Isolation and transmission of the Lyme disease spirochete from the southeastern United States.

Abstract

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

Borrelia burgdorferi Not Confirmed in Human-Biting Amblyomma americanum Ticks from the Southeastern United States

“…Vector competency studies have concluded repeatedly that B. burgdorferi is unlikely to be transmitted by A. americanum (10)(11)(12)(13)(14)(15)(16), and a potent borreliacidal agent has been identified in A. americanum saliva (17,18). From 1997 to 2010, the Department of Defense (DOD) Human Tick Test Kit Program (HTTKP), a tick identification and PCR testing service provided by the U.S. Army Public Health Command (USAPHC) for DOD personnel and dependents, tested 22,565 A. americanum ticks (21,245 adults and nymphs removed from humans, and 1,320 field-collected larvae) for borrelial agents of Lyme-like diseases using a series of nested-and real-time PCR assays.…”

Evidence ofBorrelia lonestariDNA inAmblyomma americanum(Acari: Ixodidae) Removed from Humans

“…At first, analysis targeted B. burgdorferi, because A. americanum was suspected as a vector of Lyme disease. However, vector competency studies indicated that B. burgdorferi is rarely transmitted by A. americanum (19,21,22,25,26,30,32) b Corresponds to sequence data generated in this study, under GenBank accession numbers AY237656, AY237667, AY237671, AY237677, AY237678, AY237679, and AY237707.…”

Use of a sentinel host system to study the questing behavior of Ixodes spinipalpis and its role in the transmission of Borrelia bissettii, human granulocytic ehrlichiosis, and Babesia microti.

“…3 In the southeastern states, an anomalous situation exists where Ixodes scapularis is widespread but is rarely found infected with B. burgdorferi or attaching to humans. 4 Although B. burgdorferi isolations from humans are made, 5 such isolations are uncommon in this region, although a clinically similar disease, termed Southern Tick-Associated Rash Illness (STARI), is not unusual and is associated with the bites of Amblyomma americanum ticks. 6 Lyme disease in the western United States is both less common and ecologically more complex than described on the East Coast.…”