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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research » Research » Research Project #421445

Research Project: An in Vivo Negative Selection Screen to Identify Genes and Gene Families Required for Tick Colonization

Location: Animal Disease Research

2011 Annual Report

1a. Objectives (from AD-416)
Identify metabolic and other functional pathways, genes and gene families required for tick colonization. Specific aim 1: Using Dermacentor andersoni ticks for negative selection, screen pools of Francisella tularensis subsp. novicida mutants to identify those mutants that have reduced fitness in the midgut or salivary glands. Specific aim 2: Verify the phenotype of a subset of the F. novicida mutants with reduced fitness in the midgut or salivary glands using dual infection experiments.

1b. Approach (from AD-416)
A variety of intracellular bacterial pathogens of both humans and animals are transmitted by ticks and include members of the genus Anaplasma, Ehrlichia, and Rickettsia. Not only are the molecular mechanisms by which these pathogens are able to colonize the tick largely unknown. But, identification of these mechanisms is difficult, if not impossible, as the techniques required for genetic manipulation of this group of pathogen are in the early stages of development. In contrast to this limitation for tick-borne bacterial pathogens, exploitation of genomic sequence data through mutant library screens has allowed for relatively rapid identification of genes required for specific functions in a broad array of bacterial pathogens. Similarly, a transposon mutant library of Francisella tularensis subsp. novicida has been developed and used to negatively select and identify genes required for pulmonary and systemic infection in mice. We have determined that F. novicida readily colonizes D. andersoni in a manner similar to other tick borne pathogens, including A. marginale. Through a negative selection screen and high throughput sequencing, we propose to use this mutant library to identify genes required for tick colonization.

3. Progress Report
In collaboration with colleagues at Washington State University, ARS scientists in Pullman, WA have established that Francisella tularensis subsp. novicida initially infects the midgut of 100% of acquisition fed Dermacentor andersoni nymphs with stable colonization and replication during a subsequent molt. Increased dissemination to and florid replication within the salivary gland was closely linked to a second (transmission) feed and culminated in bacterial secretion into the saliva and successful transmission. Testing multiple mutants resulted in total bacterial levels similar to that observed for single mutants. However, there was evidence of a bottleneck during colonization resulting in a founder effect in which the most successful mutant varied when comparing individual ticks. Thus, it is essential to assess mutant success at the level of the tick population rather than in individual ticks. The ability of F. novicida to recapitulate the key physiological events by which bacteria colonize and are transmitted by ixodid ticks provides a new genome-wide approach to identify required pathogen molecules and pathways. A manuscript describing these experiments and results is in review.

4. Accomplishments