Spleen microbiota compositions at phylum level.

<p>Color-coded bar plot showing the proportion of different bacterial phyla present in 35 mouse and shrew samples from Chongming Island, Shanghai city. The 4 shrew samples were marked with *, and the left 31 samples were mice; Samples marked with + were <i>Anaplasma</i>-positive.</p

The spleen microbiota of small wild mammals reveals distinct patterns with tick-borne bacteria

<div><p>Background</p><p>Wild mammals serve as reservoirs for a variety of microbes and play an important role in the enzootic cycles of these microbes. Some of them are vector-borne bacteria in the genera <i>Anaplasma</i>, <i>Ehrlichia</i> and <i>Rickettsia</i> of the order Rickettsiales, which can cause febrile illnesses in human beings as well as animals. <i>Anaplasma</i> spp., <i>Ehrlichia</i> spp. and many spotted fever group (SFG) <i>Rickettsia</i> spp. are transmitted to mammalian hosts by tick vectors during blood meals. As a powerful sequencing method, the next generation sequencing can reveal the complexity of bacterial communities in humans and animals. Compared with limited studies on blood microbiota, however, much fewer studies have been carried out on spleen microbiota, which is very scarce in wild mammals. Chongming Island is the third biggest island in China. It was unclear whether there were any vector-borne bacteria in Chongming Island. In the present study, we explored the bacterial microbiota in the spleens of wild mice and shrews from the rural areas of Chongming Island and investigated the prevalence of vector-borne bacteria.</p><p>Methodology/Principal findings</p><p>Genomic DNAs were extracted from the spleen samples of 35 mice and shrews. The 16S rDNA V3-V4 regions of the DNA extracts were amplified by PCR and subjected to the 16S rDNA-targeted metagenomic sequencing on an Illumina MiSeq platform. All the 35 spleen samples obtained data with sufficient coverage (99.7–99.9%) for analysis. More than 1,300,000 sequences were obtained after quality control and classified into a total of 1,967 operational taxonomic units (OTUs) clustered at 97% similarity. The two most abundant bacterial phyla were Firmicutes and Proteobacteria according to the analysis of rarefied sequences. Among the bacterial communities detected in this study, <i>Anaplasma</i>, <i>Rickettsia</i> and <i>Coxiella</i> were adjacently clustered by hierarchical analysis. Significant differences in many bacterial features between <i>Anaplasma</i>-positive and <i>Anaplasma</i>-negative samples were identified by LEfSe analysis and Wilcoxon rank-sum test, suggesting that the <i>Anaplasma</i>-infection of small wild mammals was associated with a specific pattern of spleen microbiota.</p><p>Conclusions/Significance</p><p>Our study has comprehensively characterized the complex bacterial profiles in the spleens of wild mice and shrews from Chongming Island, Shanghai city. This work has revealed distinct spleen bacterial communities associated with tick-borne bacteria in wild animals. The detection of tick-borne bacteria highlights the risk of contracting pathogens with public health importance upon tick-exposure in the studied areas.</p></div

Histogram of the linear discriminant analysis (LDA) scores.

<p>Differentially abundant bacterial features enriched in <i>Anaplasma</i>-positive and <i>Anaplasma</i>-negative samples, respectively. The absolute values of LDA scores were > 2 (p < 0.05). A: <i>Anaplasma</i>-positive, in red; N_A: <i>Anaplasma</i>-negative, in green.</p

Diversities in the spleen microbiota of 35 mice and shrews.

<p>Principal coordinate analysis (PCoA) plots of the spleen microbiota based on the Bray-Curtis metrics considering animal genders, types, geographic sites or infection with <i>Anaplasma</i>. (A), male versus female groups. (B), mouse versus shrew groups. (C), different geographic site groups. (D), <i>Anaplasma</i>-positive versus <i>Anaplasma</i>-negative groups. F: female; M: male. Mi: mice; Sh: shrews. A: <i>Anaplasma</i>-positive; N_A: <i>Anaplasma</i>-negative.</p

Wilcoxon rank-sum test.

<p>Differentially abundant phyla enriched in <i>Anaplasma</i>-positive and <i>Anaplasma</i>-negative samples, respectively, analyzed by Wilcoxon rank-sum test (p < 0.05; q< 0.01). A: <i>Anaplasma</i>-positive, in green; N_A: <i>Anaplasma</i>-negative, in yellow.</p

Differentially abundant bacterial taxa in the spleen microbiota between <i>Anaplasma</i>-positive and <i>Anaplasma</i>-negative samples identified by LEfSe analysis.

<p>Cladogram showed the highly represented taxa in <i>Anaplasma</i>-positive and <i>Anaplasma</i>-negative samples, respectively. A: <i>Anaplasma</i>-positive, in red; N_A: <i>Anaplasma</i>-negative, in green.</p

The detection of vector-borne bacteria, <i>Anaplasma</i>, <i>Ehrlichia</i>, <i>Rickettsia</i>, <i>Coxiella</i> and <i>Bartonella</i>, in the spleen samples of 35 small wild mammals from Chongming Island, Shanghai city.

<p>The detection of vector-borne bacteria, <i>Anaplasma</i>, <i>Ehrlichia</i>, <i>Rickettsia</i>, <i>Coxiella</i> and <i>Bartonella</i>, in the spleen samples of 35 small wild mammals from Chongming Island, Shanghai city.</p

Hierarchical cluster tree.

<p><i>Anaplasma</i>-positive and <i>Anaplasma</i>-negative samples were hierarchically clustered using UPGMA algorithm based on Bray-Curtis distances. A: <i>Anaplasma</i>-positive, in red; N_A: <i>Anaplasma</i>-negative, in green.</p

Predicted binding sites of Rv0081 selected for experimental verifications.

<p>Predicted binding sites of Rv0081 selected for experimental verifications.</p

Electrophoretic mobility shift assay for Rv0081.

<p>For each PBS, the DNA (10 mM) was incubated with increasing concentrations (0, 1nM, 1.7nM and 2.3nM) of the purified Rv0081 protein (lanes 1–4, respectively).</p