Detection of Orientia spp. Bacteria in Field-Collected Free-Living Eutrombicula Chigger Mites, United States.

Scrub typhus, a rickettsial disease caused by Orientia spp., is transmitted by infected larval trombiculid mites (chiggers). We report the molecular detection of Orientia species in free-living Eutrombicula chiggers collected in an area in North Carolina, USA, to which spotted fever group rickettsiae infections are endemic.

Mosquito Blood Feeding Prevention Using an Extra-Low DC Voltage Charged Cloth.

Mosquito vector-borne diseases such as malaria and dengue pose a major threat to human health. Personal protection from mosquito blood feeding is mostly by treating clothing with insecticides and the use of repellents on clothing and skin. Here, we developed a low-voltage, mosquito-resistant cloth (MRC) that blocked all blood feeding across the textile and was flexible and breathable. The design was based on mosquito head and proboscis morphometrics, the development of a novel 3-D textile with the outer conductive layers insulated from each other with an inner, non-conductive woven mesh, and the use of a DC (direct current; extra-low-voltage) resistor-capacitor. Blockage of blood feeding was measured using host-seeking Aedes aegypti adult female mosquitoes and whether they could blood feed across the MRC and an artificial membrane. Mosquito blood feeding decreased as voltage increased from 0 to 15 volts. Blood feeding inhibition was 97.8% at 10 volts and 100% inhibition at 15 volts, demonstrating proof of concept. Current flow is minimal since conductance only occurs when the mosquito proboscis simultaneously touches the outside layers of the MRC and is then quickly repelled. Our results demonstrated for the first time the use of a biomimetic, mosquito-repelling technology to prevent blood feeding using extra-low energy consumption.

Rickettsia amblyommatis in Ticks: A Review of Distribution, Pathogenicity, and Diversity.

Rickettsia amblyommatis is a potentially pathogenic species of Rickettsia within the spotted fever group vectored by ticks. While many studies have been published on this species, there is debate over its pathogenicity and the inhibitory role it plays in diagnosing illnesses caused by other spotted fever group Rickettsia species. Many publications have recorded the high infection prevalence of R. amblyommatis in tick populations at a global scale. While this species is rather ubiquitous, questions remain over the epidemiological importance of this possible human pathogen. With tick-borne diseases on the rise, understanding the exact role that R. amblyommatis plays as a pathogen and inhibitor of infection relative to other tick-borne pathogens will help public health efforts. The goal of this review was to compile the known literature on R. amblyommatis, review what we know about its geographic distribution, tick vectors, and pathogenicity, assess relatedness between various international strains from ticks by phylogenetic analysis and draw conclusions regarding future research needed.

Rickettsia felis and Other Rickettsia Species in Chigger Mites Collected from Wild Rodents in North Carolina, USA.

Chiggers are vectors of rickettsial pathogenic bacteria, Orientia spp., that cause the human disease, scrub typhus, in the Asian-Pacific area and northern Australia (known as the Tsutsugamushi Triangle). More recently, reports of scrub typhus in Africa, southern Chile, and the Middle East have reshaped our understanding of the epidemiology of this disease, indicating it has a broad geographical distribution. Despite the growing number of studies and discoveries of chigger-borne human disease outside of the Tsutsugamushi Triangle, rickettsial pathogens in chigger mites in the US are still undetermined. The aim of our study was to investigate possible Rickettsia DNA in chiggers collected from rodents in North Carolina, USA. Of 46 chiggers tested, 47.8% tested positive for amplicons of the 23S-5S gene, 36.9% tested positive for 17 kDa, and 15.2% tested positive for gltA. Nucleotide sequence analyses of the Rickettsia-specific 23S-5S intergenic spacer (IGS), 17 kDa, and gltA gene fragments indicated that the amplicons from these chiggers were closely related to those in R. felis, R. conorii, R. typhi, and unidentified Rickettsia species. In this study, we provide the first evidence of Rickettsia infection in chiggers collected from rodents within the continental USA. In North Carolina, a US state with the highest annual cases of spotted fever rickettsioses, these results suggest chigger bites could pose a risk to public health, warranting further study.

