Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available. To facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin. These transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.

A reduction in the use of animals in infectious disease research is desirable for animal welfare as well as for simplification and standardization of experiments. An artificial silicone-based membrane-feeding system was adapted for complete engorgement of adult and nymphal Ixodes scapularis Say (Acari: Ixodidae), and for infecting nymphs with pathogenic, tick-borne bacteria. Six wild-type and genetically transformed strains of four species of bacteria were inoculated into sterile bovine blood and fed to ticks. Pathogens were consistently detected in replete nymphs by polymerase chain reaction. Adult ticks that ingested bacteria as nymphs were evaluated for transstadial transmission. Borrelia burgdorferi and Ehrlichia muris -like agent showed high rates of transstadial transmission to adult ticks, whereas Anaplasma phagocytophilum and Rickettsia monacensis demonstrated low rates of transstadial transmission/maintenance. Artificial membrane feeding can be used to routinely maintain nymphal and adult I. scapularis , and infect nymphs with tick-borne pathogens.

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts—Rickettsia peacockii and R. buchneri—both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing R. buchneri and R. peacockii will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.

Although rickettsiae can cause life-threatening infections in humans worldwide, no licensed vaccine is currently available. To evaluate the suitability of live-attenuated vaccine candidates against rickettsioses, we generated a Rickettsia parkeri mutant RPATATE_0245::pLoxHimar (named 3A2) by insertion of a modified pLoxHimar transposon into the gene encoding a phage integrase protein. For visualization and selection, R. parkeri 3A2 expressed mCherry fluorescence and resistance to spectinomycin. Compared to the parent wild type (WT) R. parkeri, the virulence of R. parkeri 3A2 was significantly attenuated as demonstrated by significantly smaller size of plaque, failure to grow in human macrophage-like cells, rapid elimination of Rickettsia and ameliorated histopathological changes in tissues in intravenously infected mice. A single dose intradermal (i.d.) immunization of R. parkeri 3A2 conferred complete protection against both fatal R. parkeri and R. conorii rickettsioses in mice, in association with a robust and durable rickettsiae-specific IgG antibody response. In summary, the disruption of RPATATE_0245 in R. parkeri resulted in a mutant with a significantly attenuated phenotype, potent immunogenicity and protective efficacy against two spotted fever group rickettsioses. Overall, this proof-of-concept study highlights the potential of R. parkeri mutants as a live-attenuated and multivalent vaccine platform in response to emergence of life-threatening spotted fever rickettsioses.

Rickettsia spp. are found in a diverse array of organisms, from ticks, mites, and fleas to leeches and insects. Many are not pathogenic, but others, such as Rickettsia rickettsii and Rickettsia prowazeckii , can cause severe illness or death.

Background/Objectives: Incidence of vector-borne diseases, including rickettsioses and anaplasmosis, has been increasing in many parts of the world. The obligate intracellular nature of rickettsial pathogens has hindered the development of robust genetic tools for the study of gene function and the identification of therapeutic targets. Transposon mutagenesis has contributed to recent progress in the identification of virulence factors in this important group of pathogens. Methods: Combining the efficiency of the himar1 transposon method with a recombinase-mediated system, we aimed to develop a genetic tool enabling the exchange of the transposon with a cassette encoding non-native sequences. Results: This approach was used in Rickettsia parkeri to insert a himar1 transposon encoding fluorescent protein and antibiotic resistance genes for visualization and selection, flanked by mismatched loxP sites to enable subsequent recombinase-mediated cassette exchange (RMCE). RMCE mediated by a plasmid-encoded Cre recombinase was then employed to replace the transposon with a different cassette containing alternate fluorescent and selection markers and epitopes of Anaplasma phagocytophilum antigens. The resulting genetically modified R. parkeri was trialed as a live-attenuated vaccine against spotted fever rickettsiosis and anaplasmosis in mice. Conclusions: The use of this system provides a well-established and relatively efficient way of inserting non-native sequences into the rickettsial genome, with applications for the study of gene function and vaccine development.