Uniformed Services University of The:·health Sciences

Bartonella ssp. have gained importance as etiologic agents of human disease, both in temperate and tropical regions. Reports of increasing numbers of clinical cases of bartonellosis in Peru (B. bacilli/ormis), documentation of chronic bacteremia in domestic cats with cat scratch fever (B. henselae), and the association of bacillary angiomatosis and parenchymal peliosis (R. henselae and B. quintana) in AIDS patients demand improved laboratory diagnostic detection and isolation techniques for this fastidious organism. We report successful culture and polymerase chain reaction (PCR) techniques applicable for this purpose. Lyophilized B. bacilli/armis was suspended in PBS and and cultured on blood and chocolate agar plates to verify survival. Characteristic colonies were used to seed an 8% suspension of human red blood cells in RPMI 1640 media with 10% FBS and 0.7% NaHC03. Aliquots incubated at 280C in a candle jar for 3-7 days showed numerous, pleomorphic intraerythrocytic bacteria when thin smears were stained with Giemsa. Ethidium bromide staining and visualization using ultraviolet light of fixed smears of washed red cells showed numerous fluorescent organisms within the cells. B. henselae was similarly cultivated and detected after incubation at 370C. This culture system allows for early presumptive detection of Bartonella ssp., taking advantage of the organism's predeliction for intraerythrocytic habitation and the ability to stain fixed RBCs. For PCR, primers were designed to amplify regions between the16S and 23S rRNA genes of Bartonella, or the entire spacer region. In each case, the primers represented sequences conserved among Bartonella species, and the procedures amplified variable regions 40-1700 bp in length that should be useful for distinguishing species of Bartonella and for molecular epidemiology in restriction length polymorphism analysis. The author hereby certifies that the use ofany copyrighted material in the thesis manuscript entitled: "Culture, Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Smdies in Bartonella bacilliformis" beyond briefexcerpts is with the pennission ofthe copyright owner, and will save and hold harmless the Uniformed Services University ofthe Health Sciences from any damage which may arise from such copyright violations. Eve Carroll Zentrich Department ofPreventive Medicine and Biometrics Uniformed Services University ofthe Health Sciences Culture. Polymerase Chain Reaction, and Restriction ~raKmenLLen.K1hPolymoruhi~m Stydies on Bartonella