Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Entomology
Non Technical Summary
Trombiculid mites, principally Leptotrombidium species, are major vectors of scrub typhus; a potentially lethal illness to which millions of people in southern Asia, Japan, Korea and northern Australia are at risk. Approximately one million cases of scrub typhus are reported each year. Several Leptotrombidium mite species are involved as vectors but not all species are equally effective in maintaining or transmitting the pathogen. The pathogen, Orientia tsutsugamushi, is passed by mite females to the next generation through the egg stage. The pathogen is then passed vertically through successive developmental stages. Orientia tsutsugamushi is unlikely to exist as a single species microbial community within the mite host. More likely there are other microbes in the mite that exist as a complex community of bacterial species that can be expected to interact with each other and the mite host. With next-generation pyrosequencing, our research will derive novel information on changes in the composition of the bacterial community between developmental stages of the two Leptotrombidium species (L. imphalum and L. chiangraiensis) before and after infection with O. tsutsugamushi. These mite species manifest contrasting levels of vector competence. We will characterize changes in the population abundance of O. tsutsugamushi in Leptotrombidium eggs, larvae, deutonymphs and adults. Results of our exploratory investigation will provide background data needed for future research on the effect of the native microbial community on global gene expression in infected and uninfected Leptotrombidium species. This research could potentially increase our understanding of the biological basis for mite vector competence for O. tsutsugamushi.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Goals / Objectives
Trombiculid mites, principally Leptotrombidium species, are major vectors of scrub typhus; a potentially lethal illness to which millions of people in southern Asia, Japan and Korea are at risk. Approximately one million cases of scrub typhus are reported each year. Several Leptotrombidium mite species are involved as vectors but not all species are equally effective in maintaining or transmitting the pathogen. The pathogen, Orientia tsutsugamushi, is passed by mite females to the next generation through the egg stage. The pathogen is then passed vertically through successive developmental stages. Orientia tsutsugamushi is unlikely to exist as a single species microbial community within the mite host. More likely there are other microbes in the mite that exist as a complex community of bacterial species that can be expected to interact with each other and the mite host. With next-generation pyrosequencing, our research will derive novel information on changes in the composition of the bacterial community between developmental stages of the two Leptotrombidium species (L. imphalum and L. chiangraiensis) before and after infection with O. tsutsugamushi. These mite species manifest contrasting levels of vector competence. We will characterize changes in the population abundance of O. tsutsugamushi in Leptotrombidium eggs, larvae, deutonymphs and adults. Results of our exploratory investigation will provide background data needed for future research on the effect of the native microbial community on global gene expression in infected and uninfected Leptotrombidium species. This research could potentially increase our understanding of the biological basis for mite vector competence for O. tsutsugamushi.
Project Methods
Objective. Comparatively characterize bacterial communities in the whole-body microbiome of different developmental stages of laboratory-reared Leptotrombidium imphalum and L. chiangraiensis, two mite species that manifest different rates of infection with O. tsutsugamushi. We will also determine how the abundance and diversity of bacterial phylotypes vary in the developmental stages of two colony lines of mites of each Leptotrombidium species: one infected with O. tsutsugamushi and the other not infected.Experimental Approach. In the proposed research, we will take advantage of the Leptotrombidium-O. tsutsugamushi model transmission system developed in AFRIMS [Lurchachaiwong et al. 2012]. Leptotrombidium mite lines are maintained under identical insectary conditions and provided the same food, which should eliminate environmental conditions as a cause of differences in microbiome structure.Task 1. Leptotrombidium mite rearing and sample collection. We will use L. imphalum and L. chiangraiensis in the proposed research as these two species manifest different pathogen transmission dynamics. Both mite species are presently maintained in AFRIMS insectary facilities [Phasomkusolsil et al. 2009]. From these colonies, unfed Leptotrombidium larvae, deutonymphs and adults will be randomly collected and pooled by species and developmental stage (including adults) from infected and uninfected colony lines of each species. Mites will be preserved in 80% ethanol and shipped to North Carolina State University for DNA extraction.Task 2. DNA extraction. Genomic DNA will be extracted from individual mites and pools of collembolan eggs using published procedures [Ponnusamy et al. 2014]. DNA samples will be stored at -80°C for later use.Task 3. Quantitative real-time PCR to determine mite infection status. Filial infection rates may vary among infected females. Thus, the infection status of individual mites will be determined before they are pooled. Individual specimens of each life stage in the infected colony line for each mite species will be