Summary of results.

<p>Summary of results.</p

Histopathology and <i>B</i>. <i>burgdorferi</i> (Bb) within tissues of persistently-infected macaques.

<p>At 12–13 months post-infection, necropsy tissues were analyzed for evidence of tissue pathology. Frozen sections of affected tissues were then stained with fluorescently-labeled Borrelia-specific polyclonal antibodies. Shown here is: axillary lymph node hyperplasia and histiocytosis-200X (A) and Bb antigen (D) in untreated animal IN16; cervical spinal cord, focal inflammatory lesion-200X (B) and Bb antigen (E) from treated animal IH11; and a focal area of mononuclear inflammation in the myocardial interstitium-1000X (C) and Bb antigen (F) from untreated animal IN05.</p

Culture and PCR results.

<p>Culture and PCR results.</p

Amplification of <i>B</i>. <i>burgdorferi</i>-specific DNA in tissues showing histopathology.

<p>Tissue DNA was subjected to amplification of a region within the 5s-23s ribosomal genes using a nested set of primers. The positive control (+) consisted of B31.5A19 DNA and negative controls were no template control (NTC) and uninfected monkey skin tissue DNA (Ctrl mk). Tissues tested included peripheral nerves (PN), axillary (ax) and mesenteric (mes) lymph nodes (LN), lung, meninges/dura mater (dura), spleen, cerebrum, skeletal muscle (sk musc), heart, spinal cord (SC), and joint synovium (jt syno). Clear positives are marked with (*) and potential positive results are marked with (^). The treated monkeys are labeled with grey text.</p

Experimental design for evaluation of antibiotic treatment following tick-mediated infection.

<p>Xenodiagnoses were conducted with uninfected nymphal ticks.</p

Detection of <i>B</i>. <i>burgdorferi</i> within XT by immunostaining and RT-PCR.

<p>Immunostaining reveals <i>Borreliae</i> within XT from both treated and untreated monkeys. Tick midguts were stained for IFA with an anti-OspA monoclonal antibody, followed by an Alexa-IgG-488(green)-conjugated secondary antibody (B,C and D) or stained for DFA using an anti-<i>Borrelia</i> sp. FITC-conjugated polyclonal antibody (E, F). In addition, cultures of tick midgut contents were pelleted and stained for IFA (G, H). Panel A = positive control for IFA using <i>B</i>. <i>burgdorferi</i> 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 <i>B</i>. <i>burgdorferi</i>; 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). RT-PCR for <i>ospA</i> (I) and <i>ospC</i> (J) transcripts indicate the presence of viable <i>B</i>. <i>burgdorferi</i>. RNA was extracted from XT following the first xenodiagnosis (~7 months p.i.). Cohort-matched control XT derived from feeding on clean mice were used and are indicated by m1, m2 and m3.). Clear positives are marked with (*) and potential positive results are marked with (^).</p

Spirochetes identified from <i>in vivo</i> culture of monkey heart tissue.

<p>The DMC-cultured heart tissue was placed in BSK and back to <i>in vitro</i> culture for 1 week. The culture pellets were then stained with a combination of anti-OspA and anti-OspC monoclonal antibodies, followed by a red detection antibody (Panels A,B,C) or with anti-OspA followed by a green detection antibody (Panels D,E,F). Shown are differential interference contrast (Panel A) and fluorescent detection (Panels B and C) of cultures stained with monoclonal antibodies. Panels A and B: animal IN05 (untreated); Panel C: animal IH22 (treated). Panel D is a positive control <i>B</i>. <i>burgdorferi</i> culture; Panels E and F are stained specimens from heart cultures of monkeys IK14 and IL09 (both treated), respectively.</p

Longitudinal antibody responses to five <i>B</i>. <i>burgdorferi</i> antigens over the course of infection.

<p>Each graph represents one animal, with the untreated monkeys shown in the left column and the treated animals on the right. Animals were treated with doxycycline between weeks 16–20. Vertical axis: MFI = mean florescent intensity. Ncr = blood collected at necropsy. Shown is the mean ±SEM for each time point.</p

Variable manifestations, diverse seroreactivity and post-treatment persistence in non-human primates exposed to <i>Borrelia burgdorferi</i> by tick feeding

<div><p>The efficacy and accepted regimen of antibiotic treatment for Lyme disease has been a point of significant contention among physicians and patients. While experimental studies in animals have offered evidence of post-treatment persistence of <i>Borrelia burgdorferi</i>, variations in methodology, detection methods and limitations of the models have led to some uncertainty with respect to translation of these results to human infection. With all stages of clinical Lyme disease having previously been described in nonhuman primates, this animal model was selected in order to most closely mimic human infection and response to treatment. Rhesus macaques were inoculated with <i>B</i>. <i>burgdorferi</i> by tick bite and a portion were treated with recommended doses of doxycycline for 28 days at four months post-inoculation. Signs of infection, clinical pathology, and antibody responses to a set of five antigens were monitored throughout the ~1.2 year study. Persistence of <i>B</i>. <i>burgdorferi</i> was evaluated using xenodiagnosis, bioassays in mice, multiple methods of molecular detection, immunostaining with polyclonal and monoclonal antibodies and an <i>in vivo</i> culture system. Our results demonstrate host-dependent signs of infection and variation in antibody responses. In addition, we observed evidence of persistent, intact, metabolically-active <i>B</i>. <i>burgdorferi</i> after antibiotic treatment of disseminated infection and showed that persistence may not be reflected by maintenance of specific antibody production by the host.</p></div

Tick-mediated inoculation of <i>B</i>. <i>burgdorferi</i> produces different levels of erythema in monkeys.

<p>Ticks were allowed to feed to repletion on the backs of rhesus macaques for infection (Panel A). One monkey produced a bona fide erythema migrans rash, seen one week after tick removal (Panel B), whereas the other monkeys produced only small, diffuse erythema at the site of tick feeding (Panel C).</p