Performing Department
Entomology
Non Technical Summary
Effective surveillance of vectors and pathogens is critical to monitor both current and future disease outbreaks, as early detection ensures that vector control efforts are directed to the right location at the right time to avert an epidemic. Surveillance and detection of infected mosquitoes is often challenging and requires sorting, identification and testing of large numbers of specimens while maintaining a cold chain to preserve RNA. To this end, research has progressed regarding the use of trap-mounted nucleic acid preservation (FTA) cards for sentinel and remote arboviral surveillance. Thus far, these approaches are limited by the use of qPCR for detection, which requires pathogen-specific PCR primers identified a priori. With a rapidly changing vector-borne disease landscape, it may not be possible to predict which pathogens are present as new threats emerge. Here we propose to combine the utility of FTA card sample collection with a powerful next-generation shotgun sequencing approach in field studies that illustrate the wealth of information that can be gleaned about the arboviral landscape from metagenomic analyses of sentinel FTA cards. These data will facilitate identification not only of known and unknown circulating arboviruses, but also of particular mosquito species present and their microbiota.Like mosquitoes, ticks are major arthropod vectors of human disease and spread a greater diversity of pathogens than any other arthropod. The majority of tick-borne diseases have been discovered only within the last 20 years as researchers develop proper means to survey both host and vector. We have recently used shotgun metagenome sequencing to describe novel bacteria and viruses within local NJ tick populations, including the newly discovered and invasive Asian longhorned tick Haemaphysalis longicornis (H. longicornis). Continuing this molecular surveillance will provide genome data with which to assess phylogenetic placement and pathogen status of the broadening catalog of tick microbiome constituents. Such comprehensive information will provide an unprecedented tool for rapidly assessing shifts in vector-borne disease transmission in a changing world.Of note, the introduction of H. longicornis poses a threat not only for human health, but for commercial animal production in the United States. This tick is a frequent ectoparasite of livestock animals including cattle, sheep, goats, and farmed deer. This species represents a serious threat to animal production for two primary reasons: 1) parthenogenetic populations (potentially requiring only a single tick) can quickly build up large infestations on animals, causing anemia and in extreme cases exsanguination and death, and (2) transmission of livestock diseases such as Theileria orientalis. By sequencing and assembling a high-quality H. longicornis genome sequence, we can predict full-length gene sequences and thus proteins that will be used to create anti-tick vaccines. With USDA-ARS collaborators at the Cattle Fever Tick Research Laboratory, synthetic tick proteins are injected into host farm animals to illicit antibody generation. These antibodies are then imbibed by the tick via bloodfeeding, and act to neutralize gene products within the tick that are critical for survival. Our approach combines cutting-edge sequencing technology in the form of PacBio Sequel II single-molecule sequencing instrumentation and Illumina Hi-C chromosome conformation capture to produce a hybrid chromosome-anchored assembly that will results in highest-quality gene models for reverse-vaccinology.
Animal Health Component
50%
Research Effort Categories
Basic
34%
Applied
33%
Developmental
33%
Goals / Objectives
Develop and strengthen effective surveillance and monitoring of disease vectors at local and regional scales, including the development and testing of novel trapping and vector/pathogen identification techniques. Under this objective, project participants will leverage and strengthen existing surveillance programs in a coordinated fashion to yield robust comparable data across large geographic scales.
Determine the ecology and geographic distribution of invasive and native disease vectors under changing environmental conditions to enhance our ability to predict conditions leading to existing and novel animal and human diseases.
Develop novel control and management interventions and test their impacts on the transmission of human and animal diseases.
Project Methods
To assess pathogen and microbial diversity within mosquito populations, we will utilize saliva and excreta collection from Flinders Technology Associates (FTA) cards. Mosquito pools will be trapped using gravid and host-seeking (Co2-baited) traps and honey-soaked FTA paper squares housed in 35mm slide holders will be introduced into the chamber for 24hrs. During this time, mosquitoes will sugar feed and salivate upont he card, and excrete upon a second card at the base of the chamber. Both cards are stored at -80C until analysis. Cards are cut into smaller squares with sterile scissors and soaked in TE buffer at 4 degrees for 12hrs, then extracted using the Qiagen QIAamp Viral RNA mini kit, with an additional pass through a QIAshredder column for homogenization. This process co-extracts RNA and DNA; the eluted nucleic acids are divided. RNA is DNAsed, DNA is RNAsed, and template in enriched using either Phi29-mediated whole-genome amplification or whole-transcriptome amplification in the case of RNA. Illumina next-generation sequencing libraries are created from this template, and sequenced on HiSeq and/or MiSeq instrumentation.The resulting raw reads are parsed against NCBI protein databases using high-throughput homology searches e.g., DIAMOND. Taxonomic information is retrieved for each top hit using custom python scripts and results are tabulated to discern the lineages present in the metagenomic samples. When applied to ticks, this protocol utilizes whole-body homogenate from pooled larvae or nymphs, and/or dissected internal structures of adults as opposed to FTA cards. Ticks are first surface-sterilized using 1% v/v bleach solution followed by a rinse in 70% ethanol and then 2x in sterile water. Should analyses show that potential pathogens are present, the reads are mapped to a reference (e..g, West Nile virus) genome to search for genetic polymorphisms. Using this methodology, we have discovered several novel WNV genotypes circulating in NJ in 2018. The mitochondrial haplotypes of U.S and foreign Haemaphysalis longicornis populations obtained by collaborator Andrea Egizi (Monmouth County NJ tick-borne disease research lab) will be generated by mapping the reads to the H. longicornis reference mitochondrial genome and generating a consensus sequence prior to population structure modeling.The Haemaphysalis longicornis genome project proceeds by first obtaining high-quality sequencing template via extraction and isolation of intact nuclei from egg masses, or alternately, dissected adult ticks. This DNA is then prepared for sequencing on the PacBio Sequel II single-molecule sequencer via the USDA-ARS Genomic and Bioinformatics Research Unit. Concurrently, Illumina HiSeq libraries will be prepared using a Hi-C chromosome conformation capture protocol implemented in collaborator Aviva Aiden's lab (Baylor College fo Medicine). Assembled PacBio long-read scaffolds are ordered and oriented to chromosomes as informed by the Hi-C data using the Juicebox toolkit developed by the Aiden lab. The final hybrid assembly will represent a high-quality chromosome-anchored genome sequence. Genes will be predicted on this assembly using RNAseq generated from multiple individuals, life stages and dissected tissue types. These data will be mined for reverse-vaccine candidates using manual and heuristic tools e.g., Vacceed (Goodswen et al. 2014). A ranked order of in-silico predicted candidate antigens with critical functions to tick feeding and development will be expressed in a Pichia expression system and purified prior to animal stall trials for assessment of tick mortality and morbidity at the USDA-ARS Cattle Fever Tick Research Laboratory with collaborator Adalberto Perez de Leon (USDA-ARS).