Bacterial Isolates Derived from Nest Soil Affect the Attraction and Digging Behavior of Workers of the Red Imported Fire Ant, Solenopsis invicta Buren.

Populations of monogyne and polygyne red imported fire ants (RIFA), Solenopsis invicta Buren, are distributed throughout the southern United States. This ant species is hazardous to farm animals and workers, damages infrastructure, and depletes native arthropod populations. Colony expansion is affected by several biotic factors, but the effects of soil microbes on ant behavior related to soil excavation within nest sites have not been investigated. Consequently, we cultured bacteria from RIFA nest soils. The effects of individual bacterial isolates and bacterial cell densities on the choice of digging site as well as digging activity of monogyne and polygyne RIFA worker ants were evaluated in two-choice bioassays. Based on phylogenetic analysis, 17 isolates were selected and tested initially at 5 × 108 cells/mL and 20 workers per assay. Firmicutes (Bacillus, Paenibacillus, Brevibacillus) repelled the ants, but Arthrobacter woluwensis strongly attracted ants. Subsequently, the six isolates having the greatest positive or negative effects on ant behavior were evaluated at a lower bacterial cell and worker ant densities. Ant responses to these bacteria generally decreased as cell densities declined to 5 × 106 cells/mL. Observations of ant behavior during a three-hour, two-choice bioassay revealed that ants generally visited both control and bacteria-treated sand prior to making a digging site choice. Our research results indicate that soil bacteria may mediate ant nest expansion or relocation and foraging tunnel construction. Identification of bacterial metabolites that affect RIFA digging behavior merits additional research because these compounds may provide a basis for novel management strategies that repel RIFA away from sensitive infrastructure or attract fire ants to insecticidal baits.

Analyses of Bloodmeal Hosts and Prevalence of Rickettsia parkeri in the Gulf Coast Tick Amblyomma maculatum (Acari: Ixodidae) From a Reconstructed Piedmont Prairie Ecosystem, North Carolina.

Host feeding patterns and the prevalence of infection with Rickettsia parkeri were determined for the primary vector, Amblyomma maculatum Koch as well as sympatric tick species A. americanum (Linnaeus) and Dermacentor variabilis (Say) collected from a reconstructed prairie in the Piedmont region of North Carolina during 2011 and 2012. The occurrence of R. parkeri among A. maculatum adults and nymphs was 36.9% (45/122) and 33.3% (2/6), respectively. Rickettsia parkeri was detected in a single male A. americanum 2.3% (1/43). A PCR-reverse line blot hybridization assay of a 12S rDNA fragment amplified from remnant larval and nymphal bloodmeals of host-seeking ticks was used to identify bloodmeal hosts. Of the tick samples tested, bloodmeal host identification was successful for 29.3% (12/41) of adult A. americanum and 39.2% (20/51) of adult D. variabilis. For A. maculatum, bloodmeal host identification was successful for 50% (61/122) of adults collected from vegetation and 100% (4/4) of nymphs removed from cotton rats (Sigmodon hispidus Say and Ord). The cotton rat was the most common bloodmeal host with 59.0% (36/61) identified for adult A. maculatum. No statistically significant association was observed, however, between bloodmeal host and pathogen prevalence for any tick species. While the cotton rat was an important bloodmeal host for A. maculatum nymphs, this vertebrate did not appear to be the primary source of R. parkeri infection for A. maculatum.

Colonization by the Red Imported Fire Ant, Solenopsis invicta, Modifies Soil Bacterial Communities.