screened for O. tsutsugamushi in a quantitative real-time PCR assay. Quantitative real-time PCR (qPCR) is significantly more sensitive than conventional PCR in detecting O. tsutsugamushi [Kim et al. 2011]. With a CFX384 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), we will use primer pairs OtsuFP630 and OtsuRP747 to amplify a 118-bp fragment of a 47-kDa outer membrane protein gene from genomic DNA of individual mites in the infected colony line of each species [Jiang et al. 2004].Task 4. Sample processing, tag-encoded PCR amplification and pyrosequencing. After qPCR assays are completed, mite DNA samples will be separately pooled by species, stage and infection status. DNA samples will be pooled to increase the abundance of rare phylotypes. For pyrosequencing, we will use universal primers 27F and 534R, targeting the V1-V3 hypervariable region of 16S rRNA genes, to amplify an approximate 500 bp DNA fragment from each DNA sample [Lane 1991]. Bar-coded primers unique to each individual sample will give each microbial community a signature sequence tag. The resulting amplicons will be gel purified (QIAquick Gel Purification Kit, Qiagen), pooled in equal millimolar amounts and sequenced using the 454 Titanium pyrosequencing platform in the Microbiome Core Facility, which is located in the School of Medicine on the University of North Carolina campus (http://www.med.unc.edu/microbiome/) in Chapel Hill (see letter of support from facility director). There will be 3 replicate samples of pooled DNA for each of the infected and uninfected mite lines for each stage of Leptotrombidium species. This experimental design will result in the analysis of 52 DNA samples, which will require ½ of a 454 plate.Data processing and statistical analyses. Pyrosequence reads will be analyzed using the QIIME (Quantitative Insights into Microbial Ecology) pipeline [Caporaso et al. 2010]. In brief, sequences will be separated into samples by barcodes, and low quality reads will be filtered, leaving only high-quality sequences (>200-bp in length, quality scores >25 and exact barcode and primer matches). Sequences will be grouped into OTUs based on 97% sequence similarity to approximate species-level phylotypes using UCLUST [Edgar 2010]. Representative sequences from each cluster will be aligned against the Greengenes core dataset using PyNAST [Caporaso et al. 2010] and taxonomy will be assigned via the RDP-classifier [Lan et al. 2012]. Similar to our research with A. americanum [Ponnusamy et al. 2014], we will use Chao1 in rarefaction analyses to estimate richness of OTUs in the microbiomes of mite samples by species, developmental stage and infection status. Phylogenetic trees will be constructed de novo using FastTree [Price et al. 2010]. Phylogenetic analyses for each developmental stage and infection status within and between Leptotrombidium species should reveal changes in sequence structure of OTUs classified in the Rickettsiales. The UniFrac metric will be used to determine the degree of similarity between samples [Lozupone and Knight. 2005] coupled with principle coordinate analysis to graphically illustrate clustering of mite DNA samples by species, developmental stage and infection status.REFERENCES CITEDCaporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. D. Bushman, E. K. Costello, N. Fierer, A. G. Peña, J. K. Goodrich, J. I. Gordon, G. A. Huttley, S. T. Kelley, D. Knights, J. E. Koenig, R. E. Ley, C. A. Lozupone, D. McDonald, B. D. Muegge, M. Pirrung, J. Reeder, J. R. Sevinsky, P. J. Turnbaugh, W. A. Walters, J. Widmann, T. Yatsunenko, J. Zaneveld, and R. Knight. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7:335−336.Edgar, R. C. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinform. 26:2460−2461.Jiang, J., T.-C. Chan, J. J. Temenak, G. A. Dasch, W.-M. Ching, and A. L. Richards. 2004. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am. J. Trop. Med. Hyg. 70:351−356.Kim, D.-M., G. Park, H. S. Kim, J. Y. Lee, G. P. Neupane, S. Graves, and J. Stenos. 2011. Comparison of conventional, nested, and real-time quantitative PCR for diagnosis of scrub typhus. J. Clin. Microbiol. 49:607−611.Lan, Y., Q. Wang, J. R. Cole, and G. L. Rosen. 2012. Using the RDP classifier to predict taxonomic novelty and reduce the search space for finding novel organisms. PLoS ONE 7:e32491.Lane, D. 1991. 116S/23S rRNA sequencing. pp. 115-117, In: Stackebrandt, E. and Goodfellow, M. (eds.). Nucleic acid techniques in bacterial systematics. John Wiley and Sons, NY.Lozupone, C. and R. Knight. 2005. UniFrac: A new phylogenetic method for comparing microbial communities. Appl. Environ. Entomol. 71:8228−8235.Lurchachaiwong, W., T. Monkanna, S. Leepitakrat, A. Ponlawat, J. Sattabongkot, A. L. Schuster, P. W. McCardle, and A. L. Richards. 2012. Variable clinical responses of a scrub typhus outbred mouse model to feeding by Orientia tsutsugamushi infected mites. Expt. Appl. Acarol. 58:23−34.Phasomkusolsil, S., P. Tanskul, S. Ratanatham, P. Watcharapichat, D. Phulsuksombati, S. P. Frances, K. Lerdthusnee, and K. J. Linthicum. 2009. Transstadial and transovarial transmission of Orientia tsutsugamushi in Leptotrombidium imphalum and Leptotrombidium chiangraiensis (Acari: Trombiculidae). J. Med. Entomol. 46:1442-1445.Ponnusamy, L., A. Gonzalez, W. Van Treuren, S. Weiss, C. Parobek, J. J. Juliano, R. Knight, R. M. Roe, C. S. Apperson, and S. R. Meshnick. 2014. Diversity of Rickettsiales in the microbiome of the lone star tick, Amblyomma americanum. Environ. Appl. Microbiol. 79: doi:10.1128/AEM.02987-13.Price, M.N., P.S. Dehal, and A.P. Arkin. 2010. FastTree 2 -- Approximately maximum-likelihood trees for large alignments. PLoS ONE 5:e9490.