Development of a Recombinase-Mediated Cassette Exchange System for Gene Knockout and Expression of Non-Native Gene Sequences in .

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.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines.

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes.

Comparative Whole Genome Analysis of an Strain Isolated from Norwegian Sheep.

is a Gram-negative obligate intracellular tick-borne alphaproteobacteria (family Anaplasmatacea, order Rickettsiales) with a worldwide distribution. In Norway, tick borne fever (TBF), caused by , presents a major challenge in sheep farming. Despite the abundance of its tick vector, , and infections in wild and domestic animals, reports of infections in humans are low compared with cases in the U.

Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

is the principal symbiotic bacterium of the medically significant tick This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of , it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of naturally occurring in ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated.

Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding.

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.

Evidence for neuromodulation of enteropathogen invasion in the intestinal mucosa.

The extensively innervated intestinal mucosa encompasses a vast surface exposed to an array of potentially infectious microorganisms. We investigated the role of enteric nerves in modulating intracellular internalization of a multidrug-resistant Salmonella typhimurium DT104 field isolate in mucosa-submucosa sheets from the porcine ileum, a biomedical model for the human intestine. The effects of transmural electrical stimulation and drugs on intracellular internalization of Salmonella over 90 min was determined by a gentamicin-resistance assay relative to untreated tissues from the same animal serving as controls.

Catecholamines and sympathomimetic drugs decrease early Salmonella Typhimurium uptake into porcine Peyer's patches.

Peyer's patches of the small intestine serve as inductive sites for mucosal immunity as well as targets for invasive enteropathogens, including Salmonella. Because they are innervated by catecholamine-containing enteric nerves, the hypothesis that the endogenous catecholamines dopamine and norepinephrine or sympathomimetic drugs alter Salmonella Typhimurium uptake into Peyer's patches was tested. Porcine jejunal Peyer's patch explants were mounted in Ussing chambers and inoculated with a porcine field isolate of Salmonella Typhimurium DT104.

Characterization of Salmonella enterica serovar Typhimurium DT104 invasion in an epithelial cell line (IPEC J2) from porcine small intestine.

Salmonella Typhimurium DT104 is an emerging enteric pathogen in swine of increasing medical importance. In this study, the time course and the actin-dependent host signaling processes necessary for invasion of a S. Typhimurium DT104 field isolate were investigated in IPEC J2 epithelial cells derived from porcine small intestine.