Spirochetes begin to synthesize OspC during the tick blood meal, while they are still in
the tick midgut, and the expression of ospC is subsequently down - regulated after transmission of B. burgdorferi to the mammal (3, 26, 27).
During tick feeding, which lasts for several days, bacteria migrate from
the tick midgut to the salivary glands, from which they are transmitted through the saliva (2).
Larval ticks from all three groups contained increased numbers of spirochetes, suggesting that the ospC mutant, in addition to the WT and complemented clones, could infect and replicate in
the tick midgut.
These data demonstrated that OspC was not necessary for spirochetes to colonize
the tick midgut but that mice remained refractory to infection with the ospC mutant, even by tick challenge.
The tick midgut contents were also subjected to RT - PCR.
Given that our wild type B. burgdorferi acquired by xenodiagnostic ticks retain the ability to express ospC (also shown by RT - PCR in Fig 4), we can not rule out the possibility that
a tick midgut - adapted phenotype, in the absence of salivation and feeding, contributed to the failure of the B. burgdorferi acquired from XT to infect SCID mice.
Langat virus infection (bright green) in
the tick midgut (black) is shown at six days after infection in this fluorescence image.
Here,
tick midguts were pooled per animal, pelleted, washed with 5 mL HBSS and then resuspended in HBSS in a final volume of 0.25 mL for mouse injections.
Not exact matches
Panel A = positive control for IFA using B. burgdorferi culture; Panel B = XT from animal IH11 (treated); Panel C = XT from animal IK14 (treated); Panel D = XT from animal IL09 (treated); Panel E = positive control for DFA using
midgut smear of
tick that was capillary tube - fed B. burgdorferi; Panel F = XT from animal IP55 (untreated); Panel G = XT culture pellet from animal IP55 (untreated); Panel H = XT culture pellet from animal IK14 (treated).
In addition to staining the
midgut contents with FITC - labeled polyclonal anti-Borrelia species antibody, we washed and re-stained this set and stained the second set of xenodiagnostic
tick (XT) samples with an anti-OspA monoclonal antibody (CB10, obtained from J. Benach [41]-RRB-, followed by anti-mouse IgG - Alexa 488 (Molecular Probes).
(A) IFA of
midgut tissues from partially fed nymphal
ticks infected with B31 - A3 WT, ospC7 mutant, or ospC7 / ospC +4 complemented B. burgdorferi clones.
We injected four mice with homogenized
midguts from partially fed, ospC7 - infected nymphs to determine whether the ospC mutant was infectious when obtained directly from a feeding
tick.
Aspects of the
midgut environment that change during
tick feeding, such as temperature, pH, and nutrients, influence the expression of many B. burgdorferi genes, including ospC (3, 9 - 11).
IFA analysis of
midguts from a subset of
ticks immediately after immersion demonstrated that a small number of spirochetes from all three strains had been ingested.
A
tick was scored positive if at least ten spirochetes were detected per
midgut.
In this model, OspA is proposed to be an adhesin that tethers the spirochetes to the
midgut epithelium (5, 6), whereas OspC is thought to be important for movement of the spirochete within the
tick (7, 8).
IFAs were performed on dissected
midguts from fed
ticks as described in ref.
In contrast to its defective phenotype in mice, the ospC mutant is competent to colonize the
tick vector and migrate from the
midgut to the salivary glands during
tick feeding, as is required for transmission.