The long-standing association between insects and microorganisms has been especially crucial to the evolutionary and ecological success of social insect groups. Notably, research on the interaction of the two social forms (monogyne and polygyne) of the red imported fire ant (RIFA), Solenopsis invicta Buren, with microbes in its soil habitat is presently limited. In this study, we characterized bacterial microbiomes associated with RIFA nest soils and native (RIFA-negative) soils to better understand the effects of colonization of RIFA on soil microbial communities. Bacterial community fingerprints of 16S rRNA amplicons using denaturing gradient gel electrophoresis revealed significant differences in the structure of the bacterial communities between RIFA-positive and RIFA-negative soils at 0 and 10 cm depths. Illumina sequencing of 16S rRNA amplicons provided fine-scale analysis to test for effects of RIFA colonization, RIFA social form, and soil depth on the composition of the bacterial microbiomes of the soil and RIFA workers. Our results showed the bacterial community structure of RIFA-colonized soils to be significantly different from native soil communities and to evidence elevated abundances of several taxa, including Actinobacteria. Colony social form was not found to be a significant factor in nest or RIFA worker microbiome compositions. RIFA workers and nest soils were determined to have markedly different bacterial communities, with RIFA worker microbiomes being characterized by high abundances of a Bartonella-like endosymbiont and Entomoplasmataceae. Cloning and sequencing of the 16S rRNA gene revealed the Bartonella sp. to be a novel bacterium.

Mosquito-Textile Physics: A Mathematical Roadmap to Insecticide-Free, Bite-Proof Clothing for Everyday Life.

Garments treated with chemical insecticides are commonly used to prevent mosquito bites. Resistance to insecticides, however, is threatening the efficacy of this technology, and people are increasingly concerned about the potential health impacts of wearing insecticide-treated clothing. Here, we report a mathematical model for fabric barriers that resist bites from Aedes aegypti mosquitoes based on textile physical structure and no insecticides. The model was derived from mosquito morphometrics and analysis of mosquito biting behavior. Woven filter fabrics, precision polypropylene plates, and knitted fabrics were used for model validation. Then, based on the model predictions, prototype knitted textiles and garments were developed that prevented mosquito biting, and comfort testing showed the garments to possess superior thermophysiological properties. Our fabrics provided a three-times greater bite resistance than the insecticide-treated cloth. Our predictive model can be used to develop additional textiles in the future for garments that are highly bite resistant to mosquitoes.

Tick Ecdysteroid Hormone, Global Microbiota/Rickettsia Signaling in the Ovary versus Carcass during Vitellogenesis in Part-Fed (Virgin) American Dog Ticks, Dermacentor variabilis.

The transovarial transmission of tick-borne bacterial pathogens is an important mechanism for their maintenance in natural populations and transmission, causing disease in humans and animals. The mechanism for this transmission and the possible role of tick hormones facilitating this process have never been studied. Injections of physiological levels of the tick hormone, 20-hydroxyecdysone (20E), into part-fed (virgin) adult females of the American dog tick, Dermacentor variabilis, attached to the host caused a reduction in density of Rickettsia montanensis in the carcass and an increase in the ovaries compared to buffer-injected controls. This injection initiates yolk protein synthesis and uptake by the eggs but has no effect on blood feeding. Francisella sp. and R. montanensis were the predominant bacteria based on the proportionality in the carcass and ovary. The total bacteria load increased in the carcass and ovaries, and bacteria in the genus Pseudomonas increased in the carcass after the 20E injection. The mechanism of how the Rickettsia species respond to changes in tick hormonal regulation needs further investigation. Multiple possible mechanisms for the proliferation of R. montanensis in the ovaries are proposed.

Sphingobacterium phlebotomi sp. nov., a new member of family Sphingobacteriaceae isolated from sand fly rearing substrate.

