Detection limits of the DH and TSPE-UH suspension array formats.

a<p>Values displayed represent the lowest DNA concentration at which 95% of the positive samples are detected, as calculated by using probit analysis. ND = not determined.</p

DH suspension microarray measurement showing minor cross-reactivity.

<p>Three <i>Y. pestis</i> strains (Yp) and the no template control (NTC) are shown from a DH measurement. Probe <i>isf</i> showed a minor signal when a high load of <i>Y. pestis</i> genomic DNA was amplified.</p

Typical results from DH and TSPE-UH suspension microarrays detecting select pathogens.

<p>Two 17-plex bead arrays were developed for the detection of <i>B. anthracis</i> (Ba), <i>F. tularensis</i> (Ft), <i>Y. pestis</i> (Yp), <i>C. burnetii</i> (Cb) and an internal control for DNA extraction and microarray detection (Bt). The microarrays were based on (<b>A</b>) direct hybridization (DH), or (<b>B</b>) target specific primer extension combined with universal microarray hybridization (TSPE-UH) assay formats. Both microarrays make use of identical amplification products from a 16-plex asymmetric PCR. Mean fluorescence intensity (MFI) is displayed for the different probes that are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031958#pone-0031958-t001" target="_blank">Table 1</a>.</p

Oligonucleotides used for amplification and labeling of signature sequences and as fixed probes.

a<p>Excess primer = X, Limiting primer = L.</p>b<p><i>F. tularensis</i> subspecies <i>tularensis</i> yields amplicon of 307 bp, subspecies <i>novicida</i> and <i>mediasiatica</i> of 451 bp.</p

Detection of mixed pathogens by using DH and TSPE-UH suspension microarrays.

<p>Genomic DNA from <i>B. anthracis</i> (Ba), <i>F. tularensis</i> (Ft), <i>Y. pestis</i> (Yp), <i>C. burnetii</i> (Cb) was mixed in different ratios and measured by using DH (<b>A</b>) and TSPE-UH (<b>B</b>) microarrays. Mean fluorescence intensity (MFI) is displayed for the different pathogen-specific probes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031958#pone-0031958-t001" target="_blank">Table 1</a>). The detection of one pathogen is not impeded by the detection of the other targeted pathogens.</p

Table_1_Environmental Surveillance of Zoonotic Francisella tularensis in the Netherlands.DOCX

<p>Tularemia is an emerging zoonosis caused by the Gram-negative bacterium Francisella tularensis, which is able to infect a range of animal species and humans. Human infections occur through contact with animals, ingestion of food, insect bites or exposure to aerosols or water, and may lead to serious disease. F. tularensis may persist in aquatic reservoirs. In the Netherland, no human tularemia cases were notified for over 60 years until in 2011 an endemic patient was diagnosed, followed by 17 cases in the 6 years since. The re-emergence of tularemia could be caused by changes in reservoirs or transmission routes. We performed environmental surveillance of F. tularensis in surface waters in the Netherlands by using two approaches. Firstly, 339 samples were obtained from routine monitoring -not related to tularemia- at 127 locations that were visited between 1 and 8 times in 2015 and 2016. Secondly, sampling efforts were performed after reported tularemia cases (n = 8) among hares or humans in the period 2013–2017. F. tularensis DNA was detected at 17% of randomly selected surface water locations from different parts of the country. At most of these positive locations, DNA was not detected at each time point and levels were very low, but at two locations contamination was clearly higher. From 7 out of the 8 investigated tularemia cases, F. tularensis DNA was detected in at least one surface water sample collected after the case. By using a protocol tailored for amplification of low amounts of environmental DNA, 10 gene targets were sequenced. Presence of F. tularensis subspecies holarctica was confirmed in 4 samples, and in 2 of these, clades B.12 and B.6 were identified. This study shows that for tularemia, information regarding the spatial and temporal distribution of its causative agent could be derived from environmental surveillance of surface waters. Tracking a particular strain in the environment as source of infection is feasible and could be substantiated by genotyping, which was achieved in water samples with only low levels of F. tularemia present. These techniques allow the establishment of a link between tularemia cases and environmental samples without the need for cultivation.</p

Overview of monthly averaged outdoor temperature and pattern of kidding season (estimated monthly percentages of goats born in the Netherlands based on the paper of Hermans et al [22]).

<p>Overview of monthly averaged outdoor temperature and pattern of kidding season (estimated monthly percentages of goats born in the Netherlands based on the paper of Hermans et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151281#pone.0151281.ref022" target="_blank">22</a>]).</p

Univariable associations between the presence of <i>C</i>. <i>burnetii</i> in PM10 and spatial, temporal and spatial-temporal characteristics.

<p>Univariable associations between the presence of <i>C</i>. <i>burnetii</i> in PM10 and spatial, temporal and spatial-temporal characteristics.</p

Descriptive statistics of the spatial, temporal and spatial-temporal characteristics.

<p>Descriptive statistics of the spatial, temporal and spatial-temporal characteristics.</p

Significant temporal and spatial associations to the presence of <i>C</i>. <i>burnetii</i> in PM10, mutually adjusted in the multivariable model.

<p>Significant temporal and spatial associations to the presence of <i>C</i>. <i>burnetii</i> in PM10, mutually adjusted in the multivariable model.</p