Geographic distribution and genetic diversity of the Ehrlichia sp. from Panola Mountain in Amblyomma americanum

Background: A novel Ehrlichia, closely related to Ehrlichia ruminantium, was recently discovered from Panola Mountain State Park, GA, USA. We conducted a study to determine if this agent was recently introduced into the United States. Methods: We developed a sensitive PCR assay based on the conserved gltA (citrate synthase) gene and tested DNA samples extracted from 1964 field-collected and 1835 human-biting Amblyomma americanum from 23 eastern states of the USA. Results: The novel agent was detected in 36 ticks collected from 10 states between 1998 and 2006. Infected ticks were collected both from vegetation (n = 14, 0.7%) and from humans (n = 22, 1.2%). Fragments of the conserved gltA gene and the variable map1 gene were sequenced from positive samples. Two distinct clades, with 10.5% nucleic acid divergence over the 730 bp map1 sequence, were identified. Conclusion: These data suggest that the Panola Mountain Ehrlichia was not recently introduced to the United States; this agent has an extensive distribution throughout the range of its tick vector, has been present in some locations for several years, and displays genetic variability. Furthermore, people in several states were exposed to this agent through the bite of infected ticks, underscoring the potential public health risk of this emerging ehrlichiosis

Origin and Evolution of Sulfadoxine Resistant Plasmodium falciparum

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions

Detection of Ehrlichia chaffeensis in adult and nymphal Amblyomma americanum (Acari: Ixodidae) ticks from Long Island, New York

The lone star tick, Amblyomma americanum (L.), has increased in abundance in several regions of the northeastern United States, including areas of Long Island, NY. Adult and nymphal stage A. americanum collected from several sites on Long Island were evaluated for infection with Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), by using a nested polymerase chain reaction assay. Fifty-nine (12.5%) of 473 adults and eight of 113 pools of five nymphs each (estimated minimum prevalence of infection 1.4%) contained DNA of E. chaffeensis. These data, coupled with the documented expansion of lone star tick populations in the northeastern United States, confirm that E. chaffeensis is endemic to many areas of Long Island and that HME should be considered among the differential diagnoses of the many distinct tick-borne diseases that occur in this region

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

Prevalence of Ehrlichia, Borrelia, and Rickettsial agents in Amblyomma americanum (Acari: Ixodidae) collected from nine states

Ambyomma americanum (L.) (Acari: Ixodidae) is an aggressive tick that feeds on humans during all postembryonic life stages. In many regions of the United States, it is the tick most commonly found attached to humans. Public health interest has grown recently, due to the recognition of new human pathogens transmitted by A. americanum and the expanding distribution of the tick. A. americanum is a vector of several bacteria pathogenic to humans. Ehrlichia chaffeensis and Ehrlichia ewingii cause moderate-to-severe febrile illness. Rickettsia amblyommii, a member of the spotted fever group Rickettsia, also has recently been implicated as a possible human pathogen based on serologic evidence from persons recovering from illness after a tick bite. We have determined the prevalence of infection of Ehrlichia chaffeensis, E. ewingii, Borrelia lonestari, and R. amblyommii within A. americanum ticks from 29 sites in nine states. Overall infection prevalences were 4.7% for E. chaffeensis (range, 0-27%), 3.5% for E. ewingii (range, 0-18.6%), 2.5% for B. lonestari (range, 0-12.2%), and 41.2% for R. amblyommii (range, 0-84.0%). In addition, 87 ticks (4.3%) were infected with two or more bacteria. This report documents new distribution records for E. ewingii, B. lonestari, and R. amblyommii and underscores the nonhomogeneous distribution of pathogen foci of infection. Additional surveillance throughout the range of A. americanum is warranted to increase physician and public awareness of the risk of disease to humans from exposure to the agents transmitted by this tick. © 2006 Entomological Society of America

Prevalence of Ehrlichia

Ambyomma americanum (L.) (Acari: Ixodidae) is an aggressive tick that feeds on humans during all postembryonic life stages. In many regions of the United States, it is the tick most commonly found attached to humans. Public health interest has grown recently, due to the recognition of new human pathogens transmitted by A. americanum and the expanding distribution of the tick. A. americanum is a vector of several bacteria pathogenic to humans. Ehrlichia chaffeensis and Ehrlichia ewingii cause moderate-to-severe febrile illness. Rickettsia amblyommii, a member of the spotted fever group Rickettsia, also has recently been implicated as a possible human pathogen based on serologic evidence from persons recovering from illness after a tick bite. We have determined the prevalence of infection of Ehrlichia chaffeensis, E. ewingii, Borrelia lonestari, and R. amblyommii within A. americanum ticks from 29 sites in nine states. Overall infection prevalences were 4.7% for E. chaffeensis (range, 0-27%), 3.5% for E. ewingii (range, 0-18.6%), 2.5% for B. lonestari (range, 0-12.2%), and 41.2% for R. amblyommii (range, 0-84.0%). In addition, 87 ticks (4.3%) were infected with two or more bacteria. This report documents new distribution records for E. ewingii, B. lonestari, and R. amblyommii and underscores the nonhomogeneous distribution of pathogen foci of infection. Additional surveillance throughout the range of A. americanum is warranted to increase physician and public awareness of the risk of disease to humans from exposure to the agents transmitted by this tick. © 2006 Entomological Society of America

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

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

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