A Gram-stain-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium, designated type strain SSI9T, was isolated from sand fly (Phlebotomus papatasi Scopoli; Diptera: Psychodidae) rearing substrate and subjected to polyphasic taxonomic analysis. Strain SSI9T contained phosphatidylethanolamine as a major polar lipid, MK-7 as the predominant quinone, and C16 : 1ω6c/C16 : 1ω7c, iso-C15 : 0, iso-C17 : 0 3-OH and C16 : 0 as the major cellular fatty acids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that SSI9T represents a member of the genus Sphingobacterium, of the family Sphingobacteriaceae sharing 96.5-88.0 % sequence similarity with other species of the genus Sphingobacterium. The results of multilocus sequence analysis using the concatenated sequences of the housekeeping genes recA, rplC and groL indicated that SSI9T formed a separate branch in the genus Sphingobacterium. The genome of SSI9T is 5 197 142 bp with a DNA G+C content of 41.8 mol% and encodes 4395 predicted coding sequences, 49 tRNAs, and three complete rRNAs and two partial rRNAs. SSI9T could be distinguished from other species of the genus Sphingobacterium with validly published names by several phenotypic, chemotaxonomic and genomic characteristics. On the basis of the results of this polyphasic taxonomic analysis, the bacterial isolate represents a novel species within the genus Sphingobacterium, for which the name Sphingobacterium phlebotomi sp. nov. is proposed. The type strain is SSI9T (=ATCC TSD-210T=LMG 31664T=NRRL B-65603T).

Oviposition-Site Selection of Phlebotomus papatasi (Diptera: Psychodidae) Sand Flies: Attraction to Bacterial Isolates From an Attractive Rearing Medium.

Phlebotomine sand flies are worldwide vectors of Leishmania parasites as well as other bacterial and viral pathogens. Due to the variable impact of traditional vector control practices, a more ecologically based approach is needed. The goal of this study was to isolate bacteria from the most attractive substrate to gravid Phlebotomus papatasi Scopoli sand flies and determine the role of bacterial volatiles in the oviposition attractancy of P. papatasi using behavioral assays. We hypothesized that gravid sand flies are attracted to bacterially derived semiochemical cues associated with breeding sites. Bacteria were isolated from a larvae-conditioned rearing medium, previously shown to be highly attractive to sand flies. The isolated bacteria were identified by amplifying and sequencing 16S rDNA gene fragments, and 12 distinct bacterial species were selected for two-choice olfactometer bioassays. The mix of 12 bacterial isolates elicited strong attraction at the lower concentration of 107 cells per ml and significant repellence at a high concentration of 109 cells per ml. Three individual isolates (SSI-2, SSI-9, and SSI-11) were particularly attractive at low doses. In general, we observed dose-related effects, with some bacterial isolates stimulating negative and some positive dose-response curves in sand fly attraction. Our study confirms the important role of saprophytic bacteria, gut bacteria, or both, in guiding the oviposition-site selection behavior of sand flies. Identifying the specific attractive semiochemical cues that they produce could lead to development of an attractive lure for surveillance and control of sand flies.

New Mosquitocide Derived From Volcanic Rock.

Malaria, dengue, yellow fever, and the Zika and West Nile Viruses are major vector-borne diseases of humans transmitted by mosquitoes. According to the World Health Organization, over 80% of the world's population is at risk of contacting these diseases. Insecticides are critical for mosquito control and disease prevention, and insect insecticide resistance is on the increase; new alternatives with potentially different modes of action from current chemistry are needed. During laboratory screening of industrial minerals for insecticide activity against Anopheles gambiae (Giles) (Diptera: Culicidae) we discovered a novel mechanical insecticide derived from volcanic rock (MIVR) with potential use as a residual spray. In modified WHO cone tests, the time to 50% mortality was 5 h under high-humidity conditions. MIVR treated surfaces demonstrated no mosquito repellency. In field studies where the mechanical insecticide was applied to wood using standard spray equipment and then placed under stilt homes in New Orleans, LA, the residual activity was >80% after 9 wk against Aedes aegypti (L.) (Diptera: Culicidae), Aedes albopictus (Skuse) (Diptera: Culicidae) and Culex quinquefasciatus (Say) (Diptera: Culicidae) (with similar efficacy to a positive chemical insecticide control). In scanning electron microcopy studies, the MIVR was transferred as particles mostly to the legs of the mosquito. This wettable powder made from volcanic rock is a mechanical insecticide representing a potential new mode of action different from current chemistry for mosquito control and is in commercial development under the trade name Imergard™WP as an indoor and outdoor residual spray.

