Molecular Interactions that Enable Movement of the Lyme Disease Agent from the Tick Gut into the Hemolymph
Figure 5
F(ab)2 fragments of anti-BBE31 IgG interfere with B.burgdorferi migration from I.scapularis gut into hemolymph.
(A) Borreliacidal activity of BBE31 F(ab)2 fragments against B. burgdorferi. B.burgdorferi N40 were incubated in BSK medium in the absence (Control) or presence of F(ab)2 fragments prepared from normal rabbit serum [NRS F(ab)2] or BBE31 antiserum [BBE31 F(ab)2]. A borreliacidal serum from a patient with diagnosed Lyme Disease (B.burgdorferi antisera) was used as a positive control. The number of spirochetes was assessed by dark-field microscopy after 24 and 48 hours of incubation. Data are presented relative to controls without treatment. Data represent the number of spirochetes remaining viable after treatment (mean ± SD, n = 4). Difference between control and NRS F(ab)2- as well as BBE31 F(ab)2-treated samples were not statistically significant. (B) B.burgdorferi burden in I.scapularis hemolymph assayed by q-PCR. N40-infected nymphs fed on C3H mice, which had been injected with F(ab)2 fragments of either normal rabbit IgG (NRS-F(ab)2) or anti-BBE31 IgG (BBE31-F(ab)2), for 66 hours. DNA was extracted from the hemolymph for q-PCR analysis. Each dot represents hemolymph from 5 ticks. Data were collected from 2 times of tick-feedings. *: p<0.05. (C) B.burgdorferi burden in I.scapularis hemolymph assayed by confocal microscope. Tick feeding is the same as that described in part (A). The spirochetes in tick hemolymph were probed with FITC-labeled goat anti-borrelia antibodies (shown in green), the hemocyte nucleolus were stained with TO-PRO-3 (shown in blue). The FITC and TO-PRO-3 images were examined at ×25 magnifications and are presented as merged images. Scale bar represents 20 µm. (D) Spirochetes lacking bbe31 can survive in tick hemolymph and invade the salivary glands. B.burgdorferi wild-type strain MSK5 and the lp25-lacking isolate ML23 were grown in BSK medium, resuspended in PBS and microinjected into partially-fed clean nymphal hemocoel (clean nymphs fed on C3H mice for 48 hours before microinjection). Six hours later, both hemolymph (HL) and salivary glands (SG) were collected for B.burgdorferi quantification by q-PCR. Mean and SD were calculated from 5 mRNA samples and 5 ticks were grouped for 1 mRNA sample.