locations were calculated, the samples from locations with animals were positive 28.4% of the time, whereas samples from locations without animals were positive only 18.4% of the time (Fig. ). This is a statistically significant difference (Pearson's chi-square of 22.06; P
of <0.00001) and shows that the presence of livestock at the place where the samples were taken can make the presence of C. burnetii
DNA more likely. However, this analysis also shows that a large number of positive samples (187) came from locations where livestock are not normally found. There was no evidence that the presence of livestock in the surrounding area or the local climate influenced the presence of C. burnetii
in the collected samples.
FIG. 3. Percentage of Coxiella-positive samples that were taken from locations that had livestock or taken from locations without livestock. This analysis was done on all samples (A) and on highly positive samples (B). Samples were defined as highly positive (more ...)
Because a quantitative assay was used to detect C. burnetii, a more stringent threshold could be placed on the samples to look only at samples considered to be “highly positive.” If a cutoff for highly positive samples is set at a threshold cycle of less than 34, then 55 samples out of 1,622 total samples (3.4%) are highly positive. If these 55 highly positive samples are segregated based on the presence of livestock at the sampling location, a fairly striking segregation is found: 46 of the 55 highly positive samples are found at locations where livestock are expected (Fig. ). Thus, the presence of livestock makes a very significant difference in whether highly positive samples will be found at a particular location.
If the locations where highly positive samples were found are listed, the locations that had livestock and also highly positive samples are not surprising. These locations are dairies (six), farms (three), veterinary hospitals (two), ranches (two), goat facilities, a cattle feedlot, fairgrounds, an experiment station, and a livestock auction, i.e., places where the presence of C. burnetii-positive animals would not be surprising and where contamination of the local environment could be easily explained. A bit more unexpected was the presence of highly positive samples at the locations where close contact with livestock is not expected: a bank, a co-op/general store, a state government building, a community center, a county administration building, a high school, a retail store, and a city hall. The source of C. burnetii DNA at these locations is unknown, but the organisms could be carried there by human foot traffic, the wind, birds, or other small animals.
To determine if any viable C. burnetii was present in these environmental samples, six samples (5 g each) positive by both IS1111 and com1 PCR were prepared in the same way as described for a DNA extraction. However, in this case the high-speed PBS pellet was resuspended in 1 ml of PBS, and 100 μl of this mixture was injected into each of two BALB/c mice by the intraperitoneal route. Four of the samples were from the West Coast state, one was from the East Coast state, and one was from the southeastern state. The com1 PCR from these samples enabled a rough estimate of the number of genome equivalents that were injected into the mice. These ranged from 16,000 to over 1 × 106 (Table ). For two of the samples (both from the West Coast state), the mice became ill soon after the injection and had to be sacrificed. For the other four samples, the mice were sacrificed at 3 weeks after the injection of the environmental sample. Analysis of these mice indicated that they had developed antibody titers against C. burnetii Nine Mile Phase 1 and Phase 2 and had a mild splenomegaly. com1 PCR of spleen DNA indicated that the injected C. burnetii had expanded significantly in vivo (Table ). These results suggest that viable C. burnetii can be found in this collection of environmental samples.
Testing for viability in BALB/c mice