Influence of outer surface protein A antibody on Borrelia burgdorferi within feeding ticks - PubMed (original) (raw)

Influence of outer surface protein A antibody on Borrelia burgdorferi within feeding ticks

A M de Silva et al. Infect Immun. 1999 Jan.

Abstract

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick's salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 microgram/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 microgram/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induce ospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.

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Figures

FIG. 1

Expression of flaB and ospC by spirochetes within feeding ticks. RNA was prepared from _B. burgdorferi_-infected nymphal ticks that had not fed (unfed ticks) or that had fed for 60 h (feeding ticks). Quantitative RT-PCR was carried out with flaB and ospC primers to estimate the levels of flaB and ospC expression by spirochetes in ticks. Quantitative PCR was performed by adding a known constant amount of ospC or flaB competitor DNA fragments to decreasing amounts of the cDNA preparations and then performing PCR with flaB or ospC primers. Each PCR mixture for flaB (top panels) contained 14 fg of the flaB competitor, and each mixture for ospC (bottom panels) contained 3.5 fg of the ospC competitor. The cDNA preparations from unfed and partially fed ticks were diluted to obtain decreasing cDNA concentrations in the PCR. Lanes 1 to 6, samples with cDNA from unfed ticks that were undiluted and diluted 1:2, 1:4, 1:8, 1:16, and 1:32, respectively. Lanes 8 to 13, samples with cDNA from partially fed ticks that were undiluted and diluted 1:30, 1:60, 1:120, 1:240, and 1:480, respectively. No cDNA was added to lanes 7 and 14. Because the number of spirochetes increases during tick feeding, it was necessary to dilute the cDNA from feeding ticks more than the cDNA from unfed ticks to be within the sensitive range of the assay. In each panel the higher band corresponds to the amplified competitor, while the lower band corresponds to the amplified target. For each panel, in the lane in which the competitor and the target PCR products were at similar intensities, the amounts of target DNA and competitor DNA were assumed to be present at equimolar concentrations. Unfed ticks had 3 fg of flaB cDNA per tick, and feeding ticks had 88 fg of flaB per tick. Unfed ticks did not have detectable ospC cDNA, and feeding ticks had 10 fg of ospC cDNA per tick. The sensitivity of the ospC RT-PCR was 0.12 fg of cDNA.

FIG. 2

Expression of flaB and ospC by feeding ticks containing different numbers of spirochetes due to OspA antibody treatment. Groups of mice were passively immunized with 200 μl of hyperimmune OspA antiserum at dilutions of 1:10 (lanes 2 and 3), 1:25 (lanes 4 and 5), 1:50 (lanes 6 and 7), and 1:200 (lanes 8 and 9) prior to challenge with _B. burgdorferi_-infected ticks. Sixty hours into the blood meal, 60 ticks were removed from mice in each group and RNA was prepared for RT-PCR with flaB (upper panel) or ospC (lower panel) primers. Lanes 1, DNA molecular weight markers; lanes 3, 5, 7, and 9, samples used in PCR without reverse transcription; lanes 2, 4, 6, and 8, samples used in PCR after reverse transcription. Amplified products were observed only in samples that had been reverse transcribed, indicating that the RNA preparations were not contaminated with DNA. flaB expression was detected in ticks recovered from all four groups, while strong ospC expression was detected in the 1:50 and 1:200 dilution groups. Weak ospC expression was detected in the 1:25 dilution group, and ospC expression was not evident in the 1:10 dilution group. In these cDNA preparations, not enough flaB and ospC cDNAs were available for quantitative PCR studies.

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Influence of outer surface protein A antibody on Borrelia burgdorferi within feeding ticks - PubMed (original) (raw)