Characterization of the bacterial communities of life stages of free living lone star ticks (Amblyomma americanum).

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5-3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7-100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs

Multivariate analysis of the effect of sex and life stage on the relatedness of <i>Amblyomma americanum</i> bacterial communities.

<p>Tick DNAs from the archival collection (n = 27) were older, collected in a different geographical region, and processed using different protocols than tick DNAs from the Georgia collection (n = 104).</p><p>*Corrected for multiple comparisons using Holm’s method; α  = 0.05.</p

Significance of co-infection patterns between selected operational taxonomic units (OTUs).

<p>Only data from the Georgia tick DNA sample collection is shown. The upper triangle represents <i>p</i>-values of positive associations (codetection in the same sample) between OTUs and the lower triangle the <i>p</i>-values of negative associations. Black cells were not evaluated due to low expected co-occurrence (<1) between the OTUs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102130#pone.0102130-Veech1" target="_blank">[49]</a>.</p

Non-metric multi-dimensional scaling (NMDS) of a Bray-Curtis distance matrix describing archival <i>Amblyomma americanum</i> bacterial communities.

<p>Each point symbolizes a single tick’s community (n = 27); some points may overlap completely. Point and ellipse colors indicate life stage; ellipses represent 95% confidence intervals around life stage centroids. Non-overlapping centroids are considered significantly different at α = 0.05. R² values in the upper left corner of plots describe the amount of variation in the data set explained by the groupings. The stress value is a measure of the disagreement between the rank order in the original data set and that in the NMDS (lower numbers indicate better agreement). Numbers indicate the species scores for the plot as follows: (1) <i>Coxiella</i>, (2) <i>Rickettsia</i>, (3) <i>Midichloria</i> 1, (4) <i>Borrelia</i>, (5) <i>Francisella</i>, (6) <i>Midichloria</i> 2, (7) <i>Ehrlichia</i>, (8) Enterobacteriaceae, (9) <i>Pseudomonas</i> 1, (10) Burkholderiaceae, (11) Acidobacteria Gp1, (12) <i>Pseudomonas</i> 2, (13) Rhizobiales 2, (14) <i>Bacillus</i>, (15) Acetobacteraceae 1, (16) Acetobacteraceae 2, (17) Bacteria 1, (18) Bacteria 2.</p

Life stage and geographic origin of <i>Amblyomma americanum</i> ticks used in bacterial community analyses.

§<p>Nymphs collected in July 2010; adults in July 2010 and May 2011.</p><p>*Nymphs collected in August 2010; adults in May 2011.</p><p>NT = Not tested.</p

Non-metric multi-dimensional scaling (NMDS) of a Bray-Curtis distance matrix describing Georgian <i>Amblyomma americanum</i> bacterial communities.

<p>Each point symbolizes a single tick’s community (n = 104); some points may overlap completely. Point and ellipse colors indicate life stage; ellipses represent 95% confidence intervals around life stage centroids. Non-overlapping centroids are considered significantly different at α = 0.05. R² values in the upper left corner of plots describe the amount of variation in the data set explained by the groupings. The stress value given is a measure of the disagreement between the rank order in the original data set and that in the NMDS (lower numbers indicate better agreement). Brown numbers indicate the species scores for select OTUs as follows: (1) <i>Coxiella</i>, (2) <i>Rickettsia</i>, (3) <i>Midichloria</i> 1, (4) <i>Borrelia</i>, (5) <i>Ehrlichia</i>, (6) Bacillales 1, (7) <i>Wolbachia</i>, (8) Rhizobiales 1, (9) <i>Rhodobaca</i>, (10) Bacillales 2, (11) <i>Nitriliruptor</i>, (12) Bacillales 3.</p

Bacterial operational taxonomic unit (OTU) sequence abundance detected in tick samples.

<p>OTUs present in greater than one third of all tick samples were considered broadly distributed. OTUs present at a mean abundance ≥1% of the community (i.e. 10 sequences) were considered locally abundant. Sequence abundance values are slightly offset to reveal overlapping OTUs.</p

\u3cem\u3eRickettsia typhi\u3c/em\u3e Is Still Present in Southeastern Georgia, USA

Background: Rickettsia typhi is the causative agent of murine typhus, a zoonotic disease that is sustained in nature primarily through the rat to rat flea transmission cycle. Southeastern Georgia was historically associated with outbreaks of this disease; however, its current occurrence in Georgia is unknown. Rickettsia felis, another human pathogen, is primarily associated with cat fleas and can often be found on cats, dogs, and opossums. The purpose of this study was to characterize rickettsial bacteria that are associated with fleas infesting companion and wild animals in Southeastern Georgia. Methods: Fleas were collected from companion cats, dogs, and from opossums and a cotton rat that were trapped in woodland nonhuman inhabited areas in Bulloch, Fulton and Warren Counties, Georgia in 2011-2014. The fleas were identified to species and the DNA was extracted using Qiagen DNeasy kit according to the manufacturer’s directions. Flea DNA were individually tested using TaqMan assays specific for R. typhi and R. felis. Results: Only Ctenocephalides felis were collected from cats. Dogs were infested with C. felis and Pulex simulans (n=2). Opossums were mainly infested with Polygenis gwyni (n=68) while only a few C. felis (7) and Orchopeas howardi (3), were found. R. typhi DNA was detected in 4% of P. gwyni and 12% of C. felis tested, R. felis DNA was detected in 76.61% of C. felis and 14% of P. gwyni tested. Summary and Conclusions: We demonstrated that R. typhi is still present in the area and that there is a prevalent circulation of R. felis in C. felis in Georgia. This is the first report of this agent in P. gwyni. Since R. typhi is still present it may be associated with other reservoir hosts, outside of its classic urban ratrat flea cycle. Our data suggests that companion animals may become infested when coming in contact with sylvatic animals and vice versa