Development of an Insecticide-Free Trapping Bednet to Control Mosquitoes and Manage Resistance in Malaria Vector Control: A New Way of Thinking.

Mosquito-borne malaria kills 429,000 people each year with the problem being acute in sub-Saharan Africa. The successes gained with long-lasting pyrethroid-treated bednets are now in jeopardy because of wide-spread, pyrethroid resistance in mosquitoes. Using crowd modeling theory normalized for standard bednet architecture, we were able to design an attract-trap-kill technology for mosquitoes that does not require insecticides. Using three-dimensional polyester knitting and heat fixation, trap funnels were developed with high capture efficacy with no egression under worst-case laboratory conditions. Field testing in Africa in WHO huts with Gen1-3 T (trap)-Nets validated our model, and as predicted, Gen3 had the highest efficacy with a 4.3-fold greater trap-kill rate with no deterrence or repellency compared to Permanet 2.0, the most common bednet in Africa. A T-Net population model was developed based on field data to predict community-level mosquito control compared to a pyrethroid bednet. This model showed the Gen3 non-insecticidal T-Net under field conditions in Africa against pyrethroid resistant mosquitoes was 12.7-fold more efficacious than single chemical, pyrethroid-treated nets.

Association between lone star tick bites and increased alpha-gal sensitization: evidence from a prospective cohort of outdoor workers.

BACKGROUND: Alpha-gal is an oligosaccharide implicated in delayed anaphylaxis following red meat consumption. Exposure to tick bites has been correlated with development of an allergic response to alpha-gal. However, evidence prospectively linking exposure to a single tick species and an immune response to alpha-gal is lacking. METHODS: We used serum samples from a prior study cohort of outdoor workers in North Carolina, USA, with high exposure to the lone star tick, Amblyomma americanum, to prospectively evaluate the relationship between tick bites and anti-alpha-gal IgE antibodies. RESULTS: Individuals who reported exposure to one or more tick bites were significantly more likely to have a positive change in anti-alpha-gal IgE compared to individuals with no reported tick bites. This relationship was not dependent on time. A trend toward increasing number of tick bites and increased anti-alpha-gal IgE levels was observed but not statistically significant. CONCLUSION: To our knowledge, this is the first study to prospectively link documented exposure to A. americanum bites and increased sensitization to alpha-gal in a cohort of outdoor workers. Our results support the role of A. americanum as likely agents for eliciting an allergic response to red meat, and highlight the importance of preventing tick bites.

The egg and larval pheromone dodecanoic acid mediates density-dependent oviposition of Phlebotomus papatasi.

BACKGROUND: Gravid females assess the conditions of oviposition sites to secure the growth and survival of their offspring. Conspecific-occupied sites may signal suitable oviposition sites but may also impose risk due to competition or cannibalism at high population density or heterogeneous larval stage structure, respectively. Chemicals in the habitat, including chemicals emitted from other organisms, serve as cues for females to assess habitat conditions. Here, we investigated the attraction and oviposition preference of the Old World cutaneous leishmaniasis vector, Phlebotomus papatasi, to young and old conspecific stages, including eggs and evaluated the effect of a semiochemical associated with eggs and neonate larvae. METHODS: Attraction and oviposition preference of Ph. papatasi to each of various life stages (eggs, first-, second-, third-, fourth-instar larvae, pupae and male and female adults) was investigated using cage and oviposition jar behavioral assays. Identification of organic chemical compounds extracted from eggs was performed using GC-MS and chemicals were tested in the same behavioral assays in a dose-response manner. Behavioral responses were statistically analyzed using logistic models. RESULTS: Gravid Ph. papatasi females were significantly attracted to and preferred to oviposit on medium containing young life stages (eggs and first instars). This preference decreased towards older life stages. Dose effect of eggs indicated a hump-shaped response with respect to attraction but a concave-up pattern with respect to oviposition. Chemical analysis of semiochemicals from eggs and first-instar larvae revealed the presence of dodecanoic acid (DA) and isovaleric acid. Sand flies were attracted to and laid more eggs at the lowest DA dose tested followed by a negative dose-response. CONCLUSIONS: Findings corroborated our hypothesis that gravid sand flies should prefer early colonized oviposition sites as indicators of site suitability but avoid sites containing older stages as indicators of potential competition. Findings also supported the predictions of our hump-shaped oviposition regulation (HSR) model, with attraction to conspecific eggs at low-medium densities and switching to repellence at high egg densities. This oviposition behavior is mediated by DA that was identified from surface extracts of both eggs and first-instar larvae. Isovaleric acid was also found in extracts of both stages.

