The isolation of the Lyme disease spirochete (Borrelia burgdorferi) from the southeastern United States is reported. Three isolates, two from cotton mice (Peromyscus gossypinus) and one from the black-legged tick (Ixodes scapularis), were recovered from Sapelo Island, Georgia, in July and September 1991. The spirochetes were characterized by indirect fluorescent antibody assay using a battery of five monoclonal antibodies, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of whole cell lysates, and by the polymerase chain reaction (PCR) assay using primers for three DNA target sequences found in B. techniques (20). Before culturing, mouse ears were cleaned with 95% ethanol and ear clips were sliced into small pieces or several 2-mm punches were taken from each ear. Ear clips or punches were cleaned again in 95% ethanol followed by a rinse in a 1:1 mixture of 10% Clorox and 95% ethanol. Ear punches were then placed into 1.25 ml BSK II medium (18) and small ear slices were placed in 4.5 ml of medium. All cultures were incubated in a 5% CO2 atmosphere at 33-34°C and examined for spirochetes by darkfield microscopy twice weekly for 2 weeks and, if spirochetes were not detected, weekly thereafter for 6 weeks. Spirochetal isolates were tested by indirect fluorescent antibody analysis (21, 22) using five monoclonal antibodies, including two B. burgdorferi-specific anti-outer surface protein A (OspA) monoclonals (H3TS and H5332), two B. burgdorferi-specific anti-outer surface protein B (OspB) monoclonals (HSTS and H6831), and a Borrelia (genus)-specific anti-flagellin monoclonal antibody (H9724).Preparation ofeach spirochetal culture for characterization by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) involved whole spirochetal lysates (23, 24). Aliquots of 20 ml each were centrifuged at 10,000 x g for 30 min. Supernatants were removed and spirochetal pellets were suspended in 1 ml of phosphate-buffered saline (PBS: 120 mM NaCl/2.7 mM KCl/10 mM Na2HPO4/10 mM KH2PO4, pH 7.4) with 5 mM MgCl2 by vortex mixing until evenly dispersed. Samples were centrifuged again at 10,000 x g for 15 min. After a second PBS/MgC12 wash, spirochetal pellets were resuspended in 0.1 ml of sterile distilled water and then frozen at -80°C for 30 min, thawed, and refrozen. After the final thaw, the suspended spirochetal pellets were vortex-mixed and an aliquot was then removed for total Abbreviation: LD, Lyme disease.tTo whom correspondence should be addressed. 7371

No abstract

The anticonvulsant phenytoin causes a decrease in plasma concentrations of folate in epileptic patients. The mechanism underlying this depletion is unknown. To study this mechanism, phenytoin was administered to rats by addition to the diet (3 g phenytoin/kg diet) for up to 8 wk. At selected times during phenytoin administration (0, 3, 7, 10, 14, 28, 42 and 56 d), the composition of the folate pools of intestinal mucosa, liver, bile and brain was determined. The 0-d administration served as the control group. The controls were fed the same diet without phenytoin for the eight weeks of the experiment. Phenytoin administration had minimal effect on either the folate concentration or the composition of the folate pool in intestinal mucosa. Phenytoin administration did, however, cause a depletion of total hepatic folate to about 50% of control, causing the pentaglutamate derivatives of each of the pteridine derivatives to decline rapidly, with the formyl and dihydro derivatives of the pteridine moiety falling more rapidly than the methyl and methylene + tetrahydro derivatives. The monoglutamate of the methylene + unsubstituted tetrahydro derivative increased significantly with time of phenytoin treatment. The mono- and di-glutamate derivatives of the methyltetrahydrofolate increased transiently and significantly in the bile, and the polyglutamate chain length increased significantly in the brain with time of phenytoin treatment. We conclude that phenytoin inhibits the formation of polyglutamyl folates in rat liver.

We investigated the use of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi strain B-31 in human blood and urine experimentally inoculated with 5 and 1 borreliae/cm³, respectively, and to biotinylate a DNA probe specific for B. burgdorferi in the dot blot and Southern blot assays. When the blood and urine samples were subjected to PCR, a 370-bp amplified product was consistently visible on agarose gel electrophoresis after 30 and 45 cycles, respectively. The total human genomic DNA extracted from a 1-cm³ sample of inoculated blood was approximately 6.25 µg, and the total amount of B. burgdorferi DNA was estimated to be 0.01 pg/6.25 µg of the human DNA. For PCR, 2.5 µg of human DNA which contained the equivalent of 0.004 pg of borrelia DNA (approximately two borreliae) were used for enzymatic amplification. When 1/20 or 1/10 of the PCR-amplified products were used either for dot blot or Southern blot hybridization, the accessible copies of amplified B. burgdorferi DNA were sufficient for detectable hybridization to occur. PCR amplification of B. burgdorferi DNA in clinical specimens followed by dot blot hybridization may be a valuable adjunct or alternative to current but inadequate laboratory methods for the diagnosis of Lyme disease.

Larvae and nymphs of Ixodes dammini Spielman, Piesman, Clifford & Corwin from a laboratory colony were fed on two white-tailed deer, Odocoileus virginianus (Zimmerman) inoculated with either the SH2-82 or JD-1 strains of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner. Ticks were exposed to one deer 43 and 69 d after inoculation of the spirochete and to a second deer 35 and 61 d after inoculation. Polymerase chain reaction assays amplified the 158 bp OspA DNA target sequence in 11.1% (n = 9) of fed larvae and 3.3% (n = 30) of nymphs from the deer inoculated with the SH2-82 strain, and 22.7% (n = 22) of larvae and 0% (n = 21) of nymphs from a second deer inoculated with the JD-1 strain of B. burgdorferi. One of three females derived from nymphs fed on one of the inoculated deer showed presence of B. burgdorferi DNA, but none of four males was positive. Experimentally inoculated deer can serve as a source of at least two geographic strains of B. burgdorferi to I. dammini larvae and nymphs for at least several weeks.