ImergardTMWP: A Non-Chemical Alternative for an Indoor Residual Spray, Effective against Pyrethroid-Resistant Anopheles gambiae (s.l.) in Africa.

Malaria is the deadliest mosquito-borne disease and kills predominantly people in sub-Saharan Africa (SSA). The now widespread mosquito resistance to pyrethroids, with rapidly growing resistance to other insecticide classes recommended by the World Health Organization (WHO), may overturn the successes gained in mosquito control in recent years. It is of utmost importance to search for new, inexpensive, and safe alternatives, with new modes of action, that might improve the efficacy of current insecticides. The efficacy of a novel mechanical insecticidal mineral derived from volcanic rock, ImergardTMWP, was investigated to determine its efficacy as a stand-alone residual wall spray and as a mixture with deltamethrin (K-Othrine® Polyzone) in experimental huts in Cove, Benin. The evaluation was conducted with susceptible (Kisumu) and wild-type Anopheles gambiae (s.l.). Deltamethrin applied alone demonstrated 40-45% mortality (at 72 h post-exposure) during the first four months, which declined to 25% at six months for wild An. gambiae from Cove. ImergardTMWP alone and mixed with deltamethrin, under the same assay conditions, produced 79-82% and 73-81% mortality, respectively, during the same six-month period. ImergardTMWP met the 80% WHO bio-efficacy threshold for residual activity for the first five months with 78% residual activity at six months. ImergardTMWP can be used as a mixture with chemical insecticides or as a stand-alone pesticide for mosquito control in Africa.

Diel periodicity and visual cues guide oviposition behavior in Phlebotomus papatasi, vector of old-world cutaneous leishmaniasis.

BACKGROUND: Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14:10 hrs light:dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. CONCLUSION AND SIGNIFICANCE: Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies' sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed.

A Diverse Microbial Community Supports Larval Development and Survivorship of the Asian Tiger Mosquito (Diptera: Culicidae).

Laboratory microcosm experiments were conducted to evaluate effects of bacteria isolated from senescent white oak leaves on the growth and survivorship of larval Aedes albopictus (Skuse). Larvae hatched from surface-sterilized eggs were reared in microcosms containing individual bacterial isolates, combined isolates (Porphyrobacter sp., Enterobacter asburiae, Acidiphilium rubrum, Pseudomonas syringae, and Azorhizobium caulinodans), a positive control containing a microbial community from an infusion of white oak leaves, and a negative control consisting of sterile culture media. Experiments were conducted for 21 d after which microcosms were deconstructed, larval survivorship was calculated, and bacteria contained in pupae, and adults that developed were quantified to determine rates of transstadial transmission. Positive control microcosms containing diverse microbial communities had an average (±SE) pupation rate of 89.3 (±5.8)% and average larval survivorship of 96.0 (± 2.3)%. Pupation in microcosms with bacterial isolates only occurred twice among all experimental replications; average larval survivorship ranged from 19 to 56%, depending on treatment. Larval growth was not found to be dependent on bacterial isolate density or isolate species, and larval survivorship was dependent on bacterial isolate density, not on isolate species. Potential mechanisms for failed development of larvae in microcosms with bacterial isolates are discussed. Bacterial isolates alone did not support larval development. High larval survivorship in positive control microcosms suggests that a diverse microbial community is required to complete larval development. Additional studies are needed to evaluate larval growth and survivorship on nonbacterial microbes, such as fungi and protozoa.

Bacterial microbiome of the chigger mite Leptotrombidium imphalum varies by life stage and infection with the scrub typhus pathogen Orientia tsutsugamushi.

Scrub typhus is a mites-borne rickettsiosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. The disease is potentially life threatening and is prevalent in tropical Asia, islands of the western Pacific Ocean and northern Australia where an estimated one million cases occur annually. Orientia tsutsugamushi is transmitted by the bite of larval mites in the genus Leptotrombidium. In the present study, the composition of the microbiome in larvae, deutonymphs and adult males and females from laboratory colonies of L. imphalum that were infected as well as uninfected with O. tsutsugamushi were investigated by high-throughput sequencing of the bacterial 16S rRNA gene. Notably, the bacterial microbiomes of infected adult females were dominated by sequences of O. tsutsugamushi and an unidentified species of Amoebophilaceae, which together comprised 98.2% of bacterial sequences. To improve the taxonomic resolution of the Amoebophilaceae OTU a nearly full length sequence of the 16S rRNA gene was amplified, cloned, and Sanger sequenced. Infected female mites had 89 to 92% nucleotide identity with the Amoebophilaceae family, indicating that the bacterium was likely to be a species of a novel genus. The species composition of bacterial communities varied between mite life stages regardless of their infection status. Uninfected adults exhibited greater species diversity than adults infected with O. tsutsugamushi. In the infected colony, the rate of filial infection with Orientia was less than 100%. Larval and male mites that were PCR-negative for Orientia contained low numbers of sequences of Amoebophilaceae (0.01 and 0.06%, respectively) in their taxonomic profiles, suggesting that a mutualistic relationship exists between the novel species of Amoebophilaceae and O. tsutsugamushi. Our study findings provide the basis for further research to determine the influence of the novel Amoebophilaceae species on the bacterial microbiome and on vector susceptibility to and transovarial transmission of O. tsutsugamushi.

Prevalence of Rickettsia Species (Rickettsiales: Rickettsiaceae) in Dermacentor variabilis Ticks (Acari: Ixodidae) in North Carolina.

The American dog tick, Dermacentor variabilis (Say), is a vector of spotted fever group (SFG) rickettsiae, including Rickettsia rickettsii the causative organism of Rocky Mountain spotted fever (RMSF). In North Carolina, SFG rickettsioses (including RMSF) are a leading cause of tick-borne illness. Knowledge of the infection rate and geographic distribution of D. variabilis ticks infected with Rickettsia spp. provides information on the spatial distribution of public health risk. Accordingly, we extracted genomic DNA from adult D. variabilis collected from field habitats in 32 North Carolina counties from 2009 to 2013. A nested PCR assay of the 23S-5S intergenic spacer (IGS) region of Rickettsia coupled with reverse line blot hybridization (RLBH) with species-specific probes was used to detect and identify rickettsiae to species. Approximately half of the 532 tick DNA samples exhibited a band of the expected size on agarose gels, indicating infection with Rickettsia spp. RLBH analyses showed R. amblyommatis (formerly 'Candidatus R. amblyommii'), R. parkeri, and R. montanensis were predominant, while other Rickettsia species detected included R. conorii-like, R. massiliae, R. rhipicephali, R. canadensis, R. bellii, and some unknown Rickettsia spp. Some ticks were infected with more than one Rickettsia species. Notably, several Rickettsia-positive ticks harbored R. rickettsii. DNA sequencing was performed on a portion of the 23S-5S IGS amplicons and the results were concordant with RLB assay results. We conclude that Rickettsia spp. are common in D. variabilis in North Carolina. Geographic patterns in the occurrence of Rickettsia-infected D. variabilis ticks across the counties sampled